Environmental Science and Pollution Research (2021) 28:9448–9461

https://doi.org/10.1007/s11356-020-11321-x

RESEARCH ARTICLE

Nephroprotective effect of ethanol extract and fractions of a sea

lettuce, Ulva fasciata against cisplatin-induced kidney injury in rats
Nida Sohail 1 & Khan Hira 2 & Junaid Ahmed Kori 3 & Hafiza Farhat 4 & Faizah Urooj 4 & Waqas Khan 5 & Viqar Sultana 1 &
Muhammad Shaiq Ali 6 & Syed Ehteshamul-Haque 4

Received: 29 February 2020 / Accepted: 18 October 2020 / Published online: 4 November 2020
# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Cisplatin is widely used in anticancer therapy, but a substantial percentage of patients who receive the therapeutic
dose of cisplatin develop nephrotoxicity. Hepatotoxicity may also develop after a single dose or low repeated doses
of cisplatin. Ulva fasciata is an edible seaweed, commonly known as sea lettuces have also been shown various
biological activities. In this study, ethanol extract and its solvent fractions (n-hexane and chloroform) of U. fasciata
were given (orally) to different groups of rats for 10 days. Injury to the kidney was induced by administrating
cisplatin, intraperitoneally (i.p.) to rats at a dose of 7 mg/kg body weight (b.w.) dissolved in 1 mL saline, at 5th day
of the experiment. At 10th day rats were sacrificed and kidney parameters (creatinine, urea, and blood urea nitrogen
(BUN)) and electrolyte balance (Ca++, Mg++, K+, and Na+) in serum were determined, while oxidative stress markers
glutathione (GSH), catalase (CAT) and malondialdehyde (MDA), and inflammatory cytokines, tumor necrosis factor
(TNF α), and interleukin (IL-6) were determined in kidney tissues. Histological examination of the kidney was also
performed to examine the changes in kidney tissues. Cisplatin caused adverse effects on blood parameters, antiox-
idants, and inflammatory markers with severe renal tubular injury in kidney tissues. Ethanol extract of U. fasciata
and its fractions effectively improved these disorders and diminished the renal dysfunction. However, ethanol extract
was found more effective in attenuating the adverse effect of cisplatin than its fractions. n-Hexane-soluble fraction
that was subjected to GC-FID and GC-MS analysis revealed the presence of several compounds and some of them
are new from this source. It could be concluded that the U. fasciata possesses nephroprotective effect and can
attenuate cisplatin-induced renal dysfunction. Since U. fasciata is an edible seaweed, it may be used as a diet
supplement.

Keywords Urea . Creatinine . Inflammatory cytokines . Oxidative stress . Electrolytes . Hepatotoxicity

Responsible Editor: Mohamed M. Abdel-Daim

Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/s11356-020-
11321-x.

* Syed Ehteshamul-Haque 4
Department of Botany, University of Karachi, Karachi 75270,
sehaq@uok.edu.pk Pakistan

