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Research Article
Received: 19 September 2008, Revised: 11 March 2009, Accepted: 16 March 2009, Published online 14 April 2009 in Wiley Interscience

(www.interscience.wiley.com) DOI 10.1002/ffj.1929

Antioxidant and antimicrobial activities

John Wiley & Sons, Ltd.

of essential oil and extracts of fennel

(Foeniculum vulgare Mill.) seeds from Pakistan
Farooq Anwara*, Muhammad Alia,b, Abdullah Ijaz Hussaina and
Antioxidant and antimicrobial activities of essential oil and extracts of fennel

Muhammad Shahida
ABSTRACT: The present study was conducted to examine the antioxidant and antimicrobial activities of essential oil, metha-
nol and ethanol extracts of fennel (Foeniculum vulgare Mill.) seeds native to Pakistan. The seed essential oil and extract yields
from fennel seeds were found to be 2.81 and 6.21–15.63% w/w, respectively. GC and GC–MS analysis of the fennel essential
oil revealed the presence of 23 compounds, with trans-anethol (69.87%), fenchone (10.23%), estragole (5.45%) and limonene
(5.10%) as the major components. The fennel seed extracts contained appreciable levels of total phenolic contents (627.21–
967.50 GAE, mg/100 g) and total flavonoid contents (374.88–681.96 CE, mg/100 g). Fennel essential oil and extracts also
exhibited good DPPH radical scavenging activity, showing IC50 32.32 and 23.61–26.75 mg/ml, and inhibition of peroxidation
45.05 and 48.80–70.35%, respectively. Of the fennel essential oil and solvent extracts tested, 80% ethanol extract exhibited
the maximum antioxidant activity, whereas the essential oil showed appreciable antimicrobial activity against selected
strains of bacteria and pathogenic fungi. The results of the present investigation demonstrated significant (p < 0.05)
variations in the antioxidant and antimicrobial activities of fennel essential oil and extracts. Copyright © 2009 John Wiley &
Sons, Ltd.

Keywords: Foeniculum vulgare; trans-anethol; GC–MS; total phenolics; IC50; total flavonoids; MIC

Introduction spices and herbs are well known to exert antioxidant and anti-
microbial activities.[7]
Antioxidants act as radical scavengers, inhibit lipid peroxidation Fennel (Foeniculum vulgare Miller), a plant belonging to the
and other free radical-mediated processes and are able to pro- family Apiaceae, has a long history of herbal uses. Traditionally,
tect the human body as well as processed foods from oxidative fennel seeds are used as anti-inflammatory, analgesic, carminative,
damage attributed to the reaction of free radicals. The use of diuretic and antispasmodic agents.[8] Recently there has been
synthetic antioxidants, such as butylated hydroxytoluene (BHT), considerable interest in the antioxidant potential and antimicrobial
butylated hydroxyanisole (BHA) and tertiary butylhydroquinone activities of fennel seed extracts and essential oil.[8–11]
(TBHQ), in foods is discouraged due to their perceived carcino- The purpose of the present study was to evaluate the anti-
genic potential and safety concerns.[1] oxidant and antimicrobial effectiveness of the essential oil and
Currently, the use of plant-based natural antioxidants, such as methanol and ethanol extracts of fennel seeds grown in the
those of phenolic substances like flavonoids and phenolic acids central Punjab region of Pakistan.
and tocopherols in foods, as well as preventive and therapeutic
medicine, is gaining much recognition. Such natural substances
are believed to exhibit anticarcinogenic potential and offer Materials and Methods
diverse health-promoting effects because of their antioxidant
Collection and Pretreatment of Plant Material
attributes. [1,2]
Food-borne diseases are a severe health problem in the world, Fully ripened fennel (Foeniculum vulgare Mill.) seeds (var. Dulce)
even in well-developed nations.[3] The consumption of food con- were collected from cultivated plants during May–June 2006
taminated with food-borne microorganisms can pose a serious from the Botanical Garden, University of Agriculture, Faisalabad,
threat to human health. The existence of microorganisms causes
spoilage and results in reduction of the quality and quantity of
processed foods.[4] Some biologically active compounds isolated
* Correspondence to: F. Anwar, Department of Chemistry and
from spices and herbs have been in use for the inhibition of Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan. E-mail:
growth of pathogenic microorganisms because of the resistance fqanwar@yahoo.com
that microorganisms have built against antibiotics.[5]
a
Herbal spices, being a promising source of phenolics, flavonoids, Department of Chemistry and Biochemistry, University of Agriculture,
Faisalabad-38040, Pakistan
anthocyanins and carotenoids, are usually used to impart flavour
and enhance the shelf-life of dishes and processed food products.[6] b
National Institute for Biotechnology and Genetic Engineering (NIBGE),
170

