Efficiency of cape gooseberry in attenuating some biochemical disorders and oxidative stress associated with hepatocellular carcinoma

Vol 22, No. 11;Nov 2015
62 office@multidisciplinarywulfenia.org
Efficiency of cape gooseberry in attenuating some
biochemical disorders and oxidative stress associated
with hepatocellular carcinoma
Hanaa M. Serag*, Hanaa A. Hassan, Makwan S. Qadir
Physiology Division, Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt
* E-mail: dr.hanaaserag@gmail.com
Abstract
Hepatocellular carcinoma (HCC), also called malignant hepatoma is the most common type of liver
cancer. The aim of the present study is to elucidate the role of naturoceutical agent such as cape
gooseberry (CG) (Physalis peruviana) as a chemo-sensitizer for adriamycine (ADR) treatment the
hepatocellular carcinoma rats model. The present data recorded that HCC rats has a significant
increase in serum and hepatic lipids profile (TL, TC, TG, LDL-C and VLDL-C) accompanied with a
significant decrease in HDL-C level. Also, total protein (TP) and zinc (Zn) and copper (Cu) levels
were decreased. Moreover, the data illustrated a marked increase in total bilirubin, AFP level as well as
enzymes (AST, ALT, ALP, γ-GT) activity, however hepatic ALT and AST activity was significantly
decreased in HCC rats. In addition HCC rats has a significant increase in hepatic oxidative stress
markers (MDA & NO) and free radicals enzymes (AO & XO), but hepatic antioxidant status including
GSH, TAC, SOD, catalase, and GSH-PX was significantly reduced in HCC rats. On the other hand
HCC rats received ADR or CG showed an improvement in all the above parameters through the
occurred amelioration of the obtained alterations of lipids profile, liver function enzymes, oxidative
stress and antioxidant defense system. Furthermore the data evidenced that CG is more effective than
ADR and it act as a chemo-sensitizer for ADR treatment of hepatocellular carcinoma.
Keywords: Hepatocellular carcinoma, Cape gooseberry (Physalis peruviana), Liver functions,
Oxidative stress, Antioxidant defense mechanism
List of abbreviations:
TL, Total lipids; TC, Total cholesterol; TG, Triglyceride; HDL-C, High density lipoprotein- cholesterol; LDL-C, Low density
lipoprotein- cholesterol; VLDL-C, Very Low density lipoprotein- cholesterol; AST, Aspartate aminotransferase; ALT, Alanine
aminotransferase; ALP, Alkaline phosphatase; γ-GT, Gama-Glutamyle Ttransferase; AFP, Alpha –Fetoprotein; MDA,
Malondialdehyde; NO, Nitric oxide; AO, Aldehyde oxidase; XO, Xanthine oxidase; SOD, Superoxide dismutase; GSH,
Glutathione; TAC, Total antioxidant ; capacity; GSH PX, Glutathione peroxidase; CG, Cape gooseberry

Vol 22, No. 11;Nov 2015
1. Introduction
Hepatocellular carcinoma (HCC) represents the most common primary malignancy of the liver.
According to epidemiological survey, the prevalence of HCC ranks the sixth among all cancers. HCC
accounts for approximately 700,000 cancer-related deaths per year, which ranks thethird in global
cancer statistics (Jemal et al., 2011). Most cases of HCC are secondary to either a viral hepatitis
infection (hepatitis B or C) or cirrhosis (alcoholism being the most common cause of hepatic cirrhosis).
According to recent reports, the incidence of HCC has increased sharply in the last decade, especially
in Egypt, where there has been a doubling of the incidence rate during the last 10 years. This sharp rise
has been attributed to several factors such as biological including hepatitis B and C virus infection,
endemic infections in the community as schistosomiasis as well as environmental factors including
aflatoxin (Arzumanyan et al., 2013). Other factors such as cigarette smoking, occupational exposure to
chemicals as pesticides may also contribute to the etiology or progression of the disease (Anwar et al.,
2008). Liver carcinogenesis may also develop through the progressive accumulation of different
mutations (genetic) and/or gene products (protein), which eventually lead to malignant transformation
(MacPhee, 1998; Benzie, 2003 and Seufi et al., 2009).
Although, the diagnosis and treatment of HCC have significantly been improved in recent years, the
prognosis remains poor (El-Serag, 2011). Systemic chemotherapies have been evaluated for many
years. Anticancer drugs are widely used against variety of human cancer. However, while they generate
acceptable outcome in chemotherapy of some cancers, they also exhibit severe toxicity and undesirable
side effects (Minami et al., 2010). Adriamycin (an antibiotic isolated from Streptomyces peucetiusvar)
is considered one antitumor drug commonly used as single and in combination with other
chemotherapeutic agents, for treatment of wide range of human malignancies (Stewart and Ratain,
2001), such as multiple myeloma (Alberts and Salmon, 1975), osteogenic sarcoma (Wang et al., 1971),
lymphocytic leukemia (DiMarco et al., 1969), HCC patients (Carr and Nagalla, 2010) and stomach
cancer (Ogawa, 1985). Due to the increasing awareness of the side effects of conventional medicine,
the use of natural products as an alternative to conventional treatment in the healing and treatment of
various diseases, including cancer, has been increasing in the last few decades (Kaefer and Milner,
2008). Several herbal drugs have been evaluated for their potential to protect the liver against
hepatotoxicity (Ramakrishnan et al., 2006).The medicinal value of plants have assumed an important
dimension in the past few decades owing largely due to the discovery as a rich source of antioxidants
that combat oxidative stress through their redox active secondary metabolites (Hassan et al., 2014).

