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International Journal of Pharmaceutical Sciences and Drug Research

2016; 8(5): 275-280

ISSN: 0975-248X
Research Article
CODEN (USA): IJPSPP

Antidiabetic Activity of Extracts of Pithecellobium dulce Benth Leaves in

Alloxan Induced Diabetic Rats

Mule V. S.1*, Naikwade N. S.2, Magdum C. S.3, Jagtap V. A.1

1Yashwantrao Bhonsale College of Pharmacy, Sawantwadi, Maharashtra, India
2Appasaheb Birnale College of Pharmacy, Sangli, Maharashtra, India
3Rajarambapu College of Pharmacy, Kasegaon, Maharashtra, India

ABSTRACT
The objective of the present study was to study effect of Pithecellobium dulce Benth (P. dulce) leaves in alloxan
induced diabetic rats. The P. dulce leaves were extracted by maceration and soxhelation method by using water
and ethanol as solvent. Acute toxicity study was performed according to OECD 425 guidelines for both aqueous
and ethanolic extracts of P. dulce leaves. The dose of 200 mg/kg and 400 mg/kg was selected for further studies.
Animals were rendered diabetic by administration of alloxan (130 mg/kg, i.p.). The albino rats were divided in
to seven groups with five animals in each group. Diabetic animals were treated with aqueous and ethanolic
extract for 20 days. Then blood glucose, triglyceride, total cholesterol, urea, uric acid, creatinine, aspartate
aminotransferase (AST), alanine transaminase (ALT) and glycogen level in liver, muscle and kidney were
estimated according to standard procedures. The result shows significant decrease in blood glucose, triglyceride,
total cholesterol, urea, uric acid, creatinine, AST and ALT level when compared to diabetic group. The liver and
muscle glycogen level was increased significantly in extract treated groups when compared to diabetic control
group. Both extract of P. dulce posses antidiabetic and hypolipidemic potential.

Keywords: Pithecellobium dulce Benth, antidiabetic, alloxan, lipid profile, glycogen, OGTT.

INTRODUCTION microvascular damage increases the risk of retinopathy,

Diabetes mellitus (DM) is commonest form of diabetes nephropathy and neuropathy. Macrovascular
caused by a deficiency of the hormone insulin or complications like ischemic heart disease, stroke and
increase in insulin resistance, which results in peripheral vascular disease can results in reduced life
hyperglycemia. [1] Diabetes is the most serious and expectancy and significant morbidity. [3] According to
common metabolic disease all over the world and it is World Health Organization (WHO) 1.5 million peoples
caused mainly due to pancreatic β cell dysfunction and were died because of diabetes in year 2012. Globally, in
insulin resistance. [2] DM is the condition which 2014 total 422 million adult were living with diabetes;
depends on the level of hyperglycemia causing the number was 108 million in 1980. [4] In 1980 the WHO
microvascular and macrovascular complications. The also suggested to study and find out the plants which
are having potential hypoglycemic activity as the
*Corresponding author: Mr. Vinod Shivaji Mule, modern drugs has the less safety. [5] DM is difficult to
Yashwantrao Bhonsale College of Pharmacy, control with currently available drugs which are
Sawantwadi, Maharashtra, India; Tel.: +919823916005; associated with many side effects. [6] Thus today there is
E-mail: [email protected] need to search for the novel drug for treatment of DM.
Received: 12 September, 2016; Accepted: 26 September, 2016

