BIOTECHNOLOGY AND BIOENGINEERING

Chinese Journal of Chemical Engineering, 19(3) 504ü511 (2011)

Comparison of Antioxidant and Antimicrobial Activities of

Methanolic Extracts of the Artemisia sp. Recovered by
Different Extraction Techniques*

Ivana Karabegoviü1,**, Milena Nikolova2, Dragan Veliþkoviü3, Sɚša Stojiþeviü1, Vlada Veljkoviü1
and Miodrag Laziü1
1
Faculty of Technology, 124 Bulevar oslobodjenja St., 16000 Leskovac, Serbia
2
Institute of Botany, Bulgarian Academy of Sciences, 23 G. Bonchev St., 1113 Sofia, Bulgaria
3
College of Agriculture and Food Technology, 1 ûirila i Metodija St.,18400 Prokuplje, Serbia

Abstract The polyphenol content, antioxidant and antimicrobial activities of the extracts obtained by classical,
ultrasonic and Soxhlet extractions from dry aerial parts of two Artemisia species (Artemisia vulgaris and Artemisia
campestris) were compared. Ultrasound positively affected the yield of extractive substance and the kinetics of ex-
traction, but the extract obtained by the classical extraction showed the highest antioxidant activities and contained
higher total contents of phenolic compounds and flavonoids than the extracts obtained by two other extraction tech-
niques. Both flavonoid aglycones (apigenin, quercetin, quercetin 3,3ƍ-dimethyl ether) and flavonoid glycosides (ru-
tin, hyperoside and kaempferol 3-rhamnoside) were identified by thin layer chromatograph (TLC) analysis in the
extracts from both species. A. campestris extracts were richer in quercetin than A. vulgaris and its antimicrobial ac-
tivity was also better than A. vulgaris. Extracts obtained from both species were found to be more effective on the
tested yeasts than bacteria. The kinetics of the total extractive substances, such as phenolic, flavonoids and
quercetin extraction, was successfully described by the model of unsteady-state diffusion.
Keywords polyphenol content, antioxidant activity, antimicrobial activity, extraction, flavonoids, Artemisia vul-
garis, Artemisia campestris

1 INTRODUCTION of these two species have been recently shown to pos-

sess a strong antioxidant activity [13, 14]. Leaves of
Artemisia campestris L., commonly known as these plants have a characteristic taste, caused by
field wormwood or field sagewort, and Artemisia vul- monoterpenes and sesquiterpenes which in many cases,
garis L., known as mugwort, belong to the Asteraceae are the reason for their applications in folk medicine
family. Artemisia species are widely used in traditional and in human diet mostly as a spice [1, 15].
medicine all over the world with different and well- Different extraction techniques, such as hydrodis-
known therapeutic applications (stomach ache, diar- tillation, classical, Soxhlet and ultrasonic extraction,
rhoea, parasitism, intestinal and bronchial infections, are widely used for obtaining extractive substances from
angina, wounds, pimples, colds and coughs) [1, 2]. They different plant materials. As a novel technique, ultrasonic
exhibit anti-inflammatory, antitumor, antioxidant, an- extraction has recently been shown to be very promis-
tispasmodic, antimicrobial, insecticidal, antimalarial, ing and effective for obtaining bioactive substances.
antifungal and antioxidant activities [2]. These diverse The main benefits of the use of ultrasound are the in-
biological activities are manifested by different com- crease of the extraction yield, a faster process and
pounds whose main components are essential oils and even the improved quality of the extracts [16].
polyphenols. Polyphenols in aromatic plants have in- To our best knowledge, there is no report on the
hibitory effects against biological damages, excellent ultrasound extraction efficiency and kinetics of extrac-
radical scavenging ability as well as antimicrobial tive substances and flavonoid components of A. vul-
activity [37]. Flavonoid composition of A. vulgaris garis and A. campestris. The aim of present study is to
and A. campestris is well documented in the relation determine the best extraction technique (ultrasonic, clas-
of chemotaxonomy. The flavonoid profile of A. campes- sical or Soxhlet extraction) for biological active com-
tris is quite complex consisting of flavones, flavonols, pounds of both species. The quality of extracts is evalu-
flavanones, dihydroflavanols and their methyl ethers ated from their polyphenol content, antioxidant potential
whereas the profile of A. vulgaris is quite uniform con- and biological activities against seven microorganisms.
sisting mainly of simple flavonol methyl ether [811].
The antiradical efficiencies are the typical property for 2 EXPERIMENTAL
different phenolic acids (chlorogenic and syringic acid)
and flavonoids (possessing two or three hydroxyl 2.1 Materials
groups on the carbon in the B or C ring of the mole-
cules), which are present in wormwood [12]. Extracts Dried aerial parts of two Artemisia species (A.

