The hepatoprotective activity of crude extract of artemisia scoparia (aerial parts) was investigated against experimentally produced hepatic damage using carbon tetrachloride (CCl4) as a model hepatotoxin. CCl4 at the dose of 1.5 ml/kg, produced liver damage in rats as manifested by the rise in serum levels of AST and ALT to 395 +/- 110 and 258 +/- 61 IU/l (mean +/- SEM; n = 10) respectively, compared to control values of 106 +/- 15 and 26 +/- 04. Pretreatment of rats with plant extract (150 mg/kg) significantly lowered (P \u3c 0.01), the respective serum GOT and GPT levels to 93 +/- 05 and 27 +/- 03 IU/l, indicating hepatoprotective action. Pentobarbital sodium (75 mg/kg)-induced sleeping time in mice was found to be 140.8 +/- 1.5 min (n = 10) which was similar (P \u3e 0.05) to that obtained in the group of animals pretreated with the plant extract (139.9 +/- 1.8 min). CCl4 treatment extended the pentobarbital sleeping time to 212.2 +/- 19.1 min and pretreatment of animals with plant extract reversed the CCl4-induced prolongation in pentobarbital sleeping time to 143.9 +/- 5.5 min (P \u3c 0.001) which further confirms the protective action of the plant extract against CCl4-induced liver damage. These data indicate that the plant artemisia scoparia is hepatoprotective and validate the folkloric use of this plant in liver damage

