Journal of Apicultural Research and Bee World 48(4): 270-278 (2009) © IBRA 2009

DOI 10.3896/IBRA.1.48.4.07

ORIGINAL RESEARCH ARTICLE

Thermal stability of propolis from Tucumán,

Argentina

Mariela González1*, María Inés Gómez2, María Laura Tereschuk1 and Amelia Molina2
1
Facultad de Ciencias Exactas y Tecnología. Universidad Nacional de Tucumán. Av. Roca 1700, (4000), Tucumán, Argentina.
2
Facultad de Bioquímica, Química y Farmacia. UNT, Ayacucho 491. San Miguel de Tucumán (4000), Tucumán, Argentina.

Received 22 August 2008, accepted subject to revision 24 January 2009, accepted for publication 18 July 2009.

*Corresponding author: Email: magonzalez@herrera.unt.edu.ar

Summary
Propolis, the material used by bees to protect their hives, is a glue-like substance composed of plant resins, bee waxes and pollens. A large
number of flavonoids have been identified in propolis and the content of flavonoids is considered as an important index for evaluating propolis
quality. The aim of this work was to study temperature effects on propolis from Amaicha del Valle (Tucumán, Argentina) and some of the
main active flavonoids. Thermal analysis was carried out by thermogravimetric (TGA) and thermodifferential analysis (DTA). The identification
of compounds was carried out by different spectroscopic methods: Ultraviolet (UV), Infrared (IR), 1H Nuclear Magnetic Resonance (1H NMR)
and Electronic Impact Mass Spectrometry (EIMS). The propolis from Amaicha del Valle and apigenin, chrysin and galangin were found to be
stable from room temperature to 120ºC.

Estabilidad termal de propóleos del Tucumán, Argentina

Resumen
El propóleos, material usado por las abejas para proteger sus colmenas, es una mezcla gomosa, compuesta por resinas vegetales, cera de
abejas y polen. Se han identificado varios flavonoides en propóleos y su contenido se considera un índice importante para la evaluación de la
calidad del mismo. El objetivo de este trabajo es estudiar el efecto de la temperatura en propóleos de Amaicha del Valle (Tucumán, Argentina)
y en algunos flavonoides mayoritarios activos presentes en aquel. El estudio térmico se realizó mediante análisis termogravimétrico (TGA) y
análisis termodiferencial (DTA). La identificación de los compuestos fue realizada por distintos métodos espectroscópicos: ultravioleta (UV),
Infrarrojo (IR), RMN H1 y espectrometría de masa con impacto electrónico (EIMS). Se encontró que los propóleos de Amaicha del Valle y los
flavonoides apigenina, crisina y galangina son estables en el rango de temperatura desde temperatura ambiente hasta 120ºC.

Keywords: propolis, chrysin, galangin, apigenin, thermal analysis, DTA, TGA

it is used as a health food. Propolis is now found in toothpastes,

Introduction
Propolis or bee glue, the material used by bees to protect their hives,
is a glue-like substance composed of plant resins, bee waxes and
pollens. It is known to have been used in folk medicine as early as
300 BC. Since various biological activities of propolis such as
antibacterial, antiviral, anti-inflammatory and anaesthetic properties
have been found, (Ghisalberti, 1979; Marcucci, 1995; Burdock, 1998),
chocolates, shampoos, creams, tablets, etc. and world production has
increased to several tonnes per year (Ackermann, 1991).
The chemical composition of propolis is complex, and a single
propolis sample can contain more than 300 components, depending
on the plant source and its geographical origin (Ghisalberti, 1979;
Lima, et al., 2009). Fatty and phenolic acids and their esters,
aromatic aldehydes and alcohols, sesquiterpenes, naphtalene and
Thermal stability of propolis 271