Received 2009-06-02, accepted 2011-01-20.

* Supported by the Ministry of Science and Environmental Protection, Republic of Serbia (172047).
** To whom correspondence should be addressed. E-mail: icaneca@gmail.com
 Chin. J. Chem. Eng., Vol. 19, No. 3, June 2011 505

vulgaris and A. campestris) were collected at the after that time. The liquid extract was evaporated un-
blossoming stage from natural habitats, near the Ljulin der vacuum at 40 °C to constant mass. This yield of
Mountain (Bulgaria). Voucher specimens from the extractive substance (gram per 100 g dry plant mate-
plant samples were deposited at the herbarium in the rial) was also taken to represent the content of extrac-
Institute of Botany (SOM) (ɋɨ609, Co1212). The tive substance in the plant material.
plant material was milled by an electrical mill with a
fast-rotating knife (15000 r·min1, 1 min) immediatly 2.3 Determination of free radical scavenging ac-
before extraction. Moisture content was determined by tivity
drying at 105 °C to constant mass as 7.97% and
8.21% for A. vulgaris and A. campestris, respectively.
Methanol was from Merck (Darmstadt, Germany). The stable 1,1-diphenyl-2-picryl hydrazyl radical
Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazil (DPPH) was used for determination of free radi-
(DPPH), gallic acid and rutin were obtained from cal-scavenging activity of the extracts [17]. Different
Sigma (St. Louis, MO). Sodium carbonate, potasium concentrations of extracts (10, 20, 50, 100, 200, 500
acetate and aluminium chloride were purchased from and 1000 μg·ml1, in methanol) were added at an
Merck-Alkaloid (Skopje, FYR Macedonia). Seven equal volume (2.5 ml) to methanolic solution of
microorganisms were selected to test the antimicrobial DPPH (0.3 mmol·L1, 1 ml). Experiments was carried
activity: Escherichia coli ATCC 25922, Pseudomonas out in triplicates. After 30 minutes at room tempera-
aeruginosa ATCC 9027, Bacillus subtilis ATCC 6633, ture, the Ab values were measured at 517 nm on a
Staphylococcus aureus ATCC 6538, Candida albicans spectrophotometer (VARIAN Cary-100) and con-
ATCC 10231, Saccharomyces cerevisiae ATCC 9763 verted into the percentage antioxidant activity using
and Aspergillus niger ATCC 16404 (Oxoid, England). the following equation:
The bacteria tested were grown on the Trypton soya DPPH anti-radical scavenging
agar (TSA; Merck), while the Sabouraud dextrose
agar (SDA; Merck) was used to grow the yeast and the § A  Ab ·
mould. Plate count agar (Merck) was used for deter-
capacity ¨ s ¸ ˜ 100% (1)
© Ac ¹
mining the total number of microorganisms (CFU·ml1).
where As is the absorbance of the plant extract con-
2.2 Extraction of plant materials taining DPPH, Ab is the absorbance of methanol (1.0
