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International Journal of Food Properties, 14:339–355, 2011
Copyright © Taylor & Francis Group, LLC
ISSN: 1094-2912 print / 1532-2386 online
DOI: 10.1080/10942910903189256
IN VITRO ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY
OF ETHANOL EXTRACT OF THREE HYPERICUM AND
THREE ACHILLEA SPECIES FROM TURKEY
Deniz Barı¸s1, Murat Kızıl1*, Çetin Aytekin1, Göksel Kızıl1,
Murat Yavuz1, Bircan Çeken1, and A. Selçuk Ertekin2
1University of Dicle, Faculty of Science and Arts, Chemistry Department,
Diyarbakır, Turkey
2University of Dicle, Faculty of Science and Arts, Biology Department,
Diyarbakır, Turkey
The present study was conducted to determine the antimicrobial, antifungal and antioxidant
activity of the ethanol extract of Hypericum scabrum L (HSm), Hypericum lysimachioides
var. lysimachioides (HL), and Hypericum retusum Aucher (HR) and ethanol extracts of
Achillea aleppica D.C. subsp. aleppica (AA), Achillea aleppica D.C. subsp. zederbaueri
(Hayek) Hub.-Mor (AZ), and Achillea biebersteinii Afan. (AB). The antioxidant properties
of extracts were evaluated using different antioxidants tests, including reducing power, free
radical scavenging, deoxyribose assay, metal chelating activities and determination of total
phenolic compounds. The extracts obtained from Hypericum and Achillea species showed
high antioxidant properties. The protective effects of plant extracts were compared with a
well known antioxidant, Butilated Hydroxytoluen (BHT) and α-tocopherol. Total antioxi-
dant activity of ethanol extracts of plants were also tested by using ferric thiocyanate (FTC)
and thiobarbituric acid (TBA) methods. Antioxidative activities of plant extracts were found
to be comparable with Vitamin E. The results showed that the ethanol extracts of all tested
plant exhibited different activity against tested microorganisms. Since most of the studied
extracts have good antimicrobial and antioxidant activity, it might be possible to use them as
natural food additives that act both as antioxidants and as spices.
Keywords: Antimicrobial activity, Antioxidant activity, Achillea,Hypericum.
INTRODUCTION
Oxidative stress plays an important role in various degenerative diseases as well as
in the normal process of aging.[1] Oxidative stress is initiated by reactive oxygen species
(ROS), such as superoxide anion (O2−), perhydroxy radical (HOO•) and hydroxyl radical
(HO•). These radicals are formed by a one electron reduction process of molecular oxy-
gen (O2). Most living species have efficient defense system to prevent themselves against
oxidative stress induced by ROS.[2] Recent investigations have shown that the antioxidant
properties of plants could be correlated with oxidative stress defense and different diseases
like aging process, etc.[3–6] In this respect flavonoids and other polyphenolic compounds
Received 27 March 2009; accepted 15 July 2009.
Address correspondence to Murat Kızıl, University of Dicle, Faculty of Science and Arts, Chemistry
Department, 21280, Diyarbakır, Turkey. E-mail: muratk@dicle.edu.tr
339
340 BARI¸SETAL.
have received the greatest attention.[7] In view of the beneficial role of herbs in the food
industry, and the present understanding about the role of oxidative stress in pathogenesis
of multiple diseases, attempts have been made to examine the antioxidant status of some
herbal products.[8] Recently, the definition of health has no longer been restricted to the
absence of disease, but includes physical fitness, as well as mental and physiological well-
being. For the development, growth and maintenance of the body, food is required, but food
is also recognized to play a key role in the quality of life. Functional foods are those foods
that positively affect one or more target functions in the body, beyond the basic nutritional
function, in a way that it is relevant to either an improved state of health and well-being
and/or reduction of risk of disease.
Currently, many kinds of synthetic antioxidants such as BHA (butylated hydrox-
yanisol), BHT (butylated hydroxytoluene), TBHQ (tertiary butyl hydroquinone) and propyl
gallate have been used as additives for oxidation suppressant in food, cosmetic and drug
compositions. However, the use of these synthetic antioxidants for food or medicine com-
ponents has been restricted due to the toxicity and safety that can lead to the problems of
the potential health in human. Due to these reasons, many researchers have tried to find
more effective oxidation inhibitors that may be used as antioxidants for food or medicine
compositions without the side effects for the past several years. Many researchers have
paid attention to find out natural antioxidants that can be used without toxicity in human.
The genus Hypericum, which contains more than 400 species, occurs throughout the
world and is well represented in the Mediterranean and the Near East Areas. In Turkey, the
genus is represented by 89 species of which 43 are endemic.[9] Recently, there has been
increasing interest in the genus Hypericum because it is a source of a variety of compounds
with different biological activities.[10] The species Hypericum triquetrifolium Turra has
been ethonomedically used in different parts of Turkey for its sedative and antiseptic effect.
