Combined Effect of Alcoholic leaf extract of Coccinia indica and Pioglitazone (low dose) in the

Progression of Diabetes Induced Nephropathy in Rats

ABSTRACT
The present study investigated the effect of alcoholic leaf extract of Coccinia indica alone and its
combination with pioglitazone in the progression of diabetes induced nephropathy in rats. Sprague
dawley rats were fed with HFD (High fat diet) for two weeks followed by I.P injection of Streptozotocin
(35mg/kg). Blood glucose levels, triglycerides, total cholesterol, BUN (Blood urea nitrogen), serum
creatinine and urinary protein were estimated. Kidney was excised for histopathological study and anti-
oxidants were also estimated. Low dose administration of streptozotocin to dietary manipulated rats
produced diabetes which induced the renal oxidative stress, altered lipid profile, and subsequently
produced nephropathy in 7 weeks by elevating serum creatinine, blood urea nitrogen, proteinuria and
inducing glomerular damage. Treatment with the combination of Coccinia indica leaf extract (200mg/kg)
with low dose of pioglitazone (7 mg/kg) normalized all the above mentioned nephropathic markers. The
combination of Coccinia indica and low dose of pioglitazone was more effective in attenuating the
diabetes-induced nephropathy and renal oxidative stress as compared to treatment with either drug alone.
It may be concluded that diabetes-induced oxidative stress, hyperglycemia and lipid alterations may be
responsible for the induction of nephropathy in diabetic rats. The concurrent administration of Coccinia
indica leaf extract (200mg/kg) with low dose of pioglitazone (7 mg/kg) may have prevented the
development of diabetes induced nephropathy by reducing the lipid alteration, controlling the blood
glucose levels and decreasing the renal oxidative stress and certainly providing the direct renoprotective
action. Further studies are required on long term basis to evaluate the effect of the combined treatment of
Coccinia indica leaf extract with low dose of pioglitazone for the complete prevention of diabetes
induced nephropathy in newly diagnosed type 2 diabetes mellitus patients without producing their
classical side effects.
Key words: Diabetes, Oxidative stress, Nephropathy, Coccinia indica, Pioglitazone, Low dose strategy.

INTRODUCTION
Diabetes Mellitus is an endocrine disorder which is characterized by depleted insulin secretions with
altered carbohydrate, lipid and protein metabolism. Various secondary complications such as renal
failure, coronary artery disorder, cerebrovascular disease, neurological complications, blindness, limb
amputation, long term damage, failure of various organs, and eventually premature death are associated
with chronic hyperglycemia1.
Diabetic nephropathy (DN) is a condition of progressive damage of the kidney, which is characterized by
thickening of glomerular basement membrane, glomerulosclerosis, glomerular hypertrophy, podocyte
loss, expansion of mesangial cells, and tubulointerstitial fibrosis. Diabetic nephropathy is associated with
persistent elevated albuminuria, declined glomerular filtration rate, elevated arterial blood pressure, and
fluid retention2. DN is categorized into two stages; microalbuminuria and macroalbuminuria, in which the
kidney leaks more serum albumin than normal in the urine 3. Recent studies have revealed that antioxidants are
capable of neutralizing free radicals and are effective in preventing experimentally induced diabetes in animal
models4-5 as well as reduction in the severity of diabetic complications6. Reactive oxygen species (ROS) play an
important role in high glucose- induced renal injury7-8. Hence, renal morphological and functional alterations are the
specific characteristic futures of diabetic nephropathy in rats.
Coccina indica (Cuccurbitaceae) is a creeper that grows wild and in abundance in major parts of
India. Coccina indica (C.indica) has been used in ancient times for treating diabetes mellitus. It was
considered to be ‘Indian Substitute for insulin’ as it was significantly effective in diabetes treatment9.
C. indica has gained many reported scientific values for its antidiabetic property10-12. It also possess an
antioxidant activity, which may be attributed to its protective action on lipid peroxidation and to its
enhancing effect on cellular antioxidant defense contributing to the protection against oxidative damage
in streptozotocin induced diabetes13.Various parts of Coccinia indica possess different activities such as
hypoglycemia, antilipidemic, anti-inflammatory , analgesic, antipyretic, antinociceptive, hepatoprotective,
antimicrobial, antitussive, antilithitic, antimutagenic. It is also useful in curing some of the skin disorders
like ring worm, psoriasis, scabies, other itchy skin eruptions and ulcer activity 14. There is no report on the
usefulness of the combination of Coccinia indica leaf extract with pioglitazone. Therefore, the present
investigation is aimed at determining whether bioflavonoid rich extract of Coccinia indica leaf alone or in
combination with pioglitazone, an insulin sensitizer would modulate the progressive damage of kidney
induced by type 2 diabetes in rats. Low dose of pioglitazone was used to examine the synergistic activity
with Coccinia indica leaf extract because glucose uptake and tissue’s peripheral utilization by liver,
muscle, and fat, are crucial for maintaining normal blood glucose level.
2. MATERIALS AND METHODS
2.1. Experimental Animals
All the experiments were carried out with Sprague dawley rats weighing around 180 -200g after obtaining
approval from the Institutional Animal Ethical Committee (Approval Number: 2013/PCOL/001). Animals
were kept in the animal house of Krupanidhi College of Pharmacy, Bangalore, India, under controlled
conditions of temperature (23±2ºC), humidity (50±5%) and 12 h light-dark cycle. All the rats were
provided with normal pellet diet and water ad libitum, prior to the dietary manipulation. They were
acclimatized in institutional animal house.

