Omar ElBaradei Mubarak*, Naguib Faten Morsi and Mohamed Hamama. Zoology Department, Faculty of Science, Menoufiya University, Shebin El-Kom, Egypt.

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1 International Sholars Journals International Journal of Mediinal Plants Researh ISSN: X Vol. 5 (3), pp , Marh, Available online at International Sholars Journals Author(s) retain the opyright of this artile. Full Length Researh Paper Roles of H. rosa sinensis extrat in the treatment of liver against CCl4-indued hepati oxidative stress and antioxidants disorder in male albino rats Omar ElBaradei Mubarak*, Naguib Faten Morsi and Mohamed Hamama Zoology Department, Faulty of Siene, Menoufiya University, Shebin El-Kom, Egypt. Aepted 10 February, 2016 This study try to investigate the affirmation of 70% ethanol / water Hibisus rosa extrat (HRE), of the leaves, to alleviate the CCl 4- indued hepati antioxidants disorders and oxidative stress in male albino rats. For indution of early and late stages of liver intoxiation, CCl 4 (0.1ml/100gm b.w., twie a week, s.) was used for 7 and 14 weeks respetively. At the end of both stages of intoxiation, the diagnosti enzymes of liver funtion; serum aspartate transaminase (AST), and alanine transaminase (ALT) reorded highly signifiant elevation (p<0.001) with the two levels of intoxiation while hepati 5'-nuleotidase(5'-NT) reorded highly signifiant elevation (p<0.01)only with the late stage of intoxiation. On the other hand, the hepati antioxidants defense markers, redued glutathione (GSH), glutathione S-transferase (GST), glutathione peroxidase (GPx), superoxide dismutase (SOD) and atalase (CAT), showed signifiant diminish, one as ompared to its ontrol. This emphasizes the inreased oxidative stress in the liver with both early and late stages of CCl 4 -intoxiation. Consequently, the oxidative stress biomarkers, lipid peroxidation, as malondialdehyde (MDA), liver xanthine oxidase (XO) and serum advaned oxidation protein produts (AOPPs) signifiantly elevated (p<0.001) by CCl 4 liver injury, in stage dependent manner. HRE Post-treatment (100 mg/kg b.w., orally /day, for 4 weeks) for rats, whose of liver injuries, signifiantly redued the all alternations of liver funtions, antioxidant defenses and oxidative stress- markers with time dependent manner. It was lear that both CCl 4 -indued alternation and HRE amelioration effets were of hepatotoxi stage-dependene. So, it is suggested that, H. rosa sinensis extrat effetively ameliorated the hanges observed with CCl 4 injuries in liver, possibly through antioxidant and/or free radial savenging effets of its phenoli ompounds, speially flavonoids, triterpenes and tannins, whih previously were demonstrated as a phytohemial omponents in this extrat. Key words: Hibisus rosa sinensis, CCl4-hepatotoxiity, Oxidative stress, Anti-oxidants. INTRODUCTION Carbon tetrahloride (CCl 4 ) has been used extensively for deades to indue liver injury in various experimental models to eluidate the mehanisms behind hepatotoxiity (Ismail et al., 2009). This intoxiation, whih auses nerosis of hepatoytes, indues inflammation, and promotes the progression of hepati fibrogenesis, antioxidant disorder and oxidative stress (Fu *Corresponding author. omar.mubarak@gmail.om et al., 2008; Mas et al., 2008; Yin et al., 2011). It is well doumented that alteration of antioxidant status with CCl 4 may potentially ause hepatotoxiity and oxidative stress in rats (Khan et al., 2009, 2010) in whih ellular antioxidant defenses are inadequate to ompletely inativate the reative oxygen speies(ros). Oxidative stress has been impliated in the proess of liver fibrogenesis and many etiologial agents of fibrogenesis stimulate free radial reations (Kamel et al., 2010). The exessive oxidative stress is aused by reative oxygen speies and reative nitrogen speies when there is an imbalane between their formation and

2 Omar et al. 210 savenging by body antioxidants. It an promote the pathophysiology of many diseases progression through oxidation of biomoleules suh as nulei aid bases, lipids, and proteins (Wafay et al., 2012). Indeed several studies have onfirmed the potential benefit strategy of antioxidants using as a tool to prevent or slow down the progression of oxidative stress related diseases (Fu et al., 2008; Lin et al., 2008; Khan et al., 2010). They antagonize the deleterious ation of free radials and protet hepatoytes from damage. Consequently, They have emerged as potent antifibroti agents (Khan and Ahmed, 2009; Kamel et al., 2010). Attention has been developed on the natural antioxidants