Asparagus racemosus Willd (Liliaceae) ameliorates early diabetic nephropathy in STZ induced diabetic rats.
ABSTRACT Diabetic nephropathy is a major "microvascular" complication of diabetes, differs from other causes of chronic kidney diseases in its predictability, with well-defined functional progression from hyperfiltration to micro- to macroalbuminuria to renal failure. The present study was undertaken to investigate the effect of Asparagus racemosus Willd (Liliaceae) on streptozotocin-induced early diabetic nephropathy. Single i.p injection of streptozotocin (55 mg/kg) was administered to induce early diabetic nephropathy in Wistar rats and thereafter treated orally with ethanolic extract of Asparagus racemosus (EEAR) at a dose level of 100 and 250 mg/kg daily for 4 weeks. The efficacy of extract was compared with diabetic control rats. A. racemosus treatment significantly decreased plasma glucose, creatinine, urea nitrogen, total cholesterol and triglyceride levels. Renal hypertrophy, polyuria, hyperfiltration, microalbuminuria and abnormal changes in the renal tissue as well as oxidative stress were effectively attenuated by EEAR treatment. Basement membrane thickening and mesangial proliferation formation without nodules were seen in diabetic rats, whereas these structural changes were reduced in EEAR treated groups. Results of this study suggested that A. racemosus has beneficial effect in the treatment of diabetic
- SourceAvailable from: Randhir Singh Dahiya[Show abstract] [Hide abstract]
ABSTRACT: Long term hyperglycemia leads to development of complications associated with diabetes. Diabetic complications are now a global health problem without effective therapeutic approach. Hyperglycemia and oxidative stress are important components for the development of diabetic complications. Over the past few decades, herbal medicines have attracted much attention as potential therapeutic agents in the prevention and treatment of diabetic complications due to their multiple targets and less toxic side effects. To review current available knowledge of medicinal herbs for attenuation or management of diabetic complications and their underlying mechanisms. Bibliographic investigation was carried out by scrutinizing classical text books and peer reviewed papers, consulting worldwide accepted scientific databases (SCOPUS, PUBMED, SCIELO, NISCAIR, Google Scholar) to retrieve available published literature. The inclusion criteria for the selection of plants based upon all medicinal herbs and their active compounds with attributed potentials in relieving diabetic complications. Moreover, plants which have potential effect in ameliorating oxidative stress in diabetic animals have been included. Overall, 238 articles were reviewed for plant literature and out of the reviewed literature, 127 articles of were selected for the study. Various medicinal plants/plant extracts containing flavonoids, alkaloids, phenolic compounds, terpenoids, saponins and phytosterol type chemical constituents were found to be effective in the management of diabetic complications. This effect might be attributed to amelioration of persistent hyperglycemia, oxidative stress and modulation of various metabolic pathways involved in the pathogenesis of diabetic complications. Screening chemical candidate from herbal medicine might be a promising approach for new drug discovery to treat the diabetic complications. There is still a dire need to explore the mechanism of action of various plant extracts and their toxicity profile and to determine their role in therapy of diabetic complications. Moreover, a perfect rodent model which completely mimics human diabetic complications should be developed.Journal of ethnopharmacology 09/2013; · 2.32 Impact Factor
- Pharmaceutical Crops. 11/2014; 5(Suppl 1: M4):39-60.
