PREVENTIVE EFFECT OF SPIRULINA VERSICOLOR AND … · 2009; Fahim et al., 2008; Ahmed et al., 2008;...
Transcript of PREVENTIVE EFFECT OF SPIRULINA VERSICOLOR AND … · 2009; Fahim et al., 2008; Ahmed et al., 2008;...
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PREVENTIVE EFFECT OF SPIRULINA VERSICOLOR AND ENTEROMORPHA
FLEXUOSA ETHANOLIC EXTRACTS AGAINST DIETHYLNITROSAMINE/BENZO(A)PYRENE-INDUCED
HAPATOCARCINOGENCITY IN RATS OSAMA M. AHMED
1,2 MOHAMED B. ASHOUR
1 HANAA I. FAHIM
1 AYMAN M. MAHMOUD
1 NOHA A. AHMED
1
1Physiology Division, Dept. of Zoology, Faculty of Science, Beni-Suef University, Egypt
2Faculty of Oral & Dental Medicine, Nahda University, New Beni-Suef City, Beni-Suef, Egypt
ABSTRACT
This study is conducted to assess the preventive effects of Spirulina versicolor and
Enteromorpha flexuosa ethanolic extracts on hepatocarcingenesis induced by
diethylnitrosamine (DEN)/benzo(a)pyrene (BP) in albino rats. In vitro anti-proliferative
effect of both extracts on hepatocarcinoma cell lines (HepG2) was also evaluated and was
found to be moderate. After two weeks of single dose of DEN (200 mg/kg b. wt;
intraperitoneal), BP, a promotor of carcinogenesis, was intraperitoneally injected (50 mg/kg
b. wt) twice/week for 6 weeks. Spirulina versicolor and Enteromorpha flexuosa ethanolic
extracts orally administered, at dose of 25 mg/kg b. wt/day, for 2 and 8 weeks starting from
the day of DEN injection successfully counteract the carcinogenic effects of DEN and BP
as evidenced by absence of precancerous oval hepatocytes and vesicular nuclei as well as
decrease of tumor marker, alpha fetoprotein and proinflammatory cytokine, TNF-α levels
in serum. In addition, the treatments improved DEN/BP-induced elevation of AST, ALT,
LDH and γ-GGT activities, serum total bilirubin level and liver lipid peroxidation. They
increased the lowered serum albumin level and antioxidant enzymes as well. In conclusion,
Spirulina versicolor and Enteromorpha flexuosa ethanolic extracts successfully prevented
the hepatocarcinogenic and hepatoxic effects of DEN and BP via enhancement of anti-
oxidant defense system and suppression of oxidative stress and inflammation.
KEYWORDS: Dimetylnitrosamine, Benzo(a)pyrene, hepatocarcinogenesis, Spirulina
versicolor and Enteromorpha flexuosa.
INTRODUCTION Hepatocellular carcinoma (HCC) accounts for 70–85% of the primary malignant tumours of the
liver and it is the most common cause of cancer-related death in the world (Thorgeirsson
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and Grisham, 2002; Jemal et al., 2011). The major risk factors in liver cancer includes
hepatitis viral infection, environmental and industrial toxic chemicals, food additives,
aflatoxins, alcohol, drugs, water and air pollutants (Farazi and Depinho, 2006; Jemal et al.,
2009; Fahim et al., 2008; Ahmed et al., 2008; Ahmed et al., 2013).
