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Pak. J. Anal. Environ. Chem. Vol. 9, No. 2 (2008) 78 – 83
Antioxidative Activity of Extracts from Fenugreek
Seeds (Trigonella foenum-graecum)
Syeda Birjees Bukhari*, Muhammad Iqbal Bhanger and Shahabuddin Memon
National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080, Pakistan
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Abstract
Spices and herbs possess antioxidant activity and can be applied for preservation of lipid
peroxidation in biological systems. Fenugreek (Trigonella foenum-graecum) is an important spice;
its dried seeds have wide application in food and beverages as a flavoring additive as well as in
medicines. Crude extracts of fenugreek were prepared by soxhelt extraction method with different
solvents such as methanol, ethanol, dichloromethane, acetone, hexane and ethyl acetate. Extracts
were subjected for the measurement of total phenolic content (TPC) by Folin-Ciocalteu method as
well as flavonoid content, chelating activity, reducing power and antioxidant/radical scavenging
activity [1,1-diphenyl-2-picryl-hydrazyl (DPPH°) free radical scavenging activity]. Results from
different parameters were in agreement with each other. The results reveal that all extracts of the
fenugreek exhibit antioxidant activity. These findings suggest that the fenugreek extracts could act
as potent source of antioxidants.
Keywords: Fenugreek (Trigonella foenum-graecum), Antioxidant activity, Phenolic contents,
Flavonoids.
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Introduction
Herbs and spices have been extensively used as food
additives for natural antioxidants. Spices and aromatic
herbs are considered to be essential in diets or medical
therapies for delaying aging and biological tissue
deterioration [1]. The search for synthetic antioxidants
as alternatives to naturally occurring antioxidants is of
great interest both in industry as well as in scientific
research [2]. The antioxidant property of the plant
material is due to the presence of many active
phytochemicals including vitamins, flavonoids,
terpenoids, carotenoids, cumarins, curcumins, lignin,
saponin, plant sterol and etc [2-6].
Fenugreek (Trigonella foenum graecum) is an
annual herb that belongs to the family Leguminosae
widely grown in Pakistan, India, Egypt, and Middle
Eastern countries [7]. Due to its strong flavor and aroma
fenugreek in one of such plants whose leaves and seeds
are widely consumed in Indo-Pak subcontinent as well
as in other oriental countries as a spice in food
preparations, and as an ingredient in traditional
medicine. It is rich source of calcium, iron, â-carotene
and other vitamins [8]. Both leaves and seeds should be
included in normal diet of family, especially diet of
growing kids, pregnant ladies, puberty reaching girls
and elder members of family because they have
haematinic (i.e. blood formation) value [9]. Fenugreek
seed is widely used as a galactagogue (milk producing
agent) by nursing mothers to increase inadequate breast
milk supply [10]. The seeds of fenugreek contain lysine
and L-tryptophan rich proteins, mucilaginous fiber and
other rare chemical constituents such as saponins,
coumarin, fenugreekine, nicotinic acid, sapogenins,
phytic acid, scopoletin and trigonelline, which are
thought to account for many of its presumed therapeutic
effects, may inhibit cholesterol absorption and thought
to help lower sugar levels [11-13]. Therefore, fenugreek
seeds are used as a traditional remedy for the treatment
of diabetes and hypercholesterolemia in Indian and
Chinese medicines [14,15]. It’s reported to have
restorative and nutritive properties and to stimulate
digestive processes, useful in healing of different ulcers
in digestive tract [16]. Fenugreek has also been reported
to exhibit pharmacological properties such as antitumor,
antiviral, antimicrobial, anti-inflammatory, hypotensive
and antioxidant activity [17, 18].
