Protective Effects of Green tea (Camelia sinensis), Hypericum perforatum and Urtica dioica on Hepatic Injury and Lymphocyte DNA Damage Induced by Carbon Tetrachloride in Wistar Rats
Muhammet E. Guldur,
Ali Z. Karakilcik
The present study was planned to investigate the protective effects of herbal methanol extracts of Hypericum perforatum (HP) and Urtica dioica (UD) and herbal agueous extract of Camelia sinensis (CS)-containing antioxidants on carbon tetrachloride (CCl4)-induced hepatic injury and lymphocyte DNA damage. Adult male albino rats (n 50) were separated into five groups of ten each, as follows: control group; CCl4 group; CCl4+HP extract group; CCl4+UD extract group; CCl4+CS extract group. All extract groups were fed with 200 mg kg-1 extracts of HP, CS and UD, respectively once every alternate day for 7 weeks. CCl4 injections were applied to the CCl4 and extract groups at the dose of 0.4 mL kg-1. Malondialdehyde, total oxidant status and total antioxidative capacity levels and catalase, superoxide dismutase and glutathione peroxidase activities were significantly changed in the CCl4 group and indicated increased oxidative stress. The DNA damages detected via commet assay were significantly higher than in the CCl4 group. Treatment by extracts of HP, CS or UD were significantly decreased this oxidative stress and ameliorated lymphocyte DNA damage. These results indicate that the herbal extracts of HP, UC and CS have beneficial effects on liver and lymphocyte DNA damage induced by CCl4 probably due to their antioxidant properties.
to cite this article:
M. Bitiren, D. Musa, A. Ozgonul, M. Ozaslan, A. Kocyigit, O. Sogut, Muhammet E. Guldur, I.H. Kilic, Ali Z. Karakilcik and M. Zerin, 2010. Protective Effects of Green tea (Camelia sinensis), Hypericum perforatum and Urtica dioica on Hepatic Injury and Lymphocyte DNA Damage Induced by Carbon Tetrachloride in Wistar Rats. International Journal of Pharmacology, 6: 241-248.
Administration of single or repeated dose of CCl4 in rats is one
of the most common methods to investigate the mechanism of hepatic injury. This
model has been implemented in various studies for the deposition of extracellular
matrix in the fibrotic and cirrhotic liver (Luckey and Petersen,
2001; Mansour et al., 2001; Nakade
et al., 2002). The liver is an important target organ for CCl4
and the hepatocytes which can be damaged by haloalkane free radicals produced
during biotransformation of CCl4 (Comporti, 1985;
Biasi et al., 1991; Weber
et al., 2003; Ozardali et al., 2004).
Herbal medicines derived from plant extracts are being increasingly used to
treat various diseases (Amutha et al., 2010).
They are also used in the management of liver injury due to various factors
such as toxification or infection of the oriental societies (Bailey
and Day, 1989; Banskota et al., 2000; De
Feudis et al., 2003; Takeoka and Dao, 2003).
Hypericum perforatum (HP) or St. John's wort belongs to Clusiaceae family
that is widely used for the treatment of various disorders such as depression,
anxiety or nervousness. Camelia sinensis L. (CS) belongs to Theaceae
family which is a beverage of green tea and very popular in all over the world.
Urtica dioica L. (UD) belongs to Urticaceae family that is widely used
in folk medicine in cancer therapy, inflammatory and rheumatical diseases (Weisburger
et al., 1997; Thangapazham et al., 2007).
The composition of HP, CS and UD include antioxidant substances such as flavonoids
and phenolic acids which may prevent oxidative damage in cells and tissues (Konrad
et al., 2000). Moreover, it has been reported that some herbal extracts
may reduce the liver injury induced by CCl4 probably due to their
antioxidant nutrient (Thangapazham et al., 2007;
Konrad et al., 2000; Zhang
et al., 1996). Therefore, the present study was designed to investigate
the possible protective effects of herbal methanol extracts of Hypericum
perforatum (HP) and Urtica dioica (UD) and herbal agueous extract
of Camelia sinensis (CS)-containing antioxidants on carbon tetrachloride
(CCl4)-induced hepatic injury and lymphocyte DNA damage.