Diethylnitrosamine (DEN) is a well-known hepatocarcinogenic agent present in cured and
fried meals, cheddar cheese, tobacco smoke, water, agriculture chemicals and cosmetics
and pharmaceutical products (Sullivan et al., 1991; Reh and Fajen, 1996; Brown, 1999). As
established, diethylnitrosamine (N-nitrosodiethylamine; DEN) produces primary metabolic
activation resulting in initiation of liver carcinogenesis (Verna et al., 1996; Chen et al.,
2012). Other mechanisms may be involved through DNA-adduct formation, mutagenicity,
inhibition of many enzymes involved in DNA repair mechanism and tumor initiation
(Verna et al., 1996; Bansal et al., 2005). Benzo(a)pyrene (BP) is a pro-carcinogen, and its mechanism of carcinogenesis depends on
its enzymatic metabolism to the ultimate mutagen, benzo(a)pyrene diol epoxide, a molecule
that intercalates in DNA, covalently bonding to the nucleophilic guanine nucleic bases at
the N2 position. This binding distorts the DNA, inducing mutations by perturbing the
double-helical DNA structure (Volk, 2003). There are indications that benzo[a]pyrene diol
epoxide specifically targets the protective p53 gene (Shinmura, 2008). This gene is a
transcription factor that regulates the cell cycle and hence functions as a tumor suppressor
by inducing G (guanine) to T (thymidine) transversions within p53 (Shinmura, 2008). A promising new approach to cancer prevention, which is termed chemoprevention, aims to
halt, prevent or reverse the development and progression of pre-cancerous cells by
administering non-cytotoxic nutrients and/or pharmacological agents during the time period
between tumor initiation and malignancy (Sporn et al., 1976; Bishayee et al., 1995; Sayed-
Ahmed et al., 2010, Zhang et al., 2013). Numbers of investigations are being conducted
worldwide, to discover natural products that can suppress or prevent the process of
carcinogenesis during the initiation, promotion or progression stages (Lee et al., 2002;
Aggarwal et al., 2003; Cheng et al., 2004; Zhang et al., 2013). Edible seaweeds have historically been consumed by coastal populations across the globe.
Epidemiological evidence suggests regular seaweed consumption may protect against a
range of diseases of modernity (Brownlee et al., 2012). Therefore, the current study was
designed to evaluate the protective effect of both Spirulina versicolor and Enteromorpha
flexuosa hydroethanolic extracts against the cytotoxic effects of DEN and BP.
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MATERIALS AND METHODS:
Chemicals:
Diethyl Nitrosamine (DEN) and Benzo(a)Pyrene (BP) were purchased from Sigma Chemical
Company, USA. All other chemicals used for the investigation were of analytical grade. Collection of seaweeds and preparation of extracts: Enteromopha flexuosa (E. flexuosa) was obtained from El Koseir area on Red Sea (Egypt).
It was washed several times with tap water and finally with distilled water, then air-dried in
shade. Spirulina versicolor (S. versicolor) was obtained from Harraz medicinal plant
company, Cairo, Egypt (www.harrazegypt.com). Both algae were authenticated by staff
members of the Phycology Division, Botany Department, Faculty of Science, Beni-Suef
University, Egypt. Both algae were powdered with an electric grinder and extracted by
maceration in 80% aqueous ethanol until exhaustion at room temperature. After filtration,
the filtrate was concentrated under vacuum in a rotary evaporator. The residue obtained was
stored at -20 oC till use in biological evaluation.
In vitro antitumor assay: Hepatocarcinoma cell lines (HepG2) were obtained from the American Type Culture Collection
(ATCC, Minisota, U.S.A.) and were maintained in Dulbeco's Modified Eagle's Medium
(DMEM) with 10% foetal calf serum, sodium pyruvate, 100 U/ml penicillin and 100 mg/ml
streptomycin at 37oC and 5% CO2 at Pharmacology Unit, Cancer Biology Department, National
Cancer Institute, Cairo University, Egypt by serial sub-culturing. Hepatocarcinoma cell lines
were seeded in flat-bottomed microtiter 96-well plate at a cell concentration of 1x 104 cells per
well in 200 µl of growth medium. Cytotoxicity of S. versicolor and E. flexuosa ethanolic
extracts was tested using the method of Skeha et al. (1990). The microtiter plates were
incubated at 37°C in a humidified incubator with 5% CO2 for a period of 48 h. Three wells were
used for each concentration of the tested sample (0, 5, 12.5, 25 and 50 µg/ml). Control cells
were incubated without test sample and with the equivalent volume of DMSO (0.1%) for 48 hr.
At the end of the incubation period, the cells were fixed and stained with sulforhodamine B
dissolved in acetic acid. Unbound stain was removed by washing four times with 1% acetic acid
and the protein bound dye was extracted with tris–EDTA buffer.
