International Journal of Food Science and Technology 2009, 44, 269–278 269

Original article
Radical scavenging and antimicrobial activity of horsetail
(Equisetum arvense L.) extracts

Jasna M. Čanadanović-Brunet, Gordana S. Ćetković, Sonja M. Djilas*, Vesna T. Tumbas, Sladjana S. Savatović,
Anamarija I. Mandić, Siniša L. Markov & Dragoljub D. Cvetković
Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
(Received 7 June 2006; Accepted in revised form 5 September 2007)

Summary A reversed-phase high performance liquid chromatography (RP-HPLC) separation on C8 column and
quantitative method were developed to analyse hydroxyl derivatives of benzoic and cinnamic acid and
flavonoids in horsetail (Equisetum arvense L.) extracts. Total phenolic content of n-butanol, ethyl acetate
and water extracts, determined by the Folin-Ciocalteu method, was 96.4, 26.4 and 15.4 mg g)1 of dry
extracts, respectively. The antioxidative activity of horsetail extracts was tested by measuring their ability to
scavenge stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) and reactive hydroxyl radicals by electron spin
resonance spectroscopy. The results demonstrated that the free radical scavenging activity (versus both
DPPH and hydroxyl radicals) depended on the type and concentration of applied extracts; the highest DPPH
(EC50 = 0.65 mg mL)1) and hydroxyl radical scavenging activities (EC50 = 0.74 mg mL)1) were obtained
in the case of n-butanol extract. The radical scavenging activity of extracts significantly correlated with total
phenolic content. The antimicrobial tests showed that ethyl acetate and n-butanol extracts inhibited the
growth of tested bacteria.
Keywords Antimicrobial activity, electron spin resonance, free radical scavenging activity, high performance liquid chromatography
(HPLC), horsetail, phenolic compounds.

Horsetail (Equisetum arvense L.) is well known as

Introduction
Natural phytochemicals derived from fruits, vegetables
and herbs have been reported to possess a wide range of
biological effects, including antioxidant, antimicrobial
and anti-inflammatory actions (Dillard & German,
2000; Milić et al., 2000; Jayaprakasha et al., 2003; Wu
et al., 2006). Among them, phenolic acids and flavo-
noids have been the object of a great number of studies
of their antioxidative activity, which is mainly because
of their capacity to act as free radical scavengers and ⁄ or
as metal chelators (Rice-Evans et al., 1997). On the
contrary, the search for natural antioxidants of plant
origin is also being explored as an alternative to the
synthetic antioxidants used in food and pharmaceutical
industries (Ito et al., 1985).
Herbs have been used for a large range of purposes,
including medicine, nutrition, flavourings, beverages,
dyeing, repellents, fragrances, cosmetics, charms, smok-
ing and industrial uses. Nowadays, herbal, and espe-
cially herbal extracts are very attractive not only in the
modern phytotherapy but also for food industry (Halli-
well, 1999; Liu & Ng, 2000; Visioli et al., 2000).
*Correspondent: Fax: +381 21 450413;
e-mail: sdjilas@tehnol.ns.ac.yu
culinary and medicinal herb. Its fertile stems are
consumed as food in sweetened vinegar and in cooked
food (Nagai et al., 2005). As edible herb, it is very
popular in Japan, but because of pleasant horsetail’s
flavour, it is important to increase its popularity in other
parts of the world. In traditional medicine, it has been
used for urinary and prostatic disease, managing enure-
sis, managing irritable symptoms of the urinary system,
repair lung tissue after pulmonary tuberculosis and other
diseases, metabolic or hormonal oedema, haemorrhage,
wounds, rheumatism and chilblains (Grieve, 1971). In
addition, horsetail was described as anti-inflammatory
and antioxidant agent, vasorelaxant, and was highly
recommended by herbalists as haemostatic (Hoffman
Martins Do Monte et al., 2004; Oh et al., 2005). These
activities of horsetail are related to the content of several
classes of secondary metabolites such as phenolics
(flavonoids, styrylpyrones and phenolic acids), alkaloids
(equisetin, nicotine, palustrine and palustrinine), phy-
tosterols (campesterol), bitter principle and minerals
(silica, calcium, magnesium, selenium, iron, potassium,
zinc, etc.) (Veit et al., 1995; Beckert et al., 1997).
The aim of the present study was to examine the total
phenolic content of horsetail (Equisetum arvense L.)
extracts, obtained by successive extraction, and also to

