ABSTRACT
Medical plants play an
important role in the management of diabetes mellitus especially in developing
countries where resources are meager. Plant-based drugs have been used against
various diseases since a long time. The nature has provided abundant plant
wealth for all the living creatures, which possess medicinal virtues.
Therefore, there is a necessity to explore their uses and to conduct pharmacognostic and pharmacological studies to ascertain
their therapeutic properties. In fact, nowadays, diabetes is a global problem.
Hence, the present study aims to open new avenues for the improvement of
medicinal uses of Verbesina encelioides Benth. roots for the selected area
for diabetes.. Dried aqueous and alcoholic extracts were subjected for hypoglycaemic activity in swiss
albino mice (30-40g). Blood sugar level was determined using digital glucometer. The oral administration of roots extracts at
doses of 400 mg/ kg lead to a significant blood glucose reduction in normal and
in Streptozotocin, alloxan
diabetic mices significantly within 4 h. Continued,
daily administration of the drug produced a sustained effect.
KEYWORDS: Verbesina encelioides , Alloxan-induced
diabetes, Streptozotocin -induced diabetes, Hypoglycaemic activity.
INTRODUCTION:
From time immemorial
plants are used as medicine around the world and plant based medicine has been
the mainstay of traditional societies in dealing with health problems.1 The World Health Organization (WHO)
has estimated that 80% of the earth’s (6 billion) inhabitants rely upon
traditional medicine for their primary health care needs and major part of this
therapy involves the use of plant extracts or their active principles.2 Diabetes mellitus is a
chronic metabolic disorder characterized by hyperglycaemia,
polyuria, polydypsia, polyphagia, glycosuria, hyperlipidemia, negative nitrogen balance, pruritis, unexpected weight loss and some times ketonemia.3-5
At present number of diabetics worldwide is 150 million and this is likely to
increase to 300 million or more by the year 2025.6 This rise in
number is due to sedimentary lifestyle, consumption of energy rich diet,
obesity, higher life span etc.7 Regions with greatest potential are
Asia and Africa, where diabetes mellitus rates could rise 2-3 folds than the
present rates. Many medicine have been recommended for the treatment of
diabetes.8-12 The two major types of diabetes mellitus are Type-I
insulin dependent and Type-II non-insulin dependent . Type-I Insulin-dependent diabetes mellitus or juvenile-onset
diabetes (IDDM). Type-II Non-insulin dependent diabetes mellitus (NIDDM)
or maturity-onset diabetes:
Treatments of this disorder take three main
factors:
(i)
Diet
and exercise
(ii)
Insulin
replacement therapy
(iii)
Oral
hypoglycemic agents 13
Before
the advent of insulin and oral hypoglycemic drugs, the major form of treatment
involved the use of plants. Recently, some medicinal plants have been reported
to be useful worldwide and have been used empirically as antidiabetic
and remedies.14
There is an increasing demand by patients to
use the natural products with antidibetic activity
due to side effects associated with the use of insulin and oral hypoglycemic
agents such as sulfonylureas, metformin, α-glycosidase inhibitors, troglitazone.15-17
Antihyperglycemic effects of these plants are
attributed to their ability to restore the function of pancreatic tissues by
causing an increase in insulin output or inhibit the intestinal absorption of
glucose or to the facilitation of metabolites in insulin dependent processes.
More than 400 plant species having hypoglycemic activity are known to have been
recommended, and recent investigations have affirmed the potential value of
these,18-20 however searching for new antidiabetic
drugs from natural plants is still attractive because they contain substances
which take alternative and safe effects on diabetes mellitus.21
Chemical studies directed at the isolation, purification and identification of
substances responsible for the anti-diabetic activity are also being conducted.22
Most of plant contain glycosides, alkaloids, terpenoids,
flavonoids, cartenoids,
etc. that are frequently implicated as having antidiabetic
effect.23
MATERIAL AND
METHODS:
The dried root powder
(2.5kg) of Verbesina encelioides
Benth. was taken and
subjected to extraction with ethanol in soxhlet
apparatus so as to yield 4.54% w/w of extract and aqueous extract was prepared
by cold maceration yielded 2.05% w/w. Extracts were kept in a dessicator and stored in refrigerator for future biological
evaluation. Alloxan was purchased from LOBA CHEMIE,
Animals:
Swiss albino mice of
either sex (weighing 30-45 g) were used as per experimental protocols approved
by Institutional committee, The experimental protocol
has been approved by the Institutional Animal Ethics committee and by the
Regulatory body of the government. The animals were housed under standard
environmental condition (25 ± 2°C and relative
humidity 50 ± 5%) and fed with standard diet and water, ad libitum. The animals were acclimatized to laboratory
environment for a period of 14 days before performing the experiments.