MATERIALS AND METHODS
Plant materials and extraction procedure: The aerial parts of the HP, CS (Green tea) and UD stinging herb were purchased from herbal shop in Sanliurfa,Turkey. The specimens were authenticated by a plant taxonomist and the voucher specimen under No. 127P, 128D, 129S, respectively were kept at the Department of Biology, Faculty of Art and Science for future references. The dried aerial parts of each specimen were well selected, cleaned and powdered via an electrical mill. The methanol extracts of HP and UP were sited in a soxhlet apparatus containing methanol (yield 9.5%) overnight. After that, they were extracted with rotary evaporator and dried with lyofilization equipment.
Experimental design: A protocol for investigation of the protective effects of herbal methanol extracts of Hypericum perforatum (HP) and Urtica dioica (UD) and herbal agueous extract of Camelia sinensis (CS)-containing antioxidants on carbon tetrachloride (CCl4)-induced hepatic injury and lymphocyte DNA damage was introduced on November 2008. The study was approved by Ethics Board of Harran University, School of Medicine in accordance with the Helsinki Declaration. Fifty adult and healthy male albino rats weighing 225±25 g were inserted in the study. The rats were divided into five equal groups (n = 10), housed individual in polyacrylic cages with stainless covers under standard laboratory conditions (12/12 light/dark period, 22±2°C, rat chow pellets and tap water freely available). All rats were cared according to the National Academy of Sciences. Fifty adult and male albino rats were divided into five groups of ten each, as follows: control group; CCl4 group; CCl4+HP extract group; CCl4+UD extract group; CCl4+CS extract group. CCl4 (MerckR) diluted with olive oil 1/4 v/v (SigmaR) injections were applied i.p to the control and extract groups at the dose of 0.4 mL kg-1. After seven weeks of administration with CCl4, all extract groups were fed with 200 mg kg-1 extracts of HP, CS and UD by orogastric gavage, respectively once every alternate day for 7 weeks. At the end of the study, animals were fasted overnight; the blood samples were collected by heart puncturing and poured into a heparinized tube. All of the rats were sacrificed after ether anesthesia. The abdomen of rats were immediately opened. After that, the livers were removed and divided into the different of two parts. The first part was stored at -80°C for subsequent biochemical analyses and the second was fixed into 10% normal formalin over the subsequent 24 h for histopathological examination.
Histopathological evaluation: The fixed portions of the livers were
washed to remove the excess of formalin which were dehydrated in graduated ethyl
alcohol, cleared in xylol, embedded in paraffin (Paraplast FlukaR)
and sectioned as 3-5 μM. The sections were stained with haematoxylin-eosin
(H and E) and Massons trichrome for evaluation of the histopathological
changes. The evaluation of the liver biopsies were performed by conventional
light microscopy and a blinded pathologist examiner. The degree of necrotic
and fibrotic changes within the periportal tracts and lobules of the liver tissues
were assessed according to the method of Zhang et al.
(1996). In this method, four grades were recognized ranging from 0-3 as
follows: 0 (none), no fibrosis and normal liver architecture; I (mild), fatty
degenerations around portal areas and central veins, fibrosis increased in portal
areas and sinusoidal space; II (moderate), thin fibrous septa present connecting
portal areas; and III (severe), thick fibrous septa and collagen bands accompanied
by pseudo lobules.
DNA damage determination by alkaline comet assay: In the study, samples
were processed within 2 h. Lymphocyte isolation for comet assay was performed
using Lymphoprep (Amerco, Axis-Shield, Oslo, Norway). An amount of 1 mL of heparinized
blood was carefully layered over 1 mL of lymphoprep and centrifuged for 35 min
at 25°C, 500x g. The interface band containing lymphocytes were washed twice
with Phosphate-Buffered Saline (PBS) and then collected by 15 min for centrifugation
at 400x g. The cell pellets were resuspended in a small volume of PBS to obtain
approximately 2x104 cells/200 μL. Membrane integrity was assessed
by means of trypan blue exlusion method and revealed membrane integrity in 95%
of cells. Comet assay was performed according to Singh
et al. (1988). Ten microliters of lymphocyte suspension (around 2x104
cells) were mixed with 80 mL of 0.7% low-melting agarose in PBS at 37°C.
