Evaluation of the cytotoxic effect and antibacterial, antifungal, and antiviral activities of Hypericum

13 Pages·2016·0.4 MB·English

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RESEARCH ARTICLE Open Access
Evaluation of the cytotoxic effect and
antibacterial, antifungal, and antiviral activities of
Hypericum triquetrifolium Turra essential oils from
Tunisia
Zyed Rouis1*, Nabil Abid1, Sadok Koudja2, Thabet Yangui3, Ameur Elaissi4, Pier Luigi Cioni5, Guido Flamini5
and Mahjoub Aouni1
Abstract
Background: Anumberof bio-active secondary metabolites have been identified and reported for several
Hypericum species. Many studies have reported thepotentialuse of theplant extracts against several pathogens.
However, Hypericum triquetrifolium is one of the least studied species for itsantimicrobial activity. The aim ofthe
present study was to evaluate thecytotoxic effect of theessential oils ofHypericum triquetrifolium as well as their
antimicrobial potential against coxsakievirus B3 and a range of bacterial and fungal strains.
Methods: The essential oils of Hypericum triquetrifolium harvestedfrom five different Tunisian localities
(Fondouk DJedid, Bou Arada, Bahra, Fernana and Dhrea Ben Jouder) were evaluated for their antimicrobial activities
bymicro-broth dilution methodsagainst bacterialand fungal strains. In addition, the cytotoxic effect and the
antiviral activity ofthese oils were carried out using Vero cell lines and coxsakievirus B3.
Results: The results showed a good antibacterial activities against a wide range of bacterial strains, MIC values
ranging between 0.39-12.50 mg/ml and MBC values between 1.56-25.0 mg/ml. Inaddition, theessential oils
showed promising antifungal activitywith MIC values ranging between 0.39 μg/mL and 12.50μg/mL; MFC values
ranged between 3.12 μg/mL and 25.00μg/mL; a significant anticandidal activitywas noted (MIC values comprised
between 0.39 μg/mL and 12.50 μg/mL). Although their low cytotoxic effect (CC ranged between 0.58 mg/mL and
50
12.00mg/mL), the essential oils did not show antiviral activity against coxsakievirus B3.
Conclusion: The essential oils obtained fromHypericum triquetrifolium can be used as antimicrobial agents and
could be safe atnon cytotoxic doses. As shown for the testedessential oils, comparative analysis need to be
undertaken to better characterize also the antimicrobial activities ofHypericum triquetrifolium extracts withdifferent
solvents as well as theirpurifiedfractions and their pure secondary metabolites.
Keywords: Hypericum triquetrifolium, Coxsakievirus B3, Essentialoils, Bacteria, Fungi
*Correspondence:[email protected]
1LaboratoiredesMaladiesTransmissiblesetSubstancesBiologiquement
ActivesLR99ES27,FacultyofPharmacy,UniversityofMonastir,Monastir,
Tunisia
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Rouisetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Background inhabits aquatic environments and the gastrointestinal
Essentialoilsarearomaticextractswhichhavebeenused tract of healthy fish. It also commonly occurs in foods,
since ancient times as flavouring agents and constituents milk, red meats and poultry [15-17]. It causes disease and
ofseveralcommercialproducts.Thechemicalcomposition mortality mainly in freshwater fish but sometimes in mar-
of essential oils is often variable among different plants inefish[17].Thebacteriumalsoinfectshumansandcauses
and even between different plant parts. In addition, the lesionsrangingfromgastroenteritistosepticaemia[18].
composition may also differ according to the site of The genus Hypericum is a member of the Hypericaceae
collection (geographical provenance), as their components family[19,20].Anumberofbio-activesecondarymetabolites
play a major role in the plant adaptation to the ecology have been identified and reported for several Hypericum
and the environment, including biotic and abiotic factors species [21-23]. Essential oils extracted from Hypericum
[1,2]. Currently, the use of essential oils is more common species are well documented for their antimicrobial acti-
today than ever before due to their increasing demand for vities[4,24-33].
food,cosmeticsandpharmaceuticalindustries.Inaddition, Hypericum triquetrifolium Turra (H. triquetrifolium),
the interest in essential oils has increased as potential nativetoEasternEuropeandtheMediterraneanarea,has
alternatives for therapeutic purposes against common been traditionally used for its sedative, antihelminthic,
microbes. Bacterial resistance is spreading throughout the anti-inflammatory, and antiseptic effects [24,34]. In
world primarily due tothe excessive useof antibiotics and addition, several studies have reported the potential
poorinfectioncontrolpracticesinhospitals,makingitone useofitsessentialoilandcrudeextractsastherapeutic
ofourtimesbiggestissues[3].Scientificliteraturerevealed substances,mainlyinthetreatmentof burns,gastroenter-
the antimicrobial, antifungal and antioxidant potentials of itis, antinociceptive and antioxidant drugs [35-37]. How-
several essential oils [4,5]. In addition, the antiviral ever, H. triquetrifolium is one of the least studied species
potentialofessentialoilshasbeenwelldocumented[6,7]. for its antimicrobial activity. According to literature data,
MicroorganismssuchasStaphylococcusaureus(S.aureus), onlyapreviousstudyusingthegrowthinhibitionassayfor
Staphylococcus epidermidis (S. epidermidis), Entero- anumberof bacterialandcandidalstrains[38]isreported
coccus faecalis (E. faecalis), Pseudomonas aeruginosa forH.triquetrifolium.
