Lebensm.-Wiss. U.-Technol.

36 (2003) 263–271

Determination of in vitro antioxidant activity of fennel

(Foeniculum vulgare) seed extracts
.
Munir ’
Oktaya,*, Ilhami Gul . Irfan
. c,inb, O. ’ . glub
Kufrevio&
a
. University, Erzurum TR-25240, Turkey
Department of Chemistry Education, Kazım Karabekir Education Faculty, Ataturk
b
. University, Faculty of Science and Arts, Erzurum 25240, Turkey
Department of Chemistry, Ataturk
Received 23 May 2002; accepted 27 November 2002

Abstract

In this study, the antioxidant activity of water and ethanol extracts of fennel (Foeniculum vulgare) seed (FS) was evaluated by
various antioxidant assay, including total antioxidant, free radical scavenging, superoxide anion radical scavenging, hydrogen
peroxide scavenging, metal chelating activities and reducing power. Those various antioxidant activities were compared to standard
antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and a-tocopherol. The water and ethanol
extracts of FS seeds showed strong antioxidant activity. 100 mg of water and ethanol extracts exhibited 99.1% and 77.5% inhibition
of peroxidation in linoleic acid system, respectively, and greater than the same dose of a-tocopherol (36.1%). The both extracts of
FS have effective reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and
metal chelating activities. This antioxidant property depends on concentration and increasing with increased amount of sample. In
addition, total phenolic compounds in the water and ethanol extracts of fennel seeds were determined as gallic acid equivalents. The
results obtained in the present study indicated that the fennel (F. vulgare) seed is a potential source of natural antioxidant. Although,
the tests presented here show the usefulness of FS extracts as in vitro antioxidants it still needs to be that this extracts show their
activity in emulsions, biological systems, health implications or dry foods.
r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Fennel; Foeniculum vulgare; Antioxidant activity; In vitro; Seed

1. Introduction organism, various ROS can form by different ways.

Normal aerobic respiration stimulates polymorpho-
Reactive oxygen species (ROS) including free radicals nuclear leukocytes and macrophages, and peroxisomes
such as superoxide anion radicals (Od
2 ), hydroxyl appear to be the main endogenous sources of most of
radicals (OHd), singlet oxygen (1O2) and non-free- the oxidants produced by cells. Exogenous sources of
radical species such as hydrogen peroxide (H2O2) are ROS include tobacco smoke, certain pollutants, organic
various forms of activated oxygen and often generated solvents, and pesticides (Halliwell and Gutteridge, 1989;
by oxidation product of biological reactions or exogen- Davies 1994; Robinson, Maxwell, & Thorpe, 1997).
ous factors (Ceruitti 1991; Yıldırım, Mavi, & Kara, ROS can cause lipid peroxidation in foods, which leads
2001; Gul . c-in, Oktay, Kufrevioglu, & Aslan, 2002b). to the deterioration of the food (Miller, Diplock, &
ROS have aroused significant interest among scientists Rice-Evans, 1995; Sasaki, Ohta, & Decker, 1996). In
in the past decade. Their broad range of effects in bio- addition, it is well known that ROS induce some
logical and medicinal systems has drawn on the oxidative damage to biomolecules like lipids, nucleic
attention of many experimental works (Buy. ukokuroglu,
. acids, proteins, amines, deoxyribonucleic acid and
. c-in, Oktay, & Kufrevioglu, 2001; Gul
Gul . c-in, Buy-
. carbohydrates. Its damage causes ageing, cancer, and
.
ukokuroglu, Oktay, & Kufrevioglu, 2002a). In living other many diseases (Kehrer, 1993; Aruoma, 1994). As a
result of this, ROS have been implicated in more than
*Corresponding author. Tel.: +90-442-2314018; fax: +90-442-
100 diseases, including malaria, acquired immunodefi-
2360948. ciency syndrome, heart disease, stroke, arteriosclerosis,
E-mail address: [email protected] (M. Oktay). diabetes, and cancer (Tanizawa et al., 1992; Hertog,

0023-6438/03/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0023-6438(02)00226-8
264 M. Oktay et al. / Lebensm.-Wiss. U.-Technol. 36 (2003) 263–271

