Biomedicine & Pharmacotherapy 84 (2016) 430–437

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Original article

Evaluation of antioxidant and stabilizing lipid peroxidation nature of

Solanum xanthocarpum leaves in experimentally diethylnitrosamine
induced hepatocellular carcinogenesis
Periyannan Velua , Annamalai Vijayalakshmib , Perumal Iyappana,* ,
Dhananjayan Indumathib
a
Department of Biotechnology, Muthayammal College of Arts and Science, Periyar University, Rasipuram, Namakkal, Tamil Nadu, India
b
Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, Chidambaram, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Article history:
Received 8 June 2016 Solanum xanthocarpum Schrad. & Wendl, is a traditional edible leaves as a form of decoction, extracts used
Received in revised form 10 September 2016 as a herbal medicine, and consumed for health promoting profiles. The present investigation was carried
Accepted 16 September 2016 out to evaluate antioxidant status and lipid peroxidation level of anticancer activity of Solanum
xanthocarpum (SXC) on Diethylnitrosamine (DEN) induced hepato carcinogenesis in male Wistar albino
Keywords: rats. Hepatic cancer was developed on the liver of Wistar rats treated by DEN or vehicle three times a
Solanum xanthocarpum week for 16 weeks. Tumour incidence, tumour volume, tumour burden, lipid peroxidation, antioxidant,
Diethylnitrosamine liver marker enzymes and histopathological changes were assessed in DEN alone and in DEN + SXC leaves
Hepatocarcinogenesis
extract treated rats. Hundred percent tumour incidences with an imbalance in carcinogen metabolizing
Anti-oxidants
enzymes and cellular redox status were observed in rats treated with DEN alone. Oral administration of
Lipid peroxidation
SXC aqueous leaves extract treatment at a dose of 150 mg/kg b.w. to DEN treated rats were prevented
tumour incidence and restored the elevated activities of liver marker enzymes and antioxidant status to
near normal with decreased lipid peroxide levels. The biochemical consistent with histopathological
observations suggesting marked hepatoprotective effect of the leaves extract in a dose dependent
manner. These results clearly suggest that SXC aqueous leaves extract treatment prevents liver damage,
lipid peroxidation, protects the antioxidant defense system and anti-carcinogenic potential in DEN
induced hepatic carcinogenesis.
ã 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction both natives contained traces of tomatidenol. In India, glycoalka-

Solanum xanthocarpum Schrad. & Wendl (SXC), (Family:
Solanaceae), commonly known as Yellow Berried Nightshade
(syn-kantakari), also called as kantankathiri, kankari and kateri, is
a traditional edible leaves as a form of decoction and extracts used
as an herbal medicine in throughout India, mostly in dry places like
a weed on roadsides and wastelands [1,2]. In previous studies
shown that, SXC has most extensive phytochemicals and pharma-
cological studies showed SXC has alkaloids, steroids, saponins,
flavonoids and glycosides as main active components [1,3] found
that fruits from French plant (4.6%) were riches in solasodine
glycoside than those of Nepalese origin (1.6%), besides plants of
* Corresponding author at: Department of Biotechnology, Faculty of Science,
Muthayammal College of Arts and Science, Periyar University, Salem 636011, Tamil
Nadu, India.
loid content of fruits collected from Jammu Kashmir is reported to
be 3.5% (total alkaloid, 1.1%). Plant samples collected from culcutta
contained solasodine (0.0287), plant contain diosgenin [4].
Reported that SXC are widely used by practitioners of the Siddha
to treat respiratory diseases [5], and also it is one of the members of
the dasamula (ten roots) of the ayurveda, which is considered to be
a noxious weed [6], especially to treat asthma [7], diabetes [8],
rheumatism, chest pain, stone in the bladder, flatulence and
bronchospasm. In addition, report that solasodine, an alkaloidal
constituent of SXC exerts antiandrogenic activity and antiinflam-
matory [9]. Demonstrated that SXC showed DPPH-free radical
scavenging activity, hepatoprotective and antioxidant activity
galactosamine induced hepatotoxicity in Wistar albino rats.
Recently, SXC exerts to hepatoprotective activity in paracetamol
induced liver damage [2].
Hepatocelluar carcinoma (HCC) is the most common cancer
form of primary liver cancer. Globally, HCC is the fifth most

