In Vitro Test Of Antimicrobial Activity Of Foeniculum Vulgare Mill.(Fennel) Essential Oil

Abstract

The essential oil of Foeniculum vulgare was screened for antimicrobial activity against the Gram positive bacterium S.aureus, Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa, anaerobic bacterium Clostridium perfringens, and three fungal strains; Aperugillus flavus, Aperugillus niger, and Microsporum canis. The Minimum Inhibitory Concentrations (MICs) were determined by broth macro-dilution method. In the preliminary screening using disc diffusion assay against bacteria, the EO exhibited moderate inhibitory activity against the three tested bacteria. MICs and MBCs for bacteria ranged 0.781 to 25 µl /ml. The MIC values indicated that the fennel seed EO was active against all the fungal strains tested in the present study, and exhibited strong antifungal activity.

Full text

Journal of Multidisciplinary Engineering Science Studies (JMESS)
ISSN: 2458-925X
Vol. 3 Issue 4, April - 2017
www.jmess.org
JMESSP13420316 1609
In Vitro Test Of Antimicrobial Activity Of
Foeniculum Vulgare Mill. (Fennel) Essential Oil
Samah Awad AbduRahim 1, Baha Eldin Khalid Elamin 2, 3, Afra Abdelgader Ali Bashir 4, Aisha Zoheir Almagboul 5
1- Ph.D.in Medical Microbiology, Faculty of Medical Laboratory Sciences, University of Khartoum, Sudan,
samahawad90@yahoo.com.
2- Department of Microbiology, Faculty of Medical Laboratory Sciences, University of Khartoum, Sudan
3- Department of Microbiology and Parasitology, College of Medicine, University of Bisha, Saudi Arabia.
4- Department of Microbiology, Medicinal and Aromatic and Traditional Medicine Research Institute.
5- Professor of Microbiology and Phytochemistry, Medicinal Plants and Traditional Medicine Research Institute, National
center for Research, Sudan.
Abstract— The essential oil of Foeniculum
vulgare was screened for antimicrobial activity
against the Gram positive bacterium S.aureus,
Gram negative bacteria Escherichia coli and
Pseudomonas aeruginosa, anaerobic bacterium
Clostridium perfringens, and three fungal strains;
Aperugillus flavus, Aperugillus niger, and
Microsporum canis. The Minimum Inhibitory
Concentrations (MICs) were determined by broth
macro-dilution method. In the preliminary
screening using disc diffusion assay against
bacteria, the EO exhibited moderate inhibitory
activity against the three tested bacteria. MICs and
MBCs for bacteria ranged 0.781 to 25 µl /ml. The
MIC values indicated that the fennel seed EO was
active against all the fungal strains tested in the
present study, and exhibited strong antifungal
activity.
Keywords— Foeniculum vulgare, antimicrobial
activity, disc diffusion, tube dilution.
1. Introduction
The connection between man and his search for
drugs in nature dates from the far past, of which
there is ample evidence from various sources;
written documents, preserved monuments, and
even original plant medicines (1). Medicinal and
aromatic plants and their derivatives represent an
integral part of life in Sudan where flora consists
of 3137 species of flowering plants belonging to
170 families and 1280 genera. It is estimated that
15% of these plants are endemic to Sudan. The
intersection of diverse cultures and the unique
geography holds great potential for Sudanese
herbal medicine (2). The percentage of people
dependent on medicinal plants for health care is
estimated over 90%. These plants and derived
products play an important role in the primary
health care in Sudan (3).
Foeniculum vulgare is hardy, perennial–
umbelliferous herb with yellow flowers and
feathery leaves. It grows to a height of up to
2.5 m with hollow stems. The leaves grow up to
40 cm long; they are finely dissected with the
ultimate segments filiform (thread like) of about
0.5 mm wide. The flowers are produced in
terminal compound umbels. The fruit is a dry
seed 4–10 mm long (4). It is widely distributed in
most tropical and subtropical countries and have
long been used in folk medicines to treat
obstruction of the liver, spleen and gall bladder
and for digestive complaints such as colic,
indigestion, nausea and flatulence (5). In Sudan
the plant is used to treat diabetes in traditional
medicines (6). The essential oil from plants has
usually been isolated by either steam distillation
or solvent extraction (7). Trans-anethole, methyl
chavicol, fenchone, estragole, d-limonene,
neophytadiene, exo-fenchyl acetate and (E)-
phytol are the major chemical constituents (8-11).
Nineteen compounds were detected in the