Gilani, A H

Janbaz, K H

English

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eCommons@AKUDepartment of Biological & Biomedical Sciences Medical College, PakistanMarch 1994Hepatoprotective effects of artemisia scopariaagainst carbon tetrachloride: An environmentalcontaminantA H. GilaniAga Khan University, anwar.gilani@aku.eduK H. JanbazAga Khan UniversityFollow this and additional works at: https://ecommons.aku.edu/pakistan_fhs_mc_bbsPart of the Pharmacology CommonsRecommended CitationGilani, A. H., Janbaz, K. H. (1994). Hepatoprotective effects of artemisia scoparia against carbon tetrachloride: An environmentalcontaminant. Journal of Pakistan Medical Association, 44(3), 65-68.Available at: https://ecommons.aku.edu/pakistan_fhs_mc_bbs/505Hepatoprotective Effects of Artemisia Scoparia against CarbonTetrachloride: An Environmental ContaminantPages with reference to book, From 65 To 68 Anwar-ul Hassan Gilani, Khalid Hussain Janbaz ( Departments of Pharmacology and Faculty of Health Sciences, The AgaKhan University Medical College, Karachi. ) Abstract The hepatoprotective activity of crude extract of artemisia scoparia (aerial parts) was investigatedagainst experimentally produced hepatic damage using carbon tetrachloride (CCI4) as a modelhepatotoxin. CCl4 at the dose of 1.5 mI/kg, produced liver damage in rats as manifested by the rise inserum levels of AST and ALT to 395±110 and 258±61 lU/I (mean ±SEM; n=10) respectively, comparedto control values of 106± 15 and 26±04. Pretreatment of rats with plant extract (150 mg/kg)significantly lowered (P<0.01), the respective serum GOT and GPT levels to 93±05 and 27±03 IU/I,indicating hepatoprotective action. Pentobarbital sodium (75 mg/kg)-induced sleeping time in mice wasfound to be 140.8± 1.5 mm (n=10) which was similar (P>0.05) to that obtained in the group of animalspreheated with the plant extract (1 39.9±1.8 min). CCl4 treatment extended the pentobarbital sleepingtime to 212.2± 19.1 mm and pretreatment of animals with plant extract reversed the CCl4-inducedprolongation in pentobarbital sleeping time to 143.9±5.5 min (p<0.001) which further confirms theprotective action of the plant extract against CCl4-induced liver damage. These data indicate that theplant artemisia scoparia is hepatoprotective and validate the folkioric use of this plant in liver damage(JPMA 44:65, 1994).Introduction Carbon tetrachloride (CCI4), a potent hepatotoxin, has been used extensively as a cleansing agent, fireextinguisher, grain fumigant, vermifuge, solvent and as an intermediate in chlorofluorocarbonsynthesis. Its toxicity usually follows inhalation of the vapours in a poorly ventilated environment,Ingestion of contaminated food (especially grains) and ground water1. Single exposure can rapidly leadto severe centrizonal necrosis and steatosis2, whereas chronic or intermittent exposure may causecirrhosis3 and even hepatic malignancy4. In modem therapeutics, hepatic damage is regarded as a sortof medical emergency because in the absence of specific therapy one has to be contented uponconservative treatment. On the other hand, traditional healers claim certain valuable recipes based onplants constituents showing preventive as well as curative potential. One such example, is artemisiascoparia thunb. (Family: compositae; local name: Dona Than), having a folkloric reputation for itsusefulness in the treatment of jaundice and other liver disorders5. More recently, we have reported thatthe crude extract of artemisia scoparia afforded protection against acetaminophen-inducedhepatotoxicity in rats6. In the present investigation, we have tested this plant extract against carbontetrachloride (CCI4)-induced liver injury to assess whether it also affords protection against acommonly encountered environmental hepatotoxin with a different mode of hepatocellular damage7.This can also be a step forward to help elucidate the possible mechanism of its hepatoprotective action.Plant material:Whole artemisia scoparia plants were collected during the month of April, 1991 from the rural areaaround Thatta District in the province of Sindh, Pakistan and authenticated with the help of a botanist atthe University of Karachi. The plant material was shade dried, powdered and maceratect in 80%aqueous-methanol (BDH Ltd., Poole, England) for 1 week with occasional shaking. The extract wasfiltered and concentrated to dark greenish brown residue under reduced pressure on a rotary evaporator.Pharmacological materials and animals:CCl4, ketamine hydrochloride and pentobarbital sodium were obtained from Sigma ChemicalsCompany, St. Louis, Mo, USA and olive oil (P. Sasso e Figili, Oneglia, Italy) was purchased from localmarket. Swiss male mice (20-25g) and male albino Wistar rats (200-250g) housed at the animal houseof The Aga Khan University, were used for this study. The animals were housed in air conditionedquarters and had free access to tap water and food.Induction of hepatic Injury:Hepatic injurywas produced by oral administration of 1.5 ml/kg CCl4 diluted upto 20% by olive oil andthe animals in the control group received an equal volume of olive oil.Multiple dose treatment in rats:Rats were divided into three groups of 10 animals each. Group 1 served as vehicle control and receivednormal saline (3 ml/kg) and olive oil (7.5 nd/kg) orally. Group 2 served as toxic control and was given4 doses of normal saline at 12 hours intervals and CCl4 was then administered orally 1 hour post-treatment of the last dose. Group 3 was treated similar to group 2 except that plant extract (150 mg/kg)was administered instead of saline. Animals were anaesthetized