Research Article Hepatoprotective and Antiviral Efficacy ... · Research Article Hepatoprotective and Antiviral Efficacy of Acacia mellifera Leaves Fractions against Hepatitis B Virus

Research ArticleHepatoprotective and Antiviral Efficacy of Acacia melliferaLeaves Fractions against Hepatitis B Virus

Ahmed H Arbab12 Mohammad K Parvez1 Mohammed S Al-Dosari1

Adnan J Al-Rehaily1 Mohammed Al-Sohaibani3 Elwaleed E Zaroug2

Mansour S AlSaid14 and Syed Rafatullah4

1Department of Pharmacognosy College of Pharmacy King Saud University Riyadh 11451 Saudi Arabia2Department of Pharmacognosy College of Pharmacy Omdurman Islamic University Khartoum 14415 Sudan3Department of Pathology King Khalid University Hospital King Saud University Riyadh 11461 Saudi Arabia4Medicinal Aromatic and Poisonous Plants Research Center College of Pharmacy King Saud University Riyadh 11451 Saudi Arabia

Correspondence should be addressed to Mohammad K Parvez khalid parvezyahoocom

Received 5 February 2015 Accepted 25 March 2015

Academic Editor Fumio Imazeki

Copyright copy 2015 Ahmed H Arbab et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present study investigated the hepatoprotective and anti-HBV efficacy of Acacia mellifera (AM) leaves extracts The crudeethanolic-extract including organic and aqueous fractions were tested for cytotoxicity on HepG2 and HepG2215 cells (IC

50=

684 120583gmL) Of these the ethyl acetate and aqueous fractions showed the most promising dose-dependent hepatoprotectionin DCFH-toxicated cells at 48 h In CCl

4-injured rats oral administration of AM ethanol extract (250 and 500mgkgsdotbw) for

three weeks significantly normalized the sera aminotransferases alkaline phosphatase bilirubin cholesterol triglycerides andlipoprotein levels and elevated tissue nonprotein sulphydryl and total protein The histopathology of dissected livers also revealedthat AM cured the tissue lesions The phytochemical screening of the fractions showed presence of alkaloids flavonoids tanninssterols and saponins Further anti-HBV potential of the fractions was evaluated on HepG2215 cells Of these the n-butanol andaqueous fractions exhibited the best inhibitory effects on HBsAg and HBeAg expressions in dose- and time-dependent mannerTaken together while the ethyl acetate and aqueous fractions exhibited the most promising antioxidanthepatoprotective and anti-HBV activity respectively the n-butanol partition showed both activities Therefore the therapeutic potential of AM extractswarrants further isolation of the active principle(s) and its phytochemical as well as biological studies

1 Introduction

Liver injury accounts for approximately one-half of the casesof hepatic failures including all forms of acute and chronicliver diseases [1] In most of such cases toxins and drugsare involved in oxidative stress-induced hepatotoxicity [2 3]Liver infection with hepatotropic viruses including hepatitisviruses is characterized by acute and chronic hepatitis fibro-sis cirrhosis and hepatocellular carcinomas Of these hep-atitis B virus (HBV) infection continues to be an importantcause of morbidity and mortality worldwide [4] Althoughthere aremany effective therapeutic drugs available they havecertain limitations While interferon (IFN-120572) has a high inci-dence of adverse effects and nonresponse long-term therapy

with nucleos(t)ide analogues has risks of emergence of drug-resistant viral mutants [5] Therefore many effective naturalor plant products have been investigated against hepatotoxin-induced liver damages [6ndash10] In addition numerous activephytoproducts or phytochemicals (flavonoids polypheno-lic tannins terpenoids lignans saponins alkaloids andanthraquinones) of diverse geographic origin and based onlocal cultural practices have been also reported effectiveagainst HBV infections in vitro orand in vivo [11 12] Ofthese picroliv (Picrorhiza kurroa) andrographolide (Andro-graphis paniculata) artemisinin (Artemisia annua) and Sily-marin extracts for anti-HBV activities are reported long back[13] Notably the most promising anti-HBV phytoproductstested include niranthin and hinokinin (lignans) isolated

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 929131 10 pageshttpdxdoiorg1011552015929131

2 BioMed Research International

from Phyllanthus spp [14ndash16] helioxanthin from the ChineseTaiwania cryptomerioides [17] wogonin another flavonoidfrom Scutellaria radix [18] the polyphenolic extract fromGeranium carolinianum L [19] protostane triterpenes fromAlisma orientalis [20] dihydrochelerythrine alkaloids fromCorydalis saxicola [21] Saikosaponin C from Bupleurumspecies [22] and extracts from Rheum palmatum L [23]Very recently an in vitro study showing anti-HBV effect ofa compound (LPRP) isolated from Liriope platyphylla is alsopublished [24]

Acacia is the second largest genus in the Fabaceae familycomprising more than 1200 species worldwide A mellifera(AM) commonly known as ldquoBlack Thorn (English)rdquo orldquoKekadKitir (Arabic)rdquo is widely distributed in Africa andthe Arabian Peninsula including Saudi Arabia [25] It is avery thorny shrub to small tree with rounded or spreadingflat crown which may reach down to ground level While thetwigs are chewed and used as toothbrushes its pods youngtwigs leaves and flowers are highly nutritious fodder forlivestockAM leaves and roots are used in traditional Africanethnomedicine for the treatment of cold malaria [26]primary infection of syphilis sterility and bowel problems[27] including inflammation diarrhoea and bleeding [28]The published reports of various biological activities of AMinclude its antimalarial [29] and antimicrobial [30] potentialsPhytochemical studies on AM extracts have indicated thatthe main components are alkaloids saponins flavonoidstannins and triterpenoids [31ndash33] Of the studied species ofAcacia A confusa and A auriculiformis have been reportedto have hepatoprotective activity [34 35] In addition to thisA nilotica and A mellifera are also shown to have antiviralactivities against human immunodeficiency virus-1 (HIV-1) and herpes simplex virus (HSV) respectively [36 37]With this background information we therefore intended toinvestigate the hepatoprotective as well as anti-HBV activityof organic and aqueous fractions of AM leaves extract

2 Materials and Methods

21 Plant Material Leaves of AM (Family Fabaceae) werecollected from Gezira state Sudan Authentication of theplant was confirmed by a taxonomist at the herbariumof the Medicinal and Aromatic Plants Research Institute(MAPRI) Sudan as well as that of College of Pharmacy KingSaud University Riyadh Saudi Arabia A voucher specimen(number 16281) was deposited at the herbarium of College ofPharmacy King Saud University

22 Extraction and Preparation of AM Fractions The shade-dried and powdered leaves of AM (800 g) were soaked in80 aqueous ethanol (Merck USA) for two days at 25ndash30∘C and then filtered Extraction was repeated twice withthe same solvent The extract was collected passed throughWhatmann filter paper number 1 (Whatmann USA) andthen evaporated using a rotary evaporator (Buchi Switzer-land) under reduced pressure at 40∘CThe obtained greenishbrown semisolid ethanolic-extract (9747 g) was suspendedin distilled water (200mL) and then fractionated three timessuccessively with the same volume of hexane (Merck USA)

dichloromethane (Merck USA) ethyl acetate (Merck USA)and water saturated n-butanol (LobaChemie) to providethe corresponding extracts The organic solvents of thefractions were evaporated at reduced pressure using rotatoryevaporator and the aqueous extracts were lyophilized in afreeze dryer (Labconco)

