2008

NPC Natural Product Communications Vol. 3

No. 9
1515 - 1518
Comparison of Different Techniques for Extraction of
Biologically Active Compounds from
Achillea Millefolium Proa
Antoaneta Trendafilovaa,* and Milka Todorovaa
a
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences,
Sofia, Bulgaria

trendaf@orgchm.bas.bg

Received: April 28th, 2008; Accepted: August 1st, 2008

The effect of ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and conventional extraction (CE) on
the extraction of biologically active compounds from Achillea millefolium Proa has been investigated. The extracts were
analyzed by spectrophotometric methods. The influence of the extraction time and the solvent on the yields of total extract
(TEC), total phenolic compounds (TPhC), flavonoids (FC) and sesquiterpene lactones (SLC) has been studied. It was found
that TEC and TPhC increased with increasing the solvent polarity in order CHCl3, MeOH and 80% MeOH. The extraction
with MeOH gave maximum amount of flavonoids, while the SLC was found to be almost equal in all used solvents. UAE for
60 min and MAE for 60 s afforded higher or almost equal TEC, TPhC and FC than those obtained by CE for 24 h. The best
extraction of sesquiterpene lactones was achieved with CHCl3 for 20 min of ultrasound treatment and 20 s of microwave
irradiation.

Keywords: Achillea millefolium Proa, phenolic compounds, flavonoids, sesquiterpene lactones, ultrasound-assisted extraction,
microwave-assisted extraction, maceration.

Achillea millefolium Proa is a tetraploid cultivar of
yarrow rich in proazulenes and dominating in the
field of commercially available plant material for the
drug “Herba Millefolii” [1]. Yarrow (A. millefolium)
is well known medicinal plant, widely used in folk
medicine for treatment of gastrointestinal,
hepatobiliary, and gynecological disorders, against
inflammation and for wound healing [2]. Different
studies reported that the anti-inflammatory effect
is attributed to the presence of azulenogenic
sesquiterpene lactones [3]. Moreover, phenolic
compounds (flavonoids and phenolcarbonic acids)
are present in the plant and also contribute to the
pharmacological activity of yarrow. Thus, flavonoids
from yarrow mediate the spasmolytic activity [4],
whereas the choleretic effects are caused by the
dicaffeoylquinic acids [5]. Consequently, an effective
method for extraction of these biologically active
compounds from A. millefolium is important to be
developed.
Recently, ultrasound-assisted extraction (UAE) and
microwave-assisted extraction (MAE) methods are
being widely employed for extraction of various
biologically active compounds [6-11] from different
plant matrices with better yield, involving less
amounts of solvents compared to conventional
methods. However, to the best of our knowledge,
the effect of MAE and UAE methods are not reported
for the extraction of bioactive compounds from
A. millefolium.
In the present communication, we report a
comparative study of UAE, MAE and conventional
methods for the extraction of phenolics, flavonoids
and sesquiterpene lactones from cultivar A.
millefolium Proa. The effectiveness of different
extraction methods was evaluated on the bases of the
quantity of these types of secondary metabolites.
Thus, the total phenolic content (TPhC) was settled
by the Folin-Ciocalteu assay and expressed in gallic
acid equivalents [12]. The flavonoid content (FC)
was obtained by spectrophotometric method in rutin
1516 Natural Product Communications Vol. 3 (9) 2008 Trendafilova & Todorova

18 18
A B TEC [%]
15 15 TPhC [mg/g]
FC [mg/g]
12 12
SLC [mg/g]

Yield
Yield
9 9

6 6

3 3

0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Extraction time [min] Extraction time [s]

Figure 1: Influence of the extraction time on total extract content (TEC, %, g/100 g plant material), total phenolic content (TPhC, mg gallic acid/g plant
material), flavonoid content (FC, mg rutin/g plant material), and sesquiterpene lactone content (SLC, mg matricin/g plant material) in A. millefolium Proa
methanolic extracts using ultrasound power (A) and microwave irradiation (B).