ml) plus plant extract solution (2.5 ml) and Ac is the
2.2.1 Classical extraction absorbance of DPPH solution (1.0 ml) plus methanol
Ground plant material (10 g) and methanol (100 ml) (2.5 ml). The EC50 values are calculated by sigmoid
were put in a series of the Erlenmeyer flasks (100 ml), non-linear regression model using plots, where the
and the ratio of plant material mass (g) to solvent vol- abscissa represents the concentration of tested plant
ume (ml) was 1Ή10. No additional stirring was ap- extracts and the ordinate the average percent of scav-
plied. The extraction was performed at (25r0.1) °C for enging capacity from three replicates.
2.5, 5, 10, 20, 40 and 60 min. At the end of the extrac-
tion cycle the liquid extract was separated from the 2.4 Determination of total phenols
solid residue by vacuum filtration. The solid residue
was washed twice with fresh solvent (20 ml). The fil-
trates were collected and the solvent was evaporated Total phenols were determined by the Folin Cio-
in a rotary vacuum evaporator at 40 °C. calteu reagent using gallic acid as a standard [18]. Gallic
acid was used as a standard and the total phenols were
2.2.2 Ultrasound extraction expressed as mg gallic acid equivalents (GAE) per g
The sonication was performed in 2.5, 5, 10, 20, dry extract. The standard curve equation (R2 0.9994) is
40 and 60 min with methanol, at the plant material
mass (g) to-solvent volume (ml) ratio of 1Ή10 and Total phenols A765  0.0034 /12.722 (2)
temperature of (25r0.1) °C using an ultrasonic clean- Since the assay measured all phenolics, the
ing bath (Sonic, Niš, Serbia; total nominal power: choice of gallic acid as a standard was based on the
3×50 W; operating at 40 kHz frequency and internal availability of a stable and pure substance. Each of
dimensions: 30 cm×15 cm×20 cm). The temperature plant extracts (0.2 ml, 1 mg·ml1) or gallic acid was
was controlled and maintained at the desired level mixed with the Folin Ciocalteu reagent (1 ml) and an
(r0.1 °C) with water circulating from a thermostated aqueous Na2CO3 solution (0.8 ml, 7.5%). The mix-
bath by means of a pump. Separation and further tures were allowed to stand at room temperature for
treatment of the filtrates were the same as described in 30 minutes, and the absorbance of the reaction mix-
the previous section. ture was measured at 765 nm.
2.2.3 Soxhlet extraction
The ground plant material (10 g) and methanol 2.5 Determination of total flavonoids
(100 ml) were taken into the Soxhlet apparatus, and
were extracted for 7 hours. Previously, we found that The flavonoids content in extracts was determined
the yield of extractive substance was not increased spectrophotometrically using an aluminum chloride
506 Chin. J. Chem. Eng., Vol. 19, No. 3, June 2011

