Indian Journal of Pharmaceutical Education and Research Association of Pharmaceutical Teachers of India

Anti-diabetic Activity of Diplocyclos palmatus Linn. in Streptozotocin-Induced

Diabetic Mice
Jaynarayan Tripathi , Reena Kumari, Vrish Dhwaj Ashwlayan* , Parveen Bansal and Ranjit Singh
1 1 1 2 3

Centre for Advanced Research in Pharmaceutical Sciences, Shobhit University, Modipuram, Meerut
1

Department of Biochemistry, PGIMER, Chandigarh

2

Principal, Government College of Pharmacy,Rohree, Shimla, H.P

ABSTRACT Submitted: 19/11/2011 Revised: 04/03/2012 Accepted: 19/0712012

The anti-diabetic activity of methanolic extract of seed of Diplocyclos palmatus Linn. (Cucurbetaceae) was evaluated in streptozotocin-induced diabetic mice.
Methanolic extract of the seed (150 mg/kg body weight), was administered orally to male Swiss albino mice. Streptozotocin was used to induce diabetes
mellitus. The anti-diabetic potential was assessed by determining oral glucose tolerance, fasting blood glucose, urine glucose, liver glycogen content, serum
lipid profile, change in body weight and histopathology. Methanolic seed extract was administered to normal and experimental diabetic mice for 15 days.
Significant (p < 0.001) reduction in fasting blood glucose levels was observed in the treated diabetic animals from day 7 onwards. In oral glucose tolerance test,
reduction of fasting blood glucose levels was noted after 60 min of extract administration. After 15 days of treatment with extracts the maximum reduction in
fasting blood glucose (53.87%) was observed in diabetic mice treated with methanolic extract 150 mg/kg. Serum lipid levels reversed towards near normal and
the loss of body weight was controlled in treated mice as compared to diabetic control. The extract treatment also showed a significant increase in the liver
glycogen content and gradual decrease in level of urine sugar level. Microscopically examined pancreas section of mice treated with 150 mg/kg methanolic
extract showed normal architecture of pancreas. The results demonstrate that seed of Diplocyclos palmatus possesses significant anti-diabetic activity. The
results suggest that Diplocyclos palmatus has anti-diabetic activity, thereby justifying its traditional use. The plant may be used in present day systems of
medicine as an anti-diabetic drug.
Keywords: Diplocyclos palmatus, Seeds, Streptozotocin, Diabetes.

INTRODUCTION projections on both faces. This species is widely distributed

throughout India and globally distributed in tropical and sub
Diabetes is a chronic metabolic disorder characterized by
tropical region of Asia, Africa and India. Leaves are used in
abnormalities in carbohydrate and lipid metabolism1, which
inflammations and impotency6 and to treat malarial fever and
leads to postprandial and fasting hyperglycemia,
chronic colitis7. Fruits contain bitter bryonin and are used in
dyslipidemia and hyperinsulinemia2. Although many
bilious attack, flatulence and inflammation. Its roots with
synthetic drugs show significant therapeutic potential, their
roots of Michelia champacais are used in asthma and to
use has already been restricted due to several undesirable side
promote conception8. It has likewise been employed in more
effects such as hepatotoxicity, cardiomegaly and
recent times in convulsions due to the presence of worms in
hemotoxicity3, 4. Despite the presence of known anti-diabetic
the intestine, as a cathartic in dropsy, and in cases of chronic
medicines in the market, screening for new drugs from plants
inflammations, attended with glandular enlargements, or
is still attractive because of their safety and efficacy. A serous effusions. Literature survey reveals its use as an anti-
number of plant species are known worldwide to have inflammatory, anti-malarial, anti-ulcer, anti-viral and anti-
hypoglycaemic potential5. Diplocyclos palmatus Linn. diabetic agent. However, sufficient scientific data to support
(Cucurbetaceae), is annual slender herb. Its leaves are these claims are still not available. Therefore, it seemed
palmately 5-lobed, scabrous along with smooth beneath, worthwhile to assess anti-diabetic potential of seeds of
denticulate margin. Its peduncle male flowers contain calyx Diplocyclos palmatus. There is no scientific evidence to
tube 2-4×3-6 mm, spreading lobes; greenish-yellow corolla, support its use as anti-diabetic drug hence, the objective of
shortly papillose, ovate, acute; lobes. Female flowers are this study was to establish the scientific basis of the use of
fasciculate. The fruits are spherical, yellowish-green, six seeds of Diplocyclos palmatus in the management of diabetes
striped. Its seeds are grey belted, attenuated with raised using streptozotocin-induced diabetic mice.
MATERIAL AND METHODS
*Address for Correspondence:
Vrish Dhwaj Ashwlayan, Associate Prof., Pharmaceutical Technology, M.I.E.T, Collection of plant materials: Seeds of Diplocyclos palmatus
Meerut, U.P., India were collected from Haridwar Uttrakhand (India) and
E-mail: ashwlayan18@yahoomail.com authenticated by Dr. Anjula Pandey, Principal Scientist at

