Indian Journal of Traditional Knowledge

Vol. 15(3), July 2016, pp. 460-465

Evidence of the nephroprotective effect of Carica papaya L. leaves in

streptozotocin-induced diabetic rats
Inayat U Rahman1*, Mohammad Bashir2 & Khalil Ur Rahman3
*1
Gandhara College of Pharmacy, Gandhara University, Peshawar, KPK, Pakistan;
2
Department of Physiology, KIMS-Kohat, KPK, Pakistan; 3Department of Anatomy, Bannu Medical College, KPK, Pakistan
E-mails:krahman@yahoo.com; inayat@gandhara.edu.pk*; dr_mbashir@yahoo.com
Received 19 August 2015, revised 25 February 2016

Protection against diabetic nephropathy (DN) is one of the main targets in diabetes treatment and present study
evaluates the nephroprotective effect of Carica papaya L. in streptozotocin (STZ) induced diabetic rats. DN rats were
treated with 1.5 and 2.5 gm/dl C. papaya leaf extract for 6 weeks to determine its nephroprotective effect with different
parameters. Pimagedine (1 ml/mg) served as a reference drug. Compared to diabetic control group, C. papaya (1.5 and 2.5
gm/dl) treatment significantly decreased some important parameters including plasma glucose, HbA1-c, urinary AER and
albumin/creatinine ratio. Improvement in GFR was also significant by C. papaya. However, the decrease in blood urea
nitrogen (BUN), plasma creatinine, blood pressure (B.P), total cholesterol and serum albumin levels were significant only in
diabetic group treated with 2.5 gm/dl of C. papaya leaf extract. Serum triglyceride and urine volume decreased with both
low and high doses of C. papaya. Histological examination revealed marked improvement in glomerular morphology after
C. papaya treatment. The study concludes that C. papaya leaf extract may exert ameliorative effect on DN.

Keywords: Carica papaya L., Diabetic rats, Nephropathy

IPC Int. Cl.8: A61K 36/00, A01D 16/02, A01D 13/37, A01D 13/00, A01D 20/00

Since time immemorial, plant-based therapies have
been in use by diverse cultures of the world to treat
various ailments. The significance of plants in the
treatment of diabetes has long been published and
until now more than 270 plant species have been
identified as having hypoglycemic activity however,
the information regarding their usefulness in the
prevention of diabetic complications is limited.
Among them lies C. papaya L. (Caricaceae),
popularly known as pawpaw or papaya or papita1,2.
The edible portion of the plant is its pleasant fruit
which is easily digested and is a source of high
nutritional value. It is grown in almost all tropical and
many subtropical regions of the world and due to the
medicinal properties of different parts of the plant, it
is used as therapeutic remedy in the form of
infusions3. There are reports that C. papaya leaves are
useful in subsiding the signs and symptoms of
dysentery, worming and asthma4,5. In addition, the
leaf extracts of C. papaya have long been used for the
treatment of cancer and infectious diseases5. The
aqueous extract of papaya leaf is believed to
accelerate wound healing6,7, whereas the methanolic