Feskens, Hollman, Katan, & Kromhout, 1993; Duh, essence in cosmetics and perfumes industry (Stuart,
1998; Alho & Leinonen, 1999; Yıldırım, Mavi, Oktay, 1982; Marotti et al., 1993).
Kara, Algur, & Bilaloglu, 2000). Much work has recently studied the yield and
ROS are continuously produced during normal composition of fennel seed (Verghese, 1988; Arslan,
physiologic events, and removed by antioxidant defence Bayrak, & Akgul, 1989; Lawrence, 1989, 1992; Betts,
mechanisms (Halliwell, Gutteridge, & Cross, 1992). 1992; Cavaleiro, Roque, & Cunha, 1993; Piccaglia &
There is a balance between generation of ROS and Marotti, 1993; Katsiotis, 1988). In addition, influence of
antioxidant system in organisms. In pathological con- spices on protein utilisation (Pradeep & Geervani, 1994),
dition, ROS are overproduced and result in lipid microbiological survey (Satchell, Bruce, Allen, Andrews,
peroxidation and oxidative stress. The imbalance & Gerber, 1989), effects of fungal metabolites on the
between ROS and antioxidant defence mechanisms germination (Sharma & Sharma, 1983), the effect of
leads to oxidative modification in cellular membrane fertiliser treatments on yield (Abdallah, El-Gengaihi, &
or intracellular molecules (El-Habit, Saada, & Azab, Sedrak, 1978), and immune reactivity (Schwartz, Jones,
2000). Various endogenous antioxidant defence mechan- Rojas, Squillace, & Yunginger, 1997) of fennel seed was
isms play an important role in the elimination of ROS investigated. Also, the effect of fennel seed extract on
and lipid peroxides, and therefore, protect the cells the genital organs of male and female rats is performed
against toxic effects of ROS and lipid peroxides (Malini et al., 1985).
(Halliwell, 1991; Halliwell et al., 1992; El-Habit et al., Fennel is very common in northern Anatolia region
2000). (Baytop, 1999). In the Turkish folk medicine, this plant
Many antioxidant compounds, naturally occurring especially its seeds have been used as tranquilliser, tonic
from plant sources, have been identified as free radical and soporific drug (Asimgal, 1997; Baytop, 1999).
or active oxygen scavengers (Yen & Duh, 1994; Duh, The chemical composition of the essential oil obtained
1998). Recently, interest has increased considerably in from various parts of the bitter Turkish fennel plant has
finding naturally occurring antioxidant for use in foods been reported (Akgul . & Bayrak, 1988). The vascular
or medicinal materials to replace synthetic antioxidants, effect of aqueous extract of fennel leaves was tested
which are being restricted due to their side effects such using pentobarbital-anaesthetised rats. The aqueous
as carcinogenecity (Ito, Fukushima, Hasegawa, Shibata, extract of fennel inhibited the hypotensive effect in