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 Jaynarayan Tripathi et al. Anti-diabetic activity of Diplocyclos palmatus

Herbarium of National Bureau of Plant Genetic Resources Drug solution: The extract was emulsified in 0.5% w/v
(NBPGR) Pusa Campus, New Delhi. Seeds of plant were air aqueous solution of tween-80. Glibenclamide (Aventis
dried in the shade and coarsely powdered in a grinder. Pharma Ltd. Goa) was used as a standard drug. Streptozotocin
Preparation of extract:: The aqueous, methanolic and was dissolved in citrate buffer (pH 4.5).
petroleum ether extracts were prepared by extracting 100 gm Induction of non-insulin dependent diabetes mellitus
of air dried powder in a Soxhlet apparatus (Perfit, India) (NIDDM): NIDDM was induced in overnight fasted mice
which were further subjected to successive extraction using weighing 20-30 g by intraperitoneal administration of
petroleum ether (40-60°c), methanol and distilled water. streptozotocin (Sigma chemical Co. USA) solution prepared
Subsequently, the extracts were filtered; concentrated and in 0.1 M citrate buffer pH = 4.5 at the dose of 60 mg/kg body
dried using Rotary Vacuum Evaporator (Perfit, India) under weight. Diabetes was confirmed by the determination of
reduced pressure at ≤ 50°c temperature (yield 2.88, 3.62, fasting glucose concentration on the third day post
4.33 % respectively) and the residue was stored in desiccator administration of streptozotocin. Blood samples were
till subsequent use. collected after 1h of administration of streptozotocin on 1st, 4th,
Preliminary phytochemical screening: Preliminary 7th, 10th and 15thday. Elevation in blood glucose level was
phytochemical screening was carried out according to the found to be constant throughout 15 days. Serum glucose level
was determined by glucometer. Mice having serum glucose
method described by Kokate, et al8.
level between 300-400 mg/dl were selected for further study.
Animals: Swiss albino mice of either sex, weighing 25-30 g
Oral glucose tolerance test: The oral glucose tolerance test
were procured from Indian Toxicological Research Centre,
was performed in overnight fasted (18 h) normal mice. Mice
Lucknow. They were housed individually in polypropylene
divided into three groups, each consisting of six mice were
cages, maintained under standard conditions (12 hours light/
administered distilled water (10 ml/kg), glibenclamide (10
12 hours dark cycle, 25 ±1, 45-55% relative humidity). The
mg/kg, p.o.), MESDP (150 mg/kg, p.o.). Glucose (Central
animals were fed with standard rat pellet chow (Ashirwad
Drug House New Delhi) (2.5 g/kg, p.o.) was fed 0.5 h after the
Animal Feed Industries, Punjab, India) and tap water ad
administration of extract. Blood samples were collected by
libitum. All the animals were acclimatized for seven days