Essential oils, extracts and bioactive constituents of several PO Box 577, Faisalabad, Pakistan

Copyright © 2009 John Wiley & Sons, Ltd. Flavour Fragr. J. 2009, 24, 170–176

Antioxidant and antimicrobial activities of essential oil and extracts of fennel
Pakistan. The collections were made from the fully matured
plants and only brown fruits were picked. The specimens were
further identified and authenticated by a taxonomist, Dr Man-
soor Hameed, Assistant Professor, Department of Botany, Uni-
versity of Agriculture, Faisalabad, Pakistan [voucher specimen
code, Eclipta alba (L). Hassk (AS21)]. Collected specimens were
dried at 35 °C in a hot air oven (IM-30, Irmec, Germany) and
stored in polyethylene bags at –4 °C.
Chemicals and Reagents
Linoleic acid, 2,2-diphenyl-1-picrylhydrazyl, gallic acid, Folin–
Ciocalteu reagent, ascorbic acid, trichloro-acetic acid, sodium
nitrite, aluminium chloride, ammonium thiocyanate, ferrous
chloride, ferric chloride, potassium ferricyanate, butylated
hydroxytoluene (99.0%), homologous series of C9–C24 n-alkanes
and various reference chemicals (α-pinene, camphene, β-
pinene, β-myrcene, α-phellandrene, p-cymene, limonene, β-
ocimene, γ-terpinene, 1,8-cineol, fenchone, fenchyl alcohol,
estragole, fenchyl acetate, anethole, p-anisaldehyde and β-
caryophyllene) used to identify the constituents were obtained
from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals
(analytical grade), i.e. anhydrous sodium carbonate ferrous chloride,
ammonium thiocyanate, chloroform and methanol, used in this
study were purchased from Merck (Darmstadt, Germany), unless
stated otherwise. All culture media and standard antibiotic discs
were purchased from Oxoid Ltd. (Hampshire, UK).
Preparation of Fennel Seed Extracts
Ground (80 mesh) seed sample (20 g) was extracted separately
with 200 ml 100% methanol, 80% methanol (80:20, metha-
nol:water, v/v), 100% ethanol, 80% ethanol (80:20, ethanol:water,
v/v) using an orbital shaker (Gallenkamp, UK) for 8 h at room
temperature. The extracts were separated from solids by filter-
ing through Whatman No. 1 filter paper. The remaining residue
was re-extracted twice and the extracts were pooled. The solvent
was removed under vacuum at 45 °C, using a rotary vacuum
evaporator (N-N Series, Eyela, Rikakikai Co. Ltd, Tokyo, Japan)
and stored at –4 oC until used for further analyses.
Isolation of Essential Oil
The oven-dried and ground fennel seeds (80 mesh) were subjected
to hydrodistillation for 3 h using a Clevenger-type apparatus.
The obtained essential oil was dried over anhydrous sodium
sulphate, filtered and stored at –4 °C until tested and analysed.
Analysis of the Essential Oil
Gas chromatography. The essential oils were analysed using a
Perkin-Elmer gas chromatograph, Model 8700, equipped with a
flame ionization detector (FID) and an HP-5MS capillary column
(30 m × 0.25 mm i.d., film thickness 0.25 μm). Injector and detector
temperatures were set at 220 °C and 290 °C, respectively. Column
oven temperature was programmed from 80 °C to 220 °C at a
rate of 4 °C/min; initial and final temperatures were held for 3
and 10 min, respectively. Helium was used as carrier gas at a
flow rate of 1.5 ml/min. A sample of 1.0 μl was injected, using
