CHAPTER- 3 ANALYSIS OF PATHOPHYSIOLOGICAL MARKER ENZYMES, LIPID AND PROTEIN PROFILES IN CONTROL AND EXPERIMENTAL ANIMALS

Transcription

1 CHAPTER- 3

2 CHAPTER- 3 ANALYSIS OF PATHOPHYSIOLOGICAL MARKER ENZYMES, LIPID AND PROTEIN PROFILES IN CONTROL AND EXPERIMENTAL ANIMALS 3.1. INTRODUCTION The liver, hs vriety of trnsminse to synthesize nd brek down mino cids nd to interconvert energy storge molecules. The concentrtions of these in the serum (the non-cellulr portion of blood) re normlly low. However, if the liver is dmged, the heptocyte cell membrne becomes more permeble nd some of the enzymes lek out into the blood strem. The two trnsminse commonly mesured re lnine trnsminse (ALT) nd sprtte trnsminse (AST). These levels previously were clled the serum glutmte-pyruvte trnsminse (SGPT) nd the serum glutmte-oxlocette trnsminse (SGOT). Elevted levels re quite sensitive for liver injury, mening tht they re likely to be present if there is n injury. However, they my lso be elevted in other conditions. ALT is not commonly found outside the liver; AST too is most commonly found in the liver, but lso in significnt mounts in crdic (hert) nd skeletl muscle. In fct, mesurement of these used to be prt of dignosing hert ttcks, lthough newer enzymes nd proteins tht re more specific for crdic dmge hve lrgely replced this usge. In generl, ny dmge to the liver will cuse medium elevtions in this trnsminse (usully clled liver enzymes, though of course they re not the only enzymes in the liver) nd dignosis requires synthesis of mny pieces of informtion, including the ptient's history, physicl exmintion nd possibly imging or other lbortory exmintions. However, very high elevtions of the trnsminse suggests severe liver dmge, such s virl heptitis, liver injury from lck of blood flow, or injury from drugs or toxins. Most disese processes cuse ALT to rise higher thn AST; AST levels double or triple tht of ALT re consistent with lcoholic liver disese. 30

3 Ftty chnge represents the intr cytoplsmic ccumultion of triglyceride (neutrl fts). At the beginning, the heptocytes present smll ft vcuoles (liposomes) round the nucleus (microvesiculr ftty chnge). In this stge liver cells re filled with multiple ft droplets tht do not displce the centrlly locted nucleus. In the lte stges, the size of the vcuoles increse pushing the nucleus to the periphery of the cell giving chrcteristic signet ring ppernce (mcrovesiculr ftty chnge). These vesicles re well delineted nd opticlly "empty" becuse fts dissolve during tissue processing. Lrge vcuoles my colesce nd produce ftty cysts which re irreversible lesions. Bilirubin is creted by the ctivity of biliverdin reductse on biliverdin, green tetrpyrrolic bile pigment which is lso product of heme ctbolism. Bilirubin, when oxidized, reverts to become biliverdin once gin. This cycle, in ddition to the demonstrtion of the potent ntioxidnt ctivity of bilirubin, hs led to the hypothesis tht bilirubin's min physiologic role is s cellulr ntioxidnt. Bilirubin is the conventionl indictor of liver diseses nd these biochemicl restortions my be due the promotion of its glucuronidtion (Recngel et l., 1989) MATERIALS AND METHODS Determintion of optimum dosge of C. siticum nd lycorine Initilly dose fixtion study ws conducted with C. siticum nd lycorine. Mice re dministered with five different doses of C. siticum (50, 100, 150, 200, 250 mg/kg body weight) nd lycorine (3, 5, 7, 9 nd 11 mg/kg body weight) to CCl 4 induced group of mice to determine the optimum dosge. After, experimentl periods the mice were scrificed under the nesthetic condition nd blood smple ws collected for nlysis of liver mrker enzymes Selection of nimls Swiss lbino mice, weighing 30 35g, procured from the smll niml breeding centre, Agriculturl University, Mnnuthy, Kerl were used. The institutionl niml ethics committee (IAEC) pproved the reserch. Animls were cclimtized under stndrd lbortory conditions t 25 ± 2 C nd 50 ± 15% room humidity nd norml photoperiod (12 h light: drk cycle) for seven dys. The nimls were fed with commercil rt pellet diet nd wter d libitum. All the niml experimenttions were premeditted nd executed in complince with the ethicl norms pproved by Ministry 31

