Chemical Constituents and Biological Activities of Essential Oils of Hydnora africana Thumb Used

14 Pages·2017·1.74 MB·English

applied
sciences
Article
Chemical Constituents and Biological Activities of
Essential Oils of Hydnora africana Thumb Used to
Treat Associated Infections and Diseases in
South Africa
OlubunmiAbosedeWintolaandAnthonyJideAfolayan*
MedicinalPlantsandEconomicDevelopment(MPED)ResearchCentre,DepartmentofBotany,
UniversityofFortHare,Alice5700,SouthAfrica;[email protected]
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
AcademicEditor:HidenoriOtsuka
Received:21September2016;Accepted:1December2016;Published:27April2017
Abstract: Hydnoraafricana(HA)ThumbisamemberofthegenusHydnora. Therootsareusedinthe
treatmentofinfectiousdiseasesinSouthAfricafolkmedicine. Thoughtherootextractsareusedto
treatvarioushumandiseasesincludinginflamedthroat,thereisadearthofscientificdataonthe
biologicalactivitiesofessentialoilisolatedfromthisplantintheliterature. Therefore,thepresent
studywasconductedtodeterminethechemicalcomponentsandcertainbiologicalactivitiesofthe
essential oil using standard bioassay methods. The plant essential oil exhibited a moderate free
radicalscavengingactivitythatwasdependentontheradicalspecies. Similarly,theessentialoilwas
activeagainstthegrowthofallthirteenopportunisticbacteriaapartfromSalmonellatyphimurium,
PseudomonasaeruginosaandProteusvulgaris. TheessentialoilwasalsoactiveagainstAspergillusniger
amongalltheninefungiselected. Inaddition,thespeciesistypifiedbysubstantialamountsofclasses
ofcompoundsincluding;carboxylicacids(30.68%),terpenes(10.70%),alkylaldehydes(4.86%)and
esters(0.82%),identifiedasantioxidantandantimicrobialagents. TheessentialoilofH.africanacould
besaidtohavepharmacologicalproperties,andtheseagentsintheessentialoilofH.africanacould
justifythefolkloreusageofthisplantinthetreatmentofinfectionsandrelateddiseases.
Keywords: Hydnoraafricana;chemicalcomposition;biologicalactivities;infections;GC-MS
1. Introduction
MedicinalplantshavecontributedimmenselytohealthcareinAfrica. Theseplantsareeasily
accessibleandcheapsourcesoftherapeuticagents[1]. Essentialoilsfrommedicinalplantscontain
certain constituents that play a pharmacological role in the prevention and treatment of chronic
diseasessuchascancer,cardiovasculardisease,diarrhoeadiseasesandinfections[2].
Infectionistheinvasionofthebodytissuesbydisease-causingagentslikebacteria,virusand
fungi. Thehosttissue’sreactiontotheseorganismsaswellasthetoxinsproducedbytheorganisms
is attributed to inflammation in patients with HIV or AIDS, cancer, solid organ transplantation,
immunedeficiencies,chronicinflammatorygastrointestinal,liver,respiratory,urinarytractandother
infections [3]. The bacteria associated with common infections like wounds, gastrointestinal and
urinarytractinfectionsincludeKlebsiellapneumonia,StreptococcuspyogenesandSerratiamercescens[4,5].
Essential oils are known to soothe infections by acting as antimicrobial agents, or by reducing
and neutralising reactive oxygen species (ROS) generated during the pathophysiology of disease
infections[6].
Medicinalplantsplayimportantrolesasasourceofantimicrobialandantioxidant. Oneofthese
plantsisthegenusHydnoraafricana(HA)Thumb(Hydronaceae(Piperales))(Figure1A,B).Theplantis
Appl.Sci.2017,7,443;doi:10.3390/app7050443 www.mdpi.com/journal/applsci
Appl. Sci. 2016, 6, 439  2 of 14 
Medicinal plants play important roles as a source of antimicrobial and antioxidant. One of these 
plants is the genus Hydnora africana (HA) Thumb (Hydronaceae (Piperales)) (Figure 1A,B). The plant 
is also known as Jackal food, Ubnklunga (Xhosa) or Umavumbuka (Zulu) [7]. It comprises 15 species 
in South Africa. This plant, similar to about ten species of parasitic flowering plant, lives a greater 
part of its life cycle underground and is unable to create chlorophyll or perform photosynthesis. In 
traditional medicine, the root of Hydnora species, tuber, fruits, leaves and fruit pulp (such as potato) 
is used for the treatment of infectious diseases such as dysentery, diarrhoea, amenorrhoea, bladder 
and kidney complaints. Other ailments include swollen glands and inflamed throat [7]. Although 
progress has been made in validating the chemical components and biological activities of the root 
parts of the plant, the chemical composition, antioxidant and antimicrobial properties of HA 
essential oil remain obscure.  
Therefore, this study aimed at identifying the chemical constituents of H. africana essential oil 
using gas chromatography interfaced with mass spectrometry (GC‐MS). We also aimed to assess its 
antioxidant, antibacterial and antifungal potential, and to relate our findings to its possible use in 
folk medicine. 
Appl.Sci.20127., M7,a4t4e3rials and Methods  2of14
2.1. Plant Collection and Preparation 
alsoknownasTJhaec kroaoltfso oofd H,U. Abfnrikcalnuan agsa s(hXohwons ain) oFrigUurme a1Bv uwmerbeu ckoalle(cZteudl uin) [D7e].ceImtbceorm 2p01r3is eats t1h5e speciesin
SouthAfriNcats.eTlahmiasnpzli aanreta, lsoicmatiiloanr into Nakboonukotbtee Mnusnpiecicpiaelsityo,f Epaastrearns iCtiacpfle oPwroveirnicneg, Spoluathn tA,flriivcae.s Tahigs rlieeas terpartof
at the latitude and longitude as described by Masika and Afolayan [8]. The samples were collected 
itslifecycleundergroundandisunabletocreatechlorophyllorperformphotosynthesis. Intraditional
and the specimen of the voucher (Win 2014/1) was kept in the Giffen’s herbarium, University of Fort 
medicine,therootofHydnoraspecies,tuber,fruits,leavesandfruitpulp(suchaspotato)isusedfor
Hare, Alice, South Africa. The plant materials were washed using tap water and then stored in the 
thetreatmreenfrtigoefraitnorf eact t4i°oCu usndtili site wasase stimsue cfohr aesssednytisael noitle erxytr,adctiiaornr. hoea,amenorrhoea,bladderandkidney
complaints. Otherailmentsincludeswollenglandsandinflamedthroat[7]. Althoughprogresshas
2.2. Extraction of the Essential Oil 
beenmadeinvalidatingthechemicalcomponentsandbiologicalactivitiesoftherootpartsoftheplant,
The essential oil was isolated from H. Africana roots by hydro‐distillation following the 
thechemicalcomposition,antioxidantandantimicrobialpropertiesofHAessentialoilremainobscure.
