全 文 ：BIOTECHNOLOGY AND BIOENGINEERING
Chinese Journal of Chemical Engineering, 17(3) 478—483 (2009)
Antioxidant and Antimicrobial Activities of Echinacea (Echinacea
purpurea L.) Extracts Obtained by Classical and Ultrasound Extraction*
Ivana Stanisavljević1,**, Sаša Stojičević2, Dragan Veličković3, Vlada Veljković1 and Miodrag Lazić1
1 Faculty of Technology, University of Niš, Bulevar oslobodjenja 124, 16000 Leskovac, Serbia
2 Chemical Industry Nevena A.D., Djordja Stamenkovića St.,16000 Leskovac, Serbia
3 Zdravlje-Actavis, Vlajkova 199, 16000 Leskovac, Serbia
Abstract Antioxidant and antimicrobial activities of Echinacea purpurea L. (Asteraceae) extracts obtained by
classical and ultrasound solvent extraction were compared. The dry aerial part of plant was extracted by 70% etha-
nol at a solid-to-liquid ratio of 1︰10 (m/v) and 25°C. The extract obtained by classical solvent extraction contained
29% larger amounts of phenolic compounds and 20% higher content of flavonoids. 2,2-diphenyl-1-picril hydrazyl
radical (DPPH) scavenging reached 93.6% and the values of EC50 were (34.16±0.65) µg·ml－1 and (65.48±1.12)
µg·ml－1 for the extracts obtained by the classical and ultrasound extractions, respectively. The extracts, independent
of the extraction technique applied, showed a considerable growth inhibition on Candida albicans and Saccharo-
myces cerevisiae, while no growth inhibition zones were observed for Aspergillus niger. The diameters of inhibition
zone observed for all the microorganisms were larger for extracts obtained by classical extraction than those by ul-
trasound extraction.
Keywords antioxidant activity, antimicrobial activity, Echinacea purpurea L., total phenols, flavonoids, extraction
1 INTRODUCTION
Echinacea purpurea L., also known as purple
coneflower, is an herbaceous perennial and a member
of the Asteraceae family with a long, well-established
tradition of medicinal use in North America, Europe [1]
and Australia [2]. In modern cultures, E. purpurea is
used for medicinal purposes, in treating acute upper
respiratory infections, urinary tract infections, burns
and disorders such as viral infections, cutaneous af-
fections and chronic disease due to a deficiency of
immunological responses [1, 3-5]. E. purpurea stimu-
lates various immune cells including macrophages and
natural killer cells and has anti-inflammatory effects [4].
The roots, the leaves or the whole plant may be also
used in the dietary supplement preparation. The compo-
sition of the root extracts, compared to the upper plant
extracts, is very different. Root parts have more volatile
oils and pyrrolizidine alkaloids, such as tussilagine
and isotussilagine, than the aerial parts. The main active
compounds of the aerial parts are alkamides and polya-
cetylenes, caffeic and ferulic acid derivatives, polysac-
charides (such as 4-O-methylglucuronylarabinoxylans,
rhamnoarabinogalactans and acidic arabinogalactan)
and glycoproteins [5, 6]. Of the caffeic acid derivatives,
only cichoric acid, which is found to be the main
phenolic compound in E. purpurea, shows immu-
nostimulatory properties, promoting phagocyte activ-
ity in vitro and in vivo, antihyaluronidase activity and
has a protective effect on the free-radical-induced
degradation of collagen [7]. Cichoric acid shows also
antiviral activity and has recently been found to inhibit
HIV-1 integrase and replication [7]. The quantitative
analysis indicates that alkamides and caffeic acids are
present at significant concentrations in ethanol/water
extracts of this plant stored for longer than one year [8].
This revelation is important because alkamides and
caffeic acid derivatives have been identified as possi-
ble active constituents of E. purpurea.
Beside cichoric acid, typical constituents of E.
purpurea extracts are echinacoside, chlorogenic acid,
cynarine and caftaric acid. All of them are able to in-
hibit free radical production and lipid peroxidation,
involved in the development of inflammation [1, 9].
Recent studies also suggest that melanin may contrib-
ute to the activity of E. purpurea extracts [10], while
echinacoside and caffeic acid derivative do not pos-
sess immunostimulant activity, but have weak anti-
bacterial and antiviral effects and are protectants
against reactive oxygen species [11].
