Current Therapeutic Research
Available online 27 May 2015
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Chemoprevention of the Arctium lappa extract against acetaminophen-induced hepatotoxicity in rats


doi:10.1016/j.curtheres.2015.05.001
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Abstract

Background

The severe destructive hepatic injuries were induced Paracetamol (APAP) overdose and may lead to acute hepatic failure.

Objectives

The aim of the study was to investigate the ameliorative effects of the Arctium lappa root extract on acetaminophen-induced hepatotoxicity.

Methods

Rats were divided into four groups: normal control group, Arctium lappa extract group, APAP injected group and APAP treated with Arctium lappa extract group.

Results

The treatment with A. lappa extract reduced serum ALT, AST and ALP in APAP group when compared with the control group. DNA fragments in the acetaminophen injected group was also significantly increased (P <0.05). The comet assay revealed increased detaching tail length and DNA concentration during the hepatic toxicity in APAP group. The MDA contents were significantly inhibited by A. lappa treatment (12.97+0.89 nmol/mg) when compared with APAP treated only group (18.98+1.69 nmol/mg). The histopathological examination revealed APAP administration produced hepatic cell necrosis, infiltrate of lymphocytes, and vacuolation which were associated with APAP-treated animal group but the degree of reduction in APAP-induced hepatotoxicity effects was done by treatment with A. lappa extract.

Conclusions

The study showed that the A. lappa can prevent most of the hepatic tissues damage caused by acetaminophen overdose in rats.

Key words

  • Acetaminophen;
  • hepatotoxicity;
  • Arctium lappa;
  • Lipid peroxidation;
  • comet assay

Introduction

At therapeutic doses, acetaminophen (acetyl-para-aminophenol, Paracetamol, APAP) is a safe and effective analgesic and fever reducer. In fact, it is the most commonly used drug in the United States 1. In 2008 alone, more than 24.6 billion doses were sold 2. However, overdose of APAP can cause severe liver injury. The first cases of APAP hepatotoxicity were reported in 1966 3. It is now the principal cause of acute liver failure in many Western countries 4. The liver is the largest and functionally vital organ of the body that participates crucially in metabolism, excretory, secretory, synthesis and de-toxification function 5. The liver is an important target of the drugs toxicity, oxidative, and xenobiotics stress because of its distinctive metabolism and relationship to the gastro-intestinal tract 6.
The primary pathways for acetaminophen metabolism are glucuronidation and sulfation to non-toxic metabolites. Approximately 5%, however, is metabolized in the liver by the cytochrome P450 enzyme family to the toxic matter N-acetyl-p-benzoquinone imine. N-acetyl-p-benzoquinone imine combines covalently to sulfhydryl groups provided by glutathione. Following depletion of glutathione stores, N-acetyl-p-benzoquinone imine binds to cellular proteins in the hepatic tissues, leading to hepatic toxicity 7. So, the acetaminophen is regard as a predictable hepatic toxin, where biochemical signs of hepatic damage will become apparent within 24 - 48 hours after the time of overdose and produce a dose-related hepatic centrilobular necrosis 8. The base dose of acetaminophen to cause hepatotoxicity is believed to be in between 125 and 150 mg/ kg. The threshold dose to cause hepatic toxicity is 10 to 15 g and 150 mg/kg of acetaminophen for adult and child respectively. Mechanisms of acetaminophen hepatic toxicity include production of a toxic metabolite, dysfunction of mitochondria, and innate immunity alteration 9.
Experimental examinations strongly suggest antioxidant supplement as a promising therapeutic intervention for the avoidance and treatment of liver disorders 10. Therefore, there is growing interest in the evaluation of possible role of antioxidant phytochemicals capable of preventing or protecting stress of oxidative agent associated with liver disorders.
Arctium lappa Linne, common known as bardana or burdock, is a member of the Compositae (Asteraceae) family, and its carrot-like root is commonly cooked and eaten as a vegetable in Asia. A. lappa, which can be found worldwide, is used therapeutically as depurative, diuretic, diuretic, digestive stimulant and in dermatological conditions. The root of burdock has long been cultivated as a common plant for dietary use and folk medical uses 11. A. lappa extract has become a promising and important beverage, because of its therapeutic activity 12. Many health benefits of burdock have been reported due to different compounds of bioactive secondary metabolites. These compounds include, among others, lignans and flavonoids, for which A. lappa is an important natural source 13.
In addition, several investigators have been demonstrated that A. lappa displayed hepatic protective agent 12; anti-bacterial properties against gram-positive and negative bacteria 14 and anti-inflammatory effects 15, which might be due to its free radical scavenging activity 16.
Dietary consumption of antioxidants from plant materials has been associated with ignoble incidence of diseases due to decrease of stress oxidation. Thus the aim of this study was to determine the therapeutic role of burdock root extract on the acetaminophen-induced hepatotoxicity in rats.

