Centratherum anthelminticum seeds reverse the carbon tetrachloride-induced hepatotoxicity in rats

The present study is the first attempt to evaluate the hepatoprotective effect of ethanolic seeds extract (ESEt) of Centratherum anthelminticum (black cumin) in carbon tetrachloride (CCl4)-induced liver injury. The test doses (600 and 800 mg/kg) of same extract were found effective in their respective test groups by improving the body and liver weights, serum alanine and aspartate transaminases, γglutamyltranspeptidase, alkaline phosphatase, total proteins, albumin, total bilirubin, especially indirect bilirubin and uric acid levels as compared to CCl4-induced hepatotoxic control group. In addition, decreased percent inhibitions of antioxidant parameters including catalase, superoxide dismutase and reduced glutathione accompanied with increased percent inhibition of lipid peroxidation observed in both test groups. Histopathological studies also proved the liver regenerating property of ESEt by showing decrease in fatty deposition, necrosis and inflammation around the central vein of liver lobules. Therefore, the ESEt was found to be hepatoprotective and antioxidative in nature.


INTRODUCTION
Liver is the indispensable organ in maintaining the homeostasis in body by regulating metabolic and hematological functions plus bile production for fats emulsification and digestion (Hall, 2011).Besides these, diverse functions are also on its credit like storage of vitamins, iron, detoxification, removal and excretion of antibiotics, hormones, oxidative radicals and xenobiotics (Hall, 2011).However, liver's function and structure are very sensitive and can easily be affected by microorganisms (bacteria/viruses/fungi/parasites) and hepatotoxins/carcinogens (Ahmad et al., 2014).Among different liver affecting chemicals, carbon tetrachloride (CCl 4 ), which is normally used as cleaning agent in industries, in fire extinguishers, etc., is injurious for body *Corresponding author.E-mail: qureshi29@live.com. Tel: 92-21-99261300 Ext 2289, +92-300-2780017. Fax: 92-21-99261340.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License tissues especially liver.Continuous inhalation of CCl 4 vapours enhanced inflammation and necrosis of hepatocytes by producing reactive metabolites that may lead to severe cirrhosis, if the condition ignored for a long time (Bigoniya et al., 2009;Singh et al., 2011).Excluding genetic causes, environmental pollutants and chemicals used in industries are also accelerating the risk of liver problems in both developed and developing countries and Pakistan is also showing highest burden of liver disorders which is chiefly contributed by mines, mills and industrial workers (Anjum et al., 2009;Malaguarnera et al., 2012;Shah et al., 2015).
In addition to the costly conventional treatments of liver problems, medicinal plants always have prominent place in this regard especially in Asian countries as these are easily available and have no toxic effects (Guan and He, 2013).
Centratherum anthelminticum (Vernonia anthelminticum Willd) belongs to the family Asteraceae and commonly known as black cumin (Amir and Chin, 2011).It is not only widely distributed in neighboring countries of Pakistan but also popular for its culinary use in all over India and Pakistan (Singh et al., 2012).Its bitter taste seeds are popular for various medicinal purposes and their different extracts are well-reported for pharmacological activities like analgesic, antipyretic, antimicrobial, anticancer, antidiabetic, antihyperlipidemic, anti-inflammatory, antioxidant, antiurolithiatic, and skin problems (Amir and Chin, 2011;Mudassir and Qureshi, 2015).However, their hepatoprotective effect has not been documented yet.Therefore, the present study is designed to investigate the effect of ethanolic seeds extract of C. anthelminticum in carbon tetrachloride (CCl 4 ) induced hepatotoxic rats.

Animals
Female Wister albino rats (180 to 220 g) were purchased from breeding house of Dow University of Health Sciences (DUHS), Karachi and kept in animal house of Department of Biochemistry, University of Karachi (UoK) according to the international guidelines of animal handling by giving them standard laboratory diet and water ad libitum.

Plant material and extraction
Seeds of C. anthelminticum were purchased and identified by authentic taxonomist (voucher no.KU/BCH/SAQ/02) of UoK.These seeds were used to prepare ethanolic extract (ESEt) according to the procedure described by Mudassir and Qureshi (2015).