doi:10.1111/j.1365-2621.2007.01680.x
 2008 Institute of Food Science and Technology
270 Radical scavenging and antimicrobial activity of horsetail extracts J. M. Čanadanović-Brunet et al.
identify some phenolic acids and flavonoids by HPLC
analysis. In effort to establish the antioxidative activity
of different horsetail extracts against stable 2,2-diphe-
nyl-1-picrylhydrazyl (DPPH) and reactive hydroxyl
radicals, a very sensitive analytical method, electron
spin resonance (ESR) spectroscopy, was evaluated. The
contents of phenolic compounds of investigated extracts
were correlated to their antioxidative activities. Anti-
microbial activity of extracts was carried out using
disc diffusion and macrobroth dilution methods with
bacteria (Pseudomonas aeruginosa, Escherichia coli,
Staphylococcus aureus and Bacillus cereus) and
yeasts (Saccharomyces cerevisiae and Candida
pseudotropicalis).
Materials and methods
Chemicals
HPLC grade acetonitrile and acetic acid (Merck,
Darmstadt, Germany), and filtered bi-distilled water
were used for HPLC analysis. The solvents (methanol,
ethyl acetate, petroleum ether, chloroform and n-buta-
nol) used for extraction were from ‘Zorka’ Chemical Co.
(Šabac, Serbia and Montenegro). The Folin-Ciocalteu
reagent was purchased from Fluka Chemie AG (Buchs,
Switzerland). Standards of phenolic acids (gallic, pro-
tocatechuic, vanillic, caffeic, syringic, coumaric and
ferulic acid) and flavonoids (rutin and (-)-epicatechin),
peroxylamine disulphonate (Fremy’s salt), butylated
hydroxyanisole (BHA), DPPH and 5,5-dimethyl-1-pyr-
oline-N-oxide (DMPO) were obtained from Sigma
Chemicals Co. (St Louis, MO, USA). Solvents, stan-
dards of phenolic acids and flavonoids, and all other
chemicals were of analytical reagent grade.
Plant material
Aerial parts of horsetail (Equisetum arvense L., Equi-
setaceae) were collected in the period June to July 2004,
in the region of mountain Zlatibor, Serbia. Voucher
specimen (no. E-7 ⁄ 04) was authenticated by Biljana
Božin, the Department of Pharmacognosy, Faculty of
Medicine, University of Novi Sad, and deposited at the
Herbarium of the Department of Biology and Ecology,
Faculty of Natural Sciences, University of Novi Sad.
Preparation of extracts
Dried plant of horsetail (100 g) was extracted with 70%
methanol (2 · 2500 mL) at room temperature for
2 · 24 h. The obtained extract was concentrated under
reduced pressure to remove methanol, and then treated
with petroleum ether (2 · 200 mL), chloroform
(2 · 200 mL), ethyl acetate (2 · 200 mL) and n-butanol
(2 · 200 mL). The petroleum ether, chloroform, ethyl
International Journal of Food Science and Technology 2009, 44, 269–278
acetate, n-butanol and remained water extract were
evaporated to dryness under reduced pressure. The
yields, average of triplicate analysis, of extracts were:
petroleum ether, m = 1.810 ± 0.088 g; chloroform,
m = 0.128 ± 0.006 g; ethyl acetate, m = 0.204 ±
0.009 g; n-butanol, m = 1.022 ± 0.049 g and water,
m = 9.644 ± 0.453 g.
Total phenolic content
The amount of total soluble phenolics in extracts was
determined spectrophotometrically according to the
Folin-Ciocalteu method (Singleton et al., 1999). The
reaction mixture was prepared by mixing 0.1 mL of
methanolic solution (concentration 1 mg mL)1) of
extract, 7.9 mL of distilled water, 0.5 mL of Folin-
Ciocalteu’s reagent and 1.5 mL of 20% sodium carbon-
ate. After 2 h, the absorbance was measured at 750 nm
in a spectrophotometer. The total phenolic content was
expressed as gallic acid equivalents per gram dry weight.
HPLC analysis
HPLC was performed with a Hewlett-Packard Liquid
Chromatograph HP1090 equipped with diode array
detector (79880A DAD). A reversed-phase column,
Hypersil MOS (250 · 2.1 mm) with a 5-lm particle size
(Hewlett-Packard, Avondale, PA, USA), was used at the
flow rate of 0.200 mL min)1. Solvent gradient was
performed by varying the proportion of solvent A
(water-acetic acid pH 2.8) to solvent B (acetonitrile) as
follows: initial 1% B; linear gradient to 40% B in
50 min; hold at 40% B in 10 min; linear gradient to 1%
B in 5 min. The set time of recording chromatograms
and spectra was 40 min, whereas the total running time
and post-running time were 65 and 10 min, respectively.
The injected volume was 10 lL of 10 mg mL)1 water
solution of extracts and standards, and the injection was
performed manually. All solutions were filtered prior to
injection through 0.45 lm membrane filters (Millipore,
Bedford, MA, USA). The column temperature was
24 C. The spectra were acquired in the range 210–
400 nm and chromatograms plotted at 290 ⁄ 4 nm with
reference wavelength 550 ⁄ 100 nm.
Phenolic acids and flavonoids in extracts were iden-
tified by matching the retention time and their spectral
characteristics against those of standards. The purity of
the peaks was determined to ensure the identification.
The external standard method was the technique used
for quantification. For each compound, stock solution
was made by accurately weighing out commercially
standard followed by dissolving in methanol. Solutions
used for calibration were prepared by dilution of the
stock solutions. Peak areas from chromatograms were
plotted against the known concentrations of standards.
Equations generated via linear regression were used to