& Ogiso, 1983; Zheng & Wang, 2001). Natural a dose-related manner (Abdulghani & Amin, 1988).
antioxidants can protect the human body from free Ruberto et al. demonstrated that the oils of fennel had
radicals and retard the progress of many chronic in vivo antioxidant capacity (Ruberto, Baratta, Deans,
diseases as well as retard lipid oxidative rancidity in & Dorman, 2000), but there is no information about in
foods (Pryor, 1991; Kinsella, Frankel, German, & vitro antioxidant activity of water or ethanol extracts of
Kanner, 1993; Lai, Chou, & Chao, 2001). fennel seeds. However, from a toxicological point of
Fennel (Foeniculum vulgare) is a plant belonging to view, ethanol and water, as solvent, are safer than
the Umbelliferae (Apiaceae) family, known and used by acetone, methanol, and other organic solvent, and
humans since antiquity. It was cultivated in every therefore more suitable for food industry. Thus, water
country surrounding the Mediterranean Sea because of and ethanol extracts are used in the following study. The
its flavour (Muckensturm, Foechterlen, Reduron, Dan- purpose of present study was to evaluate the antioxidant
ton, & Hildenbrand, 1997). The renewed interest in activity of the water and ethanol extracts of fennel seeds
natural product rather than synthetic agents has again and to elucidate their antioxidative actions.
focused attention on plants as a source of flavouring
compounds (Yaylayan, 1991). Fennel is an aromatic
edible plant whose seed are used for savoury formula- 2. Materials and methods
tions, sauces, liqueurs, confectionery, etc. (Guilled &
Manzanons, 1996). Due to unique and preferred flavour 2.1. Chemicals
and aroma, the swollen bases of fennel are freshly
consumed in salad or cooked as kitchen vegetable Ammonium thiocyanate was purchased from E.
(Baytop, 1999; Atta-Aly, 2001). Merck. Ferrous chloride, polyoxyethylenesorbitan
The therapeutic effects and culinary utilisation of monolaurate (Tween-20), a-tocopherol, 1,1-diphenyl-
fennel were so large that it was exported from country to 2-picryl-hydrazyl (DPPHd), 3-(2-Pyridyl)-5,6-bis (4-
country for centuries (Puelo, 1980). Today, all culture of phenyl-sulfonic acid)-1,2,4-triazine (Ferrozine), nico-
fennel has given rise to a great complexity, and simple tinamide adenine dinucleotide (NADH), butylated
observation of this plant’s morphological characteristics hydroxyanisole (BHA), butylated hydroxytoluene
is not sufficient to classify (Jansen, 1981). The major (BHT), and trichloracetic acid (TCA) were purchased
constituents of fennel essential oil such as anethole and from Sigma Chemical Co. All others unlabelled che-
limonene are also used some medicinal purposes and micals and reagents were analytical grade.
 M. Oktay et al. / Lebensm.-Wiss. U.-Technol. 36 (2003) 263–271 265