colorimetric method, based on the formation of a with iron (III) chloride (FeCl3) reagent. The TLC
complex flavonoid-aluminum [19]. Each plant extract plates were scanned and the images were analyzed by
(0.5 ml, 2 mg·ml1) in methanol was separately mixed the QuantiScan 2.1® Biosoft software. The content of
with 1.5 ml of methanol, 0.1 ml of 10% aluminum compounds per sample was calculated from the den-
chloride (AlCl3), 0.1 ml of 1 mol·L1 potassium ace- sitogram peak areas by comparing to the three stan-
tate (CH3COOK) and 2.8 ml of distilled water. After dards analyzed on the same plate.
incubation at room temperature for 30 minutes, the
absorbance of the reaction mixture was measured at 2.7 Antimicrobial activity
415 nm against the distilled water blank. Rutin was
used as a standard to make the following calibration
curve (R2 0.992) and data were expressed as mg ru- The agar well-diffusion method was employed
tin equivalents (RE) per g dry extract. for the determination of antimicrobial activities of
extracts [20]. Microorganism suspension (0.1 ml),
Total flavonoids A415  0.2286 / 7.2328 (3) formed after 24 h from the culture on obliquely agar
with 10 ml sterile 0.9% NaCl, was suspended into 10
2.6 Flavonoid analysis ml of the nutritive medium (ca. 106 CFU·ml1). Petri
dish (86 mm internal diameter) was filled with this
2.6.1 Qualitative flavonoid analysis system. The wells (10 mm in diameter) were cut from
Flavonoid aglycones and glycosides were identi- the agar and 30 μl of extract solution (concentration in
fied by direct TLC comparison with markers. Two TLC methanol 20 mg·ml1) was delivered into them.
sorbents were used for the analysis of the flavonoid Erythromycin (997 μg·mg1; [114-07-8]; Approx. 98%;
aglycones. Toluene-dioxan-acetic acid (95Ή25Ή4, H2O content 4%; Sigma) and Tylosin tartarat (950
volume ratio) was used for the development of the μg·mg1; [74610-55-2]; Sigma) were used as a posi-
aglycones mixture on silica gel plates Kiselgel 60 F254 tive control (concentration in methanol 0.05 mg·ml1).
(10/20 cm, 0.2 mm layer). Toluene-methylethylketone- All dilutions were filtrated using a 0.45 μm membrane
methanol (60Ή25Ή15, volume ratio) was used for filter (Sartorius, Germany) and performed in three
DC Alufolien Polyamid 11 F254 plates (10/20 cm, replicates. After incubation at 37 °C for 24 h, agar
0.15 mm layer). Chromatograms were viewed under plates were examined for any zones of inhibition.
UV light at 254 nm before and after spraying with Diameters of zones of inhibition (mm) were measured
“Naturstoffreagenz A”, 1% solution of diphenylboric by Fisher Lilly Antibiotic Zone Reader (Fisher Scien-
acid-ethanolamine complex in methanol. tific Co., USA).
2.6.2 Identification of flavonoid glycosides
Two TLC conditions were used for analysis of 2.8 Statistical analysis
the flavonoid glycosides. Ethyl acetate-formic acid-
acetic acid-water (100Ή11Ή11Ή27, volume ratio) was All data were represented as a mean of three in-
used for silica gel plates Kieselgel 60 F254 (10/20 cm, dependent measurements. Means were compared by
0.2 mm layer). Ethyl acetate-acetic acid-water (20Ή2Ή1, Student’s t test and differences were considered to be
volume ratio) was used as the second mobile phase for significant when p<0.05 (95% confidence interval).
the same type plates. Flavonoid glycoside was identi-
fied by direct TLC comparison with markers. 3 RESULTS AND DISCUSSION
2.6.3 Quantitative flavonoid analysis 3.1 Total phenolic and flavonoid content
Quercetin (0.6, 1.3 and 1.5 μg per spot) and Ar-
temisia species extracts (10 μl, 20 mg·ml1) were ap-
plied on the DC Alufolien Polyamid 11 F254 plates The aerial parts of A. vulgaris and A. campestris
(10/20 cm, 0.15 mm layer). Toluene-methylethylketone- differ to each other considerably with respect to the
methanol (60Ή25Ή15, volume ratio) was used for total phenolic and flavonoid contents as it can be seen
the development of plates. The migration distance was in Table 1. The total phenolic content of different spe-
90 mm. Compounds were visualized after spraying cies achieved by different extraction techniques ranges

