Hepatoprotective and antioxidant activity of Zingiber chrysanthum ...

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

Hepatoprotective and antioxidant activity of Zingiber chrysanthum Rosec. rhizomes / Asian Journalof Traditional Medicines, 2011, 6 (6)Table 1. Biochemical parameters recorded seven days post- administration of CCl 4 and Z chrysanthum rhizome extracts toevaluate the hepatoprotective efficacy of AEZC and MEZCGroupsTreatment(Dose)Cholesterol(mg/dl)(mean±SE)Total Protein(g/dl)(mean±SE)Bilirubin(mg/dl)(mean±SE)Albumin(mg/dl)(mean±SE)UREA(mg/dl)(mean±SE)I Control 99.06±1.63 07.15±0.08 0.55±0.02 2.62±0.12 18.81±0.26II CCl 4 (3ml/kg.b.wt) 146.48±2.34 a 12.21±0.20 a 0.86±0.01 a 4.18±0.18 a 117.05±5.12 aIIIIVVVIVIIVIIIIXGroupsCCl 4 (3ml/kg.b.wt) +silymarin (3mg/kg. b.wt.)CCl 4 (3ml/kg.b.wt) +AEZC (250 mg/kg. b.wt.)CCl 4 (3ml/kg.b.wt) +AEZC (500 mg/kg. b.wt.)CCl 4 (3ml/kg.b.wt) +AEZC (750 mg/kg. b.wt.)CCl 4 (3ml/kg.b.wt) +MEZC (250 mg/kg. b.wt.)CCl 4 (3ml/kg.b.wt) +MEZC (500 mg/kg b.wt.)CCl 4 (3ml/kg.b.wt) +MEZC (750 mg/kg b.wt.)Treatment(Dose)114.07±3.17 b 8.78±0.09 b 0.84±0.04 b 2.89±0.04 31.50±0.33 b127.63±3.17 bc 10.46±0.15 bc 0.88±0.06 b 3.83±0.10 bc 96.15±4.96 bcd123.12±1.65 bc 10.81± 0.13 bcd 1.45±0.07 bcd 3.63±0.14 bc 90.40±1.25 bcd115.52±1.55 b 10.25±0.11 bcd 0.54±0.03 cd 3.03±0.06 bcd 76.87±0.80 bcd124.93±1.57 bc 10.22±0.18 bcd 0.73±0.04 b 3.44±0.11 bc 32.47±0.29 bd116.28±0.64 b 9.40±0.09 bcd 0.69±0.04 d 3.12± 0.10 b 34.88±0.39 bd111.45±0.90 b 9.40±0.094 bcd 0.53±0.027 cd 3.66± 0.070 bcd 38.09±0.47 bdGlucose(mg/dl)(mean±SE)Triglyceride(mg/dl)(mean±SE)ALT (U/l)(mean±SE)AST (U/l)(mean±SE)Alkalinephosphatase (U/l)(mean ± SE)I Control 47.56±1.63 94.30±3.56 13.26±0.26 52.95±0.26 384.18±10.12II CCl 4 (3ml/kg.b.wt) 110.26±2.67 a 149.22±8.05 a 17.55±0.26 a 57.66±0.37 a 418.60±0.71 aIIIIVVVIVIIVIIIIXCCl 4 (3ml/kg.b.wt) +silymarin (3mg/kg. b.wt.)CCl 4 (3ml/kg.b.wt) +AEZC (250 mg/kg. b.wt.)CCl 4 (3ml/kg.b.wt) +AEZC (500 mg/kg. b.wt.)CCl 4 (3ml/kg.b.wt) +AEZC (750 mg/kg. b.wt.)CCl 4 (3ml/kg.b.wt) +MEZC (250 mg/kg. b.wt.)CCl 4 (3ml/kg.b.wt) +MEZC (500 mg/kg b.wt.)CCl 4 (3ml/kg.b.wt) +MEZC (750 mg/kg b.wt.)54.02±2.95 101.03±4.00 14.35±0.10 b 54.72±0.16 b 378.83±18.07110.92±2.35 bcd 124.52±8.97 bc 14.15±0.57 56.41±0.31 bd 158.35±8.78 bcd119.49±1.32 bcd 190.67±7.92 bcd 14.23±0.59 54.97±0.20 b 177.17±9.47 b127.31±2.00 bcd 102.95±3.26 14.34±0.39 55.13±0.19 bc 167.07±9.42 bcd32.97±2.06 bcd 103.45±3.18 d 14.93±0.47 b 55.91±0.41 bd 319.00±8.57 bcd42.50±1.10 bcd 64.93±2.57 bcd 14.23±0.62 55.17±0.26 b 310.28±6.65 bc47.84±1.15 bd 107.08±2.8 15.39±0.32 b 55.03±0.16 b 326.80± 8.15 bcdaP

Hepatoprotective and antioxidant activity of Zingiber chrysanthum Rosec. rhizomes / Asian Journalof Traditional Medicines, 2011, 6 (6)treated group. There was no significant differencein serum cholesterol level in the higher dose groupV give AEZC and VIII and group IX given MEZCin comparison with the silymarin-treated group III.The total serum proteins levels were significantly(P