*Corresponding author
extract possesses vasodilating and antioxidant effects,
indicating its usefulness in the prevention and/or
delaying of cardiovascular diseases4. In Mexican folk
medicine, the plant is used to treat inflammation and
diabetes7,8. It also relieves diabetes associated
oxidative stress9. The evidence of hypoglycemic and
antioxidant properties of C. papaya in diabetes
mellitus diverted our attention to evaluate the
usefulness of the aqueous extract of C. papaya leaves
in diabetic nephropathy in streptozotocin-induced
diabetic rats. Diabetic nephropathy is characterized by
hyperfiltration by glomerulus and albuminuria along
with the expansion of the glomerular mesangium
which is related to the loss of renal function10. It is the
major cause of morbidity and mortality and despite
treatment with drugs like anti-hypertensives or
angiotensin converting enzyme inhibitors (ACEI),
nephrotic complications among diabetics are still on
the rise in many regions of the world11,12. Persistent
hyperglycemia and overproduction of reactive oxygen
species (ROS) are the major contributing factors in
the development of diabetic nephropathy and this
study was designed to determine the possible
antinephrotic effects of C. papaya in a rat model of
diabetic nephropathy.
 RAHMAN et al.: BENEFITS OF CARICA PAPAYA IN DIABETES MELLITUS
Methodology
Animals
A total of 30 male albino rats, 6–8 weeks of age
and weighing around160-200 gm, were purchased
from the Animal House of National Institute of
Health, Islamabad, Pakistan. The rats were housed
and maintained at 25±2 °C temperature under 12-hrs
light/12-hrs dark cycle and were fed on standard rat
diet and water ad libitum13. Experimental protocol for
the present study was approved by Institutional
Animal Ethical Committee of Gandhara University,
Peshawar.
Induction of diabetes
Diabetes was induced by intraperitonial injection of
60 mg/Kg streptozotocin freshly prepared in distilled
water14. Control group received an equivalent amount
of normal saline. After a week, rats having fasting
plasma glucose values of ≥ 300 mg/dl were
considered diabetic and were included for diabetic
nephropathy experiment for a period of six weeks.
Carica papaya leaf extracts processing
Leaves of C. papaya were collected during June–
September, 2014 from Karachi, Pakistan. The plant
was authenticated by Pharmacognosy expert at
Gandhara college of Pharmacy Peshawar, before
processing and the voucher specimen is kept at
University herbarium (Specimen No. 1322). C.
papaya leaves were first washed in 1% iodine
aqueous solution followed by washing with distilled
water and then dried. Two leaf portions weighing 10
gm and 15 gm were obtained and homogenized in
water followed by filtration through a No. 41 filter
paper with 20–25 µm retention (Whatman) to yield
1.5gm/dL and 2.5gm/dL extracts respectively. These
doses of C. papaya leaves extract selected were based
on the reported improvement in glucose tolerance and
oxidative stress15. The extracts were administered as
drinking water.
Study design
After three weeks of streptozotocin injection, rats
began to show signs of nephropathy16,17. The rats were
randomized into 5 groups with 6 rats in each group as
follows: normal control (NC) and diabetic control
(DC) groups received drinking water only; diabetic
nephropathy group treated with 1.5 gm/dL C. papaya
(C.P-1.5 gm/dL); diabetic nephropathy group treated
with 2.5 gm/dL C. papaya (C.P-2.5 gm/dL) and:
diabetic nephropathy group treated with 1 ml/mg
pimagedine bicarbonate (PMG-1ml/mg) as a
461
reference drug. The treatment was started on 22nd day
of streptozotocin injection and was considered as the
1st day of treatment.
Plasma glucose was determined weekly for 6
weeks. All other parameters were determined at the
start (Week 0) and at the end of the experiment (week
6). At the end of the study period, blood was
withdrawn by cardiac puncture and collected in tubes
to separate plasma. The body weight was measured
and 24 hrs urine samples were collected using
metabolic cages. Then the rats were anesthetized and
sacrificed. Kidneys were removed and weighed and
the kidney/body weight ratio was calculated. The
kidneys were preserved in 10% (v/v) formalin
solution for histopathological changes.
Measurement of biochemical parameters
Various blood and urine parameters including
plasma glucose, blood urea nitrogen, total cholesterol,
triglycerides, creatinine and serum albumin
concentrations were determined on a Boehringer
Mannheim ⁄ Hitachi 737 automated analyzer (Lewis,
UK). HbA1-c was measured using Fast ion-exchange
resin separation method. Blood pressure was
measured by tail-cuff method18. Albumin excretion
rate (AER) and albumin/creatinine ratios in urine
were determined according to manufacturer’s
specifications19. Glomerular filtration rate (GFR) was
calculated using the formula:
Urinary creatinine concentration Χ 24 hrs urine volume
GFR=
plasma creatinine concentration Χ1440
Differences in mean body weight, kidney weight
and water intake/100 gm of body weight were
determined at the end of the study period for all the
experimental groups.
Histopathological examination
The excised kidneys were cut into about 2 mm
thick transverse slices and fixed in 10% formalin.
After being embedded in paraffin, several transverse
sections were obtained from the kidney and stained
with Periodic acid-Schiff (PAS) for histological
examination.
Statistical analysis
Results are expressed as mean ±SD. The data was
analyzed using repeated measure analysis of variance
(ANOVA) followed by Dennett’s test and repeated
measure ANOVA followed by Tukey’s test.
462 INDIAN J TRADIT KNOWLE, VOL 15, NO. 3, JULY 2016