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Among these plants, cape gooseberry (Physalis peruviana), cape gooseberry (CG) is an annual,
herbaceous plant which belongs to Solanaceae family, its fruit, also known as golden berry, ground
cherry and in Egypt called harankash (Figure 1). The fruit of cape goosberry is highly nutritious,
having high levels of essential minerals, magnesium, calcium, potassium, sodium and phosphorus
which are classified as macronutrients, while the Iron and Zinc are considered as micronutrients
(Szefer and Nriagu., 2007);vitamins A, B and C. The main active components of vitamin A in fruits are
α-carotene, β-carotene and β cryptoxanthin. Moreover it contains biologically active components e.g.
physalins, withanolides, phytosterolsand polyunsaturated fatty acids e.g. linoleic acid andoleic acid. Its
bioactive compounds have nutritional value, medicinal properties as well as strong antioxidant
property that can prevent peroxidative damage of liver microsomes and hepatocytes (Dinan et al., 1997
and Wang et al., 1999). Cape gooseberry fruit and other aerial parts are used in the treatment of
intestinal and digestive problems and used as mutagenic, anticoagulant, antispasmodic, antileucemis
agents (Shariff et al., 2006 and Helvaci et al., 2010). Epidemiological studies from other parts of the
world indicate that increased consumption of fruits and vegetables are associated with lower risk of
cancer, leukemia, hepatitis and various chronic degenerative diseases (Reddy et al., 2010; Zhang et al.,
2013).
So, the aim of the present study is to elucidate the potential role of cape gooseberry (Physalis
peruviana) as a chemo-sensitizer for adriamycine treatement of experimental hepatocellular carcinoma
rats model.
2. Materials and methods
2.1. Animals
The healthy male albino rats (Rattus rattus), 8 weeks old, weighing 150-170g were used in this study.
Rats housed in a stainless steel cages in a windowless room with automatically regulated temperature
(22-25°C). They were kept under good ventilation under a photoperiod of 12 h light: 12 h darkness
schedule with lights-on from 06.00 am to 18.00 am. They were received a standard laboratory diet
composed of 60% ground corn meal, 15% ground beans, 10% bran, 10% corn oil, 3% casein, 1%
mineral mixture and 1% vitamins mixture, purchased from Meladco Feed Company (Aubor City,
Cairo, Egypt) and supplied with water ad libitum throughout the experimental period. Animals
received human care and the present study complies with the instruction’s guidelines. The local
committee approved the design of the experiments, and the protocol conforms to the guidelines of the
National Institutes of Health (NIH).

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2.2. Chemicals and drugs
Diethylnitrosamine (DENA) and CCl4were purchased from Sigma Chemical Co. (St. Louis, MO,
USA). DENA was freshly dissolved in sterile 0.9 % saline and given to rats at a single dose of 200
mg/kg body weight (Sarkar et al., 1997). CCL4 was given to rats at a dose of 3ml/kg/ body
weight/week (Subramanian et al., 2007; Singha et al., 2009). Adriamycin (ADR) was purchased from
Tarshouby Pharmacy, Mansoura , Egypt and subjected at a dose of 2 mg/kg body weight once per
week (Sakr et al., 2011).
2.3. Preparation of cape gooseberry juice
Cape gooseberry (Physalis peruviana) was purchased from local markets, Mansoura, Egypt. Fruits
were first washed thoroughly to remove impurities. After washing the fruits, they were cut into small
pieces, freshly prepared juice in the blender (500g Cg juice up to 500ml distled water, where each 1 ml
juice contains 1g Cg). The juice of cape gooseberry (Cg) (1 ml/kg body weight) was shaken well just
before oral administration by gavage (Arun and Asha, 2007).
2.4. Experimental design
After two weeks of acclimatization, the rats were divided into five groups 6 rats each group. Group
1(Control): the rats of this group did not receive any treatments. Group 2 (HCC): the rats treated with a
single intraperitoneal injection of freshly DENA (200 mg/kg body weight). Two weeks later, they
received a subcutaneous injection of CCl4 (3 ml/kg/week) for 10 weeks to promote the carcinogenic
effect of DENA. Group 3: (HCC+ADR): HCC rats were injected intraperitonealy with ADR at a dose
of 2 mg/kg body weight, once per week for 10 weeks. Group 4: (HCC+CG): HCC rats were daily
administered cape gooseberry (Cg) juice at a dose of 1 ml/kg/bw. Group 5: (HCC+CG +ADR): HCC
rats were injected intraperitonealy with ADR at a dose level of 2 mg/kg body weight, once per week
and were administered with cape gooseberry (CG) at a dose of 1 ml/kg/bw.
2.5. Blood and liver sampling
At the end of the experimental period (12 week), fasted overnight rats were sacrificed by cervical
dislocation and blood samples were collected into clean centrifuge tube and subjected to centrifugation
at 860 Xg for 20 min at 4O
C. The separated sera were frozen at – 20O
C for further analysis. The rats
were dissected and the livers were immediately excised, rinsed with ice-cold saline, blotted dry and