(P.aeruginosa),andEscherichiacoli(E.coli)arefrequently In the present study, the antimicrobial, cytotoxic effect
isolated from skin wounds in humans and animals. In and the antiviral activities of the essential oils extracted
addition, S. epidermidis infections are commonly acquired from H. triquetrifolium from five different Tunisian
in hospitals as a result of contamination of surgical cuts localities were evaluated in vitro. The variation in their
withmicroorganismsfromthepatientsthemselvesorfrom activities was discussed according to their chemical
the hospital personnel [8]. Infection with P. aeruginosa is compositionspreviouslyreported[39].
one of the most serious complication in burn patients
[9,10], followed by infections with E. coli, S. aureus and Methods
other microorganisms [9]. Infection with Bacillus cereus Plantmaterialandessentialoilextraction
has been well documented in the literature for over a Voucher specimens identified by Prof. Mohammed El
century and it is generally associated with gastroenteritis Hedi El Ouni (Department of Biology, Faculty of
caused by the consumption of infected food. Vibrio Sciences of Bizerte, Tunisia) have been deposited in the
alginolyticus is ubiquitous in seawater and tends to cause Herbarium of the Laboratory of Transmissible Diseases
superficialwoundandearinfections(otitismediaandotitis and Biological Active Substances (Faculty of Pharmacy of
externa) [11]; this infection can progress to bacteraemia Monastir, Tunisia), under the following accession codes:
and necrotising fasciitis, particularly in the immunocom- H. tri. 1, H. tri. 2, H. tri. 3, H. tri. 4, and H. tri. 5 for
promised patients [12]. Vibrio cholerae (V. cholerae), a Hypericum triquetrifolium from Bou Arada, Bahra, Dhrea
Gram(−)bacteriumandthecausativeagentofcholera,has BenJouder,FernanaandFondoukDjedid,respectively.
caused several pandemics since 1816, as well as sporadic Aerial parts (the top 25 cm) of the plant have been
inter-epidemicoutbreaks.V.choleraeisautochthonousina collectedduring fullblooming fromfivedifferentTunisian
region of the world where cholera never occurs and that localities between June and July 2008. In brief, plant
the human body is not an obligate environment for the samples were air-dried in darkness at room temperature
presence and dispersal of this organism [13]. Salmonella for one week. Then, samples (500 g) were cut in small
typhimurium causes typhoid fever associated with piecesandsubjectedtohydro-distillationfor3h,usingthe
gastroenteritis; the infection is caused by consuming standard apparatus recommended by the European
contaminated food or drinks. Aeromonas hydrophila Pharmacopoeia. The obtained oils were stored at +4°C in
(A. hydrophila) has been receiving increasing attention glassvialsuntilanalysis.Theresultantoilswerestudiedfor
both as an opportunistic and as a primary pathogen of their chemical variability using Gas Chromatography –
humans,aquaticandterrestrialanimals[14].A.hydrophila Electron Ionization Mass Spectrometry (GC-EIMS)
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and GC coupled with Chemical Ionization Mass againuntilfullcytopathiceffectwasobservedinfivetosix
Spectrometry (GC/CIMS). The results are reported in days.Theharvestedviruswasstoredat−70°Cuntilused.
a previous work [39].
Antimicrobialactivities
Cellsandtestedmicroorganisms Minimuminhibitoryandminimumbactericidal
Cellline concentrations(MICandMBC)
TheVero cells were derived from the kidney of a normal, The minimum inhibitory concentration (MIC) values for
adult, African green monkey (Cercopithecus) in 1962, by each essential oil against the tested bacterial strains and
Yasumura and Kawakita at the Chiba University in Japan. environmental isolatesweredetermined accordingto the
Thiscelllinehasbeenextensivelyusedforvirusreplication standardprotocols[36].Thebacterialstrainswerecultured
studies and plaque assays. Vero cells (kindly provided in tryptic soy broth (TSB) or agar (Sigma,Tunis,Tunisia)
by Pr. Bruno Pozzetto, Laboratory of Bacteriology- attheappropriattemperatureforthestrain(30°Cor37°C).