stilbene compounds, as well as a large number of flavonoids, have all structural information on small quantities of pure samples as well as
been identified in propolis (Bankova et al., 1983). Among these on mixtures. Mass spectra obtained under electron ionization (EIMS)
compounds, flavonoids have been suggested to be responsible for the conditions have been widely used for structural investigations of
biological activities (Kujumgiev et al., 1999; Bosio et al., 2000). flavonoids. The ionization technique has been applied to all classes of
Flavonoids such as pinocembrin and galangin have been found to flavonoids aglycones (Cuyckens and Claeys, 2004).
determine the antibacterial activity of propolis, and pinocembrin also The aim of this work was to carry out a preliminary study of the
has fungicidal and local anaesthetic activities. Quercetin, kaempferide temperature effect on propolis from Amaicha del Valle and on some
and pectolinaringenin have spasmolytic activity, acacetin has flavonoids present in the propolis in that area.
antiinflamatory activity and luteolin and apigenin possess antiulcer
activity (Bankova et al., 1983). The content of flavonoids is therefore
considered as an important indicator for evaluating propolis quality.
Materials and methods
Although, the chemical composition of the main flavonoids in
propolis has quantitatively or qualitatively been found to be variable, Samples
depending on the environmental plant ecology, the role of the Propolis was taken from honey bee hives at Amaicha del Valle. This
beekeeper is also very important because propolis production needs site is situated at an altitude of 2000 m above sea level and 164 km
more care than honey production. The quality of the propolis is in from San Miguel de Tucumán , the capital of the Tucumán province,
direct relation to the methods of extraction and storage. This directly Argentina.
influences its price (Ceci, 2000). In order to retain the biological Commercial flavonoids: chrysin, galangin and apigenin were
properties of propolis, beekeepers recommend keeping it in plastic purchased from Sigma-Aldrich to build a simplified model of Amaicha
bags and protected from light and high temperatures. del Valle propolis for this first study. The model chosen was based in
In order to study the behaviour of propolis in relation to the main active flavonoids present in the propolis samples (Park et al.,
temperature, thermoanalytical methods such as thermogravimetric 2001) (Fig. 1). The biological activity of those three flavonoids present
analysis (TGA) and thermodifferential analysis (DTA) have been used. in propolis is supported by literature (Ciolino and Yeh, 1999; Trochon
These methods measure the variation of some physical property of et al., 2000; Woo et al., 2005).
the sample with increase in temperature (or with time) (Dodd and
Tonge, 1987). The mass variation can be measured with TGA and the
temperature difference between the sample and a substance of inert
reference (Al2O3) measured with DTA (Artiaga Diaz, 2005; Gabbott,
2007).
The structural study of flavonoids before and after heat treatment
has been achieved using spectrophotometric, 1H Nuclear Magnetic
Resonance (1H NMR) and Electronic Impact Mass Spectrometry
(EIMS) techniques. Methanol Ultraviolet (UV) spectra of flavonoids
have been found to exhibit two major absorption peaks in the region
of 240-400 nm and the addition of different chemical reagents to the
flavonoid solution produces molecular changes that show the
presence or absence of substitutes in the different positions of the
respective rings. These molecular changes are evident in the
displacement of the absorbance peaks and in the variation of its
intensity. Their interpretation allows determination of the structure of
the respective rings (Mabry et al., 1970).
Infrared (IR) spectroscopy is most frequently used in
phytochemical studies as a “fingerprint” device, for comparing a Fig. 1. Formula of flavonoids present in propolis from Amaicha del Valle.
natural with a synthetic sample. The complexity of the IR spectrum
lends itself particularly well to this purpose and such comparisons are
very important in the complete identification of natural compounds Analysis
(Nikolovska et al., 1995). Modern mass spectrometric techniques are Samples were analyzed by DTA, TGA, UV, IR, 1H NMR and EIMS. The
very well suited to the analysis of flavonoids in plants and foodstuffs need for sample preparation depended strongly on the sample type
and play a key role in their analysis, since they can provide significant and the analytical techniques used (Cuyckens and Claeys, 2004).
272 González, Gómez, Tereschuk, Molina

