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2800
Journal of Applied Sciences Research, 8(5): 2800-2806, 2012
ISSN 1819-544X
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLES
Hepatoprotective effect of aqueous leaves extract of Psidium guajava and Zizyphus
spina – christi against paracetamol induced hepatotoxicity in rats.
Enas Ali Kamel Mohamed
Lecturer in Biochemistry and Nutrition Department Women's college, Ain Shams University
ABSTRACT
Medicinal herbs are significant source of pharmaceutical drugs. Latest trends have shown increasing
demand of some medicinal herbs have proven hepatoprotective potential. Therefore, the aim of this study was to
evaluate the hepatoprotective effect of aqueous leaves extract of psidium guajava and zizyphus spina-christi on
paracetamol induced hepatic damage . The hepatoprotective activities of the two extracts were compared with a
known hepatoprotective drug, silymarin. For this purpose, thirty adult male albino rats Wistar strain were
assigned into 5 groups. Group1: normal control group, group 2, group 3 and 4: experimentally induced
hepatotoxicity by oral administration with paracetamol (400mg/kg body) followed by oral administration of
silymarin, Psidium guajva and Zizyphus spina- christi aqueous leaf weight extracts, respectively, group 5:
experimentally induced hepatotoxicity by oral administration with paracetamol. Oral administration of
paracetamol induced liver damage in rats as manifested by a significant rise in serum levels of aspartate
aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphstase(ALP), Lactate dehydrogenase
(LDH), gamma glutamyl transferase( γ-GT) and total bilirubin. Oral administration of psidium guajava and
Zizyphus spina christi - aqueous leaf extracts, significantly attenuated hepatotoxicity induced by paracetamol
resulting in a significant decrease in serum levels of hepatic enzymes marker and total bilirubin .The results also
showed that oral administration of paracetamol induced oxidative stress in liver reflected in a significant
decrease in hepatic superoxide dismutase (SOD) and catalase (CAT) activities. Treatment with aqueous leaves
extract of either psidium guajava or zizyphus spina -christi ameliorated the oxidative stress in paracetamol
hepatotoxic rats and restored normal activities of hepatic antioxidant enzymes. It is concluded that aqueous leaf
extracts of psidium guajava or zizyphus spina-christi possesses good hepatoprotective activity.
Key words: Psidium guajava; Zizyphus spina- Christi; Paracetamol ;Hepatotoxicity; Silymarin;
Antioxidative enzyme.
Introduction
Liver is a vital organ which regulates many important metabolic functions and is responsible for
maintaining homeostasis of the body. It has an immense task of detoxification of xenobiotics, environmental
pollutants and chemotherapeutic agents. Hence, this organ is subjected to variety of diseases and disorders
Rajkpoor et al .(2008). Hepatotoxicity is one of very common ailment resulting into serious debilities ranging
from sever metabolic disorders to even mortility Patel et al. (2008). A number of chemical agents and drugs
which are used on a routine basis produce cellular as well as metabolic liver damage Meyer and Kulkarini
(2001). Paracetamol overdoses in both animals and man has been shown to produce hepatic necrosis Thomas.
(1993) and it has been used as an animal model for hepatotoxicity Visen et al .(1993). Most of hepatotoxic
chemicals damage liver cells mainly by inducing lipid peroxidation and oxidative stress in liver. Carbon
tetrachloride (CCl4) and paracetamol get converted into reactive toxic metabolites by hepatic microsomal
cytochrome P450 and cause hepatotoxicity Buwa et al. (2001).
Herbs play a major role in the management of various liver disorders. A number of plants possess
hepatoprotective property Mansour et al. (2006). One of these plants used traditionally is Psidium guajava Linn.
It is believed in Indian folklore that the water decoction of the leaves of this plant can cure jaundice within three
days. Psidium guajava contains a number of chemical constituents which are reported to posses antioxidant
activities Qian and Nihorimbere (2004). Also, Zizyphus spina-christi is known for its medicinal properties as
liver protective agent, antioxidant , hypoglycemic, hypotensive, anti inflammatory, antimicrobial, antitumor and
as an immune system stimulant Said et al. (2006). The aim of this study was to evaluate the hepatoprotective
activities of aqueous leaf extracts of Psidium guajava and Zizyphus spina-christi against paracetamol induced
hepatotoxicity in rats. The hepatoprotective activity of the two extracts were compared with a standard
hepatoprotective drug, silymarin.
