hypoglycemic potential of methanolic leaf extract
TRANSCRIPT
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Rajani et al. World Journal of Pharmacy and Pharmaceutical Sciences
HYPOGLYCEMIC POTENTIAL OF METHANOLIC LEAF EXTRACT
OF INDIGOFERA BARBERI GAMBLE AGAINST ALLOXAN INDUCED
DIABETES IN RATS
1*Dr. A. Rajani,
2Dr. K. Hemamalini and
3Dr. A. Jaswanth
1Associate Professor, Sri Indu Institutre of Pharmacy, Ibrahimpatnam.
2Professor and Vice-principal, Department of Pharmacology, Brilliant Grammar School of
Institutions- Integrated Campus, College of Pharmacy, Abdullapurmet, Hayathnagar.
3Professor and Principal, Surabhi Dayakar Rao College of Pharmacy, Gajwel, Medak.
ABSTRACT
The present study was conducted to evaluate the Anti-hyperglycemic
activity of methanolic leaf extract of Indigofera barberi Gamble
against alloxan induced diabetes in rats. The dried leaves of Indigofera
barberi Gamble were subjected to extraction by continuous hot
percolation using methanol as solvent and further evaluated for anti-
diabetic screening. Dose selection was made on the basis of acute oral
toxicity studies (4000mg/kg body weight) as per OECD and CPCSEA
guidelines. Oral administration of the test extract in the dose of 200
and 400mg/kg for 7 days resulted in significant reduction in blood
glucose levels. Alloxan induced diabetic rat model was used for the evaluation of Anti-
diabetic activity. Glibenclamide was used as the standard to compare the test results. Oral
administration of the test extract in the dose of 200 and 400mg/kg for 21 days resulted in
significant reduction in blood glucose levels. At the end of the experiment, the animals were
sacrificed and the pancreas and liver were sectioned for histopathology. The histopathological
studies of the pancreas of diabetic animals revealed the degeneration of pancreatic islet cells,
but with the restoration after treatment with the dose of the test extract. The liver showed no
histopathological change. The implications of the result obtained in the present study provide
the scientific rationale for the use of Indigofera barberi Gamble as anti-diabetic agent in the
management of diabetes.
*Corresponding Author
Dr. A. Rajani
Associate Professor, Sri Indu
Institutre of Pharmacy,
Ibrahimpatnam.
Article Received on
15 November 2017,
Revised on 05 Dec. 2017,
Accepted on 26 Dec. 2017
DOI: 10.20959/wjpps20181-10842
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 6.647
Volume 7, Issue 1, 1291-1303 Research Article ISSN 2278 – 4357
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KEYWORDS: Indigofera barberi Gamble, methanolic extract, Anti-Diabetic activity,
Alloxan, Glibenclamide.
INTRODUCTION
Diabetes Mellitus is a chronic metabolic disorder, characterized by absolute of relative
deficiency in insulin secretion and /or insulin action associated with chronic hyperglycemia
and disturbances of carbohydrates, lipid and protein metabolism. The metabolic changes
occur in diabetes lead to develop various complications including macro and micro vascular
dysfunctions1.The prevalence of diabetes mellitus has rapidly increased world wide. By the
year 2030, diabetes mellitus is expected to affect almost 30% of the world‟s population- an
estimated 366 million people.[2]
Oxidative stress has been shown to have a significant effect
in the causation of diabetes as well as diabetes related complications in human beings.[3]
Indigofera barberi Gamble has been proved to have Anti-oxidant property[4]
. So, the present
study was taken whether it had significant Anti-diabetic activity.
Indigofera barberi Gamble is a class of dicotyledons plant belonging to the family fabaceae.
It is distributed in the deciduous forests of chittor district. It is an erect plant under shrub. The
leaves are about 20-26mm long and 6-12mm wide, oblong and ovate to lanceolate, base with
a short petiole.[5]
Fig.1: Leaves of Indigofera barberi Gamble.
MATERIALS AND METHODS
Animals
Wister albino rats of either sex weighing between 100-200 g were used for this purpose. The
animals were housed in polypropylene cage and maintained at 24º±2º under 12 h light dark
cycle and were fed ad libitum with standard pellet diet and had free access to water.