with ketamine (100 mg/kg, i.m.) 24hours after the last treatment and blood (3 ml) was collected by cardiac puncture using steriledisposable syringes. Serum was separated by centrifugation (3000 rpm, for 15 mAn) and serumglutamate oxaloacetate transaminase (AST) and glutamate pyruvate transaminase MT were estimatedon the same day spectrophotometrically using Merck diagnostic kits.Modification of pentobarbital-induced sleeping time:The direct effect of plant extract on pentobarbital induced sleeping time and effect of plant extract onCCI4-induced prolongation of pentobarbital sleeping time was studied in mice by using methoddescribed by Montila and colleagues8, as shown in Table. To test the direct effect of plant extract onpentobarbital- induced sleeping time, animals were divided into two groups of 10 animals each. Group1 receive. Direct effect of artemisia scoparia on pentobarbital sleeping time as well as on CCl4-inducedprolongation of pentobarbital sleeping time im mice normal saline (3 mI/kg) while group 2 was givenplant extract (150 mg/kg) as a single oral dose and pentobarbital(75 mg/kg, i.p.) was then administeredafter 1 hour to both the groups. To assess the effect of plant extract on CCL-induced prolongation ofpentobarbital sleeping time, 2 more groups were added to the study and were treated as follows: Group3 received 4 doses of normal saline orally at 12 hrs intervals and CCLi was administered as a bolusdose (1.5 ml/kg) 1 hr post-treatment of the last dose o. saline followed after 24 hrs by pentobarbital (75mg/kg, i.p.). Animals in group 4 were treated similarly to group 3 except that plant extract (150 mg/kg)was substituted for normal saline.Statistical analysis:The results are expressed as Mean S.E.M. and all statistical comparisons were made by means ofStudent’s t-test and P <0.05 was regarded as a significant.Results Effect of plant extract on CCl4-induced toxicity:Control (saline + vehicle) serum values of GOT and GPT in rats were found to be 106±15 and 26 04IU/L resspectively (Figure 1),while a toxic dose of CCl4 (1.5 ml/kg) raised significantly (P< 0.01), the respective serum enzymevalues to 395± 110 and 258±61 IU/L. Group 3 was pretreated with plant extract (150 mg/kg, orally,twice daily for 2 days) to determine its effect on CCl4-induced rise in serum enzymes. The serumvalues in pretreated group were found to be 93±05 (GOT) and 27±03 IU/L (GPT), which aresignificantly lower (Pc 0.01) than the values of toxic control and close to normal values (P> 0.05).Effect of plant extract on pentobarbital-induced sleep:Effect of plant extract on pentobarbital sleeping time as well as on CCI4-induced prolongation of pentobarbital sleeping time was studied in mice and the results are shown in Table.Pentobarbital at a dose of 75mg/kg, i.p., caused sleep in mice for a period of 140.8±1.5 min(Mean±S.E.M., n= 10). Pentobarbital sleeping time in the group of animals pretreated with plantextract was found to be 139.9± 1.8 min which is similar to that in the control group (P>0.05). Whereaspretreatment of animals with CCl4, prolonged the pentobarbital sleeping time to 212.2±19.1 min, thevalue that is significantly higher (P <0.001) than that of control (Table). However, prior treatment ofanimals with the plant extract returned this CCl4-induced prolongation of pentobarbital sleeping timeto 143.9±5.5 min, which is close to the control sleeping time (P >0.05). Discussion The crude extract of artemisia scoparia showed heaptoprotection against CCl4-induced liver injury. Theplant extract inhibited a CCl4-induced rise in serum AST and ALT in rats. Similarly, prevention pfCCl4-induced prolongation of pentobarbital sleeping time was observed in a group of mice pre-treatedwith the plant extract. Liver injury induced by CCl4 is a commonly used model for the screening ofhepatoprotective drugs9. The rise in serum levels of AST and ALT has been attributed to the damagedstructural integrity of the liver10 because these are cytoplasmic in location and are released intocirculation after cellular damage11. CCl4 is metabolized by a specific isozyme of cytochrome P-450(IIE1) to variety of chemically reactive free radical species12. The free radical mediated toxicmanifestations can effectively be minimized by cytochrome P-450 inhibitors14, GSH precursors15, freeradical scavangers16, antioxidants17, sulfhydryl agents18 and Ca+ + channel blockers19,20. The crudeextract of artemisia scoparia plant used in this study seems to preserve the structural integrity of thehepatocellular membrane. This was evident from the significant reduction in the CC14-induced rise inserum AST and ALT levels in rats. To see whether the plant extract has inhibitory effect on hepaticmicrosomal drug metabolizing enzymes (MDME), it was administered with pentobarbital to mice andpossible change in the duration of sleep was recorded. The duration of pentobarbital-induced sleep inintact animal is considered as a reliable index for the activity of hepatic MDME21. Pentobarbital ismetabolized by the hepatic MDME to inactive metabolites and a drug with inhibitory effect on MDMEis expected to prolong pentobarbital sleep time. The fact that the plant extract did not modifypentobarbital sleeping time (P> 0.05) suggests that it is devoid of inhibitory effect on hepatic MDMEsuch as cytochrome p-450. The damage conferred by CCl4 to hepatocytes as well as hepatic MDMEcauses loss of drug metabolizing capacity of the liver, resulting in prolongation of pentobarbital-induced sleeping time22. Pretreatment of animals with plant extract prevented th CCl4-inducedprolongation of pentobarbital-sleeping time