2.2. Assessment of diabetes and lipid profile

The animals were fed with high fat diet (HFD) once a day for two weeks followed by I.P injection of
streptozotocin (35mg/kg) dissolved in ice cold citrate buffer (pH: 4.4) after overnight fasting 15. The blood
Samples were collected from tail vein and blood glucose was checked using glucose diagnostic kit
(AccuCheck). The rats with the non-fasting plasma glucose level of ≥250 mg dl-1were considered
diabetic and selected for further pharmacological studies. Oral glucose tolerance test was performed
before and after the treatment period. At the end of the experimental protocol (7 weeks after
administration of streptozotocin), the serum separated from blood samples were frozen until analyzing the
biochemical parameters. The serum glucose concentration was estimated using the commercially
available kit (Crest Biosystems, Goa, India) by glucose oxidase peroxidase(GOD-POD) method 16. The
serum total cholesterol was estimated using the commercially available kit (Crest Biosystems, Goa, India)
by cholesterol oxidase peroxidase (CHOD/PAP) method 17. The serum triglyceride was estimated using
the commercially available kit (Crest Biosystems, Goa, India) by glycerol phosphate oxidase (GPO/PAP)
method18.
2.3 Plant Material- Ethanolic extract of Coccinia indica
Coccinia indica dried leaves were charged to extractor along with ethanol. It was extracted by heating the
mass for about 5-6 hours in a closed system by re-pumping the extract to the herb bed. This process was
repeated. The extracts were combined and concentrated under reduced pressure at low temperature. This
was charged to drier unit for drying and separated the product in a powder form. This was further
powdered in a multimill to a fine mesh size. It was sieved using a sifter to make uniform particle size. The
product was blended to make a uniform and homogenous lot.
2.4. Assessment of diabetic nephropathy.
The diabetes mellitus-induced nephropathy was assessed biochemically by estimating serum creatinine,
blood urea nitrogen and proteinuria.
2.4.1. Estimation of serum creatinine.
The serum creatinine concentration was estimated by alkaline picrate method 19 using the commercially
available kit (Cresent biosystems, Goa, India). Briefly, 2.0 ml of picric acid reagent was added to 0.2 ml
of serum for deproteinization of specimen, which was mixed well and centrifuged at 3000 rpm to obtain a
clear supernatant. 100 µl of buffer reagent was added to 1.1 ml of supernatant, 0.1 ml of standard
creatinine and 0.1 ml of distilled water to prepare test, standard and blank, respectively. 1.0 ml of picric
acid reagent was added to blank and standard. The test tubes were mixed well and kept at room
temperature for 20 min. The alkaline picrate reacts with creatinine to form the orange coloured complex,
which was read at 520 nm spectophotometrically.
2.4.2. Estimation of blood urea nitrogen
Serum urea was estimated by GLDH-Urease method (end point method) with the help of Semi auto
-Analyzer Urea (BUN) Reagent. To 1000 μl of the reagent, 20 μl of standard urea (50 mg/dl) was added
and incubated for 10 min at 37οC. This incubated mixture was aspirated and concentration of standard
was calibrated to show a value of 50 mg/dl. The fasting serum urea was estimated by adding 20 μl of the
serum sample to 1000 μl of the reagent, mixed well and incubated at 37°C for 10 min. This incubated
mixture was aspirated and absorbance recorded against a reagent blankat 505 nm using Auto Analyzer 20.