 2008 Institute of Food Science and Technology
 Radical scavenging and antimicrobial activity of horsetail extracts J. M. Čanadanović-Brunet et al.
establish concentrations of phenolic acids and flavo-
noids in extracts.
DPPH radicals generation and detection
Radical scavenging activity of plant extracts and phe-
nolic compounds against stable DPPH radicals was
determined by ESR spectroscopy.
Blank probe was obtained by mixing 600 lL 0.4 mm
methanolic solution of DPPH and 200 lL methanol.
A volume of x lL of 10 mg mL)1 methanolic solution
of investigated extract or phenolic compounds was
added to (200-x) lL methanol and 600 lL 0.4 mm
methanolic solution of DPPH. The range of investigated
extract concentrations was 0.50–2.50 mg mL)1, whereas
the range of phenolic compound concentrations was
0.005–0.125 mg mL)1. In the case of p-coumaric and
vanillic acid, the range of concentrations was 0.50–
2.50 mg mL)1. The mixture was stirred for 2 min and
transferred to a quartz flat cell ER-160FT.
The ESR spectra were recorded on an ESR spectro-
meter Bruker 300E (Bruker, Rheinstetten, Germany)
under the following conditions: field modulation
100 kHz, modulation amplitude 0.256 G, receiver gain
2 · 104, time constant 40.96 ms, conversion time
327.68 ms, centre field 3440.00 G, sweep width
100.00 G, x-band frequency 9.64 GHz, power 20 mW
and temperature 23 C.
The DPPH radical scavenging activity (SADPPH) was
calculated according to the formula:
SADPPH ð%Þ ¼ 100  ðho  hx Þ=ho ;
where ho is the height of the second peak in the ESR
spectrum of DPPH free radicals of the blank and hx is
the height of the second peak in the ESR spectrum of
DPPH free radicals of the test solution.
SADPPH ⁄ concentration of extracts and phenolic com-
pounds change curves were used to find the concentra-
tion at which 50% DPPH radicals scavenging occurred
(EC50DPPH).
Hydroxyl radicals generation and detection
As hydroxyl free radicals are highly reactive, with
relatively short half-lives, the concentrations found in
natural systems are usually inadequate for direct detec-
tion by ESR spectroscopy. Spin-trapping is a chemical
reaction that provides an approach to help overcome
this problem (Rice-Evans et al., 1996).
The Fenton reaction was conducted by mixing 0.2 mL
0.3 m DMPO, 0.2 mL 10 mm H2O2 and 0.2 mL 10 mM
Fe2+ (blank). The influence of different types of extracts
on the formation and stabilisation of hydroxyl radicals
was investigated by adding the petroleum ether, chloro-
form, ethyl acetate, n-butanol and the water extracts in
the Fenton reaction system at the range of concentra-
 2008 Institute of Food Science and Technology
271
tions 0.50–3.50 mg mL)1. The range of phenolic com-
pound concentrations was 0.01–0.125 mg mL)1;
whereas in the case of (-)-epicatechin, vanillic, p-coum-
aric and ferulic acid, the range of concentrations was
0.50–2.50 mg mL)1. ESR spectra were recorded after
5 min, with the following spectrometer settings: field
modulation 100 kHz, modulation amplitude 0.512 G,
receiver gain 2 · 105, time constant 81.92 ms, conver-
sion time 163.84 ms, centre field 3440.00 G, sweep width
100.00 G, x-band frequency 9.64 GHz, power 20 mW
and temperature 23 C.
Magnetic field scanning was calibrated using Fremy’s
salt. Splitting constants were calculated from computer-
generated second derivatives of the spectra after opti-
mising signal-to-noise ratios, and were verified by
computer simulations.
The hydroxyl radical scavenging activity (SAOH) was
calculated according to the formula:
SAOH ð%Þ ¼ 100  ðho  hx Þ=ho ;
where ho is the height of the second peak in the ESR
spectrum of hydroxyl free radicals of the blank and hx is
the height of the second peak in the ESR spectrum of
hydroxyl free radicals of the test solution.
SAOH ⁄ concentration of extracts and phenolic com-
pounds change curves were used to find the concentra-
tion at which 50% hydroxyl radicals scavenging
occurred (EC50OH).
Antimicrobial activity
For these investigations, the disc diffusion and micro-
broth dilution methods were applied. From primary
isolation, medium two to three colonies of investigated
microorganisms were taken by flamed loop and sus-
pended in Mueller–Hinton or Sabouraud dextrose
broth. They were incubated at 37 C (bacteria) or
25 C (yeasts). The suspension for inoculation was
prepared from broth cultures. The number of cells in
1 mL of suspension for inoculation measured by a
McFarland nephelometer was 1 · 107 CFU ml)1. The
1 mL of this suspension was homogenised with 9 mL of
melted (45 C) Mueller–Hinton or Sabouraud dextrose
agar and poured into Petri dishes.
For screening, sterile, 6 mm discs (HiMedia, Mum-
bai, India) were impregnated with 10 lL of
100 mg mL)1 of horsetail extracts. After incubation
for 24–48 h in the thermostat at 37 C for bacteria or
25 C for yeasts, inhibition zone diameters (ZI, includ-
ing disc) were measured and expressed in mm. The
presence of inhibition zone indicated the activity of
tested extracts against bacteria or yeasts. The minimum
inhibitory concentration (MIC) was reported as the
lowest concentration of the extracts capable of inhibit-
ing the growth of the bacterium tested. The MIC was
determined by broth macrodilution test (Sahm &