Virology, Saint-Etienne, France) were used for culturing Inocula were prepared by adjusting the turbidity of each
enterovirusstrains.Vero cellsweremaintainedinRPMI bacterial culture to reach an optical density of 0.5
1640 supplemented with 10% fetal bovine serum (FBS), McFarland standards, corresponding to approximately 1 –
L-Glutamin (2 mM), penicillin (100 U/mL), and strepto- 5 × 108 CFU/mL. The concentration of spore suspensions
mycin (100μg/mL). Cells were incubated at 37°C in a 5% was determined using a haematocytometer (Thoma cell)
CO humidifiedatmosphere. andadjustedto1–5×107spores/mL.Thebrothdilution
2
method was carried out in 96-well microtitre plates using
Bacterialandfungalstrains microbial reference strains and field isolates. The essential
Gram (+) and Gram (−) bacterial strains were used in oils were prepared aseptically and transferred to sterile
the present study (Table 1). In addition, fungal and yeast 96-well microtitreplatesbytwo-fold serialdilutionsusing
strains and isolates were included for the analysis of the 5% dimethylsulfoxide (DMSO) and then diluted in TSB.
fungicidalactivityoftheessential oils(Table 2). The resultant doses of the tested essential oils ranged
between two and 250μg/mL. Eighty microliters of the
Virusstrain preparedoilsuspensionwereaddedtoeachwell,followed
Coxsakievirus B3 Nancy strain (kindly provided by Pr. by10μLofeachoildoseand10μLofresazurinindicator
Bruno Pozzetto, Laboratory of Bacteriology-Virology, solution(7-Hydroxy-3H-phenoxazin-3-one10-oxide).The
Saint-Etienne, France) was propagated in Vero cells. In latter reagent allows the detection of microbial growth in
brief, 100 μL of the virus suspension were used to infect extremely small volumes of solution in microtitre plates
a confluent monolayer of Vero cells in 75 cm2 culture without using a spectrophotometer. Two control wells
flask and adsorbed for 1 h to allow viruses to adhere wereusedforeachplate:onewellcontainingmicroorgan-
onto the cells. Non-adherent particles were washed off ism and resazurin and a second well containing only
using 2% RPMI 1640 medium and the infected cells medium and resazurin (in order to check the sterile
overlaid with 20 mL of 2% RPMI 1640 and incubated conditions of the experiment). The plates were incubated
anaerobically at 37°C for 24 h. After incubation, bacterial
Table1Bacterialreferencestrainsandtheirpathological growth was evaluated by color change from blue to pink.
effects The lowest dose indicating inhibition of growth was
Bacterialstrains Catalogue Effects recordedastheMIC.
number TodeterminetheMBC,10μLofeachculturemedium
Bacilluscereusa ATCC11778 Foodborn with novisible growthwereremovedfrom eachwell and
Escherichiacolib ATCC35218 Foodborn inoculated in TSB plates. The CFU values of surviving
Vibrioalginolyticusb ATCC17749 Intestinaldiseases,wound organisms were determined after aerobic incubation at
andearinfections theappropriated temperatureduring16–20hours [40].
Vibriocholeraeb ATCC39315 Cholera
Pseudomonasaeruginosab ATCC27853 Gastrointestinaldiseases Minimiuminhibitoryandminimiumfungicidal
Salmonellatyphimuriumb CIP104115 Typhoidfever concentrations(MICandMFC)
Aeromonashydrophilab ATCC7966 GastroenteritisandCellulitis The fungicidal activity was evaluated as discussed above.
Enterococcusfaecalisa ATCC29212 Endocardites The only differences consisted of the culture of fungi
and the yeast strains on malt extract broth (MEB) or
Staphylococcusaureusa ATCC25923 Foodborn,scalded
skinsyndrome agar (Fluka, Madrid, Spain) and incubation at 28°C. The
Staphylococcusepidermidisa CIP106510 Nosocomial essential oils (diluted in 5% DMSO) at different doses
were mixed with MEB and the plates were incubated
aGram(+)bacteria.
bGram(−)bacteria. anaerobicallyat 25°Cfor48hours.
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Table2Fungalandcandidalstrainsandtheireffects
Fungalandyeaststrains Cataloguenumber/isolates Effects
Aspergillusniger CTM10099 Blackmoldoncertainfruitsandvegetables,contaminantoffood,
aspergillosis,otomycosis,damagetotheearcanalandtympanicmembrane.
Fusariumsolani IsolatedfromTomatoplants Dampingoffoncertainfruitsandvegetables,keratitis,endophthalmitis,
cutaneousinfections,burnpatients,mycetoma,onychomycosis,sinusitis,
pulmonarydisease,endocarditis,catheterinfections,andsepticarthritis
Botrytiscinerea Isolatedfromstrawberryfruit Winegrower’slung,Hypersensitivitypneumonitis,Greymouldaffects
manyplantspecies
Candidaalbicans ATCC90028 Candidiasis,opportunisticoralandgenitalinfections
Candidaglabrata ATCC90030 Pathogenfortheurogenitaltract,andforthebloodstream(fungemia)
Candidakrusei ATCC6258 Fungemia,nosocomialpathogen
Cytotoxicityassay each essential oil for one hour at 37°C. Then 100 μL of
Theevaluationofthecytotoxiceffectoftheessentialoilsis the mixture were added to the cells cultured fluently in
based on the reduction of MTT (3-[4,5-dimethylthiazol-2- 96-well flat-bottom microtiter plate; (Experiment 2) Cells
yl]-2,5-diphenyltetrazoliumbromide),bythemitochondrial were treated with three doses (CC , 1/2 CC , 1/4 CC )
50 50 50
dehydrogenaseofviablecells,togiveablueformazanprod- of each essential oil (100 μL) for one hour at 37°C. Then,
uctthatcanbemeasuredspectrophotometrically[41].Cells 5×104TCID ofthevirus(100μL)wereadded.