1
Department of Biochemistry, University of Karachi, Karachi 75270, 5
Euronano Diagnostics-Anatolia Geneworks, 225/1, PECHS,
Pakistan Block-2, Karachi, Pakistan
2
Institute of Biomedical Sciences, Dow University of Health Sciences,
Karachi 75270, Pakistan 6
H.E.J. Research Institute of Chemistry, International Center for
3
US-Pakistan Center for Advanced Studies in Water, Mehran Chemical and Biological Sciences, University of Karachi,
University of Engineering and Technology, Jamshoro 76062, Karachi 75270, Pakistan
Pakistan
Environ Sci Pollut Res (2021) 28:9448–9461
Introduction
Cisplatin (CDDP), a cytotoxic drug, has been used for curing
from various types of cancers including tumors of bladder,
head, prostate, neck, testis, ovaries, cervix, lungs, and esoph-
agus (Akca et al. 2018). However, its overdose, even thera-
peutic dose, may have adverse effects including vomiting,
nausea, nephrotoxicity, and hepatotoxicity (Sohail et al.
2019). Although cisplatin treatment improved cancer therapy
but it causes acute kidney injury (AKI) in 20–30% patients
(Perše and Večerić-Haler 2018), its deposition in kidneys was
found 5 times higher than in other tissues, resulting in renal
dysfunction; reduced glomerular filtration with high creati-
nine, urea, and blood urea nitrogen (BUN) levels in blood;
and disturbances in electrolytes (Van Angelen et al. 2012).
Cisplatin may activate the generation of ROS (reactive oxygen
species) that enhances oxidative damage resulting in apopto-
sis, inflammatory responses, and DNA damage, resulting in
nephrotoxicity (Abdel-Daim et al. 2019; Ali et al. 2019;
Ibrahim et al. 2018). The regulation of inflammation by pro-
inflammatory and anti-inflammatory cytokines is necessary
for controlled immune response; they are minor and non-
structural proteins which have pro-inflammatory effects that
include interleukins and TNF-α (tumor necrosis factor-α)
(Ferreira et al. 2018).
Hepatotoxicity may also develop after a single dose or low
repeated doses of cisplatin (Cagin et al. 2016). Reactive oxy-
gen species produced during oxidative stress may further aug-
ment cisplatin cytotoxic effect in liver by increasing nuclear
kappa factor B expression and release of pro-inflammatory
cytokines (Cüre et al. 2016). Alrashed and El-Kordy (2019)
reported that cisplatin administration diminished the hepatic
enzymes levels like (alanine aminotransferase (ALAT), aspar-
tate aminotransferase (ASAT)) in blood.
Marine macro-algae (seaweeds) have been used as food in
many Asian countries since a long time (Namvar et al. 2013).
They contain proteins, lipids, carbohydrates, and polysaccha-
rides in high amounts such as carrageenan, alginates, ulvans,
and vitamins and minerals (Pereira 2018; Abdel-Aziz and
Ragab 2017). Interest in seaweed has also been increasing
due to their health benefits (Ambreen et al. 2012; Khalid
et al. 2018). Previous studies showed that seaweeds have pro-
tective effects against cisplatin induced nephrotoxicity (Kim
et al. 2018; Sohail et al. 2019). Green seaweed belongs to
genus Ulva commonly known as sea lettuce are broadly dis-
persed in the coastal areas of the world (Wolf et al. 2012),
where U. fasciata is used as a part of diet in many countries
(Wichard et al. 2015). In some recent studies, this seaweed has
shown extensive anti-coagulant (Govindan et al. 2012), hypo-
glycemic (Abirami and Kowsalya 2013), antioxidant
(Chakraborty and Paulraj 2010), hypocholesterolemic
(Matloub et al. 2013), and antimicrobial (Priyadharshini
et al. 2011) activities. Nowadays, attenuating the cisplatin-
9449
induced kidney injury by using new molecules is becoming
an important area of research (Perše and Večerić-Haler 2018).
Edible seaweed having nephroprotective properties may be a
possible solution of cisplatin-induced nephrotoxicity.
In present study, we have reported nephroprotective effect
of ethanol extract of U. fasciata and its solvent fractions (n-
hexane and chloroform) against cisplatin-induced nephrotox-
icity via assessing renal and liver function markers, antioxi-
dant status in kidney tissues, histopathology of kidney, and
expression of mRNA cytokine (TNF-α and IL-6). The n--
hexane fraction was also analyzed by GC-FID and GC-MS
(gas chromatography-mass spectrometry).
Materials and methods
Collection of seaweed
We collected seaweed, Ulva fasciata, at low tide from Buleji
Beach in Karachi and identified by Dr. Aisha Begum
(Associate Professor), Department of Botany. After washing
under tap water, algae was dried under shade, grinded to
coarse powder and stored at room temperature in cotton bags
until used. Herbarium sheets and voucher specimen were kept
in seaweed Herbarium, MAH Qadri Biological Research
Center, University of Karachi.
Ethanol extract
Dry powder (500 g) of seaweed (U. fasciata) was soaked in a
glass bottle in 2 L ethanol for 2 weeks and then filtered over
cotton wool by using a glass funnel. The ethanol extract was
then concentrated to a gummy mass on a rotary vacuum evap-
orator (Büchi R-200, New Castle, DE, USA) and stored at
room temperature until used.
Fractions of ethanol extract
One hundred grams (100 g) of ethanol extract of U. fasciata
was fractionated with organic solvents of increasing polarity.
The extract was first extracted with n-hexane; the n-hexane
insoluble portion was extracted with chloroform. On a rotary
vacuum evaporator the n-hexane and chloroform fractions
were concentrated to a gummy mass and used for experimen-
tal purpose.
Identification of compounds by GC-MS
About 10 g U. fasciata (powder) was washed with n-hexane to
remove excess water, then extracted in 200 mL n-hexane by
using soxhlet apparatus for 8 h. Fraction extracted from
U. fasciata was evaporated at 40 °C on rotary vacuum evap-
orator till semi gummy mass obtained. The extract was oily in
9450
nature, then exposed to GC-FID and GC-MS profiling, using
GC-MS triple quad (Agilent technologies 7890A). Gas
chromatograph connected with mass spectrometer, Jeol,
JMS- 600H. Operational mode was electron ionization (EI)
having the ion source at 50 °C, and their electron energy
was kept up at 70 eV. Individual peaks of compounds were
distinguished by looking at their retention indices and mass
spectra with computer matching against NIST Mass
Spectrometry Data Center (mainlib) NIST#: 352898 ID#:
113419 DB), USA (Farhat et al. 2019).
Experimental animals
Male Sprague Dawley rats (150–200 g body weight) were
purchased from Agha Khan University (AKU), Karachi. The
group of rats was housed in polypropylene cages under stan-
dard laboratory conditions (23 ± 2 °C and 12-h light/dark
cycles) for 1 week prior to setting them for experiment.
Animals were fed with a standard pellet diet prepared in the
laboratory following the procedure of NRC (1995) and tap
water ad libitum. Wood shaving was used for bedding, which
was changed every third day. All experiments were carried out
with the approval of the Institutional Research Ethical
Committee.
Cisplatin-induced nephrotoxicity
Cisplatin purchased from Sigma Aldrich (St. Louis, Missouri,
USA) and administered intraperitoneally (i.p.) to rats at a dose
of 7 mg/kg body weight (b.w.) dissolved in 1 mL saline on 5th
study day (SD) of the experiment. The dose was selected
according to Sayed-Ahmed et al. (2004), Osman et al.
(2000), and Sohail et al. (2019).
Study design
Fifty four rats were used in this study which were di-
vided into 9 groups (each group consisted of 6 rats) to
determine the protective effect of seaweed in cisplatin-
induced nephrotoxicity:
1. Normal control: Normal diet for rats was kept for 10 days
(SD, 1–10), without any intraperitoneal injection.
2. Vehicle control: Rats were treated with the normal saline
(i.p., 1 mL/kg b.w.) on SD 5 of the experiment.
3. Ethanol extract of U. fasciata: Ethanol extract at 200
mg/kg b.w. were administered to rats daily (p.o) for 10
days (SD 1–10).
4. n-Hexane fraction of U. fasciata: n-Hexane fraction at
100 mg/kg b.w. were administered to rats daily (p.o) for
10 days (SD 1–10).
Environ Sci Pollut Res (2021) 28:9448–9461
5. Chloroform fraction of U. fasciata: Chloroform fraction at
100 mg/kg b.w. was administered to rats daily (p.o) for 10
days (SD 1–10).
6. Cisplatin control group: Rats were intraperitoneally ad-
ministered with cisplatin (i.p., 7 mg/kg b.w.) dissolved
in 1mL saline on SD 5.
7. Ethanol extract of U. fasciata + cisplatin treated group:
Ethanol extract at 200 mg/kg b.w. was administered to
rats daily (p.o) from SD 1 to 10 and cisplatin was injected
on SD 5 of experiment.
8. n-Hexane fraction of U. fasciata + cisplatin treated group:
n-Hexane fraction at 100 mg/kg b.w. was administered to
rats daily (p.o) from SD 1 to 10 and cisplatin was injected
on SD 5 of experiment.
9. Chloroform fraction of Ulva fasciata + cisplatin treated
group: Choloroform fraction at 100 mg/kg b.w. was ad-
ministered to rats daily (p.o) from SD 1 to 10 and cisplatin
was injected on SD 5 of experiment.
On SD 10, rats were fasted overnight and decapitated and
blood was collected. Blood was centrifuged at 3000 rpm for
15 min., at 4 °C; then, supernatant was collected and trans-
ferred to new tubes for biochemical analysis. Furthermore,
kidney tissues were used for determination of antioxidant,
anti-inflammatory activities, and histopathological
examination.
Assessment of nephrotoxicity
Creatinine, urea, blood urea nitrogen (BUN), and electro-
lytes (calcium (Ca++), magnesium (Mg++), potassium (K+),
and sodium (Na+)) were determined in serum for nephro-
toxicity, while hepatic indices were determined by serum
liver enzymes (alanine aminotransferases (ALAT), aspar-
tate aminotransferases (ASAT), alkaline phosphatase
(ALP), lactate dehydrogenase (LDH)) and bilirubin levels
(total bilirubin and direct bilirubin) were determined for
hepatotoxicity on blood chemistry analyzer (Microlab-
300, Merck, France) using kits from Merck (France) and
Ecoline (Germany). For the estimation of antioxidant en-
zymes, like glutathione (GSH), catalase (CAT), and
malondialdehyde (MDA), 1 g of kidney was homogenized