split mode (split ratio, 1:100). All quantification was done by a
built-in data-handling program provided by the manufacturer of
the gas chromatograph (Perkin-Elmer, Norwalk, CT, USA). The
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essential oil composition was reported as a relative percentage
of the total peak area. Furthermore, the four major components
of the fennel essential oils, trans-anethole, fenchone, estragole
and limonene, were quantified by mean of the internal standard
addition method.[12]
Gas chromatography/mass spectrometry analysis. GC–MS ana-
lyses of the essential oils were performed using an Agilent
Technologies (Little Falls, CA, USA) 6890 N Network gas chromato-
graphic (GC) system, equipped with an Agilent Technologies
5975 inert XL mass selective detector and Agilent Technologies
7683B series auto-injector. Compounds were separated on an
HP-5 MS capillary column (30 m × 0.25 mm i.d., film thickness
0.25 μm; Agilent Technologies). A sample of 1.0 μL was injected
in the split mode with split ratio 1:100. For GC–MS detection,
an electron ionization system, with ionization energy of 70 eV,
was used. Column oven temperature programme was the same
as in GC analysis. Helium was used as the carrier gas at a flow
rate of 1.5 ml/min. Mass range was 50–550 m/z, while injector
and MS transfer line temperatures were set at 220 °C and 290 °C,
respectively.
Compounds identification. The identification of components
was based on comparison of their mass spectra with those of
NIST mass spectral library,[13,14] and those described by Adam,[15]
as well as on comparison of their retention indices either with
those of authentic compounds or with literature values.[9,15–17]
Antioxidant Activity
Determination of total phenolic contents (TPC). The total
phenolic content (TPC) in the fennel seed extracts was calcu-
lated using the Folin–Ciocalteu reagent method as described by
Sultana et al.[18]
Determination of total flavonoid contents (TFC). The total
flavonoid content (TFC) in the fennel seed extracts was deter-
mined following the procedure as described by Sultana et al.[18]
Determination of reducing power. The reducing power of the
fennel seed extracts was determined according to the proce-
dure described by Yen et al.[19]
DPPH radical scavenging assay. The 2,2-diphenyl-1-picrylhy-
drazyl radical (DPPH) assay was carried out spectrophotometri-
cally as described by Tepe et al.[20] Aliquots (50 μl) of various
concentrations (10–100 μg/ml) of the essential oil and extract
samples was added to 5 ml of a 0.004% methanol solution of
DPPH. After a 30 min incubation period at room temperature,
the absorbance was read against a blank at 517 nm:
I(%) = 100 × (Ablank – Asample/Ablank)
where Ablank is the absorbance of the control reaction (containing
all reagents except the test compound) and Asample is the absorb-
ance of the test compound. Extract concentration providing
50% inhibition (IC50) was calculated from a graph plotting per-
centage inhibition against extract concentration.
Antioxidant activity determination in linoleic acid system.
The antioxidant activities of fennel essential oil, methanol and
171
ethanol extracts were also determined in terms of measurement
www.interscience.wiley.com/journal/ffj