procedure from European Pharmacopoeia [9]. Here, 250 g of the dry roots were subjected to 
Therefore,thisstudyaimedatidentifyingthechemicalconstituentsofH.africanaessentialoil
hydro‐distillation for 3 h in an all‐glass Clevenger (model and manufacturer name). Through the 
usinggasscuhprpolmy oaf thoegatr atop thhey hienatteinrgf amcaendtlew (5it0h °Cm), athses esspseencttiarlo omil ewtarsy e(xGtraCct-eMd Sw)i.thW 4 elitarelss oofa wimateerd int o3 assessits
antioxidanht u,natnil tniob amcotreer eisaslenatniadl oailn wtaifsu cnomgainlgp ooutt.e Tnhtei aavl,eraangde pteorcreentlaagtee yoieuldr wfiansd 0.i3n%g.s Atpopriotpsrpiaotes 1s0i%bl eusein
DMSO vehicles in between 80 were utilized to dissolve the essential oil for more in vitro bioassay 
folkmedicine.
activities. 

(A)  (B)
Figure 1. Hydnora africana plant parts; Source http://www.botany.org/Parasitic_Plants/. (A) The 
Figure1. Hydnoraafricanaplantparts; Sourcehttp://www.botany.org/Parasitic_Plants/. (A)The
flower of Hydnora africana; (B) The root of Hydnora africana used. 
flowerofHydnoraafricana;(B)TherootofHydnoraafricanaused.
2.3. Chemicals and Reagents Used 
2. MaterialsanThde Mcehtehmoicdasls  used  consist  of  1,1‐diphenyl‐2‐picrylhydrazyl  (DPPH),  2,2′‐azino‐bis 
(3‐ethylbenzthiazoline‐6‐sulphonic  acid)  (ABTS),  butylated  hydroxyl  toluene  (BHT),  vanillin, 
2.1. PlantCollectionandPreparation
TherootsofH.AfricanaasshowninFigure1BwerecollectedinDecember2013attheNtselamanzi
arealocationinNkonkobeMunicipality,EasternCapeProvince,SouthAfrica. Thisliesatthelatitude
andlongitudeasdescribedbyMasikaandAfolayan[8]. Thesampleswerecollectedandthespecimen
of the voucher (Win 2014/1) was kept in the Giffen’s herbarium, University of Fort Hare, Alice,
SouthAfrica. Theplantmaterialswerewashedusingtapwaterandthenstoredintherefrigeratorat4
◦Cuntilitwastimeforessentialoilextraction.
2.2. ExtractionoftheEssentialOil
TheessentialoilwasisolatedfromH.Africanarootsbyhydro-distillationfollowingtheprocedure
fromEuropeanPharmacopoeia[9]. Here,250gofthedryrootsweresubjectedtohydro-distillation
for 3 h in an all-glass Clevenger (model and manufacturer name). Through the supply of heat to
theheatingmantle(50◦C),theessentialoilwasextractedwith4litresofwaterin3huntilnomore
essentialoilwascomingout. Theaveragepercentageyieldwas0.3%. Appropriate10%DMSOvehicles
inbetween80wereutilizedtodissolvetheessentialoilformoreinvitrobioassayactivities.
2.3. ChemicalsandReagentsUsed
The chemicals used consist of 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2(cid:48)-azino-bis
(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), butylated hydroxyl toluene (BHT), vanillin,
potassium persulphate, rutin, sodium nitroprusside (Na [Fe(CN) NO]2H O), glacial acetic acid
2 5 2
(CH COOH),sulfanilicacid,gallicacid,tannicacid,ferricchloride(FeCl ),aluminumchloride(AlCl ),
3 2 3
potassium ferricyanide [K Fe(CN) ], trichloroacetic acid (TCA), Folin-Ciocalteu reagent, sodium
3 6
carbonate(Na CO ),hydrochloricacid(HCL),sodiumdihydrogenphosphate(NaH PO ),disodium
2 3 2 4
Appl.Sci.2017,7,443 3of14
hydrogenphosphate(Na HPO ),Mueller-Hintonagar(MHA),dimethylsulfoxide(DMSO),ascorbic
2 4
acid, ciprofloxacin and amoxicillin, 2-thiobarbituric acid (TBA), potassium acetate (CH CO K),
3 2
Mueller-Hinton dextrose broth (MDB), diclofenac sodium and sodium chloride (NaCl), These
chemicals were bought from Merck and Sigma-Aldrich, Gauteng, South Africa. Every chemical
utilisedinthisstudywasofanalyticalgrade.
2.4. AntioxidantAssay
TheantioxidativecapacitiesoftheessentialoilofH.africanawereassessedintermsofDPPH,
nitricoxide,hydrogenperoxideandABTSinhibitoryassays.
2.4.1. 1,1-diphenyl-2-picrylhydrazylDPPHRadicalScavengingActivityAssay
ThemethodofLiyana-PathirananandShahidi[10]wasemployedtodetermineDPPHfreeradical
scavengingactivity. Asolutionof0.135mM2,2-diphenyl-1-picrylhydrazylradical(DPPH)inmethanol
wasbrieflyprepared. Briefly,1.0mLofthissolutionwasmixedwith1.0mLoftheHAoilandstandard
drugs(BHTandgallicacid)(0.025to0.5mg/mL).Thereactionmixturewasthenvortexedandputin
thedarkatroomtemperatureforaperiodof30min. Theabsorbanceofthemixturewasmeasured
usingaspectrophotometerat517nm. Alltestsandanalysiswererunintriplicates. Theplantextract’s
scavengingabilitywasthencalculatedusingthefollowingequation:
DPPHScavengingactivity(%)=[(Abscontrol−Abssample)/(Abscontrol)]×100
whereAbscontrolistheabsorbanceofDPPH+methanolandAbssampleistheabsorbanceofDPPH
radical+sample(sampleorstandard).
