preventive effect of spirulina versicolor and … · 2009; fahim et al., 2008; ahmed et al., 2008;...

JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.393, ISSN: 2320-5083, Volume 2, Issue 6, July 2014

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


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


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


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

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2 Weeks 8 Weeks

ALT (U/L

)

Normal Control

DEN Control



e

bcd

cde

f

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bc

b

de

0

50

100

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200

250

300

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2 Weeks 8 Weeks

LDH (U/L)

Normal Control

DEN Control



c

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ƔGT (U/L)

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DEN Control



d

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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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