Saudi Journal of Biological Sciences 30 (2023) 103571

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Saudi Journal of Biological Sciences

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Original article

Ameliorative effect of Nigella sativa conjugated silver nanoparticles

against chromium-induced hepatotoxicity and renal toxicity in mice
Tooba Nauroze a,b, Shaukat Ali a,⇑, Lubna Kanwal a,c, Chaman Ara d, Tufail Akbar Mughal e, Shagufta Andleeb b
a
Applied Entomology and Medical Toxicology Laboratory, Department of Zoology, Government College University, Lahore, Pakistan
b
Department of Zoology, University of Education, Lahore, Pakistan
c
Department of Zoology, University of the Okara, Okara, Pakistan
d
Institute of Zoology, University of the Punjab, Lahore, Pakistan
e
Department of Zoology, Women University of Azad Jammu and Kashmir, Bagh, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Hexavalent chromium induces oxidative stress in the liver and kidney. Therefore an in vivo study was
Received 11 November 2022 designed to investigate the modulatory effect of biosynthesized AgNP against Cr (VI) induced hepatotox-
Revised 24 December 2022 icity and nephrotoxicity. The organs index, serum level of ALT, AST, ALP, MDA, total protein and creatinine
Accepted 19 January 2023
were measured. The histopathology and micrometry of the liver and kidney were examined. The liver
Available online 25 January 2023
index was significantly increased (0.098 ± 0.13 g) with slight increase in kidney index in Cr exposed
group. The serum level of ALT (163.0 ± 5.5 U/L), AST (484.0 ± 10.7 U/L), ALP (337.6 ± 9.6 U/L), MDA
Keywords:
(641.2 ± 29.2 U/L), and creatinine (2.9 ± 0.2 mg/dL) were significantly increased (P
0.05) with significant
Chromium (VI)
Nigella sativa seed extract
decrease in total protein level (2.9 ± 0.2 g/dL) (P
0.05) in chromium treated group. In histopathology,
Hepatotoxicity distorted hepatic cords, necrosis, damaged glomerulus and Bowman’s capsule were observed.
Renal toxicity Micrometric studies of the liver and kidney showed significant increase in size of hepatocytes (1188.2
Histopathology ± 467.7 l2) and their nuclei (456.4 ± 206.7 l2), ACSA of Bowman’s capsule (11835.5 ± 336.7 l2) and
Micrometry glomerulus (9051.8 ± 249.8 l2) in Cr (VI) treated group. The size of brush border (10.1 ± 3.0 l) was sig-
nificantly reduced in Cr (VI) treated group however the ACSA of lumen was not significantly changed.
With the administration of NSSE and Nigella sativa AgNPs, decreased the oxidative damage caused by
Cr (V).
Ó 2023 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction harmful for animals present at higher trophic levels of the food
chain like humans (Ali et al., 2019a, 2019b). In the human body,
During human evolution, advanced technologies may become heavy metals are distributed and compartmentalized in the tissues
the source of pollution in the air, soil, and water (Onita et al., and cells, where bind to nucleic acid and proteins causing damage
2021) including heavy metals (Suchana et al., 2021). The heavy to these biomolecules, hence disrupting cellular functions and
metals (HMs), in the environment, remain stable and redistributed induce damage in the liver, kidney, lungs, central nervous system
among different components of the biological systems (Suchana (Engwa et al., 2019) and reproductive system (He et al., 2020).
et al., 2021). As HMs persist for a long time, bio-accumulates and Chromium (III) is an essential element required for the metabolism
biomagnified in the food chain (Kumar et al., 2019), especially of proteins and lipids as well as a cofactor for insulin action (Balali-
Mood et al., 2021) whereas Cr (VI) is expendable and destructive to
life (Ukhurebor et al., 2021), linked with a chain of diseases.
⇑ Corresponding author.
According to International Agency for Research on Cancer report
E-mail addresses: tooba.nauroze@ue.edu.pk (T. Nauroze), dr.shaukatali@gcu.edu.
pk (S. Ali), lubna.kanwal@uo.edu.pk (L. Kanwal), chaman.zool@pu.edu.pk (C. Ara),
(IARC 2018), Cr (VI) belongs to the group I category of an occupa-
dr_tafail@wuajk.edu.pk (T. Akbar Mughal), drshagufta@ue.edu.pk (S. Andleeb). tional cancer-causing agent (Balali-Mood et al., 2021). ROS, the
Peer review under responsibility of King Saud University. most significant free radicals produced by (Selamoğlu et al.,
2017) environmental exposure to Cr (VI), induces oxidative stress,
multi-organ toxicity including anemia, respiratory tract dysfunc-
tion, asthma, liver, kidney, and reproductive system failure in
Production and hosting by Elsevier males (Sankhla and Kumar, 2019). Oxidative stress may also result