in ice cold KCl solution (1.5%) to make 10% homogenate
using Polytron (Kinematica) PT-MR 2100 homogenizer.
The homogenate was used for measuring the antioxidant
enzymes. Furthermore, for expression of mRNA and his-
topathological studies, kidneys were dissected immediate-
ly and cut into three pieces. For histopathological studies, a
portion of the right kidney was kept in 4% formaldehyde,
whereas for the expression of mRNA 1 g kidney tissues
was kept in liquid nitrogen and RNA was extracted as
described below.
Environ Sci Pollut Res (2021) 28:9448–9461
Measurement of oxidative stress markers
Estimation of reduced glutathione
Tissue homogenate of kidney (100 μl) was mixed with 0.1 mL
of 25% TCA, 5-min incubation at room temperature to allow
proteins to precipitate. Centrifuge at 3000 rpm for 10 min, then
supernatant was obtained. About 1800 μl of 0.1 mM DTNB (5,
5′-dithiobis-2-nitrobenzoic acid in 0.3 M sodium phosphate
buffer, pH 8.0), 200 μl of supernatant was added. After incu-
bation for 10 min, we recorded the absorbance at 412 nm on the
Shimadzu UV-1800 UV-Vis Spectrophotometer, Japan
(Moron et al. 1979).
Estimation of lipid peroxidation
For lipid peroxidation 100 μl of tissue homogenate was mixed
with 100 μl of 8.1% sodium dodecyl sulphate (SDS), 750 μl of
20% acetic acid, and 750 μl of 0.8% of thiobarbituric acid (TBA)
and last volume was made 2.0 mL with distilled water. The
sample was then heated at 95 °C for an hour. The final volume
after cooling was made 2.5 mL with distilled water. After that,
2.5 mL of butanol:pyridine mixture (1:1 v/v) was included and
centrifuged for 10 min at 4000 rpm. The absorbance of organic
layer was recorded at 532 nm (Ohkawa et al. 1979).
Estimation of Catalase
For the estimation of catalase activity, 1.96 mL phosphate
buffer (0.01M, pH 7.0), 1.0 mL hydrogen peroxide (0.2 M),
and 40 μl of homogenate were mixed. One milliliter of reac-
tion mixture was mixed with 2-mL dichromate acetic corro-
sive reagent, and afterward boiled for 10 min. We recorded the
absorbance at 570 nm (Sinha 1972).
Histological analysis
The kidney tissues kept in 4% formaldehyde was dehydrated by
keeping it in distilled ethanol followed by embedding into par-
affin wax. Paraffin sections (4–5 μm thick) were obtained with
a microtome and placed on glass slides. Sections were then
stained with haematoxylin and eosin, covered with glass cover
slip and placed in an incubator (37°C) overnight, followed by
examination under light microscope (Nikon FX-35, Japan).
Determination of inflammatory cytokines
RNA extraction and cDNA synthesis
RNA was extracted from the kidney by using Trizol Reagent
(Invitrogen) according to manufacturer’s instruction, using
polytron (Kinematica) PT-MR 2100 homogenizer where tis-
sues were homogenized. The purity of RNA was determined
9451
by using Nanodrop Spectrophotometer at 280 and 260 nm,
while concentration of RNA was determined at 260 nm. The
extracted RNA was dissolved in 30-μl nuclease-free water
and stored at − 30 °C until used. cDNAs were synthesized
according to manufacturer’s instruction (Thermo
Scientific™ Revert Aid™ US). The cDNA which was newly
synthesized was stored at − 20 °C for the study of mRNA, IL-
6, and TNF-α by using real-time PCR.
Thermal cycling conditions were programmed to carry out
45 cycles, each of which consisted of three steps:
Step I: 95 °C for 15 s, 60 °C for 20 s (Denaturation)
Step II: 72 °C for 1 min, 95 °C for 15 s (Annealing)
Step III: 72 °C for 10 s, 4 °C fridge temperature
(Extension)
Determination of expression of mRNA by SYBR green
real-time quantitative ((q) PCR)
Real-time PCR was performed using the CE marked IVD kits
on Montania 4896 Real-Time PCR instrument (Anatolia Gene
works, Turkey). cDNA (1 μl) from each sample was amplified
in 20 μl of reactive mixture containing 0.25 μM of each prim-
er and 12.5 μl Thermo Scientific Maxima SYBR Green/ROX
qPCR Master Mix (2X). The forward and reverse primers for
rat are IL-6 and TNF-α which are as follows;
Primer Primer Sequence (5′–3′) Base
Name pair
Forward Reverse
TNF-α ACT GAA CTT CGG GGT GCT TGG TGG TTT 84
GAT TG GCT ACG AC
IL-6 CAG AAT TGC CAT CGT AAG TGC ATC ATC 141
ACA ACT CTT TTC TCA GTT GTT CAT
ACA
The number of cycles was 45 in a real-time PCR machine
(Applied Bio-system Thermo Fisher, USA). The expression of
mRNA that was quantified the level of targeted pro-
inflammatory cytokines including interleukin-6 (IL-6) and tu-
mor necrosis factor-alpha (TNF-α) was evaluated by determin-
ing the cycle threshold (Ct). Glyceraldehyde-3phosphate dehy-
drogenase (GAPDH) was used as a reference gene (control).
The selective cytokine gene expression was determined using
the 2- ΔΔCT according to Livak and Schmittgen (2001).
Statistical analysis
Data was analyzed on SPSS statistical software (IBM SPSS
statistics 23, USA). The data expressed as (mean ± standard
9452
deviation (SD)) (n = 6) and statistical significance was defined
by the analysis of variance (one-way ANOVA) followed by
the Tukey post hoc test with P < 0.05 was considered as
statistically significantly different from the concurrently run
controls.
Results
Preventive effect of ethanol extract of Ulva fasciata
and its fractions (n-hexane-chloroform) in kidney and
liver parameters of cisplatin intoxicated rats
Vehicle control group
In vehicle control, the values were near to normal range in
kidney parameters (creatinine, urea, and BUN) but decreased
Ca++, K+, and Na+, while liver parameters increased as com-
pared with normal control group.
Ethanol extract of U. fasciata and its fractions (n-hexane
and chloroform) treated group
Ethanol extract of U. fasciata and its fractions (n-hexane and
chloroform) showed that the kidney parameters were normal
in ranges but electrolytes level were decreased (Ca+, K+, and
Na+) as compared with normal control group, although etha-
nol extract of U. fasciata and its chloroform fraction also
increased liver enzymes (ALAT, ASAT, ALP, LDH) but bil-
irubin levels (direct bilirubin and indirect bilirubin) were in
normal ranges as compared with normal control. Beside these,
n-hexane fraction of U. fasciata raised liver enzymes and also
increased bilirubin level as compared with normal control
(Tables 1 and 2).
Cisplatin control group
Single intraperitoneal injection of cisplatin caused renal inju-
ry, showed by elevated kidney markers, i.e., urea, BUN, and
creatinine up to (99.1%), (99.1%), and (220%) while reduced
Ca++ (− 69.1%), K+ (− 36.3%), and Na+ (− 3.1%) versus with
normal control. Furthermore, cisplatin significantly (p < 0.05)
raised liver enzymes (ALAT, ASAT, ALP, LDH) up to
(157.7%, 67.5%, 61.4%, 138.3%) and total bilirubin (225%)
many folds as compared with normal control group (Tables 1
and 2).
Ethanol extract of U. fasciata and its fractions (n-hexane
and chloroform) with cisplatin treated groups
Ethanol extract of U. fasciata significantly reversed the cis-
platin induced kidney injury by decreasing the kidney param-
eters including creatinine (− 43.7%), urea (− 31.1%), and
Environ Sci Pollut Res (2021) 28:9448–9461
BUN (− 30.8%) and increased Ca++ (48.2%) and K+ (40%)
as compared with cisplatin control group. Similarly, it also
significantly attenuated hepatotoxicity by reducing the level
of liver enzymes, i.e., ALAT (− 22.7%), ASAT (− 22.9%),
ALP (− 19.7%), LDH (− 29.8%), and bilirubin (total bilirubin
(− 7.6%) and direct bilirubin (− 33.3%)) as compared with
cisplatin control group.
On the other side, n-hexane and chloroform fractions of
U. fasciata + cisplatin-treated group exhibited significant de-
crease in kidney parameters when compared with cisplatin
control group. A restoration of serum Ca++ (17.2%) showed
by n-hexane fraction while chloroform fraction significantly
increased serum Ca++ (44.8%) and K+ (34.2%) when com-
pared with cisplatin control group. Moreover, chloroform
fraction of U. fasciata + cisplatin showed improvement in
liver enzymes and bilirubin levels as evident by declined
(ALAT (− 27.3%), ASAT (− 23.9%), ALP (− 15.9%), LDH
(− 23.2%)) and (total bilirubin (− 30.7%) and direct bilirubin
(− 33.3%)) more than n-hexane as compared with cisplatin
control group (Tables 1 and 2).
Preventive effect of ethanol extract of Ulva fasciata and its
fractions (n-hexane and chloroform) on oxidative statue,
glutathione (GSH), lipid peroxidation (MDA), and catalase
levels in kidney tissue of cisplatin intoxicated rats
Lipid peroxidation was measured in terms of MDA level per g
wet kidney tissues. Ethanol extract of U. fasciata and its frac-
tions (n-hexane and chloroform) showed increase activity of
MDA as compared with normal control, whereas ethanol ex-
tract and n-hexane fraction of U. fasciata showed decrease
activity of GSH, while chloroform extract exhibited increase
activity of GSH as compared with normal control group.
Catalase activity was measured in mmol/g wet renal tissues
in which ethanol extract of U. fasciata showed no alteration
but n-hexane fraction raised catalase level as compared with
normal control rats. Chloroform extract of U. fasciata showed
decline activity of catalase as compared with normal control.
MDA level was found significantly higher (34.3%) in cis-
platin control. However, GSH and catalase level were de-
creased (− 33.5%) and (− 42.8%) respectively in cisplatin
control as compared with normal control. Ethanol extract
and chloroform fraction of U. fasciata with cisplatin signifi-
cantly decreased MDA level while no protection from n-hex-
ane fraction as compared with cisplatin control. However,
GSH reduction improved by ethanol extract of U. fasciata
and chloroform extract of U. fasciata showed highest im-
provement (61.7%) as compared with cisplatin control.
Renal tissues in the experimental group also revealed that
ethanol extract of U. fasciata with cisplatin increased catalase
level (50%) more than their fractions activity as compared
with cisplatin control (Table 3).
Table 1 Effect of ethanol extract of Ulva fasciata and its solvent fractions (n-hexane and chloroform) on kidney function and electrolyte balance in normal and cisplatin intoxicated rats