4 of Socil Justices nd Empowerment, Government of Indi nd Institutionl Animl Ethics Committee Guidelines (743/03/bc/ CPCSEA dt ) Experimentl design The mice were rndomly divided into seven groups with ech contining 6 mice. Group I served s norml nd ws given olive oil dily for period of 8 weeks. Group II nd III served s for tretment, which the mice were dministered with C. siticum (200mg/kg) nd lycorine(5 mg/kg) lone. For inducing heptotoxicity nimls of Groups IV VII were dministered orlly 1 ml/kg body weight of crbon tetrchloride (20% CCl 4 in olive oil) twice week for period of 8 weeks. Group IV served s control CCl 4. Group V dministered orlly with C. siticum (200mg/kg), VI dministered intrperitonely with lycorine 5mg/ kg of body weight Group VII ws dministered orlly with well known heptoprotective compound silymrin (100 mg/kg dily for period of 8 weeks. At the end of the experiment, nimls were scrificed by cervicl disloction. Blood ws collected nd liver smples were dissected out nd wshed immeditely with ice cold sline to remove s much blood s possible, nd immeditely stored t -20 C until nlysis. An extr smple of liver ws excised nd fixed in 10% formlin solution for histopthology nlysis Assy of sprtte trnsminse (AGAPPE Dignostics Kit) To 1000µl of working regent 100µl of smple ws dded, mixed nd incubte for 1minute t 37 o C nd the bsorbnce ws mesured. The ctivity ws clculted by using the formul. ALT ctivity in (U/L) = ( OD/min) x Assy of Alnine Trnsminse (AGAPPE Dignostics Kit) To 1000µl of working regent 100µl of serum/orgn extrct ws dded, mixed nd incubte for 1minute t 37 o C nd the bsorbnce ws mesured t 340nm.By using the following formul the AST ctivity ws mesured. AST ctivity (U/L) = ( OD/min) x

5 Assy of Alkline Phosphtse (ALP) To 1000µl of working regent 20µl of serum/orgn extrct ws dded, mixed d incubte for 1minute t 37 o C nd the bsorbnce ws mesured t 405nm. The ctivity ws clculted by using the formul. ALP Activity (U/L) = ( OD/min) x Estimtion of Glucose (AGAPPE Dignostics Kit) 10 µl of serum smple mixed with 1000 µl of enzyme regent nd incubted for 10 minutes t 37 0 c. At the sme time blnk nd stndrd solution ws prepred. The bsorbnce of smple ginst regent blnk ws red t 505 nm. The ctivity ws clculted by using the formul.glucose (mg/ml) = Abs. smple/abs. stndrd conc. of stndrd.the vlues re expressed in mg/dl Totl nd direct bilurubin Two 50 µl of serum/ tissues of smple mixed with 1000 µl of Totl bilurubin regent nd direct bilurubin regent nd then 20 µl of respective ctivtor regent ws dded, then mixed well nd incubted for 10 minutes t 37 0 C. At the sme time blnk nd stndrd solution ws prepred. The bsorbnce of smple ginst regent blnk ws red t 546 nm. The ctivity ws clculted by using the formul. Totl bilurubin = OD of smple - OD of smple blnk / OD of rtificil stndrd 10 Direct bilurubin= OD of smple - OD of smple blnk / OD of rtificil stndrd Lctte Dehydrogense: To 1000µl of working regent 10µl of serum/orgn extrct ws dded, mixed nd incubted for 1minute t 37 o C nd the bsorbnce ws mesured t 340nm. The ctivity ws clculted by using the formul. LDH P ctivity (U/L) = ( OD/min) x Estimtion of Protein Totl protein (Autozyme kit) To 0.01 ml of serum/orgn extrct 1.0ml of working solution ws dded nd incubted the ssy mixture for 5minutes t 37 o C. After completion of incubtion period the bsorbnce ws mesured t 546nm. The ctivity ws clculted by using the formul.totl protein in gm% = Absorbnce of smple/ Absorbnce of stndrd x 5. 33