https://doi.org/10.1016/j.sjbs.2023.103571
1319-562X/Ó 2023 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
T. Nauroze, S. Ali, L. Kanwal et al. Saudi Journal of Biological Sciences 30 (2023) 103571

by weak antioxidant defense system, may lead to condition where in the study and purchased from the local market in Lahore. All
antioxidant compounds from external sources must be delivered in other chemicals of analytic grade were used in the experiment.
order to maintain normal body functions (Selamoğlu et al., 2009, Distilled water was used to prepare stock solutions. All working
2015; Selamoğlu & Yılmaz, 2014). solutions were prepared fresh from stock solution with distilled
The liver is a vital organ that involves in the bioaccumulation, water.
transformation, detoxification, and excretion of toxicants, more
susceptible to damage (Suchana et al., 2021) resulting in hepato-
2.3. Preparation of Nigella sativa seed extract
cyte toxicity and cirrhosis (Ye et al., 2018). Furthermore, the main
site of serum protein especially albumin synthesis occurs in liver
The seed extract was prepared by using the method followed by
cells. Low level of protein content and low mobility present
(Kannayiram et al., 2019) with slight modification. Nigella sativa
changes in the rate of production and degeneracy of proteins. Ele-
seeds were purchased from Akbari Store, Lahore, washed with
vated activities of biochemical markers for the liver such as serum
water, and dried in air at room temperature. The seeds were
aspartate aminotransferase or glutamic-oxaloacetic transaminase,
ground into a fine powder via sieving mesh and soaked in ethanol
serum alanine aminotransferase or glutamic pyruvic transaminase,
(10 g powder into 100 mL ethanol). The mixture was allowed to
and alkaline phosphatase in blood or tissues reflect their outflow
shake well on an orbital shaker for 24 hrs. The NSSE was filtered
from the liver due to impairment in hepatocytes (Trivedi et al.,
by using Whatman filter paper No.1 and at 37 °C dried in the incu-
2021). Whereas the kidney is involved in osmoregulation as well
bator. The resultant powder was kept at 4 °C for further use. The
as metabolic excretion of toxicants leading to histopathological
dose was prepared from NSSE by using body weight, 50 mg/ kg BW.
alterations (Suchana et al., 2021). Nigella sativa (NS) belongs to
the family Ranunculaceae (Bashir et al., 2021), a traditionally used
medicinal plant (Hannan et al., 2021). Nigella sativa seed oil is a 2.4. Preparation of Nigella sativa AgNPs
notable source of bioactive compounds, vital oils, fatty acids,
polyphenols, tocopherols, and phytosterols. Thymoquinone (TQ) Nigella sativa seed powder (5 g) was mixed with 100 mL of dis-
is the most important bioactive compound with lot of health- tilled water, and boiled for 20 min at 100 °C. 10 mL seed extract
promoting properties (Mazaheri et al., 2019). Black seeds possess was added with 0.1 mM solution of AgNO3 with constant stirring
a broad spectrum of activities including antihypertensive for 10 min at 25 °C. The mixture was incubated for the next 2 h
(Maideen et al., 2021), antidiabetic (Vijayakumar et al., 2021), at 80 °C. The reaction mixture was placed at 25 °C for 24 h in a dark
antimicrobial (Hossain et al., 2021), anticancer (Almatroudi et al., room. After 24 h, the mixture was turned from light brown to a
2020), diuretic (Aghamirei et al., 2022), immunomodulatory, dark brown color indicating the reduction of Ag+ ions. To remove
anti-inflammatory (Ikhsan et al., 2018), anti-schistosomiasis, anal- impurities mixture was washed with ethanol (twice) and distilled
gesics, antioxidant, gastroprotective, hepatoprotective (Almatroudi water thrice at 12000 rpm, the pellet contained NS AgNPs. Pre-
et al., 2020) and renal protective activities (Hannan et al., 2021). pared NS AgNPs were dried at 70 °C for 72 h. in over and stored
Various studies manifested the application of Nigella sativa in at 4 °C for future use (Chand et al., 2021). The dose was prepared
diverse fields, but meager reports exist to reveal the application of from Nigella sativa AgNPs by using body weight, 50 mg/ kg BW.
Nigella sativa against the Cr (VI) induced hepatotoxicity and renal tox-
icity. Recently, their scanty investigation found regarding the protec-
2.5. Experimental design
tive action of Nigella sativa against chromium caused hepatotoxicity
and renal toxicity in mice. Hence the current study was designed to
During the experiment, Mus musculus (male) of age 2 to
find out the protective potential of Nigella sativa against chromium
3 months, weight 30 to 40 g were kept as model animals. The
(VI) induced damage to the liver and kidney in male albino mice.
albino male mice were purchased from the animal house of the
Department of Zoology, Government College University, Lahore,
2. Materials and methods Pakistan. Mice received feed no. 14 and drinking water ad-
labium. The temperature in the animal house was maintained at
2.1 Paul### 23 ± 2 °C, humidity at 45–50 %, and 12 hrs. dark-light cycle.

