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Archives • 2014 • vol.1 •90-101

Antioxidant and hepatoprotective effects of aqueous and ethanol extracts of Dendrophthoe falcata Linn leaves

Haque, A.1*; Tahmina2, Afsana, S.K.2; Sarker I.R.3; Hossain, M.3; Islam, S.3.; Islam, A.4

1Comilla University, Department of Pharmacy, Kotbari, Comilla, Bangladesh2Southeast University, Department of Pharmacy, Banani, Dhaka, Bangladesh

3Rajshahi University, Department of Pharmacy, Rajshahi, Bangladesh3Rajshahi University, Department of Biochemistry and Molicularbiology, Rajshahi, Bangladesh

*[email protected]

Abstract

Dendrophthoe falcata L. is an important parasitic plant extensively used as traditional medicine againstdifferent diseases. Therefore present study was designed to investigate the antioxidant andhepatoprotective effects of aqueous and ethanol extracts of leaves of the plant. To investigateantioxidant potentials, we have performed qualitative phytochemical screening, quantitativedetermination of total phenol, flavonoids and antioxidants levels along with free radical (DPPH)scavenging and reducing capacities (Fe3+) of the extracts. The hepatoprotective effect was examined onLong Evans rat model. Liver damage was induced by intraperitonial administration of 25% carbontetrachloride (CCl4) in olive oil (1ml/kg,b.w,i.p) and the extent of damage was measured by assessingbiochemical parameters.30 rats were divided into five groups of six animals in each group. Group I(control) was given olive oil and DMSO, while group II- V were injected CCl4 intraperitoneally. Animals ofgroup II received only CCl4. Rats of group III, IV and V received silymarin (50mg/kg p. o.), aqueous extract(200mg/kg p.o.) of D. falcata leaves (AEDFL) and ethanol extract (200mg/kg p.o.) of D. falcata leaves(EEDFL) respectively along with intraperitonial administration of CCl4 for consecutive 7 days. At the endof the experiment (7days), serum and liver samples were collected for biochemical and histopathologicalanalysis.The activities of liver markers: aspartate transaminase (AST), alanine transaminase (ALT),alkaline phosphatase (ALP), total protein (TP) and bilirubin were measured in serum of each animal.Hepatotoxicity induced by CCl4 was evidenced by significantly elevated levels of the markers. Co-administration of silymarin and the extracts (AEDFL and EEDFL) significantly (P<0.001) prevented all thechanges observed with CCl4-treated rats. The phytochemical analysis and antioxidant measurementrevealed the presence of important phytochemicals like alkaloids, tannins, resins, phenols, flavonoidsetc. and significant antioxidant effects of the extracts.

Key wards: Dendrophthoe falcate, antioxidants, Hepatotoxicity, carbon tetrachloride

April 30, 2014

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IntroductionLiver is the largest and pivotal gland the mainfunctions of which are metabolism, detoxification,excretion and generation of a variety ofcoagulation factors (1, 2). On the other hand, it isalso one of the most vulnerable organ and proneto be impaired by toxins, drugs andmicroorganisms (3,4). Millions of people are beingaffected by liver disease throughout the world (5)where drug induced hepatic injury is a majorconcern (6). It has been reported that theoxidative stress induced by free radicals is a majorcause of liver disorder such as swelling,degeneration, necrosis, and apoptosis of hepaticcells (7, 8, 9). Free radicals induce an oxidativestate that causes cellular membrane injury withthe subsequent alteration in metabolic processes.Reactive oxygen species (ROS) plays a potentialrole in pathogenesis of diverse degenerativediseases of human and have been implicated inatherosclerosis, liver disorders, lung and kidneydamage, aging and diabetes mellitus (10). In caseof liver disorders the ability of natural antioxidantsystem is impaired. Free radicals are generated incells by environmental factors such as ultravioletradiation, pollutants, x-rays, or by normalmetabolic process in mitochondria. Theintracellular concentration of ROS depends onboth their production by endogenous orexogenous factors and removal by variousendogenous antioxidants including bothenzymatic and non enzymatic components (11,12). Carbon tetrachloride (CCl4) is an extremelytoxic chemical to the liver cells. It is a widely usedmodel to evaluate hapatotoxic potential ofexperimental compounds. Histopathologicalsectioning of the liver tissues indicates the stateof cell such CCl4 induced fibrosis, cirrhosis andhepatocarcinoma (10, 13, 14). The toxic effect ofCCl4 is attributed by trichloromethyl radical (freeradical) produced during oxidative stress (15).Management of liver diseases is still a challenge tothe modern scientific community (16). Becausethe treatment options for common liver diseasesuch as cirrhosis, fatty liver and chronic hepatitisare now still problematic. There are fewconventional drugs available in market such asinterferons, colchicines, penicillamine andcorticosteroids that are inconsistent for propertreatment (17). These drugs have profound side