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Maintenance and use of animals as per the experiment was approved by the institutional
animal ethics committee. The animals and animal feed was obtained National Institute of
Nutrition (NIN), Hyderabad.
Chemicals
Alloxan monohydrate, Glibenclamide, Dextrose, Tween-80.
Plant material
Fresh leaves of Indigofera barberi Gamble plant were collected from S.V University,
Tirupati. Further the leaves were identified and authenticated by Dr. K. Madhavachetty,
Botanist & Assistant Professor, Department of Botany, S.V University, Tirupati, Chittor,
Andhra Pradesh, India.
Preparation of the plant extract
The collected leaves were shade dried and powdered in a grinder mixture to get course
powder. It was then passed through the 40 # sieve. A weighed quantity (200g) of the powder
was subjected to continuous hot extraction in Soxhlet Apparatus. The powdered leaves were
defatted with petroleum ether and later extracted with methanol. The extract was evaporated
under reduced pressure using rotary evaporator until all the solvent has been removed to give
an extract sample. Percentage yield of methanolic extract Indigofera barberi Gamble of was
found to be 16.5% w/w. A preliminary phytochemical screening of methanolic extract of
Indigofera barberi Gamble was carried out by the standard procedures.[6]
Acute oral toxicity tests[7]
Acute oral toxicity studies of the test extract were carried out as per the OECD guidelines,
draft guidelines 423 adopted and received from the Committee for the purpose of Supervision
and control of experiments on animals (CPCSEA), Ministry of social justice and
empowerness, Government of India. Administration of the dose of Indigofera barberi
Gamble up to the dose of 4000mg/kg caused considerable signs of toxicity in the animals.
One tenth of upper limit dose was selected as the level for examination of Anti-diabetic
activity.
Experimental model
Hyperglycemia was induced by intra peritoneal injection of freshly prepared aqueous solution
of alloxan monohydrate (SD fine Chemicals Pvt. Ltd., Biosar) 150 mg /kg, to overnight
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fasted rats. Control rats received similar volume of vehicle, normal saline (2 ml/kg body
weight) alone. Animals that did not develop more than 300 mg/dl glucose levels were
rejected and new animals were used. Immediately after confirmation of diabetes, rats were
classified into six groups of six rats each and one more group of normal non alloxanised
animals was also added in the study.
Experimental design
Different groups of rats were used to study the effects of methanolic extract of Indigofera
beddomei Gamble. The rats were divided into 5 groups each consisting of six rats. Group I
received normal saline and served as Control.
Group II treated with alloxan monohydrate 150 mg/kg served as diabetic Control.
Group III is treated with glibenclamide (2.5mg/kg) and served as reference standard.
Group IV& V are treated with 2 doses of Indigofera beddomei Gamble (200 and 400 mg/kg).
Treatment continued for 21 consecutive days. Before the treatment (0 day) and at the end of
7th
, 14th
and 21st day plasma levels were estimated using the glucose oxidase method.
[8]
The blood sugar level was measures using ACCU-Chek active glucose strips in ACCU-Chek
active test meter by collecting the blood from rat tail vein and for other plasma profiles blood
was collected from retro orbital plexus of the rats under light ether anaesthesia using capillary
tubes into eppendorf tubes containing heparin. The plasma was separated by centrifugation 5
min, 5000rpm and was analysed for lipid profiles (serum total cholesterol, serum
triglycerides, serum Creatinine, serum urea, haemoglobin and Total protein. The plasma
profiles were measured by standard enzymatic methods with an autoanalyzer.
Body weight measurement
Body weight was measured totally four time during the course of study[9]
(i.e., before alloxan
induction (initial values), and on the 1st, 7
th, 14
th and 21
st day of the treatment period), using a
weighing scale.
Statistical Analysis
The results of the study were subjected to one way analysis of variance (ANOVA) followed
by Dunnett‟s„t‟- test for multiple comparision. Values with p<0.05 were considered
significant.
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RESULTS
PreliminaryPhytochemical screening
The methanolic extract of Indigofera beddomei Gamble was found to contain alkaloids,
carbohydrates, glycosides, phytosterols, saponins, tannins, amino acids, flavonoids and
diterterpenes. Flavonoids in Indigofera barberi Gamble may be responsible for its Anti-
diabetic activity. The results obtained were comparable and satisfied the standard literature.
