Eid et al., 2018, AJMS 1(2):25-30 , DOI:10.5455/ajms.

12

Arabian Journal of Medical Sciences

http://www.ajms.tk

The potential ameliorative effects of morin and rutin against titanium dioxide
nanoparticles-induced hepatotoxicity in rats
Reham S. Eid a*, Mohamed H. Sherif a, Al-Shimaa M. Abas a, Mohamed M. Hussein b
a
Department of Chemistry, Faculty of Science, Zagazig University, Egypt
b
Department of Biochemistry, Faculty of Veterinary Medicine, Zagazig University, Egypt

Article Info Abstract

Background: Titanium dioxide nanoparticles (TiO2 NPs) are well known as a photocatalyst and are widely
used for photocatalysed decomposition, sensor elements, water purification and have many other applications,
however they also have toxic effect on animals exposed to them. Objectives: This study was conducted to
Keywords: investigate the potential ameliorative effect for the two potent antioxidant flavonoids morin and rutin against
Titanium dioxide nanoparticles;
TiO2 NPs-induced toxicities in rat liver. Materials and methods: Rats (n =70, weight 150-200g) were divided
Morin;
into seven groups. Group (G1) (control), G2 (treated with morin 30 mg/kg), G3 (treated with rutin 80 mg/kg),
Rutin;
G4 (fed on TiO2 NPs 100 mg/kg), G5 (TiO2 NPs+ morin), G6 (TiO2 NPs+ rutin), G7 (TiO2 NPs+ morin+
Antioxidant;
rutin). The evaluated parameters included liver damages enzymes [alanine aminotranseferase (ALT), aspartate
Hepatotoxicity
aminotranseferase (AST), alkaline phosphatase (ALP)], total protein and albumin, minerals and electrolytes
[potassium (K), phosphorous (P), sodium (Na), calcium (Ca)], antioxidant enzymes [superoxide dismutase (SOD),
catalase (CAT)], glutathione GSH and the lipid peroxidation marker malondialdehyde (MDA) in addition to DNA
Received Oct 26,
damage by comet assay and histopathological examination of liver tissues. Results: Rats administrated TiO2 NPs
Revised Nov 23,
Published Dec 7, 2018 showed hepatic toxicity as evidenced by: 1) higher serum levels of liver damage enzymes, 2) lower serum levels
of total protein and albumin, 3) higher serum levels of K and P, 4) lower serum levels of Na and Ca, 5) higher
oxidative stress as indicated by lower GSH content and antioxidant enzymes (SOD, CAT) activities and higher
MDA levels, 6) higher DNA damage of hepatocytes, and 7) higher degree of disrupted liver histopathology. All
*Corresponding author: these deteriorated parameters were reversed following administration of morin and/or rutin with best improvement
reham_soliman88@yahoo.com in the combined group. Conclusion: Treatment with the two potent antioxidant flavonoids morin and rutin
could ameliorate TiO2 NPs-induced hepatotoxicity in rats through, at least in part, inhibition of oxidative stress.