Corresponding Author: Enas Ali Kamel Mohamed, Lecturer in Biochemistry and Nutrition Department Women's college,
Ain Shams University
E-mail: [email protected]
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J. Appl. Sci. Res., 8(5): 2800-2806, 2012
Materials and Methods
Animals:
Thirty adult male Wistar strain albino rats body weight (150g  5 g) were purchased from Animal House
of El-Salam-Farm, Giza, Egypt. After acclimatization, rats were housed individually with constant
environment in controlled stainless steel cages, temperature (25C  5 C) humidity (50%  10%), and light
cycle were held constant 12/12 h. During the experimental period (4 weeks) and throughtout the trail, food
and water were provided ad libitum.
Animal's diet:
The animals were fed on the standard commercial pellets diet NRC, (1995) which obtained from Elandalous
company, Giza. The composition of pellets diet: Protein 18%, fat 4.9% and fiber 3.2%.
Preparation of aqueous extract of psidium guajava leaves:
100g of dried ground leaves powder was steeped in 300 ml of hot distilled water then bolied in water bath
for 1h.The boiled leaves extract were then filtered through whatman No4 filter paper, then the filterate was
stored at 4 C, Roy et al. (2006)
Preparation of aqueous extract of ziziphus spina christi leaves:
100g of the dried ground leaves powder were steeped in 600 ml of hot distilled water and allowed to stand
for 30 min. The extract was pooled and filtered through whatman filter paper, then the filterate was stored at 4
C, Azdu et al . (2001)
Induction of hepatotoxicity:
Hepatotoxicity was induced by oral gavage with paracetamol (400mg/kg body weight) by intragastric tube
Kanchana and sadiq (2011)
Experimental design:
The animals were randomly assigned to five experimental groups (each of 6). All international and local
rules and regulation for handling animals in experiments were followed. The experimental groups illustrated as
follows:
Group1: Healthy rats fed on pellet diet and served as normal controls.
Group2: Healthy rats fed on pellet diet and received paracetamol followed by silymarin (100mg/Kg of body
weight/day) by intragastric tube for 4 weeks Roy et al .(2006).
Group3: Healthy rats fed on pellet diet and received paracetamol followed by aqueous extract of gujava leaves
(0.5 ml /day) by intragastric tube for 4 weeks.
Group 4: Healthy rats fed on pellet diet and received paracetamol followed by aqueous extract of ziziphus
leaves (0.5 ml /day) by intragastric tube for 4 weeks.
Group5: Healthy rats fed on pellet diet and received paracetamol by intragastric tube for 4 weeks.
At the end of the experiment, the animals were anesthetized with diethyl ether after 12 hours fasting and whole
blood samples were taken from hepatic Portal vein. The blood samples left for 15 minutes at 37C for serum
separation, then centrifuged at 3000 rpm for 20 minutes, then sera were separated and kept in plastic vials at 
20C until analyses.
Biochemical assays:
Serum aspartate aminotransferase enzyme activity was determined according to the method described by
Bergmeryer et al. (1978), serum alanine aminotransferase enzyme activity was determined according to the
method described by Bermeryer et al. (1978), serum alkaline phosphatase activity was determined according to
the method described by Bowers and Mccomb (1975), gamma-glutamyl transferase activity was determined in
serum according to the method described by Schumann et al. (2002), Lactate dehydrogenase activity was
determined in serum according to the method described by Seikman et al. (2002) and serum total bilirubin was
determined according to the method as described by Domas et al. (1973). All enzymes activities and total
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J. Appl. Sci. Res., 8(5): 2800-2806, 2012
bilirubin were determined by the International Federation of Clinical Chemistry and Laboratory Medicine
(IFCC) using Dimension clinical chemistry system.
Preparation of liver homogenate:
The liver of the rats from individual group was dissected out, washed with ice-cold saline and weighed and
homogenized in 50 mM phosphate buffer (pH 7.4) using an electronic homogenizer to prepare 10% w/v
homogenate (pH 7.4). The homogenate was centrifuged at 3000 rpm g for 30 min. The supernatants were used
for measuring the activity of enzymes- superoxide dismutase (SOD) and catalase (CAT)
Superoxide dismutase (SOD) activity:
The assay of SOD was based on the ability of SOD to inhibit spontaneous oxidation of adrenaline to
adrenochrome according to the method described by Saggu et al. (1989). To 0.05 ml supernatant, 2.0 ml of
carbonate buffer and 0.5 ml of EDTA were added. The reaction was initiated by addition of 0.5ml of
epinephrine and the autooxidation of adrenaline to adrenochrome at pH 10.2 was measured by following the
change in O.D at 480 nm. The change in optical density every minute was measured at 480 nm against reagent
blank. The results are expressed as units of SOD activity (U/g wet tissue). One unit of SOD activity induced
approximately 50% inhibition of adrenaline.