Hepatoprotective and antioxidant activity of Zingiber chrysanthum Rosec. rhizomes / Asian Journalof Traditional Medicines, 2011, 6 (6)phosphatase were found in CCl 4 -treated miceindicating liver damage, although, these values werereduced significantly (P

Hepatoprotective and antioxidant activity of Zingiber chrysanthum Rosec. rhizomes / Asian Journalof Traditional Medicines, 2011, 6 (6)4.3. DPPH radical scavenging activityDPPH radical scavenging activity wasobserved for all the tested extracts at the selecteddose levels (5 mg, 10 mg, 15 mg and 20 mg),although these extracts differed in their ability toreact with and quench DPPH radicals. However,there were variations in the scavenging activity ofDPPH scavenging activity of acetone, hexane andmethanol extracts of rhizomes of Z. chrysanthumand the methanolic extract exhibited the maximumscavenging activity (74.60±0.05) followed by theacetone (65.25±0.08) and hexane extracts (60.32±0.58) at a dose level of 20 mg (Fig. 4). It is wellFig: 1known that freeFig: Reducing 1 Reducing power power activity activity of acetone,hexane of acetone,hexane and andradicals cause auto oxidationmethanol methanol extracts extracts of rhizomes of rhizomes of Z. chrysanthumof Z. chrysanthumof unsaturated lipids in food [37]. In addition,4.5 4.5antioxidants are known to interrupt the free radical4 4chain3.5reaction of oxidation and donate hydrogen3.5AEZCfrom 3phenolic hydroxyl groups, thereby formingAEZC3MEZC MEZC2.5stable free 2.5 radicals, which do not initiate HEZC HEZC or2 2BHT BHTpropagate further oxidation of [38]. Many medicinal Catechin1.5 1.5Gallic acidplants contain large amount of antioxidants other1 1than 0.5vitamins and cartenoids [39]. Various typeof ginger 0 0 in tropical countries have been shown5mg 5mg 10mg 10mg 15mg 15mg 20mg 20mgto have strong anti-oxidative Concentrations Concentrationsactivities that couldreplace α-tocopherol [40]. Recent reports havedescribed that antioxidant compounds with radicalscavenging Fig:3 activity DPPH Fig:3 DPPH radical are radical scavenging present scavenging activity onion, activity of of garlic,AbsorbanceDPPH DPPHscavenging radical scavenging effect effect (%) (%)Absorbance120100DPPHscavenging effect (%)806040200acetone,hexane acetone,hexane and methonal and methonal extracts extracts of rhizomes of rhizomes of ofZ.chrysanthum Z.chrysanthum1201008060402005mg 5mg 10mg 10mg 15mg 15mg 20mg 20mgConcentrations ConcentrationsFig:2 Fig:2 Chelating Chelating activity activity of acetone,hexane of acetone,hexane and andmethanol methanol extracts extracts of rhizomes of rhizomes of Z.chrysanthumof Z.chrysanthumAEZC AEZCMEZC MEZCHEZC HEZCBHT BHTGallic Gallic acid acidCatechin CatechinFig. 4. DPPH radical scavenging activity of acetone, hexaneand methanol c\extracts of rhizomes of Z. chrysanthumCatechinGallic acidsage and thyme extracts [41, 42]. Therefore, wedetermined of radical scavenging activity of theessential oil of zingiberaceous plants. The 2, 2´-diphenyle-1-picrylhydrazyl radical has been widelyused to evaluate the free radical scavenging capacityof antioxidants [43-45]. The determination of thescavenging stable DPPH radical is a very fast methodfor evaluating the antioxidant activity. Using thismethod, it is possible to determine the anti-radicalpower of the antioxidant activity by measurementof the reduced absorbance of the DPPH radicalat 515 nm. In the radical forms this molecule hasan absorbance at 515 nm which disappears afteracceptance of an electron or hydrogen radical from anantioxidant compound to become a stable diamagneticmolecule [46]. The data obtained revealed thatall the essential oils and extracts are free radicalsscavengers and thus, primary antioxidants thatreact with free radicals. The antioxidant activity ofnatural antioxidants has been shown to be involvedin termination of free radical reactions and theassociated Fe 3+ to Fe 2+ reducing power [19, 47].From these results it can be concluded that all theextracts exhibited significant differences in theirantioxidant potential, presumably due to differencesin the structure and concentration of the antioxidantcomponents. Among these the acetone, hexane andmethanol extract of rhizomes of Z. chrysanthumshowed clear variations in their anti-oxidantpotential. These variations in activities may bedue to differences in their chemical composition.The antioxidative activities observed for all theessential oils and extracts could be a synergisticeffect of more than the four compounds that maybe present in the system. It has been reported thatmost natural anti oxidative compound often worksynergistically with each other to produce a boardspectrum of antioxidant activity creating an effectivedefense system against free radical attack [48].These essential oils and extracts could be used asnatural antioxidants and a possible substitution70607060249

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