Table 1Effect of aqueous extract of C. papaya leaf on biochemical parameters of diabetic rats
Groups HbA1-c AER BUN Creatinine B.P Serum Serum Serum Urine
(%) (mg/day) (mg/dl) (mg/dl) (mmHg) TC (mg/dl) Albumin (g/dl) TG (mg/dl) volume (ml)
NC Baseline 4.70±0.60 0.071 18.90±1.50 0.15±0.08 100.25±2.40 91.20±5.24 3.42±0.08 87.50±4.15 2.24±0.41
Endpoint 4.67±0.29 0.070 18.91±1.10 0.16±0.05 98.50±4.10 93.40±3.70 3.60±0.06 88.30±5.00 2.65±0.30
DC Baseline 9.62±0.91 0.873 35.70±1.80 0.71±0.13 118.30±3.50 140.80±6.20 2.39±0.07 173.70±9.20 15.30±1.05
Endpoint 10±0.91 0.895 37.10±1.21 0.82±0.09 129.50±8.10 144.20±9.10 2.23±0.05 189.30±9.20 15.10±1.00
C.P-1.5 Baseline 9.70±0.24 0.869 36.22±3.43 0.73±0.41 120±8.70 135.30±6.50 2.33±0.02 177.20±7.30 15.11±1.20
Endpoint 7.55±0.80*¤ 0.469*¤ 32.10±2.70 0.70±0.06 118±15.10 132.40±7.10 2.34±0.08 142±7.35*¤ 8.75± 0.75*¤
C.P-2.5 Baseline 9.60±0.10 0.865 34.92±2.15 0.74±0.10 121.10±4.90 143.60±8.20 2.30±0.04 178.60±9.50 16.00±1.50
Endpoint 7.10±0.50*¤ 0.218*¤ 22.50±1.20*¤˚ 0.21±0.09*¤˚ 106.35±6.70*¤109.50±4.31*¤ 2.95±0.09*¤ 98±5.90*¤ 8.61± 0.60*¤
PMG Baseline 9.49±0.57 0.767 35.45±2.95 0.74±0.25 120.50±3.75 141.85±5.50 2.35±0.06 166.35±12.60 14.350±1.25
Endpoint 8.10±0.20*¤ 0.209*¤ 20.15±0.60*¤˚ 0.18±0.18*¤˚ 118.90±17.10 139.20±4.90 3.05±0.02*¤˚ 152.80±17.50 7.80± 0.90*¤

Data are mean ±SD of 6 rats in each group

NC: Normal control group; DC: Diabetic control group; C.P: C. papaya treated group; PMG: Pimagedine treated group
*p<0.05 vs. Baseline; ¤p<0.05 vs. Diabetic control; O p<0.05 vs. C.P-1.5 gm/dL

Differences with p<0.05 were considered statistically

significant.
Results
All the diabetic groups had elevated levels of
biochemical parameters than normal control (NC)
group and there were no significant differences
between these groups at baseline (Table 1). C. papaya
exerted ameliorative effect on plasma glucose
however, the extent of improvement was different
among the groups. There was a dose dependent
reduction in C.P-1.5 gm/dL and C.P-2.5 gm/dL
groups from baseline to endpoint (17.80 ±0.70
mmol/L vs. 10.0 ±0.50 mmol/L, and 17.91±0.60
mmol/L vs. 9.60±0.45 mmol/L, p<0.05). At the
Fig. 1Effect of aqueous extract of C. papaya leaf on plasma
endpoint, mean differences in plasma glucose levels glucose overtime.
of C. papaya treated groups and DC group were
significant (p<0.05). Comparison between C. papaya
treated groups revealed low levels of plasma glucose
in C.P-2.5 gm/dL group, but statistically non-
significant (Fig. 1). C. papaya treated groups showed
significant reductions in HbA1-c levels compared to
DC group at endpoint (10±0.91vs. 7.55±0.80 and
7.10±0.50, respectively, p<0.05), but the difference
between C. papaya treated groups was non-
significant. Elevated urinary AER in diabetic groups
at baseline was decreased by C. papaya and the
difference was significant when compared to DC
group at endpoint (0.895 mg/day vs.0.469 mg/day,
and 0.218 mg/day, p<0.05, respectively). However,
Fig. 2Effect of aqueous extract of C. papaya leaf on urinary
no significant difference was observed between albumin/creatinine ratio.
C. papaya treated groups (Table 1). Micro-
albuminuria as indicated by elevated group. These results were comparable to PMG-1
albumin/creatinine ratio was significantly low in C. ml/mg reference group (Fig. 2). When the effect of
papaya treated groups (p<0.05) compared to DC C. papaya on GFR was determined, significant
 RAHMAN et al.: BENEFITS OF CARICA PAPAYA IN DIABETES MELLITUS 463