International Journal of Food Science and Technology 2009, 44, 269–278
272 Radical scavenging and antimicrobial activity of horsetail extracts J. M. Čanadanović-Brunet et al.
Washington, 1991) using inoculum of
1 · 107 CFU mL)1. Final concentrations of investi-
gated extracts were 25, 50, 75 and 100 mg mL)1.
Pseudomonas aeruginosa (ATCC 27853), E. coli
(ATCC 25922), Staph. aureus (ATCC 25923), B. cereus
(ATCC 10707), S. cerevisiae (112, Hefebank Weihenst-
ephan, Freising, Germany) and C. pseudotropicalis
(clinical isolate) microorganism strains were employed
for the determination of antimicrobial activity. Penicillin
(10 IU per disc), amoxicillin (25 lg per disc), ketoco-
nazole (10 lg per disc) and nystatin (100 IU per disc)
were used as reference standards and obtained from
Bioanalyse Co., Ltd, Ankara, Turkey.
In parallel with antimicrobial investigation of horse-
tail extracts, pure solvent was also tested, and it did not
exhibit any antibacterial activity (data are not shown).
Bacteria and yeasts were obtained from the stock
cultures of Microbiology Laboratory, Faculty of Tech-
nology, University of Novi Sad.
Statistical analysis
All measurements were carried out in triplicate, and
presented as mean ± SD or SEM. Regression analysis
and significance of differences were carried out using an
SPSS Statistical Software package (SPSS for Windows,
8.0, 1997; SPSS Inc., Chicago, IL, USA). Statistical
significance level was fixed at P < 0.05.
Results and discussion
Total phenolic content
The contents of total phenolic compounds of different
horsetail extracts are shown in Table 1. The obtained
results showed that the concentration of total phenolic
compounds in n-butanol extract (96.4 ± 4.81 mg g)1)
was significantly higher (P < 0.05) than in ethyl acetate
(26.4 ± 1.32 mg g)1) and water extracts (15.4 ±
0.76 mg g)1), respectively. Petroleum ether did not
extract any of the phenolic compounds, whereas very
small quantity of the phenolic compounds (1.73 ±
0.08 mg g)1) was found in chloroform. This fact is in
correlation with polarity of the solvents used for
Table 1 Total phenolic content in horsetail extracts
Total phenolic
Extract content (mg g)1)a
Petroleum ether 0 difference between total phenolic content in ethyl acetate
Chloroform 1.73
Ethyl acetate 26.4
n-Butanol 96.4
Water 15.4
a
Data expressed as mean ± SEM.
International Journal of Food Science and Technology 2009, 44, 269–278
extraction and solubility of phenolic compounds in
them (Calliste et al., 2001).
HPLC analysis of phenolic compounds in horsetail extracts
In an attempt to identify and quantify phenolic com-
pounds in horsetail extracts, a standard mixture was
prepared containing phenolic acids and flavonoids,
which were commercially available. Gradient elution
program using acetonitrile-acetic acid–water as solvent
was chosen. The chromatogram of standard mixture is
presented in Fig. 1a. The retention times (RT) of
phenolic acids and flavonoids are reported in Table 2.
As shown in Fig. 1a, a satisfactory separation, with
sharp symmetrical peaks, was achieved for all phenolic
compounds within 22 min. Calibration curves were
established for each phenolic acid and flavonoid at
concentration ranges expected in extracts. Validation
results are summarised in Table 2.
All investigated phenolic acids and flavonoids showed
excellent linearity with correlation coefficients between
peak areas and concentrations greater than 0.995 in the
range studied.
Figure 1b–d shows the chromatograms of the ethyl
acetate, n-butanol and water extracts of horsetail.
Hydroxyl derivatives of benzoic (protocatechuic, vanil-
lic and syringic acid) and cinnamic acid (caffeic,
p-coumaric and ferulic acid) and flavonoids (rutin and
(-)-epicatechin) are identified in these extracts by com-
paring their RT and on-line ultraviolet (UV) spectra
with those of standards.
In addition, several other peaks, named unidentified
compounds (UC), had similar spectra as some flavo-
noids and that fact was used for the provisional
identification of flavonoid-glycosides not available as
standards. UC1 and UC4 to UC6 exhibited spectral
characteristics identical with that of kaempferol and
were tentatively identified as kaempferol-glycosides.
Peaks II and III have similar spectra to quercetin and
because of that UC2 and UC3 have been identified as
quercetin-glycosides. UC7 to UC9 had maxima absor-
bance wavelength of 259 and 333 nm, and were identi-
fied as apigenin-glycosides (Veit et al., 1995).
Table 3 lists UV absorption maxima of each peak and
the content of phenolic acids and flavonoids in ethyl
acetate, n-butanol and water extracts, which were
calculated by using the obtained concentrations of these
compounds from calibration curves (Table 2), expressed
as mg per gram dry weight extracts.
The results of HPLC analyses showed no significant
± 0.08
and n-butanol extracts. The total phenolic content was
23.84 mg g)1 for ethyl acetate extract and 26.00 mg g)1
± 1.32
± 4.81
± 0.76
for n-butanol extract. The ethyl acetate and n-butanol
extracts contained rutin as the major phenolic com-
pound but in different extent (13.78 mg g)1 for ethyl
 2008 Institute of Food Science and Technology
 Radical scavenging and antimicrobial activity of horsetail extracts J. M. Čanadanović-Brunet et al. 273