Indian Journal of Experimental Biology
Vol.50, July 2012 pp. 469-475
Asparagus racemosus Willd (Liliaceae) ameliorates early diabetic
nephropathy in STZ induced diabetic rats
Rahul Somania*, Abhay Kumar Singhaib, Prashant Shivgundec & Dilpesh Jainc
aDepartment of Pharmacology, Smt. Kashibai Navale College of Pharmacy, Pune 411 048, India
bDepartment of Pharmaceutical Sciences, Dr. H S Gour University, Sagar 470 003, India
cDepartment of Pharmacology, Sinhgad College of Pharmacy, Pune 411 041, India
Received 8 November 2011; revised 24 May 2012
Diabetic nephropathy is a major ‘‘microvascular’’ complication of diabetes, differs from other causes of chronic kidney
diseases in its predictability, with well-defined functional progression from hyperfiltration to micro- to macroalbuminuria to
renal failure. The present study was undertaken to investigate the effect of Asparagus racemosus Willd (Liliaceae) on
streptozotocin -induced early diabetic nephropathy. Single i.p injection of streptozotocin (55 mg/kg) was administered to
induce early diabetic nephropathy in Wistar rats and thereafter treated orally with ethanolic extract of Asparagus racemosus
(EEAR) at a dose level of 100 and 250 mg/kg daily for 4 weeks. The efficacy of extract was compared with diabetic control
rats. A. racemosus treatment significantly decreased plasma glucose, creatinine, urea nitrogen, total cholesterol and
triglyceride levels. Renal hypertrophy, polyuria, hyperfiltration, microalbuminuria and abnormal changes in the renal tissue
as well as oxidative stress were effectively attenuated by EEAR treatment. Basement membrane thickening and mesangial
proliferation formation without nodules were seen in diabetic rats, whereas these structural changes were reduced in EEAR
treated groups. Results of this study suggested that A. racemosus has beneficial effect in the treatment of diabetic
Keywords: Asparagus racemosus, Diabetic nephropathy, Microalbuminuria, Streptozotocin
Diabetic nephropathy (DN), a major long-term
microvascular complication of diabetes mellitus, is
the most common cause of end stage renal disease
(ESRD) requiring dialysis1. Further, DN has been
acknowledged as an independent risk factor for
cardiovascular disease. So, prevention or retardation
of DN has become a major goal in biomedical
research2. The injurious effects of hyperglycemia are
characteristically observed in tissues which are not
dependent on insulin for glucose entry into the cell,
hence, are not capable of down-regulating glucose
transport along with elevation of extracellular glucose
levels3. Different biochemical pathways found to be
involved in the pathogenesis and reactive oxygen
species (ROS) seem to be the common denominator
in various pathways4.
Metabolic derangements, systemic and glomerular
glyacation end products (AGEs) found to occur in the
progression of DN. In addition, kidney hypertrophy
stress and advanced
derangements adversely compounds hyperglycemia-
induced injury5 leading to the development of
long-term diabetic renal damage and increased
urinary albumin excretion rate (AER)6,7. Thickening
of glomerular basement
intertubular fibrosis and albuminuria can be found in
diabetic kidney in association with an increase in
extracellular matrix (ECM)8.
DN can be controlled by multi-targeted therapies,
which includes, intensive control of blood glucose
and blood pressure by using antihypertensive agents
with anti-proteinuric action, lipid lowering strategies
and correction of insulin resistance. Although
angiotensin converting enzyme inhibitors (ACEI) and
angiotensin receptor blockers (ARB) are effective in
DN, but not sufficient to completely prevent disease
progression, hence development of novel and effective
therapeutic strategies are therefore high priorities9.
Ayurvedic system has given knowledge about very
safe and effective medicinal plants. Ayurvedic
literature listed use of the roots of the Asparagus
racemosus in treatment of nephropathy10. It has also
hyperfiltration along with metabolic
Telephone: +91 9011093415
INDIAN J EXP BIOL, JULY 2012
been reported to possess hypoglycemic, antidiabetic11,
antioxidant12, insulin secretory13 and antilithiatic
activity14. The present investigation was undertaken to
study effect A. racemosus on streptozotocin (STZ)-
induced early DN in experimental animals.
Materials and Methods
Drugs and chemicals—Aminoguanidine hydrogen
streptozotocin, malondialdehyde (MDA), tetrabutyl
ammonium and superoxide dismutase (Sigma-Aldrich,
St. Louis), Catalase (Hi Media Laboratories Pvt. Ltd.,
Mumbai) and commercial
(Biolab, Mumbai) were purchased from local supplier.
Preparation of extract—Roots of A. racemosus
were purchased from local market of Pune, India. The
plant material was authenticated by Agharkar
Research Institute; Pune and voucher specimen
(Auth 08-132) was deposited. Roots were dried in an
oven at 40 °C and grinded into a fine powder. The
powder material was macerated with 95% ethanol,
filtered and concentrated under reduced pressure using
rotary evaporator and yield was found 6.42% w/w.
Experimental animals—Wistar rats of either sex,
weighing 150-200 g were purchased (Haffkine Bio-
Pharma Corporation Ltd., Mumbai) and maintained
under standard laboratory conditions at temperature of
23 ± 2 °C with relative humidity 55°C ± 10 % in 12 h
light and dark cycle throughout the experiments.
