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132
Vol. 26, No. 2: 132–138 Czech J. Food Sci.
Czech Medicinal Plants as Possible Sources of Antioxidants
L BUŘIČOVÁ and Z RÉBLOVÁ
Department of Food Chemistry and Analysis, Faculty of Food and Biochemical
Technology, Institute of Chemical Technology in Prague, Prague, Czech Republic
Abstract
B L., R Z. (2008): Czech medicinal plants as possible sources of antioxidants. Czech
J. Food Sci., 26: 132–138.
The antioxidant activity of 17 Czech medicinal plants was studied and compared with the antioxidant activity of green
tea. The antioxidant activities of water and ethanol extracts of the plants tested were determined by a spectropho-
tometric method using the stable free radical DPPH (2,2-diphenyl-1-picrylhydrazyl), and further the contents of the
compounds with reducing properties in water extracts were determined by flow injection analysis with amperometric
detection (using a detection potential +0.7 V). Considerable antioxidant activities were found in the extracts of plants
from the Rosaceae family (rosehips and leaves of raspberry, blackberry, and strawberry), the Lamiaceae family (oregano,
sweet balm, thyme, dead-nettle, and mint), and flowers of linden and elder.
Keywords: antioxidant activity; plant extracts; free radicals DPPH
Supported by the Ministry of Education, Youth and Sports of the Czech Republic (Project No. MSM 6046137305).
Many human diseases are caused or negatively
affected by free radicals. The natural defense of
the human organism againts free radicals is not
always sufficient mainly due to the significant
exposition to free radicals from external sources
in the modern world. The dietary intake of anti-
oxidants plays an important role in the protection
of the human organism againts free radicals. Many
clinical and epidemiological studies show a con-
nection between the antioxidant activity of the
substances present in the diet and the prevention
from such diseases as cardiovascular diseases or
carcinogenesis (H 2000; K-E
et al. 2002; L & A 2002).
Fruits, vegetables, grains, teas, wines, and some
kinds of spices are natural sources of antioxidants
(R-E et al. 1996). The intake of these food
comodities is not always satisfactory. Therefore,
the studies of possible new sources of antioxidants
have become important in the last few years. The
new sources of the antioxidants could be used for
direct consumption or for the production of food
supplements which could be used for enriching
foods with the aim of increasing their nutritional
value. Medicinal plants used in the traditional
medicine and healing are one of these sources
of antioxidants. In many countrie s, screening
studies were carried out for the comparison of
antioxidant activities of medicinal plants typical
for the respective country (C et al.
2005; I et al. 2005; K et al. 2006;
P et al. 2006; W et al. 2006). There
is no study of this kind in the available literature
concerning the Czech medicinal plants.
The aim of this work was to compare the Czech
medicinal plants in view of their antioxidant activ-
133
Czech J. Food Sci. Vol. 26, No. 2: 132–138
ity. Seventeen plants resp. parts of plants were se-
lected with respect to: popularity, the conventional
use, and the recommendation for use (they can be
taken for a long time) in the traditional medicine
and healing, their use for beverage preparation,
as well as legislation regulating the use of these
plants in food industry. The antioxidant activity
of water and ethanol extracts of the dry medicinal
plants was estimated using the free radical DPPH,
usually used in this type of screening study. To
confirm the plants antioxidant activity, the con-
tents of compounds with reducing properties were
determined by flow injection analysis (FIA) using
an amperometric detector. The antioxidant activi-
ties of the plants studied were compared with the
antioxidant activity of green tea.
MATERIAL AND METHODS
Material. The studied medicinal plants and their
parts were as follows: blueberry fruits (Vaccinium
myrtillus L.), chicory roots and tops (mixture
1:1 m/m; Cichorium intybus L.), oregano tops
(Origanum vulgare L.), fennel fruits (Foenicu-
lum vulgare Mill.), camomile flowers (Matricaria
chamomilla L.), nettle flowers (Lamium album L.),
strawberry leaves (Fragaria vesca L.), rowan fruits
(Sorbus aucuparia L.), linden flowers (Tilia cordata
Mill. and/or Tilia platyphyllos Scopoli and/or Tilia
vulgaris Hayne), blackberry leaves (Rubus idaeus
L.), mint tops (Mentha piperita L.), thyme tops
(Thymus serpyllum L.), sweet balm tops (Melissa
officinalis L.), raspberry leaves (Rubus fruticosus
L.), buckthorn fruits (Hippophae rhamnoides L.),
rosehips (Rosa canina L.) and elder flowers (Sam-
bucus nigra L.).