The antioxidant activity of E. purpurea extracts
has been already shown [7, 12-15]. Generally, the
tested E. purpurea extracts showed medium to low
activity compared to the other investigated medicinal
and aromatic plants [12]. The antioxidant activity
could be ascribed to the polyphenolic components [16],
such as flavonoids [15, 17], phenolic acids [18] or phe-
nolic diterpenes [15]. Moreover, some studies demon-
strated that the E. purpurea extracts protected immu-
nosuppressed mice against systemic infections with
Listeria monocytogenes and Candida albicans by
stimulating macrophage and neutrophil function. The
herb was non-toxic in mice, rats and humans even
when administered intravenously at high doses [19].
In previous studies, E. purpurea was reported to
increase chemotoxicity in neutrophils and bactericidal
activity against Staphylococcus and to kill tumor cells
(WEHI 164 cells) and cells infected either with the

Received 2008-06-25, accepted 2009-03-19.
* Supported by the Ministry of Science and Environmental Protection, Republic of Serbia (142073B).
** To whom correspondence should be addressed. E-mail: icaneca@gmail.com
Chin. J. Chem. Eng., Vol. 17, No. 3, June 2009 479
parasite Leishmania enriettii or with yeast Candida
albicans [6]. Also, it was found that hexane extracts of
echinacea variably inhibited growth of yeasts Saccharo-
myces cerevisiae, Candida shehata, C. kefyr, C. albi-
cans, C. steatulytica and C. tropicalis under near UV
irradiation (phototoxicity) and to a lower extent with-
out irradiation (conventional antifungal activity) [20].
So far, the classical solvent extraction with [8, 7, 21]
or without [22, 23] mechanical agitation, Soxhlet [21]
and Goldfisch [24] extraction techniques have been
applied for Echinacea sp. extraction. Recently, ultra-
sound extraction was used [5, 21, 25], but there is no
comparative study of antioxidant and antimicrobial
activities of E. purpurea extracts obtained by different
extraction techniques.
The purposes of this study are to compare the ef-
ficiency of extractive substances, antioxidant and an-
timicrobial activities of extracts obtained by ultra-
sound and classical extraction techniques. Also, total
phenolic compunds and flavonoids in E. purpurea
aqueous ethanolic extracts obtained by two methods
are compared. Antimicrobial and antiradical activity,
antioxidant capacity, total phenolics and flavonoids of
extracts are determined by in vitro assays.
2 EXPERIMENTAL
2.1 Materials
Ethanol was from Zorka-Pharma (Šabac, Serbia).
Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazil
(DPPH), gallic acid and rutin were obtained from
Sigma (St. Louis, MO). Sodium carbonate, potasium
acetate and aluminium chloride were purchased from
Merck-Alkaloid (Skopje, FYR Macedonia).
Dried aerial parts of E. purpurea L. were pur-
chased from DOO. “Adonis” (Soko Banja, Serbia).
Immediatly before being used, dry plant material was
ground by an electrical mill with a fast-rotating knife
(15000 r·min－1; 1 min). The moisture content, deter-
mined by drying at 105°C to constant mass, was
11.2% and the yield of extractive substances, obtained
by the Soxhlet extraction (9 h, 13 extraction cycles)
with 70% aqueous ethanol as extracting solvent, was
15.1 g per 100 g of dry plant material, which was
taken to represent the content of extractive substances
present in the plant material.
2.2 Extraction of plant materials
2.2.1 Classical extraction
Ground plant material (10 g) and the predeter-
mined volume of 70% aqueous ethanol were put in an
Erlenmayer flasks (100 ml) at a ratio of plant mate-
rial(g) and solvent(ml) of 1︰10. The extraction was
performed at 25°C for 2.5, 5, 10, 20, 40, 60 and 90
min. The temperature was controlled and maintained
at the desired level (±0.1°C). At the end of the extrac-
tion cycle the liquid extract was separated from the
solid residue by vacuum filtration. The solid residue
was washed twice with fresh solvent (20 ml each).
The filtrates were collected and the solvent was evapo-
rated in a rotary vacuum evaporator at 40°C.
2.2.2 Ultrasound extraction
The sonication was performed for 2.5, 5, 10, 20,
40 and 60 min with 70% aqueous ethanol, at a ratio of
plant material (g) to solvent (ml) 1︰10 and 25°C using
an ultrasonic cleaning bath (Sonic, Niš, Serbia; total
nominal power: 150 W; operating at 40 kHz frequency
and internal dimensions: 30 cm ×15 cm ×20 cm). The
temperature was controlled and maintained at the de-
sired level (±0.1°C) by water circulating from a ther-
mostated water bath. Separation and further treatment
of the filtrates were the same as described above.
