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Original Article
Extracts of Agrimonia eupatoria L. as sources of
biologically active compounds and evaluation of
their antioxidant, antimicrobial, and antibiofilm
activities
Mirjana
Z. Muruzovi
c
*
, Katarina G. Mladenovi
c, Olgica D. Stefanovi
c,
Sava M. Vasi
c, Ljiljana R.
Comi
c
Departmentof Biology and Ecology, Faculty of Science,University of Kragujevac,Radoja Domanovi
ca, Kragujevac,Serbia
article info
Article history:
Received 12 October 2015
Received in revised form
2 February 2016
Accepted 26 February 2016
Available online 25 April 2016
Keywords:
Agrimonia eupatoria
antimicrobial activity
antioxidants
biofilm
flavonoids
phenolics
tannins
abstract
In this study, we determined the concentration of total phenols, flavonoids, tannins, and
proanthocyanidins in the water, diethyl ether, acetone, and ethanol extracts of Agrimonia
eupatoria L. We also investigated the antioxidant activity of these extracts using two
methods [2,2-diphenyl-1-picrylhydrazyl (DPPH) and reducing power] and their in vitro
antimicrobial (antibacterial and antifungal) activity on some selected species of bacteria
and fungi. In addition, the effects of the acetone and water extracts on the inhibition of
biofilm formation of Proteus mirabilis and Pseudomonas aeruginosa were investigated using
the crystal violet method. The concentration of total phenols was measured according to
the FolineCiocalteu method and the values obtained ranged from 19.61 mgGA/g to
220.31 mgGA/g. The concentration of flavonoids was examined by the aluminum chloride
method and the values obtained ranged from 20.58 mgRU/g to 97.06 mgRU/g. The total
tannins concentration was measured by the polyvinylpolypyrrolidone method and the
values obtained ranged from 3.06 mgGA/g to 207.27 mgGA/g. The concentration of
proanthocyanidins was determined by the butanoleHCl method and the values obtained
ranged from 4.15 CChE/g to 103.72 CChE/g. Among the various extracts studied, the acetone
extract exhibited good antioxidant activity (97.13%, as determined by the DPPH method).
The acetone extract was active in the absorbance value range from 2.2665 to 0.2495 (as
determined by the reducing power method). The strongest antimicrobial activity was
detected on G
þ
bacteria, especially on probiotic species, and the acetone extract demon-
strated the highest activity. Biofilm inhibitory concentration required to reduce biofilm
coverage by 50% values for acetone extract was 4315 mg/mL for P. mirabilis and 4469.5 mg/mL
for P. aeruginosa. The results provide a basis for further research of this plant species.
Copyright ©2016, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan
LLC. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
*Corresponding author. Department of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovi
ca 12, 34000
Kragujevac, Serbia.
E-mail address: mirkagrujovic@gmail.com (M.
Z. Muruzovi
c).
Available online at www.sciencedirect.com
ScienceDirect
journal homepage: www.jfda-online.com
journal of food and drug analysis 24 (2016) 539e547
http://dx.doi.org/10.1016/j.jfda.2016.02.007
1021-9498/Copyright ©2016, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Agrimonia eupatoria L. (common name: agrimony) belongs to the
family Rosaceae (Tribe: Sanguisorbeae). The species is wide-
spread throughout Europe, Asia, Africa, and North America.
The common habitats of this species are along the slopes,
roadsides, and rocky areas. They are also found in dry grass-
lands and arid forests. Agrimony is a perennial herbaceous
plant characterized by upright, hairy stem with a few
branches. The leaves are leathery, plumose, and the lower
ones frequently form a rosette. The flowers are arranged in
thick, spiky bunches. The fruit grows downward [1].
The plant is well-known for its use as a raw material for the
extraction of medicinal ingredients or production of drugs in
the pharmaceutical industry. A tea made from agrimony helps
to cure digestive tract diseases. The plant is also an important
ingredient of various herbal mixtures that are used as dietary
supplements for treating increased bile production, stone in
the bile duct, and pain in the gallbladder and liver; addition-
ally, it has a positive effect on the alleviation of urinary tract
disorders [2]. According to previous studies, A. eupatoria is very
rich in secondary metabolites, including tannins, flavonoids,
phenolic acids, and triterpenoids [3e7]. The plant is recog-
nized for its traditional use and has been considerably stud-
ied. The plant possesses anti-inflammatory, neuroprotective,
antidiabetic, antiobesity, hepatoprotective, and anticancer
properties [8e13]. A high correlation between polyphenolic
contents and antioxidant activity in the waterealcoholic ex-
tracts of A. eupatoria was detected [14]. The antioxidant ac-
tivity of agrimony water extracts has been demonstrated both
in vivo and in vitro by measuring the changes in the activities of
hepatic catalase and superoxide dismutase in mice [15].A
recent study on the tea prepared from A. eupatoria, which was
given to healthy volunteers, confirmed the antioxidant prop-
erties of the plant [16]. The antiviral properties of the water
extract prepared from ground parts at four different temper-
atures (37C, 45C, 55C, and 60C) were tested on hepatitis B
virus and it was concluded that the extract prepared at 60C
had the strongest antiviral effect [17]. In another clinical
study, herbal preparations containing agrimony were suc-
cessfully used for treating 35 patients suffering from gastro-
duodenitis. Moreover, there was no documented side effects
or signs of toxicity [18].A. eupatoria is a well-known medicinal
plant, which is traditionally used in folk medicine to treat
various inflammatory diseases. Although it is known that the
plant synthesizes secondary metabolites that exhibit antimi-
crobial activity, only limited studies were carried out in this
regard. Besides, some studies have clearly demonstrated the
antioxidant activity of water and ethanol extracts of this
plant; however, the activity of acetone and diethyl ether ex-
tracts of this plant has not yet been studied. Moreover, the
antibiofilm activity of these extracts is yet to be elucidated.