the tail-vein method just prior to the drug administration
before the study. The experimental protocol was approved by
Institutional Animal Ethics Committee (IAEC) (normal fasting) and at the time intervals of 0, 30, 60 and 120
1279/ac/09/CPCSEA/05. m after glucose loading. Blood glucose level was measured
immediately by using glucose oxidase-peroxidase reactive
Sample collection: After completing the treatment of 2 strips and a glucometer (Dr. Morepen, Tai Doc Technology
weeks, the mice were anesthetized by diethylether and Corporation, Taiwan).
sacrificed. Blood samples were collected by cardiac puncture
method and intermediately by tail vein method and blood Assessment of anti-diabetic activity of methanolic seeds
glucose levels were estimated using Dr. Morepen Glucometer extract of Diplocyclos palmatus: Mice were made diabetic by
(Tai Doc Technology Corporation, Taiwan). For intraperitoneal administration of streptozotocin at the dose of
histopathological studies, pancreas and the liver were 60 mg/kg. Treatment with plant extract was started 48 h after
dissected out immediately and transferred into 10% formalin. streptozotocin injection. Blood samples were withdrawn at
three day intervals till the end of study (i.e. 2 weeks).
Experimental design: All the animals were randomly divided
into the four groups with six animals in each group. Group I, Effect of methanolic seeds extract of Diplocyclos palmatus
II, III were administered vehicle (10 ml/kg distilled water), (MESDP) on lipid profile: Blood samples were collected by
diabetic agent {streptozotocin (60 mg/kg, i.p.)} + vehicle (10 the cardiac puncture method, in the centrifuge tubes and
ml/kg, p.o. distilled water), standard {glibenclamide (10 allowed to clot for 30 m at room temperature. Blood samples
mg/kg, p.o.)} respectively. Pilot study was carried out for were centrifuged (R-8C Laboratory centrifuge, Remi India) at
selection of dose of methanolic extract. Group IV was treated 3000 rpm for 20 m. Serum was separated as supernatant and
with STZ (streptozotocin) + MESDP (methanolic seeds stored at – 20°c until analysis.
extract of Diplocyclos palmatus). The experimental group determination of triglyceride levels: Triglyceride was
was subdivided in such a manner that all sub groups estimated by method of Wako and the modifications by
concurrently received STZ (60 mg/kg, i.p.) and either McGowan and Fossati method using Accurex, triglyceride
methanolic extract of Diplocyclos palmatus seeds (150 determination kit. Working reagent was prepared by
mg/kg, p.o.) or the glibenclamide (10 mg/kg, p.o.) or vehicle. dissolving contents of reagent 2 enzymes [lipoprotein lipase,
The above treatment was given daily for 2 weeks. glycerol kinase, glycerol-3-phosphate oxidase, peroxidase, 4-