*Corresponding Author E-mail: bukhari2k4@yahoo.com
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Pak. J. Anal. Environ. Chem. Vol. 9, No. 2 (2008)
79
The purpose of this study was to evaluate
fenugreek as new potential source of natural
antioxidants. In this study, the extracts of fenugreek
were prepared in methanol, ethanol, dichloromethane,
acetone, hexane and ethyl acetate by soxhelt continuous
extraction; because organic solvents have different
polarity and therefore have different nature to extract
the compounds. The antioxidant activity of the extracts
was assessed by modification of established assays,
such as total phenolic content by Folin-Ciocalteu
reagent; total flavonoids content, chelating activity by 2,
2’ bipyridyl competition assay; antioxidant activity as
free radical scavenging by DPPH˚ and reducing power.
Material and Methods
Chemicals and Reagents
Folin-Ciocalteu reagent, methanol, ethanol,
dichloromethane, acetone, hexane and ethyl acetate
were purchased from (E. Merck). Ferrous sulphate,
Disodium ethylenediaminetetraacetate (Na2EDTA), and
butylated hydroxyanisole (BHA), were purchased from
(Fluka Riedel-de Haën). Quercetin, Gallic acid, 2, 2-
bipyridyl, HCl and 2,2-diphenyl-1-picrylhydrazyl
(DPPH) were purchased from (Sigma-Aldrich GmbH,
Germany). All other chemicals and solvents were of the
highest analytical grade and used as supplied.
Plant Material and Extraction procedures
A fenugreek seed sample was collected from
the local market of Hyderabad, Pakistan. Fenugreek
seed subjected to various treatments for investigation of
antioxidant potential. Extraction was carried out by the
reported procedure [19]. Dry fenugreek seed (10 g) was
cleaned and ground into small pieces by a waring
blender and passed through a 1-mm sieve. Methanol,
ethanol, dichloromethane, acetone, hexane and ethyl
acetate (each 150 ml) were used for extraction by
soxhelt extraction method for six hours. The extracts
were filtered. The residue was re-extracted twice under
the same condition to ensure complete extraction. The
extracts were combined, filtered and evaporated to
dryness under reduced pressure at 60 oC by a rotary
evaporator. Extracts were placed in dark bottle, and
stored at -8 °C until further analysis.
Yield Estimation
Yield was estimated by reported method [20].
Each extract (10 ml) was measured into a pre-weighed
aluminum dish. The samples were kept in an oven at 85
°C for 24 hours then followed by placing in desiccator
for 12 hours. The weight difference was used to
calculate percentage yield as well as expressed in
mg/10 ml.
Determination of Total Phenolic Content (TPC)
TPC in different solvent extracts of fenugreek
seeds was determined spectrophotometrically following
Folin-Ciocalteu method described previously [21, 22]
with minor modification. The appropriate dilution of
extract 200 µl oxidized with 1 ml of Folin-Ciocalteu
reagent, and then the reaction mixture was neutralized
with saturated 2 ml of 7.5 % sodium carbonate (w/v).
The final mixture volume was brought up to 7 ml with
deionized water. The absorbance of the resulting blue
color was measured at 765 nm on UV-Vis.
spectrophotometer with a 1 cm cell after incubation for
2 hours in dark at room temperature. Gallic acid was
used as a standard for the calibration curve. The
phenolic compound content was determined as gallic
acid equivalents using the following linear equation
based on the calibration curve.
A= 0.1786 C - 0.1739, R2 = 0.999
A is the absorbance, and C is gallic acid
equivalents (mg).
Determination of Total Flavonoid Content
The total flavonoid content was measured by
using previously reported colorimetric assay [23] with
minor modifications. Briefly 1ml of appropriately dilute
sample was added to a 10ml of volumetric flask
containing 4 ml of distilled water followed by
immediate addition of 0.6 ml of 5% NaNO2, 0.5 ml of
10% AlCl3 after 5 min, and 2 ml of 1 M NaOH after 1
min. Furthermore, each reaction flask was then
immediately diluted with 2.4 ml of distilled water and
mixed. The absorbance of pink colored solution was
noted at 510 nm. The quercetin (µg/g) was used as a
standard for the calibration curve. The total flavonoid
content of the samples was calculated by using the
following linear equation based on calibration curve.
