Nephroprotective and Antioxidant Potential of Ethanolic Extract of Flowers of Cassіa Sіamea
against Gentamicin Induced Nephrotoxicity
Mihir Y Parmar*, Mounika B, Sindhuja S and Dinesh Pore
Bharat Institute of Technology, Mangalpally, Jawaharlal Nehru Technological University, Hyderabad, Telangana, India
Submission:March 15, 2019; Published: May 16, 2019
*Corresponding author:Dr Mihir Y Parmar, Professor Pharmacology, India
How to cite this article:Mihir Y Parmar, Mounika B, Sindhuja S, Dinesh Pore. Nephroprotective and Antioxidant Potential of Ethanolic Extract of
Flowers of Cassіa Sіamea against Gentamicin Induced Nephrotoxicity. JOJ uro & nephron. 2019; 6(4): 555693. DOI: 10.19080/JOJUN.2019.06.555693
To investigate the nephroprotective and antioxidant activity of Ethanolic extract of Cassіa Sіamea (EECS) against Gentamicin (100 mg/kg, p.o. for 7 days) induced kidney damage in rats. Rats were pretreated with EECS (250 and 500 mg/kg, p.o) 30 min prior to Gentamicin ingestion for seven days. The extent of defense was measured using levels of serum enzymes like creatinine, uric acid and blood urea nitrogen (BUN). Additionally, oxidative stress parameters such as levels of Malondialdehyde (MDA), reduced glutathione (GSH) and activity of superoxide dismutase (SOD) and catalase (CAT) along with histological evaluation of kidney sections was carried out to shore up the induction of kidney scratch and nephroprotective potential. The substantially elevated kidney weight and serum enzyme levels of creatinine, uric acid and BUN. Oxidative stress parameters MDA, GSH levels and SOD, CAT activities were found to be restored towards normalization by EECS comparable with silymarin standard. Pathological changes were in same road supports finding of biochemical evidences of nephroprotection. The total phenolic content and the total flavonoid content of the extract were 21.55 ± 2.54 mg catechol equivalent/g and 24.50 ± 2.00 mg quercetin equivalent/g respectively. EECS possess an extremely hopeful antioxidant and nephroprotective potential against Gentamicin induced kidney damage.
Kidney crash is a medical event in which the kidneys fall short to adequately filter toxins and waste products from the blood. There are two forms one is acute and other is chronic; a number of other diseases or health problems may cause either form of renal failure to occur. Chronic kidney disease attacks the kidneys slowly and progressively over a period of time. It can take years for the harm to these organs to be noticeable because there are no symptoms, which is why the disease is often called the “silent killer” .
Nephrotoxicity is caused by class of drugs or xenobiotics like anticancer drug cisplatin and amino glycoside antibiotics are the chief culprit for approximately 20-40% of all acute renal failure cases in intensive care units . Gentamicin is widely used aminoglycoside antibiotics against gram-negative bacteria infections . About 30-35% of the patients, undergone gentamicin treatment for more than seven days, shows signs and symptoms of kidney toxicity . The cellular and molecular mechanism/s of Gentamicin-induced nephrotoxicity is not clearly understood. However Reactive oxygen species (ROS)
have important role in pathological mechanisms of Gentamicin-
induced acute renal failure. Production as well as amassing of ROS resulted in induction of apoptosis, tubular necrosis and increased infiltration of leukocyte . This Gentamicin-induced acute renal failure is clinically characterized by an increase in serum creatinine and uric acid levels and urea nitrogen, a reduction in the glomerular filtration rate (GFR) and urine osmolality .
There are many natural products such as plant and traditional herbal formulation available for the protective effect on kidney against damage induced by toxin and drugs. More than 600 commercial herbal products with claimed nephroprotective role are being sold in all over the world. Around 170 phytoconstituents isolated from 110 plants belonging to 55 families have been reported to show nephroprotective role. However, only a small proportion of nephroprotective plants as well as formulations used in traditional medicine are pharmacologically evaluated for their safety and efficacy [7-8].
Renal involvement has alsо been involved іn many cardіоvascular diseases, such as diabetes mellitus and regardіng the іm pact оf kіdney lesіоns іn diabetic nephropathy . Іn addіtіоn,
іt іs becоmіng highly rіsk factor tо use synthetіc drug because
оf their adverse drug reactіоn, toxicity and drug-drug interact.
Therefore, scientists are fascinated fоr new herbal molecule with
good safety and effective profile. Researches in their previous reports
reported that plants possessing pоlyphenоlіc cоmpоunds,
flavоnоіds and tannіns are useful as antіоxіdants and further іt
acts as organ protectant . Cassia sіamea (famіly: bіgnоnіaceae)
cоmmоnly knоwn as Afrіcan tulіp tree. Cassіa sіamea is useful
as a dіuretіc, antі-іnflammatоry, antіmalarіal, antі-HІV and
dіabetіc. The plant possesses quercetіn caffeіc acіds, оleanоlіc
acіd, sterоіds, pоlyphоnes, flavоnоіds, tannіns and cardіac glycоsіdes.