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accurately weighed. They were minced and homogenized in ice cold buffered saline (10% w/v). The
homogenates were centrifuged at 860 Xg for 10 min at 4 °C. Finally the supernatants were subjected to
the biochemical analysis.
2.6. Biochemical analysis
Total lipids level was estimated according to the technique of Zollner and Kirsch (1962). Total
cholesterol and triglycerides levels were estimated using kits (Biodiagnostic for laboratory services,
Egypt.) according to Allain et al. (1974) and Fassati and prencipe (1982), respectively. High-density
lipoprotein cholesterol (HDL-C) was determined by using enzymatic colorimetric method of
Lopez,(1977). Low-density lipoprotein choleseterol (LDL-C) was calculated according to the
following equation: LDL-C = TC– HDL-C – TG/5, as described by Friedewald et al. (1972). Very
low-density lipoprotein (VLDL-C) was calculated according to the following equation:
VLDL-C=TG/5, as described by Satheesh and Pari (2008). The levels of total protein , total bilirubin,
Zn and Cu were estimated using instructions of kits supplied by Bio-diagnostic Co., Mansoura, Egypt
as described by Henry (1964); Jendrassik and Grof (1938); Hayakawa and Jap (1961) and Ventura and
king, (1951), respectively.
AFP level was estimated by immunoenzymatic colorimetric method according to Acosta et al. (1983).
Aspartate transaminase (AST) alanine transaminase activity (ALT) and Alkaline phosphatase (ALP)
activity was measured using colorimetric Kits purchased by ABC diagnostic kit, Cairo, Egypt as
described by Reitman and Frankel (1957) as well as Belfield and Goldberg (1971),respectively.
Gamma-glutamyl transpeptidase (γ-GT) activity was estimated according to the method of Szasz (1969)
using γ-GT-kit purchased from SGM Diagnostic division, Roma, Italy. The content of
malondialdehyde (MDA) was estimated according to the colorimetric method of Ohkawa et al. (1982).
Nitric oxide (NO) level was determined by colorimetric method of Montgomery and Dymock (1961).
Hepatic aldehyde oxidase (AO) xanthine oxidase (XO) activity was assayed as described by the
method of Johnson et al. (1984) and Stripe and Della Corte (1969), respectively. Glutathione content
(GSH) was adopted by Beutler et al. (1963).Total antioxidant capacity was determined according to
the technique of Koracevic et al. (2001) using colorimetric Biodiagnostic Com. Kit (29 Tahreer St.,
Dokki,Giza, Egypt). Superoxide dismutase (SOD) activity was assayed by the procedure of Nishikimi
et al. (1972) .Catalase activity was determined by the colorimetric method of Aebi (1984). Glutathione
peroxidase (GPX) activity was measured by a colorimetric method of Paglia and Valentine (1967).

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2.7. Statistical analysis
Results were expressed as means ± SE. Statistical significance was calculated using one- way analysis
of variance (ANOVA) followed by Duncan′s multiple range tests (Waller and Duncan, 1969). All the
statistical analysis was carried out with the use of SPSS 12.00 software. Differences were considered
significant at P ≤0.05.
3. Results
The present data showed that HCC rats have a significant increase in serum and hepatic lipid profiles
(TL, TC, TG, LDL-C and VLDL-C) levels but only serum HDL-C level was significantly decreased if
comparing to control rats group (table 1). Also, total protein content as well as Zn and Cu level was
significantly decreased in HCC rats group compared to the control rats. Additionally, an increase was
recorded in serum total bilirubin, AFP, AST, ALT, ALP, γ-GT, hepatic ALP and γ-GT in HCC rats if
comparing to control rats group. However hepatic ALT and AST was significantly decreased in HCC
rats comparing to control rats group (table 2). Moreover, the present data indicated that HCC rats have
a significant increase in hepatic MDA and NO level as well as AO and XO enzymes activity associated
with a marked depletion in hepatic GSH content and TAC as well as SOD, catalase, and GSH-PX
enzymes activity if comparing to control rats group (table 3). Moreover, the data presented in table 1, 2,
3 illustrated that ADR treatment showed little amelioration of the obtained alterations of the liver
indicative biomarkers. On the other hand rats that received CG in single or in combination with ADR
showed marked improvement in all the above parameters as evidenced by upgrade the adverse changes
of lipid profiles, liver function enzymes, oxidative stress and antioxidant defense system.
4. Discussion
Hepatocellular carcinoma is the most frequent primary malignancy of the liver, and it accounts for as
many as one million deaths worldwide in a year. In some parts of the world it is the most common
form of internal malignancy and the most common cause of death from cancer (Jemal et al., 2005).The
main obstacles for systemic chemotherapy in HCC include chemoresistance of HCC cells, and
intolerance to the cytotoxic drugs in cirrhotic patients (Wang et al., 2010). Adriamycin is the most used
single effective agent in HCC (Lai et al., 1988). Traditional medicine, especially the herbal medicine
plays a vital role in the management of various liver disorders. Epidemiological studies have shown
that fruits, vegetables, beverages, spices, tea and medicinal herbs rich in antioxidants and other