50
wereseededin96-wellplatesataconcentrationof5×104 Altogether, the experiment aims to test the mode of
cells per well and incubated at 37°C for 24 h in a 5% CO action of the essential oils and to evaluate any effect of
2
humidifiedatmosphere.Aftertreatmentwithvariousdoses the essential oils on the virus (Experiment 1) or on the
of the essential oils (0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.50, cells before theinfection (Experiment2).
and 25.00 mg/mL), the cells were incubated at 37°C for an All the plates were incubated in a CO -incubator for
2
additional48hours.Thecellswereexamineddailyundera 48 hrs. The viability of the infected and non-infected
phase-contrast microscope to determine the minimum cells was evaluated using MTT reduction assay, as
dose of the tested essential oil that induced alterations in describedabove.Thepercentofprotectionwascalculated
cell morphology. At this stage, the medium was removed asfollows:
andcellsineachwellwereincubatedfor3–4hoursat37°C
with 100 μL of MTT solution (5 mg/mL). MTT solution Percentprotection¼½ðODTÞV (cid:2)ðODCÞV(cid:3)
was then discarded and 50 μL DMSO were added to =½ðODCÞM(cid:2)ðODCÞV(cid:3)(cid:4)100
dissolve insoluble formazan crystals. Optical density (OD)
was measured at 540 nm using a standard microplate Where (ODT) V, (ODC) Vand (ODC) M indicate the
W ™
reader (BIO-TEK EL×800 Universal Microplate Reader, absorbance of the sample, the virus-infected control
NY, USA). Cell viability was expressed with respect to the (no compound) and mock-infected control (no virus
absorbance of the control wells (untreated cells), which andnocompound),respectively[42].
were considered 100% of absorbance. The percentage of
cytotoxicity was calculated as [(A-B)/A] × 100; where A Statisticalanalysis
andBaretheOD ofuntreatedandtreatedcells,respect- Dataofantibacterialandantifungalactivitiesweresubjected
540
ively. The 50% cytotoxic concentration (CC ) was to statistiical analysis using Principal Components (PCA)
50
defined as the dose of the essential oil required for the and Hierarchical Clusters Analysis (HCA). Statistical tests
reduction of cell viability by 50%, which were were performed using STATISTICA-Pc Software 9.0
calculatedbyregressionanalysis. (StatSoftInc,www.statsoft.com).
Virusinhibitionassay Results
In this assay, essential oils were tested for their possible Antibacterialactivity
use either to cure infected cells or to protect them from AsshowninTable3,theessentialoilsexhibiteddifferent
infection. The experiment is simple, and relies on a cell antimicrobial activities with respect to the geographical
culture system able to support virus growth. Confluent regionoforigin.
Vero cells were treated with the essential oils at three The essential oil of H. triquetrifolium from Fondouk
different doses (CC , ½ CC , ¼ CC ) during and after DJedid (F.DJ.) showed a more potent antibacterial activity
50 50 50
virus infection in two sets of experiments as follows: againstthetestedstrains(MICrange:0.39–1.56mg/mL;
(Experiment 1) 5 × 104 TCID of the virus were MBC range: 1.56 – 6.25 mg/mL), with the exception of
50
exposed to three doses (CC , 1/2 CC , 1/4 CC ) of Vibriocholerae(MIC=MBC=25mg/mL).
50 50 50
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Table3AntibacterialactivityoftheessentialoilsofH.triquetrifolium(MIC/MBC;mg/mL)
Bacterialstrains H.tri.B.A.* H.tri.Bah.* H.tri.D.B.J.* H.tri.Fer.* H.tri.F.Dj.*
BacilluscereusATCC11778a 25.00/25.00 12.50/12.50 25.00/25.00 6.25/12.50 1.56/3.12
EnterococcusfeacalisATCC29212a 12.50/12.50 0.39/0.39 12.50/12.50 6.25/12.50 0.39/3.12
StaphylococcusaureusATCC25923a 3.12/3.12 25.00/25.00 12.50/25.00 6.25/25.00 0.78/3.12
StaphylococcusepidermidisCIP106510a 12.50/25.00 25.00/25.00 12.50/25.00 25.00/25.00 1.56/3.12
VibrioalginoliticusATCC17749b 12.50/25.00 12.50/12.50 12.50/12.50 6.25/12.50 1.56/3.12
EscherichiacoliATCC35218b 12.50/25.00 25.00/25.00 25.00/25.00 6.25/12.50 1.56/6.25
VibriocholeraeATCC39315b 0.39/25.00 12.50/25.00 0.78/3.12 3.12/3.12 25.00/25.00
PseudomonasaeruginosaATCC27853b 6.25/6.25 12.50/12.50 25.00/25.00 6.25/6.25 0.39/1.56
SalmonellatyphimiriumCIP104b 0.39/12.50 6.25/25.00 25.00/25.00 6.25/25.00 0.78/6.25
AeromonashydrophilaATCC7966b 0.39/6.25 0.39/3.12 12.50/25.00 6.25/12.50 3.12/6.25
aGram(+)bacteria,bGram(−)bacteria,MIC:MinimiumInhibitoryConcentration;MBC:MinimiumFungicidalConcentration.H.tri.B.A.:Hypericumtriquetrifolium
collectedfromBouArada;H.tri.Bah.:HypericumtriquetrifoliumcollectedfromBahra;H.tri.D.B.J.:HypericumtriquetrifoliumcollectedfromDhreaBenJouder;H.tri.