Catalse (CAT) activity:
The activity assay was based on the ability of CAT to induce the disappearance of hydrogen peroxide
according to the method described by Beers and Sizer (1952). A spectrophotometric method for measuring the
breakdown of hydrogen peroxide by the reaction mixture consisted of 2 ml phosphate buffer (pH 7.0). 0.95 ml
of hydrogen peroxide (0.019 M) and 0.05ml supernatant in final volume of 3 ml. Absorbance was recorded at
240 nm every10 sec for 1 min. One unit of CAT was defined, as the amount of enzyme required decomposing
1µmol of peroxide per min, at 25° C and pH 7.0. The results were expressed as units of CAT activity (U/ g wet
tissue).
Statistical Analysis:
The data were presented as means ±SD. One way analysis of variance (ANOVA) followed by post- hoc
least .Significant difference analysis (LSD) at (p< 0.05) was performed using the statistical package for social
science (SPSS) version 9 to compare all treated groups. Differences were considered to be significant when (p<
0.05).
Results:
Results reported in Table (1) and (Fig.1 and 2) showed that, levels of serum ALT, AST, ALP, γGT, LDH
and total bilirubin were markedly increased in paracetamol -hepatotoxic rats when compared with normal
control rats. oral administration of psidium guajava and Zizyphus spina- christi leaf extracts as well as
standard control drug silymarin significantly reversed these altered levels (P<0.05).
The results also demonstrated that, serum transaminase enzyme activities were significantly decreased in
the rats treated with psidium guajava aqueous leaves extract as compared with rats treated with Zizyphus spinachristi aqueous leaves extract.
The results also indicated that there was no significant difference in the levels of serum ALP, LDH, γGT
and total bilirubin between rat groups treated with pisdium guajava and Zizyphus spina-christi aqueous
leaves extract or silymarin.
Result reported in Table (2) and illustrated in Fig. 3 revealed that, the activities of hepatic antioxidative
enzymes superoxide dismutase (SOD) and catalase (CAT) were significantly decreased in paracetamol
hepatotoxic rats. Treatment with psidium guajava and Zizyphus spina- christi aqueous leaves extract or
silymarin caused a significant increase in antioxidative enzymes activities. Also, non significant difference was
observed between rat groups treated with psidium guajava and Zizyphus spina- christi aqueous leaves extract
or silymarin.
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J. Appl. Sci. Res., 8(5): 2800-2806, 2012
Table 1: Serum levels of ALT , AST , ALP , GGT, LDH activities (U/l) and total bilirubin (mg/dl) in different experimental groups (Data
are presented as mean±SD).
Rat groups
Group 1
Group 2
Group 3
Group 4
Group 5
Parameters
ALT
Abcd
359 31
Abcd
136.6  27
148.4 25.3
155.9 19.3
342  0.91
ab
241.4 14.7
255.2 33.7
263.1  24
272.2  26.27
287.80.18
Abcd
6.40.84
6.80.44
7.20.44
7.4 1.1
11.2 5.71
Abcd
117 15.4
121.6  21.17
142.5  31.3
144.1  11.64
206.6 26.4
a
0.14 5.7
0.188.36
0.16  5.4
0.18  8.36
0.2448.1
compared to: (a): group 1, (b): to group 2, (c): to group 3, (d) compared to group 4
61.61.76
AST
ALP
γGT
LDH
Total bilirubin
Significant difference (P < 0.05):
67.38.8
71.95.26
Abc
127 3.7
a bc
260.2  2
Fig. 1: Serum levels of ALT, AST, ALP and LDH (U/L) in different experimental groups.
Fig. 2: Serum levels of γ GT (IU/µL) and total bilirubin (mg/dl) in different experimental groups.
Table 2: Hepatic Catalase and SOD activities (U/g of liver homogenate) in different experimental groups.
Rat groups
Group 1
Group 2
Group 3
Group 4
Parameters
Catalase activity(U/g of liver
homogenate)
SOD activity(U/g of liver
homogenate)
Significant difference (P < 0.05):
17.944.27
14.6 4.5
17.09  8.3
17.4 2.05
Group 5
abcd
4.84  2.26
abc
13.11 3.5
26.90  5.61
20.71  5.3
23.66  5.15
22.99 6.6
compared to: (a): group 1, (b): to group 2, (c): to group 3, (d) compared to group 4
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J. Appl. Sci. Res., 8(5): 2800-2806, 2012
Fig. 3: Hepatic SOD and CAT activities (U/g of liver homogenate) in different experimental groups.