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 Jaynarayan Tripathi et al. Anti-diabetic activity of Diplocyclos palmatus

aminoantipyrine and adenosine tri phosphate (ATP)] into one Trigyceride

VDDL Cholesterol -
bottle of reagent 1 buffer (3, 5 dichloro-2-hydroxybenzene 5
sulphonate, pH 7.0). It was swirled to dissolve and allowed to For this 0.5 ml of serum was taken in a test tube and 0.5 ml of
stand for 10 m at room temperature. Serum triglycerides were precipitation reagent was added. The mixture was shaken
hydrolyzed to glycerol and fatty acids by lipase enzyme. In thoroughly and left to stand for 10 m at 25 to 30°c and then
the presence of ATP and glycerol-kinase, glycerol was centrifuged for 20 m at 4000 rpm. Within 2 h after
converted into glycerol-3-phosphate and adenosine di centrifugation, the clear supernatant was used for the
phosphate (ADP). Glycerol-3-PO4 oxidase dissociates determination of HDL-Cholesterol. The supernatant
glycerol-3-phosphate into dihydroxy-acetone phosphate and containing 0.05 ml was taken in a test tube and 1 ml reaction
hydrogen peroxide. In the presence of peroxidase, hydrogen solution was added to it. In another test tube, 0.1 ml distilled
peroxide reacts with 4-aminoantipyrine to form a coloured water was taken and 1 ml reaction solution was added.
complex. The intensity of the colour developed was
The mixtures were mixed thoroughly, incubated for 5 m at
proportional to the triglycerides concentration and was
37°c and measured for the absorbance of the sample against
measured photometrically at 505 nm. The instrument was
blank reagent at 510 nm in Biochemistry Auto analyzer
adjusted to zero with distilled water. 10µl sample was taken
(STAR 21 plus, RAPID Diagnostic Pvt. Ltd.).
with the help of pipette into a cuvette. It was mixed and
incubated for 5 m. at 37°c . The absorbance of the samples Change in body weight: Body weight was taken before and
and standard of 200 mg/dl concentration was read out against after experiment at the intervals of 1st, 4th, 7th, 10th and 15th day
the blank. The colour should be stable for 30 m. Triglyceride of study with the help of single pan balance. The change in the
was estimated by using the following formula: body weight was noted.
Urine glucose estimation: The urine glucose levels were
Triglyceride( (
Mg
dl
=
Absorbance of Samples
Absorbance of standard
x Conc. of standard (mg/dl) estimated by “Benedict's (Rankem, RFCL Ltd. New Delhi)
test for glucose as reducing sugar in urine method”. The urine
estimation of total serum cholesterol (STC): Total was collected from the STZ-induced diabetic mice
cholesterol was estimated by CHOD-POD enzymatic individually in a clean beaker on the day 1, 4, 7, 10 and 15 and
colorimetric (Photoelectric colorimeter-114, Syntronics the glucose level was determined.
Ahmedabad) end point method9 using Accurex, cholesterol Liver glycogen estimation: The determination of glycogen in
determination kit. For this method, 0.01 ml each of serum as a liver was done by solution of “anthrone reagent12”..Purified
test, standard sample and distilled water as blank along with anthrone (500 mg), thiourea (10 g) and 1 liter of 72 % sulfuric
1.00 ml reaction solution were pipetted into the reaction acid were placed in a flask. The mixture was heated up to 80-
vessels using a micropipette. 90°c . The flask was occasionally shaken to mix the contents.
estimation of serum lipids: Phosphotungstate method10 was The mixture was cooled and stored in a refrigerator. Stock
solution of standard was prepared by dissolving 100 mg of dry
used to estimate the serum lipids like very low density
glucose in 100 ml of saturated benzoic acid solution. 5 ml
lipoprotein (VLDL), low density lipoprotein (LDL) and high
stock solution was placed in a 100 ml volumetric flask and the
density lipoprotein (HDL) cholesterol level. The clear
volume was made up with saturated benzoic acid solution.
supernatant after removal of VLDL and LDL, containing
HDL was used for determination of HDL-Cholesterol Liver was blended by blender under trichloroacetic acid
(HDLc). The VLDL and LDL from serum were precipitated (TCA) and homogenized for 3 m. The homogenate was
by phosphotungstate in the presence of magnesium ions. poured into a centrifuge tube. The supernatant fluid was
centrifuged and decanted upon an acid-washed filter paper
Phosphotungustate,mg *
2
placed in a funnel and drained into a graduated cylinder. The
Serum/Plasma HDL+(LDL+VLDL+Chylomicrons)
residue was quantitatively transferred to the blender with
TCA and homogenized again for 1 m. The mixture was
VLDL-Cholesterol (VLDLc) and LDL-Cholesterol (LDLc)
centrifuged and the supernatant fluid was poured through the
were respectively calculated by using Frederickson-
same filter. Two more extractions were made in the same
Friedwald's11 formula as follows: manner. The desired volume was made up with 5 percent TCA
and the solution was mixed thoroughly. 1 ml of the TCA
Trigyceride filtrate was pipetted into a 15 ml Pyrex centrifuge tube.
LDL Cholesterol = Total Cholesterol - - HDL Cholesterol
5 Duplicate samples of each unknown were analyzed to obtain