In this study, we aimed to determine the concentration of
total phenols, flavonoids, tannins, and proanthocyanidins in
the water, diethyl ether, acetone, and ethanol extracts of A.
eupatoria. We also investigated the antioxidant activity of these
extracts using two methods [2,2-diphenyl-1-picrylhydrazyl
(DPPH) and reducing power] and their in vitro antimicrobial
(antibacterial and antifungal) activity on some selected species
of bacteria and fungi. In addition, the effects of the acetone and
water extracts on the inhibition of biofilm formation of Proteus
mirabilis and Pseudomonas aeruginosa were investigated.
2. Methods
2.1. Plant material
The aerial parts of A. eupatoria in the flowering stage were
collected from Mount Bukulja (Serbia) during the summer of
2012. Identification and classification of the plant material were
performed at the Faculty of Science, University of Kragujevac
(Kragujevac, Serbia). The voucher samples were deposited at
the Herbarium of the Department of Biology and Ecology, Fac-
ulty of Science, University of Kragujevac. The collected plant
materials were air dried in darkness at ambient temperature.
2.2. Preparation of plant extracts
The dried, ground plant material was extracted by maceration
with ethanol, diethyl ether, water, and acetone. In brief, 30 g of
the plant material was soaked in 150 mL of the solvent. The
plant material was macerated three times at room tempera-
ture using a fresh solvent every 24 hours. After every 24 hours,
the samples were filtered through a filter paper (Whatman No.
1) and the filtrates were collected and evaporated to dryness
using a rotary evaporator (IKA, Germany) at 40C. The ob-
tained amounts of crude extracts of A. eupatoria were 5.42 g of
water extract, 0.55 g of diethyl ether extract, 0.76 g of acetone
extract, and 2.4 g of ethanol extract.
2.3. Phytochemical analysis of plant extracts
2.3.1. Determination of total phenol content
The total phenolic content of the extracts was quantified ac-
cording to the FolineCiocalteu method as described by
Wootton-Beard et al [19]. Gallic acid (Sigma-Aldrich, St. Louis,
MO, USA) was used as the standard and the total phenolic
content was expressed as milligram of gallic acid equivalents/
gram of extract (mg GAE/g of extract).
2.3.2. Determination of total flavonoid content
The total flavonoid content of the extracts was determined
using the aluminum chloride method as described by
Quettier-Deleu et al [20]. Rutin (Sigma-Aldrich, St. Louis, MO,
USA) was used as the standard and the concentrations of
flavonoids were expressed as milligram of rutin equivalents/
gram of extract (mg of RUE/g of extract).
2.3.3. Determination of total extractable tannin content
The total extractable tannin content was estimated indirectly
by spectrophotometric measurement of the absorbance of the
solution obtained after the precipitation of the tannins with
polyvinylpolypyrrolidone (Sigma-Aldrich, St. Louis, MO, USA)
as described by Makkar et al [21]. The total extractable tannin
content was expressed as milligram of gallic acid equivalents/
gram of extract (mg GAE/g of extract).
journal of food and drug analysis 24 (2016) 539e547540
2.3.4. Determination of proanthocyanidin content
The proanthocyanidin content was measured by the buta-
noleHCl method with ferric ammonium sulfate as a catalyst
as described by Porter et al [22]. Cyanidin chloride (Sigma-
Aldrich, St. Louis, MO, USA) was used as the standard and the
proanthocyanidin content was expressed as milligrams of
cyanidin chloride equivalents/gram of extract (mg CChE/g of
extract).
2.4. Determination of antioxidant activity
2.4.1. DPPH radicals scavenging capacity assay
The ability of A. eupatoria extracts to scavenge DPPH free
radicals was assessed using the method described by Takao
et al [23]. The tested concentrations of plant extracts were
from 7.8 mg/mL to 250 mg/mL. Diluted solutions of extract (2 mL
each) were mixed with 2 mL of DPPH methanolic solution
(40 mg/mL). Ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA)
was used as a reference compound. The experiment was
performed in triplicate, and the absorbance of reaction
mixture was read in a spectrophotometer at 517 nm. Radical
scavenging activity was expressed as the inhibition percent-
age and calculated using the following formula:
Scavenging activity (%) ¼100 [(A
control
eA
sample
)/A
control
)](1)
where A
control
is the absorbance of the control and A
sample
is
the absorbance of the extract.