2.4.3. Estimation of protein in urine

The total protein was estimated by Biuret method. The biuret reagent is prepared by dissolving of 3 g of
CuSO4 .5 H2O in about 500 ml of distilled water and 9 g of sodium-potassium tartrate and 5 g of KI are
added to this solution. Separately 24 g of NaOH are dissolved in 300 ml of distilled water.
Both solutions are then mixed and brought up to 1,000ml by distilled water. The reagent is stable for at
least 3 months at room temperature (20–23 °C).
To each of 2 ml urine, standard and blank (distilled water) samples, 2 ml of 1.5mol/L perchloric acid are
added, vigorously mixed for several seconds and left for 10 min at room temperature. The precipitate is
then separated by centrifugation at 3,000 g for15 min, the supernatant is discarded and 1.5 ml of the biuret
reagent is added to the samples. The colour intensity is measured 12 min after mixing the samples with
the biuret reagent against the blank sample at 540 nm 21.
2.4.4 Microalbuminurea Estimation
Urine microalbuminurea was estimated by Latex Turbidimetry Method with help of Analyser using
Microalbimin Reagent.To 1000 μl of the working reagent, 7 μl of standard microalbumin (6.1 mg/l) was
added. This mixture was aspirated and concentration of standard was calibrated to show a value of 6.1
mg/l. The urine microalbumin was estimated by adding 7 μl of the urine sample to 1000 μl of the working
reagent, mixed well. Both the above mixtures were aspirated and initial absorbance (A1) read right after
mixing and final absorbance (A2) 2 min after mixing at 540 nm using Analyzer 22.
2.4.5. Histopathological study
Cross-sections of kidneys were fixed in 10% buffered formalin. After fixation, tissues were dehydrated in
ascending grades of alcohol and embedded in paraffin, cut at 5μM and stained with Hematoxylin and
Eosin (H&E) for examination of cell structure or periodic acid Schiff reagent (PAS) for evaluation of
basement membrane thickening and glycogen deposition or Masson’s trichrome for detection of collagen
deposition. The kidneys were also examined under a light microscope for the presence of renal lesions
and interstitial inflammatory cell infiltration by an observer blinded to the animal treatment group 23.
2.5. Assessment of renal oxidative stress
The oxidative stress in the kidney was assessed by estimating renal antioxidant enzymes.
2.5.1 Superoxide Dismutase (SOD)
To 100μl of kidney tissue homogenate, a mixture containing 1 ml sodium carbonate, 0.4 ml NBT and 0.2
ml EDTA was added and zero-minute reading was taken at 560 nm. The reaction was initiated by adding
0.4 ml of 1 mM hydroxylamine HCl. The reaction mixture was incubated at 250C for 5min, the reduction
of NBT was measured at 560 nm. A parallel control without the tissue homogenate was also made in
same manner. One enzymatic unit of SOD is equal to the amount in the form of protein in 100μl of 10%
tissue homogenate required to inhibit the reduction of 24 mM NBT by 50% and is expressed as units/mg
of protein24.
2.5.2 Catalase
To 100μl kidney tissue homogenate, 1.9 ml phosphate buffer was added and absorbance was measured at
240 nm. 1 ml hydrogen peroxide was added to above reaction mixture and it was allowed to stand for 1
minute. The absorbance was measured at 240 nm using phosphate buffer as blank solution. One
international unit of catalase utilized is the amount which catalyzes the decomposition of 1mM hydrogen