Table No.1: Phytochemical investigation of Indigofera barberi Gamble Methanolic leaf
extract.
S.No. Constituents Report 1 Carbohydrates +
2 Steroids ++
3 Alkaloids +
4 Flavonoids ++
5 Glycosides +
6 Proteins -
7 Tannins +
8 Volatile Oils +
+ Presence, -absent
Acute oral toxicity studies
In acute toxicity study, the methanolic leaf extract of Indigofera beddomei Gamble showed
no significant toxicity sign when observed for the parameters during the first 4hrs and
followed by daily observations for 14 days and mortality was also not observed. The test
extract was found to be safe at the tested dose level 4000mg/kg body weight. One tenth of
this dose level was taken as effective dose to evaluate the Anti-Diabetic activity in alloxan
induced diabetes in rats.
Alloxan induced diabetic model
The effect of methanolic leaf extract of Indigofera brberi Gamble in normal rats is shown in
Table-1. After 30 minutes of glucose administration, the peak glucose level increased rapidly
from fasting value and subsequently decreased. The methanolic extract of Indigofera brberi
Gamble exhibited remarkable blood glucose lowering effect at 90min.
Body weight measurement
In the present study, diabetic rats had lower body weights and high blood glucose level as
compared to normal rats. In spite of increased food consumption, loss of body weight due to
defect in glucose metabolism and excessive breakdown of tissue protein is a characteristic
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condition in diabetics. As shown in Table-5, treatment with the test extract improved the
average body weights of rats, which indicates that control over polyphagia and muscle
wasting resulted due to hyperglycemic condition.
Anti-diabetic study
Methanolic extract of Indigofera brberi Gamble leaves was subjected to anti-diabetic activity
in rats where alloxan monohydrate (120 mg/kg b.w., i.p.) used as the diabetogenic agent. A
marked rise in fasting blood glucose level observed in diabetic control compare to normal
control rats. Methanolic extract of Indigofera brberi Gamble at 200 mg/kg was insignificant
and (400 mg/kg) exhibited a significant anti-hyperglycemic activity on 7th
, 14th
and 21st day
post treatment. The antihyperglycemic effect of methanolic extract was found less effective
than the reference standard, Glibenclamide. Glibenclamide produced a significant reduction
in blood glucose compare to diabetic control. The results are shown in the Table No. 2.
Table.2 Effect of Indigofera barberi leaf extract on fasting blood glucose level in alloxan
induced diabetic rats.
Groups Fasting blood glucose level (mg/dl)
1st day 7
th day 14
th day 21
st day
Normal Control 69.833 ± 1.472 76.5 ± 1.87 82.166 ± 3.71 78.166 ± 3.488
Diabetic Control 288 ± 2.36 308.66 ± 4.84 318.33 ± 2.160 334.66 ± 2.065
Standard 269.33 ± 1.86** 230.83 ± 2.48** 170.5 ± 3.27** 131 ± 3.03**
MEIB 200mg/kg 265.5 ± 1.87* 219.16 ± 3.81* 185.5 ± 2.66* 150.33 ± 2.16*
MEIB 400mg/kg 255.16 ± 2.56** 199.33 ± 1.36** 167.8 ± 2.85** 122.667 ± 3.48**
Each value is the mean ± SEM for 6 mice, *P<0.01, **P<0.001.Compared with control, data
were analysed by using one-way ANOVA followed by Dunnett‟s t- test.
Fig.2: Effect of Indigofera barberi Rhyncosia beddomei, Trichuriella mononiae leaf
extract on fasting blood glucose level in alloxan induced diabetic rats.
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Table 3: Effect of Indigofera barberi leaf extract on biochemical parameters in alloxan
induced diabetic rats.