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Absorbance was measured in an ELISA reader. The relation between surviving fraction and
compound concentration was plotted to get the survival curve and the concentration of an agent
that causes a 50% growth inhibition for each tested extract using each cell line was obtained
from the survival curve.
In vivo investigation:
Experimental animals: Male albino rats weighting about 120-150g were used as experimental animals in the
present investigation. They were obtained from National Research Center (NRC), Dokki,
Giza. They were kept under observation before the onset of the experiment to exclude any
intercurrent infection. The chosen animals were housed in plastic good aerated cages at
normal atmospheric temperature (25±5°C) as well as normal 12 hours light/dark cycle. Rats
were given access of water and supplied daily with standard diet of known composition. All
animal procedures are in accordance with the recommendations of Canadian Committee for
care and use of animals (Canadian Council on Animal Care (CCAC), 1993).
Experimental design:
The animals were divided into 4 groups, each is 12 rats. Six rats of each group were
sacrificed at the end of 2 weeks and others at the end of 8th week. The assigned groups
were designated as follow: Group 1 (Normal): It intraperitoneally (ip) received the equivalent volume of normal saline
(vehicle 1) at 1st
day and olive oil (vehicle 2) at the end of 2nd
week of the
experiment. It was also orally administered the equivalent volume of 1%
carboxymethylcellulose (CMC) (vehicle 3) daily for 8 weeks. Group 2 (DEN/BP Control): The rats of this group received a single ip dose of 200mg/kg
b.wt. DEN (dissolved in 0.9% saline) (Saleem et al., 2013) and benzo(a)pyrene
(dissolved in corn oil) at dose level of 50 mg/kg b.wt (Lutz and Schlatter, 1979)
twice/week for 6 weeks beginning from the 3rd
week of the experiment. The rats of
this group were orally administered the equivalent volume of 1% CMC (vehicle 3)
daily for 8 weeks beginning from the starting period of the experiment. Group 3 (DEN/BP + S. versicolor ethanolic extract): It received 200mg/kg b.wt DEN and
50 mg/kg b.wt benzo(a)pyrene twice/week as group 2 and orally supplemented S.
versicolor ethanolic extract (25mg/kg b.wt/day) (AbouZid et al., 2013) dissolved in
5ml 1% CMC through the entire experimental period.
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Group 4 (DEN/BP + E. flexuosa ethanolic extract): The rats of this group received
200mg/kg b.wt DEN and 50 mg/kg b.wt benzo (a) pyrene twice/week and orally
supplemented E. flexuosa ethanolic extract (25mg/kg b.wt/day) (AbouZid et al., 2013)
dissolved in 5ml 1% CMC through the entire experimental period.
At the end of the specified periods, rats from each group were sacrificed under mild diethylether
anaesthesia. Blood samples were collected and left to coagulate then centrifuged at 3000 r.p.m
for 15 minutes. Sera were quickly removed and kept at -20°C till used. Liver were quickly
exised and perfused with ice-cold saline for each rat, 0.5 gm of liver was homogenized in 5 ml
0.9% Nacl (10% w/v) using Teflon homogenizer ( Glas-Col,Terre Haute,USA).The homogenate
was centrifuged at 3000 r.p.m and the supernatant was kept at - 20oC pending estimation of
oxidative stress parameters and anti-oxidant defense markers.
Biochemical analysis: Serum tumour necrosis factor alpha (TNF-α) and alpha fetoprotein (AFP) were determined
using reagent kit purchased from R&D Systems (USA) according to the manufacturer
instructions.
Serum ALT (Tietz, 1986), AST ((Murray, 1984), γ-GT (Persijn and van derSlik, 1976) and
LDH (Tietz, 1986) were determined using reagent kits from Spinreact (Spain). Total
bilirubin (Kaplan and Pesce, 1984) and albumin (Webster, 1974) were determined using
reagent kits purchased from Diamond Diagnostic Chemical Company (Egypt).
Liver lipid peroxidation, reduced glutathione (GSH), glutathione peroxidase (GPx),
superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (G-S-T) were
determined according to the methods of Preuss et al. (1998), Beutler et al. (1963),
Matkovics et al. (1998), Marklund and Marklund (1974), Cohen et al. (1970) and
Mannervik and Gutenberg (1981), respectively.