Journal of Multidisciplinary Engineering Science Studies (JMESS)
ISSN: 2458-925X
Vol. 3 Issue 4, April - 2017
www.jmess.org
JMESSP13420316 1610
essential oil of F. vulgare cultivated in Sudan.
Monoterpenes comprises the main constituents
(98.06%), among which (80.67%) were
oxygenated, whereas sesquiterpenes represent
only about (0.66%) of the oil. Estragole
(68.96%), D-limonene (15.41%) and anethole
(8.51%) were the main identified constituents (4).
The objective of the present study is to assess the
antimicrobial activities of the essential oil of F.
vulgare against the bacterial and fungal
pathogens.
2. Materials and methods
2.1 Plant material
The plants used in this study was purchased from
local markets (Khartoum).They were
authenticated in Medicinal and Aromatic plants
and Traditional Medicine Research Institute
(MAPTMRI). Voucher specimens were deposited
at the herbarium of the institute.
2.2 Extraction of essential oil
The powdered seeds (500 g) of F. vulgare were
hydrodistilled in a Clevenger’s type apparatus for
6 h and yellow colored oil (yield 4%), with
characteristic odor and sharp taste, was obtained.
The crude oil was dried over anhydrous sodium
sulphate to remove traces of moisture and stored
in in a refrigerator in the dark at 4°C until use.
2.3 Test microorganisms
The EO was tested against the Gram positive
bacterium Staphylococcus aureus (ATCC 25923),
two Gram negtaive organsims, Escherichia coli
(ATCC 25922), Pseudomonas aeruginosa
(ATCC 27853) and three fungi, Aperugillus
flavus, Aperugillus niger, and Microsporum
canis.
2.4 In vitro screening of EO for antibacterial
activity
The disc diffusion method was adopted to screen
the antimicrobial activity of the against standard
control bacteria. The standardized inoculum
suspension of each bacterial strain which is
equivalent to 0.5 MC farland units was swabbed
on the entire surface of Mueller-Hinton agar, then
20 μl of the EO was placed on each sterile 6mm-
diameter absorbent filter paper disc and incubated
at 37 C◦ overnight. The inhibition zones were
measured and recorded in millimeters (mm). The
scale of measurement was the following (disc
diameter included): ≥28 mm zone of inhibition
(ZI) is strongly inhibitory; < 28 to 16 mm ZI is
moderately inhibitory; < 16 to 10 is mildly
inhibitory; and <10 mm is non inhibitory. Plates
were left at ambient temperature for 15 minutes
to allow excess pre-diffusion of extracts prior to
incubation at 37°C for 24 hours (13).
2.5 Quantitative evaluation of antimicrobial
susceptibility
Minimum inhibitory concentrations (MICs) of
individual oil and the ratios decided were
determined by the tube dilution method as
described in the Clinical and Laboratory
Standards Institute (CLSI, formerly NCCLS)
against bacteria and fungi. The final twofold
dilutions of the EO were prepared volumetrically
in Brain Heart Infusion broth medium for bacteria
and Sabouruad broth for fungi. A single pipette
was used for measuring all diluents and then for
adding the stock EO to the first tube. A separate
pipette was used for each remaining dilution in
that set. Because there will be a 1:2 dilution of the