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PhOL Haque et al 91(87-101)

effects, and cannot offer hepato protection bystimulating liver function or regenerating hepaticcells (18). Inhibition of free radicals is veryimportant in terms of liver pathology. Naturalproducts from the plant kingdom are beinginvestigated as a source of anti-oxidants, and alltypes of antioxidants are capable to resist oxidativestress (11, 12, 19). These traditional products mayhave the hepatoprotective potential, and thereforecan be effectively used to treat acute and chronicliver diseases (20, 21, 22).Dendrophthoe falcata belongs to the familyLoranthaceae, commonly known as ‘Porgassa inBangla , and ‘Banda’ in Hindi (23). It is also familiaras “Bandaaka, Vrkshaadani, Vrkshruuhaa” in theIndian Ayurvedic System of Medicine (24). D.falcata is an evergreen perennial climbing woodyhemiparasitic plant with bark smooth grey, leavesopposite unequal, thick 1.6 - 25.4 cm long, flowerssingle, orange-red or scarlet softly pubescent,berries soft ovoid-oblong, 1.3cm diameter (25,26).It is found in Bangladesh and also widelydistributed in Australia, India, China, Malaysia,Myanmar, Srilanka, and Thailand (25, 26, 27, 28).The entire plant is used extensively in traditional

system of medicine as cooling, bitter, aphrodisiac,astringent, narcotic, diuretic, and isuseful in pulmonary tuberculosis, asthma,menstrual disorders, swellings, wounds, ulcers,renal and vesical calculi (24, 26, 29). Leaf paste isused in skin diseases where it is applied on boils,setting dislocated bones and extracting pus (28,23). The decoction of whole plant is used to treatjoint pains and leaf juice is used for relief fromchest pain (30, 31). D. falcata is reported to havecytotoxic, immunomodulatory activities, andwound healing potentials. In the traditional systemof medicine, D. falcata is recommended for thetreatment of epilepsy (26, 32).

To find out active constituents a number ofenzymes are separated from the leaves of D. falcatasuch as L-Threonine dehydratase, hexokinase,Glucan phosphatase (33). It has also been reportedby the isolation and identification of severalpossible active chemical constituents such as β-amyrin acetate, β-sitostirol, stigmasterol, oleanolicacid (31), kaempferol, quercetin (27), quercetin–3-O-rhamnoside, rutin, myricetin and theirglycosides, (+)-catechin, leucocyanidin, some,kaempferol-3-O-α-L-rhamnopyranoside and will

quercetin-3-O-α-Lrhamnopyranoside etc (26, 34).It also contains tannins comprising of gallic acid,chebulinic acid (28) ellagic acid (34), quercetin and(+) – catechin(28, 26). Three cardiac glycosidessuch as strospeside, odoroside F and neritalosidewere isolated from the leaves of D. falcate (26).Pentacyclic triterpenes: 3β-acetoxy-1β-(2-hydroxy-2-propoxy)-11α-hydroxy-olean-12-ene (35),kaempferol-3-O-α-L-rhamnopyranoside, quercetin-3-O-α-L-rhamnopyranoside were also reported tohave in the plant (26, 36).The study was undertaken to evaluate antioxidantand hepatoprotective activity of aqueous, ethanolextract of D. falcata leaves in Long Evans ratwhich will unveil the rationality of use of the plantas traditional medicines.Therefore, the great efforts have been invested toexploit the perfect antioxidant drug protectingliver from damage

