ABSTRACT Propolis is a very valuable bee product due to its antioxidant, bacteriostatic, antifungal and therapeutical properties which are related to the content of The propolis differed mainly in the presence of phenolic compounds being significantly largest in pampeana area propolis samples where the plant species are in the families mirthaceas and salicaceas

Enzo A 

Luis M

María C Ciappini

Santa Fe

TAPIZ Ampelio F Cazzolli

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APIACTA 41 (2006) PAGE 110-120 
 
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PHYSICO CHEMICAL CHARACTERISTICS OF PROPOLIS COLLECTED IN 
SANTA FE (ARGENTINE) 
 
TOSI, Enzo A.; CIAPPINI, María C. , CAZZOLLI, Ampelio F., TAPIZ, Luis M. 
Centro de Investigación y Desarrollo en Tecnología de Alimentos (CIDTA). 
Universidad Tecnológica Nacional. Facultad Regional Rosario. Zeballos 1341 
(S2000BQA) Rosario. Argentina e-mail: mcciappini@frro.utn.edu.ar 
 
ABSTRACT 
Propolis is a very valuable bee product due to its antioxidant, bacteriostatic, 
antifungal and therapeutical properties which are related to the content of 
phenolic and flavonoid compounds. In this work, physico chemical 
characteristics and some active compounds of 42 samples collected in different 
phytogeographical regions of Santa Fe (Argentine) were measured. Hexane 
extractable substances ranged between 14.3 and 46.2%, insoluble residues 
between 18.0 and 22.4%; resins soluble in ethanol between 26.6 and 75.8%, 
phenolic compounds between 11.3 and 38.0%,loss by heating between 1.4 and 
6.2% and ash between 1.8 and 2.4%.  Concentrations corresponding to 
phenolic and flavonoid compounds obtained by HPLC show the prevalence of 
pinocembrin, crisine, galangine and quercetine.  
The propolis differed mainly in the presence of phenolic compounds being 
significantly largest in pampeana area propolis samples where the plant species 
are in the families mirthaceas and salicaceas.  
 
Key Words: Argentine propolis/ propolis characterization 
APIACTA 41 (2006) PAGE 110-120 
 
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INTRODUCTION 
Raw propolis is an important product of the beehive produced by honeybees 
(Apis mellifera), gathering and transforming the bud exudates, by mixing it with 
waxy substances (Serra Bonhevi, 1996). It has a sweet, balsamic odour and 
presents variable consistency, depending on the origin and temperature. Its 
colour is variable, from light yellow to dark brown, with a wide range of brownish 
intermediate tones (Salamanca Grosso et al., 2002).  
The average composition of propolis shows the presence of 20-30% waxes, 
resins and aromatic balsams (40-50%), essential and aromatic oils (5-10%), 
pollen (4-5%) and other various substances, including insoluble residue (10-
30%) (Brown, 1995). Simple fractionation of propolis to obtain compounds is 
difficult due to its complex composition (Burdock, 1998). It can be separated 
into a fraction soluble in ethanol, called balsams; and into another fraction 
insoluble in alcohol, called waxy fraction. Balsam chemical pattern and propolis 
physico chemical characteristics vary according to the botanical and 
geographical origin (Montenegro et al., 2000).   
This bee product is valuable for its antioxidant, bacteriostatic, antifungal and 
therapeutic properties, related to the contents of phenolic and flavonoid 
compounds (Salamanca Grosso et al., 2002). It is applied in medicine, 
cosmetics, veterinary and food industries (Maidana, 2000). Substances 
identified in propolis are, in general, typical constituents of food and/or food 
additives, known as Generally Recognized as Safe (GRAS) substances 
(Burdock, 1998). The main difficulty is the absence of controls on the origin and 
composition of the propolis produced (Maidana, 2000). Propolis standardization 
has not been carried out in a complete and systematic way yet.  
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Argentina is the third honey world producer and the second exporter. Honey 
production comprises also other bee-hive products such as propolis, though 
with a lower incidence in the apicultural activity. The main producer provinces 
are Buenos Aires, Entre Ríos, Córdoba and Santa Fe. The most productive 
areas are in the plains. There is a large amount of agricultural activity in the 
study region. Tree communities are very scattered and limited nowadays but it 
is thought they were significantly larger in the past.  
The aim of this work is the physico-chemical characterization of propolis 
produced in the Province of Santa Fe (Argentine).  
 