Fer.:HypericumtriquetrifoliumcollectedfromFernana;H.tri.F.Dj.:HypericumtriquetrifoliumcollectedfromFondoukDjedid.
*SDvalues=0.00.
The essential oil of H. triquetrifolium collected in axisIIseparatedgroupVfromalltheothergroups.Group
Bou Arada (B.A.) was comparatively more bacteriostatic I was represented by essential oils of H. triquetrifolium
against Gram (−) bacterial strains (MIC range: 0.39 – from Bou Arada, Bahra, and Fernana. These essential
12.50mg/mL;MBCrange:6.25–25.00mg/mL).However, oils were characterized by their weak activities against
thehighestbactericidaleffectwasdetectedagainstS.aureus Bacillus cereus, Pseudomonas aeruginosa, Escherichia
(MIC=MBC=3.12mg/mL). coli, Staphylococcus epidermidis and Vibrio alginoliticus
The essential oils obtained from western regions, and their potent effects against Aeromonas hydrophila.
Bahra (Bah.) and Fernana (Fer.), were lesser active. Subgroup I , limited to the sole sample from Bahra was
b
EssentialoilsfromBahrashowedagoodactivityagainstE. distinguished from the other oils of subgroup I by its
a
feacalis and A. hydrophila (MIC=0.39 mg/mL for both good activity against Enterococcus feacalis and its weak
strains;MBCrange:0.39–3.12mg/mL,respectively). activity against Vibrio cholerae. The variability within
The essential oils from Dhrea Ben Jouder (D.B.J.) subgroup I was due to the effects against Staphylococcus
a
exhibited a weaker antimicrobial activity against the aureus and Salmonella typhimirium. These two strains
tested strains (MIC range: 12.50 – 25.00 mg/mL; MBC were resistant to the action of the oil from Fernana and
range: 12.50 – 25.00 mg/mL). On the contrary, a signifi- sensitivetotheoneofBouArada.
cant activity (MIC=0.78 mg/mL; MBC=3.12 mg/mL) GroupIIwasrepresentedbytheoilofH.triquetrifolum
wasdetected against Vibriocholerae. obtained from plants harvested in Fondouk Djedid. This
Altogether,withtheexceptionoftheessentialoilsfromF. oil was characterized for its strong activity against all the
DJ.,alltheremainingsamplesshowedabacteriostaticeffect testedstrains,exceptVibriocholeraethatwassensitivefor
against V. cholerae, with MIC values ranging between themajorityoftheotheroils.
0.39 mg/mL and 12.50 mg/mL. MBC values were Group III was limited to the essential oil of plant
3.12mg/mLand25.00mg/mLfortheessentialoilsfrom harvested in Dhrea Ben Jouder and it is distinguished
Fer./D.B.J. and B.A./Bah., respectively. In addition, B. from all the other groups by its week activity against all
cereus, S. epidermidis, E. coli and V. alginoliticus were re- thetested strains,withtheexception ofVibriocholerae.
sistant to all the essential oils, but sensitive to the essential Forthebactericidalactivity,basedonEuclideancoefficient
oilsfromF.DJ.,withMICandMBCvaluesrangingbetween matrix, the HCA classified the studied populations in four
6.25and25.00mg/mL. groups, with dissimilarity <28: group A (H. triquetrifolium
In the PCA analysis of the bacteriostatic results from D. B. J.), group B (H. triquetrifolium from F. Dj.),
(Figure 1), the horizontal axis explains 41.78% of the group C (H. triquetrifolium from Bah.), and group D
total variance, while the vertical axis a further 27.37%. (H.triquetrifoliumfromFer.andB.A.),(Figure3).
This classification was supported HCA analysis which This classification was supported by PCA that resumed
evidenced three groups (I, II, and III), identified by their 75.88%ofthetotalvariability.Inthe2-Dplan,thehorizon-
MIC with a dissimilarity≥32.0 (Figure 2). When the tal axis (axis 1) explained 46.72% of the total variance,
dissimilarity was≥30.0, group I was divided into two whereas the vertical axis (axis 2) showed further 29.16% of
subgroups (I and I ). The horizontal axis permitted the variance (Figure 4). PCA analysis showed that group A
a b
separationofgroupIVfromtheothergroupswhereasthe was characterized by its moderate activity against Vibrio
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Figure1PCAProjectionoftheH.Triquetrifoliumessentialoilsbasedupontheirbacteriostaticactivitiesagainsttestedstrains.H.tri.B.