2.4.2. 2,2(cid:48)-Aazino-bis(3-ethylbenzthiazoline-6-sulfonicacid)(ABTS)RadicalScavengingAssay
The method described by Oyedemi et al. [11] was adopted for the determination of ABTS
scavenging activity. The stock solution of 7 mM ABTS solution and 2.4 mM potassium persulfate
solutionwasprepared. Theworkingsolutionwaspreparedbymixingthetwostocksolutionsinequal
proportionsandthenkeepingitinthedarkroomfor12h. Thesolutionwasthendilutedbymixing
1mLABTS+solutionwith60mLofmethanoltogetanabsorbanceof0.708±0.001unitsat734nm
withtheuseofthespectrophotometer. Theplant’sessentialoil(1mL)andthecontrolswereallowed
toreactwith1mLoftheABTS+solutionandtheabsorbancewastakenat734nmafter7minusinga
spectrophotometer. TheABTS+scavengingcapacityoftheessentialoilwasthencomparedwiththat
ofthestandards. Thepercentageinhibitionwasthencalculatedasfollows:
Inhibition%=[(Ablank−Asample)/Ablank]×100 (1)
whereAblankistheabsorbanceofABTSradical+methanolusedascontrolandtheAsampleisthe
absorbanceofABTSradical+sampleextract/standard.
All the tests were carried out in triplicate. The activity was expressed as 50% inhibitory
concentration(IC ). ThelowertheIC value,thehighertheantioxidantactivity.
50 50
2.4.3. NitricOxideScavengingActivityAssay
Thenitricoxideradicalscavengingactivitywasdeterminedbythemodifiedmethoddescribed
byOyedemietal.[11]. Avolumeof2mLof10mMofsodiumnitroprussidepreparedin0.5mM
phosphatebuffersaline(pH7.4)mixedwith0.5mLofplantextractsandstandards, separately, to
makedifferentconcentrationsfrom0.025to0.5mg/mL.Themixturewasincubatedat25◦Cfora
periodof150minafterwhich0.5mLofincubatedsolutionwasmixedwith0.5mLofGriessreagent
(1.0mLofsulfanilicacidreagent(0.33%preparedin20%glacialacetic)acidatroomtemperaturefor
5minwith1mLofnaphthylenediaminedichloride(0.1%w/v). Afterincubatingthemixturewasfor
Appl.Sci.2017,7,443 4of14
30minandtakingabsorbanceat540nm,theamountofnitricoxideradicalsinhibitedbytheessential
oilwascalculatedbytheuseoftheequationbelow:
NOradicalscavengingactivity(%)=[(Abscontrol−Abssample)/(Abscontrol)]×100
whereAbscontrolistheabsorbanceofNOradical+methanolandtheAbssampleistheabsorbance
ofNOradical+extractorstandard.
2.4.4. HydrogenPeroxide(H O )ScavengingActivityAssay
2 2
TheH O inhibitionactivitiesoftheextractswereassessedbythemethodofOyedemietal.[11].
2 2
A solution of hydrogen peroxide (4 mM) was prepared in phosphate buffer (0.1 M; pH 7.4) and
incubated for 10 min after which 1 mL of the essential oil (0.025 to 0.5 mg/mL) was added to a
0.6 mL of hydrogen peroxide solution (4 mM). After 10 min of incubation, the absorbance of the
reactingmixturewasmeasuredspectrophotometricallyat230nm,againstablanksolutioncontaining
phosphatebuffersolutionwithouthydrogenperoxide. BHTandGallicacid(0.025to0.5mg/mL)were
usedaspositivecontrol. Belowistheformulausedtocalculatepercentagescavengingofhydrogen
peroxideofsamples:
H O inhibitioncapacity(%)=[1−(Absorbanceofsample/Absorbanceofblank)]×100 (2)
2 2
2.5. AntimicrobialActivity
2.5.1. MicroorganismsandMedia
The bacteria and fungi employed in this study were selected mainly on the basis of their
significance as opportunistic pathogens of humans with diarrheal diseases. The bacterial strains
includeShigellaflexneriKZN,Proteusvulgaris,KlebsiellapneumoniaATCC4352,Staphylococcusaureus,
Enterococcus faecalis ATCC 29212, Streptococcus pyogenes, Pseudomonas aeruginosa ATCC 19582 and
SerratiamercescensATCC9986. ThefungistrainsoftheAmericanTypeCultureCollection(ATCC)
includeTriphophytonrubrumATCC28188,TrichophytontonsuransATCC28942,Trichophytonmucoides
ATCC201382,MicrosporumcanisATCC36299,MicrosporumgypseumATCC24102,Aspergillusniger
ATCC16888,AspergillusfumigatusATTC204305,PenicilliumchrysogenumATCC10106andPenicillium
aurantiogriesum ATCC 16025. The bacteria test organisms were obtained from the Department of
BiochemistryandMicrobiology,UniversityofFortHare,SouthAfricaandfungitestorganismswere
purchasedfromDaviesDiagnostics(Pty)Ltd.,Gauteng,SouthAfrica,respectively.
TheMueller–Hintondextroseagar(MHA),Mueller-Hintondextrosebroth(MHB),potatodextrose
agar(PDA)andSabourauddextrosebroth(SDB)werepreparedaccordingtotheinstructionfromthe
manufacturer. Thenutrientagarwassuspendedindemineralizedwater,heatedandstirreduntilit
completelydissolvedandthensterilisedbyautoclavingat121◦Cfor15min. Thefungiandbacteria
were maintained at 4 ◦C on MHA and PDA. Scraped cell mass in 0.85% salt solution was diluted
andadjustedto0.5McFarlandstandardsandauthenticatedbyspectrophotometricreadingat580nm
assays[12]topreparetheinoculumsfortheassays.