http://dx.doi.org/10.1016/j.biopha.2016.09.060
0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
 P. Velu et al. / Biomedicine & Pharmacotherapy 84 (2016) 430–437
prevalent cancer and third leading cause of cancer related death,
highly aggressive. It displays a high popularity with 620,000 cases
per year testified from China, Africa and South East Asia [10]. HCC a
sharp prominent incidence of liver cancer has been form of dense
tumour observed in African countries. Almost, 0.5–1.0 million new
cases are identified every year and 595,000 deaths due to liver
cancer occur mainly in developing countries [11]. The major risk
factors are associated with development of hepatocarcinogenesis
are alcohol consumption, fungal toxins contaminated foods, toxic
industrial chemicals, air/water pollutants and hepatitis viral
infection [12]. This cancer in South East Asia and low in developed
western world countries substantially increases its incidence [13].
Diethylnitrosamine (DEN) is a representative chemicals family
of carcinogenic N- nitrosamine compounds has been distributed in
processed meat, tobacco smoke and whiskey [14]. The Interna-
tional Agency for Research on Cancer (IARC) has classified DEN is
well known hepatotoxin, hepatocarinogen and mutagenic agent
despite the lack of epidemiologic data [15]. In liver, it generates a
metabolism of progressive, proliferative, highly mutagenic tumour
lesions and neoplastic lesions. Recent findings have been
suggested that N-nitroso compound cause a wide range of tumours
in all animal models [16]. Liver has an efficient anti-oxidant
defense system to inactivate reactive oxygen species (ROS), which
Fig.1. Experimantal study.
431
are over whelmed under conditions of oxidative stress and cause
damage on critical cellular biomolecules such as lipids, proteins
and deoxyribonucleic acid (DNA). DEN is recommended to cause
an uncompromised cohort of free radicals in the liver, which in
turn increases the demand of antioxidant enzymes. Subsequently,
it leads to oxidative stress and the free radicals participate in DEN-
induced HCC [17]. In order to sustain cellular health, it is
indispensable to have a specific and effective chemical scavenger
to target multiple types of radicals. Most of the commercially based
antioxidants supplements are single oxidant [18].
There were no scientific studies on the potential of SXC against
HCC. The present investigation was carried out to evaluate the liver
marker enzymes, antioxidant status and lipid peroxidation level
histopathological analysis of solanum xanthocarpum (SXC) on
diethylnitrosamine (DEN) induced HCC in male Wistar albino rats.
2. Materials and methods
2.1. Animals
Male Wistar albino rats, weighing 130–150 g, procured from the
Small Animal Breeding Centre, Muthayammal College of Arts and
Science, Tamil Nadu, India. Animals were acclimatized under
432 P. Velu et al. / Biomedicine & Pharmacotherapy 84 (2016) 430–437

standard vivarium conditions at 25
2  C with 12 h light: dark
cycle. The animals were fed with standard rat chow and water ad
libitum. The food was withdrawn 18–24 h before the experiment.
The care and use of laboratory animals were done according to the
guidelines of the Council Directive CPCSEA, India (Reg. No. 1416/
PO/a/11/CPCSEA) about Good Laboratory Practice (GLP) on animal
experimentation.
2.2. Preparation of plant extract
The SXC leaves were collected from the habitats near to bank of
Dharmapuri, TamilNadu, India and leaves were washed with
running tap water and shade dried at room temperature
(27
2  C). An electrical blender powdered the dried leaves of
SXC from the powder; 300 g of the plant materials was extracted
with 1 L of organic solvents of ethanol for using a Soxhlet apparatus
[19] boiling point range 60–80  C for 8 h. The extracts were filtered
through a Buchner funnel with Whatman No. 1 filter paper. The
crude plant extracts were evaporated to dryness in rotary vacuum
evaporator. After evaporation of ethanol, we directly prepared a
working standard concentration for the experiment i.e. 75, 150 and
300 mg respectively.
tissues were collected and homogenized by a homogenizer in
(0.1 M) phosphate buffered saline, pH 7.4 and samples were stored
at 80  C for further assays. Biomarkers were assayed in neoplastic
tumour tissues in those animals that developed tumours and
compared with the non-neoplastic liver tissue from the animals
that did not develop tumours. Tumour volume was measured using
   
the formula. V ¼ 43p D21 D2 D3
2 2 , Where D1, D2 and D3 are the 3
diameters (mm) of the tumours. Tumour burden was calculated by
multiplying tumour volume and number of tumours/animals.
2.5. Preparation of tissue homogenate
At the end of experimental period, animals in different groups
were sacrificed under cervical dislocation between 7 am and 10 am
after an overnight fast. The liver sample was removed and washed
with 0.9% NaCl and homogenized with three volumes (w/v) of the
appropriate buffer using a Potter-Elvehjem homogenizer with a
Teflon tube and centrifuged at 1200 rpm for 20 min at 4  C. The
supernatants were used for biochemical estimations.
2.6. Macroscopic evaluation of the incidence of nodules