The trial techniques for all animals were managed and regu-
2.6. Administration of dose
lated as per local and international controls. The protocol for the
present study was allowed by the institutional bio-ethical commit-
After 15 days of acclimatization, forty mice were divided into 8
tee of the Government College University, Lahore with reference
equal groups i.e., 5 mice each, including one control group and
No. GCU-IIB-364 dated 6th October 2020. All animal trials were
seven other treatment groups. The newly prepared solutions
executed according to local and worldwide procedures followed
(0.2 mL for every one mouse of each representative group) of
by the Wet op de dierproeven (article 9) of Dutch law (interna-
Nigella sativa seed extract and Nigella sativa mediated silver
tional) and an associated rule planned via the Bureau of Animal
nanoparticles at the dose rate of 50 mg/kg BW were administered
Research Licensing, Local University as detailed in our earlier
orally employing gavage once a day. Chromium solution 1.5 mg/kg
papers (Hussain et al., 2020; Ara et al., 2020; Ali et al., 2019a,
BW, orally from K2Cr2O7 was provided orally.
2019b, 2020; Khan et al., 2019; Mumtaz et al., 2019; Mughal
Control: Group I was continued to receive chromium-free
et al., 2019; Dar et al., 2019). The rearing and use of animal were
drinking water (ad-labitum) (60 days).
carried out using NIH Publication ‘‘Guide for the Care and Use of
Cr: Group II was continued to receive chromium 1.5 mg/kg BW,
Laboratory Animals” (NRC 2011) and by the local bioethical com-
via gauge once a day (60 days).
mittee of the University on animal experimentation.
NS: Group III was continued to receive Nigella sativa seed
extract, 50 mg/kg BW, via gauge once a day (60 days).
2.2. Chemicals used NS + NP: Group IV was continued to receive Nigella sativa
AgNPs, 50 mg/kg BW, via gauge once a day (60 days).
The tested chemical K2Cr2O7 with 99% purity was procured NS (P): Group V was continued to receive NS seed extract,
from Merck (Merck, Darmstad, Germany). Black seeds were used 50 mg/kg BW + Cr, 1.5 mg/kg BW, via gauge once a day (60 days).
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T. Nauroze, S. Ali, L. Kanwal et al. Saudi Journal of Biological Sciences 30 (2023) 103571

NS + NP (P): Group VI was continued to receive Nigella sativa 2.9. Biochemical parameters
AgNPs, 50 mg/kg BW + Cr, 1.5 mg/kg BW, via gauge once a day
(60 days). Blood was centrifuged to analyze biochemical parameters at
NS (T): Group VII was continued to receive Cr, 1.5 mg/kg BW 25 °C at 2000 rpm for 10 min to separate serum. The aliquots were
(30 days) followed by Nigella sativa seed extract, 50 mg/kg BW, stored at  20 °C till further analysis. Alanine aminotransferase
via gauge once a day (next 30 days). (ALT) (Helmy et al., 2019), aspartate aminotransferase (AST)
NS + NP (T): Group VIII was continued to receive Cr, 1.5 mg/kg (Helmy et al., 2019), alkaline phosphate (ALP) (Trapero et al.,
BW (30 days) followed by Nigella sativa AgNPs, 50 mg/kg BW, via 2018, creatinine (Khamis et al., 2018) and total protein (Yılmaz
gauge once a day (next 30 days) (Fig. 1). and Ergün, 2018) were measured by commercially accessible diag-
nostic kits as described in Table 1.

2.7. Collection of Samples

2.10. Histopathology of liver and kidney
The blood sample was collected by puncturing the vein from a
For histopathological analysis, the liver and kidney of each ani-
cephalic vein or jugular vein of every mouse on the 61st day of
mal of each group, affixed with 10% formalin were processed by
the experiment. The blood collected from the mouse was shifted
the traditional method using ascension grades of alcohol and
in EDTA coated 3 mL ImuMed vacutainer and stored at 4 °C for bio-
xylene, paraffin wax embedding followed by microtomy at 5 lm.
chemical analysis. Blood was centrifuged to analyze biochemical
The tissue sections were stained by hematoxylin and eosin (H&E)
parameters at 25 °C for 10 min at 2000 rpm to separate serum.
staining for microscopic examination. The liver damage was stan-
Under deep anesthesia liver and both kidneys were removed via
dardized by steatosis, necrosis, hepatocyte degeneration, and
a long abdominal incision in the middle part. Samples were pre-
micrometric analysis. Moreover, kidney criteria were steatosis,
served in formalin for later use.
necrosis, regenerating nephron, fibrosis, nuclear fragmentation &
condensation, and micrometric analysis (Khalaf et al., 2020).