Journal of Multidisciplinary Engineering Science Studies (JMESS)
ISSN: 2458-925X
Vol. 3 Issue 4, April - 2017
www.jmess.org
JMESSP13420316 1611
EO when an equal volume of inoculum is added,
the EO dilutions were prepared at double the
desired final concentration. Within 15 minutes
after the inoculum has been standardized, 1 ml of
the adjusted inoculum was added to each tube
containing 1 ml of EO in the dilution series (and a
positive control tube containing only broth), and
mix. This results in a 1:2 dilution of each
antimicrobial concentration, and a 1:2 dilution of
the inoculum. (14).The end point (MIC) is the least
concentration of antimicrobial agent that
completely inhibits the bacterial growth after
overnight incubation in 37 C◦ (15).
2.6 Determination of minimum bactericidal
concentration (MBC)
The minimum bactericidal concentration (MBC)
of the EOs on the standard control bacteria was
carried out according to National Committee for
Clinical Laboratory Standard provision (NCCL).
One ml was pipetted from the mixture obtained in
the determination of MIC tubes which did not
show any growth and streaked on MHA and
incubated for 24 h. The least concentration of the
EO with no visible growth after incubation was
taken as the minimum bactericidal concentration
(16).
3. Results and discussion
The essential oil of Foeniculum vulgare (fennel
seeds) was tested for its antimicrobial activity
against three bacterial and four fungal strains,
Table (1) illustrates the botanical and ethno-
pharmacological information about the medicinal
plant from which the essential oil was extracted.
In the preliminary screening using disc diffusion
assay against bacteria, the EO exhibited moderate
inhibitory activity against the three tested
bacteria. The susceptibility was estimated
quantitatively by means of minimum inhibitory
concentrations (MICs) for bacteria and fungi, and
minimum bactericidal concentrations (MBCs) for
bacterial strains. MICs and MBCs for bacteria
ranged 0.781 to 25 µl /ml (Table 2).
These results revealed that the EO of fennel
possesses a strong antibacterial activity against
the aerobic control bacteria. Our result was
opposed to that obtained by Miguel et al., who
found that the EO showed a very low
antimicrobial activity (17), this might be due to
using of commercial oils from different parts of
the plant along with using agar diffusion method
which gives a general idea but it is not a reliable
method to assess the activity of the plant extracts
quantitatively, because the most antimicrobial
compounds have intermediate polarity or are non-
polar. This means that these compounds do not
diffuse easily in the aqueous agar matrix. Broth
dilution is more suitable to test the antimicrobial
activity of the plant extracts than agar diffusion
methods (18). Similar to the results of Aprotosoaie
et al., and Tarek et al., S.aureus and E. coli were
more susceptible than P.aeruginosa, the EO
exhibited a good activity at low concentration(≤ 1
µl /ml) while P. aeruginosa was less active even
at the highest concentration (>16 µl /ml) (19, 20).
MIC value of fennel oil against E.coli was
0.0781% in our study, unlike Gulfraz et al. (7),
who determined MIC value 0.8%. This high
concentration may be attributed to determination
of MIC against E.coli clinical isolate - which is
the most prevalent reported microorganism in

Journal of Multidisciplinary Engineering Science Studies (JMESS)
ISSN: 2458-925X
Vol. 3 Issue 4, April - 2017
www.jmess.org
JMESSP13420316 1612
resistant data in many countries- instead of using
standard control strain of E.coli (21). On other
hand, the MIC of the current study was more than
that recorded by Bisht et al., who determined
MIC 0.062% for E.coli (22). The major
components in fennel oil that possess
antimicrobial properties are trans-anethol and
fenchone (23). The antimicrobial properties of
essential oil of fennel and its major constituents,
anethole, have been shown to be able to suppress
several human and plant pathogenic fungi (24).
The MIC values indicated that the fennel seed EO
was active against all the fungal strains tested in
the present study, and exhibited strong antifungal
activity. For Microsporum canis, the MIC was <
0.625%. For Aperugillus flavus and Aperugillus
niger strains, the MIC value was at the same level
(Table 2). Zeng et al., investigated the antifungal
effects of fennel seed EO from varied aspects,
such as MIC and minimum fungicidal
concentration, mycelia growth, spore germination
and biomass. The results indicated that the EO
had potent antifungal activities on Trichophyton
rubrum, Trichophyton tonsurans, Microsporum
gypseum and Trichophyton mentagrophytes,
which is better than the commonly used
antifungal agents fluconazole and amphotericin B
(25). The volatile oil showed complete zone
inhibition against Aspergillus niger, Aspergillus
flavus, Fusarium graminearum and Fusarium
moniliforme at 6 lL dose. It was found to be
effective for A. niger even at 4 lL dose.
Moreover, using food poison technique, the
volatile oil and extract both showed good to
moderate zone of inhibition (26).
Table (1): Preliminary screening for antibacterial activity of fennel seed EOs by disc diffusion assay
Family/Botanical name/Synonyms/
Vernacular name
Yield
%
Conc%
Antibacterial activity
(zone of inhibition in mm)
S.aureus
E.coli
P.aeruginosa
Apiaceae
Foeniculum vulgare Mill.
Synonym: Foeniculum capillaceum Gilib.,
Anethum foeniculum L.Foeniculum officinale
All.
Vern. chemar
4%
20%
19
20
15
10%
13
17
14
Table (2): Minimum inhibitory concentrations (MICs) of fennel seed essential oil
S.aureus
E.coli
P.aeruginosa
A.flavus
A.niger
M.canis
MICs
MBCs
MICs
MBCs
MICs
MBCs
MIC
MIC
MIC
µl /ml
µl /ml
µl /ml
µl /ml
µl /ml
µl /ml
0.781
0.781
0.781
0.781
25
25
0.125
0.125
<0.0625
References
1- Petrovska BB. Historical review of medicinal
plants usage. Pharmacogn Rev. 2012; 6(11): 1–5.
2- Khalid H, Abdalla WE, Abdelgadir H, Opatz
T, Efferth T .Gems from traditional north-
African medicine: medicinal and aromatic