Finally,thecellsuspensionswerediluted1:100innutrientbrothtopresentanestimatedinoculum
of104CFU·mL−1incomparisonwithMcFarlandstandardforuseintheassays[12].
2.5.2. AntibacterialandAntifungalSusceptibilityTest
TheagarwelldiffusiontechniqueasdescribedbyPrabuseenivasanetal.[2]wasmodifiedand
usedtotestfortheantibacterialandantifungalactivity. Briefly,100µLof0.5McFarlandsolutionsof
bacterialorfungalstrainculturesin0.85%steriledistilledwater(SDW)wasplacedoverthesurfaceof
anagarplateandspreadwithasterileinoculationloop. Ineachagarplate,threewellswerecutwitha
cooled,flamedcorkborerof5mmdiameter,whileasterileneedlewasusedtoremovetheagarplugs.
A50µLoftheamoxicillin(0.0125mg/mL)ornystatin(0.03mg/mL)wereaddedtothefirstwellfor
Appl.Sci.2017,7,443 5of14
antibacterialandantifungalsusceptibilitytestrespectivelytoserveasapositivecontrol. Inthesecond
well,50µLofthecorrespondingextractsolventwereaddedwhileinthethird,50µLoftheacetone
extract/aqueousextract/essentialoil(50mg/mL)wereadded. Theessentialoilhadbeenprepared
bydissolvingin10%DMSOwithTween80(0.5%v/vforeasydiffusion)andsterilisedbyfiltration
througha0.45µmmembranefilter[2]. Preliminarystudiesconfirmedthatthecarriervehicledidnot
inhibitthegrowthofbacteria. Eachtestwasdoneintriplicate. Thecultureplateswereincubatedat
37◦C,andtheresultswereexaminedafter24hand72hforantibacterialandantifungalsusceptibility
test,respectively. Theclearzonearoundeachofthewellswasmeasuredinmm,showingtheplant
fractions’activityagainstthetestedorganisms.
2.5.3. MinimumInhibitoryConcentration(MIC)Assay
Thebrothmicrodilutionmethodusing96wellmicrotiterplateswasusedtoverifytheminimum
inhibitory concentration (MIC) of the plant extracts which indicated antibacterial or antifungal
activity [12]. After adding 120 µL of SDW into every well of the first (A) and the last (H) rows
andintoeverywellofthelastcolumn,120µLofNutrientbroth(NB)wasputinallthewellsofthe
secondrow(B)afterwhicha150µLofNBwasputinthewellsleftinthefirstcolumnand100µLin
theremainingwellsfromthesecondcolumntowardstheright. Therewasthentheadditionoffifty
microlitersoftheessentialoil(20mg/mL)intothethirdwellofthefirstcolumnaswellastheaddition
of50µLofthepositive(amoxicillinornystatin)andnegativecontrol(SDW)independentlyintothe
wellsleftinthefirstcolumn. Aserialdilutionwascarriedoutintwofolds. Thecontentsinallthe
wellsofthefirstcolumn(beginningfromthethirdrow)weremixedand100µLwastransferredinto
thesecondwellofthesamerow. Thisprocesswasreplicateduptothe11thwellafterwhichthe100
µLwasdiscarded. Consequently,thedilutionofthefractionsoftheplantandthecontrolinthewells
broughtaboutarangeofconcentrationfrom5,2.5,1.25,0.625,0.313,0.156,0.078,0.039,0.019,0.0098
and0.005mg/mL.
Subsequently,therewastheinoculationof20µLof0.5McFarlandbacterialorfungalsuspensions
intothewellsapartfromtheoneswhichhadSDW.Thetestorganism’sgrowthwasmeasuredthrough
thedeterminationoftheabsorbanceat620nmwithanautomaticELISAmicroplatereader(Synergy
Mx,BioTek®,Winooski,VT,USA)priorto,andfollowingincubation. Theplateswereincubatedat
37◦Cfor24hand72hforbacteriaandfungi,respectively. TheMIC50wasdefinedandrecordedas
theconcentrationofthetestantibacterialandantifungalagentthatrevealed50%inhibitionofbacteria
andfungigrowth,respectively.
2.6. ChemicalCompositionEvaluation
2.6.1. GasChromatography-MassSpectrometry(GC-MS)
GC-MS analyses were performed on Agilent 5977A MSD and 7890B GC System, Chemetrix
(Pty)Ltd.,Midrand,SouthAfrica;AgilentTechnologies,Deutschland,GermanywithaZebron-5MS
column (ZB-5MS 30 m × 0.25 mm × 0.25 um) (5%-phenylmethylpolysiloxane). The column and
temperatureconditionsemployedincludeGCgradeheliumataflowrateof2mL/minandsplitless
1mLinjectionwasalsoemployed. Theinjector, sourceandoventemperaturesweresetat280 ◦C,
280 ◦C and 70 ◦C, respectively. The ramp settings were; 15 ◦C/min to 120 ◦C, then 10 ◦C/min to
180◦C,then20◦C/minto270◦Candheldfor3min.
2.6.2. IdentificationofComponents
TheidentificationofthechemicalcomponentsoftheessentialoilwasinfluencedbytheirGC
retention times, percentage composition (area %) and retention indices. The interpretation and
identification of their mass spectra were confirmed by mass spectral incorporated library. The
identificationwasfurtherconfirmedbysearchusingtheNationalInstituteofStandardsandTechnology
(NIST) database (NIST/EPA/NIH mass spectral library 2014) with those of published data [13].
Appl.Sci.2017,7,443 6of14
EmpiricalsearcheswereconductedusingthePubChemProject(https://pubchem.ncbi.nlm.nih.gov/)
andDrugBank(www.drugbank.ca/)toidentifytheknownpharmacologicalpropertiesassociated
withthesecompounds.
2.6.3. StatisticalAnalysis
AllexperimentswerecarriedoutintriplicatesandtheresultswereexpressedasMean ± SD.
Whereappropriate,thedataweresubjectedtoone-wayanalysisofvariance(ANOVA)employingthe
Appl. Sci. 2016, 6, 439  6 of 14 
Minitabprogram(version12forwindows). p<0.05wereconsideredsignificant.