Histological study: After dissection, autopsy samples were taken from the liver of rats of different groups and
fixed in 10% formal saline for twenty four hours. Fixed samples were transferred to the
Pathology Department, National Cancer Institute, Cairo University, Egypt for embedding in
wax after dehydration, sectioning and staining with haematoxylin and eosin (Banchroft et
al., 1996).
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Statistical analysis: One way ANOVA (PC – STAT, 1985) was used for data analysis. Results were expressed
as mean ± standard error (SE) followed by LSD at 5% and LSD at 1% to compare between
the different groups. Values of P>0.05 were considered statistically non-significantly
different, while values of P<0.05, P<0.01 and P<0.001 were significantly, highly
significantly and very highly significantly different respectively.
RESULTS: Data describing the effect of S. versicolor and E. flexuosa ethanolic extracts on serum TNF-
α and AFP concentrations were represented in table 1. DEN/BP- administered rats showed
a highly significant (P<0.01; LSD) elevation in serum TNF-α and AFP concentrations
recording percentage increase of 123.58 and 260.78 % respectively as compared to normal
control after 8 weeks. Treatment of DEN/BP- administered rats with either S. versicolor or
E. flexuosa potentially (P<0.01; LSD) improved the altered serum levels of TNF-α and
AFP, with more potent effect offered by E. flexuosa. The effects of S. versicolor and E. flexuosa on liver function enzymes in serum, after 2 and
8 weeks of DEN administration, were represented in figures 2-5. The recorded data showed
a significant (P<0.05; LSD) elevation of ALT activity in DEN-intoxicated rats after 2
weeks in comparison to normal control rats. On the other hand, the elevation of serum AST,
LDH and γGT was highly significant (P<0.01; LSD) in DEN-intoxicated rats when
compared with normal ones. The deleterious effects on these enzymes activities were more
pronounced as the period extended from 2 weeks to 8 weeks. Treatment with S. versicolor
produced a non-significant (P>0.05; LSD) effect on serum ALT, LDH and GGT activities
after 2 weeks of DEN administration, while the effect on serum AST was highly significant
(P<0.01; LSD). On the other hand, E. flexuosa effect on ALT and LDH was highly
significant (P<0.01; LSD) and was significant (P<0.05; LSD) regarding GGT. Conversely,
treatment with E. flexuosa showed a non-significant (P>0.05; LSD) effect on serum AST
after 2 weeks period. After 8 weeks experimental period, all assayed serum enzymes were
highly significantly elevated (P<0.01; LSD) in DEN-BP intoxicated rats when compared to
the normal control group. Serum ALT, AST, LDH and GGT showed a highly significant
(P<0.01; LSD) alleviation following treatment with either S. versicolor or E. flexuosa. Regarding serum total bilirubin, there was a non-significant (P>0.05) difference between the
studied groups after 2 weeks. After 8 weeks, DEN-BP administered rats showed a highly
significant (P<0.01; LSD) elevation in serum total bilirubin concentration when compared to
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normal control rats. Treatment with both tested extracts produced a highly significant
(P<0.01; LSD) amelioration of serum total bilirubin concentration in comparison to DEN-
BP control rats. After 2 weeks, serum albumin of DEN-induced rats was highly
significantly (P<0.01; LSD) decreased in comparison to normal control group. S. versicolor
ethanolic extract protected rats against the decline of serum albumin while the effect of E.