Groups Creatinine (mg/dl) Urea (mg/dl) BUN (mg/dl) Calcium (mg/dl) Potassium (mmol/l) Magnesium (mg/dl) Sodium (mEq/l)
Environ Sci Pollut Res (2021) 28:9448–9461

Normal control 0.5 ± 0.09 24.5 ± 4 11.4 ± 1.9 9.4 ± 0.4 5.5 ± 0.5 2.3 ± 0.3 153.1 ± 3.3
Vehicle control 0.4 ± 0.05©ns (− 20%) 22.1 ± 4.6©ns (− 9.7%) 10.3 ± 2.1©ns (− 9.6%) 5.4 ± 0.3©* (− 42.5%) 5.1 ± 0.4©ns (− 7.2%) 3.1 ± 0.2©s (34.7%) 139 ± 3.3©* (− 9.2%)
U. fasciata ethanol 0.5 ± 0.08©ns (0%) 29.8 ± 2.4©ns (21.6%) 13.9 ± 1.1©ns (21.9%) 4.9 ± 0.6©* (− 47.8%) 5.1 ± 0.3©ns (− 7.2%) 3.5 ± 0.2©s (52.1%) 127±3.5©* (− 17%)
extract
U. fasciata n-hexane 0.6 ± 0.07©ns (20%) 22 ± 2.8©ns (− 10.2%) 10.2±1.3©ns (− 10.5%) 4.1 ± 0.3©* (− 56.3%) 3.7 ± 0.3©* (− 32.7%) 2.5 ± 0.2©ns (8.6%) 145.5 ± 3©* (− 4.9%)
fraction
U. fasciata chloroform 0.4 ± 0.08©ns (− 20%) 27.3 ± 3.4©ns (11.4%) 12.7 ± 1.6©ns (11.4%) 4.9 ± 0.7©* (− 47.8%) 5.1 ± 0.2©ns (− 7.2%) 3.4 ± 0.3©* (47.8%) 136.8 ± 2.7©* (− 10.6%)
fraction
Cisplatin control 1.6 ± 0.1©* (220%) 48.8 ± 6.5©* (99.1%) 22.7 ± 3.1©* (99.1%) 2.9 ± 0.5©* (− 69.1%) 3.5 ± 0.4©* (− 36.3%) 2.9 ± 0.3©ns (26%) 148.3 ± 4.1©ns (− 3.1%)
U. fasciata ethanol 0.9 ± 0.08¥* (− 43.7%) 33.6 ± 4.6¥* (− 31.1%) 15.7 ± 2.1¥* (− 30.8%) 4.3 ± 0.4¥* (48.2%) 4.9 ± 0.3¥* (40%) 2.4 ± 0.4¥ns (− 17.2%) 142 ± 3.5¥* (− 4.2%)
extract + cisplatin
U. fasciata n-hexane 0.9 ± 0.1¥* (− 43.7%) 35.3 ± 3.2¥* (− 27.6%) 16.4 ± 1.5¥* (− 27.7%) 3.4 ± 0.4¥ns (17.2%) 3.4 ± 0.2¥ns (− 2.8%) 2.1 ± 0.3¥* (− 27.5%) 143.3 ± 3.6¥ns (− 3.3%)
fraction + cisplatin
U. fasciata chloroform 0.5 ± 0.08¥* (− 68.7%) 37.5 ± 3.5¥* (− 23.1%) 17.4 ± 1.6¥* (− 23.3%) 4.2 ± 0.2¥* (44.8%) 4.7 ± 0.3¥* (34.2%) 2.7 ± 0.2¥ns (− 6.8%) 143.8 ± 2.1¥ns (− 3%)
fraction+ cisplatin

Values were expressed as means ± SD (n = 6)

Values bearing © as superscript were compared with normal control rats
Values bearing ¥ as superscript were compared with cisplatin dosed rats
Values in parenthesis are showing percent increased or decreased as compared with their respective control
ns non-significant
*Significant at p < 0.05 according to Tukey’s (post hoc) range test
9453
9454 Environ Sci Pollut Res (2021) 28:9448–9461

Total Bilirubin (mg/dl) Direct Bilirubin (mg/dl)

0.2 ± 0.05¥ns (− 33.3%)

0.9 ± 0.07¥* (− 30.7%) 0.2 ± 0.08¥ns (− 33.3%)

Histopathological analysis of cisplatin-induced renal damage

0.2 ± 0.07©ns (100%)

0.2 ± 0.08©ns (100%)
0.3 ± 0.05©ns (200%)
0.2 ± 0.08©ns (100%)
0.3 ± 0.1©ns (200%)
and its recovery by the ethanol extract of U. fasciata and its
fractions (n-hexane and chloroform)

0.4 ± 0.1¥ns
0.1 ± 0.05

(33.3%)
Light microscopic examination of the normal control
group showed normal histological appearance with normal
renal tubules (distal convoluted tubule (DCT) and proxi-

1.2 ± 0.2¥ns (− 7.6%)

Effect of ethanol extract of Ulva fasciata and its solvent fractions (hexane and chloroform) on liver enzymes and bilirubin in normal and cisplatin intoxicated rats

mal convoluted tubule (PCT)) and glomerulus (GL) struc-

0.1±0.08©* (− 75%)

1.3 ± 0.1©* (225%)

0.5 ± 0.1©ns (25%)

0.7 ± 0.1©ns (75%)

0.4 ± 0.07©ns (0%)
tural features (Fig. 1a). Cisplatin-treated control group

1.3 ± 0.2¥* (0%)

showed injury in renal cortex, degeneration of tubular ep-
0.4 ± 0.07

ithelial cells, cyst formation, and active macrophages with

infiltration of polymorphonuclear cells. Furthermore,
karyomegaly were seen (Fig. 1b). The ethanol extract of
U. fasciata and chloroform extract of U. fasciata exhibited
258.1 ± 7.7©* (138.3%)
153.6 ± 10.8©* (41.8%)

181 ± 10.6¥* (− 29.8%)

185.1 ± 8.5¥* (− 15.9%) 198 ± 11.1¥* (− 23.2%)

188.6 ± 8.8©* (74.1%)
157.8 ± 8.1©s (45.7%)

normal renal tubules; i.e., proximal tubules have brush

206 ± 5.7©* (90.2%)

borders along with uneven lumen. Furthermore, GL and

Bowman’s capsule (BM) had regular margins with normal
108.3 ± 9.1

223.8 ± 9¥*
LDH (U/L)

(− 13.2%)

mesangial cells (Fig. 1c and d). But n-hexane extract of

U. fasciata showed eosinophilic degeneration which shows
tubular necrosis along with few fatty vacuoles, however
near normal GL can be seen (Fig. 1e). In ethanol extract
176.6 ± 5.1¥* (− 19.7%)
217.3 ± 6.3¥ns (− 1.2%)
161.8 ± 7.6©* (18.7%)
155.1 ± 5.4©* (13.7%)

153.1 ± 9.5©* (12.3%)

220.1 ± 8.6©* (61.4%)

of U. fasciata + cisplatin-treated group, the GL, BM, and

179.6 ± 6©* (31.7%)

tubules (proximal and distal tubules) showed normal mor-

phologies. Glomerular degeneration and tubular epithelial
136.3 ± 7.3
ALP (U/L)

cell injury were also down versus the cisplatin control

Values in parenthesis are showing percent increased or decreased as compared with their respective control

group (Fig. 1f). Chloroform extract of U. fasciata + cis-

platin treated rats also showed normal BM and tubules
(Fig. 1g), whereas n-hexane extract of U. fasciata was
115.1 ± 6.7¥* (− 22.9%)
75.1 ± 7.7©* (− 15.7%)

142.6 ± 6.5¥ns (− 4.4%)

113.6 ± 13¥* (− 23.9%)

124.6 ± 8.2©* (39.8%)
109.5 ± 7.8©* (22.8%)
149.3 ± 6.2©* (67.5%)

found less effective in cisplatin intoxicated rats. GL and

94.8 ± 4.8©ns (6.3%)

BM showed irregular structure with a very slight loss of

*significant at p<0.05 according to Tukey’s (post hoc) range test, ns= non-significant

tubular necrosis and found cyst formation as compared

ASAT (U/L)

89.1 ± 7.5

with cisplatin control group (Fig. 1h).

Values bearing ©as superscript were compared with normal control rats.
Values bearing ¥ as superscript were compared with cisplatin dosed rats

GC-FID and GC-MS profiling of n-hexane fraction of ethanol

126.3 ± 9.9©* (157.7%)
97.6 ± 7.6¥ns (− 22.7%)
121.8 ± 8.9¥ns (− 3.5%)

U. fasciata chloroform fraction + cisplatin 91.8 ± 7.3¥* (− 27.3%)

extract of U. fasciata
67.3 ± 3.2©* (37.3%)
85.5 ± 4.7©* (74.4%)
64.8 ± 5.1©* (32.2%)
60.8 ± 9.3©ns (24%)