275
 Mule et al. / Antidiabetic Activity of Extracts of Pithecellobium dulce Benth Leaves in Alloxan…..……

Historically, many natural plant products were proved filtered by whatman filter paper and concentrated by
to be effective for treatment of DM. The natural plants heating on water bath. Concentrated aqueous extract
products have received considerable attention now was air dried and used for further study. [13-14] The
days as these products are the templates for the aqueous extracts were administered orally through
development of new molecule for DM. orogastric tube. The suspension of ethanolic extracts
P. dulce is medium sized plant which grows up to 18 m was prepared by using 0.5% CMC in distilled water. [15]
height. It is commonly known as Indian jalebi, mania Animals
tamarind or Indian sweet jalebi. [7] The common Wistar albino rats of either sex (150-300 g) were
constituents present in leaves of the plant are cyclitol, obtained from animal house of Appasaheb Birnale
dulcitol, octacosanol, α- spinasterol, kaempferol-3- college of Pharmacy, Sangli, Maharashtra, India. The
rhamnoside, quercetin and afzelin. [8] Traditionally the rats were kept at appropriate temperature of (26 ± 1°C)
leaves of the plant are used for leprosy, intestinal and light controlled (12 h light: 12 h dark) room with
disorders, peptic ulcer, tooth ache, ear ache, emollient, provision of food (Amrut Feed, Sangli). The study
abortifacient and larvicidic activity. [9] The leaves also protocol was approved by the Institutional Animal
reported to show antifungal and antibacterial activity. Ethics Committee of Appasaheb Birnale College of
Estrogenic activity was observed by isolated Pharmacy, Sangli (Approval No:
isoflavonoids from root of the plant. Literature survey ABCP/IAEC/2009/08).
also demonstrates the neuropharmacological profile of Acute toxicity study
the plant in mice. [10-11] The leaves of the plant reported Acute toxicity study was performed according to the
to have free radical-scavenging properties and Organization of Economic Cooperation and
antimycobacterial activity. The leaves of the plant have Development (OECD) guidelines 425. [16] Fifteen
reported to contain the insulin like content which may healthy male albino rats which were fasted 12 h prior to
be useful for treatment of diabetes. [9] Taking into experiment were divided into three groups with five
consideration the traditional claims, presence of insulin animals in each group. The first group serves as control
like principle in P. dulce plant and free radical group where as second and third group serves as
scavenging activity it may be useful in diabetes so aqueous and ethanolic treatment groups. Extract of P.
present study was planned to investigate the effect of P. dulce at dose of 2000 mg/kg were administered to
dulce leaves on alloxan induced diabetic rats. animals. The animals were observed for seven days as
animals had not shown any sign of toxicity,
MATERIALS AND METHODS behavioural changes and mortality the dose increased
Plant Material Collection up to 5000 mg/kg. Then animals were observed up to 7
The plant material was collected from Jaisingpur, days for toxicity, behavioural changes and mortality.
Sangli District, Maharashtra, India. The voucher Induction of Experimental Diabetes
specimen of the plant was identified by acknowledged Diabetes was induced using alloxan from Research lab
botanist, Dr. (Mrs.) U. S. Yadav at Willingdon College, Fine Chem Industries, Mumbai. The animals were
Sangli, Maharashtra, India (Voucher specimen No.: fasted overnight and diabetes was induced by a single
WILL/Bot/2009/03). The voucher specimen was intra peritoneal injection of a freshly prepared solution
deposited at same college. of Alloxan (130 mg/kg b.w.) in a saline solution. [17] 10%
The standard drug pioglitazone was obtained from dextrose was there after administered orally to combat
Aarti Drugs Ltd, Mumbai. The inducer alloxan was the immediate hypoglycemia that could occur. [18] Then
obtained from Research lab Fine Chem Industries, on 4th day fasting blood sugar level was checked in
Mumbai. All the diagnostic kits used are of Span each animal by using glucose kit and animals with
Diagnostics, Surat. fasting blood sugar above 200 mg/dl were included in
Preparation of extracts study.
The fresh leaves were subjected for observation for Experimental Design
removal of damaged leaves and any other mixed The experiment was designed for 21 days to evaluate
materials. Then the separated leaves were dried in fresh antidiabetic activity of P. dulce leaves in alloxan
circulating air under shade for seven days. The dried induced diabetes in albino rats. [19] The animals were
leaves were subjected to size reduction by dry grinder. randomly divided into seven groups as follow:
[12] The leave powder after passing through the sieve Group- I: Normal control 2 ml/kg normal saline
subjected to aqueous and ethanolic extraction. In (vehicle)
ethanolic extraction leaves powder was subjected for Group- II: Alloxan 130 mg/kg + vehicle
soxhlation extraction using ethyl alcohol as solvent. [13- Group- III: Alloxan 130 mg/kg + Pioglitazone 20
14] The extract was dried at 40°C in oven to give dried mg/kg
extract. In aqueous extraction leaves powder was Group- IV: Alloxan 130 mg/kg + Aqueous extract 200
subjected for maceration using chloroform water as mg/kg
solvent. In maceration process leaves powder was Group- V: Alloxan 130 mg/kg + Aqueous extract 400
subjected to occasional shaking on orbital shaker and mg/kg
then stands for the next 18 hours. Then the extract was
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 Mule et al. / Antidiabetic Activity of Extracts of Pithecellobium dulce Benth Leaves in Alloxan…..……