Y = 0.0205X – 1494, r = 0.9992
Y is the absorbance, and X is the flavonoid
content in µg g-1.
Chelating Activity
Chelating activity (Fe2+) was measured by 2,
2´-bipyridyl competition assay [24]. The reaction
mixture containing 0.25 ml of of FeSO4 solution (1
mM), 0.25 ml of antioxidant solution, 1 ml of Tris-HCl
buffer (pH 7.4), 1 ml 2,2´-bipyridyl solution (0.1% in
0.2 M HCl) and 2.5 ml of ethanol. The final volume was
made up 6.0 ml with distilled water. The absorbance
was measure at 522 nm and used to evaluate Fe+2
chelating activity using disodium ethylenediamine-
tetracetate (Na2EDTA) as a standard.
Pak. J. Anal. Environ. Chem. Vol. 9, No. 2 (2008)
80
Measurement of Antioxidant Properties
Reducing Power Ability (RPA)
The reducing power of fenugreek extracts was
quantified by the method described previously [25] with
minor modification. Fenugreek extract (0, 1.0, 2.0, 3.0,
5.0, 7.0, 9.0, 11.0 mg) in 1 ml of 80% methanol were
mixed with phosphate buffer (5.0 ml, 2.0 M, pH 6.6)
and potassium ferricyanide (5.0 ml, 1.0%) ; the mixtures
were incubated at 50 ˚C for 20 min. A portion (5.0 ml)
of trichloroacetic acid (10%) was added and the mixture
was centrifuged at 3000 rpm for 10 min. The upper
layer of the solution (5.0 ml) was mixed with distilled
water (5.0 ml) and ferric chloride (1.0 ml, 0.1%), and
than absorbance of the pink color mixture was measured
spectrophotometrically at 700 nm. Increased absorbance
of the mixture indicates increased reducing power. The
experiment was conducted in triplicate and results were
averaged.
Free Radical Scavenging (FRS) Activity
Free radical scavenging capacity of fenugreek
extracts was determined according to the previous
reported procedure using the stable 2, 2-diphenyl-1-
picrylhydrazyl radical (DPPHo) [26, 27]. Briefly, a
freshly prepared DPPH° solution in ethanol (0.5 ml)
was added to 3 ml of diluted each fenugreek extract to
start the radical antioxidant reaction. The final
concentration was 100 µM for DPPH°. The decrease in
absorbance was measured at different intervals, i.e. 0,
0.5, 1, 2, 5, 10 and 15 min. up to 50% at 517 nm. The
remaining concentration of DPPHo in the reaction
mixture was calculated from a standard calibration
curve. The absorbance measured at 5min of the
antioxidant-DPPHo radical reaction was used to
compare the DPPHo radical scavenging capacity of each
fenugreek extract.
% of DPPH remaining= [DPPH] T/ [DPPH] T=0 ×100
Where T is the time interval.
Statistical Analysis
Three replicates of each sample were used for
statistical analysis. Data were reported as means ± S.D.
Analysis of variance and least significant difference
tests were conducted to identify differences among
means. Statistical significance was declared at P<0.05.
Results and Discussion
The yields of the extracts obtained by the
soxhelt method were calculated as percent by weight of
the fenugreek seed. According to the chemical
composition and polar nature of phenolic compounds,
fenugreek contains a relatively high percentage yield in
ethanol and methanol while lower in hexane. Percentage
yields in ethanol and methanol are comparable but have
slight difference as shown in Table 1.
Table 1. Percentage yield of fenugreek extract in different organic
solvents as well as in mg/10ml.
Data are means (n = 3) ± SD (n = 3), (p<0.05)
The phenolic compounds may contribute
directly to the antioxidant action [28]; therefore, it is
necessary to investigate total phenolic content. The total
phenolic content was determined by following a
modified Folin-Ciocalteu reagent method. In Table 2 the
results were expressed as gallic acid equivalent. TPC
was in the range of 1.35-6.85 mg/g of the fenugreek
extract. The amounts of total phenolic compounds were
higher in ethanol extract 6.85 mg/g while lowest for
hexane 1.35 mg/g. Using a standard curve of gallic acid
(R2= 0.999). All results coincide with those of total
antioxidant capacity. In other words, the spice extract
sample shows a tendency to have high phenolic content.