23 Cell Culture and Drug Human hepatoma cells (HepG2)and HBV cell line HepG22215 (derivative of HepG2)(kind gift of Dr S Jameel International Center for GeneticEngineeringampBiotechnology NewDelhi India) were grownin RPMI-1640 medium (Invitrogen USA) supplementedwith 10 heat-inactivated bovine serum (Gibco USA) 1xpenicillin-streptomycin (Invitrogen USA) and 1x sodiumpyruvate (HyClone Laboratories USA) at 37∘C in a humid-ified chamber with 5 CO

2supply 27-Dichlorofluorescein

(DCFH Sigma USA) was used as cytotoxin on culturedHepG2 cells The approved nucleoside analog-based anti-HBV drug Lamivudine (3TC Sigma USA) was used asstandard

24 Cytotoxicity Assay AM ethanolic-extract as well as theorganic fractions was tested for cytotoxic effects if any oncultured HepG2 and HepG2215 cells Cells were seeded(05 times 105 cellswell in triplicate) in a 96-well flat-bottomplate (Becton-Dickinson Labware) and grownover nightAMtotal extract and organic fractions were dissolved in dimethylsulphoxide (DMSO 100mgmL) followed by dilutions inculture media to prepare five doses (0 25 50 100 and250 120583gmL) of each The final concentration of DMSO usednever exceeded gt01 and therefore had no cytotoxicity Theculture monolayers were replenished with complete mediacontaining a dose of AM and incubated for 48 h at 37∘Cfollowed by MTT assay (TACSMTT Cell Proliferation AssayKit Trevigen) as per the manufacturerrsquos instruction Theabsorbanceoptical density (OD) was recorded at 620 nm ina microplate reader (BioTek ELx800) and the data analyzed

25 Microscopy A direct visual observation was made underan inverted microscope (Optica 40x and 100x) to observeanymorphological changes in the cells culturedwith differentconcentrations ofAM fractions andor DCFH at 24 and 48 h

26 ExVivoHepatoprotective Activity of AMTotal Extract andFractions HepG2 cells were seeded (05 times 105 cellswell intriplicate) in a 96-well flat-bottom plate (Becton-DickinsonLabware) and grownover nightDCFHprepared (10mgmL)in DMSO was used as a cytotoxic agent (IC

50= 100 120583gmL)

(unpublished data) AM crude extract and fractions weredissolved in DMSO (100mgmL) followed by dilutionsin culture media to prepare four doses (25 50 100 and200120583gmL) of each The final concentration of DMSO usednever exceeded gt01 and therefore had no cytotoxicity Theculture monolayers were replenished with complete mediacontaining 100 120583gmL of DCFH plus a dose of AM includinguntreated as well as DCFH-treated controls The treated cellswere incubated for 48 h at 37∘C followed by MTT assay Theoptical density (OD) was recorded at 620 nm in a microplatereader (BioTek ELx800) and the data analyzed

BioMed Research International 3

27 Animals andTreatment Wister rats (male) were obtainedfrom the Experimental Animal Care Center (EACC) ofthe College of Pharmacy King Saud University RiyadhAnimals were housed in polycarbonate cages in a roomfree from any source of chemical contamination artificiallyilluminated (12 h darklight cycle) and thermally controlled(25 plusmn 2∘C) After acclimatization animals were randomizedand divided into five groups (IndashV) of six animals each GroupI animals served as untreated control and were fed orallywith normal saline 1mL Group II animals received carbontetrachloride (CCl

4) in liquid paraffin (1 1 125mLkgsdotbw)

intraperitoneally (IP) Groups III IV and V received CCl4

in liquid paraffin (1 1) 125mLkgsdotbw Groups II and III weretreated with AM total extract at a dose of 250mgkgsdotbw and500mgkgsdotbw respectively for three weeks Group V wastreated with the standard drug Silymarin [38ndash40] at a doseof 10mgkgsdotbw for three weeks After collecting the bloodthe animals were sacrificed using ether anesthesia The liverwas dissected out and used for biochemical estimations andhistological assessment All animals received human care incompliance with the guidelines of the Ethics Committee ofthe Experimental Animal Care Society College of PharmacyKing Saud University Riyadh

28 Estimation of Marker Enzymes and Bilirubin Serum glu-tamate oxaloacetate transaminase (SGOT) serum glutamatepyruvate transaminase (SGPT) [41] alkaline phosphatase(ALP) [42] and gamma-glutamyl transferase (GGT) [43] andbilirubin [44]were determined usingReflotron PlusAnalyzerand Roche kits (Roche Diagnostics GmbH MannheimGermany)

29 Estimation of Lipid Profile Total cholesterol [45] triglyc-erides [46] high-density lipoproteins (HDLC) [47] and glu-cose levels were estimated in serum using Roche diagnostickits (Roche Diagnostics GmbH Mannheim Germany)

210 Determination ofMalondialdehyde (MDA) Themethodreported by Utley et al [48] was followed In brief theliver and kidney tissues were removed and each tissuewas homogenized in 015M KCl (at 4∘C Potter-Elvehjemtype C homogenizer) to give a 10 (wv) homogenate Theabsorbance of the solution was then read at 532 nm Thecontent of MDA (nmolg wet tissue) was then calculated byreference to a standard curve of MDA solution

211 Estimation of Nonprotein Sulfhydryls (NP-SH) HepaticNP-SH were measured according to the method describedelsewhere [49] The liver tissues were homogenized inice-cold 002mM EDTA The absorbance was measuredwithin 5min of addition of 551015840dithio-bis(2)-nitrobenzoicacid (DTNB) at 412 nm

212 Determination of Total Protein (TP) Serum TP wasestimated by the kit method (Crescent Diagnostics JeddahSaudi Arabia) The absorbance (Abs) of the complex was

measured at 546 nm and TP was calculated using thestandard equation

Serum total protein

= (Abssample

Absstandard) times Concentration of standard

(1)

213 Histopathological Evaluation The animals were sac-rificed and dissected liver tissues were fixed in neutralbuffered formalin for 24 h Sections of the liver tissue werehistopathologically examined These sections were stainedwith haematoxylin and eosin using routine procedures [50]

214 Phytochemical Screening for Secondary MetabolitesQualitative phytochemical screening of AM total extract andits fractions for major secondary metabolites like alkaloidsflavonoids anthraquinones tannins and saponins was per-formed using standard procedures as described elsewhere[51ndash53]

215 In Vitro Antioxidant Activities of AM Fractions Theantioxidant activity of the fractions was evaluated usingthe 120573-carotene-linoleic acid bleaching method with minormodifications for working with 96-well plate Briefly 05mg120573-carotene (Sigma Aldrich USA) was dissolved in 1mL ofchloroform (Merck USA) and added to flasks containing25 120583g of linoleic acid (Sigma Aldrich USA) and 200mgof Tween-40 (Sigma Aldrich USA) The chloroform wasremoved at 40∘C using a rotary evaporator The resultantmixture was immediately diluted with 50mL of distilledwater and mixed for 1-2min to form an emulsion A mixtureprepared similarly but without 120573-carotene was used as ablank including a second control containing solvent insteadof extract A 200120583L aliquot of the emulsion was added towells of 96-well plate containing 40 120583L of the test sample (intriplicate) and 500 120583gmL of gallic acid (SigmaAldrich USA)was used as a standardTheplate was incubated at 50∘C for 2 hand absorbance was read (490 nm) at 30min intervals usingmicroplate reader (BioTek ELx800) The antioxidant activitywas calculated using the following equation

Antioxidant activity = 1 minus(Abs0minus Abs

119905)

