Characteristics and chemical compositions of propolis from Ethiopia

Introduction Propolis is a sticky material mixed by honeybees to utilize it in protecting their hives from infection by bacteria and fungi. The therapeutic properties of propolis are due to its chemical composition with bio-active compounds; therefore, researchers are interested in studying its chemical constituents and biological properties. The main objective of this study is to determine the chemical compositions, characteristics and relative concentrations of organic compounds in the extractable organic matter of propolis samples collected from four different areas in Ethiopia. Results The propolis samples were extracted with a mixture of dichloromethane and methanol and analyzed by gas chromatography–mass spectrometry (GC-MS).The results showed that the total extract yields ranged from 27.2% to 64.2% (46.7 ± 19.1%). The major compounds were triterpenoids (85.5 ± 15.0% of the total extracts, mainly α-, β-amyrins and amyryl acetates), n-alkanes (5.8 ± 7.5%), n-alkenes (6.2 ± 7.0%,), methyl n-alkanoates (0.4 ± 0.2%), and long chain wax esters (0.3 to 2.1%). Conclusion The chemical compositions of these propolis samples indicate that they are potential sources of natural bio-active compounds for biological and pharmacological applications.


Introduction
Honeybees collect resinous/waxy substances from exudates of plants to make a sticky material known as propolis (Ghisalberti 1979;Parolia et al. 2010). They utilize propolis to seal cracks in hives, encapsulate invader carcasses and protect their hives from infection by bacteria and fungi (Banskota et al. 2001;Simone-Finstrom & Spivak 2010). In ancient times, Egyptians, Greeks and Romans all used propolis as a remedy against some diseases (Sforcin & Bankova 2011). The therapeutic properties of propolis are due to its chemical composition with bioactive compounds; therefore, researchers are interested in studying its chemical constituents and biological properties (Sforcin & Bankova 2011;Bankova 2005;Castaldo & Capasso 2002;Sforcin 2007). The diverse chemical compositions and biological activities of propolis are attributed to geographical settings, plant sources and collecting season (Sforcin & Bankova 2011). Flavonoids, aromatic acids, diterpenoid acids, triterpenoids, and phenolic compounds are the major components of propolis (Bankova et al. 2000;Chen et al. 2008;Cursta-Rubio et al. 2007;Daugsch et al. 2008;Kumazawa et al. 2008;Markham et al. 1996;Popova et al. 2010). Some of these compounds are responsible for its biological activities (Bankova et al. 2000; Barros et al. 2007; Bassani- Silva et al. 2007;Bufalo et al. 2009;Cvek et al. 2007;Orsatti et al. 2010a;Orsatti et al. 2010b;Orsi et al. 2005;Zamami et al. 2007). There are three possible sources for the organic compounds of propolis: plants, secreted substances from honeybee metabolism, and materials that are introduced during propolis formation (Marcucci 1995). Propolis is typically composed of 50% resin and vegetable balsam, 30% wax, 10% essential and aromatic oils, 5% pollen and 5% other substances (Cirasino et al. 1987;Monti et al. 1983). Most of the studies on propolis composition and pharmacological effects have been performed on samples from Europe and Latin America (e.g. (Bankova et al. 2000;Daugsch et al. 2008;Barros et al. 2007;Monti et al. 1983)), whereas few have reported on propolis from north Africa  with none from Ethiopia. Ethiopia is located in north-eastern Africa with varied climatic and physiographic conditions that endowed the country with more than 7,000 species of flowering plants (Edwards 1976). They are considered as a potential for producing huge volume of propolis with high probabilities for various biologically active substances. However, many beekeepers in the country focus only on honey production. Therefore, the main objective of this study is to determine the chemical compositions, characteristics and relative concentrations of organic compounds in the extractable organic matter of propolis samples collected from four different areas in Ethiopia.
The relative concentrations of the n-alkenes ( Δ 1 or Δ 9 ) ranged from 0.85% to 15.92% with a mean of 6.23 ± 6.96%. The highest relative concentration (15.92%) was found in the propolis sample from the Holleta area and the minimum (0.85%) in the samples from Bako. The nalkenes ranged from C 25 to C 36 with a C max at 33. The odd carbon numbered n-alkenes were dominant with a CPI of 2.49 to 7.24 (mean 5.30 ± 2.02). The distribution of n-alkenes with major concentrations of the odd numbered homologues and C max at 33 supports an origin from insect wax (Jackson 1972;Jackson & Baker 1970), possibly from alteration of long chain n-alkanols.

Methyl n-alkanoates
The concentrations of methyl n-alkanoates were relatively low at 0.19% to 1.14% with a mean of 0.64 ± 0.40% (Table 1). They ranged from C 13 to C 29 with a C max at 17 and 25 (as acids C max = 16 and 24) (Figure 2c). Methyl n-alkanoates may be natural or form by transesterification of n-alkanoic acids during extraction as indicated by their low relative concentrations. The highest concentration (1.14%) was found for the propolis sample from Gedo and the lowest (0.19%) from Bako. The methyl n-alkanoates of these samples have a strong even carbon number predominance as the alkanoic acids (CPI > 17, except for Gedo, Table 1), indicating that they are originally from natural biota (Harwood & Russell 1984).