Table 2. Total phenolic content (TPC), flavonoid content (FC) and
chelating activity of organic solvent extracts of fenugreek
expressed as gallic acid, quercetin and Na2EDTA equivalent,
respectively.
Sample TPC Gallic
Acid eq.
(mg/g of
fenugreek)
FC Quercetin
eq.
(µg/g of
fenugreek)
Chelating Activity
EDTA eq (µg/g of f
enugreek)
Methanol 5.75 ± 0.002 607. ± 3.6 1021 ± 1.7
Ethanol
6.85 ± 0.002 653 ± 4.3 1098 ± 2.4
Dichloromethane
2.27 ± 0.003 234 ± 3.5 633 ± 2.3
Acetone 4.04 ± 0.004 416 ± 2.7 982 ± 2.1
Hexane 1.35 ± 0.002 208 ± 4.2 557 ± 3.2
Ethylacetat 3.32± 0.004 251 ± 3.3 838 ± 2.8
Data are mean (n = 3) ± Standard deviation (n=3), (p<0.05),
TPC = Total phenolic content.
Organic solvents Yield (mg/10ml) %yield of fenugreek
extract
Methanol 64.72 25.89
Ethanol 63.3 25.32
Dichloro methane 32.4 12.96
Acetone 44.1 17.65
Hexane 24.2 9.68
Ethyl acetate 40.3 16.13
Pak. J. Anal. Environ. Chem. Vol. 9, No. 2 (2008)
81
Using the AlCl3 reagent and quercetin as
standard (R2 = 0.9996), the total flavonoids are in the
range from 208-653 µg/g of quercetin equivalent (Table
2). The highest value for the ethanol was 653 µg/g and
the lowest was 208 µg/g of the fenugreek with the
following decreasing order of the extract ethanol>
methanol> acetone> ethyl acetate> dichloromethane>
hexane. Flavonoids are not easily detectable therefore,
in the extract AlCl3 was used as complexing reagent.
The chelating activity was measured against
Fe2+ and reported as EDTA equivalents as shown in
Table 2. The difference in chelating activity was
observed among the extract. The highest chelating
activity was observed in ethanol. The EDTA equivalent
was in the range of 1098-557 µg/g of fenugreek extract.
According to Ilhami et al. [29] metal chelating capacity
is significant since it reduces the concentration of
catalyzing transition metal in lipid peroxidation.
Moreover, the chelating agents, which form ó-bonds
with a metal, are effective as secondary antioxidants,
because they reduce the redox potential thereby
stabilizing the oxidized form of the metal ion, therefore
it is an important parameter. The results from this
parameter were in agreement with total phenolic
contents, the highest chelating activity were found in
ethanol (1098 µg/g of extract) while lowest in hexane
(557 µg/g of extract).
Antioxidant capacity
The antioxidant capacity of the fenugreek
extracts were analyzed by using the free radical
scavenging (DPPHo) (Fig. 1) and the ferric reducing
antioxidant power (FRAP) methods (Fig. 2).
The DPPHo test is the oldest indirect method
for determining the antioxidant activity, which is based
on the ability of the stable free radical 2, 2-diphenyl-1-
picrylhydrazyl to react with hydrogen donors including
phenols [30].
Radical scavengers may directly react and
quench with peroxide radicals to terminate the
peroxidation chain reaction and improve the quality and
stability of food product. The stable DPPH radical has
been used to evaluate antioxidants for their radical
quenching capacity [31,32] and to better understand
their antioxidant mechanism(s) each fenugreek extract
was evaluated for radical scavenging activity against
DPPHo. The decrease in absorbance of DPPH radical is
caused by antioxidant through the reaction between
antioxidant molecule and radical results in the
scavenging of the radical by hydrogen donation. As
Fig. 1a illustrates a significant (p<0.05) decrease in the
concentration of DPPHo due to scavenging activity of
fenugreek extract.