Pharmacological experiments demonstrated that Hypericum scabrum L. showed antiul-
cerogenic activity.[11] Hypericum species have received considerable interest due to the
increasing market demand for crude material of Hyperici herba. The genus Achillea is also
represented by about 85 species throughout the world and 42 of them found in the flora of
Turkey.[12] As far as ethnopharmacologic background is concerned, Achillea millefolium is
a well known species amongst the members of Achillea. It is known as ‘civanperçemi’ and
used in folk remedies as an wound healer, diuretic or carminative. Achillea biebersteinii is
called ‘Sarı çiçek’ in Turkish, and is known to be used in folk remedies for various purposes
such as the treatment of abdominal pain, particularly stomachache.[13 –14] The antimicrobial
activities of the essential oils or various extracts of several Hypericum and Achillea species
have been exhibited against gram positive and gram negative bacteria.[15–20 ] Recent studies
have demonstrated that, different species of Hypericum and Achillea contains compounds
such as flavonoids, xanthones and phenolic and can be used as antioxidants.[21–24 ]
The antioxidant activity of phenolic compounds in plants is mainly due to their
redox properties and chemical structure, which can play an important role in neutraliz-
ing free radicals, chelating transitional metals and quenching singlet and triplet oxygen,
by delocalization or decomposing peroxides.[25] These properties are linked to beneficial
health functionality of phenolic antioxidants due to their inhibitory effects against devel-
opment of many oxidative-stress related diseases such as cardiovascular disease, cancer
and diabetes. In addition some plant phenolics have shown antimicrobial and antifungal
effects.[26] Antioxidant and antimicrobial properties of several Hypericum and Achillea
species were reported in the literature.[27 –30] As a part of our continuing research project on
in-vitro antimicrobial and antioxidant activities of several Hypericum and Achillea species,
ACTIVITY OF HYPERICUM AND ACHILLEA SPECIES 341
we previously reported antioxidant and antimicrobial activities of several Hypericum
species and also the protective ability of ethanol extracts of Hypericum triquetrifolium
and Hypericum scabroides against the protein oxidation and DNA damage.[31, 15,32]
The present investigation was carried out on these two important herbal prod-
ucts. Therefore, the antimicrobial and antioxidant activities of the ethanol extracts of
three Hypericum (Hypericum scabrum L., Hypericum lysimachioides var. lysimachioides,
Hypericum retusum Aucher), and three Achillea (Achillea aleppica D.C. subsp. aleppica,
Achillea aleppica D.C. subsp. zederbaueri (Hayek) Hub.-Mor, and Achillea biebersteinii
Afan.) species collected from South East Turkey were tested.
MATERIALS AND METHODS
Collection of Plant Material
Plants Hypericum scabrum L (HSm), H. lysimachioides var. lysimachioides (HL),
Hypericum retusum Aucher (HR), Achillea aleppica subsp. aleppica (AA), Achillea alep-
pica subsp. zederbaueri (Hayek) Hub.-Mor. (AZ), and Achillea biebersteinii Afan. (AB)
were collected in Mardin and Diyarbakır in the area of South East Turkey in June 2004, by
Prof. Dr. A. Selçuk Ertekin and Dr. Zuhal Toker. Voucher specimen have been deposited at
the Herbarium of the Department of Biology, Faculty of Science and Art, Dicle University
(voucher no. DUF-2513-a, DUF-2513-b, DUF-2513-c, DUF-9504, DUF-9500 and DUF-
9505, respectively). They were identified by Prof. Dr. A. Selçuk Ertekin from the same
institution.
Preparation of Crude Extract
Aerial parts (stems, leaves and flowers) were dried for 10 days at room temperature.
A total of 20 g of each dried material was ground in an electric blender and then incubated
into a glass flask with 2000 ml (70%) ethanol for 3 days under magnetic stirrer. Solvent was
evaporated under vacuum and the crude ethanol extracts of Hypericum scabrum (5 g) with
a dark yellow colour, H. lysimachioides (5 g) with a dark reddish colour and Hypericum
retusum (5 g) with a green colour, A. aleppica subsp. aleppica (3 g) with a dark yellow
colour, A. aleppica subsp. zederbaueri (2 g) dark yellow colour and A. biebersteinii (2 g)
dark yellow colour were obtained and kept in dark glass bottles at 4◦C until use.