Herbs are repоrted tо cоntaіn phenоlіc cоmpоunds
these phenоlіc cоmpоnents are knоwn as antіоxіdants. Keeping
this in mind Present study was designed to evaluate the antіоx-
іdant, and nephrоprоtectіve actіvіty of Ethanolic extract of flоwers
оf Cassіa sіamea [11-12].
Flоwers оf Cassіa sіamea used were cоllected frоm Chіtооr
dіstrіct оf Andhra pradesh. The plant was taxonomically
identified and authenticated by Dr. Madhav shetty, Department
оf Bоtany, Srі Venkateswara Unіversіty, Tіrupathі where the
voucher specimen for the same is conserved under the reference
The flоwers were cut іntо small pіeces were cleaned and
dried under shade at room temperature for several days and
powdered. The resulting powder was then used fоr extractіоn.
The drіed pоwder оf flоwers were defatted wіth petroleum ether
and then extracted wіth 70% ethanоlіc usіng sоxhlet apparatus.
The extract was cоncentrated under reduced pressure using rate
flash evaporator which resulted with a yield of 17.54%w/w.
The extract was stored in airtight container in refrigerator.
Ethanolic extract of Cassіa Sіamea (EECS) was further used for
Silymarin were obtained from Micro Labs, Bangalore,
India and Gentamicin was procured from Piramal Health Care,
Ahmedabad, India. Urea, Uric acid and Creatinine kits were
obtained from Span Diagnostics, Surat, India. All other chemicals
used in this study were obtained commercially and were of
Wistar albino rats (200-250g) of either sex was maintained
under controlled conditions of temperature (27± 2°C) and
humidity (50±5%) and a 12-hour light–dark cycle, were used
for the experiment. The animals were housed in sanitized
polypropylene cages containing sterile paddy husk as bedding.
They had free access to standard rat pellet diet and water
ad libitum. The animals were given a week’s time to get
acclimatized with the laboratory conditions. All the experimental
procedures were performed according to the committee for the
purpose of control and supervision of experiments on animals
(CPCSEA-1015/PO/Re/S/06), ministry of social justice and
empowerment Government of India, norms and approved by the
Institutional Animal Ethics Committee (IAEC).
Rats were kept overnight fasting prior to drug administration.
Animals received a single oral dose (2000 and 5000 mg/kg,
bw) of ethanoloic extract of leaves of Cassіa Sіamea. After the
administration of Cassіa Sіamea extract, food was withheld
for further 3-4 h. Animals were observed individually at least
once during the first 30 min after dosing, periodically during
the first 24 h (with special attention during the first 4 h) and
daily thereafter for a period of 14 days. Once daily cage side
observations included changes in skin and fur, eyes and mucous
membrane (nasal) and also respiratory rate, circulatory (heart
rate and blood pressure), autonomic (salivation, lacrimation,
perspiration, piloerection, urinary incontinence, and defecation)
and central nervous system (ptosis, drowsiness, gait, tremors
and convulsion) changes. Mortality, if any, was determined over
a period of two weeks .
LD50 was done as per OECD guidelines for fixing the dose
for biological evaluation. The LD50 of Cassіa Sіamea extract as
per OECD guidelines falls under class four values with no signs
of acute toxicity at 5,000 mg/kg. The biological evaluation was
carried out at doses of 250 and 500 mg/ kg body weight .
In brief, a 1 mL of sample (1 mg/mL) was mixed with 1
mL of Folin- Ciocalteu’s phenol reagent. After 5 min, 10 mL of
a 7% Na2CO3 solution was added to the mixture followed by
the addition of 13 mL of deionized distilled water and mixed
thoroughly. The mixture was kept in the dark for 90 min at
23°C, after which the absorbance was read at 750nm. The TPC
was determined from extrapolation of calibration curve which
was made by preparing catechol solution. The estimation of the
phenolic compounds was carried out in triplicate. The TPC was
expressed as mg of catechol equivalents per g of dried sample
In a 10mL test tube, 0.3ml of extract, 3.4mL of 30%methanol,
0.15mL of NaNO2 (0.5 M) and 0.15 mL of AlCl3.6H2O (0.3 M)
were mixed. After 5 min, 1 mL of NaOH (1M) was added. The
solution was mixed well, and the absorbance was measured
against the reagent blank at 506 nm. The standard curve for
total flavonoids was made using quercetin standard solution (0
to 100 mg/L) under the same procedure as earlier described.
The total flavonoids were expressed as milligrams of quercetіn
equivalents per g of dried fraction .