2.2. Plant material and extraction upper layer of solution (2.5 ml) was mixed with distilled
water (2.5 ml) and FeCl3 (0.5 ml, 0.1%), and the
Fennel (F. vulgare) seeds (FS) were obtained from a absorbance was measured at 700 nm. Increased absor-
local market at Erzurum, Turkey. For water extraction, bance of the reaction mixture indicated increased
25 g sample of FS ground into a fine powder in a mill reducing power.
and was mixed with 500 ml boiling water by magnetic
stirrer during 15 min. Then the extract was filtered over 2.5. Superoxide anion scavenging activity
Whatman No.1 paper. The filtrates were frozen and
lyophilised in a lyophilisator at 5 mm Hg pressure and at Measurement of superoxide anion scavenging activity

501C (Labconco, Freezone 1L). For ethanol extrac- of water and ethanol extracts of FS was done based on
tion, 25 g powder of FS was mixed five times with 100 ml the method described by Liu, Ooi, and Chang (1997)
ethanol. Extraction continued until the extraction with slight modification. One millilitre of nitroblue
solvents became colourless (total solvent volume is tetrazolium (NBT) solution (156 mmol/L NBT in
500 ml). The obtained extracts were filtered over What- 100 mmol/L phosphate buffer, pH 7.4) 1 ml NADH
man No.1 paper and the filtrate was collected, then solution (468 mmol/L in 100 mmol/L phosphate buffer,
ethanol was removed by a rotary evaporator at 501C. pH 7.4) and 0.1 ml of sample solution of FS extracts in
water were mixed. The reaction started by adding 100 ml
2.3. Total antioxidant activity determination of phenazine methosulphate (PMS) solution (60 mmol/L
PMS in 100 mmol/L phosphate buffer, pH 7.4) to the
Total antioxidant activity of both FS extracts mixture. The reaction mixture was incubated at 251C for
was determined according to the thiocyanate method 5 min, and the absorbance at 560 nm was measured
(Mitsuda, Yuasumoto, & Iwami, 1996). Ten milligrams against blank samples. Decreased absorbance of the
of FS extracts was dissolved in 10 ml water. One reaction mixture indicated increased superoxide anion
milligram of FS extracts in 1 ml of water was added to scavenging activity. The percentage inhibition of super-
linoleic acid in potassium phosphate buffer (2.5 ml, oxide anion generation was calculated using the follow-
0.04 mol/L, pH 7.0). Fifty millilitre linoleic acid emul- ing formula:
sion consisting of 175 mg Tween-20, 155 ml linoleic acid, % Inhibition ¼ ½ðA0
A1 Þ=A0   100;
and 0.04 mol/L potassium phosphate buffer (pH 7.0).
On the other hand, 5.0 ml control consisting of 2.5 ml where A0 was the absorbance of the control, and A1 was
linoleic acid emulsion and 2.5 ml potassium phosphate the absorbance of the FS extracts and standards (Ye,
buffer (0.04 mol/L, pH 7.0). The mixed solution was Wang, Liu, & Ng, 2000).
incubated at 371C in a glass flask. The peroxide value
was determined by reading the absorbance at 500 nm, 2.6. Free radical scavenging activity
after reaction with FeCl2 and thiocyanate at several
intervals during incubation. The solutions without The free radical scavenging activity of water and
added extracts used as blank samples. All data are the ethanol extracts of FS was measured by 1,1-diphenyl-2-
average of triplicate analyses. The inhibition of lipid picryl-hydrazil (DPPHd) using the method of
peroxidation in percent was calculated by following Shimada, Fujikawa, Yahara, and Nakamura (1992).
equation: The 0.1 mmol/L solution of DPPHdin ethanol was
% Inhibition ¼ 100
½ðA1 =A0 Þ  100; prepared and 1 ml of this solution was added 3 ml of FS
extracts solution in water at different concentrations
where A0 was the absorbance of the control reaction and (50–250 mg). After 30 min absorbance was measured at
A1 was the absorbance in the presence of the FS extracts 517 nm. Lower absorbance of the reaction mixture
sample (Duh, Tu, & Yen, 1999). indicated higher free radical scavenging activity. The
DPPHdconcentration in the reaction medium was
2.4. Reducing power calculated from the following calibration curve, deter-
mined by linear regression (R2 : 0:9545):
The reducing power of FS extracts was determined Absorbance ¼ 0:0036  ½DPPHd:
according to the method of Oyaizu (1986). Different
amounts of FS extracts (50–250 mg) in 1 ml of distilled
water was mixed with phosphate buffer (2.5 ml, 0.2 mol/ 2.7. Metal chelating activity
L, pH 6.6) and potassium ferricyanide [K3Fe(CN)6]
(2.5 ml, 1%). The mixture was incubated at 501C for The chelating of ferrous ions by the both FS extracts
20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was estimated by the method of Dinis, Madeira, and
was added to the mixture, which was then centrifuged at Almeida (1994). Briefly the extracts samples (50–250 mg/
3000 rpm (MSE Mistral 2000, UK) for 10 min. The ml) were added to a solution of 2 mmol/L FeCl2
266 M. Oktay et al. / Lebensm.-Wiss. U.-Technol. 36 (2003) 263–271