MATERIALS AND METHODS 
Sampling was carried out considering agro-climatic features, latitude and flora 
(for honey purposes). Santa Fe (28º - 35º SL; 58º- 62º WL) was divided into its 
phytogeographical areas which closely correspond to honey production and 
bee-hive product areas. They are called chaqueña province in the north; espinal 
province in the centre and pampeana province in the south (Figure 1). 
There are no woods in the pampeana province area and the ground is covered 
by grass. Its main crops are Glicine max (soybean) and Zea mays (corn). Rows 
of Eucalyptus sp. (Mirtaceae) and Salix humboldtiana (Argentinian willow - 
Salicacceae) are also present.. 
In the chaqueña province, there are Prosopis nigra (black algarrobo - 
Fabaceae), Parkinsonia aculeata (cina-cina - Fabaceae), Acacia caven (aromito 
- Fabaceae), Erythrina crista-galli (ceibo tree - Fabaceae), Astronium Balansae 
(urunday - Anacardiaceae), Schinopsis Balansae (red quebracho from Chaco - 
Anacardiaceae) and Apidosperma quebracho-blanco (white quebracho - 
APIACTA 41 (2006) PAGE 110-120 
 
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Apocianaceae). Randomly, Sapium haematospermum  (curupí or lecherón - 
Euphorbiaceae) and Sesbania punicea (coffee plant of the coast or acacia of 
the swamps - Fabaceae) can be found. In the espinal province there are other 
species such as the Phitolacca dioica (umbra tree - Phitolacaceae), and some 
herbaceous such as Stypa neesiana (flechilla brava - Poaceae), Sporobolus 
indicus (Poaceae), Medicago spp. (clovers or alfalfas - Fabaceae) and Carex 
bonariensis (carex - Cyperaceae) (Quargnolo, 1982).  
Sampling was carried out according to Standard 
RST-RSFSR-317-77 (1977), during 2004. Samples were collected by people 
from the Provincial Apicultural Programme of the Ecological Division – Ministry 
of Agriculture, Cattle Farming, Industry and Commerce of the Province of Santa 
Fe (Argentine), who employed the scraping technique following the instructions 
of the Apicultural Programme (PROAPI) of the National Institute of Agricultural 
Technology (Instituto Nacional de Tecnología Agropecuaria - INTA). A 
representative sample of boxes and frames of at least 25% of the bee-hives in 
each apiary, composing a lot with a mass of about 500g was sent to the 
laboratory together with the corresponding data. Macroscopic impurities were 
removed from each lot. Then, 2-3g subsamples from different parts of lot were 
Figure 1 – Province of Santa Fe  (Argentine) 
 
1 North area   – Chaqueña province 
2 Center area – Espinal province 
3 South area  –  Pampeana province 
APIACTA 41 (2006) PAGE 110-120 
 
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taken for composing the 40g assay sample. These samples, previously cooled 
at -18ºC for 24 hours, were milled in a refrigerated IKA mill. The resulting 
product was packaged in glass flasks; 20g were used as a control sample and 
20g were left to carry out assays.  
Forty-two useful samples were collected; 14 from each of the three already 
described phytogeographical regions and they were submitted to the following 
determinations: 
a) Physicochemical characterization: Our methods are from the recommended 
IRAM - INTA 15935-1 Scheme 1 (2004), and they are the same techniques 
used by other authors to characterize similar samples (Woisky y Salatino, 
1998; Maidana, 2000; Maldonado, 2000).  
Sensory attributes (ISO 6658: 1985), loss by heating (in oven at 100ºC + 
2ºC, until constant weight), ash (in a muffle furnace at 550ºC + 25ºC, until 
constant weight), n-hexane extractable substances (by means of n-hexane 
extraction in a reflux Soxhlet extractor), resins soluble in ethanol (by means 
of ethanol extraction in a reflux Soxhlet extractor on the solids residue 
obtained after determining the n-hexane extractable substances)  insoluble 
residue (represented by dry solids residue obtained after determining the 
ethanol soluble resins), oxidation value (discoloration time of 0.05ml 
potassium permanganate solution 0.1N, by 2ml of hydroalcoholic 0.2% 
propolis solution), UV absorption spectrogram (by means of scanning 
spectrophotometric reading between 240nm and 420nm of the absorbance 
of an alcoholic dilution 1:1000 of ethanolic extract obtained from the soluble 
resins determination; Spectrophotometer Varian Cary 50), total phenolic 
compounds (by spectrophotometry; measuring at 765nm, the absorbance of 
APIACTA 41 (2006) PAGE 110-120 
 