(a) (b) 7
160 8
50 7
8 140
40 120
100
mRU

mRU
30
5 80 II
20 2 4 60 III V
3 6 1 3
1 2 4 6 IV VI VII
40
5 I
10
20
0
10 20 30 10 20 30
Time (min) Time (min)
(c) III (d) 4
250
50
8
200 II
40

150

mRU
7
mRU

30 7
8
100 IV 1
4 VIII 20
I V VII IX
VI 3
50 V VII
1 23 10 VIII

0
10 20 30 10 20 30
Time (min) Time (min)

Figure 1 HPLC chromatograms of the mixture of standards (a), ethyl acetate (b), n-butanol (c) and water extracts (d) at 280 nm. Peaks: 1 –
protocatechuic acid; 2 – vanillic acid; 3 – caffeic acid; 4 – syringic acid; 5 – (-)-epicatechin; 6 – p-coumaric acid; 7 – ferulic acid; 8 – rutin; I–IX:
unidentified compounds UC1–UC9.

Table 2 Retention times and calibration

curves of the phenolic acids and flavonoids Calibration curves

RT Linear range
Peak Phenolic acid (min) Regression equationa (lg mL)1) r

1. Protocatechuic acid 5.084 y = (6.14x ) 0.0017)104 1–20 0.9966

2. Vanillic acid 9.136 y = (5.24x + 0.0040)104 1–20 0.9993
3. Caffeic acid 10.243 y = (18.06x ) 0.0137)104 10–60 0.9951
4. Syringic acid 11.130 y = (11.45x ) 0.1931)104 10–60 0.9999
5. (-)-Epicatechin 12.326 y = (2.07x + 0.0003)104 20–100 0.9986
6. p-Coumaric acid 13.571 y = (19.45x + 0.0002)104 1–20 0.9995
7. Ferulic acid 16.113 y = (10.61x + 0.0366)104 1–20 0.9988
8. Rutin 21.902 y = (2.98x + 0.2118)104 2–60 0.9967

a
y, peak area; x, concentration (mg mL)1).

acetate extract and 19.23 mg g)1 for n-butanol extract).
In water extract phenolic compounds were present in
lower extent (4.13 mg g)1).
In general, the total phenolic content of the extracts
was found higher than the sum of the individual
phenolics identified using HPLC. Caution must be
taken in interpreting the results obtained by the Folin-
Ciocalteu method, as other substances in the extract
such as sugars have been shown to interfere with the
assay. In addition, different classes of phenolics con-
tribute to different extents to the absorbance produced
by reaction with the Folin-Ciocalteu reagent when
compared with chlorogenic acid (Singleton et al.,
1999). Nevertheless, the results of the Folin-Ciocalteu
test (Table 1) as well as tentatively identified peaks in
chromatograms (Fig. 1b–d) suggest that the extracts
contained some other phenolic compounds (Veit et al.,
1995; Beckert et al., 1997; Oh et al., 2005).

 2008 Institute of Food Science and Technology International Journal of Food Science and Technology 2009, 44, 269–278
274 Radical scavenging and antimicrobial activity of horsetail extracts J. M. Čanadanović-Brunet et al.