Determination of Total Phenolic Compounds
The total phenolic contents of Hypericum scabrum (HSm), Hypericum retusum
(HR), A. aleppica subsp. aleppica (AA), A. aleppica subsp. zederbaueri (AZ) and
A. biebersteinii (AB) were determined using Folin-Ciocalteus reagent (Merck, Darmstadt,
Germany) according to the method of Singleton[33] as described previously.[27] Briefly,
crude ethanol extracts of (40 μl) of HSm (1 mg/ml), HR (1 mg/ml), AA (1 mg/ml),
AZ (1 mg/ml) and AB (1 mg/ml) were mixed with 200 μl Folin-Ciocalteus reagent and
1160 μl of distilled water, followed by 600 μl 20% sodium carbonate (Na2CO3) (Sigma
Aldrich, St. Louis, MO, USA) 3 min later. The mixture was shaken for 2 h at room tem-
perature and absorbance was measured at 765 nm. All tests were performed in triplicate.
Gallic acid (Sigma Aldrich, St. Louis, MO, USA) was used as a standard. The concen-
tration of total phenolic compounds in extracts were determined as a μg of Gallic acid
342 BARI¸SETAL.
equivalents per 1 mg of extract using the following equation obtained from a standard
Gallic acid graph. R2=0.9878.
Absorbance =0.0012 ×Gallic acid (μg).(1)
Scavenging Activity of DPPH Radical
The ability of the extracts to scavenge 1,1-diphenyl-2-picryl-hydrazil (DPPH)
(Sigma Aldrich, St. Louis, MO, USA) were determined using the previously reported
procedure,[34] as described before.[27] Briefly, 0.1 mM solution of DPPH in ethanol was
prepared. Then, 1 ml of this solution was added to 3 ml of each extract solution at dif-
ferent concentrations (50–500 μg). The mixture was shaken vigorously and allowed to
stand at room temperature for 30 min. Then the absorbance was measured at 517 nm in a
spectrophotometer (Shimadzu, UV/Visible Recording). Lower absorbance of the reaction
mixture indicated higher free radical scavenging activity. The radical scavenging activity
was calculated as follows:
scavenging effect (%)=(A517 of control −A517 of sample/A517 of control)×100.
(2)
Butylated hydroxytoluene (BHT) and α-tocopherol were used as positive control. IC50
value was calculated by Linear regression analysis using Prism 2.0 version software.
Metal Chelating Activity
The chelating of ferrous ions by the ethanol extracts of HSm, HL, HR, AA, AZ, AB,
and standards were estimated by the method of Dinis.[35] Briefly, extracts (50–500 μg)
were added to a solution of 2 mM FeCl2(0.05 ml). The reaction was initiated by the
addition of 5 mM ferrozine (0.2 ml) (Sigma Chemical Co., St. Louis, MO, USA), and
the mixture was shaken vigorously and left standing at room temperature for ten minutes.
After the mixture had reached to equilibrium, the absorbance of the solution was then mea-
sured spectrophotometrically at 562 nm in a spectrophotometer (Shimadzu, UV/Visible
Recording). The percentage of inhibition of ferrozine-Fe2+complex formation was given
by the formula
%inhibition =(A0−A1)/A0×100, (3)
where A0is the absorbance of the control, and A1is the absorbance in the presence of
samples of extracts or standards. The control does not contain FeCl2and ferrozine, complex
formation molecules.
Determination of Reducing Power
The reducing power of ethanol extracts of Hypericum and Achillea species were
determined according to the method of Oyaizu.[36] The different concentrations of each
ethanol extract (50–250 μg) in 1 ml of distilled water were mixed with phosphate buffer
(2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%) (Sigma
Aldrich, St. Louis, MO, USA). The mixture was incubated at 50◦C for 20 min. A portion
ACTIVITY OF HYPERICUM AND ACHILLEA SPECIES 343
(2.5 ml) of TCA (10%) was added to the mixture, which was then centrifuged for 10 min
at 1000 ×g(Centruin 8000 Series). The upper layer of solution (2.5 ml) was mixed with
distilled water (2.5 ml) and FeCl3(0.5 ml, 0.1%), and the absorbance was measured at
700 nm in a spectrophotometer (Shimadzu, UV/Visible Recording). Higher absorbance
of the reaction mixture indicated greater reducing power. Butylated hydroxytoluen (Sigma
Aldrich, St. Louis, MO, USA) and α-tocopherol (Sigma Aldrich, St. Louis, MO, USA)
were used as a standard.
Deoxyribose Assay
The reaction mixture, containing ethanol extract of HSm, HL, HR, AA, AZ,
AB (10–100 μg/ml), was incubated with deoxyribose (10 mM) (Sigma Aldrich, St. Louis,
MO, USA), H2O2(50 mM), FeCl3(10 μM), EDTA (1 mM) (Sigma Aldrich, St. Louis,
MO, USA) and ascorbic acid (10 mM) in potassium phosphate buffer (50 mM, pH 7.4) for
60 min at 37◦C.[37] Then reaction was terminated by adding 1 ml of 10% TBA (1% w/v)
and 1 ml of TCA (2% w/v) and then heating the tubes in a boiling water bath for 15 min.