ontained in the dietary plants that are andidated for treatment or protetion of oxidative damage aused by free radials speies (Vinent et al., 2004). Several herbal drugs have been reported to have antioxidative effiay with different poteny in the liver (Mas et al., 2008). The genus Hibisus plants have different uses; some are used as food, suh as Hibisus esulenta L, and some speies as remedy in traditional mediine, suh as Hibisus sabdariffa L., as well as a olorant for herbal teas (Kili et al., 2011). Hibisus rosa has been reported as alternative of anti-diabeti ativity (Sahdewa and Khemani, 2003), against the tumor promotion stage of aner development, in mouse skin with ultraviolet radiation (Sharma et al., 2004) and finally the possible pharmaologial rationale use of this plant for onstipation and diarrhea was suggested (Gilani et al., 2005). In a previous study, the phytohemial sreening for H. rosa dry extrat identified its bioative ompound; flavonoids, glyosides, steroids, triterpenes and tannins. As well as, in vitro, we ensured the antioxidant and savenging ativities of H. rosa extrat due to these bioative ompounds (Abdel-Ghaffar and El-Elaimy, 2012). The urrent study was to evaluate the in vivo role of H. rosa sinensis extrat (HRE) in the treatment of liver against CCl 4 -idued hepati oxidative stress and antioxidants disorder in male albino rats and further, to explore its underlying mehanisms. The study was throughout CCl 4 -indued hepatotoxiity, early and late stages, and HRE post- treatment effet was assessed by investigation of liver funtion tests, hepati antioxidants and also through the biomarkers of lipid and protein oxidation. MATERIALS AND METHODS Chemials Sodium nitrite anhydrous, ammonium molybdate, ferri hloride, asorbi aid, ammonium aetate, riboflavin, methionine, nitrobluetetrazolium, sodium nitroprusside, sulfanilamide, naphthyl ethylene diamide dihydrohloride and trihloroaeti aid were obtained from Sigma- Aldrih (St. Louis, MO, USA).Thiobarbituri aid was obtained from Fluka (Berlin, Germany). All other used hemials and solvents were of high analytial grad. Plant materials The aerial parts of H. rosa sinensis were olleted in the month of April 2012 from Faulty of Agriulture Ain Shams University, Egypt. The plant was identified with the help of available literature and authentiated by the taxonomist at Department of Botany, Faulty of Siene, Menoufiya University, Egypt. The leaves of the plant were washed with tap water followed by distilled water, dried in shade for 10 days prior to extrat preparation. The well dried leaves were powdered by milling and then stored in airtight glass jars, until in use. Preparation of H. rosa extrat (HRE) The air-dried powdered leaves of the plant were repeatedly extrated in the room temperature with 70% ethanol/water until exhaustion. Animals Thirty six adult and healthy male albino rats, weighing g, were randomly hosen from the animal house of the National Researh Center, Cairo - Egypt. They were housed in standard ages and left to alimatize for 7 days to laboratory ondition at temperature of 25±3 C and a 12 h light/dark yle. The animals were maintained on standard laboratory diet and water ad libitum. Experimental design The rats were divided into six groups, 6 rats eah, as follow: Group (1): Untreated normal healthy rats. They were as ontrol group. Group (2): Rats were treated with Hibisus extrat for 4 weeks (100 mg/kg body weight, orally/day) (kumar et al., 2012). Group (3): Rats were injeted with CCl 4 (0.1ml/100gm body weight, twie a week, subutaneously) (El-Elaimy and Abdel-Ghaffar, 1997) for 7 weeks to indue early stage of liver intoxiation. Group (4): Rats were injeted with CCl 4 (with the same dose) for 7 weeks, and then were treated with Hibisus extrat (100 mg/kg body weight, orally /day) for 4 weeks. Group (5): Rats were injeted with CCl 4 (with the same dose) for 14 weeks, to indue late stage of liver intoxiation. Group (6): Rats were injeted with CCl 4 (the same used dose of it) for 14 weeks, and then were treated with Hibisus extrat (the same used dose of it) for 4 weeks. At the final day of treatment for eah group, the rats were