of percentage inhibition of peroxidation in the linoleic acid
system, following the method described by Iqbal et al.[2] with
some modifications. Essential oil and extracts (5 mg) were
added to a solution mixture of linoleic acid (0.13 ml), 99.8%
ethanol (10 ml) and 10 ml 0.2 M sodium phosphate buffer, pH 7.
The total mixture was diluted to 25 ml with distilled water. The
solution was incubated at 40 °C for 175 h. The extent of oxida-
tion was measured by the peroxide value, using the colorimetric
method as described by Yen et al.[19]
Antimicrobial Activity
Microbial strains. The fennel essential oil and extracts were
individually tested against a panel of microorganisms, including
two bacteria, Escherichia coli B10 and Bacillus subtilis SPS2, and
three pathogenic fungi, Aspergillus niger ATCC 10 575, Fusarium
solani ATCC 36 031 and Rhizopus solani. The pure bacterial and
fungal strains were obtained from the Biological Division of the
Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad,
Pakistan. The purity and identity of the strains were verified by
the Department of Veterinary Microbiology, University of Agri-
culture, Faisalabad, Pakistan. Bacterial strains were cultured
overnight at 37 °C in nutrient agar (Oxoid, Hampshire, UK) while
fungal strains were cultured overnight at 30 °C using potato
dextrose agar (Oxoid).
Disc diffusion method. The antimicrobial activity of the fennel
essential oil and extracts was determined by the disc diffusion
method.[21] The discs (6 mm in diameter) were impregnated with
15 μl essential oils or 30 mg/ml extracts (300 μg/disc) placed on
the inoculated agar. Amoxycillin (30 μg/disc) (Oxoid) and Flume-
quine (30 μg/disc) (Oxoid) were used as positive reference for
bacteria and fungi, respectively. Disc without samples was used
as a negative control. Antimicrobial activity was evaluated by
measuring the inhibition zone.
Microdilution broth method. For calculation of minimum
inhibitory concentration (MIC), which represents the concentra-
tion that completely inhibits the growth of microorganisms, a
microdilution broth susceptibility assay was used, as reported by
NCCLS.[22] A series of dilutions were prepared in the range 0.01–
72.0 mg/ml of the fennel essential oil in a 96-well microtitre
plate, including one growth control (NB/SDB + Tween 80) and
one sterility control (NB/SDB + Tween 80 + test oil). 160 μl NB
and SDB for bacteria and fungi, respectively, were added onto
the microplates with 20 μl of the tested solution. Then, 20 μl
5 × 105 CFU/ml (confirmed by viable count) of standard micro-
organism suspension was inoculated onto the microplates. The
plates were incubated at 37 °C for 24 h for bacteria and at 30 °C
for 48 h for fungi. Amoxycillin was used as a reference com-
pound for antibacterial and flumequine for antifungal activities.
The growth was indicated by the presence of a white ‘pellet’ on
the well bottom. The MIC was calculated as the highest dilution
showing complete inhibition of the tested strains.
Statistical Analysis
All the experiments were conducted in triplicate unless stated
otherwise and statistical analysis of the data was performed
by analysis of variance (ANOVA), using STATISTICA 5.5 (Stat
Soft Inc, Tulsa, Oklahoma, USA) software. A probability value of
p ≤ 0.05 was considered to denote a statistically significance
172
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F. Anwar et al.
difference. All data are presented as mean values ± standard
deviation (SD).
Results and Discussion
Essential Oil and Extracts Yields
Yields (g/100 g) of fennel seeds essential oil and various extracts
are given in Table 1. The yield of hydrodistilled fennel seed
essential oil was found to be 2.81%. Maximum extract yield was
obtained with 80% ethanol (15.63%) and the minimum with
100% methanol (6.21%). Based on these results, the extracting
ability of different solvents followed the order: 80% ethanol
> 80% methanol > absolute ethanol > absolute methanol. The
present results demonstrated a significant (p < 0.05) difference
in the extract yields among the solvent systems used. Our
results regarding higher extract yield with ethanol are in good
agreement with the findings of Oktay et al.,[8] who reported
10.95% yield of fennel seed extracts with absolute ethanol.
Conforti et al.[23] reported 10.95% and 15.78% extract yields,
respectively, from cultivated and wild fennel seeds using
methanol. Mata et al.[11] found the yield of essential oil and
ethanol extract of fennel seed from Portugal to be 0.1% and
6.9%, respectively. Mimica-Dukic et al.[9] determined the yield
of the essential oils obtained from F. vulgare seeds by steam dis-
tillation, range 2.82–3.38%.
Chemical Composition of the Essential Oil
The results of the chemical composition of fennel essential oil
are presented in Table 2. Overall, 23 compounds representing
95.53% of the oil were identified with the aid of GC–MS. The
major constituents of the essential oil tested were trans-
anethole (69.87%), fenchone (10.23%), estragole (5.45%) and
limonene (5.10%). In addition, the tested fennel essential oil also
contained considerable amounts of various minor constituents
whose contribution was <10%. Regarding the groups of chemi-
cal constituents represented, the fennel essential oil mainly
consisted of oxygenated monoterpenes (87.30%), followed by
monoterpene hydrocarbons (7.88%) and sesquiterpene hydro-
carbons (0.35%). Trans-anethole, fenchone and estragole were
the main oxygenated monoterpenes, while limonene was the
major monoterpene.
A literature search revealed trans-anethole (62.0%), fenchone
(20.3%), estragole (4.90%) and limonene (3.15%) to be the main
Table 1. Yield of fennel seeds essential oil, methanol and
ethanol extracts
Samples Yield (g/100 g)a
Essential oil 2.81 ± 0.14
100% methanol extract 6.21 ± 0.37d
80% methanol extract 12.11 ± 0.73b
100% ethanol extract 8.84 ± 0.44c
80% ethanol extract 15.63 ± 0.62a
Values are mean ± SD of three separate experiments.
a
Different letters in superscript indicate significant differences
within solvents.
Flavour Fragr. J. 2009, 24, 170–176
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Antioxidant and antimicrobial activities of essential oil and extracts of fennel