Practically all the plant materials studied (elder
flowers excepted) can be used for the preparation
of beverages resembling to tea without any limit
in accordance with the Czech food regulations
(Ministry of Agriculture 1997). Elder flowers can
be used for this purpose only in mixtures with
other plant materials up to 30% of weight. For
that reason, the medicinal plants tested (elder
flowers excepted) can be considered as safe, thus
no disagreement will probably arise about using
them in other ways than only for the preparation
of beverages.
Of every medicinal plant studied from two to
four dry samples were analysed. The samples were
evaluated in this phase of research from the con-
sumers’ point of view. Therefore, the localities
of the growth of these plants or the conditions
of their drying were not found out. One series of
dry samples (first series) was purchased from a
well known Czech producer and the other from
ordinary Czech shops. The antioxidant activity
of water extracts was established in all samples.
The antioxidant activity of ethanol extracts and
the contents of compounds with reducing prop-
erties in water extracts were determinated in the
first series.
Green tea samples were purchased from ordinary
Czech shops. Their antioxidant activities were
determined in water extracts.
The dry parts of plants and g reen teas were
kept in closed containers, in the dark, at labora-
tory temperature, and they were used within the
recommended period for their consumption.
Extraction. For the preparation of water ex-
tracts, 50 ml of deionised water (MILLI-Q-RG,
ZFMQ 050 RG, Millipore, USA) were added to 1 g
of dry ground plant material (or green tea). The
water temperature was 98°C, infusion time was
20 minutes. For the preparation of ethanol extracts,
50 ml of ethanol (96%) were added to 1 g of dry
ground plant material at laboratory temperature.
The mixture was left in a dark place. Infusion time
was 24 hours. All extracts prepared were analysed
shortly after the preparation.
Scavenging DPPH radicals. The method used
was almost the same as that used by other authors
(G et al. 1997; I et al. 2003; D
et al. 2004), but was modified in details. 2 ml of
methanol solution of DPPH radical (2,2-diphe-
nyl-1-picrylhydrazyl, Sigma Aldrich, St. Louis,
USA) in the concentration of 0.05 mg/ml and
1 ml of plant extract or dilute plant extract were
placed in cuvettes. The decrease in absorbance at
522 nm (experimentally established wavelength)
was measured using a spectrophotometer Car y
100 Bio (Varian, Palo Alto, USA) until the differ-
ence between the absorbance of the sample and
the control sample remained stable (24 h). As the
control sample, DPPH radical solution with 1 ml
of deionised water was used. Analytical grade
methanol (Penta, Chrudim, Czech Republic) was
used to zero the spectrophotometer.
The difference between the absorbance of the
DPPH radical solution containing the plant extract
and that of the control sample was expressed as mg
of -ascorbic acid (Sigma Chemical Co, St. Louis,
USA) per 1 g of dry plant material. Calibration
was used in such cases, where the plant extracts
134
Vol. 26, No. 2: 132–138 Czech J. Food Sci.
were replaced with a freshly prepared solution of
ascorbic acid in deionised water (concentration
from 0 to 1.6 mg/100 ml).
All determinations were performed in duplicates
and the particular assesment included also the
preparation of extracts.
Reduction ability. FIA equipment consisted of
a non-steel pump LCP 4020.31 (ECOM, Prague,
Czech Republic) and an amperometric detector
HP 1049A (Hewlett Packard, Avondale, USA)
equipped with a glassy-carbon electrode (operat-
ing at a potential of +0.7 V), a reference Ag/AgCl
electrode and a platinum counter electrode. Data
were recorded using a 1.6 DataApex chromatogra-
phy system (Prague, Czech Republic). A mixture
of acetonitrile (Merck, Darmstadt, Germany) and
0.2% (m/m) o-phosphoric acid (Lachema Nerato-
vice, Czech Republic) (4:1, v/v) containing so-
dium chloride (0.005 mol/l) (Lachema Neratovice,
Czech Republic) was used as the electrolyte. The
injection volume was 20 l and the flow rate of
1 ml/min was applied.
The contents of oxidable substances were ex-
pressed as mg of ascorbic acid per 1 g of dry plant
material. Calibration of freshly prepared solutions
of ascorbic acid in deionised water was used for this
purpose (concentration from 0 to 4 mg/100 ml).
All determinations were performed in duplicates
and the particular assesment included also the
preparation of extracts.
Statistical analysis. The results of the antioxi-
dant activity determination using stable synthetic
free radical DPPH were statistically analysed apply-
ing the Student t-test (on the level of probability
0.05). The correlation coefficients between the
results of the antioxidant activity determination
and the contents of compounds with reduction
properties contained in water extracts of the re-
spective medicinal plants were determined applying
the Excel software (Microsoft).
