Hepatoprotective activity of Chenopodium murale in carbon tetrachloride-induced hepatic damage in rabbits

Liver is vital organ, which plays an essential role toregulate many physiological processes in the body. It has significant functions like; metabolism, secretion and storage. It has excessive capacity for the detoxification of toxic constituents and synthesizes beneficial components. Therefore, damage to the liver imposed by hepatotoxic agents is of severe consequences (Shahani, 1999; Subramoniam and Pushpangadan, 1999). Liver diseases are primarily due to toxic chemicals, excess utilization of alcohol, infections and autoimmune diseases. Most of the hepatotoxic chemicals damage liver cells mostly by causing lipid peroxidation and other oxidative damages resulting in generation of highly toxic reactive oxygen species (Recknagel, 1983; Dianzani et al., 1991).


Introduction
Liver is vital organ, which plays an essential role to regulate many physiological processes in the body.It has significant functions like; metabolism, secretion and storage.It has excessive capacity for the detoxification of toxic constituents and synthesizes beneficial components.Therefore, damage to the liver imposed by hepatotoxic agents is of severe consequences (Shahani, 1999;Subramoniam and Pushpangadan, 1999).Liver diseases are primarily due to toxic chemicals, excess utilization of alcohol, infections and autoimmune diseases.Most of the hepatotoxic chemicals damage liver cells mostly by causing lipid peroxidation and other oxidative damages resulting in generation of highly toxic reactive oxygen species (Recknagel, 1983;Dianzani et al., 1991).
Enormous synthesis of reactive oxygen species plays a vital role in the pathogenesis and progression of several diseases involving various organs (Visioli et al., 2000).Carbon tetrachloride is biotransformed into reactive oxidative metabolites by liver microsomal enzy matic system, which results hepatotoxicity (Brent and Rumack, 1993).
Instead of remarkabledevelopments in modern medicine no effective drugs exist, which encourage liver functions and gives protection to the liver from the damage or aids to regenerate hepatocytes (Chattopadhyay, 2003).Due to lack of effective liver-protective drugs in modern medicine, hugenumber of medicinal preparations are endorsed for the treatment of liver disorders (Chatterjee, 2000) and they quite often showed significant relief.Efforts are being made internationally throughout the globe to acquire scientific proofs for these conventionally reported herbal drugs.In the present study, the carbon tetrachloride-induced acute models have been used to assess hepatoprotective activity of C. murale which is used conventionally for prevention of liver disorders.
In today practice numbers of herbal formulations are used for treating liver disorders, like Capparis spinosa, Daccus carota, Euphorbia antisyphilitica (Shirwaiker et al., 1996;Bhayee et al., 1995;Saraf et al., 1996).The present pharmacological investigation on aqueous ethanolic extract of different doses of C. murale is conducted for the determination of its hepatoprotective activity against carbon tetrachloride-induced liver damage.