(0.05 ml). The reaction was initiated by the addition of 2.10. Statistical analysis
5 mmol/L ferrozine (0.2 ml) and the mixture was shaken
vigorously and left standing at room temperature for Experimental results were mean 7S.D. of five parallel
10 min. Absorbance of the solution was then measured measurements. P values o0.05 were regarded as
spectrophotometrically at 562 nm. The percentage of significant and P values o0.01 very significant.
inhibition of ferrozine–Fe2+ complex formation was
given below formula
3. Results and discussion
%Inhibition ¼ ½ðA0
A1 Þ=A0   100;
3.1. Total antioxidant activity
where A0 was the absorbance of the control, and A1 was
the absorbance in the presence of the sample of FS
Total antioxidant activity of ethanol and water
extracts and standards.
extracts of FS was determined by the thiocyanate
method. Table 1 shows the extraction yields and gallic
2.8. Scavenging of hydrogen peroxide acid equivalents. In addition, Table 2 shows inhibition
of lipid peroxidation in percent of water and ethanol
The ability of both FS extracts to scavenge hydrogen extracts of FS, a-tocopherol, BHA and BHT. Ethanol
peroxide was determined according to the method of and water extracts of FS exhibited effective antioxidant
Ruch, Cheng, and Klaunig (1989). A solution of activity at all concentrations. The effects of various
hydrogen peroxide (2 mmol/L) was prepared in phos- amounts of water and ethanol extracts of FS (50–250 mg)
phate buffer (pH 7.4). Hydrogen peroxide concentration on peroxidation of linoleic acid emulsion are shown in
was determined spectrophotometrically from absorption Figs. 1 and 2. The antioxidant activity of ethanol and
at 230 nm with molar absorbtivity 81 mol/L
1/cm. water extracts of FS increased with increasing concen-
Extracts samples (50–250 mg/ml) in distilled water were tration. The all of doses of water and ethanol extracts of
added to a hydrogen peroxide solution (0.6 ml). FS showed higher antioxidant activities than that 100 mg
Absorbance of hydrogen peroxide at 230 nm was of a-tocopherol. As seen Table 2, the percentage of
determined after 10 min against a blank solution inhibition of 50, 100 and 250 mg doses of water extracts
containing in phosphate buffer without hydrogen of FS on peroxidation in linoleic acid system was 83.9,
peroxide. The percentage of scavenging of hy- 91.6, 96.7 and 50, 100 and 250 mg doses of ethanol
drogen peroxide of both FS extracts and standard extracts was 65.5%, 78.6%, and 91.8%, respectively.
compounds The values of both extracts were greater than that 100 mg
% Scavenged H2 O2 ¼ ½ðA0
A1 Þ=A0   100;

where A0 was the absorbance of the control, and A1 was Table 1

the absorbance in the presence of the sample of FS Yield and gallic acid equivalents (GAE) of water and ethanol extracts
of FS [FS: fennel (F. vulgare) seeds]
extracts and standards.
Solvent Yield (g/100 g) GAE (mg)
2.9. Determination of total phenolic compounds Water 16.20 21.25
Ethanol 10.95 90.00
Total soluble phenolics in the FS extracts were
determined with Folin-Ciocalteu reagent according to
the method of Slinkard and Singleton (1977) using gallic
acid as a standard phenolic compound. Briefly, 1 ml of Table 2
extract solution contains 1000 mg extract was mixed with Inhibition of lipid peroxidation in percent of water and ethanol
45 ml distilled water. One millilitre of Folin-Ciocalteu extracts of FS, a-tocopherol, BHA, and BHT [FS: Fennel (F. vulgare)
reagent was added and the content of the flask mixed seeds]
thoroughly. After 3 min 3 ml of Na2CO3 (2%) was Sample Inhibition of lipid peroxidation (%)a
added then the mixture was allowed to stand for 2 h with
Water extract 91.6
intermittent shaking. The absorbance was measured at Ethanol extract 98.6
760 nm. The concentration of total phenolic compounds a-Tocopherol 36.9
in the both FS extracts determined as microgram of BHA 94.3
gallic acid equivalent by using an equation that was BHT 97.8
obtained from standard gallic acid graph. The equation a
The antioxidant activity of the samples (100 mg) was determined by
is given below the thiocyanate method. The peroxide values were determined by
reading the absorbance at 500 nm after reaction with FeCl2 and
Absorbance ¼ 0:0008  gallic acid ðmgÞ: thiocyanate.
 M. Oktay et al. / Lebensm.-Wiss. U.-Technol. 36 (2003) 263–271 267

2.5 2

2
1.5
Absorbance (500 nm)

Absorbance (700 nm)

1.5

1
1

0.5 0.5

0
0 20 40 60 80 100 0
0 50 100 150 200 250
Time (Hour)
Amount (µg)
Fig. 1. Antioxidant activity of different doses of water extracts of FS
and a-tocopherol in the linoleic acid emulsion was determined by the Fig. 3. Reducing power of water and ethanol extracts of FS, BHA,
thiocyanate method. ’, control; B, a-tocopherol; W, water extract of BHT, and a-tocopherol. Results are mean7S.D. of five parallel
fennel (50 mg); &, water extract of fennel (100 mg); J, water extract of measurements. P value o0.01 when compared to control. (Spectro-
fennel (250 mg). photometric detection of the Fe+3–Fe+2 transformation.) [FS: fennel
(F. vulgare) seed; BHA: buthylated hydroxyanisole; BHT: Buthylated
hydroxytoluene]. ’, BHA; &, BHT; B, a-tocopherol; W, water
2.5 extract of fennel; m, ethanol extract of fennel.