1. Introduction
Nanotechnology is considered as a highly hopeful technology in
the field of molecular science that may utilize in medical, agriculture
industrial, manufacturing and military sectors (Robertson et al., 2010
and Kisin et al., 2007). The titanium dioxide nanoparticles (TiO2
NPs) used in paints, plastics, papers, cosmetics and skin care prod-
ucts such as sunscreen lotion as they spread out visible light less than
TiO2 pigments, while still providing UV protection and cause less
skin irritation than other UV absorbing chemicals (Brunet et al., 2009
and Winkler and Jochen 2003). Although these NPs have anticancer
effect against some cancer cells such as human hepatoma cells (Wang
et al., 2007), they can also damage the normal (healthy) cells after
exposure via oral and respiratory route (Cui et al., 2010).
Morin, as a natural flavonoid of Moraceae family, is abundantly
found in different herbs and fruits such as onion, seed weeds, mill,
fig, almond, red wine and osage orange (Khaki et al., 2010; Nandha-
kumar et al., 2012; Sreedharan et al., 2009). Several pharmacologi-
cal studied revealed that morin had antioxidant, anti-inflammatory,
chemoprotective, and anticancer properties (Kuo et al., 2007). Rutin,
a citrus flavonoid glycoside present mainly in buckwheat, also has
antioxidant, antiallergic, anti-inflammatory, antiangiogenic, antiviral
properties (Guruvayoorappan and Kuttan, 2007). Rutin was used in
many pharmaceutical preparations as an adjuvant for anticancer, anti-
diarrhoeal and myocardial protective drugs (Pozin et al., 1996)
The purpose of the present study was to investigate the possible
protective effect of naturally occurring antioxidants (morin and rutin)
on TiO2 NPs-induced liver toxicity in rats via determination of liver
25
damages enzymes, minerals and electrolytes, antioxidant enzymes,
GSH, MDA, DNA damage (by comet assay) and histopathological
examination of liver tissues.
2. Materials and Methods
2.1. Chemicals
Morin, rutin, and TiO2 NPs were purchased from Sigma-Al-
drich Chemical Co. (St Louis, Missouri, USA). TiO2 NPs were
supplied as a mixture of rutile and anatase white nanopowder with
particle size <100 nm and purity 99.5 %. The phase characterization
of TiO2 NPs was performed by X-ray diffraction (XRD, data not
shown).
2.2. Experimental design
Male Sprague-Dawley rats (n = 70, weight = 150-200 g) were
housed in separate metal cages with fresh drinking water supplied
ad-libtium. Rats were kept at constant environmental and nutri-
tional condition throughout the period of experiment. The animals
were left for 14 days for acclimatization before the beginning of the
experiment. The experimental study was approved by the Animal
Ethical Committee of Faculty of Veterinary Medicine, Zagazig, and
was in accordance with its rules.
Animals were divided into the following seven groups (10 rats
per group): group 1 (G1), rats were given normal saline (as a vehicle
to morin, rutin, and TiO2 NPs); G2, rats were given morin (30 mg/
kg body weight, bw) (El-Mahalaway et al., 2013); G3, rats were
 Eid et al., 2018, AJMS 1(2):25-30 , DOI:10.5455/ajms.12
administrated rutin (80 mg/kg bw) (Mirani et al., 2012); G4, rats were
treated with TiO2 NPs (100 mg/kg bw) (Vasantharaja et al., 2014);
G5, rats were treated with TiO2 NPs (100 mg/kg bw) for 4 weeks
followed by morin (30 mg/kg bw) for 4 weeks; G6, rats were given
(100 mg/kg bw)TiO2 NPs and rutin (80 mg/kg bw) for 4 weeks; and
G7, rats were treated with TiO2 NPs (100 mg/kg b.wt/day), morin (30
mg/kg b.wt/day) and rutin (80 mg/kg b.wt/day) using the same doses
and routes of previous groups. Morin, rutin, and TiO2 NPs were sep-
arately dissolved in saline (vehicle) and each rat was given 1ml daily
by stomach gavage for 4 weeks.
2.3. Sampling
Blood samples were collected by capillary puncture to the re-
tero-orbital venous plexus at the medial canthus of the eye. Clean
serum was separated by centrifugation at 3000 rpm for 15 m and
was used for biochemical assays. Rats were killed by cervical de-
capitation and livers were dissected out, rinsed in cold physiological
isotonic saline (0.9% NaCl) to remove contaminated blood then were
divided into 3 parts. The first part was used to prepare liver homoge-
nate by centrifugation at 3000 rpm for 15 m in the presence of phos-
phate buffer saline (PBS) and the clear supernatant was obtained for
determination of MDA and GSH level, and SOD, CAT activities. The
second part was stored -80 deep freezer for determination of DNA
damage using comet assay and for extraction of RNA. The third part
was cut and fixed in 10 % buffered neutral formalin solution for his-
topathological examination.
2.4. Biochemical Analysis
Serum alanine transaminase (ALT), aspartate transaminase
(AST), and alkaline phosphatase (ALP) were determined using com-
mercially available kits. Serum total protein, albumin and bilirubin
were determined according to the method of Doumas et al., (1975),
Doumas et al., (1971), and Kaplan, (1984), respectively. Serum cal-