Discussion:
Liver diseases are mainly caused by toxic chemicals, excessive consumption of alcohol, infections and
autoimmune disorders. Paracetamol toxicity like many other disease conditions is widely believed to involve
the generation of Reactive Oxygen Species (ROS).
Antioxidants have been reported to play prominent roles in prevention of ROS generation Valko et al.
(2006) and by extension may offer protection against paracetamol hepatotoxicity. These antioxidants are most
readily available in edible vegetables and other herbal plants. Hence, the evaluation of medicinal plants or herbs
with free radical scavenging potentials for protective roles against drug induced toxicity becomes relevant.
Large doses of carbon tetrachloride (CCl4) and paracetamol induces hepatic necrosis in humans and
experimental animals Bhattacharrya et al. (2003) .Observation of the preventive effect of some herbs to the liver
damage caused by paracetamol may give an indication of the hepatoprotective effect of these herbs in general.
From the results of the current study it is clear that oral paracetamol administration induced hepatotoxicity
in rats. Paracetamol hepatotoxicity was manifested by significant increase in serum liver enzymes ALT, AST,
ALP, γ GT, LDH activities and serum total bilirubin. Elevated levels of serum enzymes are inductive of
cellular leakage and loss of functional integrity of cell membrane in liver. Treatment with psidium guajava and
Zizyphus spina-christi aqueous leaf extracts or silymarin significantly reduced these elevated levels of the
enzymes towards the respective normal values indicating stabilization of plasma membrane as well as repair of
hepatic tissue damage induced by paracetamol.
Bilirubin is one of the most clinical clues to the severity of necrosis and its accumulation is a measure of
binding, conjugation and excretory capacity of hepatocyte Manokaran et al. (2008).In the present study
paracetamol hepatotoxic rats showed a significant increase in the level of serum total bilirubin when compared
with control rats. Oral administration of aqueous leaves extract with psidium guajava and Zizyphus spina-christi
or silymarin significantly restored serum total bilirubin level near normal value. This decrease in serum bilirubin
after treatment with these extracts in liver damage induced by paracetamol, indicated the effectiveness of these
extracts in normal functional status of the liver.
The results of the previous studies of (Gutierrez and Solis 2009; Mayuren, 2010 and Kanchana and sadiq,
2011) supported the results of the current study which confirmed that, paracetamol administration induced
hepatotoxicity and signficantly altered hepatic enzyme markers and total bilirubin levels in serum .
The enzymatic antioxidant defense system is the nature protector against lipid peroxidation. SOD, CAT
and GPx enzymes are important scavengers of superoxide ion and hydrogen peroxide. These enzymes prevent
generation of hydroxyl radical and protect the cellular constituents from oxidative damage Scott et al . (1991).
In the present study, it was observed that, Psidium guajava and Zizyphus spina-christi aqueous leaf extracts or
silymarin significantly prevented the diminution in the activities of hepatic SOD and CAT in paracetamol
induced liver damage rats. These findings confirmed that Psidium guajava and Zizyphus spina-christi aqueous
leaf extracts or silymarin can reduce reactive free radicals that might lessen oxidative damage to the tissues and
improve the activities of the hepatic antioxidant enzymes.
These results are in accordance with the results of (Das et al., 2007, Farghaly and Huessien, 2010 and
Marzouk et al., 2011) which demonstrated that, the activities of antioxidative enzymes SOD and CAT
significantly decreased in paracetamol hepatotoxic rats.
The hepatoprotective effect of Psidium guajava aqueous leaves extract against paracetamol hepatotoxicity
was reported by (Roy et al., 2006 and Roy and Das, 2010) which supported the results of this study. Also, the
hepatoprotective effect of silymarin against paracetamol hepatotoxicity was confirmed by previous finding of
(Kang et al., 2004 and pradeep, 2007). Moerover, results reported by (Amin and Ghoneim, 2009) are simliar to
the results of the current study which reported that , oral administration of Zizyphus spina-christi aqueous
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J. Appl. Sci. Res., 8(5): 2800-2806, 2012
leaves extract ameliorated liver injury judged by reduced ALT and AST activities in serum. In addition, it
retained control activities of endogenous antioxidant such as SOD and CAT in liver.
Conclusion:
In conclusion, the results of this study demonstrate that, both Psidium guajava and Zizyphus aqueous leaf
extracts exhibit a potent hepatoprotective action against paracetamol- induced hepatic damage in rats. It is
possible that , the mechanism of hepatoprotective action of Psidium guajava and zizyphus aqueous leaf extracts
may be due to their antioxidant and free radical scavenging properties.
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