2.6.3. Statistical Analysis 
3. ResultsandDiscussion
All experiments were carried out in triplicates and the results were expressed as Mean ± SD. 
Where appropriate, the data were subjected to one‐way analysis of variance (ANOVA) employing 
3.1. AntioxidantActivities
the Minitab program (version 12 for windows). p < 0.05 were considered significant. 
Inthisstudy,theDPPH,ABTS,nitricoxideandhydrogenperoxidescavengingactivitieshavebeen
3. Results and Discussion 
usedasinvitromodelstoevaluatetheantioxidantcapacityoftheessentialoilofHydnoraafricana,in
comparis3o.1n. Awntiitohxitdhanet sAtactnivditaiersd sBHTandGallicacid. Figure2illustratestheDPPHradicalscavenging
activity of thIne tehsiss estnutdiya,l toheil DoPfPHHy, AdnBoTrSa, naiftrriicc aonxaidce oamndp hayrderdogweni tpherBoxHidTe sacnadvenGgainllgi cacaticviidti.es Whaeveo bserved
weakDPbPeeHn iunshedib aisto irny vaitcrtoi vmitoideeslso ftot heveaelusasteen tthiae laonitliooxfidHanytd cnaopraaciatyfr iocfa nthae, cesosmenptiaalr eodil wof itHhydtnhoerar eference
africana, in comparison with the standards BHT and Gallic acid. Figure 2 illustrates the DPPH radical 
compoundsused. Previousstudieshaveindicatedthatessentialoilsfromdifferentsourcespossess
scavenging activity of the essential oil of Hydnora africana compared with BHT and Gallic acid. We 
varyingcapacitiestoscavengeDPPHradicals,probablyduetothedifferencesintheircomposition.
observed weak DPPH inhibitory activities of the essential oil of Hydnora africana, compared with the 
Factorslikestereo-selectivityoftheradicalsorthesolubilityoftheoilinthedifferenttestingsystems
reference compounds used. Previous studies have indicated that essential oils from different sources 
havebeepnorsesepsos rvteadryitnoga cffaepcatcitthiees ctaop saccaivteynogfe eDssPePnHt iaraldoicilaslsi,n pqroubeanbclyh idnuged tiof ftehree ndtifrfaerdeinccaelss i[n1 4th].eiOr urresult
wassimicloamrptoostihtioant.o FfaNctoergs uliekee sttaerle.o[‐1s5e]lewctihvoitya olsf othfeo ruanddicawls eoar kthDe PsoPluHbirliatyd iocf atlhes coailv ienn tghein dgiffaecrteinvti tyusing
testing systems have been reported to affect the capacity of essential oils in quenching different 
theessentialoilofP.brazzeana(RSA=14%for1g/L,SC50=1.5mg/mLandSC50=3.23mg/mL).
radicals [14]. Our result was similar to that of Negueet al. [15] who also found weak DPPH radical 
Incontrast,Ndoyeetal.[16]showedthattheessentialoilofAlliumsativumwasamoreeffectiveDPPH
scavenging activity using the essential oil of P. brazzeana (RSA = 14% for 1 g/L, SC50 = 1.5 mg/mL and 
radicalscavenger(SC50=7.67mg/mL).Itwasobservedthatincreasingtheconcentrationoftheoil
SC50 = 3.23 mg/mL). In contrast, Ndoye et al. [16] showed that the essential oil of Allium sativum was 
or standaa rmdosrew eaffsecntiovet DacPcPoHm rpadaincaile sdcabvyenagnery (SsCig50n i=fi 7c.6a7n mt ign/mcrLe)a. sIte wsaisn otbhseerivreDd PthPaHt inrcaredaiscinagl sthcea venging
activitiesc.oIntcaepntpraetaiorns tohfa tthteh oeill oowr estsatncdoanrdcse nwtraas tnioont sactceosmtepdan(0ie.0d2 b5ym agny/ msigLn)ifwicaansts uinfcfirecaiesenst ,inin theaeicrh  case,to
DPPH radical scavenging activities. It appears that the lowest concentrations tested (0.025 mg/mL) 
producethemaximumantioxidantcapacityofthestandards. Itispossible,therefore,thatmuchhigher
was sufficient, in each case, to produce the maximum antioxidant capacity of the standards. It is 
dosesoftheoilthanthosetestedinthisstudymayproducehigherDPPHradicalscavengingactivities.
possible, therefore, that much higher doses of the oil than those tested in this study may produce 
TheIC50hvigahleur eDsPcPoHu lrdadnicoalt sbceavdenegteinrgm aicntievditipesr. eTchisee IlCy50f ovarlubeost hcoustlda nndota rbde sdeatsertmhienyedw perreecisleelsys feorr thanthe
lowestcobontche sntatrnadtairodns ates sttheedy (w0e.0re2 5lesmsegr /thmanL t)h.eS ilomwielastr lcyo,nIcCentraftoiornt hteesteesds (e0n.0t2i5a lmogi/lmwLa).s Sgimreilaatrelyr, thanthe
50
highestcIoCn50c feonr ttrhaet eiossnentteisatl eodil (w0a.5s gmrega/temr tLha)na nthde hcoiguhledstn coontcbeentprarteiocnis teelsytedde (t0e.5r mmgin/medL.) aTnhde croeuslud lntsoto bserved
be precisely determined. The results observed in this study indicated that the Hydnora africana 
in this study indicated that the Hydnora africana essential oil hasa low effectiveness in donating a
essential oil hasa low effectiveness in donating a hydrogen proton to the lone pair electron for the 
hydrogenprotontothelonepairelectronfortheradical.
radical. 

Figure2F.iDguPrPe H2. DraPdPiHc arladsiccaavl secnagveinngginagc taicvtiivtyityo offt thhee eesssseenntitaila olilo oilf Hof. aHfr.icaafnrai.c Baanra g.rBapahrsg wraitphh dsiffwerietnht different
letters within the same concentration are significantly different (p < 0.05). 
letterswithinthesameconcentrationaresignificantlydifferent(p<0.05).