GROUPS CREATININE
(mg/dl) Sr.PROTEIN UREA (mg/dl)
Sr.CHOLESTEROL
(mg/dl) TRIGLYCERIDES
Normal
Control 0.5 ± 0.037 6.1 ± 0.60 30 ± 2 116.66 ± 2.06 81.5 ± 3.2
Diabetic
Control 1.32 ± 0.037 4.1 ± 1.0 70.83 ± 2.85 222 ± 4 140.5 ± 3.08
Standard 0.4533 ± 0.029** 6 ± 0.52** 25.83 ± 1.9408** 106.33 ± 3.502** 85.33 ± 3.14**
MEIB
200mg/kg 0.73 ± 0.026* 5.83 ± 0.6* 37.16 ± 2.92* 139.5 ± 3.27* 100.83 ± 2.85*
MEIB
400mg/kg 0.545 ± 0.03** 8.99 ± 0.5** 26.5 ± 3.08** 120.83 ± 2.85** 84.5 ± 2.25**
Each value is the mean ± SEM for 6 mice, *P<0.01, **P<0.001.Compared with control, data
were analysed by using one-way ANOVA followed by Dunnett‟s t- test.
Fig.3: Effect of Indigofera barberi leaf extract on biochemical parameters in alloxan
induced diabetic rats.
Fig.4: Effect of Indigofera barberi leaf extract on biochemical parameters in alloxan
induced diabetic rats.
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Table 4: Effect of Indigofera barberi leaf extract on body weight in alloxan induced
diabetic rats.
Groups Body weight of animals
1st day 7
th day 14
th day 21
st day
Normal Control 200.66 ± 4.4 199.83 ± 3.48 206.3 ± 3.07 208.83 ± 2.8
Diabetic Control 209.16 ± 5.07 173.66 ± 3.55 168 ± 4.09 146.66 ± 5.00
Standard 199.66 ± 48* 200.5 ± 5.04** 195.16 ± 4.57** 196.33 ± 3.32**
MEIB 200mg/kg 178.33 ± 2.16* 191 ± 2.89* 203.16 ± 2.85* 205.16 ± 2.85*
MEIB 400mg/kg 195.16 ± 2.85** 199.16 ± 2.85** 210.83 ± 3.7* 235.83 ± 3.7*
Each value is the mean ± SEM for 6 mice, *P<0.01, **P<0.001.Compared with control, data
were analysed by using one-way ANOVA followed by Dunnett‟s t- test
Fig.5: Effect of Indigofera barberi leaf extract on body weight in alloxan induced
diabetic rats.
Effect of Indigofera barberi Gamble leaf extract on body weight in diabetic rats: Normal
control animals were found to be stable in their body weight but diabetic rats showed
significant reduction in body weight during 10 days. Alloxan mediated body weight reduction
was significantly reversed by the methanolic extract in dose dependant fashion (200, 400
mg/kg). The effect of test extract at 200 mg/kg on body weight of the animals was also found
statistically not significant. Results are shown in Table No. 5.
Histopathological Result
Histopathological studies of sectioned pancreas were performed using Haemotoxylin and
Eosin stain.
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Fig: 6 Pancreatic islets of normal rats showing alpha cells and beta cells.
Fig: 7 Alloxan induced diabetes, increased damage of pancreatic damaged β– cells.
Fig: 8 Glibenclamide treated animals showing partial proliferation of pancreatic islets
of β cells.
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Fig: 9 Treatment with methanolic leaf extract of Indigofera barberi showing restoration
of beta cells of pancreatic islets of Langerhans.
Fig: 10 Treatment with methanolic leaf extract of Rhynchosia beddomei showing
restoration and normalization of beta cells of pancreatic islets of langerhans
Fig: 11 Treatment with methanolic leaf extract of Trichuriella monsoniae showing
restoration and normalization of beta cells of pancreatic islets of langerhans
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DISCUSSION
Pancreas is the primary organ involved in sensing the organism‟s dietary and energetic states
via glucose concentration in the blood and in response to elevated blood glucose, insulin is
secreted. Alloxan is one of the usual substances used for the induction of diabetes mellitus
apart from streptozotocin. Alloxan has a destructive effect on the beta cells of the pancreas.
Alloxan causes a massive reduction in insulin release by the destruction of β-cells of the islets
of langerhans, thereby inducing hyperglycaemia. Insulin deficiency leads to various
metabolic alterations in the animals viz increased blood glucose, increased cholesterol,
increased levels of alkaline phosphate and transaminases. The results of the present study
indicate that Indigofera beddomei Gamble leaf extract was found to reduce the glucose level
in animals made diabetic with alloxan. Alloxan has been shown to induce free radical
production and cause tissue injury. The pancreas is especially susceptible to the action of
alloxan induced free radical damage. In the present investigation methanolic extract of
Indigofera beddomei Gamble leaf demonstrated the significant anti-diabetic activity.