A 25 B 32
CHCl3
20 MeOH
24
80%MeOH

TPhC [mg/g]
TEC [%]

15
16
10
8
5

0 0
CE UAE1 UAE2 MAE1 MAE2 CE UAE1 UAE2 MAE1 MAE2

C 16 D8

12 6
FC [mg/g]

SLC [mg/g]

8 4

4 2

0 0
CE UAE1 UAE2 MAE1 MAE2 CE UAE1 UAE2 MAE1 MAE2

Figure 2: Comparison of total extract content (TEC), total phenolic content (TPhC), flavonoid content (FC), and sesquiterpene lactone content (SLC) in A.
millefolium Proa extracts using different extraction methods and solvents: CE (maceration for 24 h), UAE1 and UAE2 (ultrasound-assisted extraction for 20
and 60 min, respectively), MAE1 and MAE2 (microwave-assisted extraction for 20 and 60 s, respectively). Values are means of triplicate±SD for TEC, TPhC
and FC and means of duplicate±SD for SLC.

equivalents [13]. The sesquiterpene lactone content extending the extraction time, while the sesquiterpene
(SLC) was determined by IR method [14,15] and lactone content slowly decreased in MeOH after
expressed in matricin equivalents. CHCl3, MeOH and 20 min.
80% MeOH, generally employed in selective
extraction of sesquiterpene lactones, flavonoids Similar results were observed when extraction was
and phenolics have been used as extracting performed with MeOH under microwave irradiation
solvents, keeping the solid / solvent ratio constant (power 400 W) for 10, 20, 40 and 60 s (Figure 1B).
c.a. 0.05 g/mL. As can been seen, the total extract content rose up to
12.88% only for 10 s of microwave irradiation and
Initially, the kinetics of the extraction using MeOH as then slowly increased up to 13.28% for 60 s. The
a solvent for 5, 10, 20, 40 and 60 min of ultrasonic optimum extraction time also depended on the active
treatment was studied (Figure 1A). The total extract compounds. Thus, the total phenolic and flavonoid
content enhanced rapidly up to 12.37% for the first contents increased with extension of microwave
20 min of ultrasonic irradiation and then slowly irradiation time, while the amount of sesquiterpene
increased up to 13.64% for 60 min. Further, it was lactones decreased after 20 s.
found that the optimum extraction time varied for the
different types of active compounds. The total The next experiment concerned the solvent effect on
phenolic and flavonoid contents increased with the extraction process. As can be seen from Figure 2,
UAE, MAE and CE of compounds from Achillea millefolium
total extract content and the yields of active
compounds under maceration (24 h, room
temperature), UAE (20 and 60 min) and MAE
(20 and 60 s) depended on the solvent used. Thus,
TEC and TPhC increased with increasing the solvent
polarity in order CHCl3, MeOH and 80% MeOH.
Extraction with MeOH gave maximum flavonoid
content, while the content of sesquiterpene lactones
was found to be almost equal in all used solvents.
Further, a comparison of different extraction
techniques showed that TEC and the amounts of
extractable substances (TPhC, FC and SLC) under
UAE and MAE were comparable with those obtained
by conventional extraction for 24 h (CE). Thus, TEC,
TPhC and FC using ultrasonic power for 1 h (UAE2)
and microwave power for 60 s (MAE2) were higher
or almost equal to those obtained by CE for 24 h
irrespective of solvent used. The best extraction of
sesquiterpene lactones was achieved for 20 min of
ultrasonic treatment (UAE1) and for 20 s of
microwave irradiation (MAE1). It is worth to note
that chloroform, a microwave transparent solvent
gives similar results as CE and UAE methods.