Table 1 Total phenolic and flavonoid contents of A. vulgaris and A. Campestris

Extraction Total phenolic content/mg·g1 Total flavonoids/mg·g1

technique A. vulgaris A. campestris A. vulgaris A. campestris
UE 135.0±1.0 141.2±0.6 103.7±0.4 102.5±6.2
CE 141.9±0.7 164.7±0.5 110.9±2.2 118.2±3.0
SE 123.4±0.1 128.1±5.4 100.8±1.2 104.5±3.8
Note: CEüclassical extraction, UEüultrasound extraction, SEüSoxhlet extraction. 25qC, 40 min for CE and UE. Enhanced tem-
perature (close to the boiling temperature), 420 min for SE.
 Chin. J. Chem. Eng., Vol. 19, No. 3, June 2011 507

from (123.4±0.1) mg·g1 to (164.7±0.5) mg·g1, while A TLC survey of flavonoid profiles of two Ar-
the content of flavonoids, in rutin equivalents, varies temisia species extracts obtained by different extrac-
from (100.8±1.2) mg·g1 to (118.2±3.0) mg·g1. In the tion techniques showed that there were no differences
case of both Artemisia species the highest amounts of in their qualitative flavonoid composition. Quantita-
total phenolic compound and flavonoids are found in tive variations on the quercetin content of the extracts
the extracts obtained by classical extraction and the were examined by the TLC-densitometric assay and
lowest one is obtained by the Soxhlet extraction. This no significant differences in the quercetin content of
may be explained by oxidation and degradation of the extracts were established. The extracts of A. vul-
these bioactive compounds under sonication [21] or garis obtained by classical and ultrasonic extraction
the higher extraction temperature and the much longer contained quercetin in the range of 0.29 to 0.61 and
extraction time of the Soxhlet extraction [22]. The 0.32 to 0.57 ȝg·g1 (based on dry plant material), re-
plant species (A. vulgaris and A. campestris) and the spectively. The highest quercetin content (2.38 ȝg·g1,
extraction method have a statistically significant in- based on dry plant material) was found in the extract
fluence on the total phenolic and flavonoid content in obtained by the Soxhlet extraction of A. campestris.
the extracts (p<0.05). All extracts of A. campestris were richer in quercetin
Flavonoid analysis than those of A. vulgaris. For the plant species, there
TLC analysis was used for the determination of were no considerable differences in the quercetin con-
differences among flavonoid extraction efficiencies of tent among the extracts obtained at different extraction
different techniques. In these screening of A. vulgaris techniques.
and A. campestris extracts, compared to 35 flavonoids,
10 flavonoid aglycones and 3 flavonoid glycosides 3.2 Antioxidant activity
were detected (Table 2). Apigenin, quercetin and
quercetin 3,3-dimethyl ether are common flavonoid
aglycones for both species. Scutellarein 6-methyl ether, In the present study, the stable DPPH radicals
6-hydroxyluteolin-6-methyl ether, 6-hydroxyluteolin- were used to investigate the wormwood and mugwort
6,7,4ƍ-trimethyl ether and kaempferol 7-methyl ether antioxidant activity (Table 3). The interaction of a po-
are characteristic for A. campestris. Derivatives of tential antioxidant with DPPH depends on the extrac-
quercetin are identificated as main flavonoid agly- tion technique and the plant species. For the plant
cones in the extracts of A. vulgaris. These results are species the following order of antioxidant activity is
in agreement with the previously published data on established: extracts obtained by classical extraction>
the flavonoid composition of Artemisia species [8, 9]. extracts obtained by ultrasound extraction>extracts
Both species can be clearly distinguished by their pro- obtained by Soxhlet extraction. Furthermore, the an-
files of flavonoid aglycones. The flavonoid glycosides tioxidant activity is higher for the A. campestris ex-
composition is the same for both species. tracts than for the A. vulgaris extracts obtained by the