flexuosa extract was non-significant (P<0.05; LSD) as depicted in table 1. At the end of the
8th
week, DEN-BP administered rats showed a highly significant (P<0.01; LSD) reduced
serum albumin in comparison with the normal control group. On the other hand, both
treatment agents produced a highly significant (P<0.01; LSD) amelioration of serum
albumin concentration (Table 1). The effect of S. versicolor or E. flexuosa extracts on liver oxidative stress and antioxidant
defense system were represented in table 3. At both experimental periods, liver lipid
peroxidation (MDA content) showed a highly significant (P<0.01; LSD) increase in DEN-BP
administered rats when compared with normal control rats. Treatment of DEN-intoxicated rats
with either S. versicolor or E. flexuosa extracts produced a marked (P<0.01; LSD) amelioration
of the elevated MDA. Liver GSH content of DEN control rats showed a non-significant change
(P>0.05; LSD) after 2 weeks when compared to normal control rats. The treatment of DEN-
administered rats for 2 weeks with S. versicolor produced a significant (P<0.01; LSD) increase
in GSH content, while E. flexuosa extract produced a non-significant effect (P<0.05; LSD) as
compared to DEN control. After the 8 weeks of treatment, there was a non-significant (P>0.05)
difference in GSH content between all experimental groups. Liver SOD, GPx, CAT and GST
activities showed a highly significant decline in DEN-administered rats (P<0.01; LSD), at the
end of 2 weeks, in comparison to normal control rats. S. versicolor supplementation for 2 weeks
produced a highly significant (P<0.01; LSD) increase in the activities of SOD, GPx, CAT and
G-S-transferase activities. On the other hand, E. flexuosa administration for 2 weeks
significantly (P<0.01; LSD) enhanced the activities of GST and SOD while it did not
significantly affect GPx and CAT. After intraperitoneal administration of BP and as the period
extended to 8 weeks, liver activities of GPx and SOD were more deteriorated than those at the
2nd
week since the recorded percentage decreases were 58.81 and 65.42 % at the 8th
weeks and
-46.94% and -47.97% at the 2nd
week respectively. Supplementation of DEN/BP-administered
rats with S. versicolor extract produced a significant amelioration of the altered enzyme
activities. The treatment with E. flexuosa extract, on the other hand, notably alleviated
(P<0.01; LSD) the activities of SOD, GPx and CAT and non-significantly (P>0.05; LSD)
affect GST activity.
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Histopathological examination of the liver of normal control rats showed a normal
histologic structure of the hepatic lobule (Fig. 6a-c). On the other hand, liver of the
DEN/BP intoxicated group revealed dissociation of hepatocytes, oval cells hyperplasia,
vacuolated hepatocytes, karyomegaly with vesicular nuclei as precancerous changes (Fig.
7a), cytomegalic hepatocytes with foamy cytoplasm (Fig. 7b-c). The group supplemented
with S. versicolor as revealed in figure 8(a-d) fatty changes of hepatocytes, kupffer cells
activation and vacuolization of some hepatocytes. In E. flexuosa-treated group, liver tissue
showed inflammatory cells infiltration, kuppfer cell activation, binucleation of hepatocytes
and vacuolization of some hepatocytes (Fig 9a-c)
DISCUSSION Most of medical remedies for liver diseases generally have limited efficacy and hence the
use of complementary and alternative medicines (CAMs) that utilize herbal medicines have
increased, and these CAMs have attracted considerable interest from patients for treating
liver diseases (Seeff et al., 2001; Strader et al., 2002). DEN is a potent hepatocarcinogen
influencing the initiation stage of carcinogenesis during a period of enhanced cell
proliferation accompanied by hepatocellular necrosis and induces DNA carcinogen adducts,
DNA-strand breaks and in turn hepatocellular carcinomas without cirrhosis through the
development of putative preneoplastic focal lesions (Tatematsu et al., 1988; Chen et al.,
2012). The current study revealed a significant increase in serum TNF-α and AFP of DEN/BP-
administered rats. It has been reported that TNF-α is produced by macrophages and they
play an important role in tumor conditions (Moon et al., 1999). In addition, TNF-α is an
essential factor in tumor promotion (Suganuma et al., 2000). Indeed, Barton et al. (2001)
stated that TNF-α plays a causal role in the development of liver injury. Alleviation of
serum TNF-α levels produced by supplementation of either E. flexuosa or S. versicolor in
DEN/BP-intoxicated rats, may be attributed to their ant-inflammatory and antitumor
activities. Alpha fetoprotein is a fetal specific glycoprotein which falls rapidly after birth,
high level of alpha fetoprotein is suspicious of hepatocellular carcinoma but may be
elevated in chronic viral hepatitis (Patil et al., 2013). Its decrease in DEN/BP-administered
rats treated with E. flexuosa or S. versicolor ethanolic extractmay provide evidence for
decreased probability for hepatocellular carcinoma as compared with DEN/BP-
administered control rats which showed profound elevation of serum AFP.