Moreover, MAE with CHCl3 achieved the highest
amounts of sesquiterpene lactones for 20 s. These
results could be explained by the “broken cell-wall
theory”, according to which microwave transparent
solvents are better than microwave-absorbing ones
[16].
From the above-described results could be
concluding that MAE and UAE are simple, fast,
environmentally benign and relatively cost-effective
methods for extraction of biologically active
compounds from A. millefolium Proa.
Experimental
Plant material and chemicals: The dried flowering
tops of cultivar A. millefolium Proa were obtained
from Bulherba Ltd, Bulgaria. The plant material was
air-dried, ground and kept in dark place. Reagents,
standards (except matricin) and solvents were
commercially available materials of analytical grade.
Matricin was isolated from A. collina previously in
our laboratory and characterized by spectral methods.
Extraction methods
1. Conventional extraction (CE): Maceration was
performed at room temperature with MeOH, 80%
aqueous MeOH and CHCl3. Plant material (0.5 g)
was extracted with the corresponding solvent
(10 mL) for 24 h. Further, the extracts were filtered
Natural Product Communications Vol. 3 (9) 2008 1517
and solvents were removed under vacuum.
Concentrated extracts were re-dissolved in MeOH to
get a sample solution of approximately 0.6 mg/mL.
Each extraction experiment was performed in
triplicate.
2. Ultrasound-assisted extraction (UAE): UAE
experiments were carried out in a ultrasonic bath
(UST5.7-150, SIEL, Bulgaria). Plant material (0.5 g)
was extracted with MeOH (10 mL) in an Erlenmayer
flask and put in ultrasonic bath for 5, 10, 20, 40 and
60 min. The temperature was kept constant (25±1oC)
by periodical adding of ice in the bath. The extracts
were worked up as described in Section 1. The UAE
experiments were also performed with 80% MeOH
and CHCl3 for 20 and 60 min at 25±1oC. All
experiments were repeated three times.
3. Microwave-assisted extraction (MAE): The
experiments were performed with a multimodal
household microwave oven (KOR-6C2B, Daewoo) at
400 W. The plant material (0.5 g) was mixed with
MeOH (10 mL) in a roundbottom flask and exposed
to microwave irradiation (irradiation cycle: 10 s
power on, followed by 30 s power off) for 10, 20, 40
and 60 s. Superboiling of the solution did not
occur.The extracts were worked up as described in
Section 1. The MAE experiments were also
performed with 80% MeOH and CHCl3 for 20 and
60 s. All experiments were repeated three times.
Analyses of biologically active compounds
Determination of total extract content (TEC): The
corresponding extract was evaporated under reduced
pressure to dryness and a constant weight. The
determination of the total extract contents from the
plant material was calculated from the mass of dry
extract and the mass of initial dried plant. For each
extract, three measurements were performed.
Determination of total phenolic content (TPhC):
TPhC in plant extracts was determined by Folin-
Ciocalteu method [12] and expressed as mg of gallic
acid per gram of the dry plant material. For each
extract, three measurements were performed.
Determination of flavonoid content (FC): FC in
plant extracts was determined according to the
colorimetric assay [13] and expressed as mg of rutin
per gram of dry plant material. For each extract, three
measurements were performed.
1518 Natural Product Communications Vol. 3 (9) 2008 Trendafilova & Todorova