3 211 Int. J. Med. Plant Res. subjeted to light ether anesthesia and were disseted. Blood was withdrawn from the hepati portal vein, entrifuged at 3000 rpm for 10 min. at 4 C for separation of sera. Then, livers were exised, leaned and a sample were homogenized with ultrasoni homogenizer and entrifuged at 3000 rpm for 15 min. in saline (10% w/v), it was for liver 5 -Nuleotidase ativity measurement. All samples were frozen at -20 C. Post-mitohondrial supernatant preparation (PMS) Another samples of liver tissue, of the disseted rats, were removed quikly and perfused with ie-old saline. The liver was homogenized (1:10, w/v) in hilled phosphate buffer (0.1 M, ph 7.4) ontaining KCI (1.17%).The homogenate was entrifuged at 3000 rpm for 10 min to separate the nulear debris. The aliquot so obtained was entrifuged at rpm for 20 min at 4 C to obtain post-mitohondrial supernatant (PMS) for the other enzymes ativities measurements. Liver funtion tests Serum aspartate aminotransaminase (AST), alanine aminotransaminase (ALT) ativities (Reitman and Frankel, 1957) were assyed by using reagent kits purhased from BioMerieux Chemial Company (Frane). Liver 5 -Nuleotidase (5 NT) ativity (El-Aaser and El- Merzabani, 1975) was determined. The method is based on the inubation of speifi substrate, 5 -AMP with liver homogenate sample, and determination of the librated inorgani phosphorous. The enzyme ativity was expressed as μmoles of organi phosphorus liberated /min /g tissue. Antioxidant markers Liver redued glutathione (GSH) (Beutler and Kelley, 1963) The method is based on the development of yellow olor due to reation of 5, 5-dithio (bis) nitrobenzoi aid (DTNB) with ompounds ontaining sulphydryl groups and formation of produt whih is measured at 415 nm. It was expressed as nmoles/min/mg tissue. Liver glutathione S transferase (GST) ativity (Habig et al., 1974) Using1-hloro-2,4-dinitrobenzene (CDNB) as substrate. The priniple of this assay depends on measuring the onjugation of (CDNB) with redued glutathione. The onjugation is aompanied by an inrease in absorbane, at 340 nm, due to thioether formation. The rate of inrease is diretly proportional to the GST ativity in the sample. The enzyme ativity was alulated as nmol CDNB onjugate formed/min/mg tissue as using a molar extintion oeffiient of M/m. Liver glutathione peroxidase (GPx) ativity (Hafeman et al., 1974) The assay depends on inubation of PMS with H 2 O 2 and redued glutathione. The ativity of GPx was determined by quantifying the rate of H 2 O 2 -indued oxidation of redued glutathione (GSH) to oxidized glutathione (GSSH). Yellow produt whih had absorbane at 412 nm ould be formed as GSH reated with (DTNB). GPx ativity was expressed as units/g tissue, and one unit was defined as a derease in GSH of 1 μmol/min. Liver superoxide dismutase (SOD) ativity (Beauhamp and Fridovih, 1971) The nitro blue tetrazolium (NBT) is redued to blue Formosan by superoxide radials (O 2 - ). SOD ativity was assayed by measuring the ability of the enzyme extrat (PMS) to inhibit the photohemial redution of NBT. Test tubes ontaining the reation mixture, whih illuminated with a fluoresent lamp (120 W); idential tubes that were not illuminated served as blanks. After illumination for 15 min, the absorbane was measured at 560 nm. One unit of SOD was defined as the amount of enzyme ativity that was able to inhibit the rate of the redution of NBT to blue formosan by 50%. Liver atalase (CAT) ativity (Goth, 1991) In whih PMS was inubated in H 2 O 2 substrate and the enzymati reation stopped by the addition of ammonium molybdate. The intensity of the yellow omplex formed by molybdate and H 2 O 2 was measured at 405 nm.cat ativity was expressed as (ku/l). Oxidative stress biomarkers Liver lipid peroxidation (Ruiz-Larrea et al., 1994) It was measured as malondialdehyde (MDA). Thiobarbituri aid reats with malondialdehyde to yield a oloured produt that an be measured at 532 nm. The results were expressed as nmol MDA formed/hr/g tissue using a molar extintion oeffiient of M 1 m 1. Serum advaned oxidation protein produts (AOPP) (Witko-Sarsat, 1996) It was measured in serum. Its assay depends on the reation of unknown AOPP-ontaining samples or hloramine standards with reation initiator, potassium iodide that begins a olour development proess. After a brief inubation, the absorbane was immediately measured at 340 nm against blank. (AOPP) onentration was expressed as miromoles per liter