Journal of Multidisciplinary Engineering Science Studies (JMESS)
ISSN: 2458-925X
Vol. 3 Issue 4, April - 2017
www.jmess.org
JMESSP13420316 1613
plants from Sudan. Nat Prod Bioprospect.
2012; 2(3): 92–103.
3- Ibrahim FY, El-Khateeb AY. Effect of herbal
beverages of Foeniculum
vulgare and Cymbopogon proximus on
inhibition of calcium oxalate renal crystals
formation in rats. Annals of Agricultural
Sciences. 2013; 58(2):221-229.
4- Rather M, Dar AB, et al. Foeniculum vulgare:
A comprehensive review of its traditional
use,phytochemistry, pharmacology, and safety.
Arabian Journal of Chemistry. 2012 Article in
press.
5- Garga C, Khan S A, Ansari S H, Suman A,
Garg M. Chemical composition, therapeutic
potential and perspectives of Foeniculum
vulgare. Phcog Rev 2009; 3:346-52.
6- Monalisa MN, Rahmatullah M.
Antihyperglycemic, analgesic activity, and
acute toxicity studies with methanol extract of
Foeniculum vulgare seeds. World Journal of
Pharmacy and Pharmaceutical Sciences.2015;
4(9): 198-206.
7- Gulfraz M, Mehmood S, Minhas N , Jabeen N,
Kausar R, Kokab, et al. Composition and
antimicrobial properties of essential oil of
Foeniculum vulgare. African Journal of
Biotechnology.2008; 7(24):4364-68.
8- García-Jiménez N, Péerez-Alonso MJ,
Velasco-Negueruela A. Chemical composition
of fennel oil, Foeniculum vulgare Miller, from
Spain. Journal of Essential Oil Research. 2000;
12(2): 159-162.
9- Zhaoa NN, Zhoub L, Liua ZL, Duc SS, Dengd
ZW. Evaluation of the toxicity of the essential
oils of some common Chinese spices gainst
bostrychophila. Food Control. 2012;
26(2):486–490.
10- Senatore F, Oliviero F, Scandolera E,
Taglialatela-Scafati O, Roscigno G,
Zaccardelli M, et al. Chemical composition,
antimicrobial and antioxidant activities of
anethole-rich oil from leaves of selected
varieties of fennel [Foeniculum vulgare Mill.
ssp. vulgare var. azoricum (Mill.) Thell].
Fitoterapia.2013; 90:214–219.
11- Diao W, Hu Q, Zhang H, Xu J. Chemical
composition, antibacterial activity and
mechanism of action of essential oil from
seeds of fennel (Foeniculum vulgare Mill.).
Food Control. 2014; 35(1):109–116.
A. 12- Hassan OM, Elhassan IA.
Characterization of essential oils from fruits of
umbelliferous crop cultivated in Sudan
II. Coriandrum sativum L. (Coriander)
and Foeniculum vulgare Mill (Fennel). 2017;
6(1): 113-116.
13- Elgayyar M, Draughon FA, Golden DA,
Mount JR. Antimicrobial activity of
essential oils from plants against selected
pathogenic and saprophytic
microorganisms. Journal of Food and
Protection.2001; 64(7):1019-1024.
14- Clinical and Laboratory Standards Institute.
Methods for dilution antimicrobial
susceptibility tests for bacteria that grow
aerobically; approved standard_Seventh
Edition. CLSI document M7-A7 (ISBN 1-
56238-587-9). Clinical and Laboratory