Subsequently, 80 mL of mixture was layered onto a slide pre-coated with thin
layers of 1% normal melting point agarose and immediately covered with cover
slip. Slides were left for 5 min at 4°C to allow the agarose to slidfy.
After removing the cover slips, the slides were submersed in freshly prepared
cold (4°C) lysing solution (2.5 M NaCl, 100 mM ethylenediaminetetraacetic
ascid-disodium salt, 10 mM Tris-HCl, pH 10-10.5, 1% Triton X-100) and 10% dimethy
sulphoxide added just before use for at least 1 h. Slides were then immersed
in freshly prepared alkaline electrophoresis buffer (0.3 mol L-1
NaOH and 1 mmol L-1 ethylenediamine tetaaceticacid acid-disodium
salt pH>13) at 4°C for separation of DNA (50 min) and electrophoresed
(300 mA, 25 min). All the steps were carried out under minimal illumination.
After electrophoresis, the slides were stained with ethidium bromide (2 mL mL-1
in H2O; 70 mL slide-1), covered with cover slip and analyzed
using a fluorescence microscope (Olympus, Japan). Images of 100 randomly selected
cells (50 cells from each of two replicate slides) were analyzed visually from
each subject. Each image was classified into five categories according to the
intensity of the fluorescence in the comet tail and given a value of 0, 1, 2,
3 or 4 (from undamaged class 0 to maximally damaged class 4). The extent of
DNA damage was expressed between 0 and 400 arbitrary unit (Collins,
Biochemical analysis in plasma: Blood samples, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities were measured by commercially available kits using an autoanalyser (Aeroset® Abbott Laboratories, Chicago).
Measurement of TAC and TOS in liver tissues: Total Antioxidative Capacity
(TAC) and Total Oxidant Status (TOS) of supernatant fractions were measured
using a novel automated measurement method developed by Erel
(2004, 2005). The results of TAC and TOS activities
are expressed in terms of mmol Trolox Equiv. g-1 protein and μmol
H2O2 Equiv. g-1 protein, respectively. Percent
ratio of TOS level to TAC level was accepted as Oxidative Stress Index (OSI).
The index was calculated according to the following formula by Horoz
et al. (2006).
Determination of MDA, CAT, SOD and GSH-Px activities in liver tissues:
Liver catalase (CAT) activity was determined by Goths colorimetric method,
in which supernatant was incubated in H2O2 substrate and
the enzymatic reaction was stopped by the addition of ammonium molybdate. The
intensity of the yellow complex formed by molybdate and H2O2
that was measured at 405 nm (Goth, 1991). Superoxide
dismutase (SOD) activity was determined using a measurement method developed
by McCord and Fridovich (1969). This method is based
on the generation of superoxide radicals produced by xanthenes and xanthenes
oxidase, which react with 2-(4-iodophe nyl)-3-(4- nitrophenol)-5-phenyltetrazolium
chloride (INT) to form a red formazan dye. SOD activity was expressed as units
per gram protein. Glutathione peroxidase (GSH-Px) activity was measured on standard
assay conditions in 340 nm (absorbance) and at 37°C according to the method
developed by Paglia and Valentine (1967). In this measurement,
GSH-Px catalyzes the oxidation of glutathione by cumene hydroperoxide. Measurements
were performed by an autoanalyzer (Aeroset® Abbott Laboratories,
Chicago) according to the Randox application procedure. GSH-Px activity was
expressed as units per gram protein. Lipid peroxidation (LPO) was evaluated
by the spectrofluorometric method based on the reaction between malondialdehyde
(MDA) and thiobarbutiric acid (Conti et al., 1991).
Statistic analysis: Data were expressed as Mean±SD. All the statistical
analysis were performed using SPSS 11.0 (SPSS Inc., Chicago, Illinois). Kruskal
Wallis tests were used for one-way Analysis of Variance (ANOVA) and post hoc
test, bonferroni respectively (Kruskal and Wallis, 1952).
Differences between groups and pathological scoring values were compared by
χ2 (Chi-Square) test. Differences on statistical analysis of
data were considered significant at p<0.05.
RESULTS AND DISCUSSION
The level of serum liver enzymes (ALT, AST, ALP and LDH) were found to be significantly
increased in CCl4 group when compared with the control group (p<0.01).