To measure the incidence of nodules at the end of the 16 weeks,

2.3. Hepatocarcinogenesis initiation using diethylnitrosamine
rat liver were removed and flushed with saline. The livers dried
with filter paper taking care not to disturb nodules and carefully
The experimental hepatocarcinogenesis was induced by using
counted through visual macroscopic examination.
DEN (Sigma, USA). DEN is the most imperative environmental
carcinogen surrounded by nitrosamines and primarily induces
2.7. Serum liver enzymes and hepatocarcinogenesis marker assays
tumours of liver [20]. The existence of nitrosamines and their
precursors in human environment, simultaneously with the
The liver marker enzymes aspartate aminotransferase (AST)
possibility of their endogenous formation in human body from
and alanine aminotransferase (ALT) were assayed according to the
ingested secondary amines and nitrites, have led to the suggestions
method of [22]. Alkaline phosphatase (ALP) was measured by the
of their potential involvement in HCC [21]. It is now extensively
method of [22]. Lactate dehydrogenase (LDH) was determined by
used as a standard experimental model for HCC [16].
[22] and gamma glutamyltranspeptidase (gGT) were assayed by
the method of [23].
2.4. Experimental design and treatment schedule

2.8. Antioxidant status in liver

The experimental Wistar albino rats were distributed into six
groups, each group containing six animals, analyzed for a total
Superoxide dismutase (SOD, EC.1.15.1.1) was assayed by the
experimental period of 16 weeks as follows (Fig. 1):
method of [24] and catalase (CAT, EC.1.11.1.6) was measured by the
Group 1: Normal control animals fed with standard pellet diet
method of [25] glutathione peroxidase (GPx, EC.11.1.9) activity was
and water ad libitum.
determined using the method described by [26]. Glutathione
Group 2: Rats received SXC leaf extracts via intra gastric
reductase (GR, EC.1.6.4.2) activity was by the method of [27] and
intubation at a daily dose of 300 mg/kg/b.w. for 16 weeks.
glutathione-S-transferase (GST, EC. 2.5.1.18) activity was estimated
Group 3: Rats had hepatocellular carcinoma induced by
via the method given by [28].
providing 0.01% DEN through the drinking water for 16 weeks.
Group 4–6: Rats received 0.01% DEN, as in group 3, along with
2.9. Lipid peroxidation and oxidative stress markers
SXC leaf extracts via intragastric intubation at a daily dose of
75 mg/kg, 150 mg/kg, 300 mg/kg/b.w. throughout the experimental
Lipid peroxidation (LPO) was measured spectrophotometrically
period of 16 weeks.
by measuring the levels of the serum and tissue LPO by products.
At the end of 16 weeks, experimental animals (n = 6 per group)
Thiobarbituric acid reactive substances (TBARS) were estimated by
were sacrificed by cervical dislocation after an overnight fast. The
the method of [29], lipidhydroperoxides (LOOH) by the method of
blood samples were collected from experimental animals and liver
[30] and conjugated dienes (CD) by the method of [31].

Table 1
Initial and final body weight changes and growth rate of control and experimental rats.

Groups Treatment Initial body weight (g) first week Final body weight (g) last week Weight gain (g) Growth rate (g)
I Control 145.03
10.15a 236.50
18.35a 91.47
9.02a 0.81
0.06a
II SXC 300 mg/kg b.w. 148.50
13.39a 238.55
17.46a 90.47
8.36a 0.80
0.05a
III DEN (0.01%) 146.06
12.03a 190.35
15.88b 53.29
4.41b 0.47
0.02b
IV DEN + SXC 75 mg/kg b.w. 146.17
11.24a 211.98
18.12b 65.81
4.48b 0.58
0.03b
V DEN + SXC 150 mg/kg b.w. 147.51
10.17a 230.83
16.17a 93.32
7.20a 0.74
0.05a
VI DEN + SXC 300 mg/kg b.w. 148.22
10.08a 228.72
17.11a 80.50
7.25a 0.71
0.04a