2.8. General observations

2.11. Micrometric analysis
Behavioral modifications in mice for all groups were observed
and recorded on regular basis. Each animal from all groups was The micrometrical observations recorded include the size of
weighed weekly, and the dose was administered accordingly. Sim- hepatocytes and their nuclei and brush border. ACSA of Bowman’s
ilarly, the water consumption in terms of per gram body weight capsule, glomerulus, and lumen of proximal convoluted tubule
was obtained on daily basis. Liver and kidney weight were noted were measured. All the data was put into a graph pad prism. The
for each animal after collection. data was obtained from photographs at 40X from a microscope.

Fig. 1. Experimental scheme of study.

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T. Nauroze, S. Ali, L. Kanwal et al. Saudi Journal of Biological Sciences 30 (2023) 103571

Table 1
Layout for kits used in current study.

Sr No Biochemical parameter Particulars of Kit Reference

1 Alanine aminotransferase (ALT) ALT diagnostic kit (Helmy et al., 2019)
2 Aspartate aminotransferase (AST) AST diagnostic kit (Helmy et al., 2019)
3 Alkaline phosphate (ALP) Commercial double-antibody sandwich ELISA kits (Trapero et al., 2018)
4 Malondialdehyde (MDA) Commercial Photometry diagnostic kits (Teb Pazhouhan Razi (TPR), Tehran, Iran) (Amri et al., 2021)
5 Total protein Commercial test kits (Bioanalytic Diagnostic Industry, Germany) (Yılmaz and Ergün, 2018)
6 Creatinine QuantiChromTM CRNN assay kit (QC, CA) (Khamis et al., 2018)

Following formula was used to calculate the CSA in each case Cr groups. In treatment groups, ALT level was reduced significantly
(Ahmad et al., 2012): in NS (P) (92 ± 4.4 U/L), NS + NP (P) (80.4 ± 3.6 U/L), NS (T)
(121.8 ± 3.3 U/L), and NS + NP (T) (107.8 ± 3.5 U/L) (Fig. 3).
ðlength  widthÞ
CSA ¼ p AST level was significantly elevated in all Cr exposed animals
4 (484 ± 8.1 U/L) as compared to control (84.2 ± 1.5 U/L)
(P
0.05). Administration of 50 mg/kg BW dose of NSSE and Nigella
2.12. Data analysis sativa AgNPs significantly inhibited the level of AST as compared to
Cr groups. In treatment groups, AST level was reduced significantly
Data obtained were analyzed and presented as mean ± SEM. in NS (P) (188.4 ± 10.8 U/L), NS + NP (P) (145 ± 10.6 U/L), NS (T)
Kolmogorov–Smirnov test was used to evaluate the normality of (214.6 ± 13.9 U/L), and NS + NP (T) (200.2 ± 11.8 U/L) (Fig. 3).
the data and then statistically analyzed by one-way ANOVA, with ALP level was significantly elevated in all Cr exposed animals
Dunnett’s multiple comparison test, to find any significant differ- (337.6 ± 7.2 U/L) as compared to control (106.2 ± 4.9 U/L)
ence among the means of treatment groups. GraphPad Prism ver- (P
0.05). Administration of 50 mg/kg BW dose of NSSE and Nigella
sion 5.0 for Windows (GraphPad Software, San Diego, CA, USA) sativa AgNPs significantly inhibited the level of ALP as compared to
was employed for data analysis. The P
0.05 were observed as sig- Cr groups. In treatment groups ALP level was reduced significantly
nificant values. in NS(P) (189.6 ± 15.6 U/L), NS + NP(P) (187.2 ± 8.8 U/L), NS (T)
(245.8 ± 10.1 U/L) and NS + NP (T) (194.8 ± 7.1 U/L) (Fig. 3).
MDA level was significantly elevated in all Cr exposed animals
3. Results
(641.2 ± 22.1 U/L) as compared to control (158 ± 3.7 U/L)
(P
0.05). Administration of 50 mg/kg BW dose of NSSE and Nigella
3.1. Water intake
sativa AgNPs significantly decreased the level of MDA as compared
to Cr groups. In treatment groups, MDA level was reduced signifi-
Water intake was significantly different among different
cantly in NS (P) (288 ± 61.9 U/L), NS + NP (P) (253 ± 9.3 U/L), NS (T)
groups. In Cr treated group water intake was significantly
(348 ± 41.2 U/L), and NS + NP (T) (349.6 ± 31.7 U/L) (Fig. 3).
increased (0.11782 ± 0.02222 mL/g/day) as compared to control
Total protein level was significantly decreased in all Cr exposed
(0.09024 ± 0.01412 mL/g/day) (P
0.05). Administration of
animals (2.94 ± 0.2 g/dL) in comparison to control (8.42 ± 0.5 g/dL)
50 mg/kg BW dose of NSSE and Nigella sativa AgNPs decreased
(P
0.05). Administration of 50 mg/kg BW dose of NSSE and Nigella
the water intake as compared to Cr treated group. In treatment
sativa AgNPs significantly elevated the level of total proteins as
groups, water intake was reduced significantly in NS (P) (0.06935
compared to Cr groups. In treatment groups total proteins level
± 0.01357 mL/g/day) and NS (T) (0.06807 ± 0.01364 mL/g/day).
was improved significantly in NS (P) (6.16 ± 0.5 g/dL), NS + NP
However, in NS + NP (P) (0.09067 ± 0.01660 mL/g/day) and
(P) (7.96 ± 0.3 g/dL), NS (T) (4.36 ± 0.2 g/dL) and NS + NP (T) (5.2
NS + NP (T) (0.11310 ± 0.02333 mL/g/day) reduction was not up
8 ± 0.4 g/dL) (Fig. 3).
to significant level (Fig. 2).
Creatinine level was significantly increased in all Cr exposed
animals (4.0 ± 0.1 mg/dL) in comparison control (0.8 ± 0.0 mg/d
3.2. Physiological parameters
L) (P
0.05). Administration of 50 mg/kg BW dose of NSSE and
Nigella sativa AgNPs significantly decreased the level of creatinine
During the study, aggressive behavior was observed in chro-
mium exposed mice that deceased over time. However, no signs
of mortality were observed. An increase in hepatosomatic index
(0.098 ± 0.13 g) up to a significant level (p < 0.05) was observed.
However, in renal somatic index no significant difference was
observed in chromium groups vs. treatment groups after 60 days
(Table 2).