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a dilution 1:200 of ethanolic extract obtained from the determination of 
soluble resins, to which a Folin-Ciocalteu reagent is added and left to react 5 
minutes at 50ºC). The method used for determining total phenolic 
compounds is limited because of its low specificity towards certain 
polyphenols that may be present (Mosca, 2000) and the total phenolic 
compounds are only estimated.  
Two-way ANOVA were applied to determine the effects of the 
phytogeographical areas on the physico-chemical characteristics. Post-hoc 
comparisons were made by least significant difference (LSD) test. 
b) Complementary characterization studies: as colorimetric methods low-
specificity for phenolic compounds, some of them were identified and 
quantified by using HPLC. This compounds share a skeleton of dyphenil 
pyrans (C6-C3-C6), formed by two phenyl rings bounded by a C pyran ring, 
consider profit by their antioxidant capacity (Martínez Flores et al., 2002). A 
high performance liquid chromatographic (HPLC) procedure has been used 
to identify and determine some flavonoids in propolis (Bankova et al., 1982). 
A Shimadzu LC 10-AS chromatograph, equipped with a UV-visible SPD-10A 
detector and a C 18 reversed-phase column (Supelco), particle size 5µm, 
25x0.46cm ID and pre-column (4mm) of the same material. The eluent was 
water-methanol-acetic acid (60:75:5, HPLC quality), at a flow rate of 
0.7ml/min. Standard solutions were prepared by dissolving hydroxycinamic 
acids (cafeic acid, o-cumaric acid, p-cumaric acid), benzoic (syringic acid) 
and cynamic (cumaric); flavons (apigenine, acacetine, crisine), flavonols 
(quercetine, kaempferol, galangine); provided by SIGMA Chemical 
Company. Sample for the HPLC analysis involved dilutions of balsams 
APIACTA 41 (2006) PAGE 110-120 
 
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obtained in the determination of ethanol-soluble resins, applying the 
methods proposed by Markaham et al. (1996). 
 
RESULTS AND DISCUSSION 
Most propolis (64%) was dark brown or greenish irregular pieces, with intense 
but very aromatic resin odour. Less than 15% of the samples were a bright 
uniform mass; 59% of the samples presented a hot spicy or hot sweet taste. 
The results obtained are given in Table I. 
 
Table I – Physico-chemical characteristics of propolis produced in Santa Fe  
   
loss by 
heating 
 
ash 
n-hexane 
extractable 
substances  
 
insoluble 
residue 
ethanol 
soluble 
resins  
total 
phenolic 
comp. 
 
oxidation 
value 
  
Units  
 
g/100g 
 
g/100g 
 
g/100g 
 
g/100g 
 
g/100g 
 
g/100g 
 
s 
Area         
 Minimum 1.4 1.8 14.3 18.0 26.6 11.3 14 
Pampeana Maximum 1.6 2.2 46.2 22.4 75.8 22.8 19.5 
 Median 1.5 2.0 33.2 20.5 61.2 21.3 16 
 Minimum 1.5 1.9 22.1 18.5 29.0 11.4 12 
Espinal Maximum 1.7 2.3 28.4 21.4 55.3 21.3 24 
 Median 1.6 2.0 27.1 20.0 40.1 17.2 18 
 Minimum 1.7 1.8 17.6 18.5 38.8 11.3 2 
Chaqueña Maximum 6.2 2.4 30.6 29.2 55.0 38.0 48 
 Median 4.5 2.2 28.2 21.8 41.9 16.6 21 
 
Samples from chaqueña province have the highest values of loss by heating 
and differ from those of the other areas (p=0.05, LSD test).   
APIACTA 41 (2006) PAGE 110-120 
 