cium, phosphorous, potassium, and sodium ions were determined
according to the method described by Berry (1989), Farrell (1984),
and Henry (1974).
The lipid peroxidation marker MDA level, catalase (CAT), and
superoxide dismutase (SOD) activities and GSH content were deter-
mined using commercially available kits (Bio-diagnostic, Egypt) and
as previously described (kakkar et al., 1984; Moron et al., 1979;
Ohkawa et al., 1979; Sinha, 1972).
2.5. Detection of DNA fragmentation by comet assay
The comet assay was performed in Animal Reproductive Re-
search Institute (ARRI) of Agricultural Research Centre of Ministry
of Agriculture and Land Reclamation, Cairo, Egypt, according to the
method of (Singh et al., 1988).
2.6 Histopathological examination
Liver tissue samples were embedded in paraffin wax, sectioned
(4–5 µm), stained with hematoxylin and eosin (Suvarna et al., 2013),
and examined by light microscope.
2.7. Statistical analysis
All data were expressed as means ± standard error of mean
(SEM). The statistical significance was evaluated by one-way anal-
ysis of variance (ANOVA) using SPSS, 18.0 software, 2011 and
individual comparisons were obtained by Duncan’s multiple range
test (DMRT). Values were considered statistically significant when
p<0.05.
26
3. Results and discussion
3.1 Effect of morin and/or rutin administration on serum
biochemical parameters in TiO2 NPs-intoxicated rats
Administration of TiO2 NPs (G4) led to a significant increase
in serum levels of liver damage markers (ALT, AST, ALP, total and
direct bilirubin) as compared to control groups (G1-G3) (Table 1).
This indicates liver damage in G4 rats and suggests possible hepat-
ic toxicity induced by TiO2 NPs. In agreement with our findings,
Liu et al., (2009); Duan et al., (2010) and Jia et al., (2017) reported
that high doses of TiO2 NPs significantly increased liver damage
markers. In contrast, administration of morin (G5) and rutin (G6)
each alone or in combination (G7) led to a significant decrease of
these markers (Table 1). This suggests a possible ameliorative effect
to these two flavonoids against liver toxicity induced by TiO2 NPs.
Morin had been shown to decrease liver damage, prevent the escape
of liver enzymes through maintaining of cell membranes integrity
(Anbu and Saravanan, 2013; Bhakuni et al., 2017).
Administration of TiO2 NPs (G4) resulted in a significant de-
crease in serum levels of total protein and albumin as compared to
control groups (G1-G3) (Table 1). This also indicates liver damage
and loss of liver function. Again this toxic effect was reversed after
giving morin and/or rutin. Similarly, rutin improved the liver dam-
age as revealed by reduction of total protein and albumin level in
serum of intoxicated rats (Khan et al., 2012).
A significant increase in serum K and P and a significant de-
crease in serum Na and Ca were observed in TiO2NPs-treated group
(G4) as compared to the controls (G1-G3, Table 1). Administration
of morin and/or rutin with TiO2NPs caused a significant decrease
in serum K and P and a significant increase in serum Na and Ca
as compared to G4 (Table 1). In support to our findings, Canli and
Canli, (2017) reported that administration of TiO2 NPs led to a sig-
nificant decrease in Na, K-ATPase activity and a significant increase
in Ca-ATPase activity. Additionally, Khattab, (2012) also found that
morin can significantly increase Na and decrease K. Rutin-treat-
ed groups exhibited a significant increased Na concentration after
doxorubicin treatment (Parabathina et al., 2011).
3.2. Effect of morin and/ or rutin administration on liver
oxidative stress/antioxidant in TiO2 NPs-treated rats
Rats treated with TiO2 NPs exhibited a significant increase in
liver content of MDA and a significant decrease in GSH level and
the antioxidant activities of SOD and CAT as compared to con-
trol groups (G1-G3) (Fig. 1). However, treatment with rutin and/
or morin ameliorated this adverse effect. Similarly, previous studies
o reported similar toxic effect for TiO2 NPs on rat liver (Eman et
al., 2015; Mahboob et al., 2017). Moreover, Anbu and Saravanan,
(2013) and Bhakuni et al., (2017) also found that treatment with
morin showed significantly (p<0.05) attenuated lipid peroxidation
and restored levels of antioxidant enzyme glutathione. Similarly,
administration of rutin led to a significant elevation in SOD, CAT
activities and GSH level and a significant reduction in MDA level
(Magalingam et al., 2013). Taken together, TiO2 NPs could induce
hepatic toxicity through. at least in part. increasing oxidative stress
and liberation of free radicals and reducing activities of endogenous
antioxidant enzymes such as SOD and CAT with further reduction of
GSH content. However, treatment with rutin and/or morin can ame-
liorate this adverse effect, thereby relieving TiO2 NPs-induced hep-
atotoxicity. This ameliorative effect may be attributed at least in part
to the potent antioxidant activities of rutin and morin constituents.
 Eid et al., 2018, AJMS 1(2):25-30 , DOI:10.5455/ajms.12