(Abs1199000minus Abs119900

119905)times 100 (2)

where Abs0and Abs119900

0are the absorbance values measured

at zero time of incubation for sample extract and controlrespectively Abs

119905and Abs119900

119905are the absorbance values for

sample extract and control respectively at 119905 = 120min

216 InVitro Free Radical ScavengingActivity of AMFractionsThe free radical scavenging ability of the different AM frac-tions against 11-diphenyl-2-picrylhydrazyl (DPPH) radical(Sigma Aldrich USA) was evaluated as previously describedmethod [54] with minor modifications AM total extract andfractions were dissolved in DMSO (100mgmL) followedby dilutions with methanol to various concentrations (2550 100 and 500 120583gmL) Each fraction (150 120583L) was mixed


Table 1 In vivo effect of A mellifera (AM) crude ethanolic extract on CCl4-induced hepatotoxicity-related parameters

Treatment group Dose mgkg AST (UL) ALT (UL) GGT (UL) ALP (UL) Bilirubin (mgdL)Normal 10615 plusmn 436 3791 plusmn 161 326 plusmn 021 30883 plusmn 881 05 plusmn 002CCl4 125 37983 plusmn 1170a 30383 plusmn 1212a 1820 plusmn 089a 58616 plusmn 1192a 274 plusmn 01a

AM + CCl4 250 32250 plusmn 1208b 26483 plusmn 1074b 1241 plusmn 073b 5120 plusmn 1140b 166 plusmn 010b

AM + CCl4 500 28366 plusmn 982b 17816 plusmn 627 690 plusmn 038b 39850 plusmn 048b 116 plusmn 010b

Silymarin + CCl4 10 16616 plusmn 934b 9975 plusmn 355 578 plusmn 026b 325 plusmn 33 plusmn 1210b 094 plusmn 004b

All values represent mean plusmn SEM 119875 lt 005 119875 lt 001 119875 lt 0001 ANOVA followed by Dunnettrsquos multiple comparison test aAs compared with normalgroup bAs compared with CCl4 only group

with 50120583L of DPPH (0004 wv in methanol) in triplicatein a 96-well plate Appropriate blanks were prepared usingthe solvent only in addition to the same amount of DPPHreagent to get rid of any inherent solvent effect Ascorbic acidwas used as standard After 30min of incubation at 25∘Cthe decrease in absorbance was measured (490 nm) Theradical scavenging activity was calculated from the followingequation

Radical scavenging activity

= (Abscontrol minus Abssample

Abscontrol) times 100

(3)

217 Dose-Dependent Analysis of Anti-HBV Activities of AMFractions The HBV cell line HepG2215 was seeded in96-well plates (05 times 105well in triplicate) including naıveHepG2 cells as negative control Next day the old mediawere replaced with 100 120583L each of four doses (3125 625 125and 250 120583gmL prepared in culture media) of the five AMfractions including Lamivudine (20120583M) and the culturewas incubated for 2 days Culture supernatants of eachsample (triplicates) were collected and analyzed for the viralHBsAg and HBeAg using Monolisa HBsAg ULTRA Elisa Kit(BioRad USA) and HBeAgAnti-HBe Elisa Kit (DIASourceBelgium) respectively as per the manufacturerrsquos manual

218 Time-Course Analysis of HBsAg and HBeAg ExpressionsFurther antiviral activities of the most active fractions atthe highest dose were tested at days 1 3 and 5 Analysis ofinhibition of HBsAg and HBeAg secretions in the culturesupernatants was done as mentioned above

3 Results

31 Effect of AM Crude Ethanolic-Extract on Cell Mor-phology and Growth DCFH showed considerable cytotoxiceffect on the HepG2 and HepG2215 cells as reflected byaltered morphology compared to untreated cells Interest-ingly the DCFH-treated cells supplemented with 100 120583gmLand 200120583gmL of AM crude ethanolic extract and fractionswere morphologically different from the DCFH-treated cellsbut comparable to untreated cells at 48 h (data not shown)

32 Hepatoprotective Effect of AM Organic Fractions onCultured Liver Cells Hepatoprotective effect of AM crude

002040608

1121416

Surv

ival

frac

tion

HexaneDichloromethaneEthyl acetate

AqueousCrude ethanolic

200120583

gm

L

100120583

gm

L

50120583

gm

L

25120583

gm

L

DCF

(100120583

gm

L)

Unt

reat

ed

+ DCF (100120583gmL)

n-Butanol

Figure 1 MTT Cell Proliferation Assay Hepatoprotective effect ofAmellifera (AM) crude ethanolic extract and five fractions (hexanedichloromethane ethyl acetate n-butanol and aqueous) againstDCFH-induced toxicity of HepG2 cells

extract and fractions against DCFH-induced hepatotoxicitywas investigated While DCFH-toxicated cells were recov-ered to about 100 with 100 120583gmL of AM crude extractsupplementation with 200120583gmL of this further enhancedthe hepatocytes proliferation by about 20 (Figure 1) Of thefive fractions evaluated the ethyl acetate aqueous and n-butanol fractions showed themost effective hepatoprotection(Figure 1)

33 In Vivo Effect of AM Crude Extract on BiochemicalMarkers Based on the hepatoprotective activity at cellularlevel the effect of AM extract was further examined in theanimal model Administration of CCl

4dramatically elevated

the sera AST ALT GGT and ALP and bilirubin levelscompared to the normal control group (119875 lt 00001) indi-cating significant hepatotoxicity of CCl

4treatment (Table 1)

In contrast administration of AM extract significantlydecreased the above elevated parameters in CCl

4-treated

rats compared to the CCl4-treated group Moreover CCl

4-

induced toxicity caused significant elevation in lipid profileincluding cholesterol triglycerides LDL-C andVLDL-C andreduction in the HDL-C levels in serum After three weekswhile AM extract in a dose-dependent manner significantlyreduced the cholesterol triglycerides LDL-C and VLDL-C


Table 2 In vivo effect of A mellifera (AM) crude ethanolic extract on CCl4-induced lipid profile changes

Treatment group Dose mgkg TC (mgdL) TG (mgdL) HDL-C (mgdL) LDL-C (mgdL) VLDL-C (mgdL)Normal 8809 plusmn 65 7826 plusmn 409 5082 plusmn 125 7224 plusmn 634 1556 plusmn 065CCl4 125mLkg 21269 plusmn 784a 18405 plusmn 809a 2214 plusmn 056a 17580 plusmn 842a 3681 plusmn 169a



Silymarin + CCl4 10 12460 plusmn 594b 9855 plusmn 560b 3534 plusmn 181b 10474 plusmn 593b 1971 plusmn 112b


Table 3 Biochemical parameters of rat liver after treatment with A mellifera (AM) crude ethanolic extract

Treatment group Dose mgkg TP (mgdL) MDA (nmolg) NP-SH (mgdL)Normal 9584 plusmn 627 096 plusmn 011 816 plusmn 042CCl4 125mLkg 3078 plusmn 313a 855 plusmn 107a 425 plusmn 042a

AM + CCl4 250 3621 plusmn 322b 557 plusmn 077b 620 plusmn 056b

AM + CCl4 500 5663 plusmn 380b 2257 plusmn 020 b 628 plusmn 048b

Silymarin + CCl4 10 5664 plusmn 761b 287 plusmn 064b 736 plusmn 054b


levels it greatly improved HDL-C level (Table 2) Silymarinused as standard on the other hand significantly normalizedthe CCl

4-induced elevated levels of marker enzymes and

lipids Furthermore our results indicated that treatment withCCl4resulted in a significant increase in MDA but decrease

in NP-SH and TP concentration (Table 3) Treatment of ratswith AM extract resulted in a significantly diminished levelof MDA and greatly enhanced NP-SH and TP levels