Table 2. Fennel essential oil composition (%), obtained by GC–MSa

Compoundsb RIc RI from literature Composition Identificationd

Monoterpene hydrocarbons
α-Pinene 939 939[16] 0.55 ± 0.02 RT, RI, MS
Camphene 954 954[16] 0.13 ± 0.03 RT, RI, MS
Sabinene 975 975[16] 0.19 ± 0.04 RT, RI, MS
β-Pinene 979 979[16] 0.09 ± 0.02 RT, RI, MS
β-Myrcene 991 991[16] 0.87 ± 0.10 RT, RI, MS
α-Phellandrene 1003 1003[9] 0.19 ± 0.02 RT, RI, MS
p-Cymene 1025 1025[16] t RT, RI, MS
Limonene 1029 1029[16] 5.10 ± 0.10 RT, RI, MS, CI
(Z)-β-Ocimene 1037 1037[9,16] 0.60 ± 0.02 RT, RI, MS
(E)-β-ocimene 1049 1048[9] t RT, RI, MS
γ-Terpinene 1060 1059[9] 0.16 ± 0.02 RT, RI, MS
Oxygenated monoterpenes
1,8-Cineol 1031 1031[16] 0.23 ± 0.02 RT, RI, MS
Fenchone 1088 1088[16] 10.23 ± 0.20 RT, RI, MS, CI
Fenchyl alcohol 1114 1113[9] 0.40 ± 0.04 RT, RI, MS
Estragole 1197 1199[9] 5.45 ± 0.20 RT, RI, MS, CI
Fenchyl acetate (endo) 1220 1222[17] 0.12 ± 0.03 RT, RI, MS
Fenchyl acetate (exo) 1232 1232[9] 0.54 ± 0.10 RT, RI, MS
cis-Anethole 1252 1252[9] 0.27 ± 0.03 RT, RI, MS
p-Anisaldehyde 1256 1256[17] 0.19 ± 0.01 RT, MS
trans-Anethole 1288 1287[17] 69.87 ± 0.65 RT, RI, MS, CI
Sesquiterpene hydrocarbons
β-Caryophyllene 1421 1420[17] 0.26 ± 0.00 RT, RI, MS
Germacrene D 1485 1485[16] 0.09 ± 0.00 RT, RI, MS
Total 95.53
a
Values are mean ± SD of two independent experiments.
b
Compound listed in the order of elution from a HP-5MS column.
c
Retention indices relative to C9–C24 n-alkanes on the HP-5MS column.
d
RI, identification based on retention index; MS, identification based on comparison of mass spectra; RT, identifica-
tion based on retention time; CI, co-injection with authentic standards; t, trace (<0.05%).