The mice were
divided into six groups of six animals each. The first group comprised the
control. The second received the standard reference control drug. The remaining
four groups were administered test dose. The test doses were prepared in Tween 20 (1%) as suspension in distilled water to get the
desired concentration of the extracts. The dosages selected were as follows:
i)
Solvent control
1% Tween
20 in distilled water (2 ml/kg).
ii)
Reference control
Glibenclamide:2.5 mg/kg body weight in distilled water.
iii) Diabetogenic agent
Alloxan monohydrate: 120
mg/kg in normal saline.
Streptozotocin: 60 mg/kg in citrate buffer (pH 4.5)
iv) Test drugs
Ethanol extract-
200mg/kg
Ethanol extract-
400mg/kg
Aqueous extract-200
mg/kg
Aqueous extract-400
mg/kg
Swiss albino mice of either sex were used.
The animals were fasted overnight, kept under laboratory conditions and allowed
free access to water. The animals were divided in to different groups of six
animals each. The ethanolic and aqueous extracts were
administered orally at a dose of 5 mg/kg. The control group received a similar
volume of 1% Tween 20 in distilled water. Mortality in
each group was observed for 3 to 7 days. If no mortality was observed, the
procedure was repeated for higher doses such as 50, 500, 1000, 1500 and 2000
mg/kg.24 The doses of 200 and 400 mg/kg
were selected based on the results of preliminary toxicity testing.25
The animals were fasted for 18 h, but were
allowed free access to water before and throughout the duration of experiment.
At the end of the fasting period, taken as zero time (0 h), blood was withdrawn
(0.1 ml) from the tip of the tail of each mice under
mild ether anaesthesia. The blood glucose level was
measured with haemoglucostrips supplied by B. Braun
Medical Industries Sdn. Bhd.,
Figure
1: Percentage reduction in blood glucose level in normoglycaemic
mice
The acclimatized animals were kept fasting
for 24 h with water ad libitum and injected intraperitoneally
a dose of 120 mg/kg of alloxan monohydrate in normal
saline. After 1 h, the animals were provided feed ad libitum. The blood glucose level was
checked before alloxanisation and 24 h after alloxanisation. Animals were considered diabetic when the
blood glucose level was raised beyond 200 mg/100 ml of blood. This condition
was observed at the end of 48 h after alloxanisation.
The animals were then segregated in to six groups of six mice in each. Group-I
served as solvent control which received vehicle (2 ml/kg) only through oral
route. Group-II received glibenclamide (2.5 mg/kg)
and served as reference control. Group-III and IV received the ethanolic extract at a dose of 200 and 400 mg/kg body weight
respectively, through oral route. Group-V and VI received the aqueous extract
at a dose of 200 and 400 mg/kg body weight respectively, in a similar
manner. Blood glucose levels were
examined after 1, 2, 4, 8 and 10 h of administration of single dose of test
samples.22
Figure 2: Percentage reduction in blood
glucose level in alloxan induced diabetic mice.
The 18
h fasted mice were divided in to six groups of six animals each. The animals
were made diabetic by injection of a dose of streptozotocin
60 single mg/kg body weight dissolved in 0.1 ml of citrate buffer (pH 4.5) by
intravenous injection in to the tail vein. Animals were considered diabetic
when blood glucose level was raised beyond 200 mg/100 ml of blood. Group–I served as solvent control and
received only vehicle (2 ml/kg) through oral route. Group–II received glibenclamide (2.5 mg/kg). Group-III and IV received the ethanolic extract at a dose of 200 and 400 mg/kg body
weight respectively,
through oral route. Group-V
and VI received the aqueous extract at a dose of 200 and 400 mg/kg body weight
respectively. Blood glucose levels were
examined after 1, 2, 4, 8 and 10 h of administration of single dose of test
samples.5
The albino mice of either sex (20-30 g)
maintained on a standard diet (water ad libitum) and fasted overnight. The animals were killed
by decapitation and diaphragms were taken out quickly avoiding trauma and
divided in to two halves. The hemidiaphrams were then
rinsed in cold Tyrode solution (without glucose) to
remove any blood clots and were then
placed in small conical flasks containing 2ml Tyrode
solution with 2 g% glucose and incubated for 30 min at 37 oC
in an atmosphere of 95% O2-5% CO2 with shaking. Six sets
of experiments were performed. The diaphragms were exposed to:
(a)
Tyrode solution with 2g% glucose which served
as control,
(b)
Tyrode solution with 2g% glucose + insulin
(0.25 IU/ml),
(c)
Tyrode solution with 2g% glucose + ethanol
extract (200 mg/ml)
(d)
Tyrode solution with 2g% glucose + aqueous
extract (200 mg/ml)
(e)
Tyrode solution with 2g% glucose + insulin
(0.25 IU/ml) + ethanol extract (200 mg/ml).
(f)
Tyrode solution with 2g% glucose + insulin
(0.25 IU/ml) + aqueous ethanol extract (200 mg/ml).