2
Absorbance (500 nm)

both FS extracts increased with increasing amount of

1.5
sample. All of the amounts of FS extracts showed higher
activities than control and these differences were
1
statistically very significant (Po0:01). Reducing power
0.5
of water and ethanol extracts of FS and standard
compounds followed the order: BHA>BHT>water
0 extract of FS>a-tocopherol>ethanol extract of FS.
0 20 40 60 80 100
Time (Hour) 3.3. Superoxide anion scavenging activity
Fig. 2. Antioxidant activity of different doses of ethanol extracts of FS
and a-tocopherol in the linoleic acid emulsion was determined by the In the PMS/NADH-NBT system, superoxide anion
thiocyanate method. ’, control; B, a-tocopherol; W, ethanol extract derived from dissolved oxygen by PMS/NADH cou-
of fennel (50 mg); &, ethanol extract of fennel (100 mg); J, ethanol pling reaction reduces NBT. The decrease of absorbance
extract of fennel (250 mg).
at 560 nm with antioxidants thus indicates the con-
sumption of superoxide anion in the reaction mixture.
of a-tocopherol (36.9%), but lower than that same dose Fig. 4 shows the % inhibition of superoxide radical
of BHA and BHT (94.3% and 97.8%). generation of 50, 100, and 250 mg of water and ethanol
extracts of FS and comparison with same doses of BHA,
3.2. Reducing power BHT, and a-tocopherol. The both extracts of FS have
strong superoxide radical scavenging activity and
Fig. 3 shows the reductive capabilities of samples FS exhibited higher superoxide radical scavenging activity
extracts compared to BHA, BHT and a-tocopherol. For than BHT and a-tocopherol. The results were found
the measurements of the reductive ability, we investi- statistically significant ðPo0:05Þ: The percentage inhibi-
gated the Fe3+–Fe2+ transformation in the presence of tion of superoxide generation by 250 mg doses of BHA,
FS extracts samples using the method of Oyaizu (1986). water and ethanol extracts of FS was found as 98.7, 96.6
The reducing capacity of a compound may serve as a and 94.0% and greater than that same doses of BHT
significant indicator of its potential antioxidant activity and a-tocopherol (88.3 and 80.5%), respectively. Super-
(Meir, Kanner, Akiri, & Hadas, 1995). However the oxide radical scavenging activity of those samples
antioxidant activity of antioxidants have been attributed followed the order: BHA>water extract of FS>ethanol
to various mechanisms, among which are prevention of extract of FS>BHT>a-tocopherol.
chain initiation, binding of transition metal ion cata-
lysts, decomposition of peroxides, prevention of con- 3.4. Free radical scavenging activity
tinued hydrogen abstraction, reductive capacity and
radical scavenging (Diplock, 1997; Yıldırım et al., 2000). DPPHdis a stable free radical and accepts an electron
Like the antioxidant activity, the reducing power of or hydrogen radical to become a stable diamagnetic
268 M. Oktay et al. / Lebensm.-Wiss. U.-Technol. 36 (2003) 263–271

100 scavenging free radical. Free radical scavenging activity

also increased with increasing concentration. The
75 capability to scavenge the DPPH radical was calculated
Absorbance (560 nm)

using the following equation:

50 DPPHdscavenging effectð%Þ ¼ ½ðA0
A1 =A0 Þ  100;
where A0 was the absorbance of the control reaction and
25 A1 was the absorbance in the presence of the sample of
FS extracts.
0
0 50 100 150 200 250 3.5. Metal chelating activity
Amount (µg)