For thermoanalytical analysis a Thermogravimetric Analyzer

Results
Shimadzu TGA 50 and a Thermodifferential Analyzer DTA 50 were
used. Measurements were made at an inert atmosphere of nitrogen DTA samples are shown in Figs 2-4. In Fig. 2, propolis DTA shows an
3
flowing at 20 cm /min with a heating speed of 5ºC/min. Structural endothermic peak between 50ºC and 68ºC. The observed changes in
study of the methanolic extracts by UV spectrophotometry was carried the endothermic process for the propolis model in Fig. 3 is related to a
out before and after the thermal treatment. The flavonoids in a pure weight loss of 2.37% in one step between 70 and 90ºC in TGA. Data
state were also evaluated through shift reagents (Markham, 1982). from the mass loss determination for Galangin and its DTA are shown
Beckman DU7500 equipment was used for scanning from 200 nm to in Fig. 4 simultaneously. Apigenin and chrysin DTA showed neither an
400 nm. The infrared spectra were recorded with a FTIR Perkin Elmer exothermic or endothermic peak.
-1 -1
1600 IR spectrophotometer from 500 cm to 3000 cm in the Propolis and model propolis UV spectra are shown in Fig. 5. For
transmission mode using KBr pellets. Because only galangin showed pure flavonoids with shift reagents, bands II and I are typical for
an endothermic change observed in DTA its IR spectra were recorded. chrysin, apigenin, and galangin respectively before and after heat
The structural study of galangin was continued before and after treatment (Mabry et al., 1970). Fig. 6 corresponds to the IR spectra of
1
undergoing heating through H NMR (Markham and Geiger, 1994) and propolis with and without heat treatment. Because galangin shows an
Electron impact mass spectra (EIMS). EIMS were recorded by DIP/MS endothermic change observed in DTA its IR spectra were recorded.
ThermoElectron Polaris Q equipment at 200 ºC and 70eV. 1H NMR Fig. 7 shows the IR spectra for galangin with and without heat
was utilised a Bruker (400 MHz). The compounds were dissolved in treatment. Fig. 8 shows 1H NMR of galangin after heat treatment and
CD3OD. The d values were expressed in ppm and J in Hz. Table 1 shows the signal assignment. EIMS of galangin with and
without heat treatment is shown in Fig. 9.

Fig. 2. Thermodifferential analysis of propolis.

Thermal stability of propolis 273

Fig. 3. Thermodifferential analysis of the propolis model.

Fig. 4. Thermogravimetric and thermodifferential analysis of galangin.

274 González, Gómez, Tereschuk, Molina

Fig. 5. Propolis and propolis model ultraviolet spectra.

Table. 1. Galangin 1H NMR spectra with and without heat treatment.

1
H RMN δ(ppm)
J (Hz)

CD3OD CD3 3 5 6 7 8 2’ 3’ 4’ 5’ 6’

4.95 3.4 OH OH 6.19 d. OH 6.39 d. 8.16 dd. 7.47 m. 7.47 m. 7.47 m. 8.16 dd.
(2) (2)

Discussion
The endothermic process of propolis, which begins at 45ºC and ends
at about 68ºC (Fig. 2), is probably due to another component of
propolis that is not represented in this model. For the propolis model
(Fig. 3), it can be assumed that the weight loss is due to the
decomposition process of galangin because it is the only flavonoid
used in the model that lost mass over the temperature range studied.
Only one peak was observed for the thermal decomposition
process of galangin (Fig. 4). The total weight loss up to 90ºC was
6.31%, and this mass loss could correspond to water of crystallization
(theoretical weight loss 6.67%), corresponding to an endothermic
process observed in the DTA at 80.7ºC.
Both the propolis and the model show a wide band between 360
and 280 nm in their UV spectra (Fig. 5). In the propolis model there
are two shoulders and a peak at smaller wavelength, which is
characteristic of Band II of the flavones present in the model sample.
Using shift reagents the λMAX of the pure flavonoids, although having
been heat treated are coincident with the reported values for those
compounds (Marby et al., 1970; Markham, 1982).
The enthalpy variation in galangin is not demonstrated in UV
scanning. Because of this, and in order to determine whether there
was modification in the galangin structure after the thermal
treatment, an infrared spectrum was performed. The propolis sample
was also evaluated with and without temperature treatment.
The IR spectra of the propolis samples from Amaicha del Valle
with and without heat treatment were very similar. The spectra
showed the same bands in IR: a wide centred signal at 3421 cm-1
Thermal stability of propolis 275

Fig. 6. Propolis infra red spectra.