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 Jaynarayan Tripathi et al. Anti-diabetic activity of Diplocyclos palmatus

the most reliable results. To each tube, 5 volumes of 95 Where, DU = Optical density of the unknown, DS = Optical
percent ethanol were added with careful blowing. This was density of the standard, 0.1 = mg of glucose in 2 ml of standard
checked by noting the absence of an interface. The tubes were solution, 0.9 = factor for converting glucose value to glycogen
capped with clean rubber stoppers and allowed to stand value.
overnight at room temperature. After precipitation was
Histopathological studies: Isolated pancreas was preserved
completed, the tubes were centrifuged at 3000 r.p.m. for 15 m.
in 10 % formalin for 24 h. Pancreas was fixed in Bouin's fluid
The clear liquid was gently decanted from the packed
and cut in section of 3–5 μm thickness and stained by
glycogen and the tubes were allowed to drain in an inverted
hematoxyline-eosin stain. The photomicrographs of each
position for 10 m.
tissue section were taken using electron microscope.
The glycogen was dissolved by adding 2 ml of distilled water,
Statistical analysis: Values are presented as mean ± standard
the water being added in a manner that was washed down the
deviation for groups of six animals. The results were analyzed
sides of the tube. Blank reagent was prepared by pipetting 2
by one way analysis of variance (ANOVA) followed by post
ml of water into a clean centrifuge tube. A standard was
hoc Dunnett's multiple comparison test. Differences between
prepared by pipetting 2 ml of standard glucose solution,
means were considered to be statistically significant at (p<
containing 0.1 mg of glucose, into a similar tube. At this point
0.05).
10 ml of anthrone reagent was delivered into the centre of the
each tube with vigorous, but consistent, blowing to ensure RESULTS
good mixing. As each tube received anthrone reagent, it was Phytochemical testing: Preliminary phytochemical
tightly capped with an air condenser and placed in a cold tap screening revealed that saponins were present in all extracts
water bath. After the temperature of all tubes had reached the while alkaloid, carbohydrates, flavanoids, triterpenoids and
temperature of cold water, they were immersed in a boiling phenolic compounds were present in methanolic extract.
water bath to a depth a little above the level of the liquid in the Amino acids were present only in aqueous extract. Fat and
tubes for 15 m and then removed from water bath and cooled fixed oils were found only in petroleum extract.
to room temperature. The tubes and stoppers were wiped dry
and the contents of each tube were transferred to a calorimeter Fasting blood glucose determination: The effect of
tube and the absorbance was read at 620 nm after adjusting the treatment of the extracts on fasting blood glucose levels is
calorimeter with the blank reagent. Care was taken to avoid depicted in Table No. 1. Glibenclamide (GBC) treated
introduction of lint or contaminating carbohydrate into the diabetic mice of standard group III showed significant
anthrone reaction. The calculation of glycogen content was reduction in blood glucose values on day 1, 4, 7, 10 and 15
respectively in comparison to diabetic control group II. This
done by using the following formula13.
indicated that the GBC treatment successfully reduced the
blood glucose levels in the diabetic mice towards the normal
level in 15 days. Similarly, MESDP treated diabetic group IV
DU Volume of Extract
showed significant reduction in blood glucose values on day
x 0.1 x x 100 x 0.9 = mg. of glycogen per 1, 4, 7, 10 and 15 respectively as compared with diabetic
DS 100gm. of Tissue
100gm. of tissue control group II. This indicated that the MESDP treatment
could reduce the blood glucose levels in the diabetic mice
towards the normal level in the 15 days of study.