peroxide per minute at 37o C and expressed in terms of units/mg of protein25.
2.5.3 Estimation of Lipid Peroxidation
3ml phosphoric acid and 1ml of 0.6% thiobarbituric acid was added to 0.5ml of kidney tissue
homogenate. This mixture was boiled over the water bath for 45 minutes. Upon cooling, 4 ml n-butanol
was added to the above mixture and mixed for 1 minute followed by centrifugation at 20000 rpm for 20
minutes. The organic layer formed, was transferred to a fresh test tube and the absorbance was measured
at 532nm26.
2.6. Experimental Protocol
Rats were divided into 7 groups and each group comprised of 10 rats. The drugs/extract were suspended
in 0.5% w/v of carboxy methyl cellulose (CMC). After the 2 weeks of dietary (high fat diet)
manipulation, the rats (60) were treated with low dose of STZ. The high fat diet / low dose STZ induced
diabetic rats were divided into 6 groups (Group II to Group VII). Diabetic rats from Group III to VII were
treated with respective drugs/extract for 7 weeks after one week of Streptozotocin administration. Group I
(Normal control), rats were maintained on high fat diet and no treatment was given. Group II diabetic rats
were considered as diabetic control (untreated). Diabetic rats belonging to the groups (III to VII) were
treated with low dose of fenofibrate (30 mg/kg p.o.)2, low dose of Pioglitazone (7mg/kg), leaf extract of
Coccinia indica (200 mg/kg p.o.)27, combination of Leaf extract of Coccinia indica (200 mg/kg, p.o.) plus
low dose of pioglitazone and with lisinopril(1 mg/kg p.o.)2 for 7 weeks respectively.
2.7. Statistical Analysis
All values were expressed as mean ± S.D. The data obtained from various groups were statistically
analyzed using one way ANOVA, followed by Dunnett’s test. The P value of less than 0.5 was
considered to be statistically significant.
2.8. Drugs and chemicals
Streptozotocin was obtained from Sigma Aldrich Ltd. Carboxymethyl cellulose was purchased from
Quest International, Bangalore, India. Fenofibrate and pioglitazone were obtained from Ranbaxy
Laboratory Ltd, Gurgaon, India. Lisinopril was obtained from Dr.Reddy’s Laboratory Ltd, Hydrebad,
India. All other chemicals used in the present study were of analytical grade. Coccina indica was obtained
as a gift sample from Green Chem Pvt Ltd, Bangalore.
3. RESULTS
Administration of fenofibrate (30 mg/kg p.o.,7 weeks) or pioglitazone (7 mg/kg p.o., 7 weeks) at lower
doses did not produce any significant per se effect on various parameters assessed in normal rats (Results
not shown). Administration of streptozotocin (35 mg/kg, i.p., once) after post dietary manipulation
produced frank hyperglycemia. After 7days of streptozotocin administration, the rats that showed blood
glucose level of greater than 260 mg/dL were considered as diabetic
rats. Coccina indica (200mg/kg p.o.,7weeks), Coccina indica (200mg/kg p.o.,7weeks) plus pioglitazone
(7mg/kg p.o.,7weeks), Fenofibrate (30mg/kgp.o.,7weeks), pioglitazone (7mg/kgp.o.,7 weeks) and
lisinopril (1 mg/kg, p.o.,7weeks) were administered to diabetic rats after 7 days of injection of
streptozotocin and their treatments were continued for 7 weeks. All the physiological parameters were
assessed at the end of 7 weeks in normal and diabetic rats with or without drug treatments. The mortality
rate was lesser than 10 % in diabetic rats with or without drug treatments.