2.3 Determination of free radical scavenging activity
The stable 1,1-diphenyl-2-picryl hydrazyl radical
(DPPH) was used for determination of free radical-
scavenging activity of the extracts [26]. Different con-
centrations of extracts (10, 20, 50, 100, 200, 500 and
1000 µg·ml－1, in 70% ethanol) were added, at an
equal volume (2.5 ml) to an ethanolic solution of
DPPH (0.3 mmol·L－1, 1 ml). After 30 min at room
temperature, the absorbance of the plant extract with
DPPH was measured at 517 nm on a spectrophotome-
ter (VARIAN Cary-100) and converted into the percent-
age antioxidant activity using the following equation:
( )s b
c
DPPH anti - radical scavenging capacity (%) =
1001
A A
A
⎡ ⎤− ⋅−⎢ ⎥⎣ ⎦

where As is the absorbance of the plant extract con-
taining DPPH, Ab is the absorbance of ethanol (1.0 ml)
plus plant extract solution (2.5 ml) and Ac is the ab-
sorbance of DPPH solution (1.0 ml) plus ethanol (2.5
ml). The EC50 values were calculated by sigmoid
non-linear regression model using plots, where the
abscissa and the ordinate represented the concentra-
tion of tested plant extracts the average percent of
scavenging capacity from three replicates, respectively.
2.4 Determination of total phenols
Total phenols were determined by Folin Ciocal-
teu reagent using gallic acid as a standard [27]. The
total phenols were expressed as mg gallic acid
equivalents (GAE) per g dry extract [the equation of
standard curve: absorbance at 765 nm= 12.722 cgallic acid
(µg·ml － 1)+0.0034, R2 = 0.9994]. Since the assay
measures all phenolics, the choice of gallic acid as a
standard is based on the availability of a stable and
pure substance. Each of plant extracts (0.2 ml, 20
µg·ml－1) or gallic acid was mixed with Folin Ciocal-
teu reagent (1 ml) and aqueous Na2CO3 (0.8 ml, 7.5%).
The mixtures were allowed to stand at room tempera-
ture for 30 min, and the absorbance of the reaction
mixture was measured at 765 nm.
Chin. J. Chem. Eng., Vol. 17, No. 3, June 2009 480
2.5 Determination of total flavonoids
The flavonoids content in extracts was deter-
mined spectrophotometrically using an aluminium
chloride colorimetric method, based on the formation
of a complex flavonoid-aluminium [28]. Each plant
extract (0.5 ml, 20 µg·ml－1) in methanol was sepa-
rately mixed with 1.5 ml of methanol, 0.1 ml of 10%
aluminum chloride (AlCl3), 0.1 ml of 1 mol·L
－1 potas-
sium acetate (CH3COOK) and 2.8 ml of distilled water.
After incubation at room temperature for 30 min, the
absorbance of the reaction mixture was measured at
415 nm against distilled water blank. Rutin was used
as a standard to make the calibration curve and the
data were expressed as mg rutin equivalents (RE) per
g dry extract [the equation of standard curve: absorb-
ance at 415 nm = 7.2328 crutin (µg·ml－1) − 0.2286,
R2 = 0.9919].
2.6 Antimicrobial activity
An agar well-diffusion method was employed for
the determination of antimicrobial activities of ex-
tracts [29]. Seven microorganisms were selected to test
the antimicrobial activity: Escherichia coli ATCC
25922, Pseudomonas aeruginosa ATCC 9027, Bacil-
lus subtilis ATCC 6633, Staphylococcus aureus ATCC
6538, Candida albicans ATCC 10231, Saccharomyces
cerevisiae ATCC 9763 and Aspergillus niger ATCC
16404 (Oxoid, England).