components of essential oils from wild-growing fennel seed

Table 3. Amounts of the major constituents in fennel
native to the Podgorica region, central south Montenegro.[24]
Mimica-Dukic et al.[9] also reported trans-anethole (74.18%), essential oil
fenchone (11.32%), estragole (5.29%), limonene (2.53%) and α-
pinene (2.77%) as the major compounds identified in the Compounds Content (g/100 g)a
essential oil from Foeniculum vulgare Mill. Ozcan et al.[10] and trans-Anethole 68.1 ± 0.8
Ozcan and Chalchat[25] reported estragole (61.08% and 40.49%), Fenchone 9.50 ± 0.40
fenchone (23.46% and 16.90%) and limonene (8.68% and Estragole 4.92 ± 0.18
17.66%), respectively, as the major constituents in the essential Limonene 4.50 ± 0.22
oil of bitter fennel (F. vulgare spp. piperitum) grown in Turkey. a
Values are mean ± SD of three independent experiments.
Such variations in the chemical composition of essential oil
across countries might be attributed to the varied agroclimatic
(climatical, seasonal, geographical) conditions of the regions,
stage of maturity and adaptive metabolism of plants.
On a quantitative basis, the amounts of the four main compo-
Antioxidant Activity
nents, calculated using calibrated curves with pure standard
compounds, were: trans-anethole, fenchone, estragole and DPPH radical scavenging assay. We investigated the free
limonene in the essential oil tested, 68.1, 9.50, 4.92 and 4.50 g/ radical scavenging activity and lipid oxidation inhibition of fennel
100 g oil, respectively (Table 3). There are no previous data seeds essential oil and extracts. Free radical scavenging activities
available in the literature on the quantitative analysis of fennel of the fennel essential oil and extracts were measured by DPPH
essential oil’s components with which to compare our present assay. Free radical scavenging capacity increased with increasing
extract and essential oil concentration (Table 4). Fennel seed
173

results.