Values are given as mean SD of each group. Superscript letters (a–b) are used to refer and distinguish the values of the different groups.
Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).
 P. Velu et al. / Biomedicine & Pharmacotherapy 84 (2016) 430–437 433

Table 2
Effect of Solanum xanthocarpum (SXC) on the incidence of cancer nodules.
b
Groups Treatment No. of nodule bearing animals/total no. of animals Incidence of Total number Mean no. of tumours per animal
nodules (%)a of nodules
I Control 0/6 – Nil 0
II SXC 300 mg/kg b.w. 0/6 – Nil 0
III DEN (0.01%) 0/6 100 33 5
IV DEN + SXC 75 mg/kg b.w. 4/6 74.57 25 3
V DEN + SXC 150 mg/kg b.w. 2/6 36.11 13 2
VI DEN + SXC 300 mg/kg b.w. 1/6 16.05 6 1
a
(Number of nodules bearing rats/total number of rats in each group)  100.
b
(Total number of tumour/number of tumour bearing rats in each group).

2.10. Histopathological evaluation
For histopathological examination, liver samples were fixed in
10% formalin and then embedded in paraffin wax. Paraffin
embedded liver blocks were cut into 3–5 mm sections using an
ultramicrotome and processed for haematoxylin and eosin (H & E)
staining. Pathological changes in liver tissue were evaluated under
light microscopy.
2.11. Statistical analysis
Data are presented as the mean
standard deviation (SD). One
way analysis of variance (ANOVA) followed by Duncan’s multiple
range test was used to compare the means of different groups by
using SPSS 17 student’s versions. P < 0.05 was considered to be
statistically significant in all cases.
3. Results
3.1. General observation body and liver weights
There was no difference in food and water consumptions
between groups of control and experimental animals during the
entire period of the study. Tables 1 and 3 show the effect of DEN
and SXC leaves extract on body weight, liver weight and relative
liver weight in control and experimental groups of animals
respectively. The body weight was decreased significantly (P
< 0.05) in DEN-induced animals than that of untreated control
(Group I) animals where as it appeared near normal in animals
were treated with SXC aqueous leaves extract (Group IV, V and VI)
when compared with Group III animals. In Group II animals the
liver weight and relative liver weight is significantly increased
(p < 0.05) when compared with Group I animals and there is no
significant changes observed among (Group II) animals.
SXC aqueous leaves extract decreased on liver incidence and
number of tumour induced by DEN in animals. Table 2 shows the
total number of nodules and number of nodules in liver incidence
bearing animals. The SXC aqueous leaves extract treated groups V
and VI showed a significant decrease in liver incidence and number
of nodules when compared with (Group III) animals.
3.2. Serum liver enzymes and hepatocarcinogenesis marker
Table 4 indicates the effects of SXC plant leaves extract activity
of the serum liver marker enzymes such as AST, ALT, ALP, LDH and
gGT of control and experimental animals. In DEN induced hepatic
cancer bearing Group III animals exhibit remarkable increase
(P < 0.05) in the activity of these liver marker enzymes in the
serum when compared to control (Group I) animals. Whereas they
appeared to be neutralized to near normal in 150 and 300 mg SXC
aqueous leaves extract treated animals. However, the Group II drug
alone animals do not show noticeable changes in these parameters
when compared with the control (Group I) DEN induced animals
treated with SXC aqueous leaves extract (Groups IV, V and VI)
showed a drastic decline in the levels of these liver marker
enzymes when compared with DEN induced animals (Group III).
3.3. Antioxidant enzymes
These anti-oxidant levels were reverted back to near-normal
levels in HCC animals treated with SXC plant leaves extract when
compared with DEN induced animals. Thus, the drug SXC aqueous
leaves extract restored the changes to near-normal by its anti-
oxidant efficiency. Whereas the level of SOD and CAT were
noticeable in animals treated with SXC aqueous leaves extract
(Groups IV and V). Table 5 shows the levels of tissue antioxidant
enzymes (SOD, CAT, GPx, GR and GST) in control and experimental
animals. The control animals (group I) exhibit a near-normal value
of these enzymes whereas HCC induced animals (Group III)
showed significant reduce when compare to other groups. SXC
alone (Group II) given animals highlights the increased levels of
these enzymes when compared to control animals. The group IV
and VI animals restored the changes near to normal by the
potential of antioxidant efficacy of SXC.
3.4. Lipid peroxidation and oxidative stress markers
The changes in the levels of hepatic lipid peroxidation,
hydroperoxides and conjugated dienes in control and experimen-
tal animals are shown in (Table 6). The levels of TBARS, LOOH,
protein carbonyl content and CD were significantly increased
(P < 0.05) in DEN induced animals when compared with normal
control animals. Oral administration of SXC aqueous leaves extract
at different doses 75, 150, 300 mg/kg/bw/day along with DEN
significantly lowered the levels of TBARS, LOOH and CD in the liver
of animals when compared to DEN treated animals. However with
SXC alone treated animals have noticeable changes when
compared with control (Group I) animals.
3.5. Histopathological evaluation
Histopathological changes in the hepatocytes of control and
experimental animals were observed (Fig. 1A & B) the normal
architecture brought out central vein and cell preserved granulated
Table 3
Liver weight changes and relative liver weight of control and experimental rats.
Groups Treatment Liver weight Relative liver weight
I Control 8.33
0.64a 3.52
0.27a
II SXC 300 mg/kg b.w. 8.10
0.54a 3.39
0.25a
III DEN (0.01%) 10.99
1.02b 5.33
0.33b
IV DEN + SXC 75 mg/kg b.w. 10.02
0.90c 4.72
0.30c
V DEN + SXC 150 mg/kg b.w. 8.86
0.56a 3.80
0.27d
VI DEN + SXC 300 mg/kg b.w. 8.75
0.64a 3.79
0.28d
Values are given as mean SD of each group. Superscript letters (a–d) are used to refer
and distinguish the values of the different groups.
Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).
434 P. Velu et al. / Biomedicine & Pharmacotherapy 84 (2016) 430–437