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Resins content soluble in ethanol are significantly highest (p=0.05, LSD test) in 
pampeana province samples; in coincidence with higher total phenolic 
compounds content.   
Total phenolic compounds content in pampena province propolis agree with 
those reported by Woisky and Salatino (1998). Concentrations corresponding to 
phenolic and flavonoid compounds obtained by HPLC (Table II) show the 
predominance of pinocembrin, crisine, galangine and quercetine in these 
samples. Both antibacterial flavonoid pinocembrin and antiviral crisina are 
present in all the samples. Bedascarrabure et al. (2003) reported that total 
flavonoids content in pampeana province propolis is 7.87% + 0.39 and in 
chaqueña province samples is 3.61% + 0.40. 
UV spectrograms show absorbance peaks at 240nm, 270nm and 340nm.  
 
Table II – Concentration (g/100g) of some active Phenolic and Flavonoid 
compounds in Propolis collected in Santa Fe (Argentine) 
 
  Concentration Ranges 
 Cafeic acid Not detectable  - 0.18 
Phenolic Cumaric acid 0.13 – 0.56 
Compounds 
 
Syringic acid 0.06  - 2.23 
 Quercetine 1.83  - 5.13 
 Apigenine Not detectable  - 2.30 
Flavonoids Kaempferol Not detectable 
 Galangine Not detectable - 8.85 
 Acacetine Not detectable 
 Crisine 2.33  - 7.46 
 Pinocembrine 2.10  - 10.00 
 
CONCLUSIONS 
This analysis suggests that propolis in the Province of Santa Fe differ in their 
phenolic compounds. The highest phenolic compound content was found in 
APIACTA 41 (2006) PAGE 110-120 
 
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propolis from the pampeana province where the mirthaceas and salicaceas 
species grow.  
If physico-chemical specification for raw propolis are performed as stated by the 
Argentine Standardization Institute (Instituto Argentino de Normalización - 
IRAM) to be applied within this country, 20% samples exceed the highest level 
established for n-hexane substances extractable (<35%) and 5% samples 
exceed the maximum oxidation value (<22s).  
However, further sample analyse should be carried out to confirm these 
features. 
 
BIBLIOGRAFIA 
Bankova, V.S., Popov, S.S. and Marekov, N.L. (1982), High-performance liquid 
chromatographic analysis flavonoids from propolis, Journal of Chromatography, 
342, 135-143. 
  
Brown, R. (1995), Bee World 70, 109. 
 
Burdock, G. (1998), Review of the biological properties and toxicity of bee 
propolis, Food and Chemical Toxicology 36, 347-363. 
 
IRAM-INTA 15935-1 Scheme 1 (2004), Instituto Argentino de Normalización - 
Subcomité de productos agroalimentarios del NOA. Buenos Aires, Argentina 
 
ISO 6658: (1985) Sensory Analysis – Methodology – General Guidance, ISO 
copyright office, Geneva. 
 
Maidana, J. (2000), Propóleos: segunda parte, Boletín Apícola Nº 14. 
 
Maidana, Francisco Jose (2002), Influencia de los compuestos fenólicos del 
propóleos sobre el índice de oxidación, CEDIA Univ. Nacional de Sgo. Del 
Estero, Argentina. 
 
Maldonado, Luis (2000), Caracterización físico química de propóleos 
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Markham, K. R., Mitchell, K. A., Wilkins, A. L., Daldy, J. and Yinrong Lu (1996), 
HPLC and GC-MS Identification of the Major Organic Constituents in New 
Zeland Propolis, Phytochemistry, Vol. 42, Nº 1, pp. 205-211. 
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Martínez Flores, S. González Gallego, J. Culebras, J.M. y Tuñon, M. (2002), 
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Montenegro, G., Timmermann, B., Peña, R., Mujica, A. y Avila, G. (2000), 
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Mosca, L. (2000), Enzimatic assay for the determination of olive oil poliphenol 
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Argentina. 
 
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PHYSICO CHEMICAL CHARACTERISTICS OF PROPOLIS COLLECTED IN

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PHYSICO CHEMICAL CHARACTERISTICS OF PROPOLIS COLLECTED IN

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