The contents were cooled and the absorbance of the mixture was measured at 532 nm
against reagent blank. Decreased absorbance of the reaction mixture indicated decreased
oxidation of deoxyribose:
%inhibition =(Ac−As)/Ac×100, (4)
where Acis the absorbance of the control, and Asis the absorbance in the presence of
samples of extracts or standards.
Antioxidant Activity in Linoleic Acid System
Ferric thiocyanate (FTC) method. The method of ferric thiocyanate was fol-
lowed from Kikuzaki and Nakatani[38] which was slightly modified by Mitsuda et al.[39]
and Osawa and Namiki.[40] FTC method was used to determine the amount of peroxide at
the initial state of lipid peroxidation. The peroxide reacts with ferrous chloride (FeCl2)to
form a reddish ferric chloride (FeCl3) pigment. In this method, the concentration of per-
oxide decreases as the antioxidant activity increases. A mixture of 4 mg of each sample
was placed in 4 ml of absolute ethanol (Merck, Darmstadt, Germany), 4.1 mg of 2.52%
linoleic acid (Sigma, Aldrich Gmbh, Sternheim Germany) in absolute ethanol, 8 ml of
0.05 M phosphate buffer (pH 7.0) and 3.9 ml of water was placed in a vial with a screw cap
and then placed in an oven at 40◦C in the dark. To 0.1 ml of this solution, 9.7 ml of 75%
ethanol and 0.1 ml 30% ammonium thiocyanate (Sigma Aldrich, St. Louis, MO, USA)
was added. Exactly 3 min after the addition of 0.1 ml of 0.02 M ferrous chloride in 3.5%
hydrochloric acid (HCl) to the reaction mixture, the absorbance was measured at 500 nm
every 24 h until the absorbance of the control reached maximum. The control and standard
were subjected to the same procedures as the sample, except that for the control, only the
solvent was added, and for the standard, 4 mg sample was replaced with 4 mg of vitamin E.
Thiobarbituric acid (TBA) method. The method of Ottolenghi[41] was used to
determine the TBA values of the samples. The formation malondialdehyde is the basis for
the well-known TBA method used for evaluating the extent of lipid peroxidation. At low
pH and high temperature (100◦C), malondialdehyde binds TBA to form a red complex that
can be measured at 532 nm. The increase amount of the red pigment formed correlates with
344 BARI¸SETAL.
the oxidative rancidity of the lipid. 2 ml of 20% trichloroacetic acid (CCl3COOH) (Fisher
Scientific, Fair Lawn, NJ) and 2 ml TBA aqueous solution were added to 1 ml of sample
solution prepared as in the FTC procedure, incubated in a similar manner. The mixture
was then placed in a boiling water bath for 10 min. After cooling, it was centrifuged at
3000 rpm for 20 min and the absorbance of the supernatant was measured at 532 nm.
Antioxidant activity was determined as follows:
[(Absorbance of control on day maximum −Absorbance of sample on the same day)/
Absorbance of sample on the same day] ×100. (5)
All data about total antioxidant activity are the average of three replicates analyses.
Preparation of Test Microorganisms
Enterobacter aerogenes (clinical isolate, Gram-negative), Klebsiella oxytoca (clin-
ical isolate, Gram-negative), Streptococcus pyogenes (clinical isolate, Gram-positive),
Staphylococcus aureus (clinical isolate, Gram-positive) and Candida albicans (clinical
isolate) were obtained from the stock cultures of Microbiology Laboratory, Department
of Microbiology, Medical Faculty, Dicle University, Diyarbakır. Pseudomonas aerug-
inosa (ATCC 27853, Gram-negative), Klebsiella pneumoniae (ATCC 13883, Gram-
negative), Enterobacter cloacae (ATCC 23355, Gram-negative) Salmonella typhimurium
(ATCC 14028, Gram negative), Staphylococcus epidermis (ATCC 12228, Gram-
positive) and Escherichia coli (ATCC 25922, Gram-negative) were obtained from
TÜBITAK, Marmara Research Center, Gene Engineering and Biotechnology Research
Institute, Gebze, Kocaeli, Turkey. The isolates were held frozen at −70◦C in Mueller-
Hilton Broth (Difco laboratories, Detroit, Michigan, USA) containing 17% glycerol
except for C. albicans, which was deposited on Sabauraud Dextrose Broth (Oxoid,
England).
Preparation of Stock Hypericum and Achillea Solutions
Stock Hypericum and Achillea solutions were prepared by dissolving crude
Hypericum and Achillea extracts in ethanol (70%) at a concentration of 100 mg/ml. For
each ethanol extract, 100 mg/ml stock solutions were prepared in ethanol. Stock solutions
were sterilized before use by passage through a 0.22 μm-pore size polysulfone membrane
filter (Gelman Science, Ann Arbor, Michigan, USA). The stock solutions were tested for
antimicrobial activity within 10 days after preparation.