Table 1. Effect of morin and/or rutin administration on some serum biochemical parameters and minerals in TiO2 NPs intoxicated rats.
Parameter Group1 Group2 Group3 Group4 Gprou5 Group6 Group7

ALT (U/L) 19.07±0.79 de 42.00±1.28 b 40.82±1.26 b 95.50±2.52 a 25.00±0.67 cd 25.82±1.20 c 16.12±0.70 e

AST (U/L) 16.75±1.29 d 23.95±1.73 cd 25.02±2.00 c 102.97±2.39 a 37.62±1.16 b 39.62±1.22 b 19.10±1.42 cd

ALP (U/L) 183.12±5.46 d 191.15±7.9 cd 210.32±5.00 c 392.00±7.17 a 277.10±1.85 b 274.40±2.28 b 183.80±2.83 d
Total protein (g/dl) 7.05 ±.06 ab 7.32 ±0.18 a 6.97 ±0.09 ab 5.02 ±0.22 d 6.17 ±0.08 c 6.65 ±0.064b c 7.27 ±0.149 b
Albumin (g/dl) 4.15 ±0.11 a 4.10 ±0.15 ab 4.22 ±0.16 a 2.57±0.09 d 3.40 ±0.04 c 3.65 ±0.64b c 4.12 ±0.85 ab
Total bilirubin 0.76 ±0.48c 0.57±0.40 c
0.67 ±0.54 c
2.87 ±2.28 a
1.36 ±1.19 b
1.26 ±1.16 b
0.68 ±0.49 c
(mg/dl)
Direct bilirubin 0.13 ±0.19 c 0.11 ±0.19 c 0.13 ±0.23 c 0.55 ±0.27 a 0.28 ±0.17 b 0.25 ±0.13 b 0.14 ±0.18 c
(mg/dl)
Ca (mg/dl) 9.32±0.15 a 9.12±0.20 ab 9.05±0.15 ab 6.30±0.18 c 8.45±0.06 b 8.47±0.09 b 9.52±0.18 a
Na (mmol/dl) 137.32±0.46 ab 140.10±0.88 a 139.55±1.53 a 122.0±2.95 c 132.45±0.89 b 134.75±0.55 ab 139.9 ±0.50 a

K (mmol/dl) 5.75±0.06 c 3.90±0.15 c 4.12±0.18 c 6.00±0.09 a 4.97±0.08b 5.05±0.10 b 3.87±0.08 c

P (mg/dl) 4.05±0.23 b 4.30±0.19 b 4.12±0.20 b 8.02±0.59 a 5.32±0.17b 4.97±0.32 b 4.07±0.19 b

Data were presented as mean ± standard error of mean (SEM). Mean values with different superscript letters in the same row were signifi-
cantly different at (P ≤ 0.05).
3.3. Effect of morin and/ or rutin administration on DNA
fragmentation induced byTiO2 NPs

Comet assay results revealed a significant increase in DNA dam-

age of liver cells of TiO2 NPs-treated as evidenced by increase per-
centage damage, comet tail length, and comet tail moment as com-
pared to the three control groups (Figs.2 and 3). These results are in
agreement with Eman et al., (2015) and Wang et al., (2007) who also
reported a similar DNA damage in liver cells exposed to TiO2 NPs.
Other studies also revealed that TiO2 NPs can induce mutation (Xu
et al., 2009), and aberrant chromosomes (Nakagawa et al., 1997). In
contrast, some others researchers did not confirm the genotoxic ef-
fects of TiO2 NPs (Bhattacharya et al., 2009; Hackenberg et al., 2010
and Warheit et al., 2007).