Materials and methods

Materials

Dried burdock (A. lappa Linné) roots and acetaminophen were purchased from an area market and pharmacy in Damnhour town, Egypt.

Animals

Male Sprague–Dawley rats (approximate weight 200–250 g) were housed within the Animal Center of college of Science, Damanhour University. They were kept for a minimum of two weeks under environmentally controlled conditions (25 + 1° C, 55 + 5% humidity) and a 12-hour light/dark cycle was maintained with feeding on a commercial solid diet (24% protein) with free access to food and water.

Preparation of A. lappa Crude Water Extract

One hundred grams of roots of burdock (A. lappa) was ground into tiny parts and boiled with 1000 ml of distilled water for 60 min. The extract was filtered, and also the residue was filtered when boiled once more. The filtrates were mixed well, place into freeze drying bottles, keep to –60°C and transformed into powder with the freeze-dryer, the income was 24.67%. Once rats orally administered, the crude water extract powder was dissolved in normal saline with an administration dose of 300 mg/ml saline/kg weight of the animals 17.

Experimental design

Forty eight rats were irregular to four groups:
Group 1, a control group that received the same volume of saline in an acetaminophen-only group.
Group 2, a burdock group that received 300 mg/ ml saline/kg of rats.
Group 3, acetaminophen group that received single dose at 800 mg/kg oral administration
Group 4, treated group that received burdock (300 mg/ml/kg of animals) 12 h before acetaminophen injection.
Body and liver weights, blood samples and liver specimens were collected 30 days after acetaminophen treatment.

Measurement of hepatocellular enzymes

Heparinized blood samples were taken by cardiac puncture under anesthesia at the end of experiment. Aspartate transaminase (AST) , alanine aminotransferase (ALT) and alkaline phosphatase (ALP), were employing using a standard clinical automatic instrument (Beckman, Brea, CA). The results of AST, ALT and ALP, activity were expressed in international units (IU/L).

Examination of DNA fragmentation in hepatocytes

ELISA kit detection for a necrobiosis (Boehringer Mannheim, Germany), that quantitatively detects cytosolic histone associated DNA fragments, was used to assess DNA fragmentation in hepatic tissues homogenates. Briefly, the homogenized hepatic samples were collected at the end experiment. The cytosolic fraction was separated from liver tissues homogenates by centrifugation at 13,000xg at 4°C for 20 min and used as antigen supply in a sandwich ELISA with a primary anti-histone antibody coated to the microliter plate and a second anti-DNA antibody coupled to peroxidase. The %DNA fragmentation was computed in line with subsequent equation
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Single cell gel electrophoresis (Comet assay)