Preparation of Discs
Paper discs (Oxoid) with a diameter of 6 mm containing 2.0 mg of samples were pre-
pared as described below. Twenty (20) μg discs were prepared by pipetting 20 μl volumes
of stock solutions of crude ethanol extracts of samples (100 mg/ml) onto sterile blank disc.
The discs were dried and then stored at 4◦C until use within 5 to 10 days. A disc containing
solvent extracts of Hypericum and Achillea applied to inoculated plates by using flamed
forceps.
ACTIVITY OF HYPERICUM AND ACHILLEA SPECIES 345
Antimicrobial Activity Determination
Antimicrobial activity was assayed by the disc diffusion susceptibility test accord-
ing to the recommendations of the National Committee for Clinical Laboratory standards
(NCCLS).[42] The disc diffusion test was performed on Muller-Hinton Agar plates. Plates
weredriedat35to36
◦C for about 30 min in an incubator before inoculation. Three to five
freshly grown colonies of bacterial strains were inoculated into 25 ml of Muller-Hinton
Broth medium in a shaking water bath for 4 to 6 h until a turbidity of 0.5 McFarland
(1 ×108CFU/ml) was reached. Final inocula were adjusted to 5 ×108CFU/ml.
Three to five colonies of C. albicans were inoculated into 25 ml of Sabouraud Dextrose
Broth (Oxoid) in shaking water bath for 8 to 10h until a turbidity of 0.5 McFarland
standard was reached. The final inocula were adjusted to 5 ×108CFU/ml by using
a spectrophotometer.[43] The inoculum (100 μl) from the final inocula was applied to
each agar plate and uniformly spread with a sterilized cotton spreader over the surface.
Absorption of excess moisture was allowed to occur 10 min before application of dried
paper discs with a diameter of 6 mm containing 20 μg of ethanol extracts of Hypericum
and Achillea to be assayed. They were deposited on Muller-Hinton agar plates, except for
C. albicans. Antibiotic discs (all from Oxoid) containing imipenem (IPM, 10 μg/disc)
amoxicillin (AML, 25 μg/disc), erythromycin (15 μg/disc) and ampicillin/sulbactan
(SAM, 10/10 μg/disc) were used as a positive controls. For each experiment, ethanol
was applied to paper discs as a negative control.
Statistical Analysis
Data are presented as the means ±SD. Significant differences among the groups
were determined by one-way ANOVA using SPSS 12.0 software package program. The
results were considered significant if the value of pwas less than 0.05. Differences were
considered significant at p<0.05.
RESULTS AND DISCUSSION
In recent years, the studies on oxidative stress and its adverse effects on human health
have become a subject of considerable interest. It is a well-documented fact that exposure
of organisms to exogenous and endogenous factors generates a wide range of reactive
oxygen species. Plants play a significant role in maintaining human health and improving
the quality of human life. In this study, we have studied ethanol extracts of Hypericum and
Achillea species belonging to two different families in order to discover new applications
and activities.
Phenols are very important plant constituents because of their radical scavenging
ability due to their hydroxyl groups.[44] The amount of total phenolic compounds was
investigated in the plant extracts and ethanol extracts of HSm, HR, AA, AZ and AB were
determined as a Gallic acid equivalent. 262 and 226 μg Gallic acid equivalent of phe-
nols were detected in 1 mg of ethanol extracts of H. scabrum (HSm) and H. retusum
(HR), respectively. 118, 126, and 134 μg Gallic acid equivalent of phenols were also
detected in 1 mg of ethanol extracts A. aleppica subsp. aleppica (AA), 126 A. aleppica
subsp. zederbaueri (AZ) and A. biebersteinii (AB), respectively. The phenolic compounds
may contribute directly to the antioxidative action.[45] It is suggested that polyphenolic
compounds have inhibitory effects on mutagenesis and carcinogenesis in humans, when
346 BARI¸SETAL.
up to 1.0 g daily ingested from a diet rich in fruits and vegetables.[46] The Hypericum
and Achillea species seem to be a rich source of plant species containing large amounts
of phenolic acids, so it is considered to be a promising source of natural antioxidants.
Phenolic antioxidants are products of secondary metabolism in plants and are good sources
of natural antioxidants in human diets.
The use of DPPH provides an easy and rapid way to evaluate antioxidant activity.