2.4.2. Reducing power
The reducing power of the plant extracts was determined ac-
cording to the method of Oyaizu [24]. The tested concentrations
of plant extracts were from 62.5 mg/mL to 1000 mg/mL. The
absorbance of the reaction mixture was measured at 700 nm,
with each experiment performed in triplicate. Increased absor-
bance of the reaction mixture indicated increased reducing
power. Ascorbic acid was used as a reference compound.
2.5. Determination of antimicrobial activity
2.5.1. Test microorganisms
The antimicrobial activity of A. eupatoria extracts was tested
against 24 microorganisms including 18 strains of bacteria
(probiotics strains: Lactobacillus rhamnosus,Bifidobacterium
animalis subsp. lactis, and Bacillus subtilis IP 5832; standard
strains: Enterococcus faecalis ATCC 29212, Staphylococcus aureus
ATCC 25923, B. subtilis ATCC 6633, Escherichia coli ATCC 25922,
and P. aeruginosa ATCC 27853; and clinical isolates S. aureus,E.
faecalis,Bacillus cereus,B. subtilis,E. coli,Salmonella enterica,
Salmonella typhimurium,Klebsiella pneumoniae,P. mirabilis, and
P. aeruginosa) and six strains of fungi (Aspergillus flavus,
Aspergillus niger,Penicillium italicum,Penicillium chrysogenum,
Candida albicans, and C. albicans ATCC 10231). All clinical iso-
lates were a generous gift from the Institute of Public Health,
Kragujevac, Serbia. The other microorganisms (fungi and
ATCC strains) were provided by the Microbiology Laboratory,
Faculty of Science, University of Kragujevac, Serbia. The bac-
terial strains were kept in glycerol stock at 80C and the
fungal strains in paraffin oil stock at 4C.
2.5.2. Suspension preparation
Bacterial and yeast suspensions were prepared by the direct
colony method [25]. The turbidity of initial suspension was
adjusted using 0.5 McFarland densitometer (Biosan, Latvia).
Initial bacterial suspensions contain about 10
8
colony-forming
units (CFU)/mL and yeast suspensions contain 10
6
CFU/mL;
1:100 dilutions of initial suspension were additionally pre-
pared in sterile 0.85% saline. The suspensions of fungal spores
were prepared by gentle stripping of spores from slopes with
growing mycelia. The resulting suspensions were 1:1000
diluted in sterile 0.85% saline.
2.5.3. Microdilution method
The antimicrobial activity was tested by determining the
minimum inhibitory concentration (MIC) using the micro-
dilution method with resazurin [26]. Twofold serial dilutions
of the plant extracts were made in sterile 96-well microtiter
plates containing 0.1 mL of MuellereHinton broth (Torlak,
Belgrade, Serbia) per well for bacteria and 0.1 mL of Sabouraud
dextrose broth (Torlak, Belgrade, Serbia) per well for fungi.
The tested concentration range was from 0.156 mg/mL to
20 mg/mL. The microtiter plates were inoculated with the
suspensions to obtain a final concentration of 5 10
5
CFU/mL
for bacteria and 5 10
3
CFU/mL for fungi. The growth of
bacteria and yeasts was monitored by adding resazurin (Alfa
Aesar GmbH &Co., Karlsruhe, Germany), an indicator of mi-
crobial growth. Resazurin is a blue nonfluorescent dye that
becomes pink and fluorescent when reduced to resorufin by
oxidoreductases within viable cells. The inoculated microtiter
plates were incubated at 37C for 24 hours for bacteria, at 28C
for 48 hours for yeasts, and at 28C for 72 hours for molds. MIC
was defined as the lowest concentration of tested plant ex-
tracts that prevented resazurin color change from blue to
pink. For molds, MIC values of the tested plant extracts were
determined as the lowest concentration that inhibited visible
mycelia growth. Minimum microbicidal concentration (MMC)
was determined by inoculation of the nutrient agar medium
by plating 10 mL of samples from wells, where no indicator
color change was recorded. At the end of the incubation
period, the lowest concentration with no growth (no colony)
was defined as MMC.
Tetracycline, ampicillin, amphotericin B (Sigma Chemicals
Co., USA), and itraconazole (Pfizer Inc., USA), dissolved in the
nutrient liquid medium, were used as reference compounds.
Stock solutions of crude extracts were obtained by dissolving
them in 10% dimethyl sulfoxide (DMSO; Acros Organics, USA),
which was used as a control. Each test included growth con-
trol and sterility control. All tests were performed in duplicate
and mean values were presented.
2.6. Determination of antibiofilm activity
2.6.1. Tissue culture plate method
The ability of P. mirabilis and P. aeruginosa to form biofilms was
assayed as described by O'Toole and Kolter [27] with some
modifications.