The GC-FID and GC-MS profiling of n-hexane soluble frac-

ALAT (U/L)

tion of U. fasciata showed the presence of fatty acids, alco-

49 ± 9.2

hols, steroids, terpenoids, benzene derivatives, and volatile

oils. The phytochemical constituents of U. faciata are summa-
Values were expressed as means ± SD (n=6);

rized in (Table 4) (Figs. S1-S3). The characteristic peaks in the

U. fasciata n-hexane fraction + cisplatin

mass spectrum showed M-14, M-15, M-19, M-28, and M-44

U. fasciata ethanol extract + cisplatin

which confirmed the presence of methyl, hydrocarbon, car-

bonyl, hydroxyl, and carboxylic acid functional groups in the
U. fasciata chloroform fraction
U. fasciata n-hexane fraction

extract. In this study total 21 volatile compounds have been

U. fasciata ethanol extract

identified among them 6 are being reported 1st time from this
source like 2(4H)-benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-
trimethyl-, (R)-, phosphonofluoridic acid, (1-methylethyl)-,
Cisplatin control
Vehicle control
Normal control

cyclohexyl ester, 6-(3,3-dimethyl-oxiran-2-ylidene)-5,5-di-

methyl-hex-3-en-2-one, estra-1,3,5(10)-trien-17β-ol, 1,2 ben-
Table 2

Groups

zene dicarboxylic acid, di-isooctyl ester, terephthalic acid, and

di (2-ethylhexyl) ester (Table 4).
Environ Sci Pollut Res (2021) 28:9448–9461
Table 3 Effect of ethanol extract of Ulva fasciata and its solvent fractions (n-hexane and chloroform) on glutathione (GSH), lipid peroxidation (MDA),
and catalase (CAT) activity in normal and cisplatin intoxicated rats
Groups GSH (μmol/g)
Normal control 13.4 ± 0.6
U. fasciata ethanol extract 12.7 ± 0.4©ns (− 5.2%)
U. fasciata n-hexane fraction 12.8 ± 0.8©ns (− 4.4%)
U. fasciata chloroform fraction 14.9 ± 0.4©* (11.1%)
Cisplatin control 8.9 ± 0.3©* (− 33.5%)
U. fasciata ethanol extract + cisplatin 10.3 ± 0.3¥* (15.7%)
U. fasciata n-hexane fraction + cisplatin 13 ± 0.3¥* (46%)
U. fasciata chloroform fraction + cisplatin 14.4 ± 0.3¥* (61.7%)
Values were expressed as means ± SD (n = 6)
Values bearing © as superscript were compared with normal control rats
Values bearing ¥ as superscript were compared with cisplatin dosed rats
Values in parenthesis are showing percent increased or decreased as compared with their respective control
ns non-significant
*Significant at p < 0.05 according to Tukey’s (post hoc) range test
Effect of ethanol extract of U. fasciata on expression
of inflammatory cytokine
The Ct (cycle threshold) value was obtained from real time
PCR which were determined for tumor necrosis factor-α
(TNF-α), interleukin-6 (IL-6), and glyceraldehyde-
3phosphate dehydrogenase (GAPDH) transcripts in total
RNA samples from kidney (Fig; S-4 and S-5).
Analysis of the fold changes in TNF-α gene expression of
renal tissues in experimental groups revealed that higher
levels of TNF-α in cisplatin control group when compared
with normal control group after GAPDH housekeeping gene
normalization. However, ethanol extract of U. fasciata with
cisplatin decreased mRNA expression of TNF-α (Fig. 2a).
Concomitantly, fold change in IL-6 gene expression of re-
nal tissues in experimental groups revealed higher expression
of IL-6 in cisplatin control group when compared with normal
control group after GAPDH housekeeping gene normalization.
However, ethanol extract of U. fasciata with cisplatin did not
reverse the expression of mRNA of IL-6 (Fig. 2b).
Discussion
In current study the effects of ethanol extract of a green sea-
weed U. fasciata and its fractions (n-hexane and chloroform)
showed significant renoprotective effect in cisplatin-induced
nephrotoxicity. Previous studies have shown that use of cis-
platin resulted in marked renal dysfunction as verified by in-
creased level of urea and creatinine in serum (Sohail et al.
2019; Sen et al. 2018; Sherif 2015). Our findings showed that
ethanol extract of U. fasciata and its fractions significantly
9455
MDA (μmol/g) CAT (mmol/g)
10.2 ± 0.6 0.7 ± 0.04
11.3 ± 0.4©* (10.7%) 0.7 ± 0.04©ns (0%)
11.5 ± 0.6©* (12.7%) 0.8 ± 0©* (14.2%)
11.1 ± 0.4©ns (8.8%) 0.6 ± 0©* (− 14.2%)
13.7 ± 0.9©* (34.3%) 0.4 ± 0.05©* (− 42.8%)
11.3 ± 0.3¥* (− 17.5%) 0.6 ± 0.08¥* (50%)
14.2 ± 0.4¥ns (3.6%) 0.5 ± 0¥ns (25%)
13 ± 0.2¥ns (− 5.1%) 0.4 ± 0¥ns (0%)
lowered the elevated levels of kidney parameters as compared
with cisplatin control group. Several compounds have been
isolated during the last four decades from marine organisms.
Beside, edible nature of some seaweed, its secondary metab-
olites can be used for drug development (Raj et al. 2016).
Cisplatin also decreased potassium and magnesium levels,
and reduced glomerular filtration rate may cause acute renal
failure (Sherif 2015). Intoxication produced by cisplatin dur-
ing treatment may be because of the instabilities in homeosta-
sis of electrolytes alter the function of Na+-K+-ATPase and
lastly prompting cell death (Ciarimboli 2011). Our results also
showed that cisplatin reduced serum Ca++, K+, Na+, and Mg++
level. Treatment with ethanol extract of U. fasciata and its
chloroform fraction with cisplatin partially protected the ion
homeostasis by increasing level of Ca++, K+, and Mg++ which
were altered by cisplatin administration. Cisplatin causes kid-
ney harm as well as harmed different organs particularly, liver,
as evident by high levels of hepatic enzymes, i.e., ALAT,
Fig. 1 Light microscopy of renal tissue (× 200), a control, b cisplatin„
alone, c ethanol extract of U. fasciata, d Ethanol extract of U. fasciata +
cisplatin, e n-hexane extract of U. fasciata, f n-Hexane extract of
U. fasciata + Cisplatin, g Chloroform extract of U. fasciata, and h
Chloroform extract of U. fasciata + cisplatin. a Normal appearance of
renal tubules (yellow arrow), while Glomerulus (GL) and Bowman’s
capsules (BM) (blue arrow) are also normal. b Tubules show necrosis;
hemorrhage is visible (white arrow), and degenerative GL (blue arrow). c
Normal tubules and GL with regular structure. d Slight tubular degener-
ation and regular GL structure. e Due to degeneration in glomerulus,
vacuole is formed (blue arrow) and its remains is obvious (green arrow).
f Atrophic tubules show fibrosis, while GL is not normal in appearance. g
Tubules typical regular and GL with a regular structure. h Tubules do not
show a stranded structure and GL with irregular appearance
9456 Environ Sci Pollut Res (2021) 28:9448–9461

a b
BM

GL

PCT
DCT

c d

e f

g h
Table 4 GC-MS analysis of chemical constituents and fragmentation pattern of n-hexane fraction of Ulva fasciata

S/ Compound name Retention M.W Molecular Area Area (%) Characteristic mass fragments, m/z (rel.% BP) Conc.
No time (min) formula

1 2(4H)-Benzofuranone,5,6,7,7a-tetrahydro-4,4,7a-trimethyl-, (R)- 24.822 180 C11H16O2 2,141,325

0.204773163 43 (99), 111 (71), 67 (41), 137 (40), 41 (40), 109 0.041
(39), 110 (18), 53 (17), 77 (14), 79 (14)
2 Dodecanoic acid 25.774 200 C12H24O2 4,256,580 0.407053273 73 (99), 60 (98), 43 (69), 41 (56), 57 (55), 55 0.092
(46), 129 (32), 29 (32), 71 (32), 85 (29)
3 Phosphonofluoridic acid (1-methylethyl)-, cyclohexyl ester 26.039 208 C9H18FO2P 1,716,893 0.164185077 127 (99), 43 (23), 41 (17), 67 (17), 54 (99), 55 0.045
(94), 85 (75), 82 (41), 42 (33), 128 (33)
4 6-(3,3-Dimethyl-oxiran-2-ylidene)-5,5-dimethyl-hex-3-en-2-one 26.507 194 C12H18O2 1,760,520 0.168357091 151 (99), 43 (54), 41 (36), 69 (32), 109 (23), 81 0.042
(17), 194 (17), 125 (15), 152 (13), 39 (13)
Environ Sci Pollut Res (2021) 28:9448–9461