Table 2 Flavonoids in the extracts of A. vulgaris and A. campestris

A. vulgaris A. campestris
Flavonoids
CE UE SE CE UE SE
flavonoid aglycones
apigenin + + + + + +
apigenin 7-methyl ether ü ü ü + + +
6-hydroxyapigenin-6-methyl ether ü ü ü + + +
6-hydroxyluteolin-6-methyl ether ü ü ü + + +
6-hydroxyluteolin-6,7,4ƍ-trimethyl ether ü ü ü + + +
kaempferol 7-methyl ether ü ü ü + + +
quercetin + + + + + +
quercetin 3,3ƍ-dimethyl ether + + + + + +
quercetin 3,7-dimethyl ether + + + ü ü ü
quercetin 3,7,3ƍ-trimethyl ether + + + ü ü ü
flavonoid glycosides
rutin (quercetin 3-rutinoside) + + + + + +
hyperoside (quercetin-3-galactoside) traces traces traces + + +
kaempferol 3-rhamnoside + + + + + +
Note: CEüclassical extraction, UEüultrasound extraction, SEüSoxhlet extraction.
508 Chin. J. Chem. Eng., Vol. 19, No. 3, June 2011

Table 3 Total antioxidant activity of A. campestris and strong antibacterial activity and in particularly against
A. vulgaris extracts the mould (A. niger). While, independent of the ex-
Extraction EC50/μg·ml1 traction technique employed, the extracts of A. vul-
technique garis were not active against the mould. This may
A. vulgaris A. campestris
means that the extract of this species should be ap-
CE 22.2±0.3 19.8±0.2 plied at higher concentrations.
UE 26.5±0.1 20.6±0.4 No statistically significant difference (95% con-
fidence interval) was observed in antimicrobial activi-
SE 28.1±0.1 23.0±0.1
ties of A. vulgaris extracts obtained by ultrasound and
Note: CEüclassical extraction, UEüultrasound extraction, SEü classical extraction. However, the extracts of A.
Soxhlet extraction.
campestris obtained by classical extraction showed
better activities than those obtained by ultrasonic ex-
same extraction technique. Observed differences are traction with significant differences (95% confidence
statistically significant with 95% confidence interval. interval). These results might partly justify the tradi-
The amount of phenolic and flavonoid compounds in tional use of both Artemisia species. Also our test
the extracts correlates with their antioxidant activity confirmed the previously reported inhibition of S.
(e.g. the correlation coefficient between EC50 data and aureus and E. coli growth by an A. campertris extract
the total phenolic and flavonoid compound contents is at a concentration of 125 mg·ml1 [23]. Extracts and
0.81 and 0.76, respectively), confirming that this essential oils of many Artemisia sp. such as A. mexi-
compounds are likely to contribute to the radical cana [24], A. princes [25], A. diffusa, A. oliveriana, A.
scavenging activity of these plant extracts. scoparia, A. turanica [26], A. dracunculus, A. annua,
A. gypsacea, A. afra and A. khorassanica [1] have
been demonstrated to possess antimicrobial activity.
3.3 Antimicrobial activity The differences in antimicrobial activities of A.
campestris and A. vulgaris and the other Artemisia sp.
The antimicrobial effects of the Artemisia species can be attributed to differences in their qualitative and
extracts obtained by the two different extraction tech- quantitative composition caused by different growth
niques were tested against two Gram-positive bacterial and extraction conditions.
species (B. subtilis, St. aureus), two Gram-negative
bacterial species (E. coli, P. aeruginosa), two yeast 3.4 Kinetics of total extractive substances, pheno-
species (Sacc. cerevisiae, C. albicans) and one mould lic compounds, flavonoids and quercetin extraction
(A. niger). The control treatment (methanol) had no
inhibitory effect on any of the test microorganisms.
The results of these tests, as well as the effects of two The changes of extractive substances, total phe-
control antibiotics, are presented in Table 4. nols, total flavonoids and quercetin yields during clas-
All tested extracts showed better antimicrobial sical and ultrasound extraction are shown in Fig. 1.
activity against yeasts than against both Gram-positive Curves have the same shape as those for extraction of
and Gram-negative bacteria. In all cases the A. extractive substances from plantain (Plantago major)
campestris extracts were found to be more active, with [27] and sage (Salvia officinalis) [15], flavonoids and
significant differences (95% confidence interval), than active compounds from St. John’s wort (Hypericum
the extracts of A. vulgaris. The A. campestris extracts perforatum) [28] as well as pyrethrines from pyre-
obtained by both extraction techniques exhibited very thrum (Chrysanthemum cineraria) flowers [29].