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Reuben (2004) reported that elevation in transaminase levels in conjunction with a rise in
bilirubin level to more than double of its normal level, is considered as an ominous marker for
hepatotoxicity. In addition, it is well known that the elevation of ALT and ALP is credited to
hepatocellular damage and reflects the pathological alteration in biliary flow (Bulle et al., 1990;
Sivaramakrishnan et al., 2008). In the current study, serum AST, ALT, GGT and bilirubin
levels were significantly increased following DEN and BP administration. An elevated level of
serum indices of hepatocellular damage has been previously reported in many models of DEN-
induced hepatocellular degeneration (Ha et al., 2001; Lee et al., 2002; Barbisan et al., 2003;
Bansal et al., 2005; Sayed-Ahmed et al., 2010). Supplementation of either E. flexuosa or S. versicolor significantly prevented the increase
in serum liver function markers, suggesting that both agents may have protective effect
against DEN/BP-induced liver damage by stabilizing the hepatocyte membrane. The
current data run parallel to the study of Ismail et al. (2010) who demonstrated the
chemoprevention of rat liver toxicity and carcinogenesis by Spirulina platensis. In addition,
Amin et al. (2006) reported the hepatoprotective effect of Spirulina against cadmium-
induced hepatotoxicity. Moreover, Kumar et al (2005) stated that consumption of
Enteromorpha rich diets reversed DEN-hepatocellular carcinoma in rats. Recently, many studies confirmed the contribution of oxidative stress during development of
hepatocarcinogenesis and promotion of liver cancer (Gayathri et al., 2009; Al-Rejaie et al.,
2009; Janani et al., 2010; Mizukami et al., 2010; Taha et al., 2010; Yang et al., 2010). The
current data revealed a significant increase of liver MDA content with a concomitant reduction
of GSH content and activities of the antioxidant enzymes, GPx, SOD and CAT, in DEN-BP
intoxicated rats when compared with normal control rats at both experimental periods.
Treatment of DEN and BP-administered rats with either S. versicolor or E. flexuosa extracts
produced marked reduction of the elevated MDA accompanied with improved antioxidant
system. We assumed that, the hepatoprotective effects of the tested algal extracts were due to
their antioxidant properties and their ability to reduce lipid peroxidation. In this regard,
Evanprince et al. (2009) reported the hepatoprotective potential of S. fusiformis in
acetaminophen-induced hepatotoxicity in mice and attributed this finding to the antioxidant
effects of Spirulina and to its ability to potentiate the antioxidant system. In addition, Spirulina
supplementation has reduced lipid peroxidation and increased activity of the antioxidant system
in the liver of cadmium-intoxicated rats as reported by Amin et al. (2006). Similarly,
Enteromorpha has been found to exhibit potent antioxidant activity (Shanab et al., 2011;
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Narasimhan et al., 2013) and to protect against the hepatotoxic effects of DEN (Kumar et al.,
2005). In conclusion, both S. versicolor or E. flexuosa prevented DEN/BP-induced
hepatotoxicity and hepatocarcinogenesis through their anti-inflammatory, anti-carcinogenic
and antioxidant efficacies.
Table 1: Serum TNF-α and AFP Effects of S. versicolor and E. flexuosa ethanolic extracts on DEN /BP administered rats at the 8th week.
-Data are expressed as mean ± SE. Number of animals in each group is six. - Means, which share the same superscript symbol (s), are not significantly different.
Table 2: Effect of S. versicolor and E. flexuosa ethanolic extracts on serum albumin and total bilrubin concentrations of DEN /BP administered rats at the 2nd and 8th weeks.
-Data are expressed as mean ± SE. Number of animals in each group is six. - Means which share the same superscript symbol(s) are not significantly different.