For the yeast and mould, sabouraud dextrose agar
(SDA) (Merck) was used; for cultures of bacteria,
trypton soya agar (TSA) (Merck) was used, and plate
count agar (Merck) was used for determination of the
total number of microorganisms (CFU·ml－1). 0.1 ml
of microorganism suspension, formed by 24 h culture
on obliquely agar with 10 ml sterile 0.9% NaCl, was
suspended into 10 ml of the nutritive medium (ca. 106
CFU·ml－1). Petri dish (86 mm internal diameter) was
filled with this system. The wells (10 mm in diameter)
were cut from the agar and 30 µl of extract solution
(concentration 20 mg·ml－1 in methanol) was delivered
into them. As a control, methanol (30 µl) was deliv-
ered into a well for each Petri dish. Erythromycin (997
µg·mg－1; [114-07-8]; Approx. 98%; H2O content 4%;
Sigma) and Tylosin Tartarat (950 µg·mg－1; [74610-55-2];
Sigma) were used as a positive control (concentration
in methanol solution, 0.05 mg·ml－1). All dilutions
were filtrated using a 0.45 µm membrane filter (Sarto-
rius, Germany). After incubation at 37°C for 24 h,
agar plates were examined for any zones of inhibition.
Diameters of zones of inhibition (mm) were measured
by Fisher Lilly Antibiotic Zone Reader (Fisher Scien-
tific Co., USA) and each test was run in triplicate.
2.7 Statistical analysis
All of the measurements were carried out in trip-
licate and the results were expressed as mean ± stan-
dard deviation, except extract yields which were in
duplicate. Comparison of means was analyzed by
Student’s t test and differences were considered sig-
nificant when p＜0.05.
3 RESULTS AND DISCUSSION
3.1 Antioxidant capacity
The percentage of DPPH radical-scavenging ac-
tivity was plotted against the plant extract concentra-
tion (Fig. 1) to determine the concentration of extract
necessary to decrease DPPH radical concentration by
50% (so called EC50). The EC50 value was used to
measure the antioxidant activity of extracts: the lower
EC50, the higher the value of the antioxidant activity.

Figure 1 Antioxidant activity for E. purpurea extracts
obtained by classical and ultrasound extraction
● classical extraction; ▲ ultrasound extraction
As can be seen in Table 1, the extract obtained by
the classical extraction shows higher antioxidant ac-
tivity, where the differences observed are statistically
significant with 95% confidence interval. This extract
also contains larger amount of total phenolic com-
pounds per gram of either dry extract or dry plant ma-
terial. The total flavonoides content of the extract
Table 1 Total antioxidant capacity, amount of phenolic and flavonoids compounds of E. purpurea extracts (25°C, 40 min)
Total phenolics Total flavonoids Extraction
technique
EC50/
mg·ml－1 (GAE/dry extract)/
mg·g－1
(GAE/dry plant material)/
mg·g－1
(RE/dry extract)/
mg·g－1
(RE/dry plant material)/
mg·g－1
classical 65.48±1.12 60.2±0.1 3.91±0.01 32.3±0.2 2.09±0.02
ultrasound 34.16±0.65 46.8±0.3 3.73±0.02 27.0±0.4 2.15±0.03
Note: Data were expressed as the mean of three replicates ± standard deviation.
Chin. J. Chem. Eng., Vol. 17, No. 3, June 2009 481
obtained by classical extraction is higher if calculated
per gram of the extract but it is lower if calculated per
gram of the dry plant material. Thus, ultrasonic ex-
traction gave higher yield but lower purity of total
flavonoides. The total amount of these compounds is
accepted as an indication of antioxidant potential be-
cause they act in plants as antioxidants, antimicrobials
and photoreceptors [30].
The extract obtained by classical solvent extrac-
tion contained 29% larger amount of phenolic com-
pounds and 20% higher content of flavonoids. The
differences observed were statistically significant with
95% confidence interval. We believed that the ob-
served reduction of phenolic compounds and flavon-
oids in the extract obtained by ultrasound extraction
was the result of their degradation by interaction with
highly reactive hydroxyl radicals formed during soni-
cation. Sonication of water results in the formation of
hydroxyl radicals, which can combine to form hydro-
gen peroxide that may or may not be beneficial to the
extraction process itself [31]. Organic compounds in
aqueous solution exposed to an ultrasonic irradiation
behave differently according to their physical and
chemical properties [32]. In the cases of some com-
pounds with antioxidant activity, aqueous solvents
appeared to be unsuitable for ultrasonic extractions
due to the formation of free radicals from the insona-
tion of the solvent [31].
3.2 Antimicrobial activity
The antimicrobial activity of E. purpurea extracts
obtained by different extraction techniques were tested
against the seven microorganisms mentioned above by
the agar well-diffusion method.