The liver tissues of each control and rats experimental groups were separated and quickly keep at −80°C for comet assay. These specimens were homogenized in cooled buffer, pH 7.5 that containing 75 mM NaCl and 24 mM salt of disodium ethylene diamine tetraacetic acid (Na2EDTA) pH 13 to get a 10% homogenate hepatic solution. A homogenizer was utilized, then the liver samples were kept on ice throughout and when homogenization. On cold slides, six microliters of the homogenate hepatic solution were suspended on 0.5% low melting agars and sandwiched between a layer of 0.6% normal melting agars and a high layer of 0.5% low melting agars. The slides were kept on ice throughout the polymerization of every gel layer. when the solidifying of the 0.6% agars layer, the slides were immersed in a lysis solution (2.5 m NaCl, 1% sodium sarcosinate, 10 mm Tris-HCl, 1% Triton X-100, 100 mM Na2EDTA, and 10% DMSO) at 4°C. After 1 h, the slides were placed in electrophoresis buffer (1 mM Na2EDTA, 0.3 M NaOH, pH 13) for 10 min to allow DNA to unwind. Electrophoresis was performed at 300 mA and 1 V/cm for 10 min. The slides were neutralized with pH 7.5 buffer solution of Tris-HCl, and stained with ethidium bromide (20 mg/mL). Every slide was analyzed employing a Leitz Orthoplan epifluorescence microscope (Wetzlar, Germany). One hundred cells were analyzed on every slide using the comet assay II automatic digital analysis system. The tail length (mm) is that the distance of DNA migration from the center of the body of the nucleus, and it's wont to measure the extent of DNA injury. The tail moment is outlined because the product of the tail length and a fraction of the total DNA in the tail (tail Length X % DNA in tail). Each the tail length and tail intensity were measured automatically by the image analysis software 18 and 19.

Hepatic lipid peroxidation

The thiobarbituric acid reactive substances (TBARS) levels as an index of malondialdehyde (MDA) production were measured via the method of Draper and Hadley20. Briefly, hepatic tissue (0.5 g) was homogenized employing a Potter-Elvejham homogenizer with 3 ml of 0.1 M of cold phosphate buffer, pH 7.4. The homogenate of hepatic tissues was centrifuged at 300xg for 10 min. Wherever 100 µl of normal saline and 400 µl of TBA-TCA mixture were mixed with 100 µl of supernatant followed by incubation for 30 min in boiling water bath then cooled at room temperature. When centrifugation at 3,000xg for 10 min, 100 µl of 0.7% TBA was mixed with 100 µl of supernatant in cuvette and read at 535 nm. The TBARS concentrations of the samples were derived from using 1.1.3.3 tetraethoxypropane as a standard and were expressed as nmoles of MDA per mg tissue (nmol/mg).

Histopathological examination and quantification of necrotic cells

One lobe of the liver was removed at the end of experiments and was cut into longitudinal sections 3–4 mm in thickness. Liver slices were then fixed in 10% buffered formalin and processed to embed in paraffin. Hematoxylin-eosin staining was performed in line with standard histologic procedures on 4µm sections. Quantification of death cells was performed by image analysis (Interactive Image Processing, Alcatel, Paris, France) under light microscopy. Ten representative areas from every section consisting of five peri-portal and five peri-venous zones were examined. The area of every high-power field was maintained at 1.27 mm2. Areas of hepatocyte necrosis were delineated using the image analysis software, and also the percent area of every high-power field affected was determined.

Statistical analysis

All information are conferred as means + SD. Statistical analysis was performed using ANOVA. Variations between experimental animal groups were finding to be statistically significant once the P value is <0.05.

Results

Body and liver weights in the animals groups

The body and liver weights of animals injected with APAP followed by treatment with A. lappa extraction ( Table 1) were not significantly affected.
Table 1. Body and liver weights in experimental animals groups.
Animal groupsBody weights (g)Liver weight (g)
Control278+17.98+0.27
A. lappa276+27.63+0.96
APAP280+28.61+1.03
APAP +A. lappa277+48.01+0.31
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Hepatocellular enzymes levels

The indication of animals with APAP resulted in significant increases (P<0.05) in plasma ALT, AST and ALP when compared with control group suggestive of severe liver injuries (Figure 1A-C).
Effects of APAP on the ALT, AST, ALP and DNA fragments in different experimental ...
Figure 1. 
Effects of APAP on the ALT, AST, ALP and DNA fragments in different experimental animals.
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DNA fragments in experimental animals groups

Liver DNA fragments in the acetaminophen treated group was also significantly increased (P <0.05 vs control group; Fig.1D). In the present study, we found that administration of acetaminophen to rats resulted in a significant increase in liver DNA fragmentation (P< 0.05 vs. control group), indicating that APAP is capable of inducing hepatic cells apoptosis.