Journal of Multidisciplinary Engineering Science Studies (JMESS)
ISSN: 2458-925X
Vol. 3 Issue 4, April - 2017
www.jmess.org
JMESSP13420316 1614
Standards Institute, 940 West Valley Road,
Suite 1400, Wayne, Pennsylvania 19087-
1898 USA, 2006.
15- Cheesbrough M. Medical Laboratory Manual
for Tropical Countries.VolumeΙΙ. Low-
priced edition. Butterworth & Co Ltd (pub),
Cambridge, 2008, p.197-198, 243-244.
16- Doughari JH, El-mahmood AM, Tyoyina I.
Antimicrobial activity of leaf extracts of
Senna obtusifolia (L). African Journal of
Pharmacy and Pharmacology.2008;
2(1):007-013.
17- Miguel MG, Cruz C, Faleiro L, Simões MT,
Figueiredo AC, Barroso JG, et al.
Foeniculum vulgare essential oils: chemical
composition, antioxidant and antimicrobial
activities. Natural product communications.
2010; 5(2):319-28.
18- Eloff JN. A sensitive and quick microplate
method to determine the minimal inhibitory
concentration of plant extracts for bacteria.
Planta Medica.1998; 64:711–713.
19-Aprotosoaie AC, Hăncianu M, Poiată A,
Tuchiluş C, Spac A, Cioană O, et al. In vitro
antimicrobial activity and chemical
composition of the essential oil of
Foeniculum vulgare Mill. Revista medico-
chirurgicală
̆ a Societă
̆ţ̜ii de Medici ş̧i
Naturaliş̧ti din Iaş̧i. 2008; 112(3):832-6.
20- Tarek N, Hassan HM, El-Gendy AO.
Comparative chemical and antimicrobial
study of nine essential oils obtained from
medicinal plants growing in Egypt. Beni-
Suef University Journal of Basic and
Applied Sciences.2014; 3(2):149-156.
21- Aly M, Balkhy HH. The prevalence of
antimicrobial resistance in clinical isolates
from gulf corporation council countries.
Antimicrobial Resistance and Infection
Control.2012; 1:26.
22- Bisht DS, Menon KR & Singhal MK.
Comparative antimicrobial activity of
essential oils of Cuminum cyminum L. and
Foeniculum vulgare Mill. seeds against
Salmonella typhimurium and Escherichia
coli. Journal of Essential Oil Bearing Plants.
2014; 7(4): 617-622.
23- Kazemi M, Mousavi E, Kharestani H.
Chemical composition and antimicrobial
activities of essential oils of Varthemia
persica, Foeniculum vulgare and Ferula
lycia. Current Research in
Bacteriology.2012; 5:42-52.
24- Soylu S, Yigitbas H, Soylu EM, Kurt S.
Antifungal effects of essential oils from
oregano and fennel on Sclerotinia
sclerotiorum. Journal of Applied
Microbiology.2007; 103:1021-1030.
25- Zeng H, Chen X, Liang J. In vitro antifungal
activity and mechanism of essential oil from
fennel (Foeniculum vulgare L.) on
dermatophyte species. Journal of Medical
Microbiology.2015; 64: 93–103
26- Singh G, Maurya S, de Lampasona MP,
Catalan C. Chemical constituents, antifungal
and antioxidative potential of Foeniculum
vulgare volatile oil and its acetone extract.
Food Control. 2006; 17:745–752.