3.3. Biochemical parameters

The values of biochemical parameters (ALT, AST, ALP, MDA,

total protein, and creatinine) were measured after 60 days of expo-
sure to 1.5 mg/kg BW Cr and other treatments including NSSE and
Nigella sativa AgNPs. Continuous Cr intake exerted negative effects
on redox balance and treatment of NS and NS + NP modulated the Fig. 2. Water intake measurement in experimental groups, Con; Control; Cr:
toxic effects significantly. chromium treated; NS: NSSE treated; NS + NP: Nigella sativa AgNPs treated; P:
ALT level was significantly elevated in all Cr exposed animals prevention; T: treatment. Different letters showing significant different of treat-
ment groups with that of Cr treated group. Results represented in term of
(163 ± 4.1 U/L) as compared to control (42.4 ± 1.9 U/L) mean ± SEM. P
0.05. Different letters showing significant different of treatment
(P
0.05). Administration of 50 mg/kg BW dose of NSSE and Nigella groups with that of Cr treated group. Statistical icons: a,b,c,d,e,f,g = P
0.05. aa,bb,
sativa AgNPs significantly inhibited the level of ALT as compared to cc,dd,ee,ff,gg = P
0.01. aaa,bbb,ccc,ddd,eee,fff,ggg = P
0.001.

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T. Nauroze, S. Ali, L. Kanwal et al. Saudi Journal of Biological Sciences 30 (2023) 103571

Table 2
Comparison in hepatosomatic index (HIS) and renal somatic index (RSI) of treatment groups.

Treatment groups
Organ Con Cr NS NS + NP NS (P) NS + NP (P) NS (T) NS + NP (T)
HIS (g) 0.05 ± 0.07 0.098 ± 0.13aaa 0.0577 ± 0.11bbb 0.0493 ± 0.041ccc 0.0474 ± 0.031ddd 0.0548 ± 0.0307eee 0.0545±0.0793fff 0.047 ± 0.03ggg
RSI (g) 0.01 ± 0.02 0.02 ± 0.06 0.02 ± 0.004 0.015 ± 0.007 0.015 ± 0.01 0.01616 ± 0.0246 0.01551 ± 0.0244 0.015 ± 0.0184

Con; Control; Cr: chromium treated; NS: NSSE treated; NS + NP: Nigella sativa AgNPs treated; P: prevention; T: treatment. Different letters showing significant different of
treatment groups with that of Cr treated group. Results represented in term of mean ± SEM. P
0.05. Different letters showing significant different of treatment groups with
that of Cr treated group. Statistical icons: a,b,c,d,e,f,g = P
0.05. aa,bb,cc,dd,ee,ff,gg = P
0.01. aaa,bbb,ccc,ddd,eee,fff,ggg = P
0.001.

Fig. 3. Analysis of ALT, AST, ALP, MDA, Total Protein and Creatinine measurement in experimental groups. Con; Control; Cr: chromium treated; NS: NSSE treated; NS + NP:
Nigella sativa AgNPs treated; P: prevention; T: treatment. Results represented in term of mean ± SEM. P
0.05. Different letters showing significant different of treatment
groups with that of Cr treated group. Statistical icons: a,b,c,d,e,f,g = P
0.05. aa,bb,cc,dd,ee,ff,gg = P
0.01. aaa, bbb, ccc, ddd,eee,fff,ggg = P
0.001.