A.H.triquetrifoliumharvestedinBouArada,H.tri.Bah.H.triquetrifoliumharvestedinBahra,H.tri.D.B.J.H.triquetrifoliumharvestedinDhreaBen
Jouder,H.tri.Fer.H.triquetrifoliumharvestedinFernana,H.tri.F.Dj.H.triquetrifoliumharvestedinFondoukDjedid.1.Bacilluscereus,
2.Enterococcusfeacalis,3.Staphylococcusaureus.4.Staphylococcusepidermidis.5.Vibrioalginoliticus.6.Escherichiacoli.7.Vibriocholera.
8.Pseudomonasaeruginosa.9.Salmonellatyphimirium.10.Aeromonashydrophila.
cholerae. Group B was distinguished by a moderate and Staphylococcus epidermidis for Bou Arada samples,
bactericidal activity against all the tested strains, except and against Vibrio cholerae and Pseudomonas aeruginosa
Vibriocholerae. forFernanaessentialoil).
Group C was characterized by its potent activity
against Enterococcus feacalis which was resistant to the
action oftheotheroils. Antifungalactivity
Contrarytoallothergroups,groupDwascharacterized AsshowninTable4,theessentialoilsofH.triquetrifolium
by a moderate bactericidal activity against two or more exhibited a better antifungal and candidal activities than
strains (Aeromonas hydrophila, Pseudomonas aeruginosa, antibacterial activity, with MIC values ranging between
Figure2DendrogramobtainedbyhierarchicalanalysisbasedontheEuclideandistancesbetweengroupsofbacteriostaticactivitiesof
studiedessentialoils.H.tri.B.A.H.triquetrifoliumharvestedinBouArada,H.tri.Bah.H.triquetrifoliumharvestedinBahra,H.tri.D.B.J.H.
triquetrifoliumharvestedinDhreaBenJouder,H.tri.Fer.H.triquetrifoliumharvestedinFernana,H.tri.F.Dj.H.triquetrifoliumharvestedin
FondoukDjedid.
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Euclidean distances
H. tri. B.A.
H. tri. Fer.
H. tri. Bah.
H. tri. F.Dj.
H. tri. D.B.J.
24 26 28 30 32 34 36 38
Linkage Distance
Figure3DendrogramobtainedbyhierarchicalanalysisbasedontheEuclideandistancesbetweengroupsofbactericidactivitiesof
studiedessentialoils.H.tri.B.A.H.triquetrifoliumharvestedinBouArada,H.tri.Bah.H.triquetrifoliumharvestedinBahra,H.tri.D.B.J.H.
triquetrifoliumharvestedinDhreaBenJouder,H.tri.Fer.H.triquetrifoliumharvestedinFernana,H.tri.F.Dj.H.triquetrifoliumharvestedin
FondoukDjedid.
0.39μg/mL and 12.50μg/mL whereas MFC values were ThebestantifungalactivitywasexertedagainstC.glabrata
withinthe1.56μg/mLand25.00μg/mLrange. (MFC=1.56μg/mL).
The essentials oil from B.A. showed the most potent The essential oils from the Estern regions of Tunisia
fungistatic activity, with MIC values of 0.39μg/mL and (F.DJ.andD.B.J.)showedamorepotentantifungalactivity
3.12μg/mLforcandidalandfilamentousstrains(Aspergillus against the tested candidal strains, with MIC values ran-
niger, Fusarium solani and Botrytis cinerea), respectively. ging between 1.56μg/mL and 6.25μg/mL. The essential
Factor 2 : 29.16%
Factor 1 : 46.72%
Figure4PCAProjectionoftheH.Triquetrifoliumessentialoilsbasedupontheirbactericidactivitiesagainsttestedstrains.H.tri.B.A.H.
triquetrifoliumharvestedinBouArada,H.tri.Bah.H.triquetrifoliumharvestedinBahra,H.tri.D.B.J.H.triquetrifoliumharvestedinDhreaBen
Jouder,H.tri.Fer.H.triquetrifoliumharvestedinFernana,H.tri.F.Dj.H.triquetrifoliumharvestedinFondoukDjedid.1.Bacilluscereus,
2.Enterococcusfeacalis,3.Staphylococcusaureus.4.Staphylococcusepidermidis.5.Vibrioalginoliticus.6.Escherichiacoli.7.Vibriocholera.
8.Pseudomonasaeruginosa.9.Salmonellatyphimirium.10.Aeromonashydrophila.