4 Omar et al. 212 Table 1. Serum aspartate aminotransaminase, alanine aminotransaminase and liver 5'- nuleotidase ativities in different studied groups. Liver funtion enzymes Control m±se HRE m±se 7Ws.CCl4 m±se 7Ws.CCl4 +HRE m±se 14Ws.CCl4 m±se 14Ws.CCl4+HRE m±se AST ± ± ±8.78 (107.9%) ± f (32.1%) ±1.88 (151.2%) ± 4.11 f (80.9%) ALT ± ± ± ±2.20 f ± ±2.26 f (114.0%) (13.0%) (151.4%) (29.8%) 5'NT ± ± ± ± 0.66 f ± 0.41 b 22.78± 0.30 f (21.4%) (-3.3%) (37.9%) (16.6%) Values are expressed as mean ± SE (n=6), AST & ALT (U/L), 5'NT (µmol pi/min/g tissue). b (p<0.01) and (p < 0.001) )are signifiant versus ontrol group. f (p<0.001) is signifiant versus orresponding CCl 4 group. (μmol/l) of hloramine-t equivalents. Liver xanthine oxidase ativity (XO) (Athar et al., 1996) PMS was inubated with xanthine substrate and the enzymati reation was terminated by the addition of 0.5 ml ie-old perhlori aid. The oloured produt an be measured at 290 nm and XO ativity was expressed as μg uri aid formed/min/ mg tissue. Statistial analysis Results are expressed as mean ± Standard Error. Data were analyzed by SPSS software, version 19 (Chiago, IL, USA). The statistial evaluation of all data was done using analysis of variane (ANOVA) followed by Student's t-test. P value <0.05 was onsidered statistially signifiant. RESULTS Liver funtion tests Data of the present study revealed that the intoxiation of rats with CCl 4 aused, stage- dependent, signifiant inrease (p< 0.001) in serum AST and ALT ativities with perentage differene (107.9 and 151.2%) and (114.0 and 151.4%) respetively, at both early and late stages of intoxiation. While liver 5'-NT ativity elevated signifiantly (p<0.01) only with the late stage of intoxiation (37.9%), one as ompared to its ontrol. Post-treatment of H. rosa extrat showed a signifiant, stage-dependant, alleviation for ALT, AST and 5'-NT ativities. They reorded, at early stage, non- signifiant values in ase of ALT and 5'-NT but AST still of signifiant value (p<0.05). However, for the late intoxiation stage, HRE signifiantly restored the level of these biohemial markers but still of highly signifiant elevation in AST (p<0.001) and signifiant in ALT (p<0.05) (Table 1). Antioxidant biomarkers A stage-dependent highly signifiant derease (p<0.001) in the level of hepati antioxidant GSH as well as GST, GPx. CAT and SOD ativities was observed in CCl 4 intoxiated rats, of early and late stages, eah ompared with the orresponding normal ontrol level. On the other hand, post-treatment with HRE improved this adverse effet with the early stage of CCl 4 intoxiation, speially of GPx,whih is ompletely normalized, and GSH, whih beame nearer to the normal level (p<0.05).while the levels of GST, CAT and SOD still with signifiant alteration (p<0.001). However, HRE post-treatment for late stage of CCl 4- intoxiation produed a moderate improvement; the levels of all antioxidants still far from their normal values (p<0.001) (Figures 1 to 5). Oxidative stress biomarkers Intoxiated rats with CCl 4 developed a state of oxidative stress, at both stages of intoxiation, represented by the highly signifiant (p<0.001) inrease in liver lipid peroxidation with perentages (146.5 and 203.2%) at early and late stages of intoxiation respetively, measured as malondialdehyde (MDA). Signifiant elevated protein oxidation (p< 0.001) with and 274.5%, at both stages, was measured as advaned oxidation protein produts (AOPP) in serum. As well as, liver XO ativity reorded highly signifiant elevation (p<0.001, and 189.5%), all ompared to their normal ontrol levels. Hibisus extrat-treatment showed a signifiant alleviation in the intoxiated rats against these oxidative stress markers. This was manifested by the signifiant restoring (p<0.001) in the mean values of MDA, AOPP and XO as ompared to the orresponding CCl 4 intoxiated group. However, their levels still higher (p<0.001) than those of their ontrol values at the two

5 GST (nmole /min/mg tissue) GSH (mg/g tissue) 213 Int. J. Med. Plant Res. 80 GSH a,f Control Hibisus,f 7 Weeks.CCL4 7 Weeks.CCL4 + HRE 14 Weeks.CCL4 14 Weeks.CCL4 + HRE Figure 1. Liver redued glutathione ontent of rats submitted Fig. (1): Liver redued glutathione ontent of rats to different treatments. Values are expressed as mean±se submitted to different treatments. Values are expressed (n=6). a (p <0.05) and (p<0.001) are signifiant versus ontrolasmean±segroup.(n=6)f.(pa (p< <0.001)is.5)andsignifiant(p<0.001) versusare orresponding signifiantversus l4 group ontrol. group. f (p < 0.001)is signifiant versus orresponding l 4 group. 70 GST ,d,f 0 Control Hibisus 7 Weeks.CCL4 7 Weeks.CCL4 + HRE 14 Weeks.CCL4 14 Weeks.CCL4 + HRE Figure 2. Liver glutathione s- transferase ativity ontent of Fig. (2): Liver glutathione s- transferase ativity ontent rats submitted to different treatments. Values are expressed of rats submitted to different treatments. Values are as mean± SE (n=6). (p < 0.001)is signifiant versus ontrol groupexpressed.(p<as 0mean±.05)andSE f(n=6)(p< )are(p<.001)issignifianifiantversus orrespondingversusontrollgroup4group.d. (p< 0.05) and f (p < 0.001)are signifiant versus orresponding l4 group.

