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

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1 Vol. 10(26), pp , 15 July, 2016 DOI: /AJPP Artile Number: B5B4A ISSN Copyright 2016 Author(s) retain the opyright of this artile Afrian Journal of Pharmay and Pharmaology Full Length Researh Paper Centratherum anthelmintium seeds reverse the arbon tetrahloride-indued hepatotoxiity in rats Shamim A. Qureshi 1 *, Sumera Rais 2, Rabbiya Usmani 1, Syed Shabib M. Zaidi 1, Musarat Jehan 3, Tooba Lateef 1 and Muhammad Bilal Azmi 4 1 Department of Biohemistry, University of Karahi, Karahi-75270, Pakistan. 2 Department of Biohemistry, Federal Urdu University of Arts, Siene and Tehnology, Karahi-75270, Pakistan. 3 Department of Biohemistry, Sir Syed College of Medial Sienes for Girls College, Karahi, Pakistan. 4 Quality Enhanement Cell, Dow University of Health Siene, Karahi-74600, Pakistan. Reeived 4 April, 2016; Aepted 6 June, 2016 The present study is the first attempt to evaluate the hepatoprotetive effet of ethanoli seeds extrat (ESEt) of Centratherum anthelmintium (blak umin) in arbon tetrahloride (CCl 4 )-indued liver injury. The test doses (600 and 800 mg/kg) of same extrat were found effetive in their respetive test groups by improving the body and liver weights, serum alanine and aspartate transaminases, γ- glutamyltranspeptidase, alkaline phosphatase, total proteins, albumin, total bilirubin, espeially indiret bilirubin and uri aid levels as ompared to CCl 4 -indued hepatotoxi ontrol group. In addition, dereased perent inhibitions of antioxidant parameters inluding atalase, superoxide dismutase and redued glutathione aompanied with inreased perent inhibition of lipid peroxidation observed in both test groups. Histopathologial studies also proved the liver regenerating property of ESEt by showing derease in fatty deposition, nerosis and inflammation around the entral vein of liver lobules. Therefore, the ESEt was found to be hepatoprotetive and antioxidative in nature. Key words: Centratherum anthelmintium, Carbon tetrahloride, Liver funtion test and antioxidants. INTRODUCTION Liver is the indispensable organ in maintaining the homeostasis in body by regulating metaboli and hematologial funtions plus bile prodution for fats emulsifiation and digestion (Hall, 2011). Besides these, diverse funtions are also on its redit like storage of vitamins, iron, detoxifiation, removal and exretion of antibiotis, hormones, oxidative radials and xenobiotis (Hall, 2011). However, liver s funtion and struture are very sensitive and an easily be affeted by miroorganisms (bateria/viruses/fungi/parasites) and hepatotoxins/arinogens (Ahmad et al., 2014). Among different liver affeting hemials, arbon tetrahloride (CCl 4 ), whih is normally used as leaning agent in industries, in fire extinguishers, et., is injurious for body *Corresponding author. qureshi29@live.om. Tel: Ext 2289, Fax: Author(s) agree that this artile remain permanently open aess under the terms of the Creative Commons Attribution Liense 4.0 International Liense

2 534 Afr. J. Pharm. Pharmaol. tissues espeially liver. Continuous inhalation of CCl 4 vapours enhaned inflammation and nerosis of hepatoytes by produing reative metabolites that may lead to severe irrhosis, if the ondition ignored for a long time (Bigoniya et al., 2009; Singh et al., 2011). Exluding geneti auses, environmental pollutants and hemials used in industries are also aelerating the risk of liver problems in both developed and developing ountries and Pakistan is also showing highest burden of liver disorders whih is hiefly ontributed by mines, mills and industrial workers (Anjum et al., 2009; Malaguarnera et al., 2012; Shah et al., 2015). In addition to the ostly onventional treatments of liver problems, mediinal plants always have prominent plae in this regard espeially in Asian ountries as these are easily available and have no toxi effets (Guan and He, 2013). Centratherum