MethodsPlant materials:For the investigation, Dendrophthoe falcata L.leaves, mistletoe of Swietenia fabrilis tree werecollected from Joypurhat, Bangladesh inSeptember, 2012 and identified by experts of theBangladesh National Herbarium, Dhaka, where avoucher specimen has also been retained withaccession no. 39432. The collected plant partswere cleaned, dried for one week and pulverizedinto a coarse powder using a suitable grinder. Thepowder was stored in an airtight container andkept in a cool, dark, and dry place until furtheranalysis.Extract preparationApproximately 800 g of powdered material wasplaced in a clean, flat-bottomed glass containerand soaked in ethanol and similarly 500g of thepowder was soaked in distilled water. Both thecontainers with its contents was sealed and keptfor 5 days. Then extraction was carried out usingultrasonic sound bath accompanied by sonication(40 minutes). The entire mixture then underwenta coarse filtration by a piece of clean, whitecotton material. The extract then was filteredthrough Whatman filter paper (Bibby RE200,Sterilin Ltd., UK) and dried by electric oven at 450Ctemperature and continued up to obtain ethanol(12g) and aqueous (16g) extracts. The gummyextracts were stored in an air tight container.

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Drugs and chemicalsSilymarin, Diagnostic kits for serum: alanineaminotransferase (ALT) and aspartate aminotransferase (AST), alkaline phosphatase (ALP),total proteins (TP) and bilirubin were purchasedfrom Human, Germany. DPPH, quercetin, gallicacid, ascorbicacid were purchased from Sigma Aldrich, USA.Other chemicals and solvents were of highestanalytical grade commercially available.AnimalsLong Evans rat of either sex weighingapproximately 100-130 g were used for thisexperiment. The rats were purchased from theanimal research branch of the International Centrefor Diarrhoeal Disease and Research, Bangladesh(ICDDR, B). After their purchase, the rates werekept in standard environmental conditions (24.0 ±

0°C & 55-65% relative humidity and 12 h light/darkcycle) for one week to acclimate and fed ICDDR, Bformulated rodent food and water ad libitum. Theexperimental procedures involving animals wereconducted in accordance with the guidelines ofSoutheast University, Dhaka, Bangladesh. Thestudy protocol was approved by InstitutionalAnimal, Medical Ethics, Biosafety and BiosecurityCommittee of the University. The set of rulesfollowed for animal experiment were approved bythe institutional animal ethical committee (37).Acute toxicity testingSixty six rats were divided in eleven groups of sixanimals. Two extracts (AEDFL and EEDFL) wereadministrated orally at doses of 200, 400, 800, 1600and 3200 mg/kg body weight to the animal groups(one dose per group). The control group receivednormal saline (mg/kg). General signs of weaknessand symptoms of toxicity, food and water intakeand mortality were recorded for a period of 48hours and then for a period of 14 days.Phytochemical screeningQualitative tests of the AEDFL and EEDFL for thepresence of alkaloids, tannins, resins, saponins,flavonoids, steroids and terpenoids were carriedout.Test for alkaloids0.4 g of AEDFL and EEDFL were stirred with 8 ml of1% HCl in two separate test tubes. The mixtureswere warmed and filtered. 2 ml of filtrate twodifferent samples were treated separately with (a)

with few drops of potassium mercu riciodide(Mayer’s reagent) and (b) potassium bismuth(Dragendroff’s reagent). Turbidity or precipitationwith either of these reagents was taken asevidence for existence of alkaloids (38).Test for saponinsThe ability of saponins to produce emulsion withoil was used for the screening test. 20 mg of theextracts (AEDFL and EEDFL) were boiled in 20 mlof distilled water in a water bath for five min andfiltered. 10 ml of the filtrate was mixed with 5 mlof distilled water and shaken vigorously for frothformation. 3 drops of olive oil were mixed withfroth, shaken vigorously and observed foremulsion development (38).Test for terpenoidsPresence of terpenoids in the extracts was carriedout by taking 5 ml (1 mg/ml) of AEDFL and EEDFLin test tubes. Then 2 ml of chloroform, followedby 3 ml of concentrated H2SO4 were added in thetest tubes.A reddish brown coloration of theinterface confirmed the presence of terpenoids(38).Test for flavonoidsTo perform the test 50mg of AEDFL and EEDFLwere suspended in100ml of distilled water to getthe filtrate. 5 ml of dilute ammonia solution wasadded to 10 ml of filtrate followed by few drops ofconcentrated H2SO4. Presence of flavonoids wasconfirmed by yellow coloration (39).Test for tannins50 mg of AEDFL and EEDFL were boiled in 20 ml ofdistilled water then filtered. A few drops of 0.1%FeCl3 was added in filtrate and observed for colorchange. Appearance of brownish green or a blue-black coloration indicated the presence of tannins(39).Test for SteroidsOne ml of the extract was dissolved in 10 ml ofchloroform and equal volume of concentratedsulphuric acid was added by sides of the test tube.The upper layer turns red and sulphuric acid layershowed yellow with green fluorescence. Thisindicated the presence of steroids (38).Test for reducing sugarBoth the extracts (AEDFL and EEDFL) weredissolved individually in 5 ml distilled water andfiltered. The filtrates were used to test for thepresence of carbohydrates. Filtrates were treatedwith Benedict’s reagent and heated on a waterbath. Formation of an orange red precipitate_______________________________________