Group- VI: Alloxan 130 mg/kg + Ethanolic extract 200 experimental rats was confirmed by the presences of a
mg/kg high fasting glucose level when compared with normal
Group- VII: Alloxan 130 mg/kg + Ethanolic extract 400 group. Treatment of diabetic rats with AE and EE for 21
mg/kg days significantly (p ˂ 0.05) reduced their blood glucose
Oral Glucose Tolerance Test [20] levels when compared with those of diabetic control
The acclimatized animals were fasted for 24 hours with group.
water ad libitum, fasted animals were divided into Effect of extract on different biochemical parameters
seven groups of five rats. First Groups served as control in alloxan induced diabetic rats
which received distilled water. Second group serves as The effect of AE and EE on triglyceride, total
diabetic control. Third Group received Pioglitazone at cholesterol, HDL, creatinine, urea, uric acid, AST and
an oral dose 20 mg/kg and groups 4-7 received ALT in different experimental groups are presented in
aqueous and ethanolic extract at the dose of (200 and Table 2. Alloxan induced diabetic rats showed
400 mg/kg) respectively, after withdrawing the initial significant increase in triglyceride as compared to
(0 hours) blood samples and after 30 min of extracts control group. The increased triglyceride level in
administration, the rats of all groups were orally diabetic rats was significantly (p ˂ 0.05) prevented by
treated with 2 g/kg glucose. Blood glucose level was the treatment with AE and EE. The alloxan induced
checked at the interval of 30, 60, 90, 120 min, after diabetic rats showed significant increase in total
glucose loading, from tail vein by glucose strips. cholesterol level as compared to normal control group.
Biochemical Parameters The administration of AE and EE to diabetic rats
At the end of the treatment schedule, i.e. on day 21, the significantly restored the total cholesterol level. The
overnight fasted animals were anaesthetized with diabetic control group showed significant (p ˂ 0.05)
diethyl ether and blood was collected by retro orbital decrease in HDL level when compared with normal
puncture method. [21] The serum was separated from control group. Treatment with AE and EE at different
serum sample by centrifugation at 5000 rpm and stored doses significantly increased HDL levels relative to
at refrigeration temperature until use. This Serum diabetic control group. Diabetic rats treated with AE
sample was used to estimate glucose, triglyceride, total and EE at the graded dose showed statistically
cholesterol, high density lipoprotein (HDL), creatinine, significant (p ˂ 0.05) reduction in serum urea,
urea, uric acid, AST and ALT level. Further the animals creatinine, uric acid, AST and ALT levels when
were sacrificed by cervical dislocation and glycogen compared to diabetic control group.
level was estimated in collected samples of liver,
skeletal muscle and kidney by anthrone reagent. [33]
Statistical analysis
Results are expressed as mean±SEM. The statistical
analysis of data was made by analysis of variance
(ANOVA) followed by Dunnett’s test. A value of P <
0.05 was considered significant.

RESULTS
Acute toxicity
Under the presence of the experimental conditions the
absence of toxic symptoms and mortality in animals
indicates that the extract might be having the LD50
value above 2000 mg/kg p.o. body weight. Thus the Fig. 1.1: Effect of aqueous and ethanolic extract of P. dulce leaves on
glucose tolerance test in alloxan induced diabetic rats
extracts were considered to be safe for further
pharmacological study and dose of 200 mg/kg p.o. and
400 mg/kg p.o. of each extract (aqueous and ethanolic)
was selected for the further study.
Effect of extract in Oral Glucose Tolerance Test
Fig. 1.1 shows the blood glucose levels in different
groups after oral administration of glucose at the dose
of 2 g/kg b.w. Aqueous and ethanolic extract of P. dulce
showed significant decrease in glucose level from 30
min after oral glucose administration when compared
with diabetic control group.
Effect of extract on mean blood glucose level in
alloxan induced diabetic rats
Table 1 shows the effect on fasting blood glucose in Fig. 1.2: Effect of aqueous and ethanolic extract of P. dulce leaves on
different group during 21 days study in different mean fasting blood glucose level in alloxan induced diabetic rats.
experimental groups. Induction of diabetes in the
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Table 1: Effect of aqueous and ethanolic extract of P. dulce leaves on mean fasting blood glucose level in alloxan induced diabetic rats.
Mean Fasting Blood Glucose (mg/dl)
Group-I Group-II Group-III Group-IV Group-V Group-VI Group-VII
Day-0 87.87 ± 2.56 304.63 ± 2.12 306.81 ± 1.20 297.96 ± 1.56 299.99 ± 2.23 323.33 ± 3.53 366.66 ± 2.23
Day-7 95.83 ± 1.45 309.37 ± 4.23 260.34 ± 2.30* 270.00 ± 2.67* 258.75 ± 4.23* 284.84 ± 3.12* 315.14 ± 1.33
Day-14 85.53 ± 0.45 292.98 ± 3.55 204.13 ± 1.30* 215.00 ± 1.24* 201.00 ± 5.23* 185.71 ± 2.46* 182.24 ± 4.21*
Day-21 89.00 ± 2.55 287.49 ± 4.30 140.00 ± 2.12* 163.80 ± 2.78* 161.80 ± 0.24* 170.00 ± 1.23* 176.66 ± 0.98*
Values are expressed as mean ± SEM (n=5), **p ˂ 0.01, *p ˂ 0.05 when compared with control group