Following incubation, the hemidiaphrams
were taken out and weighed. Glucose uptake was calculated as the difference
between the initial and final glucose content in the incubation medium. The
glucose estimation was carried out on autoanalizer.
Data expressed as mean± S.E.M. statistical
comparison between different groups were done using one-way analysis of
variance (ANOVA) followed by the Dunnett’s ‘t’ test, to
judge the difference between various groups. Significance was accepted at P<0
In acute toxicity study, there were no
behavioral changes up to 4 h and no mortality was observed up to 7 days even at
the maximum tested dose level of 2000 mg/kg.
Figure
3: Percentage reduction in blood glucose level in streptozotocin
induced diabetic mice.
The ethanolic and aqueous extracts of Verbesina
encelioides Benth. roots produced significant decrease in the blood
glucose level when compared with the control in the normoglycaemic
mice [Table-1]. The reduction in blood glucose level was observed 2h after oral
administration of single dose of the standard drug and extracts, the reduction
in blood glucose level was reached maximum after 10 h for all the test doses
and standard drug, it was more significant (P<0.01)
for the 400 mg/kg ethanol extract and aqueous extract (200 and 400 mg/kg).
While 200 mg/kg ethanol extract showed less significance (P<0.05) when compared with the control. The percentage reduction
in blood glucose is shown in Figure-1. The maintenance of normoglycaemia
depends on the result of the interwork of islets of β-cell secretion and
insulin sensitivity in the periphery and in the liver. The results corroborated
that the test extracts showed hypoglycaemic
properties throughout the experimental period in the normoglycaemic
mice. Normal animals are often used for
potential oral hypoglycaemia in addition to diabetic
animal model as information regarding mechanism of action.
Alloxan-induced diabetic mice:
The perusals of Table-2 reveals that the ethanolic and aqueous extracts in alloxan
induced diabetic mice produced significant (p<0.01)
decrease in blood glucose level after 4h of administration of extracts when
compared with the control. The percentage decrease in blood glucose level for ethanolic and aqueous extract at the dose level of 400
mg/kg is 64.21% and 63.48 % respectively, which is comparable to standard drug glibenclamide (68.14%) as shown in Figure-2. Alloxan is a urea derivative which induces “chemical
diabetes” in a wide variety of animal species by damaging the insulin secreting
pancreatic β-cells, resulting in a decrease in endogenous insulin release.26
Numerous studies demonstrated that a variety of plant extracts effectively
lowered the glucose level in alloxan-induced diabetic
animals.27,28 In the present study, ethanolic
and aqueous extracts of V. encelioides roots effectively decrease the blood
glucose in alloxan- induced diabetic mice, which is
almost equal to that of glibenclamide.
The ethanolic and
aqueous extracts exhibited significant antidiabetic
effect in streptozotocin induced diabetic mice
[Table-3]. The standard drug, ethanol extract and aqueous extract at doses 400
mg/kg significantly (P<0.01)
reduced the blood glucose level after 2h while 200 mg/kg doses of extracts
showed P<0.01 significant result
after 4h. The maximum reduction observed at 10 h. The maximum percentage
reduction of standard drug glibenclamide is 57.25%.
It is 29.27 and 51.11% for ethanol extract at doses 200 and 400 mg/kg
respectively and 21.87 and 46.95% for aqueous extract at doses 200 and 400
mg/kg respectively [Figure-3].
Streptozotocin is cytotoxic nitrosoureido glucopyranoside derivative isolated from fermentation of Streptomyees achromogenes
and has been widely used for inducing type-I diabetes in a variety of animals
by affecting the degeneration and necrosis of pancreatic β-cells.29
The present data indicated that the ethanolic and aqueous extracts of Verbesina encelioides Benth.
roots decreased the blood glucose in streptozotocin- diabetic mice. This suggests the said
effect be due to extraintestinal action of the test
extracts.30 The extracts decreased blood glucose without stimulating
insulin secretion, in addition the extract may also have exerted hypoglycaemic effect by other mechanism such as stimulation
of glucose uptake by peripheral tissues, inhibition of endogenous glucose
production or activation of gluconeogenesis in liver
and muscles, as similar mechanism have been proposed for different plant drugs
which were reported for antidiabetic activity in streptozotocin induced diabetic animals.31
The results of the In-vitro study on glucose utilization by isolated mice hemidiaphargm are given in Table-4. The data revealed that
glucose uptake by the extracts significantly similar to that of insulin. These
findings suggest that the extracts may have direct insulin like activity which
enhances the peripheral utilization of glucose and have extra-pancreatic
effect.32
ACKNOWLEDGEMENT:
The authors are grateful to Dr. Madhu Chitkara, Director, Chitkara
Institute of Engg.
and Technology, Rajpira,
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Received on 22.10.2009
Accepted on 20.12.2009
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Research Journal of Pharmacognosy and Phytochemistry. 2(1): Jan.-Feb. 2010, 41-45