5 Tridecanoic acid 27.931 214 C13H26O2 3,273,730 0.313064129 73 (99), 60 (86), 43 (68), 41 (66), 57 (63), 55 0.06
(52), 129 (47), 29 (35), 71 (34), 69 (33)
6 Heptadecane 28.451 240 C17H36 1,710,067 0.163532312 57 (99), 43 (66), 71 (56), 85 (38), 41 (37), 29 0.042
(190), 55 (18), 99 (13), 56 (12), 70 (11)
7 Tetradecanoic acid 30.348 228 C14H28O2 12,626,835 1.20749393 73 (99), 60 (90), 43 (72), 55 (70), 41 (67), 57 0.15
(65), 129 (51), 69 (35), 71 (33), 185 (29)
8 2-Pentadecanone, 6,10,14-trimethyl- 32.215 268 C18H36O 54,470,524 5.208971772 43 (99), 58 (89), 71 (45), 57 (42), 59 (40), 41 (37), 0.085
55 (34), 69 (24), 85 (22), 95 (20)
9 Pentadecanoic acid 33.159 242 C15H30O2 18,190,321 1.739525557 43 (99), 41 (85), 57 (73), 55 (63), 82 (39), 73 (34), 0.171
69 (34), 60 (32), 71 (31), 83 (27)
10 Hexadecanoic acid, methyl ester 35.092 270 C17H34O2 69,144,870 6.612267508 74 (99), 87 (72), 43 (32), 55 (31), 41 (22), 143 0.111
(20), 75 (18), 57 (18), 69 (13), 227 (11)
11 Isophytol 35.959 296 C20H40O 3,274,510 0.31313872 71 (99), 43 (30), 41 (14), 55 (13), 57 (12), 82 (79), 0.069
69 (76), 81 (69), 72 (57), 83 (52)
12 Hexadecanoic acid, ethyl ester 37.845 284 C18H36O2 11,709,868 1.119805124 88 (99), 101 (55), 43 (36), 41 (26), 55 (23), 29 0.06
(22), 57 (21), 73 (15), 89 (13), 69 (13)
13 n-Hexadecanoic acid 39.273 256 C16H32O2 813,131,994 77.75914921 60 (99), 73 (98), 57 (84), 43 (81), 55 (76), 41 0.39
(57), 129 (43), 71 (37), 69 (35), 83 (26)
14 n-Hexadecanoic acid 41.440 256 C16H32O2 3,573,083 0.341691012 60 (99), 73 (98), 57 (84), 43 (81), 55 (76), 41 0.057
(57), 129 (43), 71 (37), 69 (35), 83 (26)
15 Nonadecanoic acid 42.033 298 C19H38O2 2,547,952 0.243658571 43 (99), 41 (98), 55 (67), 57 (50), 29 (47), 60 0.049
(46), 73 (37), 298 (32), 69 (30), 27 (23)
16 Estra-1,3,5(10)-trien-17β-ol 46.947 256 C18H24O 7773649 0.743387712 43 (99), 57 (99), 55 (71), 41 (55), 73 (47), 71 (44), 0.1
45 (39), 85 (35), 83 (35), 256 (34)
17 Estra-1,3,5(10)-trien-17β-ol 47.181 256 C18H24O 4,192,359 0.400911871 43 (99), 57 (99), 55 (71), 41 (55), 73 (47), 71 (44), 0.094
45 (39), 85 (35), 83 (35), 256 (34)
18 Methyl 20-methyl-heneicosanoate 50.263 354 C23H46O2 2,824,159 0.270072021 74 (99), 87 (75), 55 (30), 57 (28), 75 (28), 143 (28), 0.04
354 (28), 69 (21), 311 (19), 83 (13)
19 1,2-Benzenedicarboxylic acid, diisooctyl ester 50.637 390 C24H38O4 22,513,092 2.152908621 149 (99), 167 (35), 57 (34), 70 (26), 41 (22), 71 (22), 0.046
55 (21), 43 (20), 150 (10), 83 (10)
20 Terephthalic acid, di(2-ethylhexyl) ester 53.683 390 C24H38O4 1,774,733 0.169716269 261 (99), 70 (90), 112 (88), 149 ( 55), 167 (31), 279 0.034
(29), 83 (26), 113 (25), 57 (24), 71 (22)
21 Bufa-14,16,20,22-tetraenolide, 3-(acetyloxy)-, (3β,5β)- 62.681 408 C26H32O4 2,552,863 0.244128206 408 (99), 44 (84), 43 (63), 79 (35), 91 (34)), 41 (31), 0.067
55 (31), 93 (28), 241 (26), 45 (25)
9457
9458 Environ Sci Pollut Res (2021) 28:9448–9461

a
TNF α
7

Kidney gene expression 6

5
(Fold Change)

0
N.C CIS EtOH.E of U.f+CIS
TREATMENT

b IL-6
9.0
8.0
7.0
Kidney gene expression

6.0
(Fold Change)

5.0
4.0
3.0
2.0
1.0
0.0
N.C CIS EtOH.E of U.f + CIS
TREATMENT

Fig. 2 a Effect of ethanol extract of Ulva fasciata on the expression of fasciata. b Effect of ethanol extract of Ulva fasciata on the expression of
tumor necrosis factor-α (TNF-α) in kidney of cisplatin intoxicated rats. interleukin-6 (IL-6) in kidney of cisplatin intoxicated rats. NC normal
NC normal control, Cis cisplatin, EtOH. E of U. f ethanol extract of Ulva control, Cis cisplatin, EtOH. E of U. f ethanol extract of Ulva fasciata

