Preparation and Characterization of Inclusions Complexes Between

-cyclodextrin
Propolis Ethanolic Extracts and 2-hydroxypropyl-
LAVINIA LIA VLAIA1, VICENTIU VLAIA2*, IOANA VIORICA OLARIU1, ANA MARIA MUT1, CARMEN ANATOLIA GAFITANU3,
CRISTINA DEHELEAN4, DAN NAVOLAN5*, DUMITRU LUPULEASA6, GEORGETA HERMINA CONEAC1
1
Victor Babes University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Technology, 1 Eftimie
Murgu Sq., 300041, Timisoara, Romania
2
Victor Babes University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Organic Chemistry, 1 Eftimie Murgu Sq.,
300041, Timisoara, Romania
3
Grigore T. Popa University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Technology, 16
Universitatii Str. 700115, Iasi, Romania
4
Victor Babes University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Toxicology, 1 Eftimie Murgu Sq., 300041,
Timisoara, Romania
5
Victor Babes University of Medicine and Pharmacy, Faculty of Medicine, 1 Eftimie Murgu Sq., 300041, Timioara, Romania
6
Carol Davila University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Technology, 6 Traian
Vuia Str., 020956, Bucharest, Romania

The ethanolic extracts of propolis consist mainly of polyphenolics (such as flavonoids and non-flavonoids),
which are responsible for various pharmacological activities, but have poor aqueous solubility and are easily
decomposed by oxidation under the action of air, heat, and light during the processing steps and storage.
Complexation with cyclodextrins (CDs) is an effective pharmaceutical method to enhance the bioavailability
of poor water soluble compounds and to improve their chemical stability. Three different propolis sorts and
60% (v/v) ethanolic solution as extraction solvent were used to obtain the propolis ethanolic extracts. The
propolis ethanolic extract/2-hydroxypropyl--cyclodextrin inclusion complexes were prepared by kneading
method and characterized by optical microscopy and differential scaning calorimetry (DSC). Experimental
results confirmed the inclusion of polyphenolics from propolis ethanolic extracts in 2-hydroxypropyl- cyclodextrin (2HPbCD). Furthermore, the results of DSC studies demonstrated that the polyphenolics of the
studied propolis extracts were included in different concentrations in the 2HPbCD cavity. The present study
provides useful information for the potential applications of ethanolic propolis extracts/2HPbCD complexes,
as stable, effective and easy to process raw materials.
Keywords: propolis ethanolic extracts; 2-hydroxypropyl--cyclodextrin; inclusion complexes;
kneading method

Propolis, one of the by-products of the beehive, has been

used by man since ancient times for its medicinal
properties, and nowadays its constituents are used as major
active ingredients in cosmetic, pharmaceutical and health
products [1]. In the last two decades, many studies have
proved that the beneficial effects of propolis on human
health are mainly due to polyphenolic compounds, which
are responsible of a wide range of biological activities,
including antioxidant, anti-inflammatory, antibacterial,
antiviral and antitumoral [2-7].
Propolis, also called bee glue, is a lipophilic,
heterogeneous, resinous or wax-like mass, of solid
consistency, sometimes compact, sometimes becoming
malleable and adherent particles, and other times granular
or friable, taking the appearance of powdery crumbs. It
possesses a pleasant, aromatic smell, and varies in colour,
depending on its source and age, from pale yellow to dark
brown, even black [1, 8]. As for the propolis chemistry,
there are numerous literature reports which have showed
its complex chemical composition, indicating that the
main groups of compounds are waxes, resins, balsams,
aromatic and ethereal oils, pollen, and other organic matter
[9-12].
Considering the physical characteristics of propolis, as a
result of its complex structure, it is used only in the form of