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Parameter
Group
TNF-α (pg/ml)
% change AFP
(ng/ml) % change
Normal 32.1 ± 1.23d - 0.51 ± 0.05c - DEN/BP Control 71.77 ± 1.40a +123.58 1.84 ± 0.10a +260.78
DEN/BP + S. versicolor 50.5 ± 0.44b -29.64 0.97 ± 0.09b -47.28 DEN/BP + E. flexuosa 44.33 ± 1.97c -38.23 0.79 ± 0.06b -57.07
F-Probability P<0. 001 P<0.001 LSD at 5% level 3.31 0.19 LSD at 1% level 4.51 0.25
per
iod
s Parameter
Group
Total bilirubin
(mg/dl)
% change
Albumin
(g/dl)
% change
2 W
eeks
Normal control 00.52 ± 0.02d - 3.95 ± 0.02a -
DEN control 00.69 ± 0.03cd +32.69% 2.80 ± 0.15c -29.11%
DEN + E. flexuosa 00.58 ± 0.03d -15.94% 3.60 ± 0.40ab +28.57%
DEN + S. versicolor 00.62 ± 0.03d -10.14% 2.93 ± 0.10bc +4.64%
8 W
eeks
Normal control 00.59 ± 0.07d - 3.92 ± 0.25a -
DEN/BP control 1.67 ± 0.15a +183.05% 2.43 ± 0.14c -38.01%
DEN/BP + S. versicolor 1.17 ± 0.19b -29.94% 3.68 ± 0.13a +51.44%
DEN/BP+ E. flexuosa 1.01 ± 0.13bc -39.52% 3.67 ± 0.24a +51.03%
F-probability P<0.001 P<0.001 LSD at 5% level 0.34 0.697 LSD at 1% level 0.46 0.939
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Table 3: Effect of E. flexuosa and S. versicolor ethanolic extracts on oxidative stress and antioxidant defense markers in liver of DEN/BP- administered animals at the 2nd and 8th weeks.
- Data are expressed as mean ± SE. Number of animals in each group is six. - Means, which share the same superscript symbol(s), are not significantly different
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peri
ods Parameter
Group
LPO
(nmol MDA/100mg
tissue)
% change
GSH
(nmol /100mg tissue)
% change
GST
(mU/100mg tissue)
% change
GPx
(mU/100mg
tissue)
% change CAT
(k 10-2)
% change SOD
(U/g tissue)
% change
2 W
eeks
Normal control 67.25 ± 0.46bc - 19.46 ± 0.43b - 91.6 ± 8.10b - 18.94 ± 1.69ab - 40.68 ± 2.14b - 18.26 ± 0.29ab -
DEN control 87.25 ± 0.39a +29.74% 18.79 ± 0.13b -3.44% 64.67 ± 1.54d -29.42% 10.05 ± 0.32de -46.94% 15.28 ± 0.41d -62.44% 9.50 ± 0.35e -47.97%
DEN + S. versicolor
66.63 ± 0.32bc -23.63% 19.47 ± 0.54b +3.62% 91.46 ± 3.04b +41.43% 21.07 ± 1.81a +109.65% 37.61 ± 2.04bc
+146.14% 18.75 ± 0.22a +97.37%
DEN + E. flexuosa
64.88 ± 1.25c -25.64% 36.07 ± 4.88a +91.96% 85.47 ± 4.66bc +32.16% 15.67 ± 0.68bc +55.92% 20.69 ± 0.62d +35.41% 15.20 ± 0.31d +60%
8 W
eeks
Normal control 71.03 ± 1.38b - 20.34 ± 0.22b - 105.4 ± 2.53a - 16.07 ± 1.26abc - 40.46 ± 3.89b - 17.93 ± 0.63abc -
DEN/BP control 91.98 ± 3.68a +29.49% 16.49 ± 0.09b -18.93% 76.11 ± 1.88cd -27.79% 6.62 ± 0.57e -58.81% 31.54 ± 1.47c -22.05% 6.20 ± 0.70f -65.42%
DEN/BP + S. versicolor
66.95 ± 1.90bc -27.21% 21.37 ± 0.15b +29.59% 89.43 ± 2.72b +17.50% 19.63 ± 2.60ab +196.53% 43.31 ± 0.56ab +37.32% 16.47 ± 0.60cd +165.65%
DEN/BP + E. flexuosa
67.02 ± 1.38bc -27.14% 21.96 ± 0.22b +33.17% 85.19 ± 1.83bc +11.93% 13.60 ± 1.66cd +105.44% 48.60 ± 2.10a +54.09% 16.6 ± 0.66bcd +167.74%
F-probability P<0. 001 P<0. 001 P<0. 001 P<0. 001 P<0. 001 P<0. 001
LSD at 5% level 5.70 5.83 12.70 5.02 6.59 1.68
LSD at 1% level 7.62 7.85 17.37 6.76 8.87 2.27
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Figures 1-5 Fig. 1: Cytotoxicity effects of S. versicolor and E.flexuosa ethanolic extracts against HepG2 cell line. IC50 for S. versicolor = 45.62 µg/ml, IC50 for E .flexuosa = 44.95 µg/ml
Fig. 2: Effect of S. versicolor and E. flexuosa ethanolic extracts on serum AST activity of DEN/BP-intoxicated rats . LSD at 5% level: 11.77; LSD at 1% level: 15.85.