6 CAT ativity (ku/l) GPx (nmoles GSH oxidized/min/g tissue) Omar et al f GPx 30,f Control Hibisus 7 Weeks.CCL4 7 Weeks.CCL4 + HRE 14 Weeks.CCL4 14 Weeks.CCL4 + HRE Figure 3. Liver glutathione peroxidase ativity of rats Fig. (3): Liver glutathione peroxidase ativity of rats submitted to different treatments. Values are expressed submitted to different treatments. Values are expressed as mean± SE (n=6). (p < 0.001) is signifiant versus ontrolasmean±.f(pse< 0(n=6).001)is. (psignifiant<0.001) versusissignifiantorrespondingversus lont4groupl.f. (p < 0.001)is signifiant versus orresponding l 4 group CAT ,e,f Control Hibisus 7 Weeks.CCL4 7 Weeks.CCL4 + HRE 14 Weeks.CCL4 14 Weeks.CCL4 + HRE Figure 4. Liver atalase ativity of rats submitted to Fig. (4): Liver atalase ativity of rats submitted to different treatments.values are expressed as mean± SE (n=6)different.(p treatments<0.001)is.valuessignifiantre versusexpressedontrolasmean±group. e (p<0.01) and f (p < 0.001)are signifiant versus SE (n=6). (p < 0.001)is signifiant versus ontrol orresponding l 4 group. group. e (p<0.01) and f (p < 0.001)are signifiant versus orresponding l4 group.

7 SOD (% inhibition) 215 Int. J. Med. Plant Res. 90 SOD 80,f 70 b,d Control Hibisus 7 Weeks.CCL4 7 Weeks.CCL4 + HRE 14 Weeks.CCL4 14 Weeks.CCL4 + HRE Figure 5. Liver superoxide dismutase ativity of rats Fig. (5): Liver superoxide dismutase ativity of rats submitted to different treatments. Values are expressed submitted to different treatments.values are expressed as as mean±se (n=6).b (p<0.01) and (p< 0.001) are mean±se (n=6).b (p<0.01) and (p< 0.001)are signifiant versus ontrol group. d (p< 0.05) and f (p< 0 signifiant.001)are signifiant versusontrol versus group orresponding.(p<0.05) l and 4 group f(p< )are signifiant versus orresponding l4 group. Table 2. Lipid peroxidation (LPO), advaned oxidation protein produts (AOPPs) and xanthine oxidase (XO) ativities in different studied groups. Oxidative stress ontrol m±se HRE 7Ws.CCl4 7Ws.CCl4 +HRE 14Ws.CCl4 Ws.CCl4+HRE markers m±se m±se m±se m±se ±SE 48.89± ±2.93 f 60.13± ± 0.29 f LPO 19.83± ±1.07 (146.5%) (69.2%) (203.2%) (145.7%) AOPP 15.32± ± ± ±.o.39 f 57.38±1.07 (224.3%) (67.8%) (274.5%) 32.50±0.39 f (112.1%) 0.836± ± 0.01 f ± ± 0.02 f XO 0.33± ±0.03 (157.2%) (73.2%) (189.5%) (140.0%) Values are expressed as mean±se (n=6). LPO (n mol MDA / h /g tissue), AOPP (µ mol/l), XO (µg uri aid/ min/g tissue). (p < 0.001)is signifiant versus ontrol group. f (p < 0.001) is signifiant versus orresponding l 4 group. stages of intoxiation (Table 2). DISCUSSION CCl 4 is one of the environmental pollutants whih mainly ause hepatotoxi effet through generation of free radials reations due to its metabolism in the liver and subsequent initiation of lipid peroxidation (Lin et al., 2008; Ismail et al., 2009). Serum ALT, AST, and liver 5'-NT reflet the physiologial state of the liver funtion. AST and ALT are hanged aording to the distortion of liver, resulting from ellular injury aused by toxi metabolites and diseases (Patrik-Iwuanyanwu et al., 2007). 5'- nuleotidase is an intrinsi membrane glyoprotein, present as an etoenzyme in a wide variety of mammalian ells to hydrolyze 5'-nuleotides to their orresponding nuleosides (Pagani and Panteghini, 2001). The results of the present work indiated that CCl 4 produed an inrease in the ativities of the diagnosti enzymes; AST, ALT and 5'-NT in rats that reeived CCl 4 as ompared to the ontrol groups. The redutive dehalogenation of CCl 4 by Cyt-P450 enzyme system produes the highly reative trihloromethyl (CCl 3 ) whih

8 Omar et al. 216 previously reported by Fu et al. (2008) and Lin et al. (2008). CCl 3 indues injury and fibrogenesis by oxidative stress, inflammation, and ativation of hepati stellate ells. This inrease in serum ALT and AST ativities may reflet that CCl 4 intoxiation was able to reah the liver and produe ellular degeneration or destrution (Khalifa et al., 2011). Moreover, It ould potentially be attributed to more prodution of these enzymes and their releasing from the ytoplasm into the blood irulation after rupture of the plasma membrane and ellular damage (Liu et al., 2006; Shaarawy et al., 2009). On the other hand, 5'-NT is measured as an indiator of liver damage resulting primarily from