Table 3 UV absorption maxima and content

Content of phenolic acids and flavonoids of phenolic acids and flavonoids in ethyl
(mg g)1)a acetate, n-butanol and water extracts
Ethyl acetate n-Butanol Water
Peak Phenolic acid UVmax (nm) extract extract extract

1. Protocatechuic acid 258, 292 1.09 ± 0.05 0.55 ± 0.03 0.36 ± 0.02
2. Vanillic acid 260, 290 0.96 ± 0.05 0.42 ± 0.02 –
3. Caffeic acid 238, 296, 322 0.64 ± 0.03 0.26 ± 0.01 0.10 ± 0.005
4. Syringic acid 274 2.06 ± 0.10 2.80 ± 0.13 2.29 ± 0.11
5. (-)-Epicatechin 240, 278 1.24 ± 0.06 – –
6. p-Coumaric acid 294, 308 0.46 ± 0.02 – –
7. Ferulic acid 236, 294, 322 3.62 ± 0.18 2.74 ± 0.14 0.54 ± 0.03
8. Rutin 234, 254, 354 13.78 ± 0.68 19.23 ± 0.96 0.84 ± 0.04

a
Data expressed as mean ± SD.

120 Ethyl acetate n-Butanol Water The DPPH scavenging activity (SADPPH) of ethyl
f f f acetate, n-butanol and water extracts increased dose-
100 d dependently at concentrations ranging from 0.50 to
d 2.50 mg mL)1. Among them, n-butanol extract was the
80 d
most effective DPPH radical scavenger. Namely, the
SADPPH (%)

60 e total elimination of DPPH radical (SADPPH = 100%)

c c
b, c
40 b n-butanol extract, whereas the ethyl acetate and water
a
20 a and 31.6% DPPH radicals, respectively. When the BHA
0
0.5 1 1.5
Concentration (mg mL–1)
Figure 2 The scavenging activity (SADPPH) of different concentrations
of ethyl acetate, n-butanol and water extracts of horsetail on DPPH
radicals. Bars sharing the same letter (a–f) are not significantly
different (P > 0.05) from one another.
DPPH radical scavenging activity of horsetail extracts
The DPPH assay has been widely used to evaluate the
free radical scavenging activity of various polyphenolic
antioxidants commonly found in food (Llorach et al.,
2002), plant extracts (Ellnain-Wojtaszek et al., 2003;
Miliauskas et al., 2004; Pellati et al., 2004) and bever-
ages (Sánchez-Moreno et al., 1999). The reduction of
DPPH radicals is followed by monitoring the decrease in
its absorbance at characteristic wavelength during reac-
tion (Brand-Williams et al., 1995; Jimenez-Escrig et al.,
2000). But in our work, ESR spectroscopy was used to
identify DPPH radical. The typical ESR spectra of
DPPH radical in the blank and in the sample were
observed with five lines of relative intensities 1:2:3:2:1
and hyperfine splitting constant aN = 9.03 G. (Ćetk-
ović et al., 2004).
The scavenging activity (SADPPH) of different con-
centrations of ethyl acetate, n-butanol and water
extracts of horsetail is presented in Fig. 2.
was obtained in the presence of 1.5 mg mL)1 of
c
extracts, at the same concentration, scavenged 43.5%
was tested as referent pure compound, it required
2 2.5 1.5 mg mL)1 to produce SADPPH = 82.3% (data are
not shown).
However, at higher concentration (2.50 mg mL)1),
the ethyl acetate and water extracts also showed
significant SA values (76.3% for ethyl acetate and
56.9% for water extract). Within the investigated range
of concentrations, the petroleum ether and chloroform
extracts did not show any radical scavenging activity.
Hydroxyl radical scavenging activity of horsetail extracts
During the Fenton reaction, in the presence of spin
trapping agent DMPO, ESR signals with 1:2:2:1 quartet
of lines were generated (hyperfine coupling parameters
aN = aH = 14.9 G) (Čanadanović-Brunet et al., 2006).
The intensity of ESR signal, corresponding to the
concentration of formed free radicals, was decreased in
the presence of different amount of n-butanol, ethyl
acetate and water horsetail extracts. Within the inves-
tigated range of concentrations, the petroleum ether and
chloroform extracts did not show any radical scavenging
activity, as the case of DPPH assay.
It is evident that the interaction of a potential
antioxidant with hydroxyl radical depends on the type
and concentration of the investigated extract. The
following order of hydroxyl radical scavenging activity
has been established: n-butanol > ethyl ace-
tate > water (Fig. 3). In addition, the investigation

International Journal of Food Science and Technology 2009, 44, 269–278  2008 Institute of Food Science and Technology
 Radical scavenging and antimicrobial activity of horsetail extracts J. M. Čanadanović-Brunet et al. 275