DPPH Radical scavenging
0
20
40
60
80
100
120
0 5 10 15 20
Time (min)
DPPH remaining %
DPPH
Methanol
Ethanol
Dicholoro methane
Acetone
Hexane
Ethyl acetate
Figure 1 a. Kinetic behavior of radical scavenging activity of
cumin extracts as assayed by the DPPH˚ method. The final DPPH
concentration was kept 100 µM in all reaction mixtures. Values
are mean (n =3), (P < 0.05).
%DPPH 5min
0 20 40 60 80 100 120
Control
Methanol
Ethanol
Dichloro methane
Acetone
Hexane
Ethylacetate
Fenungreek Extract
% DPPH Remaining
Figure 1 II. DPPH radical scavenging activity of fenugreek
extract at 5 min. Vertical bars represents the standard deviation
of each data point. Values are mean (n = 3), (P < 0.05).
Kinetic studies of DPPHo-extract reaction were
carried out to estimate scavenging activity as a function
of time. Scavenging activity was nearly the same at first
minute of reaction and diverges with the increase in
time. Maximum difference among the extract was
observed at 5 min of the reaction and the remaining
amount (%) of DPPH° radical at 5 min after initiation of
reaction as shown in Figure 1b was 10.88, 12.42, 24.04,
18.38, 25.77 and 21.97 for ethanol, methanol,
dichloromethane, acetone, hexane and ethyl acetate
respectively. The high amount of the phenolic
compounds and reducing power having the highest
percent DPPH° scavenging activity was shown by the
ethanol extract, and the second highest activity was
determined in the methanol while lowest in hexane.
Pak. J. Anal. Environ. Chem. Vol. 9, No. 2 (2008)
82
It has been reported by Yildirim et al and
Siddhuraju et al [33, 34] that the reducing power of
bioactive compounds is associated with antioxidant
activity. Thus, it is necessary to determine the reducing
power of phenolic constituents to elucidate the
relationship between their antioxidant effects and there
reducing power [35]. The reducing power of the extracts
increases with an increase in the amount of the extract
as shown in Fig. 2. The amount of the phenolic
compounds was high in ethanol extract of fenugreek,
therefore; similar results were obtained in reducing
power activities. Hence, by correlating these results; we
can suggest that there may be relationship between the
amount of total phenolic content and reducing power.
0
0.5
1
1.5
2
2.5
3
0 0.2 0.4 0.6 0.8 1 1.2
Conce ntration mg/ml
Absorbance
BHA
Methanol
Ethanol
Dichloromethane
Acetone
Hexane
Ethylacetate
Figure 2. Reducing power of ethanol, methanol, dichloromethane,
acetone, hexane and ethyl acetate extract of fenugreek. All data is
reported as mean ± S.D (n = 3) statistically significant as P < 0.01.
The reducing power of the extracts was
compared with a known reducing agent BHA. The
reducing power of the extract was markedly lower than
that of BHA. However, among these extracts the ethanol
extract of fenugreek has shown the highest reducing
power. According to Shimon et al. [36] the fenugreek
has volatile oil, phenolic acids and flavonoids; therefore
it is a potent source of antioxidants.
Conclusion
From the present work, it could be concluded
that the solvent play a vital role in the extraction of the
plant constituents. As methanol and ethanol are highly
polar among the solvents used therefore, they contain
high yield of phenolic compounds as compared to the
other solvents. An ethanolic extract of fenugreek seeds
was shown highest antioxidant activity (% DPPH°
scavenging activity). The antioxidant activity could be
correlated with the polyphenolic components present in
the extract. The results obtained from these methods
provide some important factors responsible for the
antioxidant potential of fenugreek seeds.
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