Table 4
Effect of Solanum xanthocarpum (SXC) on the activities of liver function marker enzymes in the serum of control and experimental rats.

Groups Treatment AST (IU/L) ALT (IU/L) ALP (IU/L) LDH (IU/L) gGT (IU/L)
I Control 119.01
9.06a 38.99
2.97a 34.49
2.63a 75.99
5.79a 5.40
0.41a
II SXC 300 mg/kg b.w. 119.02
9.11a 39.01
2.99a 34.50
2.64a 76.01
5.82a 5.41
0.42a
III DEN (0.01%) 276.07
21.03b 102.82
7.83b 95.74
7.29b 143.82
10.95b 14.85
1.13b
IV DEN + SXC 75 mg/kg b.w. 227.23
17.40c 80.40
6.15c 74.14
5.68c 128.67
9.85c 11.49
0.91c
V DEN + SXC 150 mg/kg b.w. 171.90
13.16d 65.05
4.98d 57.49
4.40d 106.32
8.14d 8.35
5.29d
VI DEN + SXC 300 mg/kg b.w. 118.61
9.08a 37.81
2.90a 34.27
2.62a 75.03
5.75a 5.29
0.41a

Values are given as mean SD of each group. Superscript letters (a–d) are used to refer and distinguish the values of the different groups.
Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).

Table 5
Effect of Solanum xanthocarpum (SXC) on liver antioxidant status in control and experimental rats.

Groups Treatment SODR CAT^ GPx€ GR* GST~

a a a a
I Control 1.36
0.09 0.86
0.05 12.56
0.95 16.35
1.24 0.46
0.03a
II SXC 300 mg/kg b.w. 1.37
0.11a 0.87
0.07a 12.57
0.98a 16.36
1.26a 0.47
0.04a
III DEN (0.01%) 0.76
0.06b 0.54
0.04b 7.78
0.59b 7.69
0.59b 0.26
0.02b
IV DEN + SXC 75 mg/kg b.w. 0.81
0.06b 0.61
0.05c 8.83
0.68c 9.16
0.70c 0.28
0.02b
V DEN + SXC 150 mg/kg b.w. 0.92
0.07c 0.70
0.05d 10.09
0.77d 12.18
0.93d 0.36
0.02c
VI DEN + SXC 300 mg/kg b.w. 1.30
0.10a 0.81
0.06a 12.45
0.95a 15.26
1.17a 0.44
0.03a

50% NBT reduced/min/mg protein; ^mmol of H2O2 utilized/min/mg protein; |mmol/mg tissue; *mmol of NADPH oxidized/min/mg protein;
~
mmoles of CDNB-GSH conjugate formed/min/mg protein.
Values are given as mean SD of each group. Superscript letters (a–d) are used to refer and distinguish the values of the different groups.
Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).