The tissue culture 96-well microtiter plates (Sarstedt, Ger-
many) were prepared by dispensing 50 mL of nutrient broth
(i.e., MuellereHinton broth) into each well. From the stock
journal of food and drug analysis 24 (2016) 539e547 541
solution of tested extracts (concentration, 20 mg/mL), 50 mL
was added into the first row of the microtiter plate. Twofold
serial dilutions were then made using a multichannel pipette,
following which 50 mL of fresh bacterial suspension was added
into each well. The inoculated microtiter plates were incu-
bated at 37C for 24 hours. After incubation, the content of
each well was gently removed by tapping the microtiter
plates. The wells were washed with 200 mL of sterile 0.85%
saline to remove free-floating bacteria. Biofilms formed by
adherent cells in 96-well microtiter plates were stained with
crystal violet (0.1% w/v; Fluka AG, Switzerland) and incubated
at the room temperature for 20 minutes. Excess stain was
rinsed off by thorough washing with deionized water and then
with 200 mL of 96% ethanol. Optical densities (ODs) of stained
adherent bacteria were determined with an enzyme-linked
immunosorbent assay (ELISA) plate reader (RT-2100C, Rayto,
Shenzhen, China) at 630 nm wavelength. Biofilm inhibitory
concentration required to reduce biofilm coverage by 50%
(BIC
50
) was defined as the lowest concentration of extract that
showed 50% inhibition on the biofilm formation [28].
Only broth or broth with extracts served as control to check
sterility and nonspecific binding of media. To compensate for
background absorbance, OD readings from sterile medium,
extracts, fixative, and dye were averaged and subtracted from
all test values. All tests were performed in duplicate. Tetra-
cycline dissolved in nutrient liquid medium was used as the
reference compound.
2.6.2. Data analysis
All data were presented as means ±standard deviations
where appropriate. Pearson correlation coefficients were
determined using Microsoft Excel (Redmond, Washington, DC,
USA). For comparison of antibacterial activity between groups
of bacteria (G
e
,G
þ
, and probiotics), data were analyzed by one-
way analysis of variance using SPSS version 20 software (SPSS
Inc., Chicago, IL, USA).
3. Results
3.1. The contents of total phenols, flavonoids, total
extractable tannins, and proanthocyanidins
Secondary metabolites such as flavonoids, phenols, tannins,
and proanthocyanidins possess a high solubility in water,
acetone, ethanol, and diethyl ether and this is the criterion for
selecting solvent for plant material extraction. The results are
shown in Table 1. The acetone extract showed the maximum
measured concentration of total flavonoids (97.06 mgRU/g),
total phenols (220.31 mgGA/g), total extractable tannins
(207.27 mgGA/g), and total proanthocyanidins (103.72 CChE/g).
The concentrations in ethanol and water extracts were half as
much. The diethyl ether extract had the lowest concentration
of total phenolic content, total tannins, and
proanthocyanidins.
Based on these results, it was concluded that the highest
concentration of secondary metabolites was in the acetone
extract, whereas the lowest was in the diethyl ether extract.
The reason for the highest concentration in the acetone
extract might be because acetone is an effective extractant
with low toxicity and high extraction capacity [29].
3.2. Antioxidant activity
3.2.1. DPPH radical scavenging activity
The extracts and vitamin C used as a positive control were
tested in the concentration range from 7.8 mg/mL to 250 mg/mL
(Table 2). The antioxidant activity was highest for the acetone
extract and ranged from 97.13% to 27.73%. The ethanol and
water extracts also demonstrated a significant antioxidant
activity. Diethyl ether extract had the lowest antioxidant ac-
tivity (53.1e14.3%). Vitamin C was active in the range from
97.18% to 55.01%.
In this study, correlation between phenolic compounds
contents and DPPH radical scavenging activity was observed.
Positive linear correlation was shown for the total phenolic,
tannin, and proanthocyanidin contents (r¼0.85, 0.86, and
0.91, respectively), whereas there was a negative linear cor-
relation for flavonoids (r¼0.09).
Based on these results, it was concluded that higher con-
centrations of acetone extract and vitamin C operated in a
similar way, confirming the high antioxidant activity of agri-
mony. All tested extracts showed a concentration-dependent
antiradical activity.
3.2.2. Reducing power
The reducing power of the extracts is related to their electron-
donating ability and may serve as a significant indicator of
potential antioxidant activity. The extract concentrations
from 62.5 mg/mL to 1000 mg/mL were tested and compared with
the results gained for the equal concentrations of vitamin C,
which was used as a positive control (Table 3). The extent of
reducing the power of examined extracts was found to vary.
Among the various extracts studied, the acetone extract was
found to be the most active, followed by ethanol, water, and
diethyl ether extracts (acetone >ethanol >water >diethyl
ether). The acetone extract was active in the absorbance range
from 2.2665 to 0.2495, ethanol extract from 1.6172 to 0.0868,
water extract from 0.9475 to 0.1162, and diethyl ether extract
from 0.5059 to 0.0537. Ascorbic acid (vitamin C) was active in
the absorbance range from 2.943 to 1.19.
The correlation between phenolic compounds contents
and reducing power showed a linear correlation in relation to
the total phenolic, flavonoid, tannin, and proanthocyanidin
contents (r¼0.94, 0.56, 0.93, and 0.96, respectively).
On the basis of the obtained results, it was concluded that
the examined extracts of A. eupatoria showed moderate
reducing power compared with the positive control, and they
showed activity in all examined concentrations. It was also
concluded that the reducing power depends on concentration.
