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929
Advances in Environmental Biology, 5(5): 929-932, 2011
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Antioxidant Activity, Total Phenolic and Flavonoids Contents in Methanolic and
Aqueous Extract of Achillea millefolium L.
1
Anoosh Eghdami, 2Vahid Fateh, 2Davood Eradatmand Asli and 2Alireza Houshmandfar
1
Department of Biology, Islamic Azad University, Saveh Branch, Saveh, Iran
Department of Agronomy and Plant Breeding, Islamic Azad University, Saveh Branch, Saveh, Iran
2
Anoosh Eghdami, Vahid Fateh, Davood Eradatmand Asli and Alireza Houshmandfar; Antioxidant
Activity, Total Phenolic and Flavonoids Contents in Methanolic and Aqueous Extract of Achillea
millefolium L.
ABSTRACT
Achillea millefolium L. sensu lato (yarrow) is the best-known species of the genus Achillea due to
numerous medicinal applications both in folk and conventional medicine. Phenolic compounds such as
flavonoids and phenol carbonic acids which present in yarrow are constitute one of the most important groups
of pharmacologically active substances. In the present study, Achillea millefolium L. flowers gathered from
native populations in different locations of Saveh region of Iran were analyzed for composition of phenolic
compounds. The extract was analyzed by aluminum chloride (AlCl3) method using the Folin-Ciocalteu reagent.
The antioxidant activity was further tested by the DPPH (2,2'-diphenyl-1-picrylhydrazyl) free radical scavenging
method. High-performance liquid chromatography (HPLC) was used for chemical analyze. The extract of
Achillea millefolium L showed scavenging effects against the DPPH radical. Furthermore, the antioxidant and
total phenolic content levels were positively and significantly correlated.
Key words: Phenolic compounds, DPPH, Antioxidants, Folin-Ciocalteou
Introduction
Phenolic compounds are secondary metabolites
that are derivatives of the pentose phosphate,
shikimate, and phenylpropanoid pathways in plants
[18,9,5]. Phenolics are antioxidants with redox
properties, which allow them to act as reducing
agents, hydrogen donators, and singlet oxygen
quenchers [12]. Antioxidants are important because
they have the ability of protecting organisms from
damage caused by free radical-induced oxidative
stress [2]. Besides well known and traditionally used
natural antioxidants from tea, fruits, vegetables and
spices, some natural antioxidant are already exploited
commercially either as antioxidant additives or a
nutritional supplements [16]. In this regard, although
many of plant species have been investigated in the
search for novel antioxidants [3], there is still a
demand to find more information concerning the
antioxidant potential of plant species. Flavonoids are
a group of poly phenolic compounds with known
properties included free radical scavenging, inhibition
of hydrolytic and oxidative enzymes, and antiinflammatory action. Some evidence suggests that the
biological actions of these compounds are related to
their antioxidant activity [8,13]. According to
physiological properties of different flavonoids, the
flavonols having orto or para hydroxyl group in the
2- phenyl ring are known to have strong antioxidant
properties, while free hydroxyl at the 5,7- position
proved to have a pro-oxidant effect (Table 1).
Achillea millefolium L. has a long history of use as
traditional herb medicine even in veterinary medicine,
preparations in the form of infusions, decoctions or
fresh juices have been applied against anorexia,
stomach cramps, flatulence, gastritis, enteritis, internal
Corresponding Author
Anoosh Eghdami, Department of Biology, Islamic Azad University, Saveh Branch, Saveh, Iran
E-mail: [email protected]
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Adv. Environ. Biol., 5(5): 929-932, 2011
and external bleeding (coughing blood, nosebleed,
hemorrhoidal and menstrual bleeding, bloody urine),
wounds, sores, skin rash as well as dog and snake
bites. In the present study, Achillea millefolium L.
flowers were analyzed for composition of phenolic
compounds.
followed by 5% NaNO2 (0.03 ml). Then, AlCl3 (0.03
ml, 10%) was added, after 5 min and at 25°C. After
further 5 min, the reaction mixture was treated with
0.2 ml of 1 mM NaOH. Finally, the reaction mixture
was diluted to 1 ml with water and the absorbance
was measured at 510 nm. The results were expressed
as mg quercetin (QE) g-1 of Achillea millefolium L.
Materials and methods
Antioxidant Activity:
Plant Collection:
The plants were collected in July 2010 from
Saveh region (35°15´ N, 49°45´ E; 1680 m above
mean sea level), located in Markazi state of Iran.
Chemicals:
2,2'-diphenyl-1-picrylhydrazyl (DPPH) and
quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy4H-chromen-4-one) were purchased from Sigma
Chemical Co. (St., Louis, USA). Gallic acid, FolinCiocalteu reagent and methanol were purchased from
Merck Co. (Germany).
Sample Preparation:
The samples were first ground to fine powder.
For water extraction, 0.5 g of the fine powder was
extracted with 10 ml of ultra-filtered water at 100 °C
for 30 min in a water bath. For methanol extraction,
0.5 g of the powder was extracted with 10 ml of
80% methanol at 40 °C for 24 h. The samples were
then cooled down to room temperature and
centrifuged at 4500 rpm for 15 min. The supernatant
was recovered and used for the DPPH assay and
total phenolic analysis.