improvements were observed in C. papaya treated 178.80±4.10 gm and 182.50±5.50 gm respectively,

groups compared to DC group at endpoint (0.779 p<0.05). The ratio of kidney weight to body weight
mL/min vs. 1.671 mL/min and 2.160 mL/min, (marker of diabetic nephropathy) significantly
respectively, p<0.05). Comparison between C. papaya decreased in C.P-2.5 gm/dL group compared to DC
treated groups showed no significant difference at the group (4.61±0.16 vs. 2.90±0.08, p<0.05). In addition,
endpoint (Fig. 3). The levels of blood urea nitrogen C. papaya treatment significantly reduced water
(BUN), plasma creatinine, blood pressure (BP), serum consumption compared to DC group (894±10.40 mL
total cholesterol (TC) and serum albumin in C.P-2.5 vs. 551±6.95 mL and 478±8.50 mL, respectively,
gm/dL group showed significant improvement at p<0.05). These effects of C. papaya were comparable
endpoint upon comparison with DC group to pimagedine (Table 2).
(37.10±1.21 vs. 22.50±1.20 mg/dl; 0.82±0.09 vs. Periodic-acid Schiff (PSA) stained glomerulus of
0.21±0.09 mg/dl; 129.50±8.10 mmHg vs. diabetic control group had expanded glomerular
106.35±6.70 mmHg; 144.20±9.10 mg/dl vs. mesangium due to mesangial cell proliferation
109.50±4.31 mg/dl; 2.23±0.05 gm/dl vs. 2.95±0.09 compared to normal size glomerulus of normal
gm/dl, respectively, p<0.05). Improvement in these control group. These conditions were reversed and the
parameters of C.P-2.5 gm/dL group were also general morphology of glomerulus was much
significant from baseline (p<0.05). However, the improved by C. papaya as was the case with
changes produced by C.P-1.5 gm/dL group in BUN, pimagedine (Fig. 4A-E).
plasma creatinine, BP, TC and serum albumin did not
reach statistical significance. Moreover, comparison Discussion
between C. papaya treated groups revealed DN is one of the major causes of morbidity and
significantly low levels of BUN and plasma creatinine mortality worldwide and is the result of persistent
in C.P- 2.5 gm/dL group at the endpoint (p<0.05). hyperglycemia that causes tissue damage through
Compared to DC group, the decrease in serum AGEs formation, PKC activation and stimulation of
triglyceride (TG) was significant in C. papaya treated the aldose reductase pathway20,21. It is characterized
groups at endpoint (189.30±9.20mg/dL vs. 142±7.35 by a progressive increase in proteinuria, abnormality
mg/dL and 98±5.90 mg/dL for C.P-1.5 gm/dL and in serum creatinine, decline in GFR and hypertension.
C.P-2.5 gm/dL respectively, p<0.05). The raised urine In the present study, we experimentally induced
volume of diabetic rats at baseline was also diabetic nephropathy by injecting STZ in animal
significantly low in C. papaya treated groups at models that caused metabolic abnormalities and
endpoint compared to DC group (15.10±1.00 ml vs. subsequently kidney damage as evident by elevated
8.75± 0.75 ml and 8.61± 0.60 ml, p<0.05) but no
significant difference was observed between Table 2Effect of aqueous extract C. papaya leaf on
C. papaya treated groups (Table 1). physiological parameters of diabetic rats
C. papaya showed marked improvement in Group Body Kidney/ Water
physiological parameters. The body weight of DC weight (g) body weight consumption
group was significantly low than C. papaya treated ratio (ml)
groups at the end point (160.70±5.20 gm vs. NC Baseline 230.30±6.50 2.54±0.13 205±4.07
Endpoint 229.15±5.10 2.50±0.11 211±6.80
DC Baseline 165.10±8.70 4±0.22 710±25.30
Endpoint 160.70±5.20 4.61±0.16 894±10.40*
CP-1.5 Baseline 166±5.10 3.86±0.40 699.10±21.90
Endpoint 178.80±4.10*¤ 3.44±0.41 551±16.95*¤
CP-2.5 Baseline 168.90±7.40 4.02±0.42 692±13.50
Endpoint 182.50±5.50*¤ 2.90±0.08*¤ 478±18.50*¤˚
PMG Baseline 169.50±6.20 3.95±0.20 701±22.90
Endpoint 185.14±5.75*¤ 2.73±0.35*¤ 451.35±10.60*¤˚
Data are mean ±SD of 6 rats in each group
NC: Normal control group; DC: Diabetic control group; C.P: C.
papaya treated group; PMG: Pimagedine treated group
*p<0.05 vs. Baseline; ¤p<0.05 vs. Diabetic control; ˚ p<0.05 vs.
Fig. 3Effect of aqueous extract of C. papaya leaf on GFR. C.P-1.5 g/dL