To the best of the authors'knowledge, the reducing power of
A. eupatoria extracts has not been investigated before.
3.3. Antimicrobial activity
The results of in vitro antibacterial and antifungal activities of
water, acetone, diethyl ether, and ethanol extracts of A.
eupatoria, determined by MICs and MMCs, are shown in Tables
4 and 5. A total of 24 species of microorganisms were tested
journal of food and drug analysis 24 (2016) 539e547542
and the results were compared with the influence of ampi-
cillin and tetracycline for bacteria and with itraconazole and
amphotericin B for fungi. Tetracycline is a broad-spectrum
antibiotic effective against aerobic and anaerobic G
þ
and G
e
bacteria. Ampicillin is a beta-lactam antibiotic that attacks G
þ
and some G
e
bacteria. Amphotericin B is an antifungal drug
often used for serious systemic fungal infections and is one of
the effective treatments for fungal infections caused by
Aspergillus sp. Itraconazole prevents the growth of Candida sp.
In our experiments, it was observed that 10% DMSO did not
inhibit the growth of microorganisms.
In this experiment, the values of the MIC and MMC were in
the range from <0.156 mg/mL to >20 mg/mL. The intensity of
antibacterial activity depended on the type of extract and the
species of bacteria. Extracts were active in the following
ascending order: diethyl ether <ethanol <water <acetone.
It was noted that the extracts generally had a weak effect
on G
e
bacteria than on G
þ
bacteria and probiotics (p<0.05;
Table 4). E. coli,S. enterica, and S. typhimurium showed resis-
tance to the tested extracts (>20 mg/mL), whereas E. faecalis
showed resistance to all the tested extracts except for the
acetone extract, which was active in the concentration of
10 mg/mL for MIC and MMC. The acetone extract also showed
the highest inhibitory effect on G
þ
and G
e
bacteria. It was
most efficient on G
þ
strains of L. rhamnosus and B. animalis
subsp. lactis (MIC and MMC <0.156 mg/mL), and also on B.
cereus, B. subtilis, and B. subtilis ATCC 6633 (MIC and MMC at
0.3125 mg/mL). With regard to G
e
strains, bacteria most sen-
sitive to the acetone extract were P. aeruginosa (MIC at
0.625 mg/mL and MMC at 1.25 mg/mL) and E. coli ATCC 25922
(MIC at 1.25 mg/mL and the MMC at 2.5 mg/mL). It has been
reported that G
þ
bacteria are usually more sensitive to the
plant-origin antimicrobials, compared with G
e
bacteria,
which are usually more resistant. The resistance of the G
bacteria could be attributed to their cell wall structure. G
bacteria have an effective permeability barrier composed of a
thin lipopolysaccharide exterior membrane, which could
restrict the penetration of the active compounds from plant
extracts [30].
The examined fungi showed low sensitivity to the tested
extracts (Table 5). The acetone extract was active in the
2.5e20 mg/mL range for MIC and MMC, whereas the diethyl
ether, water, and ethanol extracts were active in the 5e20 mg/
mL range for MIC and MMC. The effect was more noticeable on
Table 1 eConcentrations of flavonoids, total phenolic content, total extractable tannins, and proanthocyanidins in the
extracts of Agrimonia eupatoria.
Type of extract Flavonoid
concentration
(mgRU/g extract)
Total phenolic
content (mgGA/g
extract)
Total extractable tannins
concentration
(mgGA/g extract)
Concentration of
proanthocyanidins
(CChE/g extract)
Water 20.58 ±0.92 118.47 ±0.72 107.52 ±0.16 55.85 ±0.75
Acetone 97.06 ±2.56 220.31 ±0.00 207.27 ±0.21 103.72 ±0.53
Diethyl ether 64.9 ±0.79 19.61 ±0.10 3.06 ±5.98 4.15 ±0.41
Ethanol 46.5 ±0.08 123.9 ±0.47 109.33 ±0.09 74.42 ±0.73
Data are presented as mean ±standard deviation.
Table 2 eComparison of antioxidant activity of different Agrimonia eupatoria extracts obtained by the 2,2-diphenyl-1-
picrylhydrazyl method.
Concentration (mg/mL) Type of extract
Water extract Acetone extract Diethyl ether extract Ethanol extract Ascorbic acid
250 93.95 ±0.27 97.13 ±0.26 53.1 ±0.40 94.88 ±0.14 97.18
125 92.87 ±0.46 94.83 ±0.22 30.46 ±0.18 94.79 ±0.13 97.18
62.5 90.25 ±1.27 91.98 ±0.24 19.6 ±0.31 94.08 ±0.16 97.15
31.25 71.86 ±0.25 87.57 ±0.04 9.7 ±0.40 59.59 ±0.07 97.06
15.6 40.91 ±0.29 50.77 ±0.53 6.88 ±0.44 33.88 ±0.10 92.01
7.8 21.9 ±0.22 27.73 ±0.36 3.14 ±0.50 19.4 ±0.43 55.01
Data are presented as mean ±standard deviation. Values represent % of the activity.
Table 3 eComparison of reducing power of various Agrimonia eupatoria extracts and ascorbic acid.