The mechanism involved in the reduction of DPPH free radicals is based on the capacity
of some compounds to donate hydrogen. Some plants are rich in secondary metabolites
derived from the shikimate pathway, such as, for example flavonoids, phenolic acids and
tannins. These phenolic compounds are able to donate hydrogen, presenting antiradical
activity. DPPH scavenging potential of extracts at varying concentrations was measured
and the results are depicted in Fig. 1. BHT showed high radical scavenging ability of
93% at 250 μg/ml. The scavenging effects for ethanol extract of H. scabrum (HSm),
A. biebersteinii (AB), H. retusum (HR), A. aleppica (AA) and A. aleppica D.C. subsp.
zederbaueri (AZ) were 90, 85, 82, 81, and 73% at the concentration of 250 μg/ml, respec-
tively. The IC50 values of HSm, BHT, HR, AB, AZ and AA in the DPPH radical scavenging
assay were 43, 41, 34, 33, 33, and 32 μg/ml, respectively. All the plants exhibited a
concentration dependent activity.
The reducing ability of a compound generally depends on the presence of
reductants,[47] which have exhibited antioxidative potential by breaking the free radical
chain, donating a hydrogen atom.[48] The presence of reductants in the extracts causes
the reduction of the Fe3+-ferricyanide complex to the ferrous form. Therefore, the Fe2+
can be monitored by measuring the formation of Perl’ Prussian blue at 700 nm. Figure 2
Figure 1 Scavenging effect of ethanol extracts of HSm, HR, AA, AZ, and AB on 1,1-diphenyl-2-picrylhydrazyl
radicals. Each value is expressed as mean 3 replicates ±the standard deviation.
ACTIVITY OF HYPERICUM AND ACHILLEA SPECIES 347
Figure 2 Reducing power of ethanol extracts of HR, HSm, HL, AA, AZ, and AB. Each value is expressed as
mean 3 replicates ±the standard deviation.
shows reducing capacities of ethanol extracts of Hypericum and Achillea species com-
pared with α-tocopherol and BHT. The reducing power of ethanol extracts of samples
and standards increased with increasing concentration of samples. At all tested concen-
trations, BHT showed higher activities than α-tocopherol and samples. These differences
were statistically significant (p<0.05). No significant differences were found between
α-tocopherol and extracts. The reducing ability of all extracts were dose dependent and
significantly higher than the control. The result obtained from this assay suggests that both
plant extracts were moderate scavengers of hydroxyl radicals.
Ferrozine can quantitatively form complexes with Fe2+, but in the presence of ion
chelating agents, the complex formation is disrupted, resulting in a decrease in the red
colour of the complex. EDTA showed very strong chelating ability (Fig. 3). The chelat-
ing effect of EDTA at all concentrations was approximately 100%. Ethanol extracts of
H. lysimachoides (HL) showed relatively higher activity when compared with that obtained
from ethanol extracts of H. scabrum (HSm), H. retusum (HR), A. biebersteinii (AB),
A. aleppica (AA) and A. aleppica D.C. subsp. zederbaueri (AZ). The chelating effect of
ethanol extract of HL, HSm and HR started to increase at a concentration of 150 mg/ml.
At that concentration, chelating ability of ethanol extract of HL, HSm and HR were 43%
39% and 25%, respectively. As shown in Fig. 3, ethanol extracts of H. lysimachoides
(HL) showed relatively higher activity when compared to the ability obtained from ethanol
extracts of H. retusum (HR) and H. scabrum (HSm). The chelating effect of ethanol extracts
of Hypericum species was dose-dependent. The Achillea extract also showed chelating
348 BARI¸SETAL.
Figure 3 Chelating effect of ethanol extracts of HR, HSm, HL, AA, AZ, and AB. Each value is expressed as
mean 3 replicates ±the standard deviation.
activity. Ethanol extract of A. biebersteinii (AB) showed higher activity than ethanol
extracts of A. aleppica (AA) and A. aleppica D.C. subsp. zederbaueri (AZ). The metal
scavenging effect of Achillea extracts have not been changed with increasing concentration
of extracts. All Achillea extracts showed concentration-independent scavenging activity.
Lipid peroxidation is of great concern to the food industry and consumers because
it leads to the development of undesirable off-flavours and potentially toxic reac-
tion products.[49] Many synthetic antioxidants, such as butylated hydroxyanisole, buty-
lated hydroxytoluene, t-butyl hydroquinone and propyl gallate, are used to retard lipid
peroxidation.[50] However, the use of synthetic antioxidants is under strict regulation due
to the potential health hazards caused by such compounds.[51, 52] This has led to a search
for and the use of naturally occurring antioxidants such as ascorbic acid, vitamin E and
certain crude plant extracts.
Hydroxyl radicals are the major active oxygen species causing lipid oxidation and
enormous biological damage.[53] The deoxyribose method is a simple assay to determine
the rate constants for reactions of hydroxyl radicals. When the mixture of FeCl3-EDTA,
H2O2and ascorbate was incubated with deoxyribose in phosphate buffer (pH 7.4), the
hydroxyl radicals generated attack the deoxyribose and resulting in a series of reactions
that cause the formation of MDA. Any hydroxyl radical scavenger added to reaction would
compete with deoxribose for the availability of hydroxyl radicals, thus reducing the amount
of MDA formation.[54] The scavenging activity of ethanol extracts of plants, positive con-
trol and DMSO was tested against OH radical generated by the UV photolysis of H2O2.