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Table4AntifungalactivityofessentialoilsofH.triquetrifoliumagainstfungalandyeaststrains(MIC/MFC;μg/mL)
Strains H.tri.B.A.* H.tri.Bah.* H.tri.D.B.J.* H.tri.Fer.* H.tri.F.Dj.*
Aspergillusniger 3.12/3.12 12.50/12.50 6.25/6.25 12.50/12.50 3.12/3.12
Fusariumsolani 3.12/3.12 12.50/12.50 6.25/6.25 3.12/3.12 3.12/3.12
Botrytiscinerea 3.12/3.12 12.50/12.50 6.25/6.25 3.12/3.12 3.12/3.12
CandidakruseiATCC6258 0.39/25.00 6.25/6.25 3.12/25.00 3.12/6.25 6.25/6.25
CandidaalbicansATCC90028 0.39/25.00 6.25/6.25 3.12/25.00 6.25/6.25 3.12/6.25
CandidaglabrataATCC90030 0.39/1.56 3.12/6.25 1.56/25.00 6.25/6.25 1.56/6.25
MIC:MinimiumInhibitoryConcentration;MBC:MinimiumFungicidalConcentration.
H.tri.B.A.:HypericumtriquetrifoliumcollectedfromBouArada;H.tri.Bah.:HypericumtriquetrifoliumcollectedfromBahra;H.tri.D.B.J.:Hypericumtriquetrifolium
collectedfromDhreaBenJouder;H.tri.Fer.:HypericumtriquetrifoliumcollectedfromFernana;H.tri.F.Dj.:HypericumtriquetrifoliumcollectedfromFondoukDjedid.
*SDvalues=0.00.
oils from F.DJ. showed a more potent antifungal activity (Figure 5).Thefirstgroupwaslimitedtothesolesample
against filamentous fungal strains (MIC=3.12μg/mL, from Bahra and it was characterized by its poor activity
MFC=3.12μg/mL) than the essential oils from D.B.J. against all mycetes (MIC=12.50 mg/ml) and its moder-
(MIC=6.25μg/mL, MFC=6.25μg/mL). In addition, the ate activity on yeast strains. The essential oil obtained
essential oils from F.DJ. (MIC range: 1.56 – 6.25μg/mL, from plants harvested in Fernana constituted Group B,
MFC=6.25μg/mL) showed slightly higher anticandidal which was characterized by a moderate activity against
activity than the essential oils from D.B.J. (MIC range: allfungistrains,exceptAspergillusnigerwhichwasmore
1.56–3.12μg/mL,MFC=25.00μg/mL). resistant to the action of this oil (MIC=12.50 mg/ml).
Compared to the eastern regions, essential oils from the The third group was formed by the oil samples from
westernlocalities(Bah.andFer.)werelessfungistatic,with FondoukDjedid, DhreaBenJouder,andBou Arada.This
MICvaluesrangingbetween3.12μg/mLand12.50μg/mL, group was separated from the others because of their
while similar fungicidal activity, with MFC values ranging strong to moderate activities against all the tested
between3.12μg/mLand12.50μg/mL. strains. When the dissimilarity was≥6.25, group III was
The HCA analysis for the five essential oils based on divided into two subgroups (III , and III ). Subgroup III
a b a
their CMI against fungi permitted to evidence three was formed by Fondouk Djedid and Dhrea Ben Jouder
groups (A, B, and C) at a distance of dissimilarity <7 samples and it was characterized by a relatively strong
Euclidean distances
H. tri. B.A.
H. tri. D.B.J.
H. tri. F.Dj.
H. tri. Fer.
H. tri. Bah.
5 6 7 8 9 10 11 12
Figure5DendrogramobtainedbyhierarchicalanalysisbasedontheEuclideandistancesbetweengroupsoffongistaticactivitiesof
studiedessentialoils.H.tri.B.A.H.triquetrifoliumharvestedinBouArada,H.tri.Bah.H.triquetrifoliumharvestedinBahra,H.tri.D.B.J.H.
triquetrifoliumharvestedinDhreaBenJouder,H.tri.Fer.H.triquetrifoliumharvestedinFernana,H.tri.F.Dj.H.triquetrifoliumharvestedin
FondoukDjedid.
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Figure6PCAProjectionoftheH.Triquetrifoliumessentialoilsbasedupontheirfongistaticactivitiesagainsttestedstrains.H.tri.B.A.
H.triquetrifoliumharvestedinBouArada,H.tri.Bah.H.triquetrifoliumharvestedinBahra,H.tri.D.B.J.H.triquetrifoliumharvestedinDhreaBen
Jouder,H.tri.Fer.H.triquetrifoliumharvestedinFernana,H.tri.F.Dj.H.triquetrifoliumharvestedinFondoukDjedid.1.Aspergillusniger.2.Fusarium
solani.3.Botrytiscinerea.4.Candidakrusei.5.Candidaalbicans.6.Candidaglabrata.