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indicates the presence of reducing sugars (40).Test for ResinAcetone-water Test: Extracts (AEDFL and EEDFL)were treated with acetone. Small amount of waterwas added and shaken. Appearance of turbidityindicates the presence of resins (40).

Determination of phytoconstituentsDetermination of total phenolsTotal phenol contents were determined usingFolin-Ciocalteu reagent as described by Yang et al.(41) with slight modifications. Total phenolic assaywas conducted by mixing 2.7 mL of deionisedwater, 0.01 ml (200µg/ml) of extracts, 0.3 ml 20%Na2CO3 and 0.10 ml Folin-Ciocalteu reagent.Absorbance of mixture was measured at 725 nm. Astandard curve was prepared with gallic acid (r2=0.945) and final results were given as mg/g gallicacid equivalent.Determination of total flavonoids1 ml of plant extract in methanol (200 mg/ml) wasmixed with 1 ml aluminium trichloride in ethanol(20 mg/ ml and a drop of acetic acid, and thendiluted with ethanol to 25 ml. The absorption at415nm was read after 40 min. Blank samples wereprepared using all the reagents with equal volumeused in the sample except extract. The totalflavonoid content was determined using a standardcurve (r2= 0.902) of quercetin (12.5-200 mg/ml)where quercetin was used as standard sample.Total flavonoid content was expressed as mg/g ofquercetin equivalent (42).

Antioxidant ability assaysDetermination of total antioxidansThe total antioxidant activity of extracts wasevaluated by phosphomolybdenum complexaccording to the method of Prieto et al. (43). 0.3 mlextracts (200µg/ml) was combined with 3ml ofreagent solution (0.6M sulfuric acid, 28 mM sodiumphosphate and 4mM ammonium molybdate). Thetubes containing the reaction solution wereincubated at 95°C for 90 min in water bath. Thenthe absorbance of the solution was measured at695nm using a spectrophotometer against blank(methanol) after cooling to room temperature.Ascorbic acid have been used as standardantioxidant (r2= 0.964) and total antioxidantcapacities of the extracts were expressed as mg/gequivalents of ascorbic acid.

DPPH• radical scavenging activity:

The DPPH free radical scavenging activity of leafextracts of D.falcata (AEDFL and EEDFL) weremeasured in term of hydrogen donating or radicalscavenging ability using the stable radical DPPH(44). Briefly, 0.004% w/v of DPPH radical solutionwas prepared in methanol and then 900 μL of thissolution was mixed with 100 μl of extract solution(12.5–200 μg/ml) and kept in a dark place for thirtyminutes. Then absorbance was measured at 517nm where methanol (98%), DPPH solution andascorbic acid were used as blank, control andstandard antioxidant respectively. Scavengingcapacity of DPPH radicals (% Inhibition) wasmeasured by the following formula and finallycalculated 50% inhibition concentration (IC50)using software.

Inhibition (%) = (A0-As)/A0 ×100

Where A0=Absorbance of control group, As=Absorbance of sample

Ferric-reducingpower assayThe Fe3+reducing power of the extracts weredetermined by the method of Oyaizu (45) withslight modifications. Different concentrations ofthe extracts and standard ascorbic acid (12.5, 25,50, 100, 200 µg/ml) were prepared. 1ml of boththe extracts and standard ascorbic acid of allconcentrations were taken in separate test tubesand were mixed with 2.5 ml of phosphate buffersolution (0.2 M, pH 6.6). 2.5 mL of potassiumferricyanide (1%) was added in each test tube, andincubated at 50°C for 30 min. Then, 2.5 mL oftrichloroacetic acid (10%) was added to themixture which was then centrifuged at 4000 rpmfor 10 min. Finally, 2.5 mL of supernatant wasmixed with 2.5 mL of distilled water and 0.1 mL ofFeCl3 (0.1%) solution followed by incubation at350C for 10 minutes. The absorbance wasmeasured at 700nm and the reducing powers of the extracts werecompared with the standared ascorbic acid.