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micronutrients protect against diverse forms of chemically-induced hepatic damage, carcinogenesis,
mutagenesis, DNA-damage and lipid peroxidation (Wattenberg, 1990 ; Hassan and Abdel-Aziz, 2010).
Liver is a major organ, regulates many important metabolic functions, and any injury causes distortion
of these metabolic functions. The liver is responsible for the metabolism of drugs and toxic chemicals,
and therefore is the primary target organ for nearly all toxic chemicals (Wolf, 1999). Most
apolipoproteins endogeneous lipids, and lipoproteins are synthesized in the liver, and depend on the
integrity of the cellular functions of this organ. Under normal physiological conditions, the liver
ensures homeostasis of lipid and lipoprotein metabolism. Hepatocellular carcinoma impairs this
process leading to alterations in the lipid and lipoprotein patterns (Jiang et al., 2006). A marker is
synthesized by the tumor and released into the circulation, but it also may be produced by normal
tissues in response to an invasion by cancer cells (Thangaraju et al., 1998). Alteration in lipid profile
manifested by a significant increase in total lipid (TL), triglycerides (TG), total cholesterol (TC), and
low density lipoprotein cholesterol (LDL-C), and a significant decrease in high density lipoprotein
cholesterol (HDL-C), was observed in HCC rats. The present increase of LDL-C, VLDL-C and
decrease of HDL-C in HCC rats model may be due to DENA induced abnormal lipid synthesis or
defective degradation of lipids are implicated in the pathological condition like cancer. Peroxidation of
lipids in biomembranes and tissues causes the leakage of these lipids into circulation and consequently
leads to hyperlipidemia. Hyperlipidemia has been shown to increase the risk of metastasis in several
cancers (Sako et al., 2004). Hepatoma is usually associated with hyperlipidemia as well as a notable
decrease in the high-density lipoprotein (HDL) fraction and an enormous increase in the VLDL and
LDL fractions (Kawasaki et al., 2004). Furthermore, the major metabolic vital organ, liver, plays a
key role in cholesterol metabolism in mammals. Reports reveal hypercholestrolemia associated with
hepatomas. The LDL cholesterol is more susceptible to oxidation in various pathologic conditions
resulting in higher lipid peroxidation (LPO) during oxidative stress (Regnstrom et al., 1992). The
occurred DENA-induced hepatic dysfunction may be attributed to its interaction with mitochondria
electron transport and the energy required for protein synthesis by the hepatocytes depending partially
on the catabolic action of glucocorticoids, which could explain the present decreased total protein
content (Dakshayani et al., 2005). Liver dysfunction is reflected by elevation in serum bilirubin
concentration. Bilirubin is a metabolic breakdown product of hem derived from senescent RBCs and
the increase in its level can be indicative to liver dysfunction (Olubunmi et al. 2011). Thus, the present
alterations in the level of TP and bilirubin may indicate liver dysfunction, which may in turn contribute
to metabolic alterations. Also, the viewed data showed increased AFP in HCC rats. This finding

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confirmed by previous study that suggested an elevated serum level of AFP in the adult animals which
are exposed to hepatocarcinogens (Sell and Beck,1978). It was reported that elevated serum protein
can be observed due to rats exposed to hepatocarcinogens and are frequently associated with HCC. Its
serum concentration can be used to confirm hepatocarcinoma and for the diagnosis of tumor response
to therapy. More than 90% of patients with hepatic cancer have increased serum AFP levels (Maideen
et al., 2012). AFP is a glycoprotein which is normally produced by the fetal liver, yolk sac, and the
gastrointestinal tract. AFP is the most commonly used tumor marker for HCC in clinical practice. It is
easily obtainable and relatively inexpensive (Bruix and Sherman.,2005). Although it is most commonly
elevated in HCC, elevations in serum AFP can be seen in various malignancies including testicular,
bile duct, pancreatic, stomach and colon cancer. Elevated AFP can also seen with non-malignant
conditions including hepatitis and cirrhosis (Di Bisceglie et al., 2005).
Moreover, the observed elevation of liver enzymes activity including AST, ALT, ALP and γ-GT in
HCC rats was supported by Shaarawy et al, (2009). These findings may be due to DENA induced
hepatic damage, hepatic dysfunction and subsequent leakage of these enzymes from the neoplastic cell
into circulation (Dakshayani et al., 2005) or may be due to the release of enzymes from normal tissue
by tumor or may be due to possible effect of tumor on remote tissue leading to the loss of its enzyme
and release into the blood (Schwartz and Bodansky, 1965). Also Rocchi et al., (1997) reported that
there is an increase in the levels of these transaminases activity in serum of HCC patients. In
concurrence with the above findings an elevated serum aminotransferase activity was observed in
animals bearing HCC. ALT, which is mainly produced in the hepatocytes, is more specific for liver
injury (Thomson, 1998). It has been reported that ALT is generally increased in situations where there
is damage to the liver cell membrane (Schumann et al 2002). Thus, when the liver is injured, the levels
of ALT in plasma usually rise (Patel et al 1994). Alkaline phosphatase (ALP) is used as a specific
tumor marker during diagnosis in the early detection of cancer (Kobayashi and Kawakubo 1994). It is
involved in transport of metabolites across cell membrane, protein synthesis, secretory activities and
glycogen metabolism. It is a membrane bound enzyme and its alteration is likely to affect the
membrane permeability and produce derangement in the transport of metabolites (Patel et al., 1994).
The present increased of ALP in HCC rats may be due to the disturbance in secretory activity or due to
altered gene expression under these conditions. The development of a tumor results in tissue damage
that lead to the release of ALP into circulation, these result confirmed by Iqbal et al. (2004). Elevation
of alkaline phosphatase is one of the signs, suggesting space-occupying lesions in the liver (Iqbal et al.,
2004). The rise in the activity of ALP in cancer bearing animals may be due to the disturbance in the

