Hepatoprotective and antioxidant activity of Zingiber chrysanthum ...

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Hepatoprotective and antioxidant activity of Zingiber chrysanthum Rosec. rhizomes / Asian Journalof Traditional Medicines, 2011, 6 (6)as active principles in the essential oil of Zingiberchrysanthum rhizome [8], there are very few scientificreports about its biological activity. In view of thesefacts, the present study was undertaken to evaluatethe hepatoprotective and antioxidant activity of theacetone and methanol extracts of the rhizome of thisplant.2. Materials and methods2.1. Collection of plant materialThe plant was collected locally from theTarai region of Uttarakhand and was identifiedtaxonomically from the Botanical Survey of India(BSI), Dehradun, Uttarakhand. The rhizomes wereseparated from the plants for the experiments carriedout in this study.2.2 Solvent extractionFor preparation of the acetone and methanolextracts of Zingiber chrysanthum (AEZC andMEZC) its rhizomes (500 gm) were shade driedand powdered. The powder was then subjected tocold extraction with 1000 ml acetone and1000 mlmethanol for 5 days. The dried acetone and methanolextracts were obtained by evaporation of the solventsusing a rotatory vacuum evaporator at 45 ºC±5 ºCand kept in a refrigerator until required for furtherinvestigation. The dried extracts were dissolved indistilled water before oral administration to differenttreatment groups.2.3. Hepatoprotective activity2.3.1. Experimental animalsFemale albino mice 2-2.5 months old weighingbetween 23-28 gm, were obtained from theExperimental Animal House, College of Veterinaryand Animal Sciences, Pantnagar, Uttarakhand.The animals were maintained under standardmanagement condition and acclimatized for twoweeks before the start of the experiment. Feed andwater were given ad libitum throughout the study.2.3.2. Experimental designTo evaluate the hepatoprotectivepotential ofAEZC and MEZC in carbon trtrachloride (CCl 4 )-induced hepatic damage, fifty four mice wererandomly divided into nine equal groups viz. GroupI to IX. Group I served as a control. Group II to IXreceived a single dose of CC1 4 (3 ml/kg. b wt., i m)(99.8% MERCK Specialties Pvt. Ltd.) the on firstday. Group III served as a positive control and wasgiven a single daily oral dose of silymarin (3 mg/kgb. wt.) for seven days. Groups IV, V and VI were fedon a diet containing AEZC at 250 mg/kg, 500 mg/kg,and 750 mg/kg b. wt. and groups VII, VIII and IXwere fed MEZC at 250 mg/kg, 500 mg/kg and 750mg/kg b. wt., respectively, for seven days. The micein each group were sacrificed humanely after sevendays and blood was collected by cardiac punctureand transferred to sterilized non-heaparinizedsyringes to separate serum for biochemicalanalysis and liver was collected and preserved forhistopathological examinations. The serum wasstored at -10 ºC until biochemical analysis whichwas carried out within 24 hrs.2.4. Biochemical profileBiochemical parameters including serumcholesterol, total protein, urea, triglyceride, albumin,glucose, bilirubin and serum AST, ALT and alkalinephosphatase [9-14] levels were determined usingdiagnostic kits (MERCK Specialties Pvt. Ltd, NewDelhi, India).2.5. Histopathological examinationThe sections of liver were processed forhistopathological examination involving tissuefixation and were then mounted using DPX formicroscopic examinations [15].243
Hepatoprotective and antioxidant activity of Zingiber chrysanthum Rosec. rhizomes / Asian Journalof Traditional Medicines, 2011, 6 (6)2.6. Antioxidant activity2.6.1. Reducing powerThe reducing power of the essential oil andAEZC and MEZC extracts were determined bythe standard methods reported earlier [16, 17].Different amounts of extracts (5, 10, 15 and 20mg) in four test-tubes, were mixed with 2.5 ml ofthe freshly prepared phosphate buffer (200 mM,pH 6.6) and 2.5 ml, 1% potassium ferricyanide.The mixture was incubated for 10 min at 50 ºCand processed separately. After incubation 2.5 ml10% trichloroacetic acid was added to the mixturefollowed by centrifugation at 650 rpm for 10 min.The upper layer (5 ml) was decanted and mixed with5ml distilled water and 1.0 ml 0.1% ferric chloride.Each tube was shaken well and the absorbanceof the resultant solution was measured at 700 nmusing a UV- Vis-spectrophotometer (Visiscon-167).The control was subjected to the same procedureand BHT, gallic acid and catechin were used asstandards.2.6.2. Effect on the chelating activity of Fe 2+The chelating activity of the essential oil onferrous ions (Fe 2+ ) was measured as describedwith slight modification [18]. Different amountsof extracts (5 mg, 10 mg, 15 mg and 20 mg) weremixed in separate test- tubes with 1 ml methanol and3.7 ml distilled water. The mixtures were allowed toreact with FeCl 2 (2 mM, 0.1ml) and ferrozine (5 mM,0.2 ml) for 10 minutes at room temperature and thenthe absorbance of each was recorded at 562 nm. Alower absorbance indicates a higher chelating power.The Fe 2+ chelating activity of the oil and extractswas compared with that of EDTA at a concentrationof 0.01 mM and citric acid at a concentration of0.025 M. The chelating activity was calculated fromthe following equation:Chelating activity (%) = [1– (A t /A 0 )]×100%where A t is the absorbance of the samples at 562nm and A 0 is the absorbance of the control at 562 nm2.6.3. DPPH radical scavenging activityThe scavenging effect on DPPH radicals wasdetermined using methods developed earlier [16, 17,19]. Different concentration of extracts (5 mg, 10 mg,15 mg and 20 mg) were mixed with 5 ml 0.004%methanolic solution of DPPH. Each mixture wasincubated for 30 minutes and the absorbance of thesamples was read at 515 nm. The DPPH solution wasfreshly prepared before use and kept in a coveredflask in a dark place at 4 ºC during the measurements.The control and standard with a lower absorbanceindicated a higher radical scavenging activity whichwas calculated from the equation:DPPH scavenging activity (%) = [1– (A t /A 0 )] ×100%where A t is the absorbance of the samples at 515nm and A 0 is the absorbance of the control at 515 nm2.7. Statistical analysisData were expressed as Mean±S.E. and analyzedstatistically for significant differences using one wayANOVA at a 1% level Statistics of significance.3. ResultsThe results of biochemical parametersrecorded seven days post-treatment to evaluatehepatoprotective efficacy of AEZC and MEZCin mice with CCl 4 -induced hepatotoxicity arepresented in Table 1. The biochemical parametersindicative of hepatotoxicity were significantlyincreased in the CCl 4 -treated group in comparisonwith the untreated control. A significant (P
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