Animals had free access to water and standard
laboratory feed ad libitum. All the experimental
procedures and protocols used in this study were
reviewed and approved (SCOP/IAEC/Approval/2008-
09/10) by the Institutional Animal Ethics Committee
of Sinhgad College of Pharmacy, Pune, constituted
under Committee for Purpose of Control and
Supervision of Experiments on Animals (CPCSEA).
Induction of early diabetic nephropathy—Single
intraperitoneal injection of STZ (55 mg/kg), prepared
in 0.1 M cold citrate buffer (pH 4.5) was administered
to induce diabetes in rats whereas, normal control rats
were received 0.1 M cold citrate buffer (pH 4.5) only.
Hyperglycemia was confirmed 48 h after STZ
injection by GOD/POD method21. After 4 weeks of
diabetes induction plasma glucose level was estimated
and those with a plasma glucose level >300 mg/dl were
selected as diabetic rats and used for further study.
Experimental design—Normal and diabetic rats
were divided into 5 groups (n=5) and were treated
daily for 4 weeks.
Group I – Normal Control (NC) received 2% gum
acacia (1 ml/kg/day, p.o
Pvt. Ltd., Mumbai),
Group II – Diabetic control (DC) received 2% gum
acacia (1 ml/kg/day, p.o)
Group III – Diabetic rats treated with EEAR
(100 mg/kg/day, p.o)
Group IV – Diabetic rats treated with EEAR
(250 mg/kg/day, p.o)
Group V – Diabetic rats treated with aminoguanidine
hydrogen carbonate (1 g/L) in drinking water with
daily fresh preparation.
After 4 weeks treatment, rats were fasted overnight
and blood sample were collected and analyzed for the
estimation of various biochemical parameters in plasma.
Rats were individually placed in metabolic cage, 24 h
total urine volume was measured and the same was used
for estimation of renal function. Rats were sacrificed and
kidneys were collected to study oxidative stress as well
as histopathological observations.
Estimations of plasma biochemical parameters—
Plasma glucose21, creatinine22, urea nitrogen23, total
cholesterol24 and triglyceride25 were determined using
commercial diagnostic kits.
Body and kidney weight and relative kidney
weight—At the end of treatment, body and kidney
weight was measured by gravimetric method using
electronic weighing balance and relative kidney
weight was calculated.
Estimations of biochemical parameters in urine—
Creatinine and albumin level were estimated using
commercial diagnostic kit and glomerular filtration
rate (GFR)26 and AER27were calculated.
Evaluation of oxidative stress—Right kidney of
individual rat was isolated, washed in cold saline and
prepared 10% w/v homogenate using 0.15 M KCl by
centrifuging at 10500 g for 10 min at 4 °C. The
supernatant obtained was used for the estimation of
lipid peroxidation (MDA)28 and catalase (CAT)29.
Homogenate was further centrifuged at 1000 g for
20 min at 4 °C and the supernatant was used for estimation
of superoxide dismutase (SOD)30 and glutathione (GSH)31.
Protein concentrations of homogenates were determined
according to Lowry et al.32.
individual rat stored in 10% formalin solution was
embedded with paraffin
Haematoxylin-Eosin (H & E). Stained samples were
observed under light microscope.
Statistical analysis—All the data were expressed as
the mean ± S.E.M. Data were subjected to one-way
analysis of variance (ANOVA) followed by the
Dunnett’s test, where P < 0.05 was considered as
and stained with
SOMANI et al.: ASPARAGUS RACEMOSUS AMELIORATES EARLY DIABETIC NEPHROPATHY
Biochemical parameters in plasma—STZ induced
diabetic rats exhibited significant increase in plasma
glucose levels compared with normal control rats
(P < 0.001). The treatment of EEAR (100 and
250 mg/kg) ameliorated plasma glucose level when
compared to diabetic control rats (Table 1). Diabetic
rats also exhibit marked increase in creatinine as well
as urea nitrogen levels as compare to normal rats
(P < 0.001). EEAR (250 mg/kg) and aminoguanidine
treatment significantly decreased plasma creatinine
and urea nitrogen level as compared to the diabetic
control rats (Table 1). Elevated level of total
cholesterol and triglyceride in the diabetic rats have
also been significantly (P < 0.001) reduced after four
weeks daily treatment with EEAR (100 and
250 mg/kg) and aminoguanidine (Table 1).