Hepatoprotective studiesHepatoprotective activity of extracts wasevaluated by CCl4 according to the method ofGerhard Vogel (46).

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Briefly, thirty animals of both sexes were dividedinto 5 groups of six animals in each group andsubjected to the following experiment. Group-Iserved as the control and received 2% gum acacia(1ml/kg p.o) daily for 7days. Group-II served as thetoxic group (CCl4 control) and received 25% CCl4 inolive oil (1ml/kg.i.p) daily for 7 days. Group-IIIserved as positive control and received thestandard drug silymarin (50mg/kg p.o.) for 7 days.Group-IV and V were treated with AEDFL andEEDFL at 200 mg /kg.p.o for 7 days. On the 7th dayanimals of each group were sacrificed afteranaesthesia carried out by chloroform. Bloodsample were collected from anticubital vain andserum was separated by centrifugation forestimation of hepatic enzymes. Then the livermarkers such as ALT, AST, ALP, TP and bilirubinwere estimated from the serum by blood chemistryanalyzer Olympus AU-400.

Histopathological examinationLiver of each animal were separated and washedwith normal saline (0.9%) solution and preservedwith 10% formalin for histopathologicalexamination. Sections (4-5mm thick) wereprepared from each liver and stained withHemotoxylin and Eosin dye for photomicorscopicobservation. The microscopic slides of the livercells were photographed at a magnification ofx100.Statistical analysisAll the data are expressed as mean ± S.E.M. (n = 6rats per group). Statistical significance (p)calculated by ANOVA done in SPSS, Version 15.0,Folled by Dunnett ´s Test. Pa<0.001 wereconsidered to be statistically significant. All thegraphs are prepared using Graph Pad Prismsoftware.

ResultsPhytochemical screeningVarious Qualitative Phytochemical tests wereperformed on AEDFL and EEDFL. ThePhytochemical screening demonstrated thepresence of saponins, tannins, flavonoids,alkaloids, reducing sugars, terpenoids and steroids.(Table 1)

Total phenol, total flavonoid and totalantioxidant contentsThe total phenol, total flavonoid and totalantioxidant contents of AEDFL and EEDFL wereexpressed as Gallic acid, Quercetin and Ascorbicacid equivalents (mg/g) respectively. (Table 2).In vitro antioxidant activityAntioxidant capacity of AEDFL and EEDFL wasexamined using following assays.DPPH free radical scavenging activityThe antioxidant activity of AEDFL and EEDFL wereassessed by the DPPH free radical scavengingassay. The leaves extracts exhibited significantDPPH free radical scavenging effects compared tostandard ascorbic acid. IC50 (50%inhibitionconcentration) value of ascorbic acid was12.55±2.35 µg/ml where as aqueous and ethanolextracts showed 19.88±2.98 µg/ml and 33.60±2.15µg/ml respectively (Figure 1).Reducing power capacityThe reducing power of a compound is related to

its electron transfer ability and may therefore;serve as an indicator of its potential antioxidantactivity. We found the dose response curves ofthe reducing powers of both the extracts ofD.falcata leaves (12.5 - 200 μg/ml) and comparedwith standard ascorbic acid. It was found that thereducing capacity of each sample increased withthe increase of concentration. Between the twoextracts the aqueous portion showed morereducing activity (Figure 2).Hepatoprotective studiesSpecific liver enzymes, total protein and bilirubinThe plasma levels of specific liver enzymes andprotein profile were assessed to determine theliver function of each rat. The liver damage,induced by hepatotoxic compound CCl4,significantly (P<0.001) elevated the plasma levelof specific liver enzymes (ALT, AST, ALP andbilirubin) and lowered total protein (TP) levels inthe hepatotoxic rats compared with the normalgroup. However co-treatment with AEDFL andEEDFL (200 mg/kg bw) reduced the elevatedlevels of liver markers such as ALT, AST, ALP andbilirubin, as well as increased protein level againstthe CCl4 group. Silymarin (50 mg/kg bw)prevented the alterations in the activity level ofALT, AST, ALP and bilirubin induced with CCl4.These data demonstrated that the effects oftoxicity induced by CCl4 on the liver function could