interferene with the bile seretion. However, the early studies showed its ativity elevation in liver diseases inluding liver irrhosis, hroni aloholism, benign biliary disease and neoplasm of the liver and bile duts (Dinkov et al., 1979). Herein, the high ativity of 5'- NT in the CCl 4 -intoxiated rats is suggestive of intrahepati obstrution of bile analiuli as a result of liver ell injury and due to inrease in fluidity of the ell membrane (Dipple et al., 1982). As muh as, Subhani et al. (2009) investigated the oxidative stress, antioxidants and their assoiation with 5'-NT ativation. They reported that its relatively higher ativity was depending on the duration of CCl 4 intake and also be ranging as the damage done to the liver ells and bile analiular membranes. Hibisus rosa extrat signifiantly redued the elevated ativities of serum AST, ALT and liver 5'NT indiating that this attenuation effets of HRE ould be attributed to its phytohemial phenolis, predominantly flavonoids, triterpenes and tannins. It has been demonstrated that these ompounds redue the risk of hepatotoxiity by ating against free radial mediated damages to restore the funtional integrity of the hepatoytes membrane, onsequently improved the liver from CCl4-indued injury (Yin et al., 2011). On the same line, Fu et al. (2008) demonstrated that a polyphenoli antioxidant, urumin, signifiantly protets the liver from CCl4-indued injury by reduing the elevated ativities of serum ALT and AST. It is well doumented that the organisms have introdued enzymati and non-enzymati antioxidant systems whih possess the ability to protet the body from damage aused by free radials by savenging or detoxifying reative oxygen speies, blok their prodution or sequester transition metals whih are the soure of free radials ( Liu et al., 2006; Halliwell and Gutteridge, 2007). With regard to the antioxidant defense system in this study, CCl 4 treatment signifiantly redued the total ontents of GSH, the main non-enzymati and naturally-ourring antioxidant in rat liver, as well as the ativities of enzymati antioxidant defenses; GST, GPx, CAT, and SOD.This indiate that the redutive CCl 3 radial initiates the proess of oxidative stress whih is onsidered to be the most important mehanism in the pathogenesis of tissue damage indued by CCl 4 (Liu et al., 2006; Fu et al., 2008). Moreover, these radials, under aerobi onditions, reat with oxygen to form trihloromethyl peroxyl radials (CCl 3 O 2 ) whih bind ovalently to ellular maromoleules leading to membrane lipid peroxidation and finally to ell apoptosis and nerosis (Koneri et al., 2008; Lee et al., 2008). Glutathione (GSH) partiipates diretly in the neutralization of free radials, reative oxygen speies and also plays a role in detoxifiation of many xenobiotis (Huang et al., 2009). It is of major importane in the redution of hydrogen peroxide and lipid peroxides in a reation that is atalyzed by selenium ontaining glutathione peroxidase and by other proteins that also exhibit glutathione-s-transferase ativity (Lin et al., 2008). Herein, the result of redued GSH may be explained on the basis of (i) its utilization in savenging CCl 3 free radials, (ii) its involvement in maintaining non-gsh ritial protein sulfhydryls in redued state, (iii) ating as a o-fator for glutathione-s-transferase during detoxifiation of CCl 4, and (iv) oxidation of glutathione to its oxidized form by glutathione peroxidase in detoxifiation of the formed hydrogen peroxide and/or lipid peroxides (Subhani et al., 2009). It was previously observed that CCl 3 radial an even reat with sulfhydryl groups of glutathione and protein thiols to alter the redox status of ells (Sheweita et al., 2001). However, we annot exlude the possibility that CCl 4 metabolite, CCl 3, may diretly reated with GSH and reduing its onentrations. Glutathione-S-transferase is thought to be the fundamental antioxidant enzyme, beause it is losely related to the diret elimination of reative oxygen speies. It atalyses the onjugation of redued glutathione, via the sulfhydryl group, to eletrophili enters on a wide variety of substrates. This ativity is useful in the detoxifiation of endogenous ompounds suh as peroxidised lipids (Valavanidis et al., 2006). GSH is onsidered an important defense against lipid