Body weight, kidney weight and relative kidney
weight—Significant (P < 0.001) decrease in body
weight and increase in kidney weight (KW) as well as
relative kidney weight of diabetic rats were observed
when compared to normal control rats. The treatment
of EEAR (250 mg/kg) and aminoguanidine in diabetic
rats significantly (P < 0.01; P < 0.001, respectively)
restored the body weight and decreased kidney weight
and relative kidney weight when compared to the
diabetic control rats (Table 2).
Biochemical parameters in urine—After 8 weeks,
significant (P < 0.001) increase in 24 h total urine
volume, creatinine, GFR and AER were observed in
the diabetic rats. EEAR
aminoguanidine treatment for four weeks significantly
(P < 0.001) prevented the rise in 24 h total urine
volume as well as creatinine level. Whereas elevated
level of GFR and AER were also been significantly
reduced in all the treated groups (Table 3).
Oxidative stress—Diabetic condition produced
significant (P < 0.001) increase in renal MDA, SOD,
GSH and decrease in CAT levels. The treatment of
EEAR for four weeks (100 and 250 mg/kg) and
aminoguanidine showed dose dependant decrease in
MDA, SOD, GSH and increase in CAT level when
compared to diabetic control rats (Table 4).
(250 mg/kg) and
Table 1—Effect of four weeks treatment of EEAR on biochemical parameters in plasma
[Values are mean ± SEM of 5 animals]
Creatinine (mg/dl) Urea nitrogen (mg/dl) Total cholesterol (mg/dl) Triglyceride (mg/dl)
0.77± 0.07 22.05±1.15
NC: normal control; DC: diabetic control, EEAR: ethanolicextract of A. racemosus; AMG: aminoguanidine hydrochloride
One way ANOVA followed by Dunnett’s test; where $P<0.001 when compared to normal control *P < 0.05, @P < 0.01, #P < 0.001 when
compared to diabetic control
Table 2—Effect of 4 weeks treatment of EEAR on body weight, kidney weight and relative kidney weight
[Values are mean ± SEM of 5 animals]
Body wt (g)
Kidney wt (g)
Relative kidney wt
Details as in Table 1
Table 3—Effect of four weeks treatment of EEAR on biochemical parameters in urine
Details as in Table 1
24 h total urine volume (mL)
50.06 ± 7.00
140.90 ± 2.29$
118.00 ± 8.00
55.90 ± 8.42#
58.37 ± 5.01#
AER (µg/24 h)
INDIAN J EXP BIOL, JULY 2012
Histopathological studies—After 8 weeks of study,
diabetic animals showed the presence of GBM
thickening and mesangial proliferation without nodules,
while normal control animals revealed no abnormalities.
Treatment with EEAR (250 mg/kg) and aminoguanidine
significantly attenuated these progressions (Fig. 1).
Hyperglycemia is the principle factor responsible
for structural alterations at the renal level. Diabetes
Control and Complications Trial Research Group
(DCCTRG) has elucidated that hyperglycemia is
directly linked to diabetic microvascular
Table 4—Effect of 4 weeks treatment of EEAR on oxidative stress
Details as in Table 1
15.63 ± 0.84
42.36 ± 1.74$
34.32 ± 1.54@
24.11 ± 1.52#
24.2 ± 1.74#
4.48 ± 0.15
7.04 ± 0.38$
6.03 ± 0.12*
4.99 ± 0.11#
5.17 ± 0.26#
9.86 ± 0.43
35.97 ± 1.08$
27.67 ± 1.57#
14.49 ± 1.55#
11.91 ± 1.32#
113.1 ± 4.24
37.46 ± 2.34$
74.73 ± 4.21#
101.9 ± 3.70#
107.7 ± 3.52#
Fig. 1— Histological section of kidneys stained with H & E- 1000 × (a)-normal control (NC) showing no abnormalities; (b)-diabetic
control (DC) showing a necrotic area in glomerulus shown by white arrows. Thickenings in basement membrane are well observed;
(c)-DC+EEAR (100 mg/kg); (d)-DC+EEAR (250 mg/kg) and (e)-DC+AMG showing features of healing like normal basement
membrane and absence of necrotic cells in glomerulus.[BC:Bowman’s capsule; G:glomerulus; P:proximal convoluted tubule]
SOMANI et al.: ASPARAGUS RACEMOSUS AMELIORATES EARLY DIABETIC NEPHROPATHY
complications33, particularly in the kidney. In the
present study, STZ induced persistent hyperglycemia
for 8 weeks and produced an early DN, characterized
by increased urinary albumin excretion and loss of
renal function. Asparagus racemosus has been
traditionally used for the treatment of diabetes and
several reports showed good results in control of
In previous study, 4 weeks treatment of EEAR in
diabetic rats significantly decreased plasma glucose
level which might be attributed to its antidiabetic12
and insulin secretory activity13. Functional alterations
such as elevated plasma creatinine and urea nitrogen
level were significantly reversed after 4 weeks
repeated dose treatment of EEAR.