Table 6
Effect of solanum xanthocarpum (SXC) on serum TBARS and liver TBARS, liver LOOH, liver CD control and experimental rats.

Groups Treatment Serum TBARSR Liver TBARS^ Liver LOOH| Liver CD€
a a a
I Control 1.65
0.11 1.82
0.13 31.89
2.41 26.71
2.03a
II SXC 300 mg/kg b.w. 1.66
0.13a 1.83
0.14a 31.90
2.43a 26.72
2.05a
III DEN (0.01%) 3.65
0.28b 4.45
0.34b 56.32
4.29b 43.66
3.33b
IV DEN + SXC 75 mg/kg b.w. 2.81
0.22b 3.61
0.28c 48.16
3.69b 38.71
2.96c
V DEN + SXC 150 mg/kg b.w. 2.25
0.17c 2.73
0.21d 39.10
2.99c 32.60
2.50d
VI DEN + SXC 300 mg/kg b.w. 1.53
0.12a 1.71
0.13e 30.95
2.37d 25.82
1.98a

Serum TBARS nmol/mL ^Liver TBARS, |LOOH, €CD Mmol/mg tissue of cell lysate.
Values are given as mean SD of each group. Superscript letters (a–c) are used to refer and distinguish the values of the different groups.
Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).

cytoplasm small uniform nuclei and nucleolus. In this study, the
hepatic histomorphology of DEN induced hepatocarcinogenesis in
animals was clearly abnormal. DEN treated (Fig. 1C) and SXC
aqueous leave extract 75 mg/kg/bw (Fig. 1D), animals induced with
DEN showed many different histological patterns, are extremely
variegated may be seen from hyper chromatic and irregular nuclei
with fibrosis, fatty infiltration, thin walled sinusoids and disar-
rangement with degeneration of hepatocytes with complete loss in
architecture. Cytologically the tumour cells slightly larger have
more irregular and malignant nucleus, mitotic, granular cyto-
plasm. Whereas (Fig. 1E) SXC aqueous leaves extract (150 mg/kg/
bw) treated animals few neoplastically transformed cells and
hepatocytes maintaining near normal architecture with less loss of
architecture. Fig. 1F SXC aqueous leaves extract (300 mg/kg/bw)
treated animals hepatocytes showing near normal structure
(Fig. 2).
4. Discussion
Liver is a vital important organ, playing an essential role in
intermediary metabolism and regulating various physiological
processes in the body [32]. By virtue of its unique vascular and
metabolic features, the liver is exposed to absorbed drugs and
xenobiotic. Drug-metabolizing enzymes detoxify many
xenobiotics but bioactive or increase the toxicity of others [33].
The possible mechanism so far, reported the chemopreventive
potential of natural products include carcinogen detoxification,
suppression of genetic mutation, suppression of cell proliferation,
induction of apoptosis and the modulation of immune system [34].
Effectively, herbal products are widely used in the treatment of
hepatic disorders all over the world.
SXC such a reputed plant prickly diffuse bright green potential
herb and distributed throughout India. In a clinical study, it was
reported that oral administration of SXC aqueous leave extract very
effective to controlling mild to moderate bronchial asthma [35,36].
Organic solvents of this plant were shown to have antispasmodic,
anti-tumour, cardiotonic, hypotensive, antianaphylactic, cytotoxic
and anti-diabetic properties [7,37].
The wide use of SXC in traditional medicine and the presence of
phyto-constituents encouraged us to explore the hepatoprotective
effect against DEN induced hepatocarcinogenesis. The percentage
of tumour beaing animals and tmour size were monitored during
the experimental period in DEN treated and DEN + SXC treated rats.
The status of phase II detoxification agents, antioxidants and lipid
peroxidation were utilized as biochemical end points, to assess the
chempreventive potential of SXC during DEN-induced hepatocar-
cinogenesis.
 P. Velu et al. / Biomedicine & Pharmacotherapy 84 (2016) 430–437 435