464 INDIAN J TRADIT KNOWLE, VOL 15, NO. 3, JULY 2016
Fig. 4Transverse slices of kidneys (PAS stainedΧ400) showing
glomerulus architecture of study groups. A) Normal control group
(NC) with normal architecture of glomerulus, B) Diabetic control
group (DC) having expanded glomerular mesangium, C) Diabetic
group treated with 1.5 gm/dL C. papaya (C.P- 1.5 gm/dL) showing
reversal of diabetes induced glomerular mesangium, D) Diabetic
group treated with 2.5 gm/dL C. papaya (C.P- 2.5 gm/dL) showing
marked improvement in glomerular morphology and expansion of
glomerular mesangium, E) Diabetic group treated with 1 ml/mg
Pimagedine (PMG-1ml/mg) showing glomerulus of almost near
normal size and configuration to that of NC group.
serum creatinine and BUN, microalbuminuria and
kidney hypertrophy22,23 in order to evaluate the
protective effect of C. papaya against diabetic
nephropathy. Plant based therapies have been in use
for the treatment of diabetes and its complications
since antiquity24 and our study suggest that C. papaya
leaves extract ameliorated blood glucose level dose
dependently along with urinary AER and
albumin/creatinine ratio, which is a key measure of
renal function. A direct relationship between
hyperglycemia and nephropathy has been reported
earlier25. We observed no dose response relationship
of C. papaya leaves on plasma creatinine, BUN, urine
volume and urine creatinine and only higher doses
showed significant improvements in plasma
creatinine, urine volume and urine creatinine. These
effects may be attributed to the greater hypoglycemic
activity of C. papaya leaves at a higher dose observed
in this study as well as earlier15. We also observed a
significant improvement in GFR of diabetic rats
treated with C. papaya leaves extract. Urine creatinine
is an index for evaluating the GFR and there is a
gradual decrease in GFR in the presence of DN26. In
normal conditions, creatinine is mainly filtered out by
kidneys and there is almost no tubular reabsorption that
results in a low plasma creatinine level. Hypertension
and dyslipidemia are the risk factors both for the
initiation and progression of diabetic nephropathy27-30
and the observed improvement in blood pressure and
blood lipids by C. papaya leaves extract particularly at
a higher dose may also be the contributing factors in
protecting against DN. Moreover, the reported
antioxidant activity of C. papaya leaves31 may inhibit
the lipids peroxidation and stabilize the membrane
lipids thereby decreasing the oxidative stress. There
was significant decrease in body weight of diabetic
control group and is the result of persistent
hyperglycemia. Treatment with C. papaya leaves
extract was associated with protection from massive
loss of body weight along with improvement in the
ratio of kidney weight/body weight and water
consumption. The study further shows that marked
expansion of glomerular mesangium due to mesangial
cell proliferation and excessive accumulation of ECM
in diabetic control group was reversed and glomerulus
returned to near normal size and configuration in
C. papaya treated groups with the higher doses
showing greater improvements. These findings suggest
that C. papaya leaves extract at higher dose may exert
nephroprotective effects by ameliorating several
pharmacological targets in DN and may be a useful
therapeutic agent for inhibiting and/or at least retarding
the initiation and progression of diabetic nephropathy.
Although, we do not have a solid reason, but the non-
protective effect of C. papaya leaves extract at a lower
dose in DN may be the result of its lesser beneficial
effects on several biochemical parameters. Moreover,
its origin and different methods used in the processing
of leaves may also be responsible for variation in
activity. Further studies, using a range of C. papaya
leaves extract (starting from 3 gm/dl) are needed to
better evaluate the nephroprotective effects in diabetes
mellitus.
Acknowledgement
Authors are thankful to Gandhara University for
providing the required facilities at Gandhara College
of Pharmacy.
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