A significant increase of the liver enzymes in the plasma was occurred after
carbon tetrachloride administration alone, which was significantly lowered by
treatment with the herbal extracts of HP, CS and UD (p<0.01) (Table
1). The value of TOS and the activity of MDA in the liver tissue were decreased
(p<0.01, p<0.05 respectively) by the treatment of HP, CS and UD than that
of the administration with CCI4. The values of TAC and OSI and the
activities of CAT, SOD and GSH-Px in the liver tissue were increased (p<0.01)
by the treatment of HP, CS and UD than that of the CCI4 group (Table
2). DNA damage in rats treated by CCI4 were significantly higher
than rats treated by the herbal extracts of HP, CS and UD (p<0.01) (Table
2, Fig. 1). Histopathological examination of liver sections
in untreated control group were evaluated as a normal histology (Fig.
2a). It was showed a typical fibrotic and cirrhotic appearance caused by
the effects of CCl4 in about 80% of the rats liver biopsies.
Broad fibro-collagen septa was bridged around portal areas and extended from
central regions to portal areas or pseudo lobule formation without central vein
(Fig. 2b, c).
of ALT, AST, ALP and LDH in the study groups
were presented as Mean±SD. a: Difference between control
and CCl4 group b: Difference between CCI4
and CCI4+HP groups, c: Difference between CCI4
and CCI4+CS groups, d: Difference between CCI4
and CCI4+UD groups. *p<0.05; **p<0.01
of Total Oxidant Status (TOS), Total Antioxidant Capacity (TAC), Oxidative
Stress Index (OSI), lymphocyte DNA damage, malondialdehyde (MDA), catalase
(CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) in
the study groups
were presented as Mean±SD. a: Difference between control
and CCl4 group, b: Difference between CCI4
and CCI4+HP groups, c: Difference between CCI4
and CCI4+CS groups d: Difference between CCI4
and CCI4+UD groups. *p<0.05; **p<0.01
obtained from image of comet assay showing classes of lymphocytes in study
groups. a: Untreated control group, class 0; b: CCl4+CS group,
class 1; c: CCl4+HP group, class 2; d: CCl4+UD, class 3 and e: CCI4 group,
In the extract groups, the liver sections obtained from rats were showed consistent
reduction regarding fibrotic or cirrhotic process compared to the CCl4
group. These specimens showed more regular liver architecture, in which only
thin fibrous bands were seen to connect portal areas (Fig. 2d-f).
Histopathological grading of liver was showed sever fibrosis in CCl4
treated group and less fibrosis in groups treated with HP, CS and UD (Table
grading of hepatic fibrozis in the study groups
Repeated intraperitoneal administration of CCl4 can cause the formation
of free radicals that generate the oxidative damage of lipids, lipoproteins
or other biochemical parameters such as enzymes, nucleic acids (Comporti,
1985). Damage of these components may be an important factor on the pathogenesis
of different liver diseases. CCl4 is metabolized in mixed function
oxidase system utilizing the nicotinamide adenine dinucleotide phosphate (NADPH)-cytocrome
P-450 electron transport chain at the level of the hepatic smooth of endoplasmic
reticulum (Biasi et al., 1991). It may occur
the hemolytic cleavage during formation of the haloalkane free radicals such
as trichloro-methyl radical (CCl3) and trichloro-methyleperoxy radical
(CCl3O2) (Biasi et al., 1991;
Weber et al., 2003). These free radicals may
bind to cellular macromolecules, so that they can react with free amino groups
on proteins and then the macromolecules may lose their physiological functions
(Garcia et al., 2004; Mac
Cay et al., 1984). This process may play an important role in increasing
of functional enzymes such as AST, ALT, ALP and LDH (Bailey
and Day, 1989; Yu et al., 2002). On the other
hand, carbon tetrachloride has been shown to induce liver injury which closely
looks like cirrhosis (Johnston and Kroening, 1998;
Terblanche and Hickman, 1991). In our study, we were determined that intraperitoneal
injection of CCl4 (0.4 mL kg-1 b.wt.) elevated levels
of oxidative stress, total oxidant status and liver enzymes.