Fig. 4. Superoxide anion radical scavenging activity of water and The chelating of ferrous ions by the extracts of FS was
ethanol extracts of FS, BHA, BHT, and a-tocopherol by the PMS/ estimated by the method of Dinis et al. (1994). Ferrozine
NADH-NBT method. Results are mean7S.D. of five parallel can quantitatively form complexes with Fe2+. In the
measurements. P value o0.05 when compared to control. [FS: Fennel
(F. vulgare) seed; BHA: buthylated hydroxyanisole; BHT: buthylated
presence of other chelating agents, the complex forma-
hydroxytoluene]. ’, BHA; &, BHT; B, a-tocopherol; W, water tion is disrupted with the result that the red colour of the
extract of fennel; m, ethanol extract of fennel. complex is decreased. Measurement of the rate of colour
reduction therefore allows estimation of the chelating
activity of the coexisting chelator (Yamaguchi, Ariga,
1000 Yoshimira, & Nakazawa, 2000). In this assay the both
extracts of FS and standard compounds interfered with
750 the formation of ferrous and ferrozine complex,
suggesting that it has chelating activity and captures
[DPPH·] (µM)

ferrous ion before ferrozine.

500
As shown in Fig. 6, the formation of the ferrozine–
Fe2+ complex is not complete in the presence of water
250 and ethanol extracts of FS, indicating that both extracts
of FS chelate the iron. The absorbance of Fe2+–
0 ferrozine complex was linearly decreased dose depen-
0 50 100 150 200 250 dently (from 50 to 250 mg). The difference between both
Amount (µg) extracts of FS and the control was statistically
Fig. 5. Free radical scavenging activity of water and ethanol extracts significant ðPo0:05Þ: The percentage of metal scaven-
of FS, BHA, BHT, and a-tocopherol by 1,1-diphenyl-2-picrylhydrazyl ging capacity of 250 mg doses of water and ethanol
radicals. Results are mean7S.D. of five parallel measurements. P extracts of FS, a-tocopherol, BHA, and BHT were
value o0.05 when compared to control. [FS: fennel (F. vulgare) seed; found as 30.7%, 26.5%, 43.0%, 74.8% and 40.6%,
BHA: buthylated hydroxyanisole; BHT: buthylated hydroxytoluene].
respectively. The metal scavenging effect of the both
’, BHA; &, BHT; B, a-tocopherol; W, water extract of fennel; m,
ethanol extract of fennel. extracts of FS and standards decreased in the order
of BHA>a-tocopherol>BHT>water extract>ethanol
extract of FS.
molecule (Soares, Dins, Cunha, & Ameida, 1997). The Metal chelating capacity was significant since they
reduction capability of DPPH radicals was determined reduced the concentration of the catalysing transition
by the decrease in its absorbance at 517 nm induced by metal in lipid peroxidation (Duh et al., 1999). It was
antioxidants. Hence, DPPHdis usually used as a reported that chelating agents, which form s-bonds with
substrate to evaluate antioxidative activity of antiox- a metal, are effective as secondary antioxidants because
idants (Oyaizu, 1986). Fig. 5 illustrates a significant they reduce the redox potential thereby stabilising the
ðPo0:05Þ decrease the concentration of DPPH radical oxidised form of the metal ion (Gordon, 1990). The data
due to the scavenging ability of soluble solids in the both obtained from Fig. 6 reveal that the both extracts of FS
extracts of FS and standards. We used BHA, BHT and demonstrate an effective capacity for iron binding,
a-tocopherol as standards. The scavenging effect of suggesting that its action as peroxidation protector
water and ethanol extracts of FS and standards on the may be related to its iron binding capacity.
DPPH radical decreased in the order of BHA>a-
tocopherol>BHT>water extract>ethanol extract and 3.6. Scavenging of hydrogen peroxide
were 88.02%, 86.38%, 64.07%, 47.49% and 36.46% at
the dose of 250 mg, respectively. These results indicated The ability of the both extracts of FS to scavenge
that the both FS extracts have a noticeable effect on hydrogen peroxide was determined according to the
 M. Oktay et al. / Lebensm.-Wiss. U.-Technol. 36 (2003) 263–271 269

0.5 40

0.4 30

Absorbance (230 nm)

Absorbance (562 nm)