extracts prepared with a suitable solvent, such as water, ethanol,

methanol, chloroform, acetone, dichloromethane, and ether.
The major components of ethanolic extracts of propolis
include polyphenolics such as flavonoids (i.e. chrysin, rutin,
apigenin, kaempherol, quercetin, pinocembrin, and
acacetin) and non-flavonoids (i.e. caffeic acid), which are
responsible for the propolis extracts bioactivities [8, 1315]. As a result, there is a growing interest in developing
nutraceutical and pharmaceutical products based on
propolis extracts. However, the application of propolis
extracts in these products is severally restricted since some
of the contained polyphenolic compounds have poor water
solubility, poor bioavailability, and are easily decomposed
by oxidation under the action of air, heat, and light during
the processing steps and storage. Therefore, to maintain
the structural integrity of the bioactive molecules, it is
necessary to develop a formulation capable to deliver them
to the physiological targets, keeping their bioactivity. One
of the most widely used method to improve chemical
stability and the water solubility of the polyphenolic agents
is the preparation of cyclodextrin (CD) inclusion complexes
[16-19]. As known, CDs presents the ability to encapsulate
molecules, acting as host molecules in the formation of
inclusion complexes with numerous hydrophobic guest
molecules, which are entirely or partially included in the

* email: vlaiav@umft.ro; navolan@yahoo.com

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REV.CHIM.(Bucharest) 67 No.2 2016

nonpolar cavity of CDs. With this property, CDs have mainly

been used in the pharmaceutical industry to increase the
hydrosolubility of active compounds poorly soluble in water,
in order to increase their bioavailability and to improve
stability. In addition, CDs can be used to reduce or eliminate
unpleasant smells or tastes, prevent drug-drug or drugadditive interactions within a formulation, based on
reduction of the free drug in solution [20, 21].
CDs are cyclic oligosacharides with six (-CD), seven
(-CD), eight (-CD) or more glucose residues linked by
(-1,4) glycosidic bonds [22]. 2-hydroxypropyl- cyclodextrin (2HPbCD) is one of the hydroxyalkylated CD derivatives, prepared through chemical modification
of the natives -CD hydroxyl group, that exhibits relatively
higher water solubility and lower toxicity than v-CD, and a
satisfactory inclusion ability [23]. At present, 2HPbCD is
the most commonly applied CD to encapsulate plant
bioactive compounds, compared to the other CDs
derivatives, due to its above mentioned favorable
properties. To the extent of our knowledge, there are only
few reports regarding the encapsulation of propolis extracts
by -CD and its hydrophilic derivatives, such as 2HPbCD
[23-26]. On the other hand, numerous published studies
on the preparation/standardization of propolis extracts
reported ethanol of different concentrations as solvent for
extraction of polyphenolics, because it allows obtaining
higher extraction yields and is less toxic than other solvents
[1, 15, 24, 27]. Therefore, alcohol was used for the obtaining
of the propolis extracts studied in this work.
The objective of this work was the encapsulation of three
different ethanolic propolis extracts within a 2HPbCD
complex, aiming to obtain a stable, effective and easy to
process material. Optical microscopy and differential
scanning calorimetry (DSC) were used to ensure the
inclusion of propolis etahnolic extracts within 2HPbCD.

Preparation of propolis extract-2HPbCD inclusion

complexes
The inclusion complex between each propolis extract
and 2HPbCD was prepared by kneading method as follows:
in a preheated to 50C glass mortar, 160 mL of each propolis
ethanolic extract (P1-Tm_60, P2-B_60 or P3-M_60) were
added over an accurately weighted amount of 2HPbCD
(126.400 g, 126.420 g and 126.470 g respectively). The
physical mixtures were kneaded throughly for an hour with
a pestle to obtain a paste, which was then dried to constant
mass in an oven at 25C, for 6 h. The dried masses were
further pulverized in a mortar, sieved through a 0.16 mm
sieve and stored in a desiccator until further evaluation.