oxidative damage in the liver whereas it has a diret antioxidant funtion by reating with superoxide radials followed by the formation of oxidized glutathione (GSSG) and other disulfides. Therefore, GSH metabolizing enzymes, GST and GPx, will be affeted when GSH level is redued in the ells (Khan and Ahmed, 2009). Consequently, the redution in the ativity of these enzymes may result in a number of deleterious effets due to the aumulation of superoxide radials and hydrogen peroxide (Khan et al., 2012). In the urrent study, post-treatment with HRE signifiantly restored GSH level as well as both GPx ativity, to be nearly normal level with early intoxiation, and GST ativity, still of signifiant elevation with both stages of CCl 4 intoxiation. This suggests that the antioxidant ativity of HRE played an important attenuation role against CCl 4 -mediated toxiity with stage dependent manner, but may be need more time and /or higher dose of HRE treatment for omplete attenuation. Catalase (CAT) and superoxide dismutase (SOD) are

9 217 Int. J. Med. Plant Res. antioxidant enzymes whih metabolize toxi oxidative intermediates. They require mironutrient as ofators suh as selenium, iron, opper, zin, and manganese for optimum atalyti ativity and effetive antioxidant defense mehanisms (Halliwell, 2001). CAT onverts hydrogen peroxide into water while SOD atalyzes the onversion of superoxide radials to hydrogen peroxide. So, the aumulation of H 2 O 2 or produts of its deomposition may also be aided by a derease in CAT, SOD and GPx ativities (Khan et al., 2010). In our study, CCl 4 has been postulated to have multiple depressed effets on CAT and SOD while HRE supplementation modulated their ativities. This indiates that, the Hibisus post- treatment regulated glutathione synthesis whih in addition to its role as an essential omponent of SOD, it interats with ell membranes to stabilize them against CCl 3 various damaging effets, inluding those due to oxidative injuries (Dulundu et al., 2007). Hand in hand, Wu (2009) reported that the low levels of GSH and SOD in ellular and extraellular fluids redue their oxygen derived free radial savenging apaity making the tissues more vulnerable to oxygen derived free radial damage. This finding was doumented by Huang et al. (2009) who found that the low level of GSH and SOD in the irrhoti patient and alohol onsumer was indiator of higher oxidative stress. It has been suggested that a derease in the ativities of primary antioxidants; CAT and SOD, may be due aumulation of reative oxygen speies whereas SOD ativity an be inhibited by hydrogen peroxide treatment (Miguel et al., 2009). So, this inhibition may ause the aumulation of H 2 O 2 or produts of its deomposition whih may also be aided by a derease in CAT, SOD and GPx ativities (Halliwell and Gutteridge, 2007; Khan et al., 2010). Goel et al. (2005) reported that highly reative oxygen metabolites, espeially hydroxyl radials, at on unsaturated fatty aids of phospholipid omponents of membranes to produe malondialdehyde (MDH), as lipid peroxidation produt. This is in agreement with our results sine CCl 4 have been reorded to indue oxidative stress, as shown by enhaned MDA prodution in liver. This may be the onsequene of an inrement in the formation of oxygen free radials generated by CCl 4 sine antioxidant defense systems are ompromised (Prisilla and Prine, 2009). The urrent study (Table 2) showed that the groups reeived CCl 4 reorded highly signifiant lipid peroxidation (146.5 and 203.2% for 7- weeks and 14-weeks of treatment respetively). Some authors suggested that, dereased ativity level of phase I and phase II enzymes, may result in less savenging of free radials leading to generation of other forms of arbon-, nitrogen- and oxygen-entered radials, ould lead to the lipid peroxidation found in liver (Liu et al., 2006) or kidney (Khan et al., 2010) after CCl 4 - administration. However, our data of signifiant dereased GST and GPx in CCl 4 -treated groups may explain this elevation of MDA, with early and late stages of CCl 4 hepatotoxiity respetively. The groups reeived the post-treatment of HRE exhibited highly signifiant alleviation, as evidened from the ameliorated MDA level (69.2 and 145.7%, for both early and late stages respetively) by