34 Histological Improvement of Injured Liver by AM Thehistological examination of rat liver tissues revealed evidenceof hepatic necrosis and fatty degenerative changes in CCl

4-

injured animals Compared to this the AM extract-treated(250mgkgday) animals exhibited congested central veinwith mild necrosis and fatty changes On the other hand thehigher dose (500mgkgday) of AM or Silymarin adminis-tration showed normal hepatocytes and central vein with fullrecovery (Figures 2(a)ndash2(e)) This finally confirmed the invivo hepatoprotective efficacy of AM that supported our exvivo data

35 Phytochemical Screening The qualitative phytochemicalscreening of the AM crude extract and organic fractionsshowed the presence of alkaloids flavonoids polyphenolictannins sterols and saponins There was however no evi-dence of anthraquinones in the fractions

36 Antioxidant Activity of AM Fractions The crude ethano-lic extract of AM was able to reduce the stable free radicalDPPH to the yellow-colored DPPH at low concentrations(100 and 500 120583gmL) almost near to the positive controlMoreover in BCBT the extract was also able to inhibit thediscoloration of 120573-carotene at a concentration of 500120583gmLThe total estimated antioxidant value was 87 comparable tothat of positive control Based on this result the four organic

fractionswere further tested for antioxidant activity (Table 4)The highest antioxidant activity was found in the ethylacetate and n-butanol extracts following dichloromethaneand hexane extracts The aqueous extract was found to havethe least activities

37 Inhibition of HBsAg Expression by AM n-Butanol andAqueous Fractions Dose- and time-dependent activities offive fractions of AM extract were tested for inhibitionof expression levels of viral HBsAg with reference tountreated controls At day 2 after treatment while the hexanedichloromethane and ethyl acetate fractions showed about25ndash35 of downregulation of HBsAg expressions at highestdoses (125 and 250 120583gmL) the n-butanol and aqueousfractions exhibited the best inhibitions by approximately sim46 and sim44 respectively (Figure 3(a)) Inhibitory effectsof n-butanol and aqueous fractions were further evaluatedin a time-course study using 125120583gmL dose Compared todays 1 and 3 post-treatment HBsAg productionwas inhibitedup to sim50 and sim40 by n-butanol and aqueous fractionrespectively on day 5 (Figure 4(a)) While prolonged treat-ment beyond day 5 did not show any significant differencefurther continuation of culture resulted in cell overgrowthand death (data not shown)

38 Downregulation ofHBVReplication byAMn-Butanol andAqueous Fractions The HBV ldquoerdquo antigen (secretory protein)is a processed product of ldquopre-Corerdquo that is cotranslatedwith ldquoCorerdquo by a bicistronic subgenomic-RNA Thereforeproduction of HBeAg is a hallmark of HBV DNA replication(except HBeAg negative chronic hepatitis B cases) This isanalogous to HIV-p24 antigen where ELISA is a valid tool tomonitor HIV replication Therefore the two most promisingAM fractions n-butanol and aqueous that greatly sup-pressedHBsAg synthesis were subjected toHBeAg analysis of


(a) (b)

(c) (d)

(e)

Figure 2 Histopathology of experimental rat liver Histograms showing (a) healthy tissues with normal hepatocytes and central vein(b) CCl

4-injured tissue with necrosis and fatty degenerative changes (c) tissue with congested central vein with necrosis and fatty changes

after 250mg of A mellifera (AM) + CCl4treatment (d) liver with normal hepatocytes and central vein with full recovery after 500mg of AM

+ CCl4treatment and (e) liver with normal hepatocytes and fully recovered central vein after Silymarin + CCl

4treatment

Table 4 Free radical scavenging and total antioxidant activities of A mellifera (AM) five fractions

AM fractions Radical scavenging activity () (DPPH) Total antioxidant activity () (BCBT)25 (120583gmL) 50 (120583gmL) 100 (120583gmL) 500 (120583gmL) 500 (120583gmL)

Hexane mdash mdash 4017 plusmn 007 683 plusmn 005 5551 plusmn 098Dichloromethane 897 plusmn 124 1015 plusmn 115 2790 plusmn 187 4439 plusmn 004 6975 plusmn 112Ethyl acetate 3000 plusmn 004 44167 plusmn 103 6583 plusmn 108 8666 plusmn 181 7816 plusmn 103n-Butanol 2051 plusmn 134 3820 plusmn 007 6253 plusmn 042 7750 plusmn 014 7233 plusmn 081Aqueous mdash 067 plusmn 005 725 plusmn 002 3887 plusmn 106 6230 plusmn 057Ascorbic acid 9097 plusmn 007 9213 plusmn 028 9215 plusmn 006 9240 plusmn 032 mdashGallic acid mdash mdash mdash mdash 8712 plusmn 105Mean plusmn standard error of mean (SEM) for triplicate experimentsNot available


0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBs

Ag

()

Hexane DichloromethaneEthyl acetateAqueous

n-Butanol

Concentration (120583gmL)

(a)

0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBe

Ag

()

Aqueousn-Butanol


(b)

Figure 3 Dose-dependent anti-HBV activities of A mellifera (AM) leaves extracts ELISA showing inhibitions of (a) HBsAg expression byAM organic and aqueous fractions and (b) HBeAg expression by AM n-butanol and aqueous fractions in HepG2215 culture supernatantsDoses used for AM 3125 625 125 and 250120583gmL Values (119910-axis) means of 3 determinations

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6

Inhi

bitio

n of

HBs

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(a)

0

10

20

30

40

50

60

0 2 4 6

Inhi

bitio

n of

HBe

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(b)

Figure 4 Time-course anti-HBV activities of A mellifera (AM) n-butanol and aqueous fractions (125120583gmL each) ELISA showinginhibitions of (a) HBsAg expression and (b) HBeAg expression in HepG2215 culture supernatants at days 1 3 and 5 Lamivudine (20 120583M)used as reference anti-HBV drug Values (119910-axis) means of 3 determinations

the culture supernatants The two fractions very clearlyshowed inhibition ofHBeAg production in a dose-dependentmanner At day 2 after treatment while 125 and 250120583gmLof n-butanol fraction downregulated HBV replication bysim40 and sim48 respectively those of aqueous fractioninhibited virus replication by sim41 and sim50 respectively(Figure 3(b)) Replication inhibitory effects of n-butanol andaqueous fractions were further evaluated in a time-coursestudy using the 125120583gmL dose Compared to days 1 and 3post-treatment HBeAg productionwas inhibited up tosim50

and sim40 by n-butanol and aqueous fractions respectivelyon day 5 (Figure 4(b)) While prolonged treatment beyondday 5 did not show any significant difference further contin-uation of culture resulted in cell overgrowth and death (datanot shown)

4 Discussion

In the present study we first evaluated the hepatoprotectiveeffects of AM total ethanolic-extract and its five fractions


against the DCFH-induced cytotoxicity on HepG2 cellsDCFH is generally used to measure in vitro oxidative stressgenerated by free radicals through the principle of oxidationof DCFH to the fluorescent DCF [55] However we usedthis agent because of its cytotoxic effect in vitro AM organicfractions not only protected the cells against DCFH-inducedtoxicity but also promoted cell recovery and proliferationThese findings were in line with our visual examination of thecell morphology under microscopeThe total ethanol extractwas however more active than the hexane dichloromethaneethyl acetate n-butanol and aqueous fractions indicatingsynergy between different secondary metabolites