activity against Candida glabrata. Subgroup III was group was further divided into two subgroups, A
b 1
limited tothe Bou Arada oil and showed a potent activity (represented by Fernana and Fondouk Djedid samples)
againstthethreetestedyeaststrains. characterized in addition by their moderate fungicidal
PCA analysis performed with MIC values of the five activities against Fusarium solani and Botrytis cinerea,
essential oils against the tested fungi explained 85.28% and A (limited to Bahra essential oil) which showed a
2
of total variance using the two first factors (Figure 6). In weak fungicidal activity against mycetes strains. Group
particular, the first axis explained 59.57% of the total B,limitedtotheessentialoilofplantharvestedinDhrea
variance, while the vertical axis a further 26.25%. Ben Jouder, presented a moderate activity on mycetes
According to this analysis, the essential oils of plants strains and a very weak activity against yeasts. Group C,
harvested from Bou Arada, Bahra, and Dhrea Ben Jouder represented exclusively by the sample from Bou Arada,
werenegativelycorrelatedwithaxis1andwithyeaststrains showedagoodactivityonCandidaglabrata,amoderate
(which were positively correlated with the horizontal activity against mycetes, and a poor fungicidal action on
factor); this group of oils correlated positively with CandidaalbicansandCandidakrusei.
mycetes strains (negatively correlated with horizontal PCA analysis based on MFC values showed, explained
axis). This observation suggests that these oils were 90.61% of the total variability using the first two axes. In
relativelymorepotentagainstyeaststrainsthanmycetes detail, PCA horizontal axis explained 70.61% of the total
strains. The essential oil of plant collected from variance, while the vertical axis a further 20.00%
Fondouk Djedid correlated negatively with the second (Figure 8). While essential oils obtained from plant
factor and it was characterized by its weak activity harvested from Bou Arada, Dhrea Ben Jouder and
againstCandidakrusei.SamplefromFernanacorrelated Fondouk Djedid negatively correlated with the first axis
positively with the second axis. It was distinguished by and with mycetes strains, they positively correlated with
its relatively good activity on Candida krusei and its yeast strains. This means that they were more effective on
weakactionagainstAspergillusniger. mycetes than against yeast strains. Essential oil obtained
Statistical analysis based on MFC values against fungi from plant harvested from Bahra positively correlated with
strains showed that while essential oils were classified the first axis, but it correlated negatively with yeast strains.
according to their activities against mycetes and yeast Sample obtained from plant harvested from Fernana
strains respectively using the PCA analysis, they were negatively correlated with the second axis and positively
discriminated according to the total of their activities correlated with Aspergillus niger (Strain 1 in Figure 8)
with theHCAanalysis. whichsupportitsweakactivityagainstthisstrain.
HCA analysis classified the essential oils into three
groups (A, B, and C) within a dissimilarity≥15.0 Cytotoxicitytestandantiviralactivity
(Figure 7). Samples of Group A were characterized by The cytotoxic effect of the essential oils was dose-
their moderate activities against all yeast strains. This dependent(Figure9).TheHypericumessentialoilsshowed
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Euclidean distances
H. tri. B.A.
H. tri. D.B.J.
H. tri. Bah.
H. tri. Fer.
H. tri. F.Dj.
5 10 15 20 25 30
Figure7DendrogramobtainedbyhierarchicalanalysisbasedontheEuclideandistancesbetweengroupsoffongicidactivitiesof
studiedessentialoils.H.tri.B.A.H.triquetrifoliumharvestedinBouArada,H.tri.Bah.H.triquetrifoliumharvestedinBahra,H.tri.D.B.J.H.
triquetrifoliumharvestedinDhreaBenJouder,H.tri.Fer.H.triquetrifoliumharvestedinFernana,H.tri.F.Dj.H.triquetrifoliumharvestedin
FondoukDjedid.
differentcytotoxicprofiles.Themostcytotoxicessentialoil incubated with cells before the inoculation. We cannot
was the one from D.B.J. (CC =0.58 mg/mL), followed by exclude the antiviral activity of these essential oils against
50
Fer. (CC =1.12 mg/mL), Bah. (CC =2.50 mg/mL) and other viruses, mainly the enveloped particles, which are
50 50
F.DJ. (CC =4.17 mg/mL). The least cytotoxic effect was knowntobemoresensitivetoenvironmentalconditions.
50
shownfortheessentialoilfromB.A.(CC =12.00mg/mL)
50
(Table 5). Unfortunately, the essential oils did not show an Discussion
evident antiviral activity against coxsakievirus B3 Nancy The antibacterial activity of the essential oils of Hypericum
strain, whether incubated with virus prior to infection or species is well documented in the literature for H.
Figure8PCAProjectionoftheH.Triquetrifoliumessentialoilsbasedupontheirfongicidactivitiesagainsttestedstrains.H.tri.B.A.H.
triquetrifoliumharvestedinBouArada,H.tri.Bah.H.triquetrifoliumharvestedinBahra,H.tri.D.B.J.H.triquetrifoliumharvestedinDhreaBen
Jouder,H.tri.Fer.H.triquetrifoliumharvestedinFernana,H.tri.F.Dj.H.triquetrifoliumharvestedinFondoukDjedid.1.Aspergillusniger.2.Fusarium
solani.3.Botrytiscinerea.4.Candidakrusei.5.Candidaalbicans.6.Candidaglabrata.