Concentration (mg/mL) Type of extract
Water extract Acetone extract Diethyl ether extract Ethanol extract Ascorbic acid
1000 0.9475 ±0.00 2.2665 ±0.00 0.5059 ±0.00 1.6172 ±0.09 2.943
500 0.6899 ±0.00 1.2968 ±0.02 0.2789 ±0.00 0.8843 ±0.01 2.943
250 0.3759 ±0.00 0.7859 ±0.05 0.1703 ±0.00 0.3603 ±0.13 2.843
125 0.2682 ±0.00 0.4614 ±0.00 0.0941 ±0.00 0.1169 ±0.00 2.667
62.5 0.1162 ±0.00 0.2495 ±0.00 0.0537 ±0.00 0.0868 ±0.00 1.190
Data are presented as absorbance mean value ±standard deviation measured at 700 nm.
journal of food and drug analysis 24 (2016) 539e547 543
yeasts, than on filamentous fungi. C. albicans was partially
sensitive to the activity of all the extracts, in the concentration
of 5 mg/mL for MIC and MMC, and C. albicans ATCC 10231 was
the most sensitive to the acetone extract in the concentration
of 2.5 mg/mL for MIC and 5 mg/mL for MMC. The acetone
extract was the most effective against P. italicum and P. chrys-
ogenum in the concentration of 2.5 mg/mL for MIC and MMC.
3.4. Antibiofilm activity
Biofilms were quantified by measuring the absorbance of
stained biofilms at 630 nm with an ELISA plate reader. The
results indicated that P. aeruginosa and P. mirabilis have the
ability to form biofilms better than other bacteria studied. The
absorbance values were 1.047 for P. aeruginosa and 0.285 for P.
mirabilis (Table 6).
In vitro activity of water and acetone A. eupatoria extracts on
the inhibition of biofilm formation was examined. Absorbance
values of controls were subtracted from the absorbance
values of the tested samples (fixed and dyed) in order to
recompense the background absorbance. The absorbances are
shown in Table 6. The absorbance values for acetone extract
ranged between 1.123 and 0.411 for P. aeruginosa and between
0.258 and 0.094 for P. mirabilis. The absorbance values for
water extract ranged between 1.163 and 0.714 for P. aeruginosa
and between 0.493 and 0.275 for P. mirabilis. Decrease of the
absorbance values of tested samples in relation to the absor-
bance of growth control demonstrated the ability of tested
extracts to prevent the biofilm formation.
BIC
50
was defined as the lowest concentration of extract
that showed 50% inhibition on the biofilm formation. The re-
sults are shown in Table 7. The acetone extract showed a
Table 4 eAntibacterial activities of water, acetone, diethyl ether, and ethanol extracts of Agrimonia eupatoria.
Species Water extract Diethyl ether extract Acetone extract Ethanol extract Tetracycline Ampicillin
MIC MMC
a
MIC MMC MIC MMC MIC MMC MIC MMC MIC MMC
Escherichia coli ATCC 25922 2.5 3.75 2.5 10 1.25 2.5 1.25 2.5 4 6 0.37 0.5
E. coli >20 >20 >20 >20 10 10 20 >20 2 6 2.1 1.2
Salmonella enterica >20 >20 >20 >20 10 10 20 20 2 2 1 1
Salmonella typhimurium >20 >20 >20 >20 7.5 10 10 20 2 2 2 2
Pseudomonas aeruginosa
ATCC 27853
10 20 >20 5 2.5 2.5 5 >20 4 32 >128 >128
P. aeruginosa 10 20 20 3.75 0.62 1.25 1.25 >20 >128 >128 >128 >128
Proteus mirabilis 520 >20 5 2.5 2.5 2.5 20 >128 >128 >128 >128
Klebsiella pneumoniae >20 >20 >20 10 10 10 20 >20 4 32 >128 >128
Staphylococcus aureus
ATCC 25923
2.5 5 5 5 1.25 2.5 1.25 2.5 1.5 3 0.25 0.75
S. aureus 0.62 2.5 0.31 1.25 0.12 0.62 0.62 1.25 <0.06 <0.06 <0.06 <0.06
Bacillus subtilis
ATCC 6633
1.25 5 0.62 1.87 0.31 0.31 0.62 1.25 0.25 0.37 3 4
B. subtilis 1.25 5 0.62 1.25 0.31 0.62 0.62 1.25 <0.06 0.25 16 128
Bacillus cereus 1.25 1.25 0.62 0.62 0.31 0.31 0.62 0.62 0.25 0.5 4 6
Enterococcus faecalis
ATCC 29212
5>20 5 20 2.5 5 1.87 5 8 12 1 2
E. faecalis >20 >0>20 >20 10 10 20 20 1 6 4 6
Lactobacillus rhamnosus 1.25 2.5 0.31 0.31 <0.16 <0.16 <0.16 <0.16 0.16 1 64 13
Bifidobacterium animalis
subsp. lactis
0.16 1.87 <0.16 <0.16 <0.16 <0.16 <0.16 <0.16 4 8 <0.06 0.12
B. subtilis
IP 5832
2.5 2.5 0.62 0.62 0.31 0.31 0.31 0.62 <0.06 <0.06 8 16
MIC ¼minimum inhibitory concentration; MMC ¼minimum microbicidal concentration.
a
Data are given as mg/mL for plant extract and mg/mL for antibiotics.
Table 5 eAntifungal activities of water, acetone, diethyl ether, and ethanol extracts of Agrimonia eupatoria.