It has been found that the ethanol extracts of samples showed concentration-independent
scavenging activity on hydroxyl radicals as shown in Fig. 4. Hypericum lysimachoides
exhibited 84% scavenging capacity at the 80 μg/ml concentration. DMSO, a well known
hydroxyl radical scavenger, had 81% scavenging activity at the same concentration. At that
concentration H. scabrum (77%) and H. retusum (71%) showed similar scavenging activity
ACTIVITY OF HYPERICUM AND ACHILLEA SPECIES 349
Figure 4 Effect of ethanol extracts of HR, HSm, HL, AA, AZ, and AB on deoxyribose degradation assay. Each
value is expressed as mean 3 replicates ±the standard deviation.
with A. biebersteinii and A. aleppica, respectively. A. aleppica D.C. subsp. zederbaueri
showed the lowest scavenging activity (67%) at this concentration. The extracts inhibition
were found to be statistically significant than the control (p<0.05).
Peroxide is gradually decomposed to lower molecular compounds during the oxidation
process and these compounds were here measured by FTC and TBA methods. The amount
of peroxide at the primary stage of linoleic acid peroxidation was measured by FTC method,
whereas TBA method measures at the secondary stages. The total antioxidant activity of
ethanol extracts of samples were determined by peroxidation of linoleic acid using the FTC
and TBA methods. During linoleic acid peroxidation, peroxides were formed, and these
compounds oxidized Fe2+to Fe3+. The Fe3+ion formed a complex with SCN−, which had
a maximum absorbance at 500 nm. Thus a high absorbance value was an indication of high
peroxide formation during the emulsion incubation. As shown in Fig. 5, the absorbance of
the control at 500 nm increased to a maximal value of 0.95 on day 14, whereas vitamin E
and ethanol extracts of H. retusum (HR), H. scabrum (HSm), A. aleppica (AA), A. aleppica
D.C. subsp. zederbaueri (AZ), and A. biebersteinii (AB), increased to 0.38, 0.40, 0.44, 0.59,
0.52 and 0.47, respectively, on the same day. These differences were found statistically
significant than control (p<0.05). Figure 6 shows the total antioxidant activity of ethanol
extracts of samples by FTC and TBA methods. Vitamin E had the highest antioxidant
activity (60%), followed by ethanol extracts of HR (58%) and ethanol extracts of HSm
(54%), AZ (51%), AB (45%), and AA (38%). No significant difference was found between
the total antioxidant activity of all tested samples and Vitamin E in both methods.
The high phenolic content and antioxidant activity correlated well with high antimi-
crobial activity against microorganisms. In this study, 11 microorganisms were used to
screen the possible antimicrobial activities of Hypericum and Achillea species. Table 1
350 BARI¸SETAL.
Figure 5 Absorbance value of ethanol extracts of of HR, HSm, AA, AZ and AB in the linoleic acid emulsion
using FTC method. Each value is expressed as mean 3 replicates ±the standard deviation.
Figure 6 The total antioxidant activity of ethanol extracts of samples by using FTC and TBA method. Each value
is expressed as mean 3 replicates ±the standard deviation.
shows the result of antibacterial activity of the standard antibiotics against tested microor-
ganisms. All the isolates were susceptible to imipenem. Five (5) of the 12 and 4 of the 12
isolates were resistant to amoxicillin and ampicillin/sulbactan, respectively.
Table 2 shows antimicrobial activities of Hypericum and Achillea extracts against
different microorganisms. Achillea species showed a broad spectrum of strong antibacterial
activity all tested bacteria and fungi species. The diameters of growth inhibition zones
ranged from 8 to 30. The activity of all Achillea species exhibited moderate activity against
ACTIVITY OF HYPERICUM AND ACHILLEA SPECIES 351
Tab le 1 Antimicrobial activity of standard antibiotics.∗
Zones of inhibition (mm)∗∗
IPM AML SAM
Microorganisms (10 μg/paper disc) (25 μg/paper disc) (10/10 μg/paper disc)
Klesiella pneumoniaea24 – 10
Enterobacter cloacaea24 18 18
Salmonella typhimuriuma24 20 16
Staphylococcus epidermisa30 28 24
Escherichia colia30 20 14
Enterobacter aerogenesb26 – –
Staphylococcus aureusb30 14 16
Klebsiella oxytocab30 – –
Streptococcus pyogenesb18 12 18
Pseudomonas aeruginosab26 – –
Candida albicansbNT NT NT
∗: Values are the mean of 3 replicates. ∗∗: Diameter of inhibition zone including well diame-
ter of 6 mm. IPM: Imipenem (10 μg); AML: Amoxycillin (25 μg); SAM: Ampicillin/Sulbactam
(10/10 μg); –: not active; NT: not tested; a: The isolates obtained from TÜBITAK, Marmara Research
Center, Gene Enginering and Biotechnology Research Institute, Gebze, Kocaeli. b: The isolates
obtained from the stock cultures of Microbiology Laboratory, Department of Microbiology, Medical
Faculty, Dicle University, Diyarbakir.