RESULTS AND DISCUSSION
The antioxidant activity in water extracts of all
samples of the medicinal plants studied is shown
in Table 1. Antioxidant activities are expressed as
milligrams of ascorbic acid per 1 g of dry plant
material. These data are mismatched w ith the
Table 1. Antioxidant activity of water extracts determined using DPPH radical
Plant material Number of samples Antioxidant activity (mg/g)
Strawberry leavesa4 123.0 ± 32.4
Oregano topsa3 116.9 ± 4.7
Blackberry leavesa3 111.5 ± 15.7
Raspberry leavesa, b 3 90.6 ± 10.6
Sweet balm topsa, b 4 87.5 ± 23.7
yme topsa, b, c 3 83.4 ± 22.8
Rosehipb, c 3 69.7 ±11.5
Netlle flowersb, c 3 64.9 ± 0.8
Mint topsb, c 4 61.7 ± 21.3
Elder flowersc3 60.2 ± 3.3
Linden flowersc3 58.8 ± 6.3
Chamomile flowersd3 31.8 ± 4.0
Blueberry fruitsd3 31.8 ± 2.7
Buckthorn fruitsd, e 2 21.4 ± 4.6
Rowan fruitsd, e 3 15.6 ± 8.1
Chicory roots and topse3 13.2 ± 1.1
Fennel fruitsf2 3.2 ± 1.4
a, b, c, d, e, f
statistically significant differences were not found in groups of plants marked, with the same letters data was
expressed as mean ± SD as milligrams of ascorbic acid per 1 g of dry plant material
135
Czech J. Food Sci. Vol. 26, No. 2: 132–138
contents of antioxidants, because one molecule of
ascorbic acid is able to inactivate two free DPPH
radicals while phenolic acids, e.g., are able to in-
activate 4 to 6 free DPPH radicals per molecule
(B-W et al. 1995). Therefore, the
antioxidant contents are probably lower.
Antioxidants are secondary metabolites and
their contents in plants depend on varied stress
conditions of vegetation (V et al. 1999).
However, considerable differences were not found
between the antioxidant activities of the samples of
the same plant. Water extracts of strawberry leaves,
oregano tops, raspberry leaves, blackberry leaves,
sweet balm tops, and thyme tops had generally a
high antioxidant activity. Rosehips, dead-nettle
flowers, mint tops, elder flowers, and linden flowers
had a medium antioxidant activity, and camomile
flowers, blueberry fruits, buckthorn fruits, rowan
fruits, chicory roots and tops and fruits of fennel
had a low antioxidant activity.
Green tea is known as one of the richest sources
of natural antioxidants (K-E et al.
2002). Therefore, the antioxidant activities of the
plants tested were compared with the antioxidant
activity of green tea. All investigated plants had a
lower antioxidant activity than the water extracts
of green tea (a typical result with three samples
was 313.3 ± 15.2 mg of ascorbic acid per 1 g of
dry plant material) prepared in the same way as
water extracts of medicinal plants. However, the
plants studied contain no caffeine, which is why
the plants with a lower antioxidant activity than
that of green tea could be an interesting source
of natural antioxidants.
Antioxidant activity depends on the manner
in which the extracts are prepared (and in which
antioxidant activity is determined, see next). There-
fore, antioxidant activity was established in the
first series, including the samples of all the plants
studied, in both their water and ethanol extracts
(and, further, the contents of the compounds with
reducing properties in water extracts of these
samples were determined, see next). The extrac-
tion of substances with the antioxidant activity
Table 2. Antioxidant activity of water and ethanol plant extracts (first series of samples) determined using DPPH
radical
Plant material Antioxidant activity (mg/g)
water extract ethanol extract
Strawberry leaves 121.6 ± 14.2 33.6 ± 3.9
Oregano tops 118.2 ± 7.0 11.8 ± 0.6
Blackberry leaves 114.8 ± 2.9 30.1 ± 1.3
yme tops 109.4 ± 0.7 21.4 ± 0.3
Raspberry leaves 102.7 ± 1.2 17.1 ± 0.7
Sweet balm tops 100.0 ± 11.2 6.5 ± 0.5
Mint tops 72.2 ± 2.4 21.3 ± 2.3
Netlle flowers 65.8 ± 3.6 14.3 ± 0.4
Linden flowers a 63.0 ± 3.8 36.7 ± 1.8
Rosehip 62.7 ± 1.1 6.3 ± 1.1
Elder flowers 60.8 ± 0.1 13.5 ± 0.5
Chamomile flowers 34.8 ± 0.2 5.5 ± 0.2
Blueberry fruits 34.2 ± 0.0 14.9 ± 0.1
Rowan fruits 25.0 ± 2.5 9.4 ± 0.1
Buckthorn fruits 24.6 ± 0.5 0.9 ± 0.1
Chicory roots and tops 12.4 ± 0.4 4.0 ± 0.5
Fennel fruits 4.2 ± 0.2 1.3 ± 0.1
a value is the mean of four determinations, the other values are the mean of two determinations; data was expressed as
mean ± SD as milligrams of ascorbic acid per 1 g of dry plant material
136
Vol. 26, No. 2: 132–138 Czech J. Food Sci.
was markedly more efficient using hot water than
using ethanol at laboratory temperature with all
plants studied (Table 2).