120 Ethyl acetate n-Butanol Water 3.5

DPPH radical assay
h h h 2 Hydroxyl radical assay
100 g, h 3 r = 0.9269
g
2.5

EC50 (mg mL–1)

80 f
SAOH (%)

e, f e 2
60 d, e r2 = 0.8792
d
d
c, d 1.5
40 c
c
b 1
b
20 a
a 0.5
0
0.5 1 1.5 2 2.5 3 3.5 0
0 20 40 60 80 100 120 140 160 180
Concentration (mg mL–1)
Total phenolic content - Folin Ciocalteu method (mg g–1)

Figure 3 The scavenging activity (SAOH) of different concentrations of

4
ethyl acetate, n-butanol and water extracts of horsetail on DMPO-OH
3.5 r2 = 0.7195 DPPH radical assay
spin adduct. Bars sharing the same letter (a–h) are not significantly Hydroxyl radical assay
different (P > 0.05) from one another. 3

EC50 (mg mL–1)

2.5 r2 = 0.7892
Table 4 EC50DPPH and EC50 OH
values of ethyl acetate, n-butanol and 2
water extracts
1.5
Extract EC50DPPH (mg mL)1)a EC50OH (mg mL)1)a
1
Ethyl acetate 1.52 ± 0.07 2.29 ± 0.11 0.5
n-Butanol 0.64 ± 0.03 0.74 ± 0.03
Water 2.27 ± 0.11 3.29 ± 0.16 0
0 5 10 15 20 25 30
a Total phenolic content - HPLC method (mg g–1)
Data expressed as mean ± SEM.
DPPH
Figure 4 Relationship between EC50 (EC50 ; EC50OH) and total
showed that the hydroxyl radical scavenging activity phenolic content obtained by the Folin-Ciocalteu and HPLC methods.
increased with increasing the concentration of all
extracts.
The concentration of 1 mg mL)1 of n-butanol extract On the contrary, linear regression analysis showed that
is less effective in scavenging hydroxyl radicals the correlation of EC50DPPH and EC50OH values were
(SE = 65.3%) in comparison with the DPPH radical rather poorly (r2 < 0.5, P < 0.05) linked to the indi-
scavenging activity. Hence, at the concentration of vidual phenolics (data not shown). It appears, therefore,
2.50 mg mL)1, n-butanol extract inhibits completely that the differences between the correlation of individual
the formation of hydroxyl radical. At this concentra- phenolics and total phenolics with the EC50DPPH and
tion, ethyl acetate extract produced SA = 55.9% and EC50OH suggest that the free radical scavenging activity
water extract SA = 38.3%. BHA eliminated 85.3% of of extracts is rather the consequence of synergistic
hydroxyl radicals at 2.5 mg mL)1 (data are not shown). phenomena among the individual components. A sim-
The EC50 value is a parameter widely used to measure ilar relationship has been established by Soleas et al.,
the free radical scavenging activity (Cuvelier et al., 1997; Makris et al., 2003.
1992). A lower EC50 indicates a higher antioxidant
activity. The EC50DPPH and EC50OH values of ethyl
DPPH and OH radical scavenging activity of standards
acetate, n-butanol and water extracts are presented in
Table 4. The radical scavenging activity of phenolic compounds
Among the three extracts, n-butanol extract was the is correlated to some structure features, such as the
richest in total phenolics (Table 1) and revealed as the number and arrangement of hydroxyl groups, resonance
most effective at scavenging both DPPH and hydroxyl delocalisation of aroxyl radicals (ArOd) and steric
free radicals (Table 4). hindrance derived from bulky groups substituting
The values of total content of phenolic compounds hydrogen in aromatic ring (Cotelle et al., 1996; Rice-
obtained by the Folin-Ciocalteu method and HPLC Evans et al., 1996).
method were correlated with EC50DPPH and EC50OH, In effort to prove that the phenolics identified by
respectively (Fig. 4). In both cases, the correlation HPLC were particularly responsible for SADPPH and
coefficients were sufficiently high (r2 > 0.7, P < 0.05). SAOH, we also presented the DPPH and OH radical

 2008 Institute of Food Science and Technology International Journal of Food Science and Technology 2009, 44, 269–278
276 Radical scavenging and antimicrobial activity of horsetail extracts J. M. Čanadanović-Brunet et al.