Fig. 2. Histopathological examination. The histopathological profiles of representative liver tissues control from various experimental wistar albino rats are shown in A)
Normal untreated rat liver (Group I) showing normal cellular architecture brought outcell with preserved granulated cytoplasm, small uniform nuclei and nucleolus
andnormal hepatocytes. B) Group II SXC aqueous leaves extracts (300 mg/kg bw.) alone treated rat liverdemonstrating characteristics of normal liver. C & D) SXC aqueous
leaves extract (75 mg/kg bw.) (Group III and IV) with DENcontrol group showing loss of architecture many histological pattern are cancerousappearance, extremely
variegated, irregular sinusoids, hyperchromatic, irregularnucleic with fibrosis, fatty acid infiltration, and disarrangement with desertion, venouscongestion, neoplastically
transformed hepatocytes. E) Liver section from SXC aqueous leaves extract (150 mg/kg bw.) + DEN showingmoderate improvement of hepatic histopathology over group V. F)
Liver section from SXC aqueous leaves extract (300 mg/kg bw.) + DEN showinghepatocytes maintaining near normal architecture group VI.

Our findings demonstrate that SXC leaf extracts exerts its
tumour inhibitory effects by modulating antioxidants and lipid
peroxidation in the target organ as well as in the liver, and plasma.
Among these doses tested, the medium dose 150 mg/kg b.w.
showed significant anti-oxidant enhancing and tumour suppress-
ing effects compared to other two doses.
The result of the present study is consistent with anticarcino-
genic properties of SXC products reported in literature. However
some chemicals prevented cancer by enhancing anti genotoxic
activity. For example, apigenin, lupenol and solamargine exert a
chemopreventive effect against cancer through the anti-genotoxic
potential against anti-cancerous drugs from SXC aqueous leaves
extract [1].
In DEN treated animals, tumour formation (100%) with mean
tumour incidence, volume and burden was observed. Hepatic
tumour was observed in DEN treated animals with SXC leaves
extract, it was dis-appeared. Oral administration of SXC leaves
extract completely prevented tumour incidence, volume and
burden that which is appeared in DEN treated animals. In this
study and findings of the pathological as well as neoplastic changes
were documented that DEN treatments showed loss of architec-
ture many histological pattern are cancerous appearance, ex-
tremely variegated, irregular sinusoids, hyperchroamtic, irregular
nuclei with fibrosis, fatty acid infiltration and disarrangement with
desertion, venous conjection and neoplastically transformed
products.
In this present studies have shown that hepatic metabolism of
DEN generates ROS resulting in oxidative stress and cellular injury
[38]. High serum concentrations of transaminases are taken as an
index of hepatic injury where elevation of ALT is regarded as a more
sensitive indicator [39] and this tendency is also known to be
distinct in rodents [40]. Hepatic damage is clearly evidenced by the
marked elevation in the activity of serum AST, ALT, ALP, LDH and
gGTa simultaneous fall in the liver tissue [41]. Elevated activity of
transaminases in serum observed in this study might be due to the
release of these enzymes from the cytoplasm, into the blood
circulation rapidly after rupture of the plasma membrane and
cellular damage in hepatocyte. This is substantiated by a
simultaneous fall in the activity of these markers in the liver
tissue. The discharge of LDH reflects a nonspecific alteration in the
plasma membrane integrity and/or permeability. LDH is a familiar
sensitive marker of solid neoplasm [42] and many studies revealed
increased LDH activity in various types of tumour [43]. gGT is an
enzyme embedded in the hepatocyte plasma membrane, mainly in
the canalicular domain; again the release of this enzyme into
serum indicates damage to the cell and thus injury to the liver. It is
pointing out that serum gGT activity is considered to be one of the
best indicators of liver damage [44]. It is reported that SXC aqueous
leaves extract helps in parenchymal cell regeneration in liver, thus
protecting membrane integrity and thereby minimizing enzyme
leakage [45].
Oxidative stress caused by ROS has been reported in membrane
lipid peroxidation, DNA damage and mutagenesis associated with
various stages of tumour formation process [46] and main
mechanism behind the DEN initiated promoted hepatocarcino-
genesis. ROS may cause lipid, protein or DNA damage, leading to
genetic mutations, chromosome instability and/or modulation of
cell growth that may result in cancer and also potentially
dangerous by-products of cellular metabolism that have direct
effect on cell development, growth and survival. Increased cellular
oxidants within the cell leads to the induction of lipid peroxidation,
xanthine oxidase activity, glutathione depletion, decrease in