of liver sections (a) untreated control group showing normal histology;
(b) CCl4 administrated group showing necrosis and fibrotic
septums developed around portal areas and between portal areas-central
veins; (c) pseudo lobule formation without central vein; (d and e) representative
histological appearances in CCl4+CS (d) and CCl4+HP (e) extract
groups showing a significant reduction of necrosis and fibrosis in liver
when compared with CCl4-administrated group and (f) treated
with CCl4+UD group showing a reduction of necrosis and fibrosis
in liver when compared with CCl4-administrated group (a and b, H and E
stain, x100; c, d, e and f, Massons trichrome stain, x100)
MDA constitutes an important link between tissue injuries and liver fibrosis.
It is postulated that primary mechanism link of CCl4 is widely due
to its active metabolite CCl3. Radicals (Weber
et al., 2003; Parola et al., 1993).
It has been reported that CCl3 radicals are one of the highly reactive
species which react with cellular components such as lipids and lipoproteins
(Srivastava et al., 1990; Yau-Huei
and Hsin-Chen, 2002).
The enzymatically antioxidant defense systems are natural protective barriers
against free radicals. The SOD and CAT are important scavengers of superoxide
ion and H2O2. These enzymes prevent the generation of
hydroxyl radicals and protect the cellular constituents from oxidative damage
during biotransformation of CCI4 (Weisburger
et al., 1997). The methanolic extract of Ginko biloba has been administered
to the rats against the liver damage caused by CCl4. It was determined
that increased the activities of GSH-Px (De Feudis et
al., 2003). In addition, Capparis decidua had increased the activities
of SOD, CAT, GSH-Px in diabetic rats (Yadav et al.,
1997). In our study, it was observed that treatment by extracts of HP, CS
or UD with herbal caused a significant increase in the levels of TAC and antioxidant
enzyme activities. The antioxidant enzyme activities such as GSH-Px, SOD, CAT
in the CCl4 damaged liver of rats were scavenged and prevented the
reactive free radicals. Thus, the oxidative damage to the tissues was reduced.
The liver cells innate ability to awake and maintain defense against oxidants
by secreting more antioxidants. In addition, the cells are overpowered by the
oxidative stress or damage caused by CCl4. GSH-Px is an intracellular
reductant enzyme which plays a major role in catalysis, metabolism or transport.
It protects cells against free radicals and drug toxification. Furthermore,
it is known that the reduction of GSH-Px in living organisms may cause tissue
injury (Jollow, 1980). Thus, the administration of CCl4
elicit a hepatotoxic affect which decreases GSH-Px and increases the LPO level
in the liver tissues of rats (Luckey and Petersen, 2001).
However, in a study, ıt has been demonstrated that GSH-Px level could be
elevated in rats which pretreated with hepatoprotective Nigella sativa
(Mansour et al., 2001). Similarly, in our study
the liver GSH-Px content was reduced in the CCl4-administered rats
and it was significantly enhanced after the treatment by the extracts of HP,
CS and UD that confirmed its capacity of removing free radicals and reducing
peroxidation. Treatment with the extracts HP and CS overpower the onslaught
by suppressing the formation of reactive oxygen species and protecting the antioxidant
mechanism. Meanwhile, treatment with UD can arrest the hepatocytes from proliferation
due to its activity in prevention mitosis of hepatocytes (Konrad
et al., 2000).
There are no more information related to the hepatoprotective effects of Hypericum
perforatum and Urtica dioica. However, many investigations depended
on the antidepressant activity of HP and treatment of prostatic carcinoma
by UD (Cvejic et al., 2007; Lichius
and Muth, 1997). It has been noted that silymarin and garlic has synergistic
effect and could be used as hepatoprotective agents against hepatotoxicity (Shaarawy
et al., 2009). Previously investigations have been reported that
green tea had antioxidant effect and chemopreventive activity against CCl4-induced
by lipid peroxidation in liver (Giardiello, 1996). It
has been shown that excessive lipid peroxidation accompanied increase in ALT,
AST, ALP and LDH levels and decrease in antioxidant levels (Chen
et al., 2006). Our studys main findings also have demonstrated
harmony with the findings of previous researches mentioned above. In our study,
while the treatments with the herbal extracts of HP, CS and UD were decreased
the elevation of MDA and liver enzyme levels, administration of CCl4 increased
the oxidative stress and lymphocyte DNA damage. Treatment by extracts of HP,
CS or UD were significantly decreased OSI and ameliorated lymphocyte DNA damage.