Thirty Wistar albino rats of either sex were assigned to five
groups (n= 6): Group I: Rats in this group were injected with
normal saline, intraperitoneally and served as control; Group II:
Rats in this group were injected with gentamicin (100mg/ kg,
i.p) for seven consecutive days; Group III: Rats in this group were
injected with gentamicin (100mg/kg, i.p) and administered
extract of EECS (250mg/ kg, p.o) for seven consecutive days;
Group IV: Rats in this group were injected with gentamicin
(100mg/kg, i.p) and administered extract of of EECS (500mg/
kg, p.o)for seven consecutive days. Group V: Rats in this group
were injected with gentamicin (100mg/kg, i.p) and administered
silymarin (200mg/ kg,p.o) for seven consecutive days. After
the last dosing of seventh day after 24h the blood sample were
collected by puncturing retro-orbital plexus and serum was
separated by centrifugation. Rats were sacrificed; kidneys were
excised, rinsed clean in saline and preserved in 10% formalin for
Portions of the kidney from all the experimental groups were
fixed in 10% formalin, dehydrated in alcohol and then embedded
in paraffin. Microtome sections (5μm thick) were prepared from
each liver sample and stained with hemtoxylin-eosin (H&E) dye.
The sections were examined for the pathological findings.
The experimental outcomes were expressed as Mean ±
SEM for six animals in all groups. All parameters were analyzed
statistically using one-way analysis of variance (ANOVA),
followed by Dunnett’s multiple comparison test (DMCT) using
Graph Pad prism 5.0 software . Data were considered
statistically significant at P < 0.05.
The Ethanolic extract of Cassіa Sіamea (EECS) was found to
contain total phenolic content and the total flavonoid content of
the extract were 21.55±2.54 mg catechol equivalent/g and 24.50
± 2.00 mg quercetіn equivalent/g respectively
Kidney weight was significantly higher in gentamicin treated
group. Administering EECS (250&500 mg/kg, p.o) and Silymarin
(200 mg/kg, p.o) significantly decrease the kidney weight In
gentamicin induced nephrotoxicity, Gentamicin treated group
showed a significant elevated level of blood urea nitrogen,
serum creatinine and serum uric acid compared to normal
control. Treatments with EECS (250 & 500 mg/kg, p.o) as well
as Silymarin (200mg/kg, p.o) for 7 days caused significant
decline in above serum biomarkers (Figure 1A to 1D). Values are
expressed as mean ± SEM for six rats in each group. a Different
from Normal, b Different from Gentamicin (*p< 0.05, **p< 0.01,
Oxidative stress parameters of kidney homogenate were
measured. A significant (P<0.001) increase in MDA while
declines in GSH levels (P<0.01), SOD and CAT activities were
found in Gentamicin treated group as compared to normal
control. Treatments with EECS (250 & 500 mg/kg, p.o) and
Silymarin (200 mg/kg, p.o) significantly decrease MDA levels
while significant elevation in GSH level, SOD and CAT activity as
compared to Gentamicin control (Figure 2 A to D). Values are
expressed as mean ± SEM for six rats in each group. a Different
from Normal, b Different from Gentamicin (*p< 0.05, **p< 0.01,
Figure 3 Histopathological Changes IN Kidney Tissue of
Different groups. In gentamicin induce nephrotoxicity model
normal control rats (Figure 3A) showed normal glomerular
and tubular histology whereas gentamicin treated group was
found to cause distorted tubular shape, cellular infiltration of
the tubules (tubulitis), glomerular and blood vessel congestion,
and also result in the presence of inflammatory cells in kidney
sections (Figure 3B). The concurrent treatment with the EECS
(250 & 500 mg/kg, p.o) reduced such changes in kidney histology
(Figure 3C & 3D, 3E).
Nephrotoxicity induced by gentamicin is characterized by a
decrease in the glomerular filtration rate and tubular injury due
to the formation of reactive oxygen species (ROS), which may
be directly involved in membrane lipid peroxidation, mesangial
cells contraction, which alters the filtration surface area and
modify the ultrafiltration coefficient and decrease the glomerular
filtration rate . Management of such renal hemodynamic
abnormality and reduction of the same are important to prevent
the deterioration of normal functions of kidney. Formation of
non-protein nitrogenous compound such as urea, uric acid and
creatinine takes place due to degradation of these protein and
nucleic acids . Changes in the levels of serum urea, creatinine
and uric acid concentrations strongly suggested impairment
of kidney function in nephrotoxicity. Administration of EECS
decreased the levels of serum urea, creatinine and uric acid in
treated groups significantly.
Several experimental evidences have suggested that gentamicin
causes cell damage in the kidney by stimulating ROS production
[20-21]. Moreover, when exposed to ROS, the kidneys
of the rats that received gentamicin, suffered more because of
reduced antioxidant defense system enzymes . In the present
study Oxidative stress parameters of kidney homogenate
were measured. A significant increase in MDA while declines in
GSH content, SOD and CAT activities were found in Gentamicin
treated group. Administration of with EECS (250 & 500 mg/kg,
p.o) significantly decrease MDA levels while significant elevation
in GSH level, SOD and CAT activity dose dependent manner as
compared to Gentamicin control. Therefore, it seemed that EECS,
due to its antioxidant properties, reduced cellular damages in
kidneys. This nephroprotective activity of the EECS may be due
to antioxidant activity which may be due to the presence of flavonoids
and phenolic compounds as reported in our study. The
results of our study reveal the nephroprotective activity of EECS.
The probable mechanism for its protection against cellular damage
may be due to its antioxidant activity as evident by its in vivo
stress parameters improvements.