anthelmintium (Vernonia anthelmintium Willd) belongs to the family Asteraeae and ommonly known as blak umin (Amir and Chin, 2011). It is not only widely distributed in neighboring ountries of Pakistan but also popular for its ulinary use in all over India and Pakistan (Singh et al., 2012). Its bitter taste seeds are popular for various mediinal purposes and their different extrats are well-reported for pharmaologial ativities like analgesi, antipyreti, antimirobial, antianer, antidiabeti, antihyperlipidemi, anti-inflammatory, antioxidant, antiurolithiati, and skin problems (Amir and Chin, 2011; Mudassir and Qureshi, 2015). However, their hepatoprotetive effet has not been doumented yet. Therefore, the present study is designed to investigate the effet of ethanoli seeds extrat of C. anthelmintium in arbon tetrahloride (CCl 4 ) indued hepatotoxi rats. MATERIALS AND METHODS Animals Female Wister albino rats (180 to 220 g) were purhased from breeding house of Dow University of Health Sienes (DUHS), Karahi and kept in animal house of Department of Biohemistry, University of Karahi (UoK) aording to the international guidelines of animal handling by giving them standard laboratory diet and water ad libitum. Animal grouping and proedure Experimental rats were divided into four groups (6 rats/group) inluding normal and hepatotoxi ontrols (group I and II), eah of them treated with distilled water (1 ml/kg), positive ontrol (group III) treated with silymarin (200 mg/kg) and test groups (groups IV and V) treated with ESEt (600 and 800 mg/kg). Eah treatment was done orally one per day in early morning for 5 days onseutively. However, hepatotoxiity was indued in group II to V by intraperitoneal injetion of CCl4 (3 ml/kg in 1:1 ratio with olive oil) on 3rd and 5th day of trial after 1 h of their respetive treatments. After 24 h of last dose of CCl4, rats were sarified to ollet blood and serum to analyze biohemial parameters. In addition, livers were disseted out arefully to weigh them, estimate antioxidant parameters and for histologial examination. The present experimental proedure was approved by Board of Advane Study and Researh (BASR) of UoK. Physial, biohemial and antioxidant parameters Perent body weight hange (BWC) of rats in eah group was alulated by using formula (Azmi and Qureshi, 2013) after measuring the body weights of eah rat on initial (IBW) and final (FBW) days of trial. Beside this, the livers of eah group were also weighed as LW (g). Perent BWC = FBW IBW IBW Biohemial parameters inluding alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyltranspeptidase (GGT), total bilirubin (TBR), diret bilirubin (DBR), indiret bilirubin (IBR), total protein (TP), albumin (ALB) and uri aid (UA) were measured in serum through ommerially available enzymati kits (Randox, UK). Whereas antioxidant parameters inluding, atalase (CAT), superoxide dismutase (SOD), redued glutathione (GSH) and lipid per oxidation (LPO) were measured in liver homogenate by standard manual methods (Lateef and Qureshi, 2013). Perent protetion in ALT, AST, and ALP of positive ontrol and test groups against hepatotoxi ontrol was alulated 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 respetive enzyme. Perent Protetion = 1 X 100 At Ao Ax Ao X 100 Similarly, perent gain/loss in LW and TBR, IDBR, TP, ALB and UA levels in positive and test groups was alulated with formula desribed by Azmi and Qureshi (2012). Plant material and extration Seeds of C. anthelmintium were purhased and identified by authenti taxonomist (vouher no. KU/BCH/SAQ/02) of UoK. These seeds were used to prepare ethanoli extrat (ESEt) aording to the proedure desribed by Mudassir and Qureshi (2015). HISTOLOGY Disseted out liver tissues from eah group were immersed in 10% formaldehyde solution separately and sent to Dr. Essa s diagnosti laboratory, AbulHasan Isfahani Road, Karahi Pakistan for histologial studies. Positive ontrol and vehile Silimyrin (Siliver 200 mg) and dimethyl sulphoxide (DMSO; 0.05%) of Abbott Laboratories (Pakistan) Ltd and Fisher Sientifi, UK, respetively were used as positive ontrol in the present study and vehile for administering the doses of ESEt in experimental rats. Statistial analysis One way analysis of variane (ANOVA) was used to analyze the results of present study and mentioned as mean ± SD (standard deviation). The differene of mean of eah parameter was ompared among all groups and found signifiant at p<0.05 through