Table 4 Antimicrobial activity of A. vulgaris and A. campestris extracts (40 min, 25 °C) and
antibiotic sensitivity of microorganisms (zone size/mm)
Extracts, 20 mg·ml1 Antibiotics, 0.05 mg·ml1
Microorganisms A. vulgaris A. campestris Tylosin
Erythromycin
UE CE UE CE tartarat

Escherichia coli ATCC 25922 12.1±0.2 12.7±0.3 18.0±0.5 20.0±0.4 21.2±0.1 18.4±0.0
Pseudomonas aeruginosa ATCC 9027 12.2±0.3 12.5±0.1 18.4±0.2 21.1±0.3 25.2±0.9 17.6±0.1
Bacillus subtilis ATCC 6633 12.0±0.2 12.1±0.2 18.2±0.4 20.5±0.3 19.1±0.1 17.3±0.7
Staphylococcus aureus ATCC 6538 12.2±0.2 12.7±0.2 18.3±0.6 20.6±0.2 23.6±0.0 18.5±0.6
Candida albicans ATCC 10231 24.2±0.1 26.5±0.2 saa saa 23.0±0.0 16.2±0.3
Saccharomyces cerevisiae ATCC 9763 30.5±0.3 30.6±0.2 saa saa naa naa
Aspergillus niger ATCC 16404 naa naa 32.5±0.1 33.1±0.1 20.5±0.7 18.1±0.1
Note: CEüclassical extraction, UEüultrasound extraction. Control treatment (methanol) had no inhibitory effect on any of the test
microorganisms. saaüstrong antimicrobial activity, inhibition zone >35 mm. naaüno antimicrobial activity.
 Chin. J. Chem. Eng., Vol. 19, No. 3, June 2011 509

Independent of the recovery technique applied

and the type of plant material, the extraction of extrac-
tive substances, total phenols, total flavonoids and
quercetin occured in two main stages: first, dissolution
of material near the surface characterized by a rapid
increase in the extractive substances yield in the be-
ginning of the process (washing or fast extraction),
and second, diffusion of the solute through plant par-
ticles into the solution (slow extraction). The optimum
time for both extraction techniques was approximately
20 minutes, ensuring nearly the maximum yields of
total extractive substances, total phenols, total flavon-
(a) Extractive substances oids and quercetin.
For the purpose of mathematical modeling of
classical and ultrasonic extraction of total extractive
substances, total phenols, total flavonoids and quercetin
from A. vulgaris and A. campestris, the model of un-
steady diffusion through plant material was applied. The
basic equation of this kinetic model is as follows [30]:
q0  q
1  bc ˜ e k ct (4)
q0
where bc is the washing coefficient; k c is the slow
extraction coefficient, min1; q is the content of ex-
tractive substances in the plant material during the
extraction, g·(100 g)1; and t is time, min. The differ-
(b) Total phenols ence (q0q) is the amount of extractive substances
dissolved in the solvent. The yields of extractive sub-
stances, total phenols, total flavonoids and quercetin
obtained by the Soxhlet extraction were adopted as its
initial content (q0) (Table 5).