6 Totl Albumin (Autozyme Kit) To 0.01 ml of serum/orgn extrct 1.0ml of working solution ws dded nd incubted the ssy mixture for 1minute t 37 o C. After completion of incubtion period the bsorbnce ws mesured t 600nm. The ctivity ws clculted by using the formul.totl lbumin in gm% = Absorbnce of smple/ Absorbnce of stndrd x Estimtion of lipid profile (ENSURE Kit). To 1ml of Enzyme regent 10µl of serum/orgn extrct ws mixed well nd kept t 37 o C for 5 minutes t room temperture nd the bsorbnce ws mesured by using spectrophotometer t 505nm. The ctivity ws clculted by using the formul. Cholesterol conc mg/dl = Absorbnce of Test/ Absorbnce of Stndrd x Conc of Std (200) Estimtion of Triglycerides 10 µl of serum/ tissues smple mixed with 1000 µl of enzyme regent nd incubted for 5 minutes t 37 0 c. At the sme time blnk nd stndrd solution ws prepred. The bsorbnce of smple ginst regent blnk ws red t 540 nm. The ctivity ws clculted by using the formul. Triglycerides conc. (mg/ml) = bsorbnce of smple/ bsorbnce of stndrd HDL cholesterol: To 200µl of serum/ orgn extrct smple 300µl of HDL ppt regent ws dded nd mixed well nd kept t room temperture for 10minutes then centrifuged t 3000rpm for 10minutes. Then the pellet ws discrded nd 1ml of enzyme regent ws dded to 100 µl of superntnt, then incubted for 5minutes t 37 o C nd the bsorbnce ws red t 505 nm. The ctivity ws clculted by using the formul. HDL Cholesterol conc.mg/dl: Abs of Test/Abs of stndrd x Conc of Std (50) LDL Cholesterol: The reson for choosing LDL cholesterol s trget for lipid profile is tht it represents the frction of cholesterol, which is most deleterious nd hs been mostly directly correlted with clinicl studies. The LDLws clculted by using the formul. LDL = (Totl cholesterol) - (HDL Cholesterol) (Triglyceride/5) LDL cholesterol levels were expressed s mg/dl serum. 34

7 VLDL- cholesterol The frction of VLDL is to trnsport endogenously synthesized triglyceride nd cholesterol into the peripherl tissue. VLDL cholesterol vlues were clculted using the following formul, The vlues re expressed in mg/dl. VLDL = Triglyceride/ RESULTS Tbles 3.1 nd 3.2 show the optimum dosge for heptoprotective ctivity of C. siticum nd lycorine. At the dose of 200 mg nd 5 mg ther ws significnt (P < 0.05) ltertion in the ctivities of pthologicl mrker enzymes such s AST, ALT, ALP,LDH nd bilurubin in serum smple nd the observtions evidenced tht C. siticum nd lycorine effectively rescue the heptocytes from CCl 4 induced oxidtive dmge without disturbing its cellulr metbolic function nd structurl integrity. Hence, the dose of 200 mg nd 5 mg/kg bw hs been chosen for this study. Tble 3.1. Determintion of optimum dosge of C. siticum Conc. of C. siticum mg/kg AST(U/L) ALT(U/L) ALP(U/L) LDH(U/L) Totl bilurubin mg/dl Control 095 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.01 Tble 3.2. Determintion of optimum dosge of lycorine Conc. of Lycorine mg/kg AST(U/L) ALT(U/L) ALP(U/L) LDH(U/L) Totl bilurubin mg/dl Control 095 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.07 Tble 3.3. shows the body weight nd reltive orgn weight (liver, kidney nd spleen) of the control nd experimentl nimls. Mice treted with CCl 4 showed reduction in the body weight with reltive orgn weight elevtion. It shows tht the 35