Determination of Total Phenolic Content:
The total phenolic content of the Achillea
millefolium L. extracts was determined using the
Folin-Ciocalteu reagent [19]. The reaction mixture
were contained 200 μl of diluted rice bran extract,
800 μl of freshly prepared diluted Folin-Ciocalteu
reagent and 2 ml of 7.5% sodium carbonate. The
final mixture was diluted to 7 ml with deionized
water. Mixtures were kept in dark at ambient
conditions for 2 h to complete the reaction. The
absorbance at 765 nm was measured. Gallic acid was
used as standard and the results were expressed as
mg of Gallic acid equivalents (GAE) g-1 of Achillea
millefolium L.
Determination of Total Flavonoid Content:
Total flavonoid content was determined using
aluminium chloride (AlCl3), and quercetin (standard)
as described by Ordon Ez et al. [11]. The plant
extract of 0.1 ml was added to 0.3 ml distilled water
The antioxidant activity was determined using
DPPH free radical scavenging method. A dilution of
1 M DPPH was prepared and the absorbance of a
mixture of 1 ml of the extract and 1 ml of the DPPH
solution was measured at 517 nm. The radical
scavenging activity was calculated from the following
equation [1]:
HPLC Analysis:
The analysis was performed with a flow rate of
0.75 mL min-1, using 0.2% trifluoroacetic acid (TFA)
as solvent A and Methanol as solvent B, with a
linear gradient from 5 to 80% methanol in 50 min
(Table 2). All solvent used were filtered on Acetate
Plus (0.22) before analysis. The selected flavonoid
standards required a greater concentration of
Methanol (80%) and a longer HPLC run for their
proper elution than phenolic acids. Each standard was
first injected individually to determine the exact
retention time and chromatographic characteristics
(.max, absorbance ratio) followed by the analysis of
the standard mixture. The methanolic extract of
Achillea millefolium L was filtered on qualitative
circle, Whatman filter paper No. 1 (Whatman
International Ltd., UK) under vacuum and
subsequently on Acetate Plus filters (0.22 μm) to
remove the undesirable contaminants. HPLC analysis
was performed using a liquid chromatographic
Agilent 1200 equipped with UV detector G1314B.
Separations were carried out using an analytical
column (150 × 4.6 mm, 5 μm) and with G1311A
quaternary pump. All the analysis was performed in
triplicate.
Results and discussion
Table 3 indicated the total phenolic and
flavonoids contents and antioxidant activities of
methanol and aqueous extract of Achillea millefolium
L. Furthermore, HPLC chromatograms for some of
polyphenol from methanolic exteract of Achillea
millefolium L. and for some of polypehnol standard
were presented in Figure 1 and 2 respectively. The
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Adv. Environ. Biol., 5(5): 929-932, 2011
Table 1:. Some of the flavonoids compounds name and structure
Table 2:. Gradient elution method performed with binary solvent system using
0-5 5-8 8-11 11-14 14-17 17-20 20-23 23-26 26-29 29-32
A
5
10 15
20
25
30
35
40
45
50
B
95
90 85
80
75
70
65
60
55
50
as mobile phase
32-35 35-38
55
60
45
40
38-41
65
35
41-44
70
30
44-47
75
25
47-50
80
20
Table 3; Total phenolic and flavonoids contents and antioxidant activities of methanol and aqueous extract of Achillea millefolium L.
Variety
Phenolic content
Flavonoids content
Antioxidant activity by
(QE g-1)
DPPH (Inhibition %)
(mg GAL g-1)
Methanolic extract
123.9 ± 2.6
41.2 ± 1.7
75%
Aqueous extract
48.4 ± 2.7
13.15 ± 1.8
50.8%
Fig. 1: HPLC chromatogram for some of polypehnol standard (gallic acid, vanillic acid, coumaric acid and
benzoic acid respectively).
Fig. 2: HPLC chromatogram for some of polyphenol from methanolic exteract of Achillea millefolium L.
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Adv. Environ. Biol., 5(5): 929-932, 2011
analysis of the extract of Achillea millefolium L.
showed scavenging effect against DPPH radical. The
hierarchy for antioxidant capacity with respect to
their EC50 values was methanolic extract > aqueous
extract. Correlation coefficient showed that total
phenolic content was responsible for antiradical
efficiency in the extract of Achillea millefolium L.
The antioxidant and total phenolic content levels are
also positively and significantly correlated. It has
been recognized that flavonoids show antioxidant
activity and their effects on human nutrition and
health are considerable [13]. The mechanisms of
action of flavonoids are through scavenging or
chelating process [7,3]. Phenolic compounds are a
class of antioxidant agents which act as free radical
terminators [17]. The results strongly suggested that
the extract of Achillea millefolium L. can be a
promising source of potential antioxidants. The
antioxidant activities observed can be ascribed both
to mechanisms exerted by phenolic compounds and
also to synergistic effects of different
phytocompounds.
Each value in the table was obtained by
calculating average of three experiments; SE (±),
standard error.
7.
8.
9.
10.
11.
12.
13.
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