3.1. Effect of therapeutic interventions on serum glucose

There was a significant increase in the serum concentration of glucose in diabetic rats as compared to
normal rats. Treatment with low dose of fenofibrate (30mg/kgp.o.,7weeks) did not show any change in
the serum glucose concentration in diabetic rats. Low dose pioglitazone treated diabetic rats showed a
partial decrement in the glucose level in diabetic rats. However, the combined treatment of Coccinia
indica (200 mg/kg p.o., 7 weeks) with low dose of pioglitazone (7 mg/kg p.o., 7 weeks) showed
significant control in serum glucose levels as compared with groups treated with pioglitazone or Coccinia
indica alone. Lisinopril (1mg/kg p.o.,7 weeks) treated diabetic rats showed a slight decrease in glucose
level, but not statistically significant. (Table 1)
3.2. Oral Glucose Tolerance Test (OGTT):

3.2.1. Before Treatment

The effect of the single dose of Alcoholic leaf extract of Coccinia indica (200 mg/kg) and pioglitazone on
oral glucose tolerance test was recorded in the results of Table 2. It is important to note that in Table 2,
the blood glucose value in the control group rose to a peak value at 1 h after glucose load and then fell to
near 0 h fasting level almost by the end of 2 h. Close study of Table 2 clearly indicates that the Coccina
indica leaf extract showed definite lower peak blood glucose values, 2 hr after glucose load indicating
more pronounced antihyperglycemic activity. On the other hand, a single dose of pioglitazone (10mg /kg)
treatment showed more pronounced antihyperglycemic activity than Coccina indica leaf extract
treatment. (Table 2)
3.2.2. After Treatment
Results (table 3) show the mean fasting plasma glucose values of normal, diabetic untreated and diabetic
treated rats subjected to glucose tolerance test after 7 weeks. The fasting blood glucose values for normal,
diabetic untreated and diabetic treated with pioglitazone, Coccinia indica and the combined therapy of
pioglitazone plus Coccinia indica was 79.83 ± 3.25, 407.83 ± 3.15, 106.17 ± 4.20, 120.00 ± 3.01 and
108.33 ± 3.91 respectively. In the case of normal animals, the peak values were obtained in 1 hr and
returned back to almost initial values in 2 hours. The untreated diabetic rats showed a 10% increase in
blood glucose level at the end of 2 hr. The treated diabetic rats showed definite lower peak blood glucose
values, 2 hr after glucose load indicating more pronounced antihyperglycemic activity. The combined
therapy of pioglitazone an d Coccinia indica leaf extract with pioglitazone showed a better
antihyperglycemic effect than when treated alone. (Table 3)

3.3. Effect of therapeutic interventions on serum lipid profile

Increase in serum concentrations of total cholesterol and triglycerides levels were noted in diabetic rats as
compared to normal rats. Low dose fenofibrate (30 mg/kg p.o.,7 weeks) treatment significantly attenuated
diabetes-induced alteration in lipid levels. But treatment with low dose of pioglitazone (7 mg/kg p.o.,7
weeks) did not significantly alter the lipid levels in diabetic rats. Diabetic rats treated with Coccina indica
(200mg/kg p.o.,7 weeks) showed a significant reduction in lipid levels when compared with untreated
diabetic rats. On the other hand, the combined treatment with Coccina indica with low dose of
pioglitazone did not show a significant decrease in lipid levels when compared with the groups of rats
treated with Coccina indica alone but showed significance when compared with rats treated with
pioglitazone alone. However, treatment with lisinopril (1 mg/kg, p.o.,7 weeks) partially prevented the
diabetes-induced increases in triglycerides levels. (Table 4)

3.4. Effect of therapeutic interventions on serum creatinine and blood urea nitrogen

The serum levels of creatinine and blood urea nitrogen were markedly increased in diabetic rats as
compared to normal rats. Diabetic rats treated with Coccina indica (200mg/kg p.o.,7 weeks), low dose of
fenofibrate (30 mg/kg p.o.,7 weeks) and low dose of pioglitazone (7 mg/kg p.o.,7 weeks) partially reduced
the serum creatinine and blood urea nitrogen. Moreover, the combined therapy of Coccina indica
(200mg/kg p.o.) and pioglitazone (7 mg/kg p.o.,7 weeks) markedly reduced the elevated levels of serum
creatinine and blood urea nitrogen as compared to treatments with either extract/drug alone or lisinopril (1
mg/kg, p.o.,7 weeks) in diabetic rats. (Table 5)
3.5. Effect of therapeutic interventions on proteinuria

There was a significant increase in proteinuria in diabetic rats when compared to normal rats. Coccina
indica (200mg/kg p.o.,7 weeks), low dose of fenofibrate (30 mg/kg p.o.,7 weeks) and low dose of
pioglitazone (7 mg/kg p.o.,7 weeks) partially reduced the incidence of proteinuria in diabetic rats. On the
other hand, the combined therapy of Coccina indica (200mg/kg p.o.,7 weeks) with pioglitazone (7 mg/kg
p.o.,7 weeks) significantly reduced the occurrence of proteinuria as compared to treatments with either
alone or lisinopril (1 mg/kg, p.o.,7 weeks) in diabetic rats. (Table 6)
3.6. Effect of therapeutic interventions on renal oxidative stress