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Table 4. Antioxidant activities of fennel seeds essential oil, ethanol and methanol extractsa
Antioxidant assays Essential oil
100% Methanol
DPPH, IC50 (μg/ml) 32.32 ± 0.77a 26.75 ± 1.06b
Inhibition in linoleic 45.05 ± 1.01d 48.80 ± 0.90c
acid system (%)
Total phenolic contentsb — 627.21 ± 18.36c
(mg/100 g of seed extracts)
Total flavonoid contentsc — 374.88 ± 12.89d
(mg/100 g of seed extracts)
a
Values are mean ± SD of three separate experiments.
b
Total phenolic contents expressed as gallic acid equivalent.
c
Total flavonoid contents expressed as catechin equivalent
Different letters in superscript indicate significant differences within solvents.
essential oils and extracts showed excellent radical scavenging
activity, with IC50 (the extract concentration providing 50% of
inhibition) values of 32.32 and 23.61–26.75 μg/ml, respectively.
The free radical scavenging activity of ethanol extract was superior
to that of essential oil. Furthermore, 80% ethanol and methanol
extracts exhibited more scavenging activity than absolute
ethanol and methanol extracts. When compared with the syn-
thetic antioxidant BHT, both essential oil and extracts offered
slightly lower antioxidant activity.
No earlier reports are available regarding the DPPH radical
scavenging activity of fennel seed essential oils with which to
compare the results of our present analysis. However, according
to Conforti et al.[23] the IC50 value for methanol extract of wild and
cultivated fennel seeds was determined to be 31 and 83 μg/ml,
respectively. Mata et al.[11] reported that ethanol extract of
fennel seeds exhibited stronger radical scavenging activity
(IC50 = 12.0 μg/ml) than the synthetic antioxidant BHT (IC50 =
15.7 μg/ml).
Percentage inhibition of linoleic acid oxidation. Table 4 shows
the percentage inhibition of linoleic acid oxidation as exhibited
by the fennel essential oil and extracts. 80% ethanol extract
offered significantly (p < 0.05) higher inhibition of peroxidation
(70.35%) than fennel essential oil (45.05%) and other extracts
(48.8–55.31%). When the inhibitions of linoleic acid oxidation of
fennel essential oil and extracts were compared with BHT, all the
extracts and essential oil exhibited significantly (p < 0.05) lower
antioxidant activity than that shown by BHT (92.1%). The order
of inhibition of linoleic acid oxidation offered by essential oil
and various extracts of fennel seeds were as follows: BHT
> 80% ethanol > 80% methanol > absolute ethanol > absolute
methanol > essential oil.
Due to lack of data on the percentage inhibition of linoleic
acid oxidation of fennel seed essential oil, we could not compare
the results of our present study with the literature. However, the
percentage inhibitions of fennel seed extracts determined in our
study are in close agreement with the findings of Parejo et al.,[26]
who reported the linoleic acid peroxidation for crude fennel
extract as being in the range 45.35–45.79% and that of BHT as
89.24%. Contrary to our results, Ozcan et al.[25] reported that the
inhibition of an ethanol extract of fennel seed was found to be
superior (98.6%) to that of BHT (97.8%).
174
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F. Anwar et al.
Extracts BHT
80% Methanol 100% Ethanol 80% Ethanol
24.25 ± 0.72bc 26.10 ± 0.90b 23.61 ± 0.89c 19.00 ± 0.95d
55.31 ± 1.04c 50.76 ± 0.99c 70.35 ± 1.74b 92.07 ± 2.24a
635.84 ± 12.01b 870.93 ± 20.82b 967.50 ± 35.51a —
454.99 ± 16.33b 529.41 ± 15.81c 681.96 ± 24.11a —
Total phenolic and total flavonoid contents. The total phe-
nolic contents (TPC) and total flavonoid contents (TFC) of fennel
seed extracts are presented in Table 4. The amounts of TPC and
TFC extracted from fennel seeds in different solvent systems
were in the ranges 627.21–967.50 GAE (mg/100 g) and 374.88–
681.96 CE (mg /100 g), respectively. Ethanol extract (80%) of the
fennel seeds showed the highest TPC and TFC, 967.50 and
681.96 mg/100 g, respectively. These differences in the amount
of TPC and TFC may be due to varied efficiency of the extracting
solvents to dissolve endogenous compounds. The ability of
different solvents to extract TPC and TFC was of the order:
80% ethanol > 80% methanol > absolute ethanol > absolute
methanol. Ethanol is preferred for the extraction of antioxidant
compounds mainly because of its lower toxicity.[27,28]
Conforti et al.[24] reported TPC (chlorogenic acid equivalents) from
extracts of cultivated and wild fennel to be 100 and 151 mg/g extract,
respectively. According to Mata et al.,[11] the ethanol extract of
fennel seed revealed 63.1 mg/g TPC (pyrogallol equivalents). No
earlier reports are available on the TFC of fennel seeds extracts
with which to compare the results of our present analysis.
Reducing power. The trends of reducing potential of different
fennel seed extracts are presented in Figure 1; the greater the
intensity of the colour, the greater will be the absorption; conse-
quently, the greater will be the antioxidant activity.[18] The reduc-
ing potential of the tested fennel extracts was observed at
concentrations of 2.5–10 mg/ml. The absorbance recorded for
the tested extract solutions in this assay were noted to be in the
range 0.20–1.85, indicating a high correlation index (r 2 = 0.9416–
0.9954). The maximum absorbance value (1.85) was recorded for
80% ethanol, while the minimum was for absolute methanol
(0.20). The reducing power of different solvent extracts lowered
in the order: 80% ethanol > 80% methanol > absolute methanol
> absolute ethanol. The variations in the reducing powers of
different fennel seed extracts were statistically significant
(p < 0.05). When these results were compared with standard
ascorbic acid, all the extracts showed significantly (p < 0.05) less
reducing power.
Our results are comparable with the investigations of Oktay
et al.,[8] who found the reducing power of ethanol and water
extracts of fennel seeds at a concentration of 250 μg/ml to be
1.09 and 0.509, respectively.
Flavour Fragr. J. 2009, 24, 170–176
 10991026, 2009, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ffj.1929 by HINARI-LEBANON, Wiley Online Library on [21/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Antioxidant and antimicrobial activities of essential oil and extracts of fennel