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be effectively counter balanced by the treatmentof D.falcata leaf extracts (AEDFL and EEDFL).(Table3).Histopathological evaluationThe microscopic assessment (H&E staining) of liversections in the experimental Groups 1–5 are shownin Figures 3. Figure 3(a) shows that there was nopathological abnormality observed in the liver ofnormal rat and thus showing the absence ofvascular or necrosis changes. Figure 3(b) showsthat CCl4 induced severe necrosis changes andsubstantial changes in liver section such asballooning, microvesicular steatosis, fattydegeneration, increase in sinusoidal space (SS)dilation and central vein, in CCl4 treated group ascompared to normal group. On the other hand,livers of rats in all treatment groups (AEDFL andEEDFL and silymarin) showed noticeable recoveryfrom CCl4 -induced liver damages with lessmicrovesicular steatosis and hepatocytes necrosisfeatures compared to CCl4 control group (Figure 3c,3d and 3e) (Figure 3).

DiscussionModern drugs, hazardous chemicals and pollutantsare the main factors of liver damage (47). So it isvery much important to find out safe and effectivetherapeutic options to minimize the situation. CCl4induced- hepatotoxicity is one of the mostimportant methods for conducting thehepatoprotective study in animal model (48).Medicinal plants are sources of diverse nutrients,many of which display antioxidant properties thatare capable of shielding the body against bothcellular oxidative stress and harmful pathogens(49); hence these are considered an alternativetherapeutic approach in folk medicine (50).In thepresent research, AEDFL and EEDFL wereexamined as a promising therapy for treating liverdamage induced by CCl4 in rat model. The protocolinduced cirrhosis with similar pathology andetiology pattern to the human liver cirrhosis withthe same biochemical values for typical humancirrhosis markers (51). The CCl4, afteradministrations to rats, is biotransformed in liver bycytochrome P450 (CYP2E1) of endoplasmicreticulum to the trichloromethyl free radical(NCCl3) and then further converted to a peroxyradical (CCl3O2N) (52, 53). radicals lead to auto-oxidation (cascade of reactions) of cellular lipidis

and proteins and , thereby not only change thestructures of endoplasmic reticulum and othermembrane but also generates endogenoustoxicants that can readily react with adjacentmolecules like membrane proteins or diffuse tomore distant molecules like DNA, which may leadto more hepatic complications and functionalanomalies such as liver cell necrosis, apoptosis,fibrosis, or cirrhosis (48, 54, 55 ,56) andsignificantly lower activities of antioxidantenzymes such as Catalase (CAT), peroxidase(POD), superoxide dismutase (SOD), glutathioneperoxidase (GSH-Px), glutathione reductase(GSR), glutathione-S-transferase (GST), andquinone reductase (QR) (56). As a consequence,the cell membranes of hepatocytes become morepermeable, and enzymes such as ALT and AST,ALP, generated by cell membrane andmitochondrial damages in liver cells, (57) caneasily leak out into the blood circulation. So,amounts of these enzymes are increased in theplasma compared with normal subjects (1). A highconcentration of bilirubin in serum is an indicationfor increased erythrocyte degeneration rate. Theliver is known to play a significant role in theserum protein synthesis. So reduced level of TP isthe consequence of defective biosynthesis ofprotein in liver (48). These changes of markerslevel reflect the degree of hepatocyte damageand necrosis (53) and vice versa. So, the extant ofliver damage can be effectively assessed byestimating the activities of the liver markers: ALT,AST, ALP, TP and bilirubin.Antioxidants are the prominent bioactivecompounds that can effectively protect cells ofliver or other organs from damage by opposingthe activities of the free radicals (NCCl3, CCl3O2N)(58). The coordinate action of antioxidant systemis very critical for the detoxification of freeradicals. SOD reduces the concentration of highlyreactive superoxide radical by converting it toH2O2 whereas CAT and GSH-Px decomposes H2O2

and protect the tissues from highly reactivehydroxyl radicals. Studies have reported that CCl4reduces the activities of antioxidant enzymes andcauses hepatopathy (59, 60).Phytochemical screening of AEDFL and EEDFLrevealed the presence of alkaloids, tannins, resins,saponins, flavonoids, terpenoids, and steroidsthat are biologically active compounds.