Long chain wax esters
Long chain wax esters were also detected in these samples with relative concentrations of 0.29% to 2.08%, and consisting mainly of docosanyl-, tetracosanyl-, hexacosanyl-and octacosanyl hexadecanoates. The major compound of the wax esters was tetracosanyl hexadecanoate in all samples (Table 1, Figure 2f). They are likely derived from lipid components of terrestrial plants (Baker 1982;Kolattukudy 1976;Hamilton 1995) of the region or from waxes secreted by the bees (Tulloch 1971). Subsequent reports have shown that the components of waxes in some younger plants are generally alcohols (40%) and they are mainly wax esters (42%) in older plants (Avato et al. 1990;Bianchi et al. 1989). The vegetation wax ester composition depends not only on plant species, but also on the geographical location (Sforcin & Bankova 2011). Waxes secreted by bees contain more than 15% of wax esters (Katzav-Gozansky et al. 1997). Bee wax esters generally include tetradecyl-dodecanoate, tetradecanoate and hexadecanoate, as well as hexadecyltetradecanoate and hexadecanoate (Katzav-Gozansky et al. 1997).

Unique composition
It has been reported that propolis components, which are complex, have biological properties including antimicrobial, antioxidant and anticancer activities (Lustosa et al. 2008;Naito et al. 2007;Diaz-Carballo et al. 2008). Propolis was also reported to have effects against cariogenic bacteria (de Castro Ishida et al. 2011). Triterpenoids are major and to date unique components of these propolis samples from different regions in Ethiopia, indicating a high potential as sources of biologically active substances. Further studies are needed to investigate the biological activities of these propolis samples, and the correlations between their chemical compositions and botanical origins.

Conclusion
The solvent-extractable organic matter (DCM:MeOH) of propolis samples from four regions in Ethiopia have been characterized using GC-MS techniques. The mixed solvent was used to extract both polar and non-polar compounds of proplis samples. The major compounds were in order: triterpenoids > > n-alkanes~n-alkenes > long chain wax esters > methyl n-alkanoates. The predominant triterpenoids were αand β-amyrins, αand β-amyryl acetates, followed by lupeol, and αand β-lupeyl acetates. n-Alkanes and n-alkenes ranged from C 21 to C 31 and C 25 to C 35 with C max at 27 and 33, respectively. Long chain wax esters and methyl n-alkanoates were minor components in these samples. The sources of the major triterpenoids are from the regional Acacia waxes and gums. Phenols (e.g. flavonoids) or other antioxidants were not detectable in these samples.
The variation in the identities of propolis components among various reports is likely due to diverse environmental source vegetation, and different extraction methods and solvents used. Therefore, a standardized analytical method should be adopted in order to be able to compare results obtained by different investigators.

Sampling
The propolis samples were collected from the central parts of Ethiopia representing highlands and midland areas. Acacia, Euphorbiaceae sp. Croton macrostachys, and Boraginaceae sp. Cordia africana. The propolis samples were collected using a stainless steal spatula (>30 g of each) in a Teflon-caped glass container, labeled and kept in a freezer until analysis.

Extraction
About 20 g of each sample was broken up and extracted three times using ultrasonic agitation for a 15 min period each with a mixture of dichloromethane (DCM) and methanol (MeOH, 40 mL, 3:1 v:v) mixture to make certain that both polar and non-polar compounds were extracted. The extraction was carried out in a precleaned beaker. The extract was then filtered using a filtration unit containing an annealed glass fiber filter for the removal of undissolved particles. The filtrate was first concentrated on a rotary evaporator and then reduced using a stream of dry nitrogen gas to a volume of approximately 2 mL. The volume was then adjusted to exactly 2 mL by addition of DCM:MeOH (3:1, v:v). A 50-μL aliquot of each total extract was derivatized with silylating reagent [N,O-bis(trimethylsilyl)trifluoroacetamide, BSTFA, Pierce Chemical Co.] by the standard procedure (Knapp 1979), before analysis by gas chromatography-mass spectrometry (GC-MS). This derivatizing agent replaces the H in hydroxyl groups with a trimethylsilyl [(CH 3 ) 3 Si, i.e. TMS] group for better GC resolution of polar compounds.

Chemical analysis
Instrumental analysis by GC-MS was carried out with an Agilent 6890 gas chromatograph coupled to a 5973 Mass Selective Detector, using a DB-5MS (Agilent) fused silica capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) and helium as carrier gas. The GC was temperature programmed from 65°C (2 min initial time) to 310°C at 6°C min −1 (isothermal for 55 min final time) and the MS was operated in the electron impact mode at 70 eV ion source energy. Mass spectrometric data were acquired and processed using the GC-MS ChemStation data system.

Identification and quantification
The identification of n-alkanes was based on the GC-MS data. Retention times were compared with those of external standards. The identities of triterpenoids, n-alkanes, n-alkenes, n-alkanoic acids, methyl n-alkanoates, and long chain wax esters are based primarily on their mass spectra (i.e. key ions at m/z 191/189, 85, 83, 117, 87, and 257, respectively), comparison with those of standards or in the literature, and gas chromatographic retention times. Average response factors were calculated for each compound. All quantifications were based on the compound peak areas derived from the ion fragmentograms correlated with the total ion current (TIC) trace.