Tab le 2 Antimicrobial activitiy of the ethanol extracts of Hypericum and Achillea species from
Turkey.∗
Zones of inhibition (mm)∗∗
HR HSm HL AA AZ AB
Microorganisms (2 mg/paper disc)
Klesiella pneumoniaea–8–101010
Enterobacter cloacaea8 8 10 10 10 10
Salmonella typhimuriuma8 8 10 10 10 10
Staphylococcus epidermisa14 14 12 30 28 24
Escherichia colia–81081010
Enterobacter aerogenesb–81081010
Staphylococcus aureusb20 14 16 10 10 10
Klebsiella oxytocab–8–8108
Streptococcus pyogenesb16 14 18 10 10 10
Pseudomonas aeruginosab14 16 18 10 12 14
Candida albicansb8 8 8 8 10 10
∗: Values are the mean of 3 replicates; ∗∗: Diameter of inhibition zone including well diameter of
6 mm. HR: Hypericum retusum; HSm: Hypericum scabrum L; HL: Hypericum lysimachioides var.
Lysimachioides; AA: Aucher Achillea aleppica D.C. subsp. Aleppica;AZ:Achillea aleppica D.C.
subsp. Zederbaueri (Hayek) Hub.-Mor; AB: Achillea biebersteinii Afan; –: not active. a: The isolates
obtained from TÜBITAK, Marmara Research Center, Gene Enginering and Biotechnology Research
Institute, Gebze, Kocaeli. b: The isolates obtained from the stock cultures of Microbiology Laboratory,
Department of Microbiology, Medical Faculty, Dicle University, Diyarbakir.
medically important pathogen S. epidermis and P. aeruginosa. The activity of Achillea
extracts was greater or similar to the tested standard antibiotics. The antimicrobial activity
of ethanol extracts of Hypericum species showed different antibacterial activity against
tested microorganisms. The diameters of growth inhibition zones of Hypericum species
352 BARI¸SETAL.
ranged from 8 mm to 20 mm. Among all the Hypericum extracts studied, ethanol extract
of HSm demonstrated the most robust antimicrobial activity against all tested microorgan-
isms. S. epidermis,S. aureus,S. pyogenes and P. aeruginosa were very sensitive to all
Hypericum extracts.
Of the species used, P. aeruginosa is a common opportunistic human pathogen that
is associated with life-threatening acute infections and chronic airway colonization during
cystic-fibrosis[55] and S. aureus is one of the most common of the Gram-positive bacteria
causing food poisoning. Its source is not the food itself but the humans who contami-
nate foods after they have been processed.[56] All of the extracts belong to Hypericum or
Achillea showed strong antibacterial activity against these bacterium. All extracts were
found to have moderate activity against C. albicans is the microbe responsible for most
clinical yeast infections, e.g., in mouth infections.
CONCLUSIONS
In recent years, the studies on oxidative stress and its adverse effects on human health
have become a subject of considerable interest. Free radicals play an important role in some
pathogenesis of serious diseases, such as neurodegenerative disorders, cancer, liver cir-
rhosis, cardiovascular diseases, atherosclerosis, cataracts, diabetes, and inflammation.[57]
Compounds that can scavenge free radicals have great potential in ameliorating these
disease.[58] It is reported that phenolic compounds in plants possess strong antioxi-
dant activity and may help to protect cells against the oxidative damage caused by free
radicals.[59] Due to the presence of the conjugated ring structures and hydroxyl groups,
many phenolic compounds have the potential to function as antioxidants by scavenging
superoxide anion,[60] singlet oxygen,[61] lipid peroxy radicals,[62] and stabilizing free rad-
icals involved in oxidative processes through hydrogenation or complexing with oxidizing
species.[63] In this study, extract of both Hypericum and Achillea species showed strong
antioxidant activity, reducing power, DPPH radical and metal chelating activities when
compared with different standards such as BHT and α-tocopherol. In addition all extracts
posed noticeable antimicrobial activity against gram positive and gram negative bacteria.
The results of this study show that the extracts can be used as easily accessible source
of natural antioxidants and as a possible food supplement or in pharmaceutical industry.
Further investigations on the isolation and identification of antioxidant components in the
plants may lead to chemical entities with potential for clinical use.
ACKNOWLEDGMENT
This work was supported by research grants from the Dicle University Research Council (DUAPK,
project number 03-FF-63).
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