Species Water extract Diethyl ether extract Acetone extract Ethanol extract Amphotericin B Itraconazole
MIC MMC
a
MIC MMC MIC MMC MIC MMC MIC MMC MIC MMC
Candida albicans
ATCC 10231
20 20 5 5 2.5 5 5 5 0.49 1.95 1.95 1.95
C. albicans 5 5 5 5 5 5 5 5 0.98 1.95 1.95 1.95
Penicillium italicum 20 20 7.5 7.5 2.5 2.5 10 10 7.81 7.81 0.98 0.98
Penicillium chrysogenum 20 20 5 5 2.5 2.5 10 10 7.81 7.81 0.98 0.98
Aspergillus flavus 20 20 20 20 10 10 20 20 0.98 15.62 0.98 0.98
Aspergillus niger 20 20 20 20 20 20 20 20 0.98 0.98 15.62 15.62
MIC ¼minimum inhibitory concentration; MMC ¼minimum microbicidal concentration.
a
Data are given as mg/mL for plant extract and mg/mL for antimycotics.
journal of food and drug analysis 24 (2016) 539e547544
greater effect than water on the inhibition of biofilm forma-
tion of P. aeruginosa and P. mirabilis. In fact, the water extract
showed no effect on the tested concentrations.
The acetone extract was effective at a concentration of
4469.5 mg/mL for P. aeruginosa and 4315 mg/mL for P. mirabilis.
To the best of the authors'knowledge, the antibiofilm activity
of water and acetone A. eupatoria extracts was examined for
the first time.
4. Discussion
Because of its widespread use in folk medicine, agrimony
provokes a great scientific interest. Many A. eupatoria phyto-
chemical composition assays have focused on alcoholic and
waterealcoholic extracts. In this study, chemical composi-
tion, antioxidant activity, and antibacterial properties of the
acetone and diethyl ether extracts have been analyzed for the
first time. In addition, the in vitro activity of water and acetone
A. eupatoria extracts on biofilm formation was examined for
the first time.
According to previous studies, A. eupatoria is very rich in
secondary metabolites, and was reported to contain 3e11% of
tannin and about 1.9% of flavonoids, phenolic acids, and tri-
terpenoids [3e7]. Therefore, in this paper, we carried out a
phytochemical analysis of the tested plant extracts. The
highest concentrations of secondary metabolites were in the
acetone extract, and the lowest was in the diethyl ether
extract. Shabana et al [31] reported that the major compounds
in the waterealcoholic extract were flavonoids (0.33%),
tannins (10.08%), and phenolic acids [2.26% luteolin 7-O-
sophoroside, luteolin 7-O (600-acetyl glucoside), acacetin 7-O-
glucoside, luteolin 7-O-glucoside and apigenin 7-O-glucoside,
protocatechuic acid, vanillic acids, and p-hydroxybenzoic
acid]. The polyphenolic profile of plant extracts was charac-
terized mainly by the high-performance liquid chromatog-
raphy (HPLC) method. Dulger and Gonuz [32] isolated
flavonoids, tannins, and terpenoids from ground parts of this
plant. Kubinova et al [33] reported a high percentage of quer-
cetin and apigenin glycoside in the HPLC profile of species
from the Agrimonia genus, with the highest concentration of
flavonoids being detected in the methanol extract of A. eupa-
toria. Our study confirmed that A. eupatoria is very rich in
secondary metabolites.
4.1. Antioxidant activity
The high correlation between polyphenolic content and total
antioxidant activity has been demonstrated in water-
ealcoholic agrimony extracts [14]. Ivanova et al [11] indicated
that the antioxidant activity of A. eupatoria might be attributed
to the chemical structure of polyphenols and/or to the result
of activation of endogenous antioxidant defense systems. In
this paper, the antioxidant activity of acetone, diethyl ether,
ethanol, and water extracts of A. eupatoria was investigated
and the results were compared with vitamin C. The higher
concentrations of acetone extract and vitamin C operated in a
similar way. All the tested extracts and the control substance
showed a very strong antioxidant activity, and thus, this study
further confirms the high antioxidant activity of agrimony.
In this study, the reducing power of all four tested extracts
was examined for the first time, and they showed moderate
reducing power compared with the positive control. In this
case, the acetone extract was found to be the most active.
4.2. Antimicrobial and antifungal activities
Extracts of A. eupatoria possess powerful antibacterial activity.
Dulger and Gonuz [32], using the disk-diffusion method,
investigated the antimicrobial activity of the ethanol extract of
some plant species, including A. eupatoria. Theirresults showed
that A. eupatoria extracts exhibited antimicrobial activity on all
kinds of microorganism strains studied,and the inhibition zone
Table 6 eAntibiofilm activity of water and acetone extracts of Agrimonia eupatoria.
a
Concentration (mg/mL) Species
Pseudomonas aeruginosa Proteus mirabilis
Growth control
b
Water extract Acetone extract Growth control
b
Water extract Acetone extract
5000 1.047 0.714 0.511 0.285 0.276 0.121
2500 0.735 0.586 0.281 0.156
1250 0.735 0.611 0.281 0.193
625 0.744 0.609 0.298 0.205
312.5 0.865 0.703 0.301 0.215
156.25 0.755 0.838 0.348 0.251
78.12 1.016 0.925 0.362 0.258
39.06 1.163 1.235 0.493 0.312
a
Data represented absorbance at 630 nm.
b
Data shown is the mean value of the eight repetitions.