Positive control and vehicle
Silimyrin (Siliver 200 mg) and dimethyl sulphoxide (DMSO; 0.05%) of Abbott Laboratories (Pakistan) Ltd and Fischer Scientific, UK, respectively were used as positive control in the present study and vehicle for administering the doses of ESEt in experimental rats.

Animal grouping and procedure
Experimental rats were divided into four groups (6 rats/group) including normal and hepatotoxic controls (group I and II), each of them treated with distilled water (1 ml/kg), positive control (group III) treated with silymarin (200 mg/kg) and test groups (groups IV and V) treated with ESEt (600 and 800 mg/kg).Each treatment was done orally once per day in early morning for 5 days consecutively.However, hepatotoxicity was induced in group II to V by intraperitoneal injection of CCl4 (3 ml/kg in 1:1 ratio with olive oil) on 3rd and 5th day of trial after 1 h of their respective treatments.After 24 h of last dose of CCl4, rats were sacrificed to collect blood and serum to analyze biochemical parameters.In addition, livers were dissected out carefully to weigh them, estimate antioxidant parameters and for histological examination.The present experimental procedure was approved by Board of Advance Study and Research (BASR) of UoK.

Physical, biochemical and antioxidant parameters
Percent body weight change (BWC) of rats in each group was calculated by using formula (Azmi and Qureshi, 2013) after measuring the body weights of each rat on initial (IBW) and final (FBW) days of trial.Beside this, the livers of each group were also weighed as LW (g).
Biochemical parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyltranspeptidase (GGT), total bilirubin (TBR), direct bilirubin (DBR), indirect bilirubin (IBR), total protein (TP), albumin (ALB) and uric acid (UA) were measured in serum through commercially available enzymatic kits (Randox, UK).Whereas antioxidant parameters including, catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH) and lipid per oxidation (LPO) were measured in liver homogenate by standard manual methods (Lateef and Qureshi, 2013).Percent protection in ALT, AST, and ALP of positive control and test groups against hepatotoxic control was calculated with the help of following formula (Al-Qarawi et al., 2004) where At, Ax and Ao are the mean readings of group III/IV/V, II and I of respective enzyme.
Similarly, percent gain/loss in LW and TBR, IDBR, TP, ALB and UA levels in positive and test groups was calculated with formula described by Azmi and Qureshi (2012).

HISTOLOGY
Dissected out liver tissues from each group were immersed in 10% formaldehyde solution separately and sent to Dr. Essa's diagnostic laboratory, AbulHasan Isfahani Road, Karachi Pakistan for histological studies.

Statistical analysis
One way analysis of variance (ANOVA) was used to analyze the results of present study and mentioned as mean ± SD (standard deviation).The difference of mean of each parameter was compared among all groups and found significant at p<0.05 through

Effect of ESEt on percent body weight change and liver weights
ESEt in doses of 600 and 800 mg/kg significantly decreased (p<0.05 and p<0.0001) the percent reduction in body weights in group IV and V, respectively as compared to hepatotoxic control group (group II) where administration of CCl 4induced marked reduction in body weights (Figure 1).Similarly, the liver weights (g) in group II were clearly increased (p<0.0001) as compared to group III, IV and V where silymarin and test doses of ESEt were found effective in normalizing the weights of livers (Table 1).

Effect of ESEt on liver and non liver-specific parameters
Liver-specific enzymes including ALT, AST and ALP (U/l) were drastically elevated in group II whereas all same enzymes were significantly decreased (p<0.01 and p<0.0001) in groups III, IV and V which were treated with silymarin (100 mg/kg) and ESEt (600 and 800 mg/kg), respectively and showed significant percent protection especially in ALT (95 to 98%), AST (73 to 81%) and ALP (37 to 100%) (Figure 2).Whereas GGT (U/l) level was found decreased in group II (Table 1).Similarly, ESEt and silymarin were found decreasing TBR level especially indirect BR (mg/dl) in their respective groups as compared to group II (Table 1).On the other hand, TP (g/dl) and ALB (g/dl) were decreased in group II and became significantly increased (p<0.01 and p<0.0001) in group III, IV and V (Table 1).Beside these, non-liver-specific parameter uric acid (mg/dl) was found decreased prominently (p<0.0001) in the last three groups (Table 1).