suggestive of its protective effect against CCl4-induceddamage to hepatocytes including MDME. The possible mechanism of the protective effect of artemisiascoparia would be rather speculative at this stage, however, it is clear from the results of sleeping timestudy that the protective effect is not mediated through inhibition of hepatic MDME. We have recentlyshown that the crude extract of artemisia scoparia exhibits Ca++ channel blocking activity in isolatedtissue experiments23. Calcium content in the liver cells are increased during the process ofexperimental hepatic damage19,24 and calcium channel blocking drugs, i.e., nifedipine, diltiazem andverapamil were found to inhibit the development of hepatic damage, induced by different hepatotoxinsincluding acetaminophen25 and CCl419. Similarly, hepatoprotective activity of the plant extract,against acetaminophen reported previously6 and ccl4-induced liver damage reported in this study maybe attributed to its Ca++ channel blocking activity. Moreover, hepatoprotective action of the plantextract may also be due to the occurrence of different types of plant constituents. It is interesting thatpure compounds such as rutin and other flavonoids from artemisia scoparia26 have already beenreported to have antioxidant, free radical scavenger and Ca++ channel blocking-like (non-specificsmooth muscle relaxant) activity in isolated tissue preparations27,28, though their hepatoprotectiveactivity was not studied. The plant extract afforded hepatoprotectivity both against acetaminopher6 andCCl4 (this study) at the dose of 150 mg/kg, whereas higher dose (500 mg/kg) was required for thisactivity in case of other plants tested in our laboratory such as, cyperus scariosus20, artemisiaabsinthium29 and cichorium intybus30. The effectiveness of the crude extract of artemisia scoparia at adose, distinctly lower than that used for other plants may be due to the fact that this plant containsmultiple constituents which may have cumulative beneficial effect in liver injury. In conclusion, thisstudy provides the scientific basis for its traditional use in liver damage and further studies on theconstituents of this plant is in progress to explore its possible mode of hepatoprotective action.Acknowledgements Khalid Hussain Janbaz is on study leave from Bahauddin Zakariya University, Multan.References 1. Jagielski, 3., Scudamore, LA., Heuser, 5.0. Residues of carbontetrachloride and 1.2-dibromoethanein cereals and processed foods afterliquid fumigant grain treatment for peatcontrol. Peatic.Sci.,1978;9:117-11&2. Recknagel, Ito. and tilende, BA. Carbontetrachloride hepatotoxicity: an example of lethal clevage.CRC. CriL Rev.Toxicol., 1973;2:263-97.3. Cameron, OR. and Karunaratne, W.A.E. Carbontetrachioride cirrhosia in relation to liverdegeneration. J.Pathol.Bacteriol., 1936;42:1-21.4. Reuber, M.D. and Clover, EL. Hyperplastic and early neoplastic lesions of the liver in buffalo strainrats ofvarioua ages given subcutaneous carbontetrachloride. J.Natl.Can cer Inst., 1967;38:891-95.5. Perry, L.M. ed. Medicinal plants of East and South-East Asia. London, M.I.T. Press, 1980, p. 84.6. Noguchi, T., Fong. ICL, Lai, BK. et al. Specificity of a phenobarbisal-indueed cytochrome P-450 formetabolism of carbontetrachloride tothe trichloromethyl radical. Biochem. Pharmacol., 1982;31:615-24.7. Montilla, M.P., Cabo, 3., Navarro, M.C. et al. The protective and curative action of withaniafruteacena leaf extract against CCl4-induced hepatoxicity. Phytotherapy Rn., 1990;4:212-15.8. Plaa, C.L and I-lewis, W.R. eda. Quantitativeevaluation of indices of hepatotoxicity. In, Toxicologyof the liver, New York, Raven Press, 1982; pp. 103-120.9. Chenoweth, MB. and Hake, CL. The smaller halogenated aliphatic hydrocarbons. Annu. Rev.Pharmacol., 1962;2:363-98.10. SaIlie, R., Tredger, J.M. and William, R. Drugs and the liver. Biopharm. Drug Diapoa., 1991; 12:251-59.11. Packer, J.E., Slaser, T.F. and Wilson, ltL. Reactionsof the carbon tetrachloride-related peroxy freeradical (CC13O1/2) with amino acids: pulae radiolyais evidence. Life Sci., 1978;23:2617- 20.12. Rao, KS. and Recknagel, ItO. Early incorporation of carbon- Isbelled carbontetrachioride in ratliver particular lipids and proteins. Exp.Mol.Pathol., 1969;10:219-28.13. Castro, iA., de Ferreyra, G.C., deCastro, C.R.e et al. Prevention ofcarbontesrachlorideinducednecrosia by inhibitora of drug metabolism. Furtheratudieson the mechanism of their action. Biochem.Pharmacot, 1974;23:295-305.14. DeFerreyra, E.C., Castro, J.A., Diaz-Gomez, M. et al. Prevention and treatment ofcarbontetrachloride hepatotoxicity by cysteine: studies about its mechanism. Toxicol. Appl. Pharmacol.,1974;27:558-68.15. Mailing, MM., Eichelbaum, F.M., Saul, W. et al Nature of the protection againstcarbontetrachloride-induced hepstocoxicity produced by pre-treatment with dibenamine N-chloroeshyl-dibenzylamine. Biochem. Pharmacol, 1974;23:1479-91.16. Yaauda, H., lzumi, N., Shimada, 0. et al The protective effect of tinoridine againstcarbontetrachloride hepatotoxicity. Toxicol.Appl.Pharmacol., 1980;52:407-13.17. Mark, T., Freedman, AM., Dickens, B.F. et al. Protective effects of sulfhydryl-containingangiotensin converting enzyme inhibitora against free radical injury in endothelial cells. Biochem.Pharmacol., 1990;40:2169-75.

Hepatoprotective effects of artemisia scoparia against carbon tetrachloride: An environmental contaminant

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Hepatoprotective effects of artemisia scoparia against carbon tetrachloride: An environmental contaminant

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