HYPOGLYCEMIC POTENTIAL OF METHANOLIC LEAF EXTRACT

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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


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**


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


Fig.4: Effect of Indigofera barberi leaf extract on biochemical parameters in alloxan




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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*


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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HYPOGLYCEMIC POTENTIAL OF METHANOLIC LEAF EXTRACT

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