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Significant amount of total antioxidants (329.79 ±

8.12, 297.97 ± 7.73), total phenols (178.72 ± 3.49,121.79 ± 4.39) and total flavonoids (153.80 ± 6.39,132.50 ± 7.38) were also presents in the AEDFL andEEDFL respectively. These phytoconstituents havebeen reported to exert major antioxidant activitiesby scavenging of free radicals that cause lipidperoxidation (9, 55). These antioxidant effects ofAEDFL and EEDFL were assessed by DPPH radicalscavenging and reducing power methods.DPPH radical, rapid and sensitive freeradical is considered to be a model of antioxidantsactions of scavenging activity of free radicals bydonating hydrogen atom (58). Median inhibitionconcentration (IC50) of AEDFL and EEDFL were19.88±2.98 µg/ml and 33.60±2.15 µg/mlrespectively. Furthermore, reducing power of anantioxidant depicts the neutralizing of oxidants bydonating electrons (58). AEDFL has showed morereducing ability than the EEDFLCo-treatment of AEDFL and EEDFL prevented thetoxic effects of CCl4 by restoring the activities ofantioxidant enzymes towards the level of controlanimals. AEDFL and EEDFL which have significantantioxidant activities ameliorate the liver injuries byscavenging of free radicals, or by preventingcytochrom P450 enzyme which is furtherconfirmed by the reduced amount ofhistopathological injury (55). A number of studieshave revealed that GSH conjugates play a majorrole in eliminating the CCl4-induced toxicmetabolites which are the main cause of liverinjuries. Repeat administration of CCl4 depletesGSH level (61). The maintenance of sufficientglutathione level is important for the prevention ofCCl4-induced damages (62). So alternatively, themechanism of hepatoprotection of AEDFL andEEDFL against the CCl4 toxicity might be due torestoration of GSH concentration in the liver ofexperimental animal (63). Between the twoextracts AEDFL showed better protection as itposes more antioxidant activity than EEDFL.These in vitro and in vivo assays indicate that theplant extracts (AEDFL and EEDFL) are significantsource of natural antioxidants which might preventthe progression of oxidative stress on variousorgans especially on hepatic tissues from itsdamage by free radicals. However, the specificcompounds of the extracts which are responsiblefor the protection by antioxidant activity are stillunclear.

Therefore, further studies are necessary to isolateand identify the antioxidant compounds and toclarify the actual mode of protection ofhepatotoxicity.

AcknowledgmentsThe authors are grateful to Pharmacy departmentof Southeast University, Dhaka, Bangladesh forproviding all required facilities to conduct theresearch in their lab. The authors are also gratefulto authority of Arms forces Medical CollegeHospital, Dhaka, Bangladesh for providinghistological examination of rat liver in their lab.The authors are also thankful to animal division ofthe International Centre for Diarrhoeal Diseaseand Research, Bangladesh (ICDDR, B) for timelyproviding sufficient experimental rats.Conflict of interest: Authors have no conflict ofinterest.

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Table 1: Phytochemical test for aqueous and ethanol extracts of Dendrophthoe falcata leaves (AEDFL and EEDFL).

Table 2: Quantitative estimation of phytochemicals and antioxidant activities of aqueous and ethanol extracts ofD.falcata leaves (AEDFL and EEDFL).

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AA AEDFL EEDFL0

10

20

30

40

IC50

(µ

g/m

L)

AA= Ascorbic acid AEDFL=Aquous extract of Dendrophthoe falcata leaf

EEDFL=Ethanol extract of Dendrophthoe falcata leaf

12.55±2.35

19.88 ±2.98

33.60±2.15

Figure-1: Determination of IC50 of AEDFL and EEDFL. Data expressed as mean ± SEM(n =3) for all tested

Figure 2: Reducing power of the aqueous and ethanol extract of D.falcata leaves (AEDFL and EEDFL) and ascorbic acid.n = 3. Error bars indicate standard error of mean.

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Table 3: Effects of AEDFL and EEDFL on liver function tests in rat.

Figure 3: Micrograph of liver of rats (H & E stain). (a) Representative section of liver from the control group showing thenormal histology. (b) CCl4 (1ml/kg bw, 25% in olive oil) induced hydropic necrosis, lymphocytes infiltration, and ballooning ofhepatocytes. (c) CCl4+silymarin (50 mg/kg bw) repairing of hepatocytes. (d) CCl4+AEDFL (200 mg/kg bw) repairing ofhepatocytes (e) CCl4+EEDFL (200 mg/kg bw) repairing of hepatocytes.

antioxidant and hepatoprotective effects of aqueous and ...€¦ · ) in olive oil (1ml/kg,b.w,i.p)...

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