ASAT, LDH, ALP, and bilirubin levels (total bilirubin and
direct bilirubin) (Koyuncu et al. 2017; Ben Saad et al. 2017).
The current study showed that ethanol extracts of U. fasciata
with cisplatin also significantly (p < 0.05) attenuated the hep-
atotoxicity by decreasing hepatic enzymes (ALAT, ASAT,
ALP, and LDH) and bilirubin levels as compared with cisplat-
in control rats. n-Hexane and chloroform fractions of
U. fasciata showed considerable decreased in hepatic markers
as compared with cisplatin control rats.
Oxidative stress due to cisplatin in kidneys may result in
generation of higher amount of reactive oxygen products
which leads to disappearance of the antioxidant defense sys-
tem (Kucuk et al. 2009). In current study, increased MDA
level indicates extent of lipid peroxidation in cisplatin control
rats; however, ethanol extracts and chloroform fraction of
U. fasciata with cisplatin reversed this level in rats. Previous
studies also reported that increased level of MDA due to cis-
platin may induce nephrotoxicity (EkinciAkdemir et al. 2017;
Mi et al. 2018), though n-hexane fraction of U. fasciata did
not reverse the MDA levels in cisplatin-induced oxidative
damage. Presumably, cisplatin diminished the renal antioxi-
dants activities in the medulla and cortex of the kidney, as
evident by decreased antioxidant enzymes including CAT,
GSH-Px, and SOD (Amirshahrokhi and Khalili 2015).
Ethanol extract of U. fasciata and its fractions increased
GSH levels while ethanol extract of U. fasciata significantly
increased catalase activity as compared with cisplatin control
group.
Histopathological study regarding cisplatin intoxication re-
vealed that its administration causes interstitial hemorrhage,
interstitial inflammation, atrophy, and tubular dilatation (Akca
et al. 2018). Our histopathological findings indicated morpho-
logical changes in tubules (proximal and distal convoluted
tubules), which is an indication of vascular segment decay
and tubular necrosis. Comparative results have also been re-
ported by Ravindra et al. (2010). The kidney tissue samples of
ethanol extract and chloroform fraction of U. fasciata in cis-
platin treated groups exhibited diminished tubular necrosis,
Environ Sci Pollut Res (2021) 28:9448–9461
normal GL structure, while n-hexane fraction with cisplatin
showed minor alteration with no better protection as compared
with ethanol extract and chloroform fraction of U. fasciata.
Ethanol extract of U. fasciata provides significant protection
when administered before and after cisplatin injection and is
also capable of preventing the cisplatin induced intoxication
as compared with its fractions. Kuriakose and Kurup (2010)
reported the effect of Aulosira fertilisima against cisplatin-
induced alterations by histopathological examination of kid-
ney tissue.
In the present study GC-FID and GC-MS analysis of n-
hexane fraction of U. fasciata showed that hexadecanoic acid
(Palmitic acid) to be one of the main component followed by
dodecanoic acid, tridecanoic acid, tetradecanoic acid, and
pentadecanoic acid, respectively. The hexadecanoic acid has
been previously found that it has antimicrobial, anticancer,
and anti-angiogenic activities (Usha and Maria 2015; Van
Angelen et al. 2012). Heptadecan has been reported to atten-
uate the oxidative stress and also have anti-inflammatory ef-
fect on the kidney (Kim et al. 2013). α-Linolenic acid,
stearidonic acid, sphingosine-type compound, and guaiane
sesquiterpene derivatives have been previously isolated from
U. fasciata (Chakraborty and Paulraj 2010). Cyto-protective
and anti-genotoxic effects of U. fasciata have been reported
(Rodeiro et al. 2015). Rodeiro et al. (2015) reported that Ulva
seaweed possesses dynamic characteristics and should be giv-
en due consideration to nutritional deficiencies, food prob-
lems, and attenuating the drug-induced nephrotoxicity.
The overall study showed that the ethanol extract of
U. fasciata has more protective effect than their fractions so
we further investigated its effect on anti-inflammatory activi-
ty. In response to nephrotoxicity, different types of inflamma-
tory proteins like intracellular adhesion molecules and cyto-
kines are produced. In cytokines, TNF-α expression was in-
creased in the kidney as early as the first day after cisplatin
administration (Lee et al. 2006). Tan et al. (2016) observed
raising levels of IL-6, TNF-α, and IL-1β which are collective-
ly called pro-inflammatory cytokines with hepatic fibrosis in
mice treated with CCl4. Ma et al. (2017) reported cisplatin-
induced significant elevation in inflammatory mediators
(TNF-α, IL-6). In the present study, up-regulation of TNF-α
and IL-6 mRNA expressions were seen in the cisplatin control
group as comparison with normal control rats. Oral feeding of
ethanol extract of U. fasciata produced significant down-
regulation of TNF-α expression.
Conclusions
Chemotherapy of various types of cancer is an extraordinary
challenge for modern medicine, since most of the anti-cancer
drugs have serious side effects. Cisplatin, a widely used anti-
cancer drug, has adverse effects on other organs, particularly,
9459
the kidney. There is no alternative to chemicals in cancer
therapy, but their side effects can be minimized by the appli-
cations of nutraceuticals. Some of the algae have been report-
ed to attenuate the adverse effects of the chemicals on kidney,
like spirulina normalized the elevated level of urea, creatinine,
uric acid, liver, and cardiac enzymes in deltamethrin-
intoxicated rats (Abdel-Daim et al. 2013). In this study,
U. fasciata demonstrated a protective effect against nephro-
toxicity induced by cisplatin. It attenuated the elevated level of
kidney, liver function markers, oxidative stress, anti-
inflammatory cytokines, and also stabilized cellular morphol-
ogy distorted by cisplatin. The presence of various bioactive
compounds in U. fasciata may have a nephro-protective role
against cisplatin-induced kidney damage. Role of sea lettuce
or other edible seaweed in attenuating the drug-induced tox-
icity needs further investigation.
Acknowledgements Support of Prof. Dr. Viqar Uddin Ahmad (now
late), H.E.J. Research Institute of Chemistry, University of Karachi, for
GC-MS analysis and Dr. Aisha Begum, Department of Botany,
University of Karachi for seaweed identification are acknowledged.
Authors’ contributions NS and KH collected the seaweed, extracted and
fractionated the extracts, and wrote the manuscript. Experiments on ani-
mal were conducted by NS and FU. Histopathology was carried out by
NS and HK. Molecular work on anti-inflammatory cytokine was con-
ducted by NS, JA, and WK. GC-MS profiling of n-hexane extract was
done by NS, HF, and MSA. VS and SE helped in the seaweed collection,
supervised research work, and improve the quality of final version of the
manuscript. All authors read and approved the final manuscript.
Funding Financial assistance provided by the Higher Education
Commission, Pakistan (Grant # nrpu-4505) is sincerely acknowledged.
Data availability The data will be provided on request.
Compliance with ethical standards
Competing interests The authors declare that they have no conflict of
interest.
Ethics approval and consent to participate All experiments were con-
ducted according to the rules of the Institutional Animal Ethics
Committee (IAEC), University of Karachi.
Consent for publication Not applicable.
References
Abdel-Aziz MFA, Ragab MA (2017) Effect of use of fresh macro algae
(Seaweed) Ulva fasciata and Enteromorphaflaxusa with or without
artificial feed on growth performance and feed utilization of
Rabbitfish (Siganusrivulatus) fry. J Aquac Res Dev 8(482):2.
https://doi.org/10.4172/2155-9546.1000482
Abdel-Daim MM, Abuzead SM, Halawa SM (2013) Protective role of
Spirulina platensis against acute deltamethrin-induced toxicity in
rats. PLoS One 8(9):e72991
9460
Abdel-Daim MM, Abushouk AI, Donia T, Alarifi S, Alkahtani S, Aleya
L, Bungau SG (2019) The nephroprotective effects of allicin and
ascorbic acid against cisplatin-induced toxicity in rats. Environ Sci
Pollut Res Int 26(13):13502–13509. https://doi.org/10.1007/
s11356-019-04780-4
Abirami RG, Kowsalya S (2013) Antidiabetic activity of Ulva fasciata
and its impact on carbohydrate metabolism enzymes in alloxan in-
duced diabetic rats. Int J Res Phytochem Pharmacol 3(3):136–141
Akca G, Eren H, Tumkaya L, Mercantepe T, Horsanali MO, Deveci E,
Yilmaz A (2018) The protective effect of astaxanthin against
cisplatin-induced nephrotoxicity in rats. Biomed Pharmacother
100:575–582
Ali S, Khan MA, Hussain MM, Qamar A, Bashir S (2019) Evaluation of
cisplatin induced genotoxicity in male Sprague dawley rats. Pak J
Physiol 15(1):21–24
Alrashed AA, El-Kordy EA (2019) Possible protective role of panax
ginseng on cisplatin-induced hepatotoxicity in adult male albino rats