As shown in Table 2, independent of the extrac-
tion technique, the ethanolic E. purpurea extracts
show activity against almost all of the tested microor-
ganisms, exception is only mould A. niger. The con-
trol treatment (methanol) has no inhibitory effect on
any of the test microorganisms. Sensitivities of S. aures,
C. albicans, and S. cerevisiae are higher for both
echinacea extracts than the case of tested antibiotic
(Erytromycin and Tylosin tartarat).
The diameters of inhibition zone observed for all
microorganisms were larger for extracts obtained by
classical extraction than those by ultrasound extraction.
The differences observed were statistically significant
(with 95% confidence interval) in the case of E. coli,
B. subtilis, C. albicans and S. cerevisiae. No growth
inhibition zones were observed for A. niger for both
tested extracts.
3.3 Kinetics of extraction
The changes of the extract yield from the aerial
parts of E. purpurea L. during the classical and ultra-
sound extraction are shown in Fig. 2. Independent of
the extraction method, the extraction occurs in two
main stages: first, dissolution of material near the sur-
face characterized by a rapid increase in the extractive
substance yield in the beginning of the process
(washing or fast extraction), and second, diffusion of
the solute from the porous plant residue into the solu-
tion (slow extraction). The optimum time for both ex-
traction techniques was approximately 40 min, ensur-
ing nearly the maximum oil yield.
As can be seen in Fig. 2, the main benefits of ul-
trasound included the increase of extractive substance
yield and faster extraction. In recovering the extractive
substances from E. purpurea L. the ultrasound extrac-
tion was more efficient than the classical solvent ex-
traction, but less efficient than the Soxhlet extraction.
The total extract content obtained by ultrasound ex-
traction after 40 min was 22.8% higher than that ob-
tained by the classical extraction. At the same time, it
was 52.8% of the yield obtained by the Soxhlet ex-
traction, which was 15.1 g·(100 g)－1. However, a
shortcoming of the Soxhlet extraction is higher oper-
ating temperature and in this case more than tenfold
longer extraction time.
Table 2 Antimicrobial activity of E. purpurea extracts (40 min, 25ºC) and antibiotic sensitivity of
microorganisms (zone size, mm)
Extracts (20 mg·ml－1) Antibiotics (0.05 mg·ml－1)
Test microorganisms
Ultrasound Classical Erytromycin Tylosin tartarat
Escherichia coli ATCC 25922 10.5±0.3 11.2±0.2 21.2±0.1 18.4±0.0
Pseudomonas aeruginosa ATCC 9027 11.3±0.3 a 11.9±0.4 a 25.2±0.9 17.6±0.1
Bacillus subtilis ATCC 6633 10.5±0.1 10.9±0.1 19.1±0.1 17.3±0.7
Staphylococcus aureus ATCC 6538 11.0±0.1 b 11.2±0.1 b 23.6±0.0 18.5±0.6
Candida albicans ATCC 10231 18.2±0.3 21.0±0.1 23.0±0.0 16.2±0.3
Saccharomyces cerevisiae ATCC 9763 21.2±0.1 25.7±0.1 naa naa
Aspergillus niger ATCC 16404 naa naa 20.5±0.7 18.1±0.1
Note: Control treatment (methanol) had no inhibitory effect on any of the test microorganisms. Differences between values for pa-
rameters designated with the same letters (a and b) were not statistically significant with 95% confidence interval (Student’s t test).
“naa” means no antimicrobial activity.
Chin. J. Chem. Eng., Vol. 17, No. 3, June 2009 482

Figure 2 Variation of the extractive substance yield (based
on dry plant meterial) from the E. purpurea L.
△ ultrasound extraction; ▲ classical extraction
4 CONCLUSIONS
The present study suggests that 70% aqueous
ethanolic extracts of E. purpurea is a potential source
of active natural and non-toxic substances, which have
functions as antioxidants, antimicrobials and antibiot-
ics. The extract obtained by the classical extraction
from aerial parts of E. purpurea L. contained larger
amount of bioactive compound (total phenols and fa-
vonoid). Also, it showed stronger antioxidant and an-
timicrobial activities than the extract obtained by ul-
trasound extraction. Independent of the extraction
technique, the ethanolic E. purpurea extracts showed
antimicrobial activity against all tested microorgan-
isms except in the case of A. niger. Ultrasound had a
positive effect on the extractive substance yield from
E. purpurea L., but negative effects on the content of
total phenolic compounds and flavonoids. Some bio-
active compounds were probably degradated by inter-
action with highly reactive hydroxyl radicals formed
during sonication.
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