After 7 weeks, the untreated diabetic rats showed a marked increase in renal thiobarbituric acid reactive
substances as compared to normal rats. In addition, the renal concentration of SOD and catalase was also
decreased in these untreated diabetic rats as compared to the normal rats. Treatment with Coccina indica
alone (200mg/kg p.o.,7 weeks), low dose of fenofibrate (30 mg/kg p.o.,7 weeks) and low dose of
pioglitazone (7 mg/kg p.o.,7 weeks) partially prevented the diabetes-induced increase in renal
thiobarbituric acid reactive substances and decrease in renal SOD and catalase levels. Moreover, the
concurrent administration of Coccina indica (200mg/kg p.o.,7 weeks) and pioglitazone (1 mg/kg p.o., 7
weeks) markedly attenuated the diabetes-induced increase in renal thiobarbituric acid reactive substances
and decrease in renal SOD and catalase levels as compared to treatments with either extract/drug alone or
lisinopril (1 mg/kg, p.o.,7 weeks) in diabetic rats. (Table 7)
3.7. Histopathological Analysis
Effect of Coccinia indica alone and in combination with low dose of pioglitazone on renal histological
study. The sections of 5 μm thickness were made and stained with hematoxylin and eosin to assess the
pathological changes of glomeruli using the light microscopy (10X). The kidney of the diabetic rat
developed pathological changes in the glomeruli such as parenchymal edema, tubular congestion and
extracellular mesangial expansion as compared to the kidney of the normal rat. The concurrent
administration of Coccinia indica and low dose of pioglitazone markedly reduced these pathological
changes in glomeruli as compared to treatment with either drug alone. Treatment with combination of
Coccinia indica and low dose of pioglitazone was observed to be as effective as treatment with Lisinopril
(Nephropathy standard).