Experimental part
Materials and methods
Propolis, as raw material, was obtained from three
different apiaries located in the West region of Romania:
an alpine apiary located in Barzava (4607 00 N; 2158
59 E; altitude 218 m), and two river meadow apiaries
located in Timisoara (4544 57 N; 2113 37 E) and Minis
(460800" N; 213600" E) respectively. The three sorts of
propolis were codified P1-Timisoara (P1-Tm), P2-Barzava
(P2-B) and P3-Minis (P3-M). Ethanol p.a. (Chimopar,
Bucharest, Romania) and distilled water were used for
propolis extraction. 2-Hydroxypropyl--cyclodextrin (with
purity greater than 99%, an average Mw 1310 and an
average degree of substitution of 4.6) was purchased from
CycloLab R&D Ltd (Budapest, Hungary).

Results and discussions

Preparation of propolis extract-2HPbCD inclusion
complexes
The average yields of the propolis ethanolic extracts/
2HPbCD inclusion complexes, codified as P1-Tm_60/
2HPbCD, P2-B_60/2HPbCD and P3-M_60/2HPbCD, were
97.6% (w:w), 96.5% (w:w) and 98.2% (w:w) respectively.

Preparation of propolis ethanolic extracts

The three propolis ethanolic extracts, corresponding to
P1-Tm, P2-B and P3-M propolis sorts, were obtained using
a 60% (v/v) ethanolic solution as extraction solvent, by the
method described in our previous study [26]. Briefly, 5 g of
each pulverized propolis sort was extracted for 96 h with
20-fold volume of 60% ethanol solution using a magnetic
stirrer. Extraction was carried out at room temperature, in
a sealed flask and protected from light. The obtained
ethanolic extracts were filtered to remove insoluble
materials, the residues were washed with 5 mL of the
same solvent and the final propolis extracts were stored at
4C until encapsulation within 2HPbCD. These propolis
extracts (codified P1-Tm_60, P2-B_60 and P3-M_60) were
physico-chemical characterized by HPLC method in our
previous studies [26, 28].
REV.CHIM.(Bucharest) 67 No.2 2016

Characterization of propolis ethanolic extract/2HPbCD

inclusion complexes
Optical microscopy
Morphological evaluation of the propolis ethanolic
extracts/2HPbCD inclusion complexes was performed by
optical microscopy, using Nikon Eclipse E8000 Confocal
Microscope, connected to the NIKON Coolpix E4500 digital
camera, with a 40-600x optical zoom (Nikon, Japan).
Differential scanning calorimetry (DSC)
The crystallinity of propolis ethanolic extracts/2HPbCD
inclusion complexes was characterized by DSC on a
Differential Scaning Calorimeter DSC 204 ( Netzsch Geratebau GmbH, Germany), provided with the specific
software DSC Netzsch 204 Acquisition Soft/2000 for data
aquisition and the software Netzsch Proteus-Thermal
Analysis version 4.0/2000 for data processing. Samples of
approximately 102 g were weighed and sealed in
aluminum pans. The samples were heated at a rate of 4
C/min, in a range of 20-500C, the cooling being performed
with liquid nitrogen.

Characterization of propolis ethanolic extract/2HPbCD

inclusion complexes
Macroscopic examination
In figure 1 is shown, as example, the image of P2-B_60/
2HPbCD in solid state.
The optical microscopy analysis
The results of optical microscopy analysis of inclusion
complexes obtained after the complexation of propolis
ethanolic extract from P1-Tm, P2-B and P3-M sorts with
2HPbCD are presented in figure 2a, 2b and 2c.

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Fig. 1. Photograph of P2-B_60/2HPbCD

inclusion complex in solid state
379

Fig. 2. The optical microscopy images

(magnitude 400 x) of the studied inclusion
complexes: P1-Tm_60/2HPbCD (a), P2-B_60/
2HPbCD (b), and P3-M_60/2HPbCD (c)

Fig. 3. DSC thermograms for the P1-Tm_60/

2HPbCD (a), P2-B_60/2HPbCD (b), and P3M_60/2HPbCD (c) inclusion complexes

DSC analysis
The DSC thermograms of the prepared inclusion
complexes between propolis ethanolic extracts and
2HPbCD are shown in figure 3.
Preparation of propolis extract-2HPbCD inclusion
complexes
The obtaining of propolis extract/2HPbCD inclusion
complexes through the kneading method was performed
with massive yields of 97-98%, losses being caused mainly
by the preparation technology.
380

Characterization of propolis ethanolic extract/2HPbCD

inclusion complexes
Macroscopic examination
The inclusion complexes between propolis ethanolic
extracts and 2HPbCD, obtained through the kneading
method were amorphous yellow powders, with a pleasant
flavoured taste and odour, characteristic of that of propolis.