Table 1: Effect of MESDP on fasting blood glucose level in diabetic mice

Groups Treatment Fasting blood glucose level(mg/dl)
Day 1 Day 4 Day 7 Day 10 Day 15
I Distilled water (10 ml/kg, p.o.) 120.83± 12 113.16± 9.19 107± 15.53 116.83± 16.26 118.83± 14.02
II STZ (60 mg/kg, i.p.) + distilled water 312.67± 34.46 325.17± 40.93 326.4± 37.48 308.5± 51.86 305± 47
(10 ml/kg, p.o.)
III STZ (60 mg/kg, i.p.) + GBC
(10 mg/kg, p.o.) 363.5± 35.55** 215± 30.96*** 168.17± 40.65*** 150.5± 27.04*** 129.33± 19.21***
IV STZ (60 mg/kg, i.p.) + MESDP 320.67± 28.82 ns
264.17± 27.20** 202.17± 29.52*** 171.67± 17.60*** 140.67± 21.79***
(150 mg/kg, p.o.)
***p< 0.001 vs. diabetic control group II, **p< 0.01 vs. diabetic control group II, MESDP= methanolic extract of Diplocyclos palmatus seed,
GBC= glibenclamide.

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 Jaynarayan Tripathi et al. Anti-diabetic activity of Diplocyclos palmatus

Biochemical parameters estimation of lipid profile parameters: MESDP exhibited

oral glucose tolerance test (OGTT) in normal mice: significant reduction (p< 0.001) in all tested lipid parameters.
Treatment with GBC significantly improved the glucose A marked increase in total cholesterol and decrease in HDLc
tolerance at normal fasting levels at 0, 30, 60 and 120 m, were observed in untreated diabetic mice. MESDP
respectively. Further, treatment with MESDP significantly administration decreased serum triglycerides (STG); total
reduced sugar glucose level at 120 m compared to normal cholesterol (STC), LDL and VLDL levels and increased
control. These data suggested that treatment with MESDP HDLc level. The markers of dyslipidemia such as STC/HDLc
showed tolerance to glucose administration (Table No. 2). and LDLc/HDLc ratios were found to be significantly
urine glucose estimation: The urine sugar levels in normal elevated in the diabetic group (Table No. 5).
and diabetic group of mice are given in Table No. 3. The Table 4: Effect of MESDP on liver glycogen content in STZ-induced
normal control mice showed absence of sugar in urine. The diabetic mice
urine sugar levels of the different groups of diabetic animals Groups Treatment Liver glycogen
treated with standard drug (glibenclamide) and MESDP for content on day 15
15 days decreased towards the normal level. (mg/100 gm)
I Normal control Distilled water (10 ml/kg, p.o.) 542.86 ± 31.64
estimation of liver glycogen content: There was significant
increase in liver glycogen level to 473.25 ± 64.91 (p< 0.001) II Diabetic control STZ (60 mg/kg, i.p.) + 263.95 ± 8.62
Distiled water (10 ml/kg p.o)
on day 15 in glibenclamide treated diabetic control group III.
Similarly MESDP treatment significantly (p< 0.001) III Standard GBC (10 mg/kg, p.o.) 473.25 ± 64.91***
increased the glycogen content to 383.85 ± 42.96 (p< 0.001) IV Treated MESDP(150 mg/kg, p.o.) 383.85 ± 42.96***
in STZ-induced diabetic group IV (Table No.4). *** p< 0.001 compared to diabetic control, MESDP= methanolic extract of
Diplocyclos palmatus seed, STZ= streptozotocine, GBC= glibenclamide

Table 2: Effect of MESDP on OGTT in normal mice

Groups Treatments Normal fasting values(mg/dl) Blood glucose concentration(mg/dl)
0m 30 m 60 m 120 m
I Glucose(2.5 g/kg) 124.66 ± 5.92 269.83± 3.43 373.66± 4.08 250.33± 4.5 174.5± 4.5
II GBC(10 mg/kg) 104.83 ± 2.31*** 181.83± 5.07*** 237.5 ± 2.73*** 165.16± 3.31*** 147± 2.82***
III MESDP (150 mg/kg) 108.67 ± 4.93*** 190.33± 4.13*** 266.67± 4.55*** 186.5± 5.43*** 156.17± 8.30***
***p<0.001 vs. normal control, MESDP= methanolic extract of Diplocyclos palmatus seed, OGTT= oral glucose tolerance test, GBC= glibenclamide