Appl.Sci.2017,7,443 7of14
TheABTSradicalscavengingactivityofH.africanaessentialoilisillustratedinFigure3.TheABTS
Appl. Sci. 2016, 6, 439  7 of 14 
radicalscavengingactivityoftheessentialoilwasalsosignificantlylower(p>0.05)whencompared
The ABTS radical scavenging activity of H. africana essential oil is illustrated in Figure 3. The 
tothoseofBHTandGallicacidatalltheconcentrationsinvestigated(Figure3). The%inhibitionof
ABTS radical scavenging activity of the essential oil was also significantly lower (p > 0.05) when 
ABTSbytheessentialoilwasfoundtobeconcentration-dependent. TheIC valuefortheABTS
compared to those of BHT and Gallic acid at all the concentrations investigated (Fi5g0ure 3). The % 
radicalscavinehnibgiitniogn aofc tAivBTitSy boyf ththe eesessensetinalt ioaill woails wfouansd0 .t5o 5bem cogn/cmenLtr,atwiohni‐ ldeetpheondseenot.f Tthhee ICst5a0 nvadluaer dfosr couldnot
bedetermitnhee dApBTreS criasdeilcyala sscathveenygiwnge raectlivoiwty eorf ththaen etshseentlioalw oeils twcaos n0c.5e5n tmrag/tmioLn, twehsitlee dth.oTshe iosf rethseu lthereis
standards could not be determined precisely as they were lower than the lowest concentration tested. 
similartotheworkbyMamadalievaaetal.[17]whofoundthatthreeUzbekScutellariaspeciesalso
This result here is similar to the work by Mamadalievaa et al. [17] who found that three Uzbek 
exhibitedweakABTSradicalscavengingactivity.
Scutellaria species also exhibited weak ABTS radical scavenging activity. 

Figure3.AFBigTuSrer 3a. dAiBcTaSl sracdaivcaeln sgcaivnegngaicntgi vacittiyviotyf otfh tehee esssseennttiiaall ooili olfo Hf.H af.riacafrniac.Banara g.rBapahrsg wraithp hdisffwereitnht different
letters within the same concentration are significantly different (p < 0.05). 
letterswithinthesameconcentrationaresignificantlydifferent(p<0.05).
The nitric oxide radical scavenging activities of the essential oil and the reference compounds 
Thenaitrrei pcroesxeindteedr iand Fiicgaulres 4c.a Avte anllg tihneg coanccteinvtirtaiteiosnos ftetshteed,e tshsee enssteiantliaol iolial nprdodtuhceedr eaf seirgennificceanctolym  pounds
higher (p < 0.05) nitric oxide radical scavenging activity, when compared with both BHT and Gallic 
arepresentedinFigure4. Atalltheconcentrationstested,theessentialoilproducedasignificantly
acid. However, the capacities of all the tested compounds to scavenge nitric oxide were largely 
higher(p<co0n.0ce5n)trnaititornic‐doepxeidndeernat.d Nicitarilc socxaidvee, ning iitnseglfa, ccatniv ailttye,r wcehll esntruccotumrep aanrde dfunwctiitohn bboy tmhoBdHifyTinga ndGallic
acid. Howdeifvfeerre,ntt hsiegncaallpinagc iptaiethswoayfsa. lIlts thtoexictietys tuesduaclloym repsuolutsn wdhsento its ccoamvbeinnegse wnitihtr tihce osxuipdeeroxwideer e largely
concentrataionino-nd teo pfoernmd peenrot.xyNniittrritiec, woxhiicdhe i,s ian vietrsye sltfr,ocnag noxaidltaenrt [c1e8l]l. Tshtreu acbtiluitrye oaf nthde efsusenncttiaiol onil boyf Hm. odifying
africana to effectively scavenge nitric oxide radicals in this study suggests it could be useful against 
differentsignallingpathways. Itstoxicityusuallyresultswhenitcombineswiththesuperoxideanion
oxidative conditions involving this radical. 
toformperoxynitrite,whichisaverystrongoxidant[18]. TheabilityoftheessentialoilofH.africana
toeffectivelyscavengenitricoxideradicalsinthisstudysuggestsitcouldbeusefulagainstoxidative
conditionsAipnpvl. Socli.v 20in16g, 6,t 4h39is  radical. 8 of 14 

Figure4. NFiigtruirce o4.x Niditericr aodxiidcea rlasdcicaavl escnagvienngginagc taicvtiivtiyty ooff tthhee eesssesnetniatli oaill oofi lHo. fafHric.anaafr. iBcaarn gar.apBhasr wgirtha phswith
differentledttifefresrewnt iltehttienrst whiethsianm thee scaomnec ceonntcreanttiroatnioan raeres sigignniiffiiccaanntltyl ydidffeifrfeenrt e(pn <t (0p.05<). 0.05).
The scavenging activities of the essential oil and the standards against hydrogen peroxide are 
depicted in Figure 5. For both the oil and the standards, the amount of hydrogen peroxide remaining 
in the reaction mixtures, decreased as their concentrations were increased. There was no significant 
difference observed between the oil and BHT at nearly all the concentrations (p < 0.05) except at 0.05 
mg/mL where BHT showed a significantly higher activity than the oil (p > 0.05). At all the 
concentrations tested (0.025 to 0.5 mg/mL), the essential oil showed lesser hydrogen peroxide 
antioxidant activity as compared to the standards (BHT and gallic acid), only able to reduce 
hydrogen peroxide by less than 20%, while gallic acid had the highest activity, reducing the H2O2 by 
over 80%. 

Figure 5. Hydrogen peroxide scavenging activity of the essential oil of H. africana. Lines with 
different letters within the same concentration are significantly different (p < 0.05). 
3.2. Antimicrobial Analysis 
The essential oil was tested against thirteen opportunistic bacteria. The results showed that the 
plant was active against the growth of all organisms except Salmonella typhimurium, Pseudomonas
Appl. Sci. 2016, 6, 439  8 of 14 

Figure 4. Nitric oxide radical scavenging activity of the essential oil of H. africana. Bar graphs with 
different letters within the same concentration are significantly different (p < 0.05). 