3 Qureshi et al. 535 Table 1. Effet of ESEt on physial and biohemial parameters. Groups LW (gm) GGT (U/l) TBR (mg/dl) DBR (mg/dl) IBR (mg/dl) TP (gm/dl) ALB (g/dl) UA (mg/dl) I: Normal Control 7.46± ± ± ± ± ± ± ± 0.21 II: Hepatotoxi Control 15.31± ± ± ± ± ± ± ± 0.34 III: Positive Control 11.93±1.19 (-22%) 0.71 ± ± 0.01 b (-55.8%) 0.10 ± ± (-73.6%) 5.26 ± 0.19 (52%) 4.10 ± 0.04 (41%) 8.86 ± 0.12 (-13.13%) IV: Test group 11.30±1.43 (-26.1%) 1.36 ± ± 0.01 b (-53.4%) 0.10 ± ± (-73.6%) 5.10 ± 0.08 (37%) 4.35 ± 0.29 (49%) 9.17 ± 0.55 (-11.2%) V: Test group 10.60±1.07 (-30.7%) 3.05 ± ± 0.01 (-39.5%) 0.19 ± ± (-73.6%) 5.90 ± 0.08 (59%) 4.56 ± 0.06 (56%) 7.60 ± 0.64 (-25.4%) Eah value represents the mean ± SD (n=6). b & = p<0.01 & p< when ompared with group II. Values in parenthesis represent the perent gain (+) / loss (-) in parameters. least signifiane (LSD) test (SPSS, version 17.0). RESULTS Effet of ESEt on perent body weight hange and liver weights ESEt in doses of 600 and 800 mg/kg signifiantly dereased (p<0.05 and p<0.0001) the perent redution in body weights in group IV and V, respetively as ompared to hepatotoxi ontrol group (group II) where administration of CCl 4 - indued marked redution in body weights (Figure 1). Similarly, the liver weights (g) in group II were learly inreased (p<0.0001) as ompared to group III, IV and V where silymarin and test doses of ESEt were found effetive in normalizing the weights of livers (Table 1). Effet of ESEt on liver and non liver-speifi parameters Liver-speifi enzymes inluding ALT, AST and ALP (U/l) were drastially elevated in group II whereas all same enzymes were signifiantly dereased (p<0.01 and p<0.0001) in groups III, IV and V whih were treated with silymarin (100 mg/kg) and ESEt (600 and 800 mg/kg), respetively and showed signifiant perent protetion espeially in ALT (95 to 98%), AST (73 to 81%) and ALP (37 to 100%) (Figure 2). Whereas GGT (U/l) level was found dereased in group II (Table 1). Similarly, ESEt and silymarin were found dereasing TBR level espeially indiret BR (mg/dl) in their respetive groups as ompared to group II (Table 1). On the other hand, TP (g/dl) and ALB (g/dl) were dereased in group II and beame signifiantly inreased (p<0.01 and p<0.0001) in group III, IV and V (Table 1). Beside these, non-liver-speifi parameter uri aid (mg/dl) was found dereased prominently (p<0.0001) in the last three groups (Table 1). Effet of ESEt on antioxidant parameters Perent inhibitions of CAT, SOD and GSH were signifiantly dereased in positive ontrol (group III) and test groups (IV and V) as ompared to hepatotoxi ontrol group II (Figure 3 and 4). Whereas, in the ase of perent inhibition of LPO, an entirely opposite piture was observed in positive and test groups (Figure 4). Effet of ESEt on histology of liver tissues Histopathologial studies were done by preparing slides of liver tissues stained with hematoxylin and eosin (Figure 5). Liver tissue (slide A) of CCl 4 - indued hepatotoxi group showed degeneration of hepatoytes aompanied with fatty deposition (ballooning) and infiltration of inflammatory ells around dilated entral vein in liver lobule. However, all these features of liver injury were gradually reovered in tissues (slide C and D) of groups treated with ESEt in doses of 600 and 800 mg/kg. DISCUSSION Liver problems, beause of aquired auses, are ontributing the major portion of death burden globally (Malaguarnera et al., 2012). The harateristi