8 CCl 4 induced hypertrophy in liver tissues. However, the body weight nd reltive orgn weight hs been mintined by tretment with ethnolic extrct of C. siticum (200mg/kg of b.w), lycorine (5 mg/kg b.w) nd silymrin (100 mg/kg b.w) to CCl 4 induced group of mice. This investigtion suggests tht possibility of C. siticum nd lycorine to confer defense mechnism ginst CCl 4 induced oxidtive stress. No significnt devitions observed in the mice treted with C. siticum nd lycorine compred with control group of mice. Tble 3.3. Effect of ethnolic extrct of Crinum siticum nd lycorine on body weight nd orgn weight (Reltive weight (g/g of the body weight, %) Groups Body weight Liver Kidney Spleen (g) (g) (g) (g) Control ± ± ± ± 0.02 C.siticum lone ± ± ± ± 0.05 Lycorine lone ± ± ± ± 0.32 CCl 4 lone ± ± ± ± 0.05 C.siticum+ CCl ± 0.20 b 5.71 ± 0.19 b 1.68±0.18 b 0.48 ± 0.03 b CCl 4 + lycorine ± 1.43 b 5.98 ± 0.20 b 1.77±0.19 b 0.52 ± 0.02 b CCl 4 +Silymrin ± 0.32 b 5.62 ± 0.20 b 1.41±0.17 b 0.42 ± 0.02 b Results were expressed s Men± S.E.M (n=6). P<0.05 compre with control group of mice. b P<0.05 compre with CCl 4 induced group of mice Tbles 3.4 exemplify the effect of C. siticum nd lycorine on serum glucose, ure, totl bilurubin nd direct bilurubin in control nd experimentl group of mice. The level of serum glucose, ure, totl bilurubin nd direct bilurubin elevted in CCl 4 induced group of mice. Moreover, mice dministered with C. siticum nd lycorine the level of glucose, ure, totl bilurubin nd direct bilurubin were reduced compred with CCl 4 induced group of mice. Similrly, silymrin lso reduced the level of glucose, ure, totl bilurubin nd direct bilurubin s compred to CCl 4 induced group of mice.but, no significnt chnges were observed in the C. siticum nd lycorine lone treted group of mice in comprision to control group of mice. 36

9 Tble 3.4 Effect of ethnolic extrct of Crinum siticum nd lycorine on serum glucose, ure, bilurubin. Groups Control C. siticum lone Lycorine lone CCl 4 lone C. siticum+ CCl 4 CCl 4 + lycorine CCl 4 +Silymrin Results were expressed s Men± S.E.M (n=6). b P<0.05 compre with CCl 4 induced group of mice Totl Direct Glucose Ure bilurubin bilurubin mg/dl mg/dl mg/dl mg/dl 099 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2.4 b 18 ± 0.8 b 0.38 ± 0.06 b 2.1 ± b 125 ± 6.2 b 19 ± 0.4 b 0.43 ± 0.03 b 2.2 ± b 105 ± 7.2 b 18 ± 0.6 b 0.33 ± 0.04 b 2.0 ± b P<0.05 compre with control group of mice. Fig. 3.1 nd 3.2 represent the effect of C. siticum nd lycorine on serum nd liver sprtte trnsminse, lnine trnsminse, lctte dehydrogense, lkline phosphtse in control nd experimentl mice. Mice treted with CCl 4 showed higher in serum nd liver ALT, AST, ALP nd LDH level s compred to tht of control group of mice (p< 0.05). It proves the CCl 4 induced hepto toxicity in experimentl nimls. However, mice treted with C. siticum, lycorine nd silymrin during CCl 4 induction cused significnt decrese in serum nd liver levels of ALT, AST, ALP, LDH compred with CCl 4 induced group (p < 0.05). No significnt sttisticl chnges observed in mice treted with C. siticum nd lycorine lone. These results suggested tht the possibility of C. siticum nd lycorine to give protection ginst CCl 4 induce oxidtive stress in mice. AST(U/I) ALT(U/L) ALP(U/L) LDH(U/L) b b b b b b b b b Fig. 3.1: Effect of C.siticum nd lycorine on pthophysiologicl enzyme in serum. Results were expressed s Men± S.E.M (n=6). P<0.05 compre with control group of mice. b P<0.05 compre with CCl 4 induced group of mice. 37