On the basis of the contents of the compounds
possessing reducing properties, the medicinal
plants studied were lined up in the following order:
sweet balm tops > thyme tops > oregano tops >
strawberry leaves > mint tops > elder flowers >
raspberry leaves > nettle flowers > blackberr y
leaves > camomile flowers > linden flowers > blue-
berry fruits > rosehips > rowan fruits > buckthorn
fruits > chicory roots and tops > fennel fruits. The
comparison between the antioxidant activities in
water extracts and the contents of compounds
with reducing properties contained in these ex-
tracts is shown in Figure 1. There is a significant
linear correlation (P < 0.01; correlation coefficient
r = 0.602 (resp. r = 0.745) where the trendline goes
from the origin of coordinates (resp. does not go
from the origin of coordinates) between these two
parameters. Although all oxidable substances do
not have antioxidant activity (the opposite, some
compounds without reduction abilities can act
as antioxidants, e.g. some substances that are
able to bind metal ions), a close relation between
the antioxidant activity and the content of com-
pounds with reducing properties was described
by other authors (B et al. 2001; C et
al. 2006).
On the basis of the presented results, we can
consider the studied plants of the Lamiaceae family
(oregano, sweet balm, thyme, dead-nettle, mint) and
the Rosaceae family (leaves of raspberry, blackberry,
and strawberry) to be good sources of antioxidants.
These results agree with the results published for-
merly. A good antioxidant activity of the Lamiaceae
family was found by many authors (Z & A-
2000; D et al. 2004; C et al.
2005). The plants of the Rosaceae family were not
investigated in detail from the antioxidant point
of view unlike those of the Lamiaceae family. K-
et al. (2006) determined the antioxidant
activities and phenolic contents of 70 medicinal
plants. The leaves of raspberry, blackberr y, and
strawberry were among eleven of the most effective
plants. In another study (W & L 2000), the
antioxidant activities of leaves and berries of these
plants were compared. The leaves were a richer
source of antioxidants and had a greater content
of phenolic substances than berries.
A medium antioxidant activity was found in
the extracts of rosehips, elder flowers, and linden
flowers in the present study. The majority of these
results also agree with the results published for-
merly. For instance, the study by S et al. (2007)
established rosehips as a potential source of natural
antioxidants. A significant antioxidant activity
of linden flowers was established in a study by
Y et al. (2000). In this study, the authors
compared the antioxidant activities of linden flow-
ers, black tea, and sage. Water extract of linden
flowers had a higher antioxidant activity than water
extract of sage. But the study of Italian authors
(G et al. 2003) detected poor antioxidant
activity in the essential oil of elder flowers while
their ethanol extract was inactive.
A low antioxidant activity was found in the ex-
tracts of camomile flowers, blueberry fruits, buck-
thorn fruits, rowan fruits, and chicory roots and
tops (althought the extract of camomile flowers
showed medium activity in the determination of
compounds with reducing properties). The fruits
tested, particulary buckthorn (E et al.
2002), are rich in the content of vitamin C. During
drying, the content of vitamin C probably decreases
as described with rosehips by E et al.
(2005). Contrary to this, dry rosehips were classi-
fied as a plant material with medium antioxidant
activity in this study. It may be due to the presence
of other antioxidants in rosehips apart from sole
vitamin C (X et al. 2000).
0
20
40
60
80
100
120
140
160
0 10 20 30 40 50
FIAȱ(mg/g)
DPPHȱ(mg/g)
Figure 1. Relationship between antioxidant activities
(determined using DPPH radicals) and the contents
of compounds with reduction ability (determined by a
flow injection analysis with amperometric detection)
in medicinal plants water extracts (r = 0.602). Data was
expressed in both methods as milligrams of ascorbic acid
per 1 g of dry plant material
137
Czech J. Food Sci. Vol. 26, No. 2: 132–138
Conclusion
On the basis of our results, Czech medicinal plants
from the Rosaceae family (rosehips and leaves of
raspberry, blackberry and strawberry), the Lamia-
ceae family (oregano, sweet balm, thyme, dead-nettle
and mint), and flowers of linden (Tiliaceae family)
and elder (Loniceraceae family) appear to be good
and safe sources of antioxidants. These plants could
be used for direct consumption as various kinds of
beverages or as extracts to increase the nutritional
value of different foods and diets.
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Received for publication June 8, 2007
Accepted after corrections August 27, 2007