Comet assay (Genomic single DNA fragmentation)

In the Figure 2 and Figure 3, the comet assay revealed increased detaching tail length and DNA concentration during the hepatotoxicity by APAP in different experimental animal groups when compared with the control animals.
Comet assay of the hepatic tissues in different animal groups, (A) the control ...
Figure 2. 
Comet assay of the hepatic tissues in different animal groups, (A) the control group, (B) A. lappa group, (C) APAP group, and (D) APAP treated with A. lappa. Asterisks (*) stretched hepatocyte with DNA damage with become more abundant in APAP group.
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The head and tail DNA percentage in different animal groups.
Figure 3. 
The head and tail DNA percentage in different animal groups.
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Hepatic lipid peroxidation (MDA)

The lipid peroxidation has been reported to be related to APAP-induced hepatotoxicity, the content of malondialdehyde (MDA) is the end product of the lipid peroxidation which was detected at the end of the experiment. The low levels of MDA concentration were investigated in the control and A. lappa animal groups (9.81+0.99, 8.98+1.09 nmol/mg tissue respectively), but an increase significant was present in APAP-treated rats (18.98+1.69 nmol/mg tissue). The MDA contents were significantly inhibited by A. lappa treatment (12.97+0.89 nmol/mg tissue, Figure 4).
A. lappa treatment inhibits hepatic lipid peroxidation (malondialdehyde ...
Figure 4. 
A. lappa treatment inhibits hepatic lipid peroxidation (malondialdehyde production) after acetaminophen induction.
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Histopathological evaluation

APAP administration produced hepatic cell necrosis in a zone around the terminal hepatic venules. The intense infiltrate of inflammatory lymphocytes and vacuolation were associated with APAP-treated animal group (Fig. 5C-E). The A. lappa extract reduced the severity of all APAP-induced hepatic responses, but the degree of reduction in APAP-induced necrosis was incomplete, where the dilated and congested central vein and some necrotic area were present in APAP treated with A. lappa extract animal group ( Fig. 5F).
Hematoxylin-eosin stained hepatic sections from different animal groups. A and B ...
Figure 5. 
Hematoxylin-eosin stained hepatic sections from different animal groups. A and B are the hepatic tissues of control and A. lappa extract groups showing that the normal features of hepatic tissue and central vein (CV) structure. C, D and E, are the sections from APAP animal group showing that the infiltration of inflammatory lymphocytes (IC), necrosis area, fat vacuolation (V). F is the section from APAP injected animals treated with A. lappa extract showing normal structure hepatocytes and central vein (CV). Original magnification X 250 H&E.
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Discussion