(Biochemical and Histological Study). J Microsc Ultrastruct 7(2):
84–90
Ambreen, Hira K, Tariq A, Ruqqia, Sultana V, Ara J (2012) Evaluation of
biochemical component and antimicrobial activity of some sea-
weeds occurring at Karachi coast. Pak J Bot 44:1799–1803
Amirshahrokhi K, Khalili AR (2015) Thalidomide ameliorates cisplatin-
induced nephrotoxicity by inhibiting renal inflammation in an ex-
perimental model. Euro J Inflamm 38(2):476–484
Ben Saad H, Gargouri M, Kallel F, Chaabene R, Boudawara T, Jamoussi
K, Magné C, MounirZeghal K, Hakim A, Ben Amara I (2017)
Flavonoid compounds from the red marine alga
Alsidiumcorallinum protect against potassium bromate-induced
nephrotoxicity in adult mice. Environ Toxicol 32:1475–1486
Cagin YF, Atayan Y, Sahin N, Parlakpinar H, Polat A, Vardi N, Yildiz A
(2016) Beneficial effects of dexpanthenol on mesenteric ischemia
and reperfusion injury in experimental rat model. Free Radic Res
50(3):354–365
Chakraborty K, Paulraj R (2010) Sesquiterpenoids with free-radical-
scavenging properties from marine macroalga Ulva fasciata
Delile. Food Chem 122(1):31–41
Ciarimboli G (2011) Role of organic cation transporters in drug-induced
toxicity. Expert Opin Drug Metab Toxicol 7(2):159–174
Cüre MC, Cüre E, Kalkan Y, Kırbaş A, Tümkaya L, Yılmaz A, Yüce S
(2016) Infliximab modulates cisplatin-induced hepatotoxicity in
rats. Balk Med J 33(5):504
EkinciAkdemir F, Albayrak M, Çalik M, BayirGülçin I (2017) The pro-
tective effects of p-coumaric acid on acute liver and kidney damages
induced by cisplatin. Biomed Pharmacother 5(2):18. https://doi.org/
10.3390/biomedicines5020018
Farhat H, Urooj F, Tariq A, Sultana S, Ansari M, Ahmad VU,
Ehteshamul-Haque S (2019) Evaluation of antimicrobial potential
of endophytic fungi associated with healthy plants and characteriza-
tion of compounds produced by endophytic Cephalosporiumand
Fusarium solani. Biocatal Agric Biotechnol 18:101043. https://
doi.org/10.1016/j.bcab.2019
Ferreira VL, Borba HHL, Bonetti ADF, Leonart L, Pontarolo R (2018)
Cytokines and interferons: types and functions. In: Auto-antibodies
and cytokines. IntechOpen. https://doi.org/10.5772/intechopen.
74550.
Govindan SM, Thomas J, Pratheesh PT, Kurup GM (2012) Ex vivo
anticoagulant activity of the polysaccharide isolated from Ulva
fasciata. Int J Life Sci Biotechnol Pharma Res 1:194–197
Ibrahim A, Al-Hizab FA, Abushouk AI, Abdel-Daim MM (2018)
Nephroprotective effects of benzyl isothiocyanate and resveratrol
against cisplatin-induced oxidative stress and inflammation. Front
Pharmacol 9:1268. https://doi.org/10.3389/fphar.2018.0126
Khalid S, Abbas M, Saeed F, Bader-Ul-Ain H, Suleria HAR (2018)
Therapeutic Potential of Seaweed Bioactive Compounds. In:
Environ Sci Pollut Res (2021) 28:9448–9461
Seaweed Biomaterials. Intech Open. https://doi.org/10.5772/
intechopen.74060
Kim DH, Park MH, Choi YJ, Chung KW, Park CH, Jang EJ, An HJ, Yu
BP, Chung HY (2013) Molecular study of dietary heptadecane for
the anti-inflammatory modulation of NF-kB in the aged kidney.
PLoS One 8(3):e59316
Kim IH, Kwon MJ, Jung JH, Nam TJ (2018) Protein extracted from
Porphyra yezoensis prevents cisplatin-induced nephrotoxicity by
down regulating the MAPK and NF-κB pathways. Int J Mol Med
41(1):511–520
Koyuncu I, Kocyigit A, Gonel A, Arslan E, Durgun M (2017) The pro-
tective effect of Naringeninoxime on cisplatin-induced toxicity in
rats. Biochem Res Int 2017:1–9. https://doi.org/10.1155/2017/
9478958
Kucuk A, Kabadere S, Tosun M, Koken T, Kinaci MK, Isikli B, Erkasap
N (2009) Protective effects of doxycycline in ischemia/reperfusion
injury on kidney. J Physiol Biochem 65(2):183–191
Kuriakose GC, Kurup MG (2010) Effects of Aulosira fertilisima against
cisplatin-induced nephrotoxicity and oxidative stress in rats. Ren
Fail 32(2):224–233
Lee S, Kim W, Moon SO, Sung MJ, Kim DH, Kang KP, Park SK (2006)
Rosiglitazone ameliorates cisplatin-induced renal injury in mice.
Nephrol Dial Transplant 21(8):2096–2105
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression
data using real-time quantitative PCR and the 2−ΔΔCT method.
Methods 25(4):402–408
Ma X, Yan L, Zhu Q, Shao F (2017) Puerarin attenuates cisplatin-induced
rat nephrotoxicity: The involvement of TLR4/NF-κB signaling
pathway. PLoS One 12(2):e0171612
Matloub AA, El-Sherbini M, Borai IH, Ezz MK, Rizk MZ, Aly HF,
Fouad GI (2013) Assessment of anti-hyperlipidemic effect and
physco-chemical characterization of water soluble polysaccharides
from Ulva Fasciata Delile. Res J Appl Sci 9(4):2983–2993
Mi XJ, Hou JG, Wang Z, Han Y, Ren S, Hu JN, Li W (2018) The
protective effects of maltol on cisplatin-induced nephrotoxicity
through the AMPK-mediated PI3K/Akt and p53 signaling path-
ways. Sci Rep 8(1):1–12
Moron MS, Depierre JW, Mannervik B (1979) Levels of glutathione,
glutathione reductase and glutathione S-transferase activities in rat
lung and liver. Biochim Biophys Acta Gen Subj 582(1):67–78
Namvar F, Mohamad R, Baharara J, Zafar-Balanejad S, Fargahi F,
Rahman HS (2013) Antioxidant, anti-proliferative and anti-
angiogenesis effects of polyphenol-rich seaweed
(Sargassummuticum). Biomed Res Int 2013:1–9. https://doi.org/
10.1155/2013/604787
NRC (National Research Council) (1995) Nutrient requirements of labo-
ratory animals, Fourth Revised Edition, The National Academies
Press, Washington, D.C. https://doi.org/10.17226/4758
Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal
tissues by thiobarbituric acid reaction. Anal Biochem 95(2):351–
358
Osman AM, El-Sayed EM, El-Demerdash E, Al-Hyder A, El-Didi M,
Attia AS, Hamada FMA (2000) Prevention of cisplatin-induced
nephrotoxicity by methimazole. Pharmacol Res 41(1):113–119
Pereira L (2018) Seaweeds as source of bioactive substances and skin
care therapy cosmeceuticals, algotheraphy and thalassotherapy.
Cosmetics 5(4):68. https://doi.org/10.3390/cosmetics5040068
Perše M, Večerić-Haler Ž (2018) Cisplatin-induced rodent model of kid-
ney injury: characteristics and challenges. Biomed Res Int 2018:1–
29. https://doi.org/10.1155/2018/1462802
Priyadharshini S, Bragadeeswaran S, Prabhu K, Ran SS (2011)
Antimicrobial and hemolytic activity of seaweed extracts Ulva
fasciata (Delile 1813) from Mandapam, Southeast coast of India.
Asian Pac J Trop Biomed 1(1):S38–S39
Raj GA, Chandrasekaran M, Jegan S, Venkatesalu V (2016)
Phytochemical analysis and antifungal activity of Ulva species from
Environ Sci Pollut Res (2021) 28:9448–9461
the Kanniyakumari Gulf of Mannar, South Coast India. Nat Prod:
Indian J Appl Res 12(3):104
Ravindra P, Bhiwgade A, Kulkarni S, Rataboli PV, Dhume CY (2010)
Cisplatin induced histological changes in renal tissue of rat. J Cell
Anim Biol 4(7):108–111
Rodeiro I, Olguín S, Santes R, Herrera JA, Pérez CL, Mangas R,
Camacho-Carranza R (2015) Gas chromatography-mass spectrom-
etry analysis of Ulva fasciata (Green Seaweed) extract and evalua-
tion of its cyto-protective and anti-genotoxic effects. Evid Based
Complement Alternat Med 2015:1–11. https://doi.org/10.1155/
2015/520598
Sayed-Ahmed MM, Eissa MA, Kenawy SA, Mostafa N, Calvani M,
Osman AMM (2004) Progression of cisplatin-induced nephrotoxi-
city in a carnitine-depleted rat model. Chemotherapy 50(4):162–170
Sen S, Chakraborty R, Kalita P (2018) Dillenia indica fruit prevents
cisplatin-induced kidney injury in experimental rats through modu-
lation of oxidative stress, marker enzyme, and biochemical changes.
Nutrire 43(1):15. https://doi.org/10.1186/s41110-018-0074-1
Sherif IO (2015) Amelioration of cisplatin-induced nephrotoxicity in rats
by triterpenoidsaponin of Terminaliaarjuna. J Clin Exp Nephrol 19:
591–597
Sinha AK (1972) Colorimetric assay of catalase. Anal Biochem 47(2):
389–394
Sohail N, Hira K, Tariq A, Sultana V, Ehteshamul-Haque S (2019)
Marine macro-algae attenuates nephrotoxicity and hepatotoxicity
9461
induced by cisplatin and acetaminophen in rats. Environ Sci Pollut
Res 26(24):25301–25311
Tan H, He Q, Li R, Lei F, Lei X (2016) Trillin reduces liver chronic
inflammation and fibrosis in carbon tetrachloride (ccl4) induced
liver injury in mice. Immunol Investig 45:371–382
Usha R, Maria VRS (2015) Gas chromatography and mass spectrometric
analysis of Padina pavonia (L.) Lamour. Bio Disc 6(1):1–5
Van Angelen AA, Glaudemans B, van der Kemp AW, Hoenderop JG,
Bindels RJ (2012) Cisplatin-induced injury of the renal distal con-
voluted tubule is associated with hypomagnesaemia in mice.
Nephrol Dial Transplant 28(4):879–889
Wichard T, Charrier B, Mineur F, Bothwell JH, Clerck OD, Coates JC
(2015) The green seaweed Ulva: a model system to study morpho-
genesis. Front Plant Sci 6–72. https://doi.org/10.3389/fpls.2015.
00072
Wolf MA, Sciuto CA, Andreoli Moro I (2012) Ulva (Chlorophyta,
Ulvales) biodiversity in the North Adriatic Sea (Mediterranean,
Italy): Cryptic species and new introductions. J Appl Phycol 48(6):
1510–1152
Publisher’s note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.