Table 2: Effect of aqueous and ethanolic extracts of P. dulce on biochemical parameters in alloxan induced diabetic rats
Parameter Group-I Group-II Group-III Group-IV Group-V Group-VI Group-VII
Triglyceride (mg/dl) 118.62 ± 8.05 257.94 ± 6.94 131.00 ± 2.03* 136.00 ± 3.33* 135.60 ± 4.45* 139.99 ± 1.92* 129.88 ± 5.88*
Total Cholesterol
139.75 ± 5.23 216.51 ± 6.66 121.66 ± 8.33* 137.77 ± 5.88* 127.66 ± 8.77* 129.34 ± 1.13* 127.68 ± 2.99*
(mg/dl)
HDL (mg/dl) 52.44 ± 2.56 26.62 ± 1.40 48.54 ± 4.56* 38.26 ± 6.45* 44.60 ± 5.20* 33.20 ± 2.68* 39.42 ± 6.12*
Serum Urea (mg/dl) 38.40 ± 0.46 53.70 ± 1.23 36.66 ± 1.04* 37.92 ± 3.38* 34.60 ± 0.44* 42.38 ± 0.34* 42.73 ± 1.05*
Serum Creatinine
1.04 ± 0.08 1.43 ± 0.02 0.79 ± 0.09* 1.10 ± 0.66* 0.96 ± 0.08* 1.36 ± 0.39* 1.12 ± 0.02*
(mg/dl)
Serum Uric acid
1.12 ± 0.03 3.28 ± 0.08 1.27 ± 0.05* 1.79 ± 0.23* 1.30 ± 0.04* 1.82 ± 0.07* 1.46 ± 0.09*
(mg/dl)
AST (U/l) 140.00 ± 3.44 456.00 ± 6.30 144.00 ± 5.34 * 234.00 ± 7.34 * 184.00 ± 8.20 * 248.00 ± 3.43 * 204.00 ± 6.62*
ALT (U/l) 63.00 ± 4.32 380.00 ± 6.48 82.00 ± 4.20* 196.00 ± 6.24* 146.00 ± 3.08* 208.00 ± 5.08* 182.00 ± 4.30*
Values are expressed as mean ± SEM (n=5), **p ˂ 0.01, *p ˂ 0.05 when compared with control group

Table 3: Effect of aqueous and ethanolic extracts of P. dulce on liver, muscle and kidney glycogen levels in alloxan induced diabetic rats
Organ Group-I Group-II Group-III Group-IV Group-V Group-VI Group-VII
Liver 16.59 ± 2.54 8.07 ± 1.20 20.22 ± 2.03* 23.11 ± 0.32* 22.87 ± 0.15* 17.8 ± 0.18* 18.12 ± 0.04*
Skeletal Muscle 3.17 ± 0.27 1.53 ± 0.24 2.48 ± 0.31* 2.54 ± 0.31* 2.17 ± 0.03* 3.10 ± 0.45* 2.16 ± 0.09*
Kidney 0.72 ± 0.08 1.40 ± 0.32 1.18 ± 0.21* 1.41 ± 0.24* 1.59 ± 0.15* 1.11 ± 0.18* 1.57 ± 0.04*
Values are expressed as mean ± SEM (n=5), **p ˂ 0.01, *p ˂ 0.05 when compared with control group