4. DISCUSSION
Our study observed that the combined therapy of Coccina indica leaf extract with low dose of
pioglitazone had renoprotective effect in diabetic induced nephropathy in rats.
Creatine phosphate metabolism by skeletal muscles produces creatinine, a non-protein waste product
which is freely filtered by the kidney and thus the serum creatinine level depends on the glomerular
filtration rate (GFR). A raise in the level of serum creatinine is considered to be an index of renal
dysfunction. The blood urea nitrogen measures the amount of urea nitrogen, a waste product of protein
metabolism. A significant raise in the level of blood urea nitrogen indicates kidney’s dysfunction. The
presence of proteinuria correlates with the progression of glomerulosclerosis and tubulointerstitial
fibrosis. Diabetes-induced proteinuria often induces inflammation in tubular cells, which can facilitate the
development of interstitial fibrosis and tubular atrophy28.
The presence of small quantities of albumin (Microalbuminuria) predicts development of clinical diabetic
nephropathy29. The abnormalities in blood, urine and pathological changes in glomeruli have been
documented to be an index of experimental diabetic nephropathy. In our study, the untreated diabetic rats
for 7 weeks presented abnormalities in serum creatinine, blood urea nitrogen and proteinuria, thus
suggesting the presence of diabetes induced nephropathy. The therapeutic intervention with either
Coccina indica leaf extract or pioglitazone partially prevented the diabetes induced nephropathy by
decreasing the serum creatinine, blood urea nitrogen, proteinuria, and the pathological changes in
glomeruli was also reverted back to normal. It is interested to note that the concurrent administration of
Coccina indica leaf extract with low dose of pioglitazone showed a synergistic effect in delaying the
progression of diabetes-induced nephropathy as compared to treatments with either alone.
Previous studies suggest that hyperlipidemia is an independent risk factor for the induction and
progression of diabetes-induced nephropathy. In diabetic condition the expressions of LDL receptors on
the cell surface were shown to be decreased and there by altering the circulating lipid profile which in
turn contributing to renal disease progression. In the renal, the circulating lipids are trapped by
extracellular matrix molecules. These trapped lipids undergo oxidation and generates reactive oxygen
species, which may deteriorate the structure and function of the diabetic kidney. Thus, the elevated
circulating lipids may play a pivotal role in the induction and progression of nephropathy. The same is
supported by the results obtained in the present study which showed a marked increase in serum
cholesterol and triglyceride levels in untreated diabetic rats with nephropathy.
Low dose treatment with fenofibrate prevented the development of diabetic nephropathy. Fenofibrate acts
as an hypolipidemic through an activation of PPAR-α. Increase in circulating lipids indicates PPAR-α
deficiency that can aggravate the severity of diabetic nephropathy in diabetic mice. In our study the
renoprotective effect of fenofibrate may be attributed to PPAR-α activation-mediated reduction in
circulating lipids in diabetic rats. Diabetic rats when treated with a combination of Coccinia indica and
low dose of pioglitazone showed a significant effect on lowering the triglyceride and cholesterol levels
when compared to untreated diabetic rats.
Induction and progression of nephropathy in diabetic rats is due to uncontrolled hyperglycemia.
Hyperglycemia upregulates the renal generation of angiotensin-II and TGF-β and there by accelerating
the process of renal advanced glycation end products formation. Moreover, hyperglycemia induces
oxidative stress by generating reactive oxygen species through an activation of NADPH oxidase to
damage the structure and function of the diabetic kidney. Thus, hyperglycemia contributing as a pivotal
role in the progression of nephropathy.
Hyperglycemic condition reduces the mRNA levels of PPARγ in the glomeruli but for the glucose
metabolism, the activation of PPAR-γ is involved30. Treatment with combination of Coccinia indica and
low dose of pioglitazone had significant effects on lowering the blood glucose levels. Oral glucose
tolerance test conducted in our study supports the antidiabetic effects of Coccinia indica leaf extract and
pioglitazone, because of the changes in the percentage of glucose level at the end of 2hr post glucose load
due to an increase in peripheral utilization of glucose. Pioglitazone, exhibits its antihyperglycemic action
by acting through PPARγ. Similarly, Coccinia indica leaf extract is well documented for its
antihyperglycemic activity9-12. Long term therapy of pioglitazone causes hypoglycemia and cardiovascular
toxicity. These adverse effects of pioglitazone can be prevented by reducing the dose of pioglitazone and
combining with herbal extracts to synergize its antidiabetic effect. Similarly, our study has also shown the
synergistic effect of Coccinia indica leaf extract with low dose of pioglitazone in reducing the glucose
levels.
The increase in renal thiobarbituric acid reactive substances and decrease in SOD and catalase levels
indicates the development of oxidative stress 31. Diabetic rats showed an increase in the renal
thiobarbituric acid reactive substances and consequently decreased the SOD and catalase levels in the
kidney, damaging renal architecture. Treatment with Coccinia indica or pioglitazone had partially
reduced the oxidative stress in diabetic rats with nephropathy by reducing renal thiobarbituric acid
reactive substances and concurrently elevating renal SOD and catalase levels. Moreover, the combination
of Coccinia indica and low dose of pioglitazone markedly prevented the development of renal oxidative
stress in diabetic rats with nephropathy that is supported by the histopathological findings.
The overall observed beneficial effect of combination of Coccinia indica and pioglitazone in preventing
the development of diabetic nephropathy may be attributed to reduction in high circulating lipids, blood
glucose level and prevention of renal oxidative stress. Lisinopril has been employed as a standard drug
due to its diabetic renoprotective effect which is well documented in basic and clinical studies 32.
The renoprotective effect of Coccinia indica leaf extract with low dose of pioglitazone observed in our
study was as good as the effect produced by lisinopril in ameliorating diabetes-induced nephropathy
which is supported by the histopathological studies.

5. CONCLUSION
In the present study, it may be concluded that the concurrent administration of Coccinia indica and low
dose of pioglitazone may have prevented the progression of diabetes-induced nephropathy by controlling
the blood glucose levels, normalizing the altered lipid profile and decreasing the renal oxidative stress. In
addition, their combination strategy may provide synergistic renoprotective effect against diabetic
nephropathy with simultaneous prevention of hypoglycemia caused by pioglitazones when administered
for a longer period.
So we conclude that the treatment with the alcoholic leaf extract of Coccinia indica ameliorates
hyperglycemia, hyperlipidemia and oxidative stress associated with high fat diet / low dose STZ induced
diabetes.
Regular consumption of food containing these properties might prevent the development of diabetes and
delay in the progression of diabetes complication, especially in case of pre-diabetic state. Further studies
are required on long term basis to evaluate the effect of the combined treatment of Coccinia indica leaf
extract with low dose of pioglitazone for the complete prevention of diabetes induced nephropathy in
newly diagnosed type 2 diabetes mellitus patients without producing their classical side effects.
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