features of liver problems are loss of appetite and weight, elevation in liver-speifi parameters and redution in hepati funtions (Singh et al., 2011). Interesting, the same features was learly observed in the present arbon tetrahloride (CCl 4 )-indued hepatotoxi rat model. CCl 4 is a well-known liver toxiant (Adewale et al., 2014). It severely damaged ellular integrity of hepatoytes by onverting itself into reative metabolites inluding trihloromethyl (.CCl 3 ) and peroxytrihloromethyl (.OOCCl 3 ) radials after passing through hepati ytohrome P 450 enzyme responsible for the detoxifiation of xenobioti or hemials (Khan et al., 2012). Elevated levels of both ALT and AST are the best indiators of liver damage whereas GGT and

4 Enzyme Ativity 536 Afr. J. Pharm. Pharmaol. Figure 1. Effet of ESEt on Perent BWC. Eah bar represents the mean ± SD (n=6). a and = p<0.05 and p< respetively when ompared with group II ALT (U/l) AST (U/l) ALP (U/l) Group I Group II Group III Group IV Group V Figure 2. Effet of ESEt on Liver-Speifi Enzymes. Eah bar represents the mean ± SD (n=6). = p< when ompared with group II. ALP together reflet the damage in bile dut. However, isolated elevation of AST and ALP is the warning of ardia and bone problems as their hief onentrations are present in both these tissues, respetively (Bishop et al., 2013). In the present study, CCl 4 -indued hepatotoxi ontrol rats showed a drasti inreased in ALT, AST, ALP and dereased in GGT levels. On the other hand, ALT, AST and ALP were found extensively improved in positive ontrol 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 intraellular enzymes and their abundane presene in serum above 100 to 1000 folds of their normal levels is the eho of the alteration in ell membrane intatness (Bishop et al., 2013) while dereased levels of GGT are the refletion of intrahepati holestasis and dereased bile aid prodution (Hyder et al., 2013). The alteration in ell

5 Perent Inhibition Perent Inhibition Qureshi et al CAT SOD Group I Group II Group III Group IV Group V Figure 3. Effet of ESEt on Perent Inhibition of CAT and SOD. Eah bar represents the mean ± SD (n=6). = p< when ompared with group II GSH LPO Group I Group II Group III Group IV Group V Figure 4. Effet of ESEt on Perent Inhibition of GSH and LPO. Eah bar represents the mean ± SD (n=6). = p< when ompared with group II. membrane was learly proved by observing severe nerosis along with inflammation and steatosis (fatty deposition) in hepatoytes around entral vein of lobules in mirosopi examination of liver tissues of hepatotoxi group whih was gradually healed up and improved by observing almost normal arhiteture of liver tissues that were disseted out from test groups treated with both doses of ESEt, respetively. Important point is that the ESEt of C. anthelmintium showed muh better results as ompared to silymarin in bringing the liver anatomy bak to normal. The ellular protetive effet of ESEt of C. anthelmintium was also onfirmed by observing dereased levels of uri aid (UA) in both test groups as ompared to hepatotoxi ontrol group whih showed high onentration of same parameter. UA is the end produt of purine metabolism and its abnormally inreased onentration in serum is the alarming sign not only for the presene of kidney dysfuntion but also for the degradation of body tissues or ells (Kutzing and

6 538 Afr. J. Pharm. Pharmaol. Nerosis & ballooning Dilated Central Vein A B C D Figure 5. Effet of ESEt on Liver Tissue. A= CCl 4-indued hepatotoxi ontrol group that showed fatty deposition (ballooning), nerosis and inflammation around entral vein. These toxi features are greatly improved in test groups treated with 600mg and 800mg/kg (C & D). However, inflammation and ballooning an be observed in liver slide of silymarin (100mg/kg) treated group (B). Firestein, 2008). This benefiial effet of ESEt was more strengthen by observing a signifiant derease in perent redution in body weights of both