CCl4is a well-known hepatotoxin and is frequently used

as a chemical inducer of liver damage Its metabolic trans-formation by cytochrome P450 to trichloromethyl and tri-chloromethyl peroxyl free radicals causes covalent bindingto macromolecules and lipid peroxidation resulting in cellinjury [2 3] Liver damage by acute exposure to CCl

4

therefore causes clinical symptoms such as jaundice andelevated levels of liver enzymes in the blood [53] The liverenzymes such as AST ALT and ALP found within organsand tissues are released into the bloodstream followingcellular necrosis and cell membrane permeability and areused as a diagnostic indicator of liver damage in vivo Inthe present study treatment of CCl

4-injured rats with 250

and 500mgkg of AM total extract significantly reduced thesera ALT AST GGT and ALP and bilirubin levels in a dose-dependent manner Similar trend was observed for the seracholesterol TG and HLD levels The effect of AM extractwas comparable to Silymarin used as standard drugs thatsuggested a hepatoprotective effect of AM extract in vivoFurther the significant reduction in levels of LDLP-C LDLP-C and total cholesterol in theAM-treated rats and an increasein HDL-C level supported the hepatoprotective potential ofthe AM extract

MDA is a metabolite that is produced during lipid perox-idation of cell membrane and is used as an indicator for celldamage [56]The level ofMDAwas reduced in bothAM- andSilymarin-treated rats which also suggested the protectiveand curative activities of AM against liver damages NPSHare involved in several defense processes against oxidativedamage In the current study the liver NP-SH levels in CCl

4-

treated rats were significantly diminished when comparedwith the control group Treatment of CCl

4-injured rats with

AM or Silymarin replenishedNPSH concentration comparedto untreated animals that further suggested free radicalscavenging activity ofAM extract in vivoMoreover the levelsof TP in rat serumwere related to the function of hepatocytesDiminution of TP is a further indication of liver damage inCCl4-injured animals The level of TP would be decreased in

hepatotoxic condition due to defective protein biosynthesisin liver In our study the level of TP was restored to normalvalue indicating its hepatoprotective activity of AM that wascomparable to Silymarin

In vitro antioxidant activity of AM total ethanolic-extractand organic fractions revealed strong antioxidant activity inDPPH test The antioxidant activity could be thus attributedto the presence of antioxidant and free radical scavenging

factors for example phenolic compounds flavonoids andsaponins which were reported to have hepatoprotectiveactivity [57ndash60] Hepatoprotective activity of flavonoids isdue to their ability to scavenge free radicals [58] Such highactivity observed for ethyl acetate and n-butanol fractionscompared to the corresponding hexane dichloromethaneand aqueous fractions suggested that the antioxidant com-pound(s) is of high to intermediate polarity Furthermore thehigher activity observed for n-butanol fraction compared tothe aqueous one can be explainedMost of the active phenoliccompounds and glycosides were taken from aqueous phaseby n-butanol and metal salts which if present could bedissolved in the aqueous phase that could have inhibited theactivity of antioxidants [61] Nevertheless there is a linearrelationship between the hepatoprotective and the antioxi-dant activity HBV andHIV share some biological propertiesincluding mechanism of genome replication While HIVand HSV are retroviruses HBV is called pararetrovirus thatreplicates its DNA genome through a unique RNA inter-mediate step using reverse-transcriptase Therefore manyof the licensed drugs originally developed for HIV andHSV have been also effective against HBV The antiviralactivities of A mellifera and the aqueous extract of A niloticaagainst HSV and HIV respectively are previously reported[36 37] In line with this our anti-HBV evaluations ofthe different fractions at noncytotoxic concentrations then-butanol and aqueous fractions showed very promisingantiviral activitiesThe variable activities shown by the differ-ent fractions may be attributed to the diversity of structureandor uneven distribution of phytochemical constituentspresent in these fractions Nevertheless this further suggeststhat the bioactive compounds present in tested AM fractionsare of high polarity Identification of alkaloids flavonoids andpolyphenols showing antioxidant and anti-HBV activities inour study is in accordance with previously published reports

5 Conclusions

Our results revealed very promising hepatoprotective andcell-proliferative effects of AM leaves fractions in bothex vivo and in vivo experimental conditions Interestinglyfurther ex vivo evaluations of the n-butanol and aqueousfractions also exhibited anti-HBV efficacy Taken togetherwhile the ethyl acetate and aqueous fractions exhibited themost promising antioxidanthepatoprotective and anti-HBVactivity respectively the n-butanol fraction showed bothactivitiesTherefore the therapeutic potential of AM extractswarrants further isolation of the active principle(s) and itsphytochemical as well as biological studies

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This Project was funded by the National Plan for ScienceTechnology and Innovation (MAARIFAH) King Abdulaziz


City for Science and Technology Kingdom of Saudi ArabiaAward Number (MED11-1585-02)

References

[1] N Kaplowitz ldquoDrug-induced liver injuryrdquo Clinical InfectiousDiseases vol 38 no 2 pp S44ndashS48 2004

[2] A Bala P K Haldar B Kar S Naskar and U K MazumderldquoCarbon tetrachloride a hepatotoxin causes oxidative stress inmurine peritoneal macrophage and peripheral blood lympho-cyte cellsrdquo Immunopharmacology and Immunotoxicology vol34 no 1 pp 157ndash162 2012

[3] I Kus M Ogeturk H Oner S Sahin H Yekeler and MSarsilmaz ldquoProtective effects of melatonin against carbon tetra-chloride-induced hepatotoxicity in rats a light microscopic andbiochemical studyrdquo Cell Biochemistry and Function vol 23 no3 pp 169ndash174 2005

[4] S Locarnini M Littlejohn M N Aziz and L Yuen ldquoPossibleorigins and evolution of the hepatitis B virus (HBV)rdquo Seminarsin Cancer Biology vol 23 no 6 pp 561ndash575 2013

[5] K Lacombe A Boyd J Gozlan F Lavocat P-M Girard and FZoulim ldquoDrug-resistant and immune-escape HBV mutants inHIV-infected hostsrdquo Antiviral Therapy vol 15 no 3 pp 493ndash497 2010

[6] S I Alqasoumi and M S Abdel-Kader ldquoScreening of tradi-tional Saudi plants for hepatoprotective effectrdquo Planta Medicavol 74 no 9 p 1139 2008

[7] P M Dandagi M B Patil V S Mastiholimath A Gadadand R Dhumansure ldquoDevelopment and evaluation of hepato-protective polyherbal formulation containing some indigenousmedicinal plantsrdquo Indian Journal of Pharmaceutical Sciencesvol 70 no 2 pp 265ndash268 2008

[8] J Kinjo M Hitoshi R Tsuchihashi et al ldquoHepatoprotectiveconstituents in plants 15 protective effects of natural-occurringflavonoids and miscellaneous phenolic compounds as deter-mined in an HepG2 cell cytotoxicity assayrdquo Journal of NaturalMedicines vol 60 no 1 pp 36ndash41 2006

[9] S A Ganie B A Zargar A Masood andM A Zargar ldquoHepat-oprotective and antioxidant activity of rhizome of podophyllumhexandrum against carbon tetra chloride induced hepatotoxic-ity in ratsrdquo Biomedical and Environmental Sciences vol 26 no3 pp 209ndash221 2013

[10] M S Al-Said R A Mothana M A Al-Yahya et al ldquoEdible oilsfor liver protection hepatoprotective potentiality of Moringaoleifera seed oil against chemical-induced hepatitis in ratsrdquoJournal of Food Science vol 77 no 7 pp T124ndashT130 2012