Table 3: Effect of MESDP on urine glucose level in STZ-induced diabetic mice

Groups Groups Treatments Intensity of glucose in urine (colour change of the precipitate)
1 (days) 4 (days) 7 (days) 10 (days) 15 (days)
I Normal control Distilled water(10 ml/kg, p.o.) Nil Nil Nil Nil Nil
II Diabetic control STZ (60 mg/kg, i.p.) +
Distilled water(10 ml/kg p.o) +++ +++ +++ +++ ++++
III Standard GBC (10 mg/kg, p.o.) +++ ++ ++ + +
IV MESDP MESDP(150 mg/kg, p.o.) +++ +++ +++ ++ +

Keys: (+) = mild, (++) = moderate, (+++) = higher, (++++) = severe, MESDP = methanolic extract of Diplocyclos palmatus seed,
STZ = streptozotocine, GBC= glibenclamide.

Table 5: Effect of MESDP on lipid profile in STZ- induced diabetic mice

Treatment STG STC HDLc LDLc VLDLc STC/HDLc LDLc/HDLc
groups (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) ratio ratio
I Normal control 90.67± 4.5 82.33± 3.79 38.87± 1.02 25.33± 2.27 18.13± 0.9 2.11± 0.06 0.67± 0.02
II Diabetic control 132.67± 4.16 157± 5.29 18.67± 1.52 111.8± 6.6 26.53± 0.83 8.45± 0.89 6.03± 0.8
III MESDP (150 mg/kg) 117± 2.64* 133.33± 5.51* 33.67± 3.05** 76.27± 4** 23.4± 0.52* 3.97± 0.23** 2.27± 0.19**
*p< 0.05, **p< 0.01 compared to diabetic control, MESDP= methanolic extract of Diplocyclos palmatus seed,
STZ= Streptozotocine, STG =total serum triglyceride, STC=Total serum cholesterol, HDLc = high density lipoprotein cholesterol,
LDLc= low density lipoprotein cholesterol.

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Change in body weight: The body weight was slightly Histopathology: Photomicrographs showed normal
increased (28 ± 1.22 g) in the normal control group I as architecture of pancreas with acini of serous epithelial cells
compared to initial body weight. Whereas in diabetic control along with nest of endocrine cells separated by
group II, there was marked decrease (24.5 ± 0.5 g) in the body fibrocollagenous, stroma into lobules of vehicle-treated mice
(Fig.1a). Extensive damage to the acini of serous epithelial
weight. Group III treated glibenclamide and the group IV
cells and islets of langerhans (Fig.1b), restoration of normal
pretreated with MESDP increased body weight significantly architecture of pancreas with acini of serous epithelial cells by
to 27.5 ± 1 g (p< 0.01) and 27.58 ± 0.58 g (p< 0.05) glibenclamide (Fig.1c) are also shown. The partial restoration
respectively. Although there was a marginal reduction in the of normal cellular population and normal architecture of
body weight of animals in these groups, compared to the final pancreas with acini of serous epithelial cells along with nest
weight of normal control mice (Table No.6). of endocrine cells separated by fibrocollagenous, stroma into

Table 6: Effect of MESDP on body weight (gm) in STZ-induced diabetic mice

Groups Treatments Body weight(g)
Day 1 Day 15
INormal Control Distilled water(10 ml/kg, p.o.) 26.83 ± 0.98 28 ± 1.22
II Diabetic Control STZ (60 mg/kg, i.p.)+ Distiled water(10 ml/kg p.o) 27.91 ± 0.66 24.5 ± 0.5
III Standard GBC (10 mg/kg, p.o.) 28.91 ± 0.86 27.5 ± 1**
IV Treated MESDP(150 mg/kg, p.o.) 29.08 ± 0.92 27.58 ± 0.58*
**p< 0.01,*p< 0.05 compared to diabetic control, MESDP = methanolic extract of Diplocyclos palmatus seed,
STZ= streptozotocine GBC= glibenclamide