treating against CCl 4 - oxidative stress and suppressed antioxidants. In onurrent study, in rat primary hepatoytes ulture, Yin et al. (2011) found that Hibisus sabdariffa extrat has also been shown to derease the formation of MDA. It has been suggested that the diminished lipid peroxidases, generated after CCl 4 treatment, is eliminated by the alleviated GPx in the presene of restored glutathione, thus urbing the propagation of lipid peroxidation. Teli and his olleagues (2000) explained that, in vivo, damage to proteins may be more important than damage to lipids in oxidative stress. A marker of protein oxidation, advaned oxidation protein produts (AOPP), attrated the attention of various investigators (Kaneda et al., 2002; Woods et al., 2003). It is well established that exposure of proteins to reative oxygen speies an alter the physial and hemial struture of the target ausing onsequent oxidation of side hain groups, protein sission, bakbone fragmentation, ross-linking unfolding, and formation of new reative groups. The latter inlude oxidation of hydrophobi amino ayl residues to hydroxyl (-OH) and hydroperoxy (P-OOH) derivatives, protein arbonylation (PCO), oxidation of thiol (-SH) groups, nitrotyrosine (NT) formation, and many others (Stadtman, 2004). Our result (Table 2) onfirmed all these truths whereas it revealed that there was a dramati elevation in serum AOPP with CCl 4 intoxiation represented by and 274.5% at early and late stages of liver injury, respetively. This elevation of AOPP is due to protein damaged by CCl 3 - indued oxidative stress. It is lear that, the onformational hanges that result from this omplex of reations lead to the derease or loss of protein biologial funtion as well as its AOPP aumulation. Moreover, Khan and Sultana (2004) added that oxidized proteins are funtionally inative and their unfoldings are assoiated with enhaned suseptibility to proteinases thus, ells an generally remove oxidized proteins by proteolysis. This may postulate that the enhaned xanthine oxidase ativity, whih is an oxidized enzyme for purines, protein and amino aid atabolism, in the urrent study with and 189.5% at both stages of CCl 4 intoxiation, respetively, has been attributed to inreased plasma damage by the generated oxidative stress. This may be onsidered as a reason for the aumulation of oxidized proteins. However, ertain oxidized proteins are poorly handled by ells together with possible alteration in the rate of prodution of oxidized proteins; this may ontribute to their observed aumulation suh as in diabetes and atheroslerosis (Wood et al., 2003). As usual in this study, HRE

10 Omar et al. 218 treatment aused stage dependent redution for elevated AOPP ontents that reorded 67.8 and 112.1%, and xanthine oxidase ativity, 73.2 and 140.0%, at the early and late stages, respetively, when ompared with ontrol (Table 2). So, in general, H. rosa extrat ameliorated, with stage dependent manner, CCl 4 -mediated inhibition of antioxidant enzymes ativities and suppressed free radial mediated lipid and protein oxidation. We an suggest that the attenuation effets of HRE against the CCl 4 -indued hepatotoxiity and oxidative stress was by ating against free radial and CCl 4 - mediated damages. This ould be attributed to its phytohemials; phenolis, predominantly flavonoids, triterpenes and tannins whih well doumented as antioxidants savenging ativities produers (Abdel-Ghaffar and El-Elaimy, 2012), and their ability to stabilize hepatoellular membranes by dereasing membrane fluidity, whih ould sterially hinder diffusion of free radials and thereby derease the kinetis of free radial reations (Khan and Sultana, 2004). Consequently, It is suggested that this ompound redue the risk of oxidative stress through dereased prodution of free radial derivatives as well as inreased the defense mehanism against the reative oxygen speies. Thereby, the antioxidant potential of Hibisus extrat will restore antioxidant apaity within the hepati tissue to at against the oxidative stress. Overall, the urrent results emphasized that H. rosa sinensis extrat reorded a moderate attenuation against CCl 4 hepatotoxiity but may be need more time and/or higher dose for omplete attenuation. 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