Materials and Methods
Preparation of extract: C. murale whole plant was collected from peripheries of Bahawalpur.Plant was recognized and validated by a texanomist Prof. Tayeb Qureshi.
The voucher specimen (Collection No. CM-WB-05-12-051 ) were kept in the herbarium of Department of Pharmacology, Faculty of Pharmacy and Alternative Medicine, the Islamia University of Bahawalpur Pakistan for future reference.1.5 kg of powdered material of the extract was soaked in 4.0 L of 70%aqueous alcohol.Mixture was shaken occasionally with glass rod for 12 days.On each fourth day of shaking, straining was carried out by sterilized muslin cloth.Menstrum was filtered with filter paper and residue left i.e. marc was again soaked and same process was repeated.The filtrate from each straining was stored in air tight container at 4°C in refrigerator to avoid its deterioration.Finally, whole filtrate was again filtered.Collected filtrate was dried under reduced pressure on rotary evaporator (Laborata 4000 Heidolph, Japan) at 40 °C till dryness.130 grams of crude extract was obtained.
Percentage yield of dried extract was calculated as 8.66%.Extract was stored in air tight container at 4°C till its experimental evaluation.
Experimental animals: Healthy adult local breed both male and female rabbits were used (each weighing about 1.0-1.5 kg).Animals were held in cages at room temperature (23 ± 12°C).Ten rabbits/cage were accommodated in animal house at Faculty of Pharmacy and Alternative Medicine, the Islamia University of Bahawalpur.Humidity was maintained as 55 ± 15%.Animals were fed properly according to a fixed time Acute toxicity studies: The method adapted from Litchfield and Wilcoxon was used for acute toxicity studies.Male and female mice of either sex were used.Each was weighing 35 ± 5.6 g.During the test, mice were put in the laboratory conditions: standardized boxes, food made of granules, water ad libitum, natural light and ambient temperature at 25-30°C.
Seven groups were made each including six mice.Extract in doses of 1 g/kg, 2 g/kg, 4 g/kg, 8 g/kg, 10 g/kg and 12 g/kg body weight were administered and one untreated control group that received only normal saline.Animals of each group were receiving a specific dose of the extract to be tested and normal saline by oral route using feeding tube.All mice were observed for mortality for a total time period of 72 hours.The dose at which animals died was considered to be the toxic dose (Liechtfield and Wilcoxon, 1949).Animals were died at 8 g/kg.
Hepatoprotective activity of ethanolic extract of C. murale in carbon tetrachloride-induced toxicity: Induction of liver toxicity was carried out by subcutaneous administration of carbon tetrachloride (suspended in olive oil at 1:1) at dose of 0.75 mL/kg body weight.Animals were divided into five groups.Each group consists of 10 rabbits.Total study period was eight days.Carbon tetrachloride was administered 30 min after extract administration on the 7th day of total 8 days study period to all groups except Group I which served as normal control and it received only normal saline.
Group II-V received the following treatments from 1st to 7th day of the study (Ahmad and Eram, 2011).
Assessment of liver function: After 24 hours administration of carbon tetrachloride, rabbits were anaesthetized by ketamine and collection of blood samples (3 mL) was carried out particularly by heart puncture technique (Wright, 1973).Blood samples were allowed to stand at room temperature for coagulation (usually for 45 min) in sterile disposable centrifuge tubes.For obtaining serum, the coagulated blood samples were centrifuged at 2,500 rpm for 15 min.A clear supernatant layer was collected (called serum) from the tubes with the help of micropipette, added into eppendorf tubes and was kept in refrigerator for biochemical parameters like SGOT, SGPT, alkaline phosphatase and total bilirubin (Ahmad and Eram, 2011).
Histopathological studies: For histopathological study, the livers were quickly removed and washed with normal saline and fixed in 10%formalin.Liver sections were made by microtome, dehydrated in ethyl-alcohol and cleaned with xylene.Finally these were embedded in paraffin wax.The sections of about 56 mm were cut and paraffin was removed from the embedded section with xylene.Sections were rehydrated with ethyl alcohol and dehydrated with 0.9%NaCl solution.Sections were stained with hematoxylin and Eosin dye.Eventually, these were mounted by Canada balsam.These were then used for the histopathology studies of liver cells architecture under microscope.Their microphotographs were taken (Humason, 1979).

Statistical analysis:
The results were presented as mean ± SEM.Unpaired t-test was applied by using 'Graph Pad Prism' software.Differences were significant when p<0.05 (Woodson, 1987).

Results
The in vivo and in vitro hepatoprotective effects of various medicinal plants are generally determined by utilization of acute hepatoxicity model i.e. carbon tetrachloride-induced (Weber et al., 2003).Due to subcutaneous administration of carbon tetrachloride (0.75 mL/kg), there was considerable elevation in the enzyme i.e.SGOT, SGPT and alkaline phosphatase.
Total bilirubin level was also raised when compared with normal control.The hepatoprotective activity of extracts on acute hepatotoxicity model induced by carbon tetrachloride has been outlined in Table I.Serum SGOT, SGPT and alkaline phosphatase elevation reflect liver damage due todisintegration of cell membrane and necrosis.This causes cellular leakage and ultimately functional integrity of cell is lost (He and Aoyama, 2005).Also, elevated level of serum total bilirubin directs jaundice (Sturgill and Lambert, 1997).Serum enzyme level and histopathological findings direct the intensity and kind of liver injury.Above mentioned enzymes level was almost restored by C. murale extract showing hepatoprotective activity.