[11] C Wohlfarth and T Efferth ldquoNatural products as promisingdrug candidates for the treatment of hepatitis B and Crdquo ActaPharmacologica Sinica vol 30 no 1 pp 25ndash30 2009

[12] J Liu H McIntosh and H Lin ldquoChinese medicinal herbs forchronic hepatitis B a systematic reviewrdquo Liver vol 21 no 4 pp280ndash286 2001

[13] R Mehrotra S Rawat D K Kulshreshtha G K Patnaik andB N Dhawan ldquoIn vitro studies on the effect of certain naturalproducts against hepatitis B virusrdquo Indian Journal of MedicalResearch vol 92 pp 133ndash138 1990

[14] R-L Huang Y-L Huang J-C Ou C-C Chen F-L Hsuand C Chang ldquoScreening of 25 compounds isolated fromPhyllanthus species for anti-human hepatitis B virus in vitrordquoPhytotherapy Research vol 17 no 5 pp 449ndash453 2003

[15] M Ott S P Thyagarajan and S Gupta ldquoPhyllanthus amarussuppresses hepatitis B virus by interrupting interactionsbetween HBV enhancer I and cellular transcription factorsrdquoEuropean Journal of Clinical Investigation vol 27 no 11 pp 908ndash915 1997

[16] R Mehortra S Rawat D K Kulshreshtha P Goyal G KPatnaik and B N Dhawan ldquoIn vitro effect of Phyllanthusamarus on hepatitis B virusrdquo Indian Journal ofMedical Researchvol 93 pp 71ndash73 1991

[17] Y-C Cheng C-X Ying C-H Leung and Y Li ldquoNew targetsand inhibitors of HBV replication to combat drug resistancerdquoJournal of Clinical Virology vol 34 no 1 pp S147ndashS150 2005

[18] Q Guo L Zhao Q You et al ldquoAnti-hepatitis B virus activity ofwogonin in vitro and in vivordquo Antiviral Research vol 74 no 1pp 16ndash24 2007

[19] J Li H Huang M FengW Zhou X Shi and P Zhou ldquoIn vitroand in vivo anti-hepatitis B virus activities of a plant extractfrom Geranium carolinianum Lrdquo Antiviral Research vol 79 no2 pp 114ndash120 2008

[20] Z Y Jiang XM Zhang F X Zhang et al ldquoA new triterpene andanti-hepatitis B virus active compounds fromAlisma orientalisrdquoPlanta Medica vol 72 no 10 pp 951ndash954 2006

[21] Y-R Wu Y-B Ma Y-X Zhao et al ldquoTwo new quaternaryalkaloids and anti-hepatitis b virus active constituents fromCorydalis saxicolardquo Planta Medica vol 73 no 8 pp 787ndash7912007

[22] L-C Chiang L T Ng L-T Liu D-E Shieh and C-C LinldquoCytotoxicity and anti-hepatitis B virus activities of saikos-aponins from Bupleurum speciesrdquo Planta Medica vol 69 no8 pp 705ndash709 2003

[23] Z Li L-J Li Y Sun and J Li ldquoIdentification of naturalcompounds with anti-hepatitis B virus activity from Rheumpalmatum L ethanol extractrdquo Chemotherapy vol 53 no 5 pp320ndash326 2007

[24] T-J Huang Y-C Tsai S-Y Chiang et al ldquoAnti-viral effect of acompound isolated from Liriope platyphylla against hepatitis Bvirus in vitrordquo Virus Research vol 192 pp 16ndash24 2014

[25] M G Hagos and G N Smit ldquoSoil enrichment by Acaciamellifera subsp detinens on nutrient poor sandy soil in a semi-arid southern African savannardquo Journal of Arid Environmentsvol 61 no 1 pp 47ndash59 2005

[26] A Koch P Tamez J Pezzuto and D Soejarto ldquoEvaluation ofplants used for antimalarial treatment by the Maasai of KenyardquoJournal of Ethnopharmacology vol 101 no 1-3 pp 95ndash99 2005

[27] C Mutai D Abatis C Vagias D Moreau C Roussakis andV Roussis ldquoCytotoxic lupane-type triterpenoids from Acaciamelliferardquo Phytochemistry vol 65 no 8 pp 1159ndash1164 2004

[28] A H Fatima and A M Mamoun ldquoThe relationship be-tween seed polymorphism and germination of Acacia mellif-era(Vahl)Benth seedsrdquo International Journal of Scientific andResearch Publications vol 3 no 5 2013

[29] C Mutai G Rukunga C Vagias and V Roussis ldquoIn vivoscreening of antimalarial activity of Acacia mellifera (Benth)(Leguminosae) onPlasmodiumberghei inmicerdquoAfrican Journalof Traditional Complementary and AlternativeMedicines vol 5no 1 pp 46ndash50 2008

[30] C Mutai C Bii G Rukunga et al ldquoAntimicrobial activityof pentacyclic triterpenes isolated from Acacia melliferardquo Af-rican Journal of Traditional Complementary and AlternativeMedicines vol 6 no 1 pp 42ndash48 2009


[31] S Lalitha K Rajeshwaran P S Kumar K Deepa and KGowthami ldquoIn vivo screening of antibacterial activity ofAcaciamellifera (BENTH) (Leguminosae) on human pathogenic bac-teriardquo Global Journal of Pharmacology vol 4 pp 148ndash150 2010

[32] C Mutai D Abatis C Vagias D Moreau C Roussakis andV Roussis ldquoLupane triterpenoids from Acacia mellifera withcytotoxic activityrdquoMolecules vol 12 no 5 pp 1035ndash1044 2007

[33] H F Abdel-Razik A O Enayat S A A El-Toumy and E AA Wafaa ldquoEvaluation of hepatoprotective activity of the Acacianilotica (L) Wild ex Delile leaves on carbon tetrachloride-induce liver damage in ratsrdquo Planta Medica vol 72 no 11 p986 2006

[34] Y-T Tung J-H Wu C-C Huang et al ldquoProtective effect ofAcacia confusa bark extract and its active compound gallic acidagainst carbon tetrachloride-induced chronic liver injury inratsrdquo Food andChemical Toxicology vol 47 no 6 pp 1385ndash13922009

[35] A Sathya andP Siddhuraju ldquoProtective effect of bark and emptypod extracts from Acacia auriculiformis against paracetamolintoxicated liver injury and alloxan induced type II diabetesrdquoFood and Chemical Toxicology vol 56 pp 162ndash170 2013

[36] G Hussein H Miyashiro N Nakamura et al ldquoInhibitoryeffects of Sudanese plant extracts onHIV-1 replication andHIV-1 proteaserdquo Phytotherapy Research vol 13 no 1 pp 31ndash36 1999

[37] F M Tolo G M Rukunga F W Muli et al ldquoThe anti-viraleffect of Acacia melliferaMeliaazedarach and Prunus Africanaextracts against herpes simplex virus type 1 infection in micerdquoJournal of Tropical Microbiology and Biotechnology vol 2 no 1pp 3ndash9 2006

[38] R Parveen S Baboota J Ali A Ahuja S S Vasudev andS Ahmad ldquoEffects of silymarin nanoemulsion against carbontetrachloride-induced hepatic damagerdquo Archives of PharmacalResearch vol 34 no 5 pp 767ndash774 2011

[39] I S Chen Y C Chen C H Chou R F Chuang L Y Sheenand C H Chiu ldquoHepatoprotection of silymarin against thioac-etamide-induced chronic liver fibrosisrdquo Journal of the Science ofFood and Agriculture vol 92 no 7 pp 1441ndash1447 2012