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secretory activity or in transport of metabolites, or may be due to altered synthesis of certain enzymes
in these conditions. Also γ-GT t is over expressed in tumor cells (Bailey et al 2001). It is a
membrane-bound enzyme that exhibits a tissue-specific expression and is influenced by various
physiological and pathological conditions, including fetal liver development and hepatic
carcinogenesis (Yao et al., 2004; Tang et al., 1999). γ-GT has been noted to be useful as a specific
HCC marker, and its sensitivity has been reported to be 74% in detecting any size of HCC and 43.8%
in detecting small HCC (Cui et al., 2004). It is involved in the transport of amino acids and peptides
into cells (Hanigan and Pitot, 1985).γ-GT has been shown to play an important role in the metabolism
of foreign substances, and during cell growth and differentiation (Thusu et al., 1991). The observed
increased of γ-GT in HCC rat may be due γ-GT was strikingly activated during the course of
hepatocarcinogenesis induced by several hepatocarcinogens in animals, these result confirmed by Fiala
and Fiala (1973) who suggested that chemical carcinogens may initiate some systematic effects that
induce γ-GT synthesis (Vanisree and Shyamaladevi, 1998; Farombi et al., 2009).
Other related studies indicated that HCC is associated with oxidative stress, as reflected by increased
lipid peroxides with decreases in the antioxidants. Oxidative stress is known to be associated with
increased lipid peroxidation and reduced antioxidant activity (Manimaran and Rajneesh, 2009). An
elevation of both MDA and No level in HCC rats may be due to oxidative conversion of cellular
poly-unsaturated fatty acids to toxic products known as malondialdhyde (MDA) or lipid peroxides
(Dewa et al., 2009). MDA owing to its cytotoxicity and inhibitory action on cellular protective
enzymes is suggested to act as a tumor promoter and a carcinogenic agent (Manimaran and Rajneesh,
2009). MDA is a major end product of lipid peroxidation which can crosslink with DNA and other
protein molecules, thereby it promotes tumoriogenesis (Luczaj and Skrzydlewska, 2003(. In the
present investigation, an increase in MDA formation was presumably associated with increased ROS,
consistent with the observation that these free radicals reduce the activity of hepatic SOD. These result
confirmed by Robak and Glyglewsi (1988). In addition, the occurred increased of NO in HCC rats may
be due to hepatocytes themselves in response to tissue damage and inflammation induced by various
xenobiotics including CCl4. In addition, its role in oxidative stress cannot be neglected, since high
levels of NO have been associated with oxidative injury via lipid peroxide. (Breikaa et al., 2013). NO
plays crucial roles in inflammation and liver injury (Leung et al., 2011). It is produced in large
quantities by Kupffer cells, endothelial cells, and the (Breikaa et al., 2013). NO is a potent cellular
signal, used in a variety of regulatory physiological pathways. However, increased generation of NO is
considered cytotoxicand can lead to tissue damage (Cooper and Magwere, 2008).

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The observed oxidative stress in HCC associated with the recorded elevation of free radicals enzymes
such as AO and XO. The changes in the activity of aldehydes produced by lipid peroxidation have also
been reported in a variety of tumor cells is a metabolizing enzyme, located in the cytosolic
compartment of tissues in many organisms. AO catalyzes the oxidation of aldehydes into carboxylic
acid, and in addition, catalyzes the hydrozylation of some heterocycles (Gordon et al., 1940). Also XO
produce oxidative stress by generating ROS. XO is the most important cellular source of enzymatic
radicals, XO leads to the formation of oxygen radicals and hydrogen peroxide during the 2 steps of
hypoxanthine and xanthine utilization these enzymes catalyze. The oxidation of hypoxanthine
to xanthine and can further catalyze the oxidation of xanthine to uric acid. These enzymes play an
important role in the catabolism of purines in some species, including humans. Xanthine oxidase is an
enzyme involved in several pathways. Some recent studies had noticed that xanthine oxydase
expression is augmented in milk and lower in colostrums; this fact can be involved in the transition
from colostrum to milk production (Hille, 2005). Free radicals react with body tissues and generate
lipid peroxidation, DNA lesions and enzyme inactivation, thus leading to the alteration and impairment
of function of all cellular components leading to apoptosis (Ciriolo, 2005). In the present study
changes in the activity of catalase (CAT), Glutathione peroxidase (GPx), and Superoxide dismutase
(SOD) glutathione reductase (GRD) were investigated. Antioxidants are substances that either directly
or indirectly protects cells against adverse effects of xenobiotics, drugs, carcinogens and toxic radical
reactions (Sen, 1995). The observed decrease in SOD activity suggests inactivation of the enzyme
possibly due to increased superoxide radical production or an inhibition by the H2O2 as a result of
corresponding decrease in the activity of catalase which selectively degrades H2O2 (El shahat, 2013).
The occurred decreased GSH, SOD and CAT in HCC rat groups may be due to accumulation of lipid
peroxidation that emphasized to increase during carcinogenesis with accompanying reduction in
activity of SOD, CAT and depletion of GSH content, suggesting induction of oxidative stress. SOD is
considered the first line of defense against deleterious effects of oxygen free radicals in the cells by
catalyzing dismutation of superoxide radicals (O2¯) to H2O2 and molecular oxygen. CAT is
responsible for detoxification of H2O2, which is an effective inhibitor of SOD (De Duve and
Baudhhuin, 1996). Kregel and Zhang, (2007) attributed the significant decrease in the activity of SOD
and CAT might be due to the excess of ROS, which interacts with the enzyme molecules causing their
denaturation and partial inactivation. The reduction in the activity of CAT may be due to reflect
inability of tissues to eliminate H2O2. SOD enzyme requires zinc and copper for its antioxidant role
(Roughead et al., 1999). The significant changes in total SOD (MnSOD and CuZnSOD) can be
supported by the present deficiency of serum level of Zn and Cu (being essential for regulating cellular