Effect of ESEt on antioxidant parameters
Percent inhibitions of CAT, SOD and GSH were significantly decreased in positive control (group III) and test groups (IV and V) as compared to hepatotoxic control group II (Figure 3 and 4).Whereas, in the case of percent inhibition of LPO, an entirely opposite picture was observed in positive and test groups (Figure 4).

Effect of ESEt on histology of liver tissues
Histopathological studies were done by preparing slides of liver tissues stained with hematoxylin and eosin (Figure 5).Liver tissue (slide A) of CCl 4 -induced hepatotoxic group showed degeneration of hepatocytes accompanied with fatty deposition (ballooning) and infiltration of inflammatory cells around dilated central vein in liver lobule.However, all these features of liver injury were gradually recovered in tissues (slide C and D) of groups treated with ESEt in doses of 600 and 800 mg/kg.

DISCUSSION
Liver problems, because of acquired causes, are contributing the major portion of death burden globally (Malaguarnera et al., 2012).The characteristic features of liver problems are loss of appetite and weight, elevation in liver-specific parameters and reduction in hepatic functions (Singh et al., 2011).Interesting, the same features was clearly observed in the present carbon tetrachloride (CCl 4 )-induced hepatotoxic rat model.CCl 4 is a well-known liver toxicant (Adewale et al., 2014).It severely damaged cellular integrity of hepatocytes by converting itself into reactive metabolites including trichloromethyl (.CCl 3 ) and peroxytrichloromethyl (.OOCCl 3 ) radicals after passing through hepatic cytochrome P 450 enzyme responsible for the detoxification of xenobiotic or chemicals (Khan et al., 2012).Elevated levels of both ALT and AST are the best indicators of liver damage whereas GGT and  ALP together reflect the damage in bile duct.However, isolated elevation of AST and ALP is the warning of cardiac and bone problems as their chief concentrations are present in both these tissues, respectively (Bishop et al., 2013).In the present study, CCl 4 -induced hepatotoxic control rats showed a drastic increased in ALT, AST, ALP and decreased in GGT levels.On the other hand, ALT, AST and ALP were found extensively improved in positive control and test groups treated with silymarin (100 mg/kg) and two doses of ESEt (600 and 800 mg/kg) while GGT remained low or below normal in these groups.ALT, AST and ALP are the intracellular enzymes and their abundance presence in serum above 100 to 1000 folds of their normal levels is the echo of the alteration in cell membrane intactness (Bishop et al., 2013) while decreased levels of GGT are the reflection of intrahepatic cholestasis and decreased bile acid production (Hyder et al., 2013).The alteration in cell  membrane was clearly proved by observing severe necrosis along with inflammation and steatosis (fatty deposition) in hepatocytes around central vein of lobules in microscopic examination of liver tissues of hepatotoxic group which was gradually healed up and improved by observing almost normal architecture of liver tissues that were dissected out from test groups treated with both doses of ESEt, respectively.Important point is that the ESEt of C. anthelminticum showed much better results as compared to silymarin in bringing the liver anatomy back to normal.
The cellular protective effect of ESEt of C. anthelminticum was also confirmed by observing decreased levels of uric acid (UA) in both test groups as compared to hepatotoxic control group which showed high concentration of same parameter.UA is the end product of purine metabolism and its abnormally increased concentration in serum is the alarming sign not only for the presence of kidney dysfunction but also for the degradation of body tissues or cells ( Firestein , 2008).This beneficial effect of ESEt was more strengthen by observing a significant decrease in percent reduction in body weights of both test groups treated with same extract (600 and 800 mg/kg) as compared to hepatotoxic control which was only treated with CCl 4 (3 ml/kg) and showed an extreme percent loss in body weight up to -12%.Even silymarin, the well-known hepatoprotective medicine, did not prove to be statistically efficient in this respect in positive control group.
The possible involvement of ESEt of C. anthelminticum in regenerating liver tissue was also fortified by estimating the normal levels of total protein (TP), albumin (ALB) and total bilirubin (TBR) both direct and indirect in extract treated test groups as compared to CCl 4 -induced hepatotoxic control group that showed decreased levels of TP, ALB and elevated levels of TBR particularly indirect/ unconjugated one.Literature declared that 90% of total protein, except immunoglobulin and 100% albumin are synthesized in liver (Murphey et al., 2007).However, TP and ALB can also be decreased in renal functions and malnutrition but these are considered as the best meter for evaluating the functionality of liver (Thapa and Walia, 2007).Similarly, increased serum IDBR level was not only observed in severe hemolysis but also in liver problems including hepatitis and cirrhosis with decreased or no conjugation reaction taking place in liver (Hall, 2011).Another study stated that depletion of TP induced intense mitosis in hepatocytes which leads to the enlargement of liver and this condition persists till the TP concentration becomes normal in blood (Bishop et al., 2013;Hall, 2011).Interestingly, the same happened in the present study as decreased TP and ALB was observed in hepatotoxic control group accompanied with increased liver weights, whereas improvement in TP and ALB levels in positive control and test groups also normalized the liver weights in these groups.The observed liver regenerating ability of ESEt in the present study might be by inhibiting the factors that hindered liver tissue regeneration like tumor necrosis factor alpha (TNFα), etc (Kang et al., 2012).Amazingly, chloroform fraction of C. anthelminticum seeds was claimed to inhibit TNF-α in human tumor cells (Arya et al., 2012).
The was already well-reported in hyperlipidemic rabbits (Lateef and Qureshi, 2013) and again became beneficial in contributing to the hepatoprotective action of same extract in the present study.In vivo induction of CCl 4 stimulates severe oxidative stress by producing two trichloromethyl radicals in the presence of hepatic mixed function oxidase (Cyto P 450 ), which reported to induce lipid peroxidation, alteration in cell membrane permeability and mitochondrial function, thereby producing reactive oxygen species (ROS) and creating tissue inflammation and necrosis (Thanh et al., 2015).ESEt displayed radical scavenging activity in both test groups treated with same extract (600 and 800 mg/kg) by showing decrease in percent inhibition of CAT, SOD, reduced GSH and increase in percent inhibition of LPO whereas an entirely opposite picture was observed in CCl 4 -induced hepatotoxic group for these four parameters.The antioxidant potential of C. anthelminticum seeds might be residing in their polyphenolic content, especially flavonoids.Different extracts of C. anthelminticum seeds proved the presence of polyphenols and flavonoids having radical scavenging abilities in in-vitro assay (Ani and Naidu, 2011;Mudassir and Qureshi, 2015;Shah et al., 2007).