[40] C-C Li C-Y Hsiang S-L Wu and T-Y Ho ldquoIdentificationof novel mechanisms of silymarin on the carbon tetrachloride-induced liver fibrosis in mice by nuclear factor-120581B biolumi-nescent imaging-guided transcriptomic analysisrdquo Food andChemical Toxicology vol 50 no 5 pp 1568ndash1575 2012

[41] S Reitman and S Frankel ldquoA colorimetricmethod for the deter-mination of serum glutamic oxalacetic and glutamic pyruvictransaminasesrdquoTheAmerican Journal of Clinical Pathology vol28 no 1 pp 56ndash63 1957

[42] E J King and A R Armstrong ldquoCalcium phosphorus andphosphaterdquo in Practical Clinical Biochemistry H Varley EdCBS Publishers New Delhi India 1998

[43] S Fiala A E Fiala and B Dixon ldquoGlutamyl transpeptidase intransplantable chemically induced rat hepatomas and lsquospon-taneousrsquo mouse hepatomasrdquo Journal of the National CancerInstitute vol 48 no 5 pp 1393ndash1401 1972

[44] A Stiehl ldquoHyperbilirubinaemia in liver diseaserdquo Fortschritte derMedizin vol 100 no 18 pp 842ndash845 1982

[45] P N M Demacker A G M Hijmans H E Vos-Janssen Avanrsquot Laar and A P Jansen ldquoA study of the use of polyethyleneglycol in estimating cholesterol in high-density lipoproteinrdquoClinical Chemistry vol 26 no 13 pp 1775ndash1779 1980

[46] L B Foster and R T Dunn ldquoStable reagents for determinationof serum triglycerides by a colorimetric Hantzsch condensationmethodrdquo Clinical Chemistry vol 19 no 3 pp 338ndash340 1973

[47] M Burstein and H R Scholnick ldquoTurbidimetric estimation ofchylomicrons and very low density lipoproteins in human seraafter precipitation by sodium lauryl sulfaterdquo Biomedicine vol19 no 1 pp 16ndash19 1973

[48] H G Utley F Bernheim and P Hochstein ldquoEffect of sulfhydrylreagents on peroxidation in microsomesrdquo Archives of Biochem-istry and Biophysics vol 118 no 1 pp 29ndash32 1967

[49] J Sedlak and R H Lindsay ldquoEstimation of total protein-bound andnonprotein sulfhydryl groups in tissuewith Ellmanrsquosreagentrdquo Analytical Biochemistry vol 25 pp 192ndash205 1968

[50] C F Culling Handbook of Histopathological and HistochemicalTechniques Butterworth-Heinemann LondonUK 3rd edition1974

[51] D Satyajit Z L Sarkar and A I Gray Natural ProductsIsolation Humana Press Totowa NJ USA 2nd edition 2006

[52] A Kar Pharmacognosy and Pharmacobiotechnology New AgeInternational New Delhi India 2nd edition 2007

[53] P Tiwari M Kaur G Kaur and H Kaur ldquoPhytochemicalscreening and extraction a reviewrdquo Internationale Pharmaceu-tica Sciencia vol 1 pp 98ndash106 2011

[54] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo LWTmdashFoodScience and Technology vol 28 no 1 pp 25ndash30 1995

[55] C Rota C F Chignell and R P Mason ldquoEvidence for free rad-ical formation during the oxidation of 21015840-71015840-dichlorofluorescinto the fluorescent dye 21015840-71015840-dichlorofluorescein by horseradishperoxidase possible implications for oxidative stress measure-mentsrdquo Free Radical Biology and Medicine vol 27 no 7-8 pp873ndash881 1999

[56] M Suhail S Suhail B K Gupta and V Bharat ldquoMalondialde-hyde and antioxidant enzymes in maternal and cord blood andtheir correlation in normotensive and preeclamptic womenrdquoJournal of Clinical Medicine Research vol 1 no 3 pp 150ndash1572009

[57] G Ai Q C Liu W Hua Z M Huang and D W WangldquoHepatoprotective evaluation of the total flavonoids extractedfrom flowers of Abelmoschus manihot (L) Medic in vitro andin vivo studiesrdquo Journal of Ethnopharmacology vol 146 no 3pp 794ndash802 2013

[58] S S Saboo G G Tapadiya I A Farooqui and S S KhadabadildquoFree radical scavenging in vivo antioxidant and hepatopro-tective activity of folk medicine trichodesma sedgwickianumrdquoBangladesh Journal of Pharmacology vol 8 no 1 pp 58ndash642013

[59] Q L Tran I K Adnyana Y Tezuka et al ldquoHepatoprotectiveeffect of majonoside R2 the major saponin from Vietnameseginseng (Panax vietnamensis)rdquo PlantaMedica vol 68 no 5 pp402ndash406 2002

[60] YWang Z Lou Q-BWu andM-L Guo ldquoA novel hepatopro-tective saponin from Celosia cristata Lrdquo Fitoterapia vol 81 no8 pp 1246ndash1252 2010

[61] I I Koleva T A van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002


Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


from Phyllanthus spp [14ndash16] helioxanthin from the ChineseTaiwania cryptomerioides [17] wogonin another flavonoidfrom Scutellaria radix [18] the polyphenolic extract fromGeranium carolinianum L [19] protostane triterpenes fromAlisma orientalis [20] dihydrochelerythrine alkaloids fromCorydalis saxicola [21] Saikosaponin C from Bupleurumspecies [22] and extracts from Rheum palmatum L [23]Very recently an in vitro study showing anti-HBV effect ofa compound (LPRP) isolated from Liriope platyphylla is alsopublished [24]

Acacia is the second largest genus in the Fabaceae familycomprising more than 1200 species worldwide A mellifera(AM) commonly known as ldquoBlack Thorn (English)rdquo orldquoKekadKitir (Arabic)rdquo is widely distributed in Africa andthe Arabian Peninsula including Saudi Arabia [25] It is avery thorny shrub to small tree with rounded or spreadingflat crown which may reach down to ground level While thetwigs are chewed and used as toothbrushes its pods youngtwigs leaves and flowers are highly nutritious fodder forlivestockAM leaves and roots are used in traditional Africanethnomedicine for the treatment of cold malaria [26]primary infection of syphilis sterility and bowel problems[27] including inflammation diarrhoea and bleeding [28]The published reports of various biological activities of AMinclude its antimalarial [29] and antimicrobial [30] potentialsPhytochemical studies on AM extracts have indicated thatthe main components are alkaloids saponins flavonoidstannins and triterpenoids [31ndash33] Of the studied species ofAcacia A confusa and A auriculiformis have been reportedto have hepatoprotective activity [34 35] In addition to thisA nilotica and A mellifera are also shown to have antiviralactivities against human immunodeficiency virus-1 (HIV-1) and herpes simplex virus (HSV) respectively [36 37]With this background information we therefore intended toinvestigate the hepatoprotective as well as anti-HBV activityof organic and aqueous fractions of AM leaves extract

2 Materials and Methods

21 Plant Material Leaves of AM (Family Fabaceae) werecollected from Gezira state Sudan Authentication of theplant was confirmed by a taxonomist at the herbariumof the Medicinal and Aromatic Plants Research Institute(MAPRI) Sudan as well as that of College of Pharmacy KingSaud University Riyadh Saudi Arabia A voucher specimen(number 16281) was deposited at the herbarium of College ofPharmacy King Saud University