as compared to Cr groups. In treatment groups creatinine level was nuclei of hepatocytes, derangement in hepatic cords and abnormal
reduced significantly in NS (P) (2.0 ± 0.1 mg/dL), NS + NP (P) (1.7 sinusoids, microvesicles, and microvesicles in hepatocytes indicat-
± 0.1 mg/dL), NS (T) (2.8 ± 0.1 mg/dL) and NS + NP (T) (2.3 ± 0.2 ing steatosis, massive vacuolar degeneration of hepatocytes with
mg/dL) (Fig. 3). necrosis, a large number of Kupfer cells (Fig. 4) and bile duct meta-
plasia were the notable features (Supplementary Fig. 1). The differ-
3.4. Histopathology of liver and kidney ent tissue section of kidney in the Cr group represented numerous
histological alterations including degeneration of renal tubules,
Histological slides of liver and kidney tissue of control groups glomerular degeneration and disorganized bowman’s capsule,
showed well-organized histological structures including hepato- necrosis (Fig. 5), fatty cell infiltration and microvesicles indicating
cytes, hepatic cords, central vein, glomerulus was encircled by steatosis, fibrocytes infiltration and fibrosis in interstitial tissues,
bowman’s capsule, proximal convoluted tubules were with brush nuclear fragmentation, and condensation, eccentric nuclei, and
border, however distal convoluted tubule with intact endothelium. vacuolation (Fig. 6) were major features observed. However, in
In Cr treated group abnormalities in liver and kidney sections were the treatment groups, liver and kidney structures recovered
observed. During the microscopic examination, different alter- significantly.
ations were found including nuclear fragmentation, atrophid
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T. Nauroze, S. Ali, L. Kanwal et al. Saudi Journal of Biological Sciences 30 (2023) 103571

Fig. 4. Histopathology of Liver (40X) A: control; B: Cr; C: NS; D: NS + NP; E: NS (P); Fig. 5. Histopathology of Kidney (40X). Yellow arrows: Bowman’s capsule: black
F: N + NP (P); G: NS (T); H: N + NP (T) treated groups. CV: central vein, Yellow arrow: glomerulus: orange arrow: distal convoluted tubule, blue arrow: proximal
arrows: sinusoids, purple arrow: normal hepatocytes, white arrow: kupfer cells: convoluted tubule, white arrow: micro vesicles and eccentric nucleus, black arrow:
black arrow: necrosis, red arrow: micro vesicles in hepatocytes, green arrow: distorted glomerulus, green arrow: necrosis.
degenerating hepatocytes, blue arrows: hepatic cords.
was not up to significant level (Fig. 8). In glomerulus size was also
reduced in treatment groups NS (P) (6291.00 ± 631.46 l2), NS + NP
(P) (5676.29 ± 326.25 l2) and NS (T) (6070.99 ± 213.25 l2), how-
3.5. Micrometry of liver and kidney

The micrometric analysis of the liver includes the size of hepa-

tocytes and size of hepatocytes nucleus in Fig. 7. Whereas kidney
micrometric analysis includes measurement of ACSA of Bowman’s
capsule, ACSA of the glomerulus, ACSA of the lumen of proximal
convoluted tubule, size of brush border in Fig. 7. A significant
increase in the size of hepatocytes and their nucleus were observed
in Cr treated groups 1188.191 ± 467.698 l2 and 456.419 ± 206.739
l2 (P
0.05) as compared to control (836.778 ± 223.984 l2, 254.
269 ± 105.453 l2) respectively. With the administration of
50 mg/kg BW dose of Nigella sativa seed extract and Nigella sativa
AgNPs, the size of hepatocytes was reduced up to a significant level
in treatment groups including NS (P) (631.595 ± 155.019 l2),
NS + NP (P) (669.454 ± 165.959 l2), NS (T) (707.928 ± 233.903
l2) and NS + NP (T) (707.928 ± 707.928 l2) up to significant level
(Fig. 7).
A significant increase in bowman’s capsule and glomerulus was
observed in Cr treated groups 11835.47 ± 336.66 l2 and 9051.82
± 249.81 l2 (P
0.05) as compared to control (9106.81 ± 522.18 Fig. 6. Selected regions of the histological section of Cr (VI) treated kidney showing
l2,7136.26 ± 261.61 l2) respectively. Administration of 50 mg/kg histopathology (40X). Blue arrows: bowman’s capsule: yellow arrow: atrophy of
BW dose of Nigella sativa seed extract and Nigella sativa AgNPs, glomerulus: white arrow: fibrocytes, dark green arrow: fibrosis in interstitial
the size of Bowman’s capsule was reduced up to the significant tissues, light green arrow: micro vesicles and nucleus disappeared, black arrow:
macrovesivles, green arrow: necrosis, light blue arrow: brush border, dark blue
level in treatment groups including NS (P) (7481.91 ± 455.10 l2), arrow: necrosis, red arrow: nuclear fragmentation and condensation, dark pink
NS + NP (P) (9304.56 ± 658.89 l2) and NS (T) (7128.01 ± 320.03 arrow: eccentric nucleus, light pink arrow: vacuolation, orange arrow: mage
l2), however in NS + NP (T) (10586.26 ± 871.53 l2) reduction nucleus.