After the induction of diabetes by injecting freshly prepared alloxan through intraperitonial
cavity, it was confirmed by testing of glucosuria in the urine using glucose indicator sticks.
The changes in the body weight of different experimental groups were noted. The body
weight of the alloxan induced diabetic group II rats was found to be reduced. On treatment
with Indigofera beddomei Gamble and Glibenclamide on Group III and IV, the body weight
was gained comparing to the normal and control rats of Group I. These shows Indigofera
beddomei Gamble exhibited considerable gain of body weight, increased fluid intake and
food is the one of the symptoms of Diabetes which has been normalize on treatment effect of
herbal.
The results from the present study also indicate that Indigofera beddomei Gamble leaf extract
can reduce the levels of serum urea, serum creatinine, serum cholesterol and increase the
serum protein and confirms the possibility that the major function of the extract are on the
protection of vital tissues (Kidney and liver) including the pancreas, thereby reducing the
causation of diabetes in the experimental animals. Insulin is potent inhibitor of lipolysis.
During diabetes, activity of lipase enzyme increases lipolysis and release more free fatty
acids in the circulation because of lack of insulin. Increase in fatty acid concentration in turn
increases the beta-oxidation of fatty acids by increasing the activity of HMG-CoA reductase
for producing more cholesterol. Insulin also increases the receptor-mediated removal of LDL-
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cholesterol and decreased activity of insulin during diabetes causes hypercholesterolemia.
Moreover, cholesterol is a powerful risk factor for many coronary heart diseases. The degree
of hypercholesterolemia is directly proportional to severity in diabetes. In our study, we have
observed higher levels of cholesterol in plasma of diabetic rats. Further, alloxan induced
diabetic rats when treated with methanolic extract significantly reduced the serum cholesterol
level. It has been reported that plant extract exert their cholesterol lowering effect seems to be
a decrease in cholesterol absorption from the intestine, by binding with bile acids within the
intestine and increasing bile acids excretion. A significant increase in serum cholesterol and
triglycerides observed in alloxan induced diabetic rats in our experiment is in agreement with
the findings of the fore mentioned studies. The marked hyperlipidemia that characterizes the
diabetic state may therefore be regarded as a consequence of the uninhibited actions of
lipolytic hormones on the fat depots. From our study, it is assumed that the administration of
methanolic extracts of Indigofera beddomei Gamble to surviving diabetic rats might have
reduced the pancreatic lipase activity, which is responsible for the hydrolysis of non-
absorbable dietary triglycerides into absorbable monoglycerides and free fatty acids, which,
in turn, leads to the decrease of plasma cholesterol and triglycerides levels.[10]
The literature review indicated hepatoprotective property of Indigofera beddomei Gamble
leaf extract, the improvement of liver function and subsequent increase in uptake of blood
glucose and its utilization may be another mechanism of action of the extract. Other possible
mechanism includes the stimulation of β-cells and subsequent release of insulin and
activation of the insulin receptors. Estimation of insulin level and insulin receptor may give
more insight into the mechanism of the anti-diabetic activity exhibited by the extract. The
present study also indicates that Indigofera beddomei Gamble can partially inhibit alloxan
renal toxicity as observed from serum urea and creatinine levels. The literature reports reveal
that flavonoids and tannins present in the plant extract known to possess antidiabetic activity.
In the present investigation also the observed antidiabetic potential of test extract may be due
to presence of similar phytoconstitutes which was evident by preliminary phytochemical
screening.
CONCLUSION
In the recent times, traditionally used medicinally important plants were tested for their anti-
hyperglycemic potential by various investigators due to the serious side effects of
conventional medicines. The present experiment was continuous post treatment for 21days
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with methanolic leaf extract of the plant Indigofera beddomei Gamble against alloxan
induced diabetic model, it has beneficial effects on blood glucose levels as well as improving
hyperlipidemia and other metabolic aberrations.
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