antioxidant (catalase, glutathione reductase and glutathione
peroxidase) and phase-II detoxifying (glutathione-s-transferase
436 P. Velu et al. / Biomedicine & Pharmacotherapy 84 (2016) 430–437
and quinone reductase) enzyme activities [47]. Antioxidants
possess the induction of drug metabolizing enzymes, inhibition
of carcinogen induced mutagenesis and scavenging of free radicals
[48].
Endogenous antioxidant system may counteract with the ROS
and reduce the oxidative stress with the enzymic antioxidants
SOD, CAT and GPx. SOD and CAT provide the first defense against
oxygen toxicity by catalyzing the dismutation of superoxide anion
to hydrogen peroxide and the decomposition of hydrogen peroxide
to water and molecular oxygen was carried out by GPx [49]. GPx is
a selenoprotein endogenous anti-oxidant enzyme present in the
cytosol and mitochondrial matrix that participate in the defense
mechanism. Running down in the activity of these three
intracellular antioxidant enzymes can be owed to an enhanced
radical fabrication during DEN and Phenobarbital metabolism.
Earlier, it was reported that there was a decrease in the activities of
enzymatic anti-oxidants during hepatoma conditions [50].
Tumour cells have been reported to sequester essential anti-
oxidants from the circulation, in order to meet the demands of the
growing tumour [51]. Previous report revealed that these non-
enzymatic anti-oxidants levels were also decreased in hepatoma
bearing experimental animals [52]. In the present study, tumour
bearing rats showed diminish activities of enzymatic antioxidants
(SOD, CAT, GPx, GST, and GR) and non-enzymatic antioxidants
(GSH, Vit E and Vit C) in both plasma and liver tissue of findings
data are correlates with previous finding [53,54].
Measurement of plasma TBARS in pathological conditions could
help to assess the extent of tissue damage. Increased levels of
plasma TBARS could therefore be over production of ROS in the
cancerous conditions with subsequent leakage in rats treated with
DEN. In the present study, the elevation of LOOH in liver of animals
treated with DEN was observed. The increase in LPO levels in liver
suggest enhances lipid peroxidation leading to tissue damage and
failure of antioxidant defense mechanisms to prevent the
formation of excessive free radicals. Thus, SXC aqueous leaves
extract can be evidenced for its free radical scavenger potency,
extinguishing LPO and a proficient to safeguard the membranes
from free radicals mediated cellular damage. Increased level of LPO
was recently reported during DEN-induced HCC. This dynamic
action may further lead to uncompromised production of free
radicals overwhelming the cellular antioxidant induced HCC. LPO
generation at the initiation stage can be prevented by free radicals
scavengers and antioxidant action of SXC aqueous leaves extract,
animals treated with SXC aqueous leaves extract (75, 150 and
300 mg/kg b.w.) exhibited significantly lowered the levels of LPO,
both in liver and serum, when compared with animals induced
with DEN. This shows the anti-lipid peroxidative role of SXC
aqueous leaves extract that is probably mediated by its ability to
scavenge free radical generation.
In the present investigation, there was a significant increase in
the level of TBARS, LOOH and CD in the liver of DEN exposed
animals which confirm the onset of hepatic oxidative stress.
Decreased levels of these oxidative stress markers in the liver of
DEN treated animals administered with SXC aqueous leave extract
revealed the radical scavenging activity which could be due to the
presence of OH group in their compounds structure [55].
Histopathological view of reactions in DEN treated group
showed the degeneration, many different histologic patterns are
extremely variegated and loss of architecture when compared to
control group. Neoplastically transformed cells and hepatocytes
maintaining moderate changes were considerably mild in the
groups treated with DEN + SXC 75 mg and SXC 150 mg i.e. animals
treated with SXC 300 mg/kg/b.w. showed mild in the group while
hepatocyte changes (Fig. 2).
5. Conclusions
In conclusion, the results of our present investigation demon-
strated that SXC aqueous leaf extract exerts a chemopreventive
effect against experimentally induced in vivo hepatic cancer in a
dose dependent manner in rats. The dose responsive chemo-
preventive properties of SXC have been reflected in its ability to
abrogate the development of preneoplastic hepatic nodule
formation and restoring the normal biochemical and histological
nature of system. Hence, SXC aqueous leaves extract can be
considered as a potent drug for the treatment of hepatocellular
carcinoma. Further studies are warranted to delineate their
mechanisms of action responsible for the inhibition of hepatic
tumourogenesis.
Conflict of interest
None declared.
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