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T. Nauroze, S. Ali, L. Kanwal et al. Saudi Journal of Biological Sciences 30 (2023) 103571

There was an inexplicable change in the body weight with Cr

(VI) administration (Shil and Pal, 2019) whereas the weight of
the liver was increased (El-Demerdash et al., 2021). According to
already published data Cr (VI) caused lesions, chronic inflamma-
tion, an increase in Kupfer cells, and fatty degeneration resulting
change in the weight of the liver (Suljević et al., 2021), resulting
in high HIS whereas RSI was not changed up to the detectable level
when compared to control. However, mice treated with NSSE
showed a detectable recovery in liver weight. It is established that
Cr (VI) toxicity causes oxidative stress and cellular damage
(Awasthi et al., 2018). The liver is involved in detoxification, Cr
(VI) exposure induces ROS synthesis to result in oxidative damage
that is visible in liver tissue sections (Yang et al., 2022). Liver dys-
trophy and inflammation were observed, and hepatocytes with fat
vesicles were related to poor regulation of lipid reserves due to
toxicity. As similar to previous studies hepatocytes atrophy,
nuclear condensation and fragmentation, derangement of hepatic
cords, and lateral side arrangement of the nucleus were seen in tis-
sue section that may be due to deposition of lipids and glycogen
precipitation. Necrosis in hepatocytes was because of the increased
number of neutrophils and lymphocytes (Buchko and Havryliak,
2021). With the administration of NSSE and biosynthesized AgNP
in the present study, the liver structure was improved.
Evaluation of the kidney functions had been made in the cur-
Fig. 7. Analysis of Hepatocyte and their nucleus size measurement in experimental rent study by estimating ALT, AST, ALP, and total protein. In the
groups. Con; Control; Cr: chromium treated; NS: NSSE treated; NS + NP: Nigella
current study the elevated level of ALT, AST, and ALP can be attrib-
sativa AgNPs treated; P: prevention; T: treatment. Results represented in term of
mean ± SEM. P
0.05. Different letters showing significant different of treatment uted to a high level of ROS and pointed out liver damage that mod-
groups with that of Cr treated group. Statistical icons: a,b,c,d,e,f,g = P
0.05. aa,bb, ified the membrane permeability and transport function along
cc,dd,ee,ff,gg = P
0.01. aaa, bbb, ccc, ddd,eee,fff,ggg = P
0.001. with the outflow of enzymes from hepatocytes into the blood-
stream a clear indication of hepatotoxicity (El-Demerdash et al.,
2021). With the administration of NSSE levels of these enzymes
ever in NS + NP (T) (7951.64 ± 1134.21 l2) reduction was not up to were reduced (Mazhar, 2022) due to the action of TQ (Fadishei
significant level. et al., 2021). Cr (VI) decreases the protein level due to impaired
In the glomerulus, size was also reduced in treatment groups NS protein synthesis machinery, most probably due to bioaccumula-
(P) (20.249 ± 3.130 l2), NS + NP (P) (19.324 ± 4.897 l2), NS (T) (19. tion of Cr (VI) (Trivedi et al., 2021), NSSE and biosynthesized AgNP
070 ± 8.116 l2), and NS + NP (T) (17.819 ± 3.766 l2) up to signif- treatment improved the level of total proteins.
icant level (Fig. 8). The kidney is the vital target organ for the accumulation of Cr
The lumen of the proximal convoluted tubule was also not (VI), and the elimination of toxicant (Kakade et al., 2020) reactive
changed up to the significant level in chromium treated group species synthesized by Cr (VI) effect cell membrane lipids and
(1049.63 ± 168.57 l2) as compared to the control group (841.85 ± induce peroxidation of unsaturated fatty acids (Suljević et al.,
114.96 l2) (p > 0.05) (Fig. 8). In the proximal convoluted tubule, 2021) and necrosis in kidney tissue (Yin et al., 2019). Upon expo-
the brush border decreased in Cr group and changed up to a signif- sure to Cr (VI), massive destruction was found in the renal cortex
icant level in chromium treated group (10.070 ± 3.031 l) as com- including fatty cell infiltration representing lipid accumulation
pared to the control group (20.086 ± 2.593 l) (Fig. 8). due to ROS species (Hanan et al., 2019). Regarding renal filtration,
in our study, there were inconsistent forms of histopathological
changes were observed such as glomerulus degeneration, necrosis,
4. Discussion fibrosis, sloughing of tubular epithelial cells, and cytoplasmic vac-
uolation. According to previous studies, cytoplasmic vacuolation is
Chromium compounds switch their oxidative state and do not the job of reactive species that aid the discharge of lysosomal
withdraw into the ecosystem. Cr (VI) in comparison to Cr (III) is enzymes and upcoming protein oxidation of the cell, convert them
more toxic and easily passes through cell membranes with the into fragments, and set apart them into vacuoles as part of the cel-
aid of anion carriers (Xueting et al., 2018). The current study on lular defense mechanism, protecting the interference with cell
albino mice aimed to evaluate whether Nigella sativa seed extract metabolism (Sorour and Abd-Elgalil, 2019). As nephritis in the
and Nigella sativa AgNPs supplementation can turn down the tubule and glomerulus damage are linked with irregular flow of
effects of Cr (VI) toxicity (by examining the level of ALT, AST, the glomerular filtrate. The slow or blocked condition of glomeru-
ALP, MDA, total protein, creatinine, and histopathology of liver lar filtrate results in shrinkage of the glomerulus (Hanan et al.,
and kidney). We observed that Nigella sativa seed extract and 2019). In Cr treated animals water consumption was maximum,
Nigella sativa AgNPs supplementation played a protecting role by as ammonia execration requires more water and animals drink
turning down the Cr (VI) induced toxicity in the liver and kidney. more water compared to other groups. This indicates reno-
Although Nigella sativa is a top-ranked herbal medicine with mirac- hepatic toxicity (Abbas et al., 2016). With the administration of
ulous healing power (Kazmi et al., 2019) effective in various NSSE in the present study, the architecture of the renal cortex
chronic diseases (Rashidmayvan et al., 2019). This is the foremost was improved (Baloch et al., 2020) and water consumption was
study on the ameliorative potential of Nigella sativa seed extract reduced as observed in our study.
and Nigella sativa AgNPs on water intake HIS, RSI, ALT, AST, ALP, Evaluation of the kidney functions had been made in the cur-
MDA, creatinine, and histopathology of liver and kidney against rent study by estimating MDA and creatinine. In our study Cr
Cr (VI). That’s why data is not sufficiently available for comparison. (VI) exposure elevated the level of MDA and creatinine in serum.
7
T. Nauroze, S. Ali, L. Kanwal et al. Saudi Journal of Biological Sciences 30 (2023) 103571