Table 7 eBiofilm inhibitory concentration of water and
acetone extracts of Agrimonia eupatoria.
Species Water
extract BIC
50a
Acetone
extract BIC
50
Tetracycline
BIC
50
Pseudomonas
aeruginosa
>5000 4469.50 1522.50
Proteus mirabilis >5000 4315 Not determined
BIC
50
¼biofilm inhibitory concentration required to reduce biofilm
coverage by 50%.
a
Data given as mg/mL.
journal of food and drug analysis 24 (2016) 539e547 545
diameters were 8e16 mm. In addition, S. aureus, Listeria mono-
cytogenes, and Micrococcus luteus were found to be the most
sensitive. Compared with S. aureus, the extract had greater ef-
fect than conventional antibiotics, except for ofloxacin and
tetracycline. In the case of L. monocytogenes, the extract was
more effective than the antibiotic ampicillin. In our research,
the sensitivity of S. aureus was noticed in all tested extracts.
Ghaima [34] studied the antibacterial effect of the water and
ethanol A. eupatoria extracts on the strains of some pathogenic
bacteria (S. aureus, P. aeruginosa,andE. coli). The results showed
that the ethanol extract was more effective than the water
extract, and this result was confirmed in our study. Cwikla et al
[35] examined the effects of watereethanol extract and essen-
tial oils of some plant species to determine their influence on
Helicobacter pylori using the microdilution method and the re-
sults were compared with the inhibitory action of antibiotics.
They concluded that the extracts of A. eupatoria,Hydrastis can-
adensis,Filipendula ulmaria,andSalvia officinalis were the most
active in inhibiting the growth of H. pylori.Petkov[36] has been
reported antibacterial activity for agrimony extracts against S.
aureus and a-hemolytic streptococci.
In our research, the effect of water, acetone, diethyl ether,
and ethanol extracts of A. eupatoria on fungi was closely
examined for the first time and the results were compared
with the effect of antifungal drugs. Compared with the posi-
tive controls, it could be concluded that all extracts exhibited
low antifungal effect. According to Soliman and Badeaa [37],
extracts of A. eupatoria leaves belong to the category of plant
with low antiaflatoxin effect.
4.3. Antibiofilm activity
Bacterial biofilms are structures consisting of extracellular
polymeric substances and single or multiple species of bac-
teria that form clusters and adhere to surfaces [38]. Biofilms
are a self-protection growth pattern of bacteria, which are
different from planktonic cells. They have been of consider-
able interest in food hygiene because biofilms may contain
spoilage and reduce the risk of contamination by pathogenic
bacteria, which increases postprocessing contamination and
risk to public health [39]. The mechanisms of the effect of
extracts on biofilm are different among plants. For example,
curcumin (the main active compound of Curcuma longa), pre-
vents the adherence of bacterial cells in the biofilm, making
them more sensitive to antimicrobial agents rather than in
biofilm state [40]. The effect of various plant extracts on the
inhibition of biofilm formation has been documented for
Melilotus albus,Dorycnium herbaceum,Calendula officinalis, and
Nigella sativa [28,41,42].
In this study, for the first time, the antibiofilm activity of
the acetone and water extracts of A. eupatoria was investigated
on two bacteria, namely, P. aeruginosa and P. mirabilis, which
have demonstrated the ability to form biofilm. We also
investigated the effect of these extracts on inhibition on bio-
film formation. The acetone plant extract was found to be very
effective in inhibiting the bacterial biofilm formation. The
antibiofilm activity could be related to the higher proportion of
tannins and flavonoids in the plant [43]. This result is
confirmed in our study, because the acetone extract showed
the highest effect on the inhibition on biofilm formation, and
it also contained the higher proportion of tannins and flavo-
noids than the other tested extracts of A. eupatoria.
In this paper, it was shown that the extracts of A. eupatoria
are a good source of bioactive compounds, what can be used in
the food and pharmaceutical industries. Our study demon-
strates the high phenolic content and antioxidant potential of
A. eupatoria extracts and the study results may help in the
development of beverages that could protect against free
radical damage or safe food products and additives with
appropriate antioxidant properties. Further studies must be
carried out to include isolates of active compounds, elucidate
the structures, toxicity testing, and also to evaluate the effects
of plant extracts on the inhibition of biofilm formation.
4.4. Conclusion
Based on the study of acetone, diethyl ether, ethanol and
water extracts of A. eupatoria, it could be concluded that all
these extracts, especially acetone, are an important source of
biologically active compounds. The study results also confirm
the antioxidant and antibacterial properties of A. eupatoria;
besides, the inhibition on biofilm formation by the acetone
extract was significant. Additional effects of A. eupatoria ex-
tracts and their fine mechanisms of action are to be revealed
by future studies.
Conflicts of interest
The authors declare that they have no competing interests.
Acknowledgments
The authors are grateful to Professor Dr Marina Topuzovi
c for
identification and classification of the plant material. This
investigation was supported by the Ministry of Education and
Science of the Republic of Serbia (Grant Nos. 41010 and
173032).
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