0.3
20

0.2
10
0.1

0
0
0 50 100 150 200 250
0 50 100 150 200 250
Amount (µg)
Amount (µg)
Fig. 7. Hydrogen peroxide scavenging activities of water and ethanol
Fig. 6. Metal chelating effect of different amount of water and ethanol extracts of FS, BHA, BHT, and a-tocopherol. Results are mean7S.D.
extracts of FS, BHA, BHT, and a-tocopherol on ferrous ions. Each of five parallel measurements. P value o0.05 when compared to
value is expressed as mean7S.D. of five parallel measurements. P control. [FS: fennel (F. vulgare) seed; BHA: buthylated hydroxyani-
value o0.05 when compared to control. [FS: fennel (F. vulgare) seed; sole; BHT: buthylated hydroxytoluene]. ’, BHA; &, BHT; }, a-
BHA: buthylated hydroxyanisole; BHT: buthylated hydroxytoluene]. tocopherol; W, water extract of fennel; m, ethanol extract of fennel.
’, BHA; &, BHT; B, a-tocopherol; W, water extract of fennel; m,
ethanol extract of fennel.

method of Ruch et al. (1989). The scavenging ability of between phenolic content and antioxidant activity (Yen,
water and ethanol extracts of FS on hydrogen peroxide Duh, & Tsai, 1993; Yang, Lin, & Mau, 2002), whereas
is shown Fig. 7 and compared with BHA, BHT and a- the others found no such relationship, since other
tocopherol as standards. The both FS extracts were compounds are responsible for the antioxidant activity
capable of scavenging hydrogen peroxide in an amount- (Bocco, Cuvelier, Richard, & Berset, 1998; Maillard
dependent manner. 250 mg of water and ethanol extracts & Berset, 1995; Heinonen, Lehtonen, & Hopia, 1998;
of FS exhibited 53.8% and 45.5% scavenging activity on Kahkonen et al., 1999). The phenolic compounds may
hydrogen peroxide, respectively. In the other hand, at contribute directly to antioxidative action (Duh et al.,
the same dose, BHA, BHT, and a-tocopherol exhibited 1999). It is suggested that polyphenolic compounds have
37.5%, 86%, and 57% hydrogen peroxide scavenging inhibitory effects on mutagenesis and carcinogenesis in
activity. These results showed that the both FS extracts humans, when up to 1.0 g daily ingested from a diet rich
had stronger hydrogen peroxide scavenging activity. in fruits and vegetables (Tanaka, Kuei, Nagashima, &
Those values close to BHA, but lower than that BHT Taguchi, 1998).
and a-tocopherol. There was statistically significant As a conclusion, the water and ethanol extracts
correlation between those values and control of FS showed strong antioxidant activity, reducing
ðPo0:05Þ: The hydrogen peroxide scavenging effect of power, DPPH radical, superoxide anion scavenging,
250 mg of the both extracts of FS and standards hydrogen peroxide scavenging, and metal chelating
decreased in the order of BHT>a-tocopherol>water activities when compared to standards such as BHA,
extract of FS>BHA>ethanol extract of FS. Hydrogen BHT, and a-tocopherol. The results of this study
peroxide itself is not very reactive, but it can sometimes show that the water and ethanol extract of FS can be
be toxic to cell because of it may give rise to hydroxyl used as easily accessible source of natural antioxidants
radical in the cells (Halliwell, 1991). Thus, the removing and as a possible food supplement or in pharmaceutical
of H2O2 is very important for antioxidant defence in cell industry. It can be used in stabilising food against
or food systems. oxidative deterioration. However, the polyphenolic
compounds or other components responsible for the
3.7. The total phenolic compounds antioxidant activity of water and ethanol extracts
of FS are already unknown. Therefore, it is suggested
The phenolic compounds are very important plant that further work could be performed on the iso-
constituents because of their scavenging ability due to lation and identification of the antioxidant components
their hydroxyl groups (Hatano, Edamatsu, Mori, Fujita, in FS.
& Yasuhara, 1989). It was determined that there were
21, 25 and 90.0 mg gallic acid equivalent of phenolic
compounds in the 1 mg of the water and ethanol extracts
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