Fig. 8. Analysis of ACSA of Bowman’s capsule, glomerulus, lumen of proximal convoluted tubule and brush border size measurement in experimental groups. Con; Control;
Cr: chromium treated; NS: NSSE treated; NS + NP: Nigella sativa AgNPs treated; P: prevention; T: treatment. Results represented in term of mean ± SEM. P
0.05. Different
letters showing significant different of treatment groups with that of Cr treated group. Statistical icons: a,b,c,d,e,f,g = P
0.05. aa,bb,cc,dd,ee,ff,gg = P
0.01. aaa, bbb, ccc,
ddd,eee,fff,ggg = P
0.001.

MDA is the biomarker for lipid peroxidation and protein damage biochemical parameters, and histological alterations in the liver
results in an elevated level of MDA in serum (Muller et al., 2022). and kidney. Furthermore, Nigella sativa supplementations to chro-
MDA, the final product of lipid peroxidation, used as indicator for mium Cr (VI) treated mice modulated oxidative damage and reha-
liver injury (Selamoğlu et al., 2014). Serum creatinine level indi- bilitate the disturbance in the majority of the measured
cates acute tubular necrosis caused by renal toxicity parameters. Its effect is noticeable in the prevention group. So,
(Alsuhaibani, 2018) and also provides a rough estimate of the rate Nigella sativa had strong antioxidant potential in amelioration of
of glomerular filtration (Franca and Prince, 2019), the glomerulus Cr (VI) toxicity by reduction of free radicals synthesis and enhanc-
injury leads to a reduction in the diffusion process by thickening ing the antioxidant defense system; additionally, our data reinforce
of capillary basement membrane resulting slower rate of filtration the use of Nigella sativa as a hepatic protective and renal protective
(Deshmukh and Manjalkar, 2021) and NSSE and biosynthesized natural-born product.
AgNP administration reduced level of creatinine.
As per the micrometric analysis of the liver showed in Cr group Declaration of Competing Interest
the size of hepatocytes and their nucleus were increased may be
due to disruption of nuclear function, which might be due to pro- The authors declare that they have no known competing finan-
tein DNA interaction and transcriptional activity of hepatocytes cial interests or personal relationships that could have appeared
(Fedchenko et al., 2022). In the kidney, an increase in glomerular to influence the work reported in this paper.
size increase may be due to glomerulus damage, and crescent for-
mation (Bouteldja et al., 2021) may increase in the size of the Bow- Appendix A. Supplementary material
man’s capsule. In NSSE and biosynthesized AgNP treated groups
size were significantly reduced. Supplementary data to this article can be found online at
Our results indicated that the administration of Nigella sativa https://doi.org/10.1016/j.sjbs.2023.103571.
and Nigella sativa mediated AgNPs in mice produced marked mod-
ulatory effects against chromium (VI) and caused hepatic, renal,
and biochemical alterations. Based on the results, Nigella sativa
and Nigella sativa mediated AgNPs seems to have an ameliorative References
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