In the present study, administration of morin and/or rutin signif-

icantly lowered DNA damage as revealed by reduction in the level
of DNA tail percentage damage, tail length, and tail moment (Figs.2
and 3). Similarly, Khan et al., (2012) also found that administration of
rutin significantly decreased DNA damage in a dose dependent way in
rat treated with (KBrO3) as compared to the control group.

Overall, these findings suggest that TiO2 NPs could induce hepat-
ocyte genotoxicity (as revealed by DNA fragmentation) probably via
induction of oxidative stress. However, administration of rutin and/or
morin can relieve this genotoxic effect.

3.4. Effect of morin and/ or rutin administration on liver

histology of TiO2 NPs-treated rats

In control groups (G1-G3), liver sections showed normal hepatic

parenchyma with preserved lobular pattern, portal triads structures,
vascular tree, kupffer cells and stromal component (Fig.4A-C). In
contrast, TiO2 NPs-treated group (G4) showed hydropic degeneration
in most hepatic parenchyma (arrow) with congested hepatic blood
vessels (arrowhead), besides hemorrhagic area in some examined
sections (curved arrow) (Fig.4D, E).

On the other hand, morin-treated group (G5) showed slight vacu-

Fig.1. Effect of morin and/ or rutin administration on liver tissue olar degeneration in few areas of hepatic parenchyma (arrow, Fig.4F),
MDA and GSH concentrations, SOD and CAT activities in TiO2 NPs while rutin-treated group (G6) and the co-treated group (G7) exhib-
intoxicated rats. ited normal hepatic parenchyma with slight round mononuclear cells
27
 Eid et al., 2018, AJMS 1(2):25-30 , DOI:10.5455/ajms.12

Fig.2. Photomicrographs representation of liver DNA damage, us-

ing comet assay in different animal groups; A, Group1 (control); B,
Group2 (morin); C, Group3 (rutin); D and E, Group4 (TiO2 NPs);
F, Group5 (TiO2 NPs+ morin); G, Group6 (TiO2 NPs+ rutin); H,
Group7 (TiO2 NPs+ morin+ rutin).

infiltration in portal area (arrow, Fig.4G, H). Similar to our findings,

Eman et al., (2015) also found congestion and lymphocytic aggrega-
tion in liver tissue of rats administrated TiO2 NPs.

Although we did not find fibrosis, as our study was acute and last-
ed short time (4 weeks), another chronic study (8 weeks) reported that
TiO2 NPs could induce fibrosis around the central vein where TiO2
NPs always aggregated (Chen et al., 2009). In consistent with our re-
sults, Anbu and Saravanan, 2013 also showed that administration of
morin could reduce the severity of the damage in the hepatocytes and
preserves the structural integrity of the liver by virtue of its antioxi-
dant properties. Similarly, treatment of rutin showed a significant res-
toration of the hepatic lesions and architecture of the liver following
intoxication (Ashraf et al., 2012).

Throughout the experiment, the combined group treated by both

morin and rutin gave better improvement against TiO2 NPs-induced
hepatotoxicity than each alone. This suggests presence of a possi-
ble kind of synergism between the two flavonoids and so their com-
bined administration is recommended to give best effect against TiO2
NPs-induced hepatotoxicity. Further investigations are needed to Fig.3. Effect of morin and/ or rutin administration on comet assay pa-
check the actual mode of action of these two flavonoids on molecular rameters in liver tissue of TiO2 NPs intoxicated rats.
basis and whether their effect is limited to this type of NPs or against

28
 Eid et al., 2018, AJMS 1(2):25-30 , DOI:10.5455/ajms.12
Fig.4. Histopathological examination of liver exposed to TiO2-NPs
and co-administrated with morin and rutin during the whole period of
experiment. A (G1), B (G2), C (G3), D and E (G4), F (G5), G (G6),
and H (G7). H&E, 300x
Conclusion
Morin and/or rutin could improve the damaged liver tissues and
the associated disrupted function that induced by TiO2 NPs toxicity.
The results suggest that morin and rutin may be appropriate for clini-
cal application in the treatment of liver disorders induced by TiO2 NPs
toxicity and the use of TiO2 NPs should be conjugated with flavonoids
to overcome their hepatotoxic effect.
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