Hypercholesterolemia and hypertriglyceridemia have
been reported to occur in diabetic rats34. Furthermore,
kidneys are involved in catabolism of lipoprotein[a];
therefore, clearance of lipoprotein[a] in patients with
nephropathy is decreased. Increased levels of plasma
triglycerides and total cholesterol were significantly
reduced by EEAR and aminoguanidine treatment. DN
is characterized by increased AER and 24 h total urine
volume, hyperfiltration occurred in the setting of
normoalbuminuria as well as microalbuminuria and
that loss of renal function began in the context of
proteinuria35. Urinary albumin level is a selective
marker of glomerular injury and elevated AER
represents progressive nephropathy, results into a loss
of auto-regulation and inflammation27. Several studies
have shown that reduction of microalbuminuria
indicates a better prognosis36. EEAR
aminoguanidine treatment effectively reversed
microalbuminuria and attenuated hyperfiltration,
suggests EEAR attenuates the progression of early
Diabetes caused excessive break down of tissue
protein, results into loss of body weight37. DN has
also been known to produce hypertrophy and increase
in kidney weight38. Therefore, the relative kidney
weight in diabetic rats was significantly increased
than normal rats. EEAR (250) and aminoguanidine
treatment significantly restored body weight and
decreased kidney weight, suggest EEAR has
preventive effect on kidney hypertrophy. This is in
agreement with previous findings39.
Increased formation of reactive oxygen species is a
major cause for development and progression of
diabetic microvascular complications such
nephropathy and it has been demonstrated that
modulation of oxidative stress through treatment with
antioxidants effectively reduced diabetic snag40.
Preventive antioxidants like superoxide dismutase
(SOD), catalyses the dismutation of superoxide to
H2O2 and catalase (CAT) breaks it into water. Lipid
peroxidation (LP) is a free radical mediated process,
initiates chain of reaction that gives rise to many
products of toxicological
(4-HNE) and various 2-alkenals41. Oxidative stress
was found to be inhibited dose dependently by EEAR
as assessed by decrease in renal MDA levels. Diabetic
rats exhibited elevated levels of enzymic antioxidants
SOD, non-enzymic antioxidant GSH and reduced
CAT. EEAR treatment reversed these changes;
whereas effect of EEAR (250) was comparable to
aminoguanidine. Elevations in GSH and SOD activity
may be compensatory mechanisms for the chronic
overproduction of free radicals and oxidative stress40.
Pathogenesis of DN results in expansion of
mesangial matrix and thickening of GBM, due to
accumulation of extracellular
components42. Young et al.43 reported that mesangial
cell proliferation occurs in experimentally induced
DN exhibit detectable changes in mesangial ECM
deposition. In the present
abnormalities in glomerulus such as basement
membrane thickening and mesangial proliferation
without nodules were observed in the diabetic kidney
as compared to normal control rats. However nodular
lesions, capsular drops, mesangiolysis, fibrin cap and
mesangial sclerosis was not observed in the present
study. Glomerulus is a principle site for the action of
reactive oxygen species, leading to glomerulonephritis44.
EEAR treatment significantly prevented the GBM
thickening and mesangial proliferation.
In conclusion, ethanolic extract of A. racemosus
reduced hyperglycemia, creatinine, urea nitrogen
level, AER, GFR and oxidative stress in diabetic rats,
which are important factors relating to the progression
of DN. Therefore, these findings showed that
A. racemosus has beneficial effect in the treatment of
early diabetic nephropathy.
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