Fig. 7. Galangin infra red spectra.

276 González, Gómez, Tereschuk, Molina

Fig. 8. Galangin before and after heat treatment 1H Nuclear Magnetic Resonance ( 1H RMN).

Fig. 9. Galangin before and after heat treatment Electronic Impact Mass Spectrometry (EIMS).
Thermal stability of propolis 277

assigned to the tension variation O-H; in the region under 3000 cm-1
two bands were observed at 2918 and 2853 cm-1 assigned to a vibra-
tion tension (asymmetric and symmetric) of the C-H bond; a signal
corresponding to the tension vibration of the carbonyl group at 1738
cm-1; a signal corresponding to tension variation of C=C group at a
1607 - 1466 cm-1; and other slightly wider bands of bonds C-O and
deformation vibrations of bonds C-H were observed between 1400
and 1000 cm-1.
The IR spectra of galangin with and without heat treatment
were also very similar. The same signals were observed in them: a
wide centred band at 3403 cm-1 assigned to the tension vibration of O
-H group; an intense band at 1642 cm-1 corresponding to the tension
vibration of the carbonyl group; at 1613 cm-1 a band that can be as-
signed to tension vibration of C=C group; and several bands corre-
sponding to tension vibrations of the C-O group and deformation
vibration of the C-H bonds are observed at 1512, 1469, 1372, 1310
and 1167 cm-1. Before heat treatment, galangin shows the presence Fig. 10. Fragmentation data of aglycone flavonoids.
of weakly bonded water molecules in the structure which is evident
from the broad ν(OH) band below 3500 cm-1 (Fig. 7 a). After heat
treatment the band appears more defined, suggesting the presence of
more strongly coordinated water molecules (Fig. 7 b).
The values of chemical displacement for the 1H NMR of
galangin with heat treatment are consistent with those reported in the
literature for this flavonoid (Markham and Geiger, 1994). A multiplet
corresponding to the non substituted protons of B rings is observed at
about 7.5 ppm, whilst a signal corresponding to two H of B ring (2’ Fig. 11. Mechanism of Retro- Diels-Alder (RDA) breaking for B2+
and 6’), is at about 8 ppm, which can be attributed to the interference fragment formation.
that would cause the OH in position 3 of the C ring regarding those
protons.
EIMS for galangin and galangin after heat treatment were also 80.7°C for the range of temperatures studied. The study of galangin
strikingly similar. The only difference between the EIMS spectra of with and without heat treatment through UV, IR, 1H NMR and EIMS
galangin and galangin with heat treatment were the values of relative demonstrated no chemical change.
1
abundance for some fragments. Although IR, UV, H RMN and EIMS The endothermic process of propolis, which begins at 45ºC
measurements of galangin before and after heating do not show dif- and ends at about 68ºC, is probably due to another component of the
ferences, the mass loss detected in TGA is probably due to a loss of propolis absent in this simplified model. The endothermic change for
crystallization water. the propolis model is assigned to the ΔT of galangin present in it.
For mass spectra for free aglycones (Ma et al., 1997), the Likewise, it can be assumed that the weight loss of the propolis model
primary fragmentations are shown in Fig. 10. The more important is due to galangin.
peaks of the spectrum for galangin as well as for galangin with heat We can conclude that over the range of temperatures studied, the
treatment were at a m/z 270 [M+, 100%], 269 [M-H]+, 241 [M-CHO] propolis from Amaicha del Valle is stable to heat treatment with
+ +
, 242 [M-CO] , 197 [M + H - H2O – 2 CO], 144 [ 0,4 +
B - H2O], 105 regard to the flavonoids that we studied.
[B2+], 129 [ 1,4 + +
B + H - H2O] y 87 [B2+ - H2O]. Fig. 11 shows the
fragmentation mechanism for B2+, which begins because of a Retro-
Diels-Alder (RDA) like-breaking.
The IR and UV spectra of propolis with and without heat
treatment do not show differences in the characteristic bands. Among
the three studied flavonoids with and without heat treatment, only
galangin shows an endothermic change in DTA with a weight loss of
6.31% which may be due to the loss of water of crystallization at
278
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