Table 5 Yields of extractive substances, quercetin, total

phenols and flavonoid compound from A. vulgaris and
A. campestris obtained by the Soxhlet extraction
Extractive Total
Plant Quercetin Total phenolic
substances 1 1 flavonoids
material /mg·g content/mg·g
/mg·g1 /mg·g1
A. vulgaris 205.4 1.49 2.17±0.003 2.07±0.025
A. campestris 221.2 2.38 2.83±0.119 2.41±0.084
(c) Total flavonoids

To check the proposed mathematical model, the

dependence of ln(1q/q0) versus time is shown in Fig. 2.
The linear shape of all the curves corresponding to
different plant materials (A. vulgaris and A. campes-
tris aerial parts), extraction techniques (classical and
ultrasound extraction) and extractive compound (total
extractive substances, total phenols, total flavonoids
or quercetin) illustrates a very good agreement be-
tween the model and the experiment only in the later
stage of the extraction period, while a significant de-
viation from the model equation is observed in the
initial period. For the practical use of the model equa-
tion, for instances for designing the extraction process,
(d) Quercetin
this is not a problem as it is usually completed in the
Figure 1 Variation of extractive substances, total phenols, slow extraction stage. Parameters of the kinetic model
total flavonoids and quercetin yields obtained by classical are calculated from the experimental data by means of
(triangles) and ultrasound (circles) extraction from A.
campestris (open symbols) and A. vulgaris (black symbols) the linear regression method using Eq. (4) and the co-
efficient of linear correlation is higher than 0.91.
510 Chin. J. Chem. Eng., Vol. 19, No. 3, June 2011

(a) A. campestris (a)

extractive substances; total flavonoids; total phe-
nolic compounds; quercetin

(b) A. vulgaris
Figure 2 The linearized form of the kinetic equation (b)
(classical extraction: open symbols, ultrasound extraction: black
symbols; extractive substances: circles, total phenols: triangles, extractive substances; total flavonoids; total pheno-
total flavonoids: squares and quercetin: quadrangle) lic compounds; quercetin
Figure 3 The kinetic parameters of unsteady diffusion
(CEüclassical extraction, UEüultrasound extraction)
Values of the kinetic parameters are compared in
Fig. 3. Independent of the extraction technique and the
type of extractive substances, the effect of ultrasound quercetin 3,3-dimethyl ether, rutin, hyperoside and
on the washing coefficient related to the A. vulgaris kaempferol 3-rhamnoside are identified by TLC
extraction is somewhat higher than that related to the analysis in both species, while A. campestris extracts
A. campestris extraction. In the case of both plant ma- are richer on quercetin. The A. campestris extracts are
terials, ultrasound affects washing of total extractive more active than the extracts of A. vulgaris, while all
substances and quercetin much more than those of extracts from both species show better antimicrobial
total phenolic and flavonoid compounds. The first activity against yeasts than bacteria. Antioxidant ac-
stage of extraction is positively affected by sonication tivity of the extracts is related to their total flavonoid
attributed to cell destruction, better solvent penetration and phenol contents.
and mass transfer intensification [31]. Ultrasound affects positively both the kinetics of
The slow extraction coefficient seems to be nega- extraction and the yield of extractive substances from
tively affected by ultrasound. This decrease may be both Artemisia species. The contents of bioactive
caused by oxidation and degradation of these bioac- compounds are reduced under sonication, probably
tive compounds under prolonged sonication, which due to their degradation by interaction with highly
has been already noticed in the case of Plantago ma- reactive hydroxyl radicals formed by ultrasound action.
jor [27]. The effects of other extraction conditions on Therefore, classical extraction seems to be more fa-
the slow extraction coefficient appear to be very com- vored from the practical and economical points of
plex and could not be easily seen. view since it is easer to perform with less capital and
energy costs than the other two techniques.
Classical and ultrasound extraction of total ex-
4 CONCLUSIONS tractive substances, total phenols, total flavonoids and
quercetin occur via the same two-step mechanism,
The present study suggests that methanolic ex- namely washing followed by slow extraction (diffu-
tracts of two tested Artemisia sp. aerial parts are poten- sion). Based on this very simplified mechanism, the
tial source of active natural substances such as natural extraction kinetics was modeled by unsteady state
antioxidants and antimicrobials. Apigenin, quercetin, diffusion model through plant material.
 Chin. J. Chem. Eng., Vol. 19, No. 3, June 2011 511

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