Appl.Sci.2017,7,443 8of14
The scavenging activities of the essential oil and the standards against hydrogen peroxide are 
depicTthede isnc aFvigeunrgei n5g. Faocrt ibvoitthie sthoe fotilh aeneds sthene tsitaalnodialradnsd, ththe eamstoaunndta ordf shyadgraoingestn hpyedrorxoigdeen repmeraoinxiidnge 
ainre thdee preiactcetidonin mFiixgtuurrees5, .deFcorerabsoedth atsh teheoiirl caonndcetnhterasttiaonnds awrderse, tihnecreaamseodu.n Tthoefreh ywdarso gneon sipgenriofixciadnet 
rdeimffearienninceg oinbstehrevreeda cbteiotwnemenix tthuer eosi,l daencdr eBaHseTd aats ntehaerilryc aolnl cthene tcroanticoennstrwateiroenisn (cpr e<a 0s.e0d5.) Tehxceerpetw aat s0.n0o5 
smiggn/mifiLca nwthdeirfefe rBeHncTe oshbosewrveded  ab estiwgneiefincatnhtelyo ilhaignhderB HacTtiavtitnye athrlayna ltlhteh eoiclo n(pc e>n tr0a.0ti5o)n. sA(tp  a<ll0 .t0h5e) 
ecxocnecpetnatrta0t.i0o5nsm gte/smteLd w(0h.0e2re5 BtHo T0.s5h omwge/dmaL)s,i gtnhiefi ceasnstelnythiailg hoeilr aschtoivwiteydt hleasnsethr ehoyild(rpog>e0n. 0p5)e.rAoxtiadlel 
tahneticooxnidcaenntt raatcitoivnistyte satse dco(0m.0p2a5retdo 0t.o5 mthge/ smtaLn)d,athrdese s(sBeHntTia lanodil sghaolwlice daclieds)s,e ronhlyyd raobglee ntop erreodxuidcee 
ahnytdiorxoigdeann ptearcotixviidtye absyc loemssp tahraend 2t0o%th, ewshtailned gaarldlisc (aBcHidT haandd tghael lhicigahceidst) ,aocntilvyitayb, lreetdourceidnug cteheh yHd2rOo2g beny 
poevreorx 8id0%eb. ylessthan20%,whilegallicacidhadthehighestactivity,reducingtheH O byover80%.
2 2

Figure5.HydrogenperoxidescavengingactivityoftheessentialoilofH.africana.Lineswithdifferent
Figure 5. Hydrogen peroxide scavenging activity of the essential oil of H. africana. Lines with 
letterswithinthesameconcentrationaresignificantlydifferent(p<0.05).
different letters within the same concentration are significantly different (p < 0.05). 
3.2. AntimicrobialAnalysis
3.2. Antimicrobial Analysis 
The essential oil was tested against thirteen opportunistic bacteria. The results showed that
The essential oil was tested against thirteen opportunistic bacteria. The results showed that the 
theplantwasactiveagainstthegrowthofallorganismsexceptSalmonellatyphimurium,Pseudomonas
plant was active against the growth of all organisms except Salmonella typhimurium, Pseudomonas 
aeruginosa and Proteus vulgaris. The essential oil of H. africana showed activity against growth of
somepathogenicbacteriathatcauseinfectionsassociatedwithdiarrhoealdiseases(Tables1and2).
Theantibacterialactivitycouldalsobeattributedtothepresenceofsomechemicalconstituentsin
the essential oil fraction of H. africana. For example, Terpinen-4-ol, both in aqueous solutions and
vapour form, acts as an antimicrobial agent against bacteria of Legionella genus [19]. β-cymene
possesses antibacterial and antifungal activities [20]. Napthalene has antibacterial and antifungal
properties against various human pathogens [21]. Diphenyl ether possesses bactericidal activity
againstgram-positiveandgramnegativebacteria[22]. (−)-Spathulenol,anoxygenatedsesquiterpene,
hasantifungal,antibacterial,antiviralandcytotoxicactivities[23].Thezonesofinhibitionvariedfrom
10to25mm(Table1). TheHAessentialoilshowedasignificantgrowthinhibitoryeffectagainsttheten
organisms(p<0.05)havingIC valuesrangingfrom5to0.005mg/mL(Table2). ThelowestMICwas
50
observedwithEnterococcusfaecalis(p<0.05)(Table2). Oftheselectedandtestedninefungalisolates,
theplantspeciesinhibitedonlythegrowthofAspergillusnigerwhoseMICactivitywassignificantly
similartothecontroldrug(p<0.05)(Tables3and4).
Appl.Sci.2017,7,443 9of14
Table 1. Inhibition zone diameters caused by the essential oil (50 mg/mL) in the tested
opportunisticbacteria.
Organism EssentialOil PositiveControl(Amoxicillin)
Salmonellatyphimurium− NA 36±1.1a
Enterococcusfaecalis− 17±0.2a 34±1.2b
Escherichiacoli− 15±1.3a 35±0.2b
Pseudomonasaeruginosa− NA NA
Bacilluscereus+ 10±1.6a 32±1.2b
Shigellasonnei− 15±0.3a 35±2.2b
Streptococcuspyogens+ 22±0.2a 35±2.1b
Bacillussubtilis+ 22±0.2a 40±0.2b
Shigellaflexneri− 19±0.2a 40±1.1b
Klebpneumoniae− 23±2.1a 33±2.2b
Staphylococcusaureus+ 25±3.1a 37±0.2b
Proteusvulgaris− NA 38±1.1a
Serretiamercenscens− 25±3.1a 35±2.2b
Thebacteriaaredenotedthusly: St(Salmonellatyphimurium),Ef(Enterococcusfaecalis),Ec(Escherichiacoli),
Pa(Pseudomonasaeruginosa), Bc(Bacilluscereus), Ss(Shigellasonnei), Sp(Streptococcuspyogens), Bs(Bacillus
subtilis),Sf(Shigellaflexneri),Kp(Klebpneumoniae),Sa(Staphylococcusaureus),Pv(Proteusvulgaris)andSm
(Serratiamarcescens). Dataexpressedasmeans±SD;n=3;valuesalongarowwithdifferentsubscriptsare
significantlydifferent(p≤0.05).