22 Extraction and Preparation of AM Fractions The shade-dried and powdered leaves of AM (800 g) were soaked in80 aqueous ethanol (Merck USA) for two days at 25ndash30∘C and then filtered Extraction was repeated twice withthe same solvent The extract was collected passed throughWhatmann filter paper number 1 (Whatmann USA) andthen evaporated using a rotary evaporator (Buchi Switzer-land) under reduced pressure at 40∘CThe obtained greenishbrown semisolid ethanolic-extract (9747 g) was suspendedin distilled water (200mL) and then fractionated three timessuccessively with the same volume of hexane (Merck USA)

dichloromethane (Merck USA) ethyl acetate (Merck USA)and water saturated n-butanol (LobaChemie) to providethe corresponding extracts The organic solvents of thefractions were evaporated at reduced pressure using rotatoryevaporator and the aqueous extracts were lyophilized in afreeze dryer (Labconco)

23 Cell Culture and Drug Human hepatoma cells (HepG2)and HBV cell line HepG22215 (derivative of HepG2)(kind gift of Dr S Jameel International Center for GeneticEngineeringampBiotechnology NewDelhi India) were grownin RPMI-1640 medium (Invitrogen USA) supplementedwith 10 heat-inactivated bovine serum (Gibco USA) 1xpenicillin-streptomycin (Invitrogen USA) and 1x sodiumpyruvate (HyClone Laboratories USA) at 37∘C in a humid-ified chamber with 5 CO

2supply 27-Dichlorofluorescein

(DCFH Sigma USA) was used as cytotoxin on culturedHepG2 cells The approved nucleoside analog-based anti-HBV drug Lamivudine (3TC Sigma USA) was used asstandard

24 Cytotoxicity Assay AM ethanolic-extract as well as theorganic fractions was tested for cytotoxic effects if any oncultured HepG2 and HepG2215 cells Cells were seeded(05 times 105 cellswell in triplicate) in a 96-well flat-bottomplate (Becton-Dickinson Labware) and grownover nightAMtotal extract and organic fractions were dissolved in dimethylsulphoxide (DMSO 100mgmL) followed by dilutions inculture media to prepare five doses (0 25 50 100 and250 120583gmL) of each The final concentration of DMSO usednever exceeded gt01 and therefore had no cytotoxicity Theculture monolayers were replenished with complete mediacontaining a dose of AM and incubated for 48 h at 37∘Cfollowed by MTT assay (TACSMTT Cell Proliferation AssayKit Trevigen) as per the manufacturerrsquos instruction Theabsorbanceoptical density (OD) was recorded at 620 nm ina microplate reader (BioTek ELx800) and the data analyzed

25 Microscopy A direct visual observation was made underan inverted microscope (Optica 40x and 100x) to observeanymorphological changes in the cells culturedwith differentconcentrations ofAM fractions andor DCFH at 24 and 48 h

26 ExVivoHepatoprotective Activity of AMTotal Extract andFractions HepG2 cells were seeded (05 times 105 cellswell intriplicate) in a 96-well flat-bottom plate (Becton-DickinsonLabware) and grownover nightDCFHprepared (10mgmL)in DMSO was used as a cytotoxic agent (IC

50= 100 120583gmL)

(unpublished data) AM crude extract and fractions weredissolved in DMSO (100mgmL) followed by dilutionsin culture media to prepare four doses (25 50 100 and200120583gmL) of each The final concentration of DMSO usednever exceeded gt01 and therefore had no cytotoxicity Theculture monolayers were replenished with complete mediacontaining 100 120583gmL of DCFH plus a dose of AM includinguntreated as well as DCFH-treated controls The treated cellswere incubated for 48 h at 37∘C followed by MTT assay Theoptical density (OD) was recorded at 620 nm in a microplatereader (BioTek ELx800) and the data analyzed












Serum total protein

= (Abssample


(1)





119905)


119905)times 100 (2)




119905and Abs119900















(3)



3 Results



002040608

1121416

Surv

ival

frac

tion



200120583

gm

L

100120583

gm

L

50120583

gm

L

25120583

gm

L

DCF

(100120583

gm

L)

Unt

reat

ed

+ DCF (100120583gmL)

n-Butanol








4-treated


4-




















4-








(a) (b)

(c) (d)

(e)





4treatment





0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBs

Ag

()


n-Butanol


(a)

0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBe

Ag

()

Aqueousn-Butanol


(b)


0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6

Inhi

bitio

n of

HBs

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(a)

0

10

20

30

40

50

60

0 2 4 6

Inhi

bitio

n of

HBe

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(b)




4 Discussion






4





4-


4-injured rats with





5 Conclusions




Acknowledgment




References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Serum total protein

= (Abssample


(1)





119905)


119905)times 100 (2)




119905and Abs119900















(3)



3 Results



002040608

1121416

Surv

ival

frac

tion



200120583

gm

L

100120583

gm

L

50120583

gm

L

25120583

gm

L

DCF

(100120583

gm

L)

Unt

reat

ed

+ DCF (100120583gmL)

n-Butanol








4-treated


4-




















4-








(a) (b)

(c) (d)

(e)





4treatment





0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBs

Ag

()


n-Butanol


(a)

0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBe

Ag

()

Aqueousn-Butanol


(b)


0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6

Inhi

bitio

n of

HBs

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(a)

0

10

20

30

40

50

60

0 2 4 6

Inhi

bitio

n of

HBe

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(b)




4 Discussion






4





4-


4-injured rats with





5 Conclusions




Acknowledgment




References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























(3)



3 Results



002040608

1121416

Surv

ival

frac

tion



200120583

gm

L

100120583

gm

L

50120583

gm

L

25120583

gm

L

DCF

(100120583

gm

L)

Unt

reat

ed

+ DCF (100120583gmL)

n-Butanol








4-treated


4-




















4-








(a) (b)

(c) (d)

(e)





4treatment





0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBs

Ag

()


n-Butanol


(a)

0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBe

Ag

()

Aqueousn-Butanol


(b)


0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6

Inhi

bitio

n of

HBs

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(a)

0

10

20

30

40

50

60

0 2 4 6

Inhi

bitio

n of

HBe

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(b)




4 Discussion






4





4-


4-injured rats with





5 Conclusions




Acknowledgment




References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































4-








(a) (b)

(c) (d)

(e)





4treatment





0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBs

Ag

()


n-Butanol


(a)

0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBe

Ag

()

Aqueousn-Butanol


(b)


0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6

Inhi

bitio

n of

HBs

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(a)

0

10

20

30

40

50

60

0 2 4 6

Inhi

bitio

n of

HBe

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(b)




4 Discussion






4





4-


4-injured rats with





5 Conclusions




Acknowledgment




References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e)





4treatment





0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBs

Ag

()


n-Butanol


(a)

0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBe

Ag

()

Aqueousn-Butanol


(b)


0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6

Inhi

bitio

n of

HBs

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(a)

0

10

20

30

40

50

60

0 2 4 6

Inhi

bitio

n of

HBe

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(b)




4 Discussion






4





4-


4-injured rats with





5 Conclusions




Acknowledgment




References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBs

Ag

()


n-Butanol


(a)

0

10

20

30

40

50

60

0 50 100 150 200 250 300

Inhi

bitio

n of

HBe

Ag

()

Aqueousn-Butanol


(b)


0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6

Inhi

bitio

n of

HBs

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(a)

0

10

20

30

40

50

60

0 2 4 6

Inhi

bitio

n of

HBe

Ag

()

Time (days)

AqueousLamivudine

n-Butanol

(b)




4 Discussion






4





4-


4-injured rats with





5 Conclusions




Acknowledgment




References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















4





4-


4-injured rats with





5 Conclusions




Acknowledgment




References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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