Different extraction methods of biologically active components from propolis: a preliminary study
Chemistry Central Journal
Preliminary communication Different extraction methods of biologically active components from propolis: a preliminary study
Boryana Trusheva 0
Dorina Trunkova 0
Vassya Bankova 0
0 Address: Institute of Organic Chemistry with the Centre of Phytochemistry, Bulgarian Academy of Sciences , Acad. G. Bonchev str., bl. 9, 1113 Sofia , Bulgaria
Background: Propolis is widely used in apitherapy, preparations, and food and beverage additives. Various extraction techniques were applied in the extraction of the biologically active constituents of poplar type propolis in order to compare their efficiency. The methods employed were: traditional maceration extraction, ultrasound extraction (UE), and microwave assisted extraction (MAE). Results: The total amounts of extracted phenolics and flavonoids were determined, and the effectiveness of the methods compared. MAE was very rapid but led to the extraction of a large amount of non-phenolic and non-flavonoid material. UE gave the highest percentage of extracted phenolics. Conclusion: Compared to the maceration extraction, MAE and UE methods provided high extraction yield, requiring short timeframes and less labour. UE was shown to be the most efficient method based on yield, extraction time and selectivity.
The use of propolis as a remedy has a long history . In
addition, preparations, as well as food and beverage
additives containing propolis extracts can be found on the
market in numerous countries . It is generally accepted
that the solvent of choice for the extraction of biologically
active components of propolis (mainly phenolics,
including different types of flavonoids) is 70% ethanol, with
most commercial products extracted using this solvent
system [3,4]. The traditional method (maceration),
however, is time consuming, requiring timeframes from 2 to
10 days [4,5]. Recently, modern extraction methods have
been developed for the fast and efficient extraction of
organic compounds from solid matrices, with microwave
assisted extraction (MAE) and ultrasonic extraction (UE)
among the most promising for the extraction of natural
products [6,7]. MAE is the process of using microwave
energy to heat solvents in contact with a sample in order
to partition some chemical components from the matrix
into the solvent. The benefits of UE are thought to be due
mainly to the mechanic effects of acoustic cavitation. Both
methods have recently demonstrated the potential to
reduce extraction times significantly and increase
extraction yields in a number of studies on medicinal plants .
The aim of this preliminary study is to compare the
effectiveness of the extraction of bioactive components from
poplar propolis (phenolics, flavones/flavonols and
flavanones/dihydroflavonols  using the maceration
method, MAE and UE. We have chosen to quantify groups
of active compounds with the same or similar chemical
structure rather than individual substances. Recently, it
has been demonstrated that the concentration of such
compound groups in propolis extracts correlates much
better with the levels of antibacterial activity and is more
informative than the concentration of individual
components [9,10]. We believe that this study is the first
application of MAE to propolis.
Results and discussion
Three extraction methods were employed in order to
obtain the biologically active components of poplar type
(European) propolis. The results are summarized in Table
In all three extraction techniques the amount of solvent
used (10:1 or 20:1) does not significantly influence the
extraction yield (ANOVA). This is an important finding
because it demonstrates that the use of solvent/propolis
ratios larger than 10:1 (v/w) is unnecessary, leading only
to solvent and energy losses.
In the case of ultrasound extraction, the yield of
biologically active constituents increases with the time. It is clear
that a 30 min sonication should be sufficient to extract the
available amount of phenolics and flavonoids. In general,
this confirms the results of a previous study .
With MAE the situation was different. Surprisingly, an
additional cycle of irradiation (3 × 10 s instead of 2 × 10
s) resulted in a drastic decrease in the yield of phenolics
and flavanones/dihydroflavonols. The small increase in
percentage of the extracted flavones/flavonols was not
statistically significant. Presumably, the high energy influx in
this case leads to chemical changes (most probably
oxidation) of the phenolic compounds (including flavonoids)
and to lower amounts of these substances detected in the
The three extraction methods differed mainly in the total
percentage of phenolics extracted, with UE giving the
highest yield. UE led also to the extraction of the highest
amount of flavanones and dihydroflavonols, while in the
total extraction of flavones/flavonols, UE and MAE were
did not show statistically significant differences. The
results demonstrate that the use of ultrasound and of
microwave extractions greatly reduce the extraction time.
The percentage of total extract obtained with a 70%
ethanol solvent system also varies significantly with the
extraction method, much more so than the amount of extracted
active compounds. This is important, because the greater
percentage of total extract and similar percentage of
extracted active compounds means that larger amounts of
waxes have been extracted. This was especially the case
with MAE where the extracted phenolics constituted 40%
of the mass of the raw propolis while the total extract was
70% of the raw propolis. UE resulted almost only in the
extraction of active components and minimum wax.