Conclusion
The results concluded that ethanolic seeds extract of C. anthelminticum has potent activity to reverse the harmful effects of carbon tetrachloride on liver tissues.However, further work on the same theme has to be done on its isolated compounds to know which would be the active principle in same extract having hepatoprotective activity.

Figure 1 .
Figure 1.Effect of ESEt on Percent BWC.Each bar represents the mean ± SD (n=6).a and c = p<0.05and p<0.0001 respectively when compared with group II.

Figure 2 .
Figure 2. Effect of ESEt on Liver-Specific Enzymes.Each bar represents the mean ± SD (n=6).c = p<0.0001when compared with group II.

Figure 3 .
Figure 3.Effect of ESEt on Percent Inhibition of CAT and SOD.Each bar represents the mean ± SD (n=6).c = p<0.0001when compared with group II.

Figure 4 .
Figure 4. Effect of ESEt on Percent Inhibition of GSH and LPO.Each bar represents the mean ± SD (n=6).c = p<0.0001when compared with group II.

Figure 5 .
Figure 5.Effect of ESEt on Liver Tissue.A= CCl4-induced hepatotoxic control group that showed fatty deposition (ballooning), necrosis and inflammation around central vein.These toxic features are greatly improved in test groups treated with ESEt @ 600mg and 800mg/kg (C & D).However, inflammation and ballooning can be observed in liver slide of silymarin (100mg/kg) treated group (B).
antioxidant property of ESEt of C. anthelminticum

Table 1 .
Effect of ESEt on physical and biochemical parameters.Each value represents the mean ± SD (n=6).b & c = p<0.01& p<0.0001 when compared with group II.Values in parenthesis represent the percent gain (+) / loss (-) in parameters.