Table2.Minimuminhibitoryconcentrations(MIC)oftheH.africanaessentialoilsagainstthetested
opportunisticbacteria.
InhibitionZoneDiameter(mm)
Solvent
St Ef Ec Pa Bc Ss Sp Bs Sf Kp Sa Pv Sm
Essential
NA 2.5a >5a NA >5a >5a 0.02a 0.02a 0.02a 2.5a 0.02a NA 0.02a
oil
Positive
0.01 0.01b 0.01b NA 0.01b 0.01b <0.01b 0.01b 0.01b <0.01b 0.01b 0.01b 0.01b
control
Thebacteriaaredenotedthusly:St(Salmonellatyphimurium),Ef(Enterococcusfaecalis),Ec(Escherichiacoli),Pa
(Pseudomonasaeruginosa),Bc(Bacilluscereus),Ss(Shigellasonnei),Sp(Streptococcuspyogens),Bs(Bacillussubtilis),
Sf(Shigellaflexneri),Kp(Klebpneumoniae),Sa(Staphylococcusaureus),Pv(Proteusvulgaris)andSm(Serratia
marcescens).Dataexpressedasmeans±SD;n=3;valueswiththedifferentsuperscriptinthesamecolumnare
significantlydifferent(p≤0.05).NAindicatesnotactive.
Table 3. Inhibition zone diameters caused by the essential oil (50 mg/mL) in the tested
opportunisticfungi.
Organism EssentialOil PositiveControl(Nystatin)
Aspergillusfumigates NA 30±1.1
Aspergillusniger 20±1.2a 20±1.2a
Microsporiumgypsum NA 25±0.2
Triphophytonrubrum NA 25±0.2
Triphophytontonsurans NA 30±1.2
Triphophytonmucoides NA 25±2.2
Penicilliumaurantrognereum NA 30±2.1
Penicilliumchrysogenum NA 25±0.2
Microsporumcanis NA 35±1.1
Thefungiaredenotedthusly: Af(Aspergillusfumigatus),An(Aspergillusniger),Mg(Microsporiumgypsum),
Tr(Triphophytonrubrum),Tt(Triphophytontonsurans),Tm(Triphophytonmucoides),Pa(Penicilliumaurantrognereum),
Pc(Penicilliumchrysogenum)andMc(Microsporumcanis).Dataexpressedasmeans±SD;n=3;valuesalonga
rowwithdifferentsubscriptsaresignificantlydifferent(p≤0.05).NAindicatesnotactive.
Appl.Sci.2017,7,443 10of14
Table4.Minimuminhibitoryconcentrations(MIC)oftheH.africanaessentialoilsagainstthetested
opportunisticfungi.
InhibitionZoneDiameter(mm)
Solvent
Af An Mg Tr Tt Tm Pa Pc Mc
Essentialoil NA 2.5a NA NA NA NA NA NA NA
Positivecontrol 0.01 2.5a 0.02 2.5 <0.01 0.02 0.01 0.02 0.01
Thefungiaredenotedthusby:Af(Aspergillusfumigatus),An(Aspergillusniger),Mg(Microsporiumgypsum),
Tr(Triphophytonrubrum),Tt(Triphophytontonsurans),Tm(Triphophytonmucoides),Pa(Penicilliumaurantrognereum),
Pc(Penicilliumchrysogenum)andMc(Microsporumcanis).Dataexpressedasmeans±SD;n=3;valuesalonga
rowwithdifferentsubscriptsaresignificantlydifferent(p≤0.05).NAindicatesnotactive.
Althoughcompoundsintheoilareknowntohaveantimicrobialactivityindependently,they
couldbeactingindependentlyorincombinationtopotentiatetheplants’potentials[24]. Therefore,
itiscrucialtoisolateandelucidatethebioactivecompoundsanddeterminetheirpharmacological
properties. Thisshouldfacilitatetheidentificationofnovelantimicrobialagents.
GC-MS analysis of the essential oil fraction revealed fraction revealed the presence of
67compoundsindifferentchemicalclassesincludingterpenoids,aldehydes,ketones,fattyacidesters
andcarboxylicacids(Table5). Thedetailsofcertaincompoundscould,however,notbeconfirmedin
theNISTdatabase. Amongidentifiedcompounds,carboxylicacids,dominatedbyn-Hexadecanoic
acid and cis-9-hexadecanoic acid, constituted the largest group of compounds (30.68% of total oil
composition). Thehighercontentofcarboxylicacidsintheessentialoil,comparedtoothergroups
ofcompounds, isanunusualfinding, asmostotheressentialoilsreportedlycontainterpenesand
otherhydrocarbons. Theresultsofthisstudy,however,bearasimilaritytothoseobservedforthe
compositionofessentialoilsfromTetrapleuratetrapteraUdouriohandEtkudoh,[25],whoalsoreported
ahighcontentofcarboxylicacidsintheoil.
Terpenoids identified in the oil made up 10.70% of the total oil composition. They included
Beta-Myrcene, o-limonene, Terpien-4-ol, pulegone, Beta-bisabolene, (−)-spathulenol, cis-lanceol,
alloaromadendrene,Longifolene,Phenanthrene,7-ethenyl-1,2,3,4,4a,4b,5,6,7,8,8a,9-dodecahydro-1,
1,4b,7-tetramethyl-, [4aS-(4a.alpha.,4b.beta., 7.alpha.,8a.alpha.)]- and Bicyclo [2.2.1] heptan-2-one,
1,7,7-trimethyl-,(1S)-Bicyclo. Aldehydes(4.86%);Ketones(2.85%)andfattyacidesters(1.82%)were
also among compounds whose identity was confirmed from the library. The low percentage of
terpenoidsandrelativelyhighcontentofcarboxylicacidsintheoilsextractedfromHydnoraafricana
maysuggestthattheoilmaynotbeconsideredasatrueessentialoil[25]. Thismaybethereasonfor
theweakantioxidanteffectsshownbytheoilagainstDPPH,ABTSandnitricoxide. However,the
potentialapplicationsoftheoilinthecosmeticindustries,duetothepresenceofsometerpenoidsmay
beveryuseful.