10 AST(U/L) ALT(U/L) ALP(U/L) LDH(U/L) b b b b b b b b b b b b Fig. 3.2: Effect of C.siticum nd lycorine on pthophysilogicl enzyme in liver. Results weree expressed s Men± S.E.M (n=6). P<0.05 compre with control group of mice. b P<0.05 compre with CCl 4 induced group of mice The effect of C. siticum nd lycorine on serum nd liver cholesterol, triglyceride, low density lipoprotein (LDL), high density lipoprotein (HDL) nd very low density lipoprotein (VLDL) is presented in Tbles 3.5. nd 3.6. Nevertheless, mice treted with CCl 4 exhibited higher serum nd liver cholesterol, triglyceride, LDL, VLDL nd decresed HDL level in serum nd liver compred to tht of control group of mice (p< 0.05). However, dministrtion of C. siticum (200mg/kg) nd lycorine (5mg/kg) to CCl 4 induced group of mice showed significnt reduction in serum nd liver level of cholesterol, triglyceride, very low density lipoprotein nd low density lipoprotein, Where s HDL level significntly incresed compred to those group induced by CCl 4. Moreover, no significnt sttisticl chnges were observed in mice treted with C. siticum nd lycorine lone when compred to tht of control group of mice. Tble 3.5. Effect of ethnolic extrct of C. siticum nd lycorine serum lipids profiles Groups Cholesterol (mg/dl) Triglyceride (mg/dl) HDL (mg/dl) LDL (mg/dl) VLDL (mg/dl) Control 074 ± ± ± ± ±0.9 C. siticum lone 075 ± ± ± ± ±0.6 Lycorine lone 072 ± ± ± ± ±1.5 CCl 4 lone 146 ± ± ± ± ±0.4 C. siticum+ CCl ± 1.5 b 103 ± 15 b 33.1 ±1.7 b b 42.22± ±1.6 b Lycorine+ CCl ± 6.9 b 116 ± 8.6 b 31.7± 6.3 b b 49.02± ±0.6 Silymrin+ CCl ± 2.5 b 94.8 ±3.2 b 34.7 ±1.6 b b 44.04± ± 0.1 b Results were expressed s Men± S.E.M (n=6). P<0.05 compre with control group of mice. b P<0.05 compre with CCl 4 induced group of mice. 38