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redox state) which may be related to the reduction of SOD activity in liver of HCC rats exerting
mitochondrial impairments and dysfunction confirming the above explanations. Zinc is thought to have
its own antioxidant property. It thus induces endogenous antioxidants, such as the metallothionines
(DiSilvestro, 2000). Zinc salts have also been reported to exert radical scavenging properties in vitro
(Bagchi et al., 1997). It seems possible that the increased mean serum Zn levels found with the
supplemental intakes of oats may thus improve the antioxidant status by possibly synthesizing zinc-
containing proteins (Bagchi et al., 1997). Moreover, catalase protects SOD against inactivation by
H2O2, while SOD protects catalase against inhibition by (O2¯). Thus, the balance of this enzyme
system may be essentialto get rid of ROS generated in the tissues. In this concern, GSH represents an
important defense mechanism in protecting cells against ROS (Linder, 1995). The decreased of GSH
in HCC rat may be due to the diminished activity of glutathione reductase (GR) (Pulpanova et al.,
1982).The decrease in GSH content in circulation has been reported in malignant states which may
contribute to increased susceptibility to lipid peroxidation (Pasupathi et al., 2009). Synthesized GSH
can be translocated to enter blood circulation and to be taken by organs possessing γ-GT that helps to
metabolize GSH. It has shown that reduction in activity of γ-GT leads to inability to maintain the
constituent amino acids of GSH, resulting in its loss (Griffith and Meister, 1980). The occurred
decreased GPx in HCC rat may be due to of GPx was owed by an enhanced free radical production
during DENA and CCl4 metabolism (Prince., 2004).
On the other hand, the treatment of HCC has suggested in recent years via chemotherapies. Anticancer
drugs are widely used against variety of human cancer. However, while they also exhibit severe
toxicity and undesirable side effects (Minami et al., 2010). Adriamycin is one of anticancer drug. Two
main mechanisms are proposed to explain the adriamycin cytotoxicity. One suggests that the drug is
bio activated by NADPH cytochrome-P450-reductase and interacts with DNA leading to the inhibition
of both replication and transcription of DNA (Sawamura et al., 1996).Also it can serve as an electron
acceptor from microsomal and nuclear flavoproteins. The other mechanism suggests that adriamycin
induces an oxygen free radical formation causing an oxidative stress in cells and hence nucleic acid
cleavage (Feinstein et al., 1993; Stewart and Ratain, 2001).
There are a number of evidences indicating that natural substances from edible and medicinal plants
exhibited strong antioxidant activity that could act against hepatic toxicity caused by various toxicants
(Hassan et al., 2014 ; Othman et al., 2014). One of those candidate plants is cape gooseberry that has
various bioactive compounds (withanolides and phenolics) (Fang et al., 2012). Some of these

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compounds have a strong antioxidant property and prevent peroxidative damage to liver microsomes
and hepatocytes (Wang, et al., 1999; Hassan and Abdel-Aziz, 2010). Natural antioxidants could
prevent the deleterious effects of toxic agents by scavenging free radicals and other reactive oxygen
species or by modulation of the inflammatory response. Liver-protective herbal drugs contain a variety
of chemical constituents like phenols, coumarins, lignans, essential oil, monoterpenes, carotinoids,
glycosides, flavanoids, organic acids, lipids, alkaloids and xanthones derivatives (Qiu et al., 2007and
Sindhu et al., 2012). The present decreased of TG, LDL-c and VLDL-c in HCC rats received cape
gooseberry may be due to effects of cape gooseberry on lipid profile and HDL-c could be related to
antioxidant activity which might attribute to those identified compounds of cape gooseberry,
flavones, alkaloids, (Osho et al., 2010 ; Osman et al., 2013). Various evidence indicates that many
medicinal plants have been found to be useful to successfully manage hyperlipidemia (Lin et al., 2005;
Rashwan, 2012).This is probably due to the wealth of phytosterols in the cape gooseberry fruit which
induce a decrease in lipoprotein cholesterol levels in total plasma (Ramadan et al., 2011). The
observed decreased of cholesterol in HCC+ cape gooseberry rats may be due to due to the
hypocholesterolemic effects of cape gooseberry are mainly due to the lycopene existing in the plant
which is a strong antioxidant which inhibits the production of LDL and presumably increases the
escritoires through releasing cholesterol; therefore, it reduces blood cholesterol level and controls
cholesterol synthesis (Zarei et al., 2011; Ramadan, 2012). A significant an increase of total protein at
cape gooseberry supplementation may be due to cape gooseberry rich in phenolic compounds and
flavonoids that are widely used as antioxidant (Abd El-Ghany and Nanees, 2010), or may be due to
cape gooseberry contains zeaxanthin and beta cryptoxanthin esters or carotenoid esters which can be
used as food additives or nutraceuticals (Pintea et al., 2005). Also, the decreased of total bilirubin
(Tb) in HCC+ cape gooseberry rats may be due to the prevention of the leakage of intracellular
enzymes by its membrane stabilizing activity according to Chang et al. (2008 ;Osman et al., 2013).
An amelioration of AFP after supplementation of cape gooseberry juice to HCC rats may be attributed
to CG antioxidant activity. Additionally, the observed improvement of the occurred alterations in liver
enzymes including ALT, AST, ALP, and γ-GT in HCC+ cape gooseberry rat groups may be due to
alterations by cape gooseberry juice is a clear indication of the improvement of the functional status of
hepatocytes with preservation of cellular architecture leakage of intracellular enzymes by its
membrane stabilizing activity (Chang et al., 2008; Tatiya et al., 2012; Al-Olayan et al., 2014). The
successful of CG supplementation to HCC rats model in amelioration of oxidative stress and
improvement of antioxidants defense mechanism is consider a good indicator for its anti-lipid
peroxidative property and antioxidant activity through its high levels of antioxidants compounds such