Compared to the maceration extraction, MAE and UE
methods provided high extraction yield, requiring short
timeframes and less labour
MAE of bioactive phenolics and flavonoids from poplar
type propolis was found to be a very fast extraction
method, compared to maceration and even UE. However,
the extraction selectivity was low, with significant
amounts of unwanted wax was found to have been
extracted. In addition, longer irradiation times resulted in
a decrease in the percentage of extracted active
components, presumably owing to degradation processes.
UE has been shown to be the most efficient extraction
method, taking into consideration yield, short extraction
time (30 min) and high selectivity.
Table 1: Comparison of the extraction yields (percentage of extracted matter from raw propolis) for the maceration extraction, UE
Extraction type Propolis/solvent (m/V)
Total phenolics, % Total flavones/flavonols, %
• Mean values of three extraction ± SD;
• a values compared with those obtained via maceration, (propolis/solvent 1:10),
• b values significantly different from those obtained by maceration (propolis/solvent 1:10), p < 0.05 (Student's t-test),
• c values compared and found significantly different, p < 0.05 (Student's t-test)
The propolis sample was obtained from the National
Institute of Apiculture in Bologna, Italy, and its poplar
origin proved using TLC test . The whole sample (51 g)
was frozen, ground and homogenized prior to beginning
2 g of propolis were used in each of the extraction
experiments. The extraction solvent was 70% ethanol. After
extraction the sample was filtered and the filtrate diluted
to 100 mL with 70% ethanol in a volumetric flask.
Analysis then followed according to the methods described
later. Each extraction experiment was performed in
For maceration extraction, the material was placed in an
Erlenmayer flask, the corresponding amount of solvent
was added (details in Table 1) with the sample left to
macerate in the dark for 72 h at room temperature.
Ultrasound extraction (UE)
The UE was carried out using a 300 W ultrasonic bath. The
sample was placed in an Erlenmayer flask with the
corresponding amount of solvent and was treated with
ultrasound at 25°C for a given duration (Table 1).
Microwave assisted extraction (MAE)
MAE was performed using a multimodal household
microwave oven Panasonic NN-S255W at 800 W and a
100 mL flask exposed to microwave irradiation
(irradiation cycle: 10 s power on, followed by 10 s power off) for
a given duration (Table 1).
Analyses of the biologically active compound
The analysis was performed as described in a previous
study . In brief, it consisted of the following:
Total amount of extract
2 mL of each extract were evaporated in vacuo to dryness
and a constant weight. The percentage of dry extracts was
calculated and the mean of the three replicates was
Total flavone and flavonol content
An aliquot (2 mL) of each extract, 20 mL methanol and 1
mL 5% AlCl3 in methanol (m/V) were mixed in a
volumetric flask and the volume made up to 50 mL with
methanol. The mixture was left for 30 min and the absorbance
at 425 nm was measured. Calibration was performed
using galangin as reference compound. All the values
found were within the linear range of the methods,
Total flavanone and dihydroflavonol content
An aliquot (1 mL) of each extract and 2 mL of DNP
(2,4dinitrophenylhydrazine) solution (1 g DNP in 2 mL 96%
sulfuric acid, diluted to 100 mL with methanol in a
volumetric flask) were heated at 50°C for 50 min. After
cooling to room temperature, the mixture was diluted to 10
mL with 10% KOH in methanol (m/V). 1 mL of the
resulting solution was added to 10 mL methanol and was
diluted to 50 mL with methanol (volumetric flasks).
Absorbance was measured at 486 nm. Calibration was
performed using pinocembrin as reference compound. All
the values found were within the linear range of the
method, recovery .108%. For each extract, three
measurements were performed.
Total phenolics content
An aliquot (1 mL) of each extract was transferred to a 50
mL volumetric flask, containing 15 mL distilled water. To
this were added 4 mL of the Folin-Ciocalteu reagent and 6
mL of a 20% sodium carbonate solution (m/V). The
volume was made up with distilled water to 50 mL. The
sample was left for 2 h and the absorbance at 760 nm was
measured. Calibration was performed using a 2:1 (w/w)
reference mixture of pinocembrin and galangin as a
standard. All the values found were within the linear range of
the method, recovery .90% [using other calibration
standards, e.g. gallic acid or caffeic acid, the recovery was much
lower ]. For each extract, three measurements were
BT performed the extractions and participated in the
chemical analyses. DT participated in the chemical
analyses. VB conceived of the study, participated in its design
and coordination and performed the statistical analysis.
All authors read and approved the final manuscript.
The authors wish to thank Dr. Milena Popova and Dr. Vanya Kurteva for
fruitful discussions. We also gratefully acknowledge the partial support
offered by the National Science Fund (Bulgaria), Contract TKX-1609.
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