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REV.CHIM.(Bucharest) 67 No.2 2016

The optical microscopy analysis

The optical microscopy analysis of inclusion complexes
obtained after the complexation of propolis ethanolic
extract from P1-Tm, P2-B and P3-M sorts with 2HPbCD
revealed in all cases a parallelepipedic crystallin product,
with micrometric-sized particles (fig. 2a, 2b and 2c).
DSC analysis
There are two endothermic peaks at 90 C (H = -27.29
J/g) and 135 C (H = -577.2 J/g) in the DSC thermogram
of P1-Tm_60/2HPbCD inclusion complex (fig. 3a),
corresponding to the dehydration/dissociation process,
most probably due to the release of the water crystallization
molecules. Moreover, in the release area of polyphenolics
(up to ~ 280C), the endothermic effect of dissociation
was only 13 J/g and after that temperature (above 290oC)
the decomposition of the complex occurred. This results
indicated the relatively low content of polyphenolic
bioactive compounds incorporated into the cavity of
2HPbCD.
The DSC curves of the P2-B_60/2HPbCD and P3-M_60/
2HPbCD inclusion complexes showed significant
endothermic effects between 150 and 280C,
corresponding to the inclusion complex dissociation (fig.
3b and 3c). Thus, in the case of the P2-B_60/2HPbCD
inclusion complex the loss by dehydration up to about 135140oC was of 676 J/g, while the dissociation of the inclusion
complex was indicated by a loss of 75.24 J/g between 150
and 280oC (fig. 3b).
In the thermogram of the P3-M_60/2HPbCD inclusion
complex the two endothermic peaks, indicating its
dehydration and dissociation, were slightly shifted to lower
temperatures (82.6 and 145.9 C) and their corresponding
areas were higher (-881.7 J/g and -102.7 J/g) than that of
P2-B_60/2HPbCD inclusion complex (fig. 3c). Although the
absolute dissociation effect was greater for the P3-M_60/
2HPbCD complex, its high water content makes this
product unsuitable for pharmaceutical processing,
compared with P2-B_60/2HPbCD complex, for which the
molecular inclusion of compounds from extract is more
effective (much lower content of water of crystallization
in the finite complex).
Conclusions
In this study, three propolis/2HPbCD inclusion
complexes were succesfully prepared from three ethanolic
extracts of propolis and 2HPbCD by kneading method. After
the complexation with 2HPbCD, the three propolis sorts
were transformed from solid, heterogenous, resinous and
sticky masses to amorphous, parallelipipedic forms with
micrometric-sized particles, as characterized by optical
microscopy.
The DSC analysis revealed that the polyphenolics of the
studied propolis extracts were included in different
concentrations in the 2HPbCD cavity to form the
corresponding propolis extract/2HPbCD inclusion complex,
the lowest content of the bioactive compounds was found
in the case of P1-Tm_60/ 2HPbCD complex. Also, the
differential scanning calorimetry indicated that the P2B_60/2HPbCD inclusion complex is the most suitable for
pharmaceutical processing, due to the lowest content of
crystallization water and highest efficient molecular
inclusion. However, in order to confirm the enhanced watersolubility and to explain in detail the inclusion mode of
polyphenolics from propolis extracts in the 2HPbCD cavity
further studies using phase solubility measurement, proton

REV.CHIM.(Bucharest) 67 No.2 2016

nuclear magnetic resonance ( 1H-NMR), and X-ray

diffractometry (XRD) need to be performed.
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Manuscript received: 26.10.2015

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