Effect of extract on liver, skeletal muscle and kidney langerhan’s causing partial destruction and induces
glycogen diabetic rats diabetes mellitus through reduced insulin secretion. [23]
The effect of extract on liver glycogen level shows that The intraperitoneal administration of alloxan at the
there was significant increase in liver glycogen level. dose of 130 mg/kg destroys pancreatic β cells giving
The reduced Liver glycogen level in diabetic rats was rise to diabetic rats. [24] The induction of diabetes was
increased significantly by both AE and EE. The muscle confirmed by fasting blood glucose level above 200
glycogen level was reduced in alloxan induced diabetic mg/kg on fourth day of administration of alloxan
rats. The AE and EE showed significant (p ˂ 0.05) intraperitoneally using glucose strips.
increase in muscle glycogen level when treated for 21 The significant reduction in blood glucose level was
days. The effect on kidney glycogen level was found to observed in extracts treated rats when compared with
increase glycogen level in alloxan induced diabetic rats. diabetic control group. The rats treated with alloxan
Standard drug, AE and EE reduce significantly shows continuous rise in blood glucose level for 21
glycogen level in kidney. days. Traditionally many plants are used for the
treatment of diabetes mellitus and which were found to
DISCUSSION be very effective alternative for present drugs which
Considering the presence of insulin like principle in the are having many side effects. So considering this we
leaves of P. dulce, the present study was conducted to made an attempt to evaluate the antidiabetic potential
study the antidiabetic activity of P. dulce leaves extracts. of P. dulce leaves extracts in alloxan induced diabetic
The present era is gaining the more focus on plant rats. Both aqueous and ethanolic extracts on single dose
drugs as it is having less side effects and lower price as administration were not able to reduce the blood
compared to present allopathic drugs. [22] The present glucose level. The repeated administration of P. dulce
study reveals that the treatment with aqueous and leaves extracts for 21 days significantly reduces the
ethanolic extract of P. dulce leaves for 21 days shows blood glucose level when compared with diabetic
significant antidiabetic effect on alloxan induced control group. In present study we found that both
diabetic rats. extract of P. dulce when administered to diabetic rats
The acute toxicity study indicates that aqueous and reduces blood glucose level when compared with
ethanolic extract of P. dulce leaves when administered diabetic control group. The extracts might be produced
orally at the dose of 5000 mg/kg did not produced any their antidiabetic activity by increasing production or
sign of toxicity or death in treated animals. This release of insulin from pancreas or reducing insulin
suggests that LD50 of plant extracts is above 5000 resistance. [25] It is well known that the pioglitazone
mg/kg indicating the extract is non toxic for further use produces hypoglycemia by decreasing the peripheral
in study. Alloxan is commonly used chemical agent to resistance to insulin and increasing glucose transporter
induce diabetes in rats. It acts on pancreatic β cells of activity in cells. In our study both extracts of P. dulce
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 Mule et al. / Antidiabetic Activity of Extracts of Pithecellobium dulce Benth Leaves in Alloxan…..……

reduces blood glucose level which may be possibly due and ALT. The extract of P. dulce decreases significantly
to increase in insulin release or due to increase in AST and ALT level when compared with diabetic
peripheral glucose uptake. The extracts may be acting control group supporting its protective effect on the
on some of the surviving pancreatic β cells and liver.
increases the release of insulin responsible for The result suggests that in treated groups there is
antidiabetic potential. [26-27] significant improvement in liver and muscle glycogen
In oral glucose tolerance test blood glucose level was level while kidney glycogen level was decreased
estimated at the intervals of 0, 30, 60, 90 and 120 significantly when compared with diabetic control
minutes. This test shows the glucose utilization by group. This improvement in liver and muscle glycogen
body and which is used to diagnose the diabetes. The level may be due to increase in uptake of glucose from
blood glucose level by administration of two different blood. This effect on glycogen level support the
doses of P. dulce aqueous and ethanolic extract were probable increase in glucose uptake due to treatment
found to be dose dependent when compared to diabetic with P. dulce extracts.
control group. Our study reveals that both aqueous and ethanolic
In lipid metabolism alloxan produces hyperlipidemia extract produces its antidiabetic effect in alloxan
in diabetic rats which may be due to increase in release induced diabetic rats. The effect on lipid profile
of free fatty acids from stored fats. [28-29] Our present indicates the hypolipidemic activity of both extract
study also reveals that the lipid profile i.e. triglyceride which is dose dependent. Oral glucose tolerance test
and total cholesterol were significantly decreased by concludes the aqueous and ethanolic extract of P. dulce
aqueous and ethanolic extracts of P. dulce when promotes glucose tolerance. The effect of extracts on
compared to diabetic control group. In contrast, the blood urea, uric acid and creatinine indicates protective
mean HDL level of P. dulce treated rats was effect on kidney. As the extracts of P. dulce decreases
significantly higher as compared to diabetic control significantly AST and ALT level it may have protective
group. Thus the treatment with aqueous and ethanolic effect on liver. P. dulce leaves extracts also increases
extract of P. dulce may acts on stored fats which inhibits significantly liver and muscle glycogen which may be
the release of free fatty acids and decreases the due to increase in glucose uptake. Further detail study
triglyceride and total cholesterol level. The extracts also is required to identify the mechanism of action of
increase HDL level in diabetic rats after treatment for extracts which have antidiabetic potential.
21 days.
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