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0
10
20
30
40
50
60
70
80
90
100
2 Weeks 8 Weeks
AST ( U
/L)
Normal Control
DEN Control
DEN+ S.versicolor extract
DEN+ E.flexuosa extract
d
c
d
bc
d
a
bcb
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Fig. 3: Effect of S. versicolor and E. flexuosa ethanolic extracts on serum ALT activity of DEN/ BP-intoxicated rats. LSD at 5% level: 10.11; LSD at 1% level: 13.62
Fig. 4: Effect of S.versicolor and E.flexuosa ethanolic extracts on serum LDH activity of DEN/ BP-intoxicated rats. LSD at 5% level: 59.33; LSD at 1% level: 79.90.
Fig. 5: Effect of S. versicolor and E . flexuosa ethanolic extracts on serum γGT activity of DEN/BP-intoxicated rats. LSD at 5% level: 7.13; LSD at 1% level: 9.60.
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0
10
20
30
40
50
60
70
2 Weeks 8 Weeks
ALT (U/L
)
Normal Control
DEN Control
DEN+ S.versicolor extract
DEN+ E.flexuosa extract
e
bcd
cde
f
a
bc
b
de
0
50
100
150
200
250
300
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450
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LDH (U/L)
Normal Control
DEN Control
DEN+ S.versicolor extract
DEN+ E.flexuosa extract
c
a
a
b
c
a
bb
0
5
10
15
20
25
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ƔGT (U/L)
Normal Control
DEN Control
DEN+ S.versicolor extract
DEN+ E.flexuosa extract
d
a
a
b
d
a
cd
bc
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Legend for figures from 6 to 9
Fig. 6a-c: Photomicrograps of liver sections of normal control groups showing the normal
histological structure of hepatic lobule, CV: (central vein); S: (sinusoids); T:
(trabecula); KC: (Kupffer cells). (H and E x400).
Fig. 7: Photomicrographs of liver sections of DEN- BP administered rats showing
dissociation of hepatocytes, oval cells hyperplasia (OV), vacuolated hepatocytes
(Vh), karyomegaly with vesicular nuclei as precancerous changes. (Fig.7a),
cytomegalic hepatocytes (CM) with foamy cytoplasm. (Fig.7b) and vesicular
nuclei as precancerous changes (Fig.7c) (H and E x400).
Fig. 8a-d: Photomicrographs of liver sections of DEN/BP administered rats treated with S.
versicolor showing fatty change (FC) of hepatocytes, kupffer cells (KC)
activation and vacuolization (V) of some hepatocytes (H and E X400).
Fig. 9: Photomicrographs of liver sections of DEN/BP administered rats treated with E.
flexuosa showing inflammatory cells infiltration (IF) in hepatic sinusoids (Fig.
9a), kupffer cell (KC) activation and binucleation of hepatocytes (Fig. 9b) and
kupffer cell activation and vacuolization (V) of some hepatocytes (Fig. 9c). (H
and E X400).
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