test groups treated with same extrat (600 and 800 mg/kg) as ompared to hepatotoxi ontrol whih was only treated with CCl 4 (3 ml/kg) and showed an extreme perent loss in body weight up to -12%. Even silymarin, the well-known hepatoprotetive mediine, did not prove to be statistially effiient in this respet in positive ontrol group. The possible involvement of ESEt of C. anthelmintium in regenerating liver tissue was also fortified by estimating the normal levels of total protein (TP), albumin (ALB) and total bilirubin (TBR) both diret and indiret in extrat treated test groups as ompared to CCl 4 -indued hepatotoxi ontrol group that showed dereased levels of TP, ALB and elevated levels of TBR partiularly indiret/ unonjugated one. Literature delared that 90% of total protein, exept immunoglobulin and 100% albumin are synthesized in liver (Murphey et al., 2007). However, TP and ALB an also be dereased in renal funtions and malnutrition but these are onsidered as the best meter for evaluating the funtionality of liver (Thapa and Walia, 2007). Similarly, inreased serum IDBR level was not only observed in severe hemolysis but also in liver problems inluding hepatitis and irrhosis with dereased or no onjugation reation taking plae in liver (Hall, 2011). Another study stated that depletion of TP indued intense mitosis in hepatoytes whih leads to the enlargement of liver and this ondition persists till the TP onentration beomes normal in blood (Bishop et al., 2013; Hall, 2011). Interestingly, the same happened in the present study as dereased TP and ALB was observed in hepatotoxi ontrol group aompanied with inreased liver weights, whereas improvement in TP and ALB levels in positive ontrol 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 fators that hindered liver tissue regeneration like tumor nerosis fator alpha (TNFα), et (Kang et al., 2012). Amazingly, hloroform fration of C. anthelmintium seeds was laimed to inhibit TNF-α in human tumor ells (Arya et al., 2012). The antioxidant property of ESEt of C. anthelmintium

7 Qureshi et al. 539 was already well-reported in hyperlipidemi rabbits (Lateef and Qureshi, 2013) and again beame benefiial in ontributing to the hepatoprotetive ation of same extrat in the present study. In vivo indution of CCl 4 stimulates severe oxidative stress by produing two trihloromethyl radials in the presene of hepati mixed funtion oxidase (Cyto P 450 ), whih reported to indue lipid peroxidation, alteration in ell membrane permeability and mitohondrial funtion, thereby produing reative oxygen speies (ROS) and reating tissue inflammation and nerosis (Thanh et al., 2015). ESEt displayed radial savenging ativity in both test groups treated with same extrat (600 and 800 mg/kg) by showing derease in perent inhibition of CAT, SOD, redued GSH and inrease in perent inhibition of LPO whereas an entirely opposite piture was observed in CCl 4 -indued hepatotoxi group for these four parameters. The antioxidant potential of C. anthelmintium seeds might be residing in their polyphenoli ontent, espeially flavonoids. Different extrats of C. anthelmintium seeds proved the presene of polyphenols and flavonoids having radial savenging abilities in in-vitro assay (Ani and Naidu, 2011; Mudassir and Qureshi, 2015; Shah et al., 2007). Conlusion The results onluded that ethanoli seeds extrat of C. anthelmintium has potent ativity to reverse the harmful effets of arbon tetrahloride on liver tissues. However, further work on the same theme has to be done on its isolated ompounds to know whih would be the ative priniple in same extrat having hepatoprotetive ativity. Conflit of interest The authors have not delared any onflit of interest. REFERENCES Adewale OB, Adekeye AO, Akintayo CO, Onikanni A, Saheed S (2014). Carbon tetrahloride (CCl4)-indued hepati damage in experimental sprague dawley rats: Antioxidant potential of Xylopia aethiopia. J. 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