11 Tble 3.6. Effect of ethnolic extrct of C. siticum nd lycorine liver lipids Profiles Groups Cholesterol (mg/dl) Triglyceride ( mg/dl) HDL ( mg/dl) LDL ( mg/dl) VLDL ( mg/dl) Control ± ± ± ± ±0.3 C. siticum lone ± ± ± ± ±0.5 Lycorine lone ± ± ± ± ±0.5 CCl 4 lone 165.6± ± ± ± ±0.2 C. siticum+ccl ±2.6 b 165.1±11 b 26.43±0.5 b 46.54±0.1 b 33.04±0.6 b Lycorine+CCl ±7.5 b 168.3±9.2 b 25.31±1.3 b 57.15±0.5 b 33.60±0.5 b Silymrin+CCl ±1.2 b 120.6±1.5 b 27.63±0.8 b 46.79±0.1 b 24.11±0.8 b Results were expressed s Men± S.E.M (n=6). P<0.05 compre with control group of mice b P<0.05 compre with CCl 4 induced group of mice The protein content is decresed in CCl 4 induced group of mice orgn viz liver, spleen nd kidney incresed in hert s compred to control group of mice. However, mice dministered with C. siticum nd lycorine showed significnt increse in protein content in liver, spleen, kidney nd decrese in hert tissue s compred to CCl 4 induced group of mice (Tble 3.7). Tble Effect of ethnolic extrct of C. siticum nd lycorine on tissues protein profiles. Groups Protein concentrtion (mg of protein/gm of tissue) Hert Liver Spleen Kidney Control 33.15± ± ± ±2.9 C. siticum lone 32.23± ± ± ±3.2 Lycorine lone 32.52± ± ± ±3.2 CCl 4 lone 35.42± ± ± ±2.5 C. siticum+ CCl ±0.2 b 48.36±3.2 b 88.23±1.2 b 34.26±1.6 b CCl 4 + lycorine 33.36±0.3 b 46.02±3.9 b 85.32±0.6 b 30.89±1.5 b CCl 4 +Silymrin 32.12±0.4 b 52.41±3.1 b 91.26±0.8 b 36.02±1.2 b Results were expressed s Men± S.E.M (no =6). P<0.05 compre with control group of mice b P<0.05 compre with CCl 4 induced group of mice. 39

12 Tble: 3.8. Level of serum protein, lbumin nd globulin in control nd experimentl mice. Groups Protein Albumin Globulin (gm/dl) (gm/dl) (gm/dl) Control 6.1 ± ± ± 0.65 C. siticum lone 6.0 ± ± ± 0.43 Lycorine lone 6.1 ± ± ± 0.82 CCl 4 lone 4.0 ± ± ± 0.26 C. siticum+ CCl ± 0.05 b 1.46 ± 0.18 b 3.15 ± 0.54 b CCl 4 + lycorine 5.5 ± 0.26 b 1.51 ± 0.51 b 3.31 ± 0.31 b CCl 4 +Silymrin 5.8 ± 0.04 b 1.25 ± 0.81 b 3.12 ± 0.42 b Results were expressed s Men± S.E.M (no =6). P<0.05 compre with control group of mice. b P<0.05 compre with CCl 4 induced group of mice Tble 3.8. describes the level of serum protein, lbumin nd globulin in control nd experimentl mice. It shows the reduced level of serum totl protein, lbumin, nd incresed level of serum globulin in CCl 4 induced mice. But, fter the mice treted with C. siticum nd lycorine, the ltered level ws reversed to nerly norml level. However, no significnt sttisticl chnges observed in mice treted with C. siticum nd lycorine lone when compred to tht of control group of mice DISCUSSION Crbon tetrchloride is xenobiotic tht produce heptotoxicity in humn s well s in vrious experimentl nimls nd it ws biotrnsformed by Cytochrome P450 (CYP) into trichloromethyl rdicl CCl - 3 nd trichloromethyl peroxyl rdicl which is initited the lipid peroxidtion (Reckngel et l., 1989) nd involved in the pthogenesis of liver (Reckngel, 1967). Both rdicls re cpble of binding to proteins or lipids, leding to membrne lipid peroxidtion nd finlly cell necrosis (Brttin et l., 1985 nd Reckngel et l., 1989). Severl studies hve shown tht crucil mechnism of the heptoprotective effects my be relted to the ntioxidnt cpcities to scvenge rective oxygen species (Kodi et l., 2007, Nik nd Pnd, 2007). A vst rry of literture hve reported tht numerous ntioxidnts such s vitmin E (Sodergren et l., 2001) vitmin C nd A (Zuluet et l., 2007) nd vitmin D ws reducing cpble of CCl 4 induced 40