Discussion
Cytochrome i.e.CYP 2E1, CYP 2B1 or CYP 2B2 and possibly CYP 3A activate carbon tetrachloride to form CCl3* (trichloromethyl radical) which is a free radical.
Free radicals induces oxidative stress that leads to cell membrane injury i.e. plasma membrane disintegration which result in alteration in metabolic processes.
Reactive oxygen species play an important role in the pathogenesis of various degenerative diseases and have been found to implicate in atherosclerosis, liver disorders, lung and kidney damage, aging and diabetes mellitus.In liver disorders, the ability of natural anti-oxidant system is impaired (Johnston and Korening, 1998).Reactive oxygen species is constantly removed from the cell as it is produced, however, when its concentration is beyond the limits it results in deleterious effects on cell.Eventually peroxidation and alkylation take place (Halliwel and Gutteridge, 1990), resulting in destruction of basic skeleton of the cell, alteration in the functional capability of mitochondria and disturbance in the homeostasis of ions (Brattin et al., 1984).
The activities of serum marker enzymes like SGOT, SGPT and alkaline phosphatase can make assessment of liver function.When liver cell membrane is damaged, these enzymes normally located in the cytosol are released in to blood stream.Their estimation in the serum is a useful quantitative marker of the extent and type of hepatocellular damage (Sallie, 1999).In common, SGOT and alkaline phosphatase are present in high amount in liver due to hepatic necrosis (Shah et al., 2002).Bilirubin is a break down product of heme and present in the cytoplasm of hepatic cells.Whenever there is damage to the liver cells, it leaks into blood stream which authenticates liver damage (Nkosi et al., 2005).
In the current study, enzymes level (SGOT, SGPT and alkaline phosphatase) was elevated considerably in negative control group with damaged liver morphology.The extract at both doses (500 mg/kg and 750 mg/kg) reduced the elevated level of enzymes and restored the normal morphology.This alleviation of enzymes and restoration of damaged liver cell morphology to normal is because of decrease in lipid peroxidation, aggravated by free radicals i.e.CCl3• ( t r i c h l o r o m e t h y l r a d i c a l ) , C C l 3 O O • (trichloromethylperoxy radical) (Weber et al., 2003).The decrease in enzymes level is due to the presence of anti-oxidants in C. murale which has free radical scavenging activity.This reflects the protection of structural and functional integrity of carbon tetrachloride damaged liver cells.Also, restoration of serum alkaline phosphatase and total bilirubin level nearer to normal shows consistency of biliary functions.Phytochemical analysis shows that C. murale contains; volatile oils, saponines, gerniol, flavonoids, alkaloids, sterols, and coumarins (Ahmad and Eram, 2011).Flavonoids and saponins are known to hold hepatoprotective action in animals (Tran et al., 2001).
Flavonoids block lipid peroxidation in cell membranes due to their high anti-oxidant activity and free radical scavenging property (Halliwel and Guitreg, 1990).Kaempferol is areported flavonoid found in C. murale and has strong anti-oxidant activity which helps to prevent oxidative harm to cells, lipids and DNA.It also contains beta-setosterol which is a reported hepatoprotective agent.(Ahmad and Eram, 2011).Coumarins in C. murale have potent anti-oxidant effect and hence strong hepatoprotective activity (Amaca et al., 2011).
In the current study, extract caused a substantial inhibition in SGOT, SGPT and alkaline phosphatase activities towards the respective normal range with contemporary reduction of raised bilirubin level (Mukherjee, 2002).This shows that extract restored the structural integrity of the hepatocellular membrane damaged by carbon tetrachloride which was authenticated by histopathological investigation.The occurrence of saponins, flavonoids and tannins in extract may participate the hepatoprotective activity.
The hepatoprotective effect of the extract of C. murale may be because of its capability to halt the bioactivation of carbon tetrachloride and its powerful anti-oxidant potential by scavenging the free radicals.
It is concluded that aqueous ethanolic extract of C. murale have potent hepatoprotective actions upon carbon tetrachloride-induced hepatic damage in rabbit and can be used in hepatic disorders.

Figure 1 :
Figure 1: Histopathological slides of liver of different animal groups.Normal control (A); Carbon tetrachloride negative control (B); Standard control (C); Test group 1 (D); Test group 2 (E) . It basically belongs to African and European continents.In

table (
o.).The remaining groups received the different amount of extracts (500 mg/kg and 750 mg/kg p.o.) and the hepatotoxin.

Table I : Effects of ethanolic extract of C. murale (whole plant) on rabbits serum biochemical parameters after carbon tetrachloride administration
Values are represented as Mean ± S.E.M. (n=10).0 = Normal, +1 = Mild, +2 = Moderate and +3 = Severe.Microphotographs of liver sections of different groups