In developing countries, 80% of the indigenous populations depend on a local traditional medicine. Within the European countries, medicinal herbs represent an important pharmaceutical market with annual sales of US $7000,000 21. Herbs have long been utilized as medical aims, and of the many available drugs are directly or indirectly derived from herbs. Recently, the A. lappa tea is derived from Arctium lappa which has become an important and promising beverage, because of the A. lappa exhibits the free radical scavenging effects and anti-inflammatory 22, therefore, it can be used to avoidance the tissue damage by free radicals inducers. Recently, the chemical components, researches of clinical and pharmacological applications of Arctium lappa Linne had been recorded. There are six compounds of the Arctium lappa Linne seeds which could be isolated 23. One of them is a new lignan called neoarctin B. The other five components were identified as lappaol F, arctiin, arctigenin, matairesinol and daucosterol 23. In traditional medicine of Chinese for the treatment of common cold caused by heat and wind, the herbal drug of Arctium lappa Linne fruit is an often-used. This drug includes many components, principally arctiin with a small amount of arctigenin 24. In the our study, the phytoprotective effects of Arctium lappa against acetaminophen-induced hepatocytes damage were investigated in rat.
Acetaminophen (APAP) toxicity is one of the most widespread drug induced side-effects worldwide and damage to the hepatic and renal tissues is a major complication with overdose of APAP. The metabolites of APAP created in the hepatic and other organs tissues are likely to be the main contributor into the mechanism of its toxicity. The effects of APAP is the earnest necrotic centri-lobular hepatocytes 25. In the present study, an oral single dose administration of APAP (800 mg/kg) was hepatotoxic in rats as proved by the significant increases in plasma ALT and AST activities as well as ALP concentration. The release of abnormal levels of hepatic enzymes in plasma are believe to be indicators of hepatocytes injury 26. The high concentrations of serum liver enzymes referred to hepatocytes damaged, since these enzymes are located in the cell cytoplasm and released into the blood stream following hepatic cells damage 27. The associated with the lipid peroxidation and APAP-induced hepatotoxicity was reported in case APAP overdose, the end product of the lipid peroxidation is the malondialdehyde (MDA) which was detected at the end of the present experiment.
In the present study, the comet assay was employed to assess the hepatotoxicity of APAP group and APAP treated with A. lappa group or in other groups. The most important tools one is comet assay which used for the assessment of DNA damage in the cells 28. The different types of DNA damage and calculating the DNA migration.was detected by the comet assay. The level of double and single-strand breaks and alkali- unstable sites is positively correlated with the extent of DNA migration. However, the crosslinks of DNA can be detected by the comet assay, as observed by the reduction of DNA migration when comparison with the control cells 29. From the present data, the decreased of the DNA migration had due to the antioxidant properties of the A. lappa extract, which would have decreased the level of intracellular free radicals with the consequent decrease in the occurrence of breaks due to oxidative stress causing damages in the DNA strand. The A. lappa was able to decrease the DNA migration, in a way that the decrease in the migration of DNA extension observed in the A. lappa extract treatment could result from a synergic effect of crosslinks between A. lappa extract components and APAP. The current experiment has shown that the treatment of APAP-induced hepatotoxicity with A. lappa extract acts retarding the DNA migration of the hepatocytes.
Hepatic injury is a common pathological feature which exists in many hepatic diseases. Therefore, treatment and prevention of hepatic injury is a key to clinical liver diseases treatment. These results are consistent with the histopathological findings in APAP group where the acetaminophen induced hepatocytes damage 30. The histopathological changes induced by APAP overdose, these changes includes necrosis, a infiltration lymphocytes and fatty liver, may be relieved by A. Iappa extract. Binuclear hepatocytes were also present in A. lappa-treated groups, which indicated the regeneration of hepatic cells ( Fig. 5B & F).
Lin et al. 22 reported that A. lappa has the free radical-scavenging properties. The IC50 levels were the 2.06 and 11.8 mg/ml of A. Iappa extract on superoxide-scavenging and hydroxyl radical activity, respectively. In the current study, Perhaps the phytoprotective effect of A. lappa can be returned to its free radical-scavenging activity and antioxidant effect, thus the removing of the injurious effects of toxic acetaminophen metabolites induces the hepatocytes regeneration
In conclusion, the present study showed that the A. lappa can mitigate most of the hepatic tissues damage caused by the overdose of acetaminophen in rat. The current study suggested that the phytoprotective activities of A. lappa demonstrated here and the fact that the tea of A. lappa has been used by humans, make it a potential candidate for therapy of acetaminophen-induced hepatoxicity in humans.

Conflict of Interest

The authors declare that there are no conflicts of interest.

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Address correspondence to: Dr. Attalla Farag El-Kott, Deanship of Scientific Research, King Khalid University, Saudi Arabia.
Copyright © 2015 Published by Elsevier Inc.
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