Vol 22, No. 11;Nov 2015
as polyphenols and other like flavonoids ( Abdel Moneim and El-Deib, 2012 ; Hassan and Ghoneim,
2013). Also the observed decreased of MDA and NO in HCC rats received cape gooseberry may be
due to free radicals scavengers, potential mechanism by which cape gooseberry juice can act as
anti-inflammatory as well as the inhibition of the induction of inducible nitric oxide synthase (iNOS)
protein/enzyme and thus protect the liver. Therefore, dietary consumption of cape gooseberry extract
may be used as best potential antioxidant and hepatoprotective in liver toxicity (Rashwan, 2012;
El-Gengaihi et al., 2013). NO plays crucial roles in inflammation and liver injury (Leung et al., 2011).
It is produced in large quantities by Kupffer cells, endothelial cells, and the hepatocytes themselves in
response to tissue damage and inflammation induced by various xenobiotics including CCl4. In
addition, its role in oxidative stress cannot be neglected, since high levels of NO have been associated
with oxidative injury via lipid peroxide (Breikaa et al., 2013; Al-Olayan et al., 2014). Also the
decreased of AO and XO in cape gooseberry administered rat may be due to the presence of the
active compounds in CG that have biological significance in the elimination of reactive free radicals,
aldehydes are highly reactive molecules that may have a variety of effects on biological systems. They
can be generated from a virtually limitless number of endogenous and exogenous sources. Although
some aldehyde-mediated effects such as vision are beneficial, many effects are deleterious, including
cytotoxicity, mutagenicity, and carcinogenicity. A variety of enzymes have evolved to metabolize
aldehydes to less reactive forms. Changes in aldehydes activity have been observed during
experimental liver carcinogenesis and in a number of human tumors, including some liver, colon, and
mammary cancers. Changes in aldehydes define at least one population of preneoplastic cells having a
high probability of progressing to overt neoplasms (Wu et al., 2005; Saada et al., 2010; Osman et al.,
2013). Cape gooseberry supplementation showed a significant antioxidant status as manifested by
elevation of GSH, TAC, SOD, CAT and GSH-PX ase. Many plant secondary metabolites act as potent
antioxidants have shown that free radical scavenger/antioxidant such as superoxide dismutase (SOD),
catalase (CAT), reduced glutathione (GSH) and glutathione peroxidase (GPX), reduce and prevent
the tissue damage induced by different hepatotoxins (Hassan et al., 2014). The first line of defense
against superoxide anion (O2), H2O2 and (OH -
)are the major ROS, which induce, cell degeneration by
increasing LPO of cell membrane lipids are SOD, CAT GPX are the family of metallo enzymes that
convert O2- to H2O2. The toxic end products of peroxidation induce damage of the structural and
functional integrity of cell membranes, break DNA strands and denature cellular proteins. The natural
cellular antioxidant enzymes include SOD, is an important enzyme as because it is found virtually in
all aerobic organisms. O2- is the only known substrate for SOD and it is considered to be a stress
protein, which is synthesized in response to oxidative stress. It scavenges superoxide radicals by

Vol 22, No. 11;Nov 2015
speeding up their dismutation (Kanimozhi and Prasad, 2009; Vásquez-Garzón et al., 2009).
In conclusion, the present data indicated the efficacy of CG juice supplementation as an
anti-hepatocellular carcinoma in addition to its ability as a chemosensitizer for ADR treatment. This is
mediated by intracellular pathways, involving improvement the alterations in liver functions as well as
other aspects of HCC, the suppression of oxidative stress and modulation of antioxidant defense
mechanism. Thus, supplementation with edible CG may help in safe application of cancer technology
in medicine as well as in many other aspects of nowadays life. Fractionation guided evaluation could
help in the development of ideal anticancer in the near future.
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Vol 22, No. 11;Nov 2015
Table 1: Serum and hepatic biochemical parameters in control and different treated rat groups.
Results are presented as means ±E (n=6 for each group).
C: Control, HCC: hepatocellular carcinoma, HCC+ADR: hepatocellular carcinoma+ Adriamycin,
HCC+CG: hepatocellular carcinoma+ Cape gooseberry, HCC+ADR+CG: hepatocellular carcinoma+
Adriamycin + Cape gooseberry. Mean with different letters (a- e) are significantly difference. Mean with the same letters are non-significantly difference.
Animal groups
Serum
HCC+A+CGHCC+CGHCC+AHCCCParameters
512.09
±0.52e
498.1
±0.55d
610.29
±0.72c
768.6
±0.65b
490.69
±0.35a
Mean
± SE
TL
mg/dl
118.52
±1.12e
110.96
±1.14d
126.61
±1.12c
136.0
±1.16b
102.45
±1.05a
Mean
± SE
TC
mg/dl
88.27
±0.69d
86.79
±0.78ad
96.1
±0.76c
126.6
±0.8b
84.31
±0.71a
Mean
± SE
TG
mg/dl
32.52
±0.81ac
33.91
±0.81ac
31.84
±0.79c
25.08
±0.43b
34.67
±0.98a
Mean
± SE
HDL
mg/dl
68.35
±1.16e
59.21
±1.12d
75.57
±1.39c
85.66
±1.44b
50.86
±1.08a
Mean
± SE
LDL
mg/dl
17.65
±0.41a
17.35
±0.43a
19.22
±0.58a
25.32
±0.66b
16.92
±0.49a
Mean
± SE
VLDLmg/dl
6.28
± 0.55ed
6.35
± 0.48d
4.83
± 0.3cb
4.45
±0.2b
7.3
± 0.7a
Mean
± SE
TP
(mg/dl)
1
± 0.03ab
0.95
±0.02a
1.1
± 0.03ab
1.23
±0.06b
0.93
±0.01a
Mean
± SE
T-bilirubin
mg/dl
95.55
± 0.88d
100.65
± 0.86d
80.09
± 0.62c
71.85
± 0.52b
119.78
± 0.93a
Mean
± SE
Zn
(µg/ml)
295.8
± 0.63ac
303.43
± 0.55a
270.81
± 0.51cb
250.84
± 0.25b
310.79
± 0.53a
Mean
± SE
Cu
(µg/ml)
30.27
±0.52d
28.46
±0.15d
35.82
±0.82c
55.08
±1.15b
24.03
±0.46a
Mean± SE
TL mg/g wet
tissue
Liver 120.88
±0.59d
118.57
±0.36a
123.13
±0.36c
128.2
±0.49b
117.24
±0.59a
Mean
± SE
TC mg/g wet
tissue
52.19
±0.52ed
53.93
±0.53d
49.1
±0.38c
63.9
±0.68b
39.29
±0.38a
Mean
± SE
TG mg/g wet
tissue
1.29
±0.02dc
1.95
±0.05a
1.28
±0.03c
1.06
±0.01b
2.05
±0.06a
Mean
± SE
TP
g/g wet tissue