DPPH
Table 5 EC50 and EC50OH values of standards activity. Keto group at C4 and double bond between
DPPH )1 a OH )1 a
C2 and C3 in the pyrone ring can enhance this activity
Compound EC50 (mg mL ) EC50 (mg mL )
of rutin (Jovanovic et al., 1996).
)3
Protocatechuic acid 0.04 ± 1.80 · 10 0.05 ± 2.50 · 10)3
Vanillic acid 1.72 ± 0.09 2.08 ± 0.10
Antimicrobial activity
Caffeic acid 0.01 ± 0.49 · 10)3 0.02 ± 1.00 · 10)3
Syringic acid 0.05 ± 2.42 · 10)3 0.07 ± 3.50 · 10)3 The values of antimicrobial activity of petroleum
(-)-Epicatechin 0.09 ± 4.42 · 10)3 1.02 ± 0.05 ether, chloroform, ethyl acetate, n-butanol and water
p-Coumaric acid >2.50 >2.5 extracts of horsetail and standards are shown in
Ferulic acid 0.09 ± 4.37 · 10)3 1.21 ± 0.06
Table 6.
Rutin 0.07 ± 3.49 · 10)3 0.09 ± 4.50 · 10)3
The obtained results showed that ethyl acetate and n-
a
Data expressed as mean ± SEM. butanol extracts inhibited the growth of all tested
bacteria, except E. coli. Both extracts showed similar
antimicrobial activity against Gram-positive and Gram-
negative bacteria. The MIC with ethyl acetate extract
scavenging activity of standards, expressed as EC50 was found to be 50 mg mL)1 for P. aeruginosa, and with
values (Table 5). n-butanol extract 75 mg mL)1. It is an important fact
The relationship between structure and activity can that ethyl acetate extracts showed strong antibacterial
be proposed from this series of the phenolics. These activity against Staph. aureus (MIC is 25 mg mL)1), one
results are in agreement with the fact that phenolic of the most common Gram-positive bacterium causing
compounds with o-dihydroxyl group in aromatic ring food poisoning. On the contrary, very weak antimicro-
possess stronger antioxidant activity than monophen- bial activity (MIC ‡ 100 mg mL)1) of water extract was
olics (Bors et al., 1990). For example, caffeic acid found against the tested bacteria. Petroleum ether and
reacted better both with DPPH and OH radicals than chloroform extracts did not show any activity (data are
p-coumaric acid. In addition, methoxy substitution of not shown). All horsetail extracts did not inhibit the
the hydroxyl group in the ortho position of the growth of E. coli (MIC > 100 mg mL)1). In addition, it
diphenolics, as in ferulic or vanillic acid, resulted in is important to note that this pathogenic bacteria
to a decrease in the free radical scavenging reaction showed the lowest sensibility on Penicillin
(Rice-Evans et al., 1996). According to literature data, (ZI = 10 mm). None of the horsetail extracts showed
rutin and (-)-epicatechin possess marked free radical activity on the yeasts.
scavenging activity (Bouchet et al., 1998; Ellnain-Wo- In conclusion, the results of ESR analysis indicate
jtaszek et al., 2003). In both compounds, the presence that n-butanol, ethyl acetate and water extracts have
of o-dihydroxyl group in B-ring is fundamental for significant DPPH and OH scavenging activity, which is
their scavenging properties, but m-hydroxyl groups at in correlation with the contents of total phenolic
C5 and C7 in ring A also contribute to scavenging compounds. The various antioxidant mechanisms of

Table 6 Antimicrobial activity of different

Microorganisms horsetail extracts and reference standards
P. aeruginosa E. coli Staph. aureus B. cereus

ZI MIC ZI MIC ZI MIC ZI MIC

Extracts
Ethyl acetate 10.5 ± 0.6 50 n.i. >100 13.3 ± 0.6 25 8.7 ± 0.6 100
n-Butanol 9.3 ± 1.0 75 n.i. >100 8.7 ± 0.6 100 8.0 ± 0.0 100
Water 8.0 ± 0.0 100 n.i. >100 n.i. >100 7.0 ± 0.0 100
ZI ZI ZI ZI

Standards
Amoxicillin 22.7 ± 1.5 28.0 ± 0.0 29.0 ± 0.0
21.3 ± 1.15
Penicillin 23.3 ± 0.8 32.7 ± 1.15 34.0 ± 0.0
10.0 ± 0.0

ZI expressed as mean ± SD (mm); MIC expressed as mg mL)1.

n.i., no inhibition.

International Journal of Food Science and Technology 2009, 44, 269–278  2008 Institute of Food Science and Technology
 Radical scavenging and antimicrobial activity of horsetail extracts J. M. Čanadanović-Brunet et al.
these three extracts, which contain hydroxybenzoic and
hydroxycinnamic acid derivatives and flavonoids with
different chemical behaviours, may be attributed to
strong hydrogen donating ability, a metal chelating
ability, and their effectiveness as good scavengers of
DPPH and hydroxyl radicals. All of these results
obtained show that the horsetail extracts can be used
as a easily accessible source of natural antioxidants and
as a possible food supplement. The results of anti-
microbial activity may provide support for some of the
uses of horsetail as a phytochemical. Therefore, it is
suggested that further works should be performed on
identification of in vivo antioxidant activity of horsetail
extracts.
Acknowledgment
This research is part of the project No. BTN-371012B,
which is financially supported by the Ministry of Science
and Environmental Protection of the Republic of
Serbia.
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