Meanwhile, hepatocellular degenerative and necrotic changes were very low without
advanced fibrosis and cirrhotic process in HP, CS, also in UD treated groups
(Table 3, Fig. 2d-f).
HPLC analysis of green tea (CS) has shown that it is composed of several polyphenols
(as much as 30% by dry weight), most of which are catechins as epigallocatechin
galbate (15.1%), epigalbocatechin (6.9%), epicatechin gallate (3.0%), epicatechin
(1.8%) and caffeine (8.1%) (Shim et al., 1995).
Polyphenol constituents of CS and its preventive mechanism remain to be defined.
The catechins are known free radical scavengers; galbocatechins and the catechin
gallates exhibit the strongest antioxidant properties (Uchida
et al., 1987). Furthermore, all catechins significantly inhibit cytochrome
P-450-dependent monooxygenase(s). On the basis of the structure-activity relationship
between epicatechins, epigallocatechin gallate is the most potent inhibitor,
suggesting that the galloyl or hydroxyl groups may bind to a cytochrome P-450
catalytic site and interfere with the activation of precarcinogens (Wang
et al., 1988). The protective effects of CS may be attributed to
interaction of polyphenolic catechins with cytochnome P-450 monooxygenase(s)
or scavenging of reactive oxygen species by catechins to prevent the harmfull
effects of CCl4 at critical target sites in the liver. These considerations
are in accordance with the results of the studies showing the interaction of
polyphenolic catechins with cytochrome P-450-dependent monooxygenase(s) or scavenging
of reactive oxygen species (Shim et al., 1995;
Wang et al., 1988; Bray et
al., 2002). Urtica dioica has been successfully used for the
management of benign disorders in traditional medicine in China, India, Turkey
and Europa. Lymphocytes play a crucial role in the generation of the immune
response. Urtica dioica extract stimulate moderately proliferation and
proportion of lymphocyte, without using any kind of mitogen. Immunostimulation
may be responsible for the traditional usage of the aerial parts of UD. Its
protective effects have been attributed to its phyto-chemicals containing lignans,
flavonoids, polysaccharides, lectins and steroids. These phyto-chemicals may
play a significant role in cells and tissues as antioxidants and other constituents
interaction (Harput et al., 2005).
Clinical trials have provided evidence of cytocrome P4503a (CYP3A) induction
by St. John's Wort (SJW) extracts. Many of the case studies or clinical trials
report interaction of SJW and drugs which are substrates of CYP3A. Therefore,
potential interaction between SJW and theophylline (a substrate metabolized
by both CYP3A and CYP1A) has also been reported. The effect of SJW on the other
isoform of hepatic CYP450 is also important. Bioactive components of HP cantains
the hiperforin, adhyperpforin and hypericin which are responsible for its therapeutic
effects. It was detected that HP-ingestion caused a decrease in the plasma concentrations
of various drugs metabolized by CYP3A (Bray et al.,
2002). The preventive effects of HP and theophylline may be attributed to
interaction of polyphenolic catechins with cytochnome P-450 monooxygenase(s)
or scavenging of reactive oxygen species by catechins to prevent the harmfull
effects of CCl4 at critical target sites in the liver. These observations
resemble with the results of the investigations performed with CS (39), HP (41),
UD (43), nigella sativa and vitamins (Shim et al.,
1995; Wang et al., 1988; Harput
et al., 2005; Turkdogan et al., 2001).
In conclusion, we suggest that the herbal extracts of Hypericum perforatum,
Urtica dioica and Camelia synensis have beneficial effects on
CCl4-induced liver and lymphocyte DNA damage. The protective benefits
of these herbal extracts may be due in part to their potent antioxidant properties
and ability to reduce oxidative stress. They should be given over a significant
period to reduce the risk of hepatic damage caused by free radicals. Thus, these
antioxidant herbals may be used for protective purpose which exposed to hepatotoxic
agents. However, further researches are required to better understand the relationship
between the hepatotoxicity and protective effects of herbal antioxidants.
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