Vol 22, No. 11;Nov 2015
Table 2: AFP and liver function enzymes in control and different treated rat groups.
Animal groupsParameters
Serum
HCC+A+CGHCC+CGHCC+AHCCC
1.25
±0.66a
1
±0.94a
1.33
±0.7a
2.57
±0.28b
0.99
±0.1a
Mean
± SE
AFP
ng /ml
45
± 0.63d
41.83
±0.6ad
49.16
±0.7c
60.5
±0.99b
40.16
±0.62a
Mean
± SE
AST
units/ml
37.33
±1.05acd
33.16
±1.06ad
38
±1.51ac
50.5
±1.76b
34.66
±1.02a
Mean
± SE
ALT
U/ml
105.65
±0.38ec
97.56
±0.5ad
109.4
±0.56c
153.11
±1.68b
93.63
±0.38a
Mean
± SE
ALP
IU/L
28.97
±1.02dc
23.7
±0.41a
27.04
±0.67c
32.02
±1.07b
21.53
±0.5a
Mean
± SE
γ-GT
U/L
16.68
±0.31e
17.8
±0.28d
14.54
±0.21c
15.6
±0.23b
18.77
±0.34a
Mean
± SE
AST U/g wet
tissue
Liver
46.07
±0.54e
57.13
± 0.61d
42.74
±0.56cb
42.41
±0.51b
60.77
±0.67a
Mean
± SE
ALT U/g
wet tissue
40.16
±1.09ec
43.5
±1.1dc
42.5
±1.04c
54.9
±1.14b
33.03
±0.76a
Mean
± SE
ALP IU/g wet
tissue
27.72
±0.85dc
25.84
±0.79acd
26.72
±0.87cb
28.63
±0.89bd
23.82
±0.68a
Mean
± SE
γ-GT
U/g
C: Control, HCC: hepatocellular carcinoma, HCC+ADR: hepatocellular carcinoma+ Adriamycin, HCC+CG: hepatocellular
carcinoma+ Cape gooseberry, HCC+ADR+CG: hepatocellular carcinoma+ Adriamycin+ Cape gooseberry. Mean with different
letters (a- e) are significantly difference. Mean with the same letters are non-significantly difference.

Vol 22, No. 11;Nov 2015
Table 3: Hepatic oxidative stress and antioxidant biomarkers in control and different treated rat
groups.
C: Control, HCC: hepatocellular carcinoma, HCC+ADR: hepatocellular carcinoma+ Adriamycin, HCC+CG: hepatocellular
carcinoma+ Cape gooseberry, HCC+ADR+CG: hepatocellular carcinoma+ Adriamycin+ Cape gooseberry. Mean with different
letters (a- e) are significantly difference. Mean with the same letters are non-significantly difference.
Animal groupsParameters
Liver
HCC+A+CGHCC+CGHCC+AHCCC
530.5520.38581.07701.12512.11Mean
± SE
MDA
nmol/g wet tissue ±0.49a
±0.59a
±0.77c
±0.82b
±0.64a
626.04611.3652.81724.41590.16Mean
± SE
NO
µmol/g ±0.81d
±0.78d
±0.89c
±0.88b
±0.72a
2.28
±0.042e
1.61
±0.03d
3.28
±0.04c
3.67
±0.06b
0.84
±0.02a
Mean
± SE
AO
umol/min
15.83
±0.47e
13.33
±0.45d
18.43
±0.66c
25.13
±0.95b
11
±0.28a
Mean
± SE
XO mmole/hour
/gm tissue
15.9217.8412.7810.0119.11Mean
± SE
GSH mmol/g
wet issue ±0.41d
±0.44ad
±0.35cb
±0.26b
±0.53a
110.18118.12101.195.26128.1Mean
± SE
TAC
mM/g ±0.71d
±0.63d
±0.69cb
±0.47b
±0.78a
817.28841.71836.97416.54871.54Mean
± SE
SOD
U/g wet tissue ±0.63a
± 0.79a
±0.73a
±0.42b
±0.88a
183.52180.68150.28132.3190.7Mean
± SE
Catalase
U/g wet tissue ±0.91ed
±0.64d
±0.59c
±0.23b
±0.85a
781.44797.08760.95745.04801.94Mean
± SE
GSH-PX
U/gt ±1.63e
±1.59d
±1.65c
±1.75b
±1.83a

Efficiency of cape gooseberry in attenuating some biochemical disorders and oxidative stress associated with hepatocellular carcinoma

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