13 heptotoxicity effects by preventing lipid peroxidtion. Heptic cells prticipte in vriety of metbolic ctivities nd contin host of enzymes. In tissues, Asprtte trnsminse (AST) nd Alnine trnsminse (ALT) were found in higher concentrtions in cytoplsm nd mitochondri. In liver injury, the trnsport function of the heptocytes is disturbed; resulting in the lekge of plsm membrne, thereby cusing n incresed enzyme level in serum nd soluble enzymes like tht AST will lso be relesed. The elevted level of AST nd ALT in serum indictes of cellulr lekge nd loss of functionl integrity of cell membrnes in liver (Rjesh nd Lth, 2004). Similrly, the present study hs shown tht level of AST nd ALT ws incresed fter CCl 4 dministrtion. However, tretment with C. siticum nd lycorine to CCl 4 induced group of mice declined the ctivity of AST nd ALT, which my stbilize the plsm membrne s well s repir of heptic tissue dmge cused by CCl 4. This is supported by the view tht serum level of trnsminses come bck to norml with the heling of heptic prenchym nd regenertion of heptocytes, stimultion of heptic regenertion ws known to mke the liver more resistnt to dmge by toxins (Thbrew et l., 1987).The ctivity of serum lctte dehydrogense, lkline phosphtse, cholesterol, triglyceride, very low density lipoprotein nd low density lipoprotein indicted deteriortion in heptic function due to prenchyml injury fter CCl 4 dministrtion. However, tretment with C. siticum nd lycorine significntly declined the CCl 4 induced liver dmge s evidenced by decresed serum ctivity of lctte dehydrogense, lkline phosphtse, level of cholesterol, triglyceride, very low density lipoprotein, low density lipoprotein, globulin nd increse in serum HDL nd lbumin level. Bilirubin is one of the most useful clinicl prmeter to dignose the severity of heptic necrosis. It is n importnt degrdtion product of hemoglobin nd is normlly excreted into bile. If heptic prenchyml dmge is severe, less bilirubin will be excreted nd hyperbilirubinemi is observed tht reflects pthophysiology of liver dmge (Klssen nd Wtkins, 1984). Increse in totl serum bilirubin concentrtion fter CCl 4 dministrtion might be ttributed to the filure of norml uptke, conjugtion nd excretion by the dmged heptic prenchym. A noticeble observtion with C. siticum nd lycorine showed decresed level of serum bilurubin, which suggests tht it cn be used in the cute condition of jundice. In the present 41

14 investigtion there ws significnt rise in serum ure concentrtion fter toxicnt dministrtion. It my be due to dysfunctionl nd dystrophic chnges in the liver nd kidney. Due to severe renl impirments, ure excretion flls nd its concentrtion in serum rises rpidly (Abuelo, 2007). Accumultion of ure hs been reported in liver diseses like cirrhosis nd encephlopthy. The present investigtion reveled tht C. siticum nd lycorine significntly provided protective effects on serum ure, leding to norml heptic physiology s well s improved glomerulr filtrtion rte. The totl protein levels including lbumin levels will be decresed in heptotoxic conditions due to defective protein biosynthesis in liver (Clwson, 1989). The CCl 4 intoxiction cuses disruption nd disssocition of polyribosomes on endoplsmic reticulum nd thereby reduces the biosynthesis of protein. The tretment of C. siticum nd lycorine well restored the proteins synthesis by protecting the polyribosomes. In conclusion, the findings of this study demonstrted tht the C. siticum nd lycorine ws effective in prevention of CCl 4 induced heptic dmge in mice. Present result show tht the C. siticum nd lycorine effectively normlized the level of pthophysiologicl mrker enzymes, lipid nd protein profiles. This my be due to the inhibition of lipid peroxidtion processes. The inhibitory effects of C. siticum nd lycorine my be useful s heptoprotective gent ginst chemicl-induced heptotoxicity. 42