Determination of sesquiterpene lactone content CHCl3 as a reference. A standard curve was

(SLC): SLC was determined on Bruker Tensor 27 by
an IR spectroscopic method [14,15] with some
modifications and expressed as matricin equivalents
per gram of dry plant material. In this method, a
portion of each extract solution (10 mL) was
evaporated to dryness using a rotary vacuum
evaporator. The resulting residue was dissolved in 10
mL of distilled H2O and then extracted with CHCl3 (3
x 10 mL). The combined CHCl3 extracts were dried
(anhydrous Na2SO4), and concentrated under reduced
pressure. The obtained lactone fractions were further
dissolved in CHCl3 (10 mL), yielding basic solutions
for quantitative analyses. The measurements were
carried out within a range 1600-1800 cm-1 with
prepared with matricin in CHCl3 (ranging from 0.19
to 0.79 mg/mL, regression equation of the calibration
curve y = 0.02106x + 0.58602, R= 0.9971). For each
extract, two measurements were performed.
Statistical analysis: Results are presented as mean ±
SD. Correlation analysis was carried out with
GraphPadPrism version 5.0 for Windows, GraphPad
software.
Acknowledgments - The authors acknowledge the
financial support of project TK-X-1609 provided by
the National Science Fond of Bulgaria.

References
[1] Bugge G. (1991) Untersuchungen der Sippen des Achillea millefolium Komplexes auf Azulengehalt und Ploidiegrad. Angewandte
Botanik, 65, 331-339.
[2] Ivanov I, Landgev I, Neshev G. (1977) Achillea millefolium L. In Bilkite v Balgaria i izpolzvaneto im (Medicinal plants in
Bulgaria and their applications). Ivanov I, Landgev I, Neshev G.(Eds). Sofia, p. 82-83.
[3] Kastner U, Sosa S, Tubaro, A, Breuer J, Rücker G, Loggia R Della, Jurenitsch J. (1993) Anti-Edematous Activity of Sesquiterpene
Lactones from Different Taxa of the Achillea millefolium Group. Planta Medica, 59, A669.
[4] Lemmens-Gruber R, Marchart E, Rawnduzi P, Engel N, Benedek B, Kopp B. (2006) Investigation of the spasmolytic activity of the
flavonoid fraction of Achillea millefolium s.l. on isolated guinea-pig ilea. Arzneimittelforschung/Drug Research, 56, 582-588.
[5] Benedek B, Geisz N, Jaeger W, Thalhammer T, Kopp B. (2006) Choleretic effects of yarrow (Achillea millefolium s.l.) in the
isolated perfused rat liver. Phytomedicine, 13, 702-706.
[6] Vinatoru M. (2001) An overview of the ultrasonically assisted extraction of bioactive principles from herbs¸ Ultrasonics
Sonochemistry, 8, 303-313.
[7] Mandal V, Mohan Y, Hemalatha S. (2007) Microwave assisted extraction – an innovative and promising extraction tool for
medicinal plant research. Pharmacognosy Reviews, 1, 7-18.
[8] Huie CW. (2002) A review of modern sample preparation techniques for the extraction of medicinal plants. Anaytical and.
Bioanalytical Chemistry, 373, 23-30.
[9] Sharma A, Verma SC, Saxena N, Chadda N, Singh NP, Sinha AK. (2006) Microwave- and ultrasound-assisted extraction of
vanillin and its quantification by high-performance liquid chromatography in Vanilla planifolia. Journal of Separation Science, 29,
613-619.
[10] Sharma UK, Sharma K, Sharma N, Sharma A, Singh HP, Sinha AK. (2008) Microwave-assisted efficient extraction of different
parts of Hippophae rhamnoides for the comparative evaluation of antioxidant activity and quantification of its phenolic constituents
by reverse-phase high-performance liquid chromatography (RP-HPLC). Journal of Agricultural and Food Chemistry, 56, 374-379.
[11] Proestos C, Komaitis M. (2008) Application of microwave-assisted extraction to the fast extraction of plant phenolic compounds.
LWT - Food Science and Technology, 41, 652-659.
[12] Asami KD, Hong Y-J, Barett DM, Mitchell AE. (2003) Comparison of the total phenolic and ascorbic acid content of freeze-dried
and air-dried marionberry, strawberry, and corn grown using conventional, organic and sustainable agricultural practices. Journal
of Agricultural and Food Chemistry, 51, 1237-1241.
[13] Miliauskas G, Venskutonis PR, Van Beek TA. (2004) Screening of radical scavenging activity of some medicinal and aromatic
plant extracts. Food Chemistry, 85, 231-237.
[14] Gromek D, Kisiel W, Stojakowska A, Kohlmuenzer S. (1991) Attempts of chemical standardizing of Chrysanthemum parthenium
as a prospective antimigraine drug. Polish Journal of Pharmacology and Pharmacy, 43, 213-217.
[15] Bloszyk E, Geppert B, Drozdz B. (1978) Quantitative determination of sesquiterpene lactones in plant material by infrared
spectroscopy. Planta Medica, 34, 79-86.
[16] Escribano-Bailon MT, Santos-Buelga C. (2003) Polyohenol extraction in foods. In Methods in Polyphenol Analysis. Santos-Buelga,
C. and Williamson G. (Eds), UK, pp. 1-16.