Figure 1. Reducing potential of methanol and ethanol extracts of fennel seeds

Table 5. Antimicrobial activity in terms of inhibition zones and minimum inhibitory concentration of fennel essential oil, ethanol
and methanol extracts against the selected strains of bacteria and fungia

Tested Essential Extracts Amoxicillin Flumequine

microorganisms oil
100% methanol 80% methanol 100% ethanol 80% ethanol
Diameter of inhibition zoneb
Escherichia coli 14 ± 1 — — — — 28 ± 2 —
Bacillus subtilis 29 ± 1 — — — — 32 ± 2 —
Aspergillus niger 28 ± 2 — — — — — 31 ± 2
Fusarium solani 26 ± 1 — — — — — 29 ± 1
Rhizopus solani 19 ± 1 — — — — — 28 ± 2
Minimum inhibitory concentration (MIC)c
Escherichia coli 259.3 ± 4.9 — — — — 80.3 ± 3.2 —
Bacillus subtilis 62.6 ± 1.9 — — — — 20 ± 0.9 —
Aspergillus niger 80.6 ± 1.4 — — — — — 30.3 ± 1.3
Fusarium solani 91.1 ± 5.0 — — — — — 40.4 ± 1.2
Rhizopus solani 110.3 ± 3.3 — — — — — 43.9 ± 1.7
a
Values are mean ± SD of three separate experiments.
b
Diameter of inhibition zone (mm) including disc diameter of 6 mm.
c
Minimum inhibitory concentration, MIC (mg/ml).

In agreement with our results, Cantore et al.[29] reported that

Antimicrobial Activity
The antibacterial activity of the essential oil and extracts from
fennel seeds against a panel of food-borne and pathogenic
microorganisms were assessed. The results are presented in
Table 5. Fennel essential oils exhibited considerable anti-
microbial activity against all the strains tested, particularly against
Gram-positive bacteria. The results from the disc diffusion
method, followed by measurement of minimum inhibitory con-
centration (MIC), indicated that B. subtilis and A. niger were the
most sensitive microorganisms tested, showing the largest
inhibition zones (29 and 28 mm) and the lowest MIC values
(62.6 and 80.6 μg/ml), respectively. Least activity was exhibited
against E. coli, with the smallest inhibition zones (14 mm) and
the highest MIC value (259.3 μg/ml). In general, the anti-
microbial activity of the tested fennel essential oil is comparable
with the standard drugs, amoxicillin and flumequine.
the Gram-negative strains of bacteria, especially E. coli, have less
sensitivity to fennel essential oils. Ozcan et al.[30] found that
fennel essential oils exhibit an inhibitory effect against a wide
range of Bacillius species. Mimica-Dukic et al.[9] also reported that
the essential oils of fennel are active against Aspergillus species.
Ozcan et al.[10] reported the antifungal activity of essential oils
from bitter fennel. As expected, fennel seed extracts offered no
antimicrobial activity.
The results of the present study demonstrated that essential
oil and various extracts from fennel (F. vulgare) show good anti-
oxidant and free radical scavenging activities. Furthermore,
fennel essential oil also exhibited appreciable antimicrobial
activity. The production of such essential oil and bioactive com-
ponents from indigenous resources and their utilization as
potential natural food preservatives could be of economic value.
However, further investigations involving more detailed in vitro
175

Flavour Fragr. J. 2009, 24, 170–176 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/ffj

and in vivo studies to establish which components of the essential
oil or extracts offer the best antioxidant activity are recom-
mended. Overall, this study presents valuable information on
the composition and antioxidant attributes of fennel essential
oil from Pakistan. It advocates its consumption in food and
pharmaceutical preparations local industries. In addition,
fennel showing antioxidant activity might be explored for
functional food and nutraceutical applications, besides its
traditional uses.
Acknowledgements
Authors are highly thankful to Higher Education Commission
(HEC), Islamabad, Pakistan for funding this research under the
PYI programme. We would also like to extend our sincere
gratitude to Professor Dr. Muhammad lqbal Bhanger, Director,
National Centre of Excellence in Analytical Chemistry, University
of Sindh, Jamshoro, Pakistan for providing us GC-MS facility.
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