Fig.1: Histological study of pancreas of isletes of Langerhans

a. Control: Distilled water (10 ml/kg, p.o) b. Diabetic control: STZ (60 mg/kg, i.p.)

c. Standard: Glibenclamide (10 mg/kg, p.o) d. Treated: MESDP (150mg/kg, p.o)

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 Jaynarayan Tripathi et al. Anti-diabetic activity of Diplocyclos palmatus

lobules was shown by methanol extract. No fibrosis or reached its maximum at 120 m. Administration of the extract
inflammation was noted (Fig. 1d). to diabetic mice showed a significant decrease in the fasting
blood glucose. Hence, the possible mechanism of anti-
DISCUSSION
hyperglycemic action of MESDP is the potentiation of the
The aim of present study was to investigate the anti-diabetic insulin effects of plasma by increasing either the pancreatic
potential of methanolic extract of seed of Diplocyclos secretion of insulin from the existing beta cells or by its
palmatus (MESDP), using STZ-induced diabetic mice release from the bound form.
model. Hyperglycaemia produced by STZ exhibited marked
The decrease in hepatic glycogen content in diabetes is
increase in serum triglycerides and total cholesterol. Under
probably due to lack of insulin in the diabetic state which
normal conditions, the enzyme lipoprotein lipase hydrolyses
results in the inactivation of glycogen synthase enzyme15. The
triglycerides. Diabetes mellitus results in failure to activate
significant increase in the glycogen content of the treated
this enzyme thereby causing hypertriglyceridemia. Elevated
groups may be because of reactivation of the glycogen
serum total cholesterol, triglycerides and decreased high
synthase enzyme. Hence, improvement of glycogenesis may
density lipoprotein level were observed in diabetic control
be another probable way of anti-diabetic action16. The anti-
mice. Chronic administration of the extract for 15 days to the
hyperglycemic activity caused by glibenclamide and MESDP
STZ-induced diabetic mice significantly (p < 0.05) produced
in streptozotocin-induced diabetic mice indicates
a fall in blood glucose level and lipid profile. Hence the
normalization of serum lipid profile and stimulation of insulin
methanolic extract may be considered to have good anti-
secretion from beta cells. Flavonoids, sterols/triterpenoids,
hyperglycemic activity and did not cause any hypoglycemic
alkaloids and phenolic compounds are known to be bioactive
effect unlike insulin and other synthetic drugs. Normalization
anti-diabetic principles17. Flavonoids are known to regenerate
of the blood glucose level resulted in significant reduction in
the damaged beta cells in the alloxan-induced diabetes in
the level of serum cholesterol and triglycerides. The anti-
rats18. Phenolic compounds are found to be effective anti-
hyperglycemic activity caused by glibenclamide and MESDP
in streptozotocin-induced diabetic mice indicates hyperglycemic agents19. The anti-diabetic effect of MESDP
normalization of serum lipid profile and stimulation of insulin may be due to the presence of more than one anti-
secretion from beta cells. The observed hypolipidaemic effect hyperglycemic constituent and their synergistic properties.
may be because of decreased cholesterogenesis and fatty acid CONCLUSION
synthesis. Significant lowering of total cholesterol and
It is thus concluded that Diplocyclos palmatus (MESDP) has
elevation of HDL cholesterol are very desirable biochemical
promising anti-diabetic effect, which potentially improved
states for prevention of atherosclerosis and ischemic
abnormalities of diabetic conditions in streptozotocin-
conditions.
induced diabetic mice. The probable hypoglycemic effect of
In diabetic control group, the characteristic loss of body MESDP may be attributed to increase in serum and pancreatic
weight is caused by an increase in muscle wasting and loss of insulin levels. However, longer duration studies on chronic
tissue proteins14. The difference in the body weight observed models are required to elucidate the exact anti-diabetic
during the period of treatment of the mice treated with mechanism of action. As well as there is a need to isolate
MESDP was less as compared to the diabetic control group, bioactive principles which can be developed as potent anti-
which may be due to its protective effect in controlling muscle diabetic drug.
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