Recent trends and important developments in propolis research
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The newest developments in propolis pharmacological research are summarized. The problem regarding biological studies, caused by the chemical variability of propolis, is discussed. The most important trends and developments in recent propolis research are outlined: biological studies performed with chemically characterized samples, bioassay-guided studies of active principles and comparative biological studies of propolis of different origin and chemical composition. These types of studies are extremely valuable with respect to propolis standardization and practical applications in therapy. They will allow scientists to connect a particular chemical propolis type to a specific type of biological activity and formulate recommendations for practitioners.
For this reason, propolis has become the subject of intense pharmacological and chemical studies for the last 30 years. As a result, much useful knowledge has been gathered. However, it is important to note that in the last decade, the paradigm concerning propolis chemistry radically changed. In the 1960s, propolis was thought to be of very complex, but more or less constant chemistry, like beeswax or bee venom ( 2 , 3 ). In the following years, analysis of numerous samples from different geographic regions led to the disclosure that the chemical composition of bee glue is highly variable. This circumstance was soon understood by seasoned chemists, such as Popravko ( 4 ) and Ghisalberti ( 5 ). Nevertheless, most of the scientists studying the biological properties of propolis continued to assume that the term ‘propolis’ was as determinative with respect to chemical composition as the botanical name for a medicinal plant. Numerous studies, carried out with the combined efforts of phytochemists and pharmacologists, led in recent years to the idea that different propolis samples could be completely different in their chemistry and biological activity.
Bees have been in existence for >125 million years and their evolutionary success has allowed them to become perennial species that can exploit virtually all habitats on Earth. This success is largely because of the chemistry and application of the specific products that bees manufacture: honey, beeswax, venom, propolis, pollen and royal jelly. As the most important ‘chemical weapon’ of bees against pathogenic microorganisms, propolis has been used as a remedy by humans since ancient times. It is still one of the most frequently used remedies in the Balkan states ( 1 ), applied for treatment of wounds and burns, sore throat, stomach ulcer, etc.
The Problem of Chemical Variability of Propolis
To understand what causes the differences in chemical composition, it is necessary to keep in mind the plant origin of propolis. For propolis production, bees use materials resulting from a variety of botanical processes in different parts of plants. These are substances actively secreted by plants as well as substances exuded from wounds in plants: lipophilic materials on leaves and leaf buds, gums, resins, latices, etc. (6). The plant origin of propolis determines its chemical diversity. Bee glue's chemical composition depends on the specificity of the local flora at the site of collection and thus on the geographic and climatic characteristics of this site. This fact results in the striking diversity of propolis chemical composition, especially of propolis originating from tropical regions.
Nowadays, it is well documented that in the temperate zone all over the world, the main source of bee glue is the resinous exudate of the buds of poplar trees, mainly the black poplar Populus nigra (7). For this reason, European propolis contains the typical ‘poplar bud’ phenolics: flavonoid aglycones (flavones and flavanones), phenolic acids and their esters (8). Poplar trees are common only in the temperate zone; they cannot grow in tropical and subtropical regions. For this reason, in these habitats, bees have to find other plant sources of propolis to replace their beloved poplar. As a result, propolis from tropical regions has a different chemical composition from that of poplar type propolis. In the last decade, Brazilian propolis attracted both commercial and scientific interest. The main source of Brazilian bee glue turned out to be the leaf resin of Baccharis dracunculifolia (9,10). Among the main compound classes found in Brazilian propolis are prenylated derivatives of p-coumaric acid and of acetophenone. Diterpenes, lignans and flavonoids (different from those in ‘poplar type’ propolis) have also been found (9). However, in Brazil, several types of propolis were registered in recent studies (11,12), that come from plant sources different from B.dracunculifolia and containing compounds other than those mentioned above. Recently the chemistry of Cuban propolis caught the attention of scientists. Its main components are polyisoprenylated benzophenones, and this makes Cuban propolis different from both European and Brazilian bee glue. The plant source of this propolis type was detected to be the floral resin of Clusia rosea, from whence came the prenylated benzophenones (13). There is no doubt that in other ecosystems, propolis plant sources and the chemical composition of propolis will continue to surprise scientists.
The distinct chemistry of propolis from different origins leads to the expectation that the biological properties of different propolis types will be dissimilar. However, in most cases. this is not true! Actually, propolis is the defense of bees against infections, and the antibacterial and antifungal activity of all samples is not surprising. The similarity in many of the other types of activity is less obvious but it is a fact. Of course, the responsible compounds are different, as shown in : mainly favanones, flavones, phenolic acid and their esters in poplar type (European) propolis, prenylated p-coumaric acis and diterpenes in Baccharis type (Brazilian) propolis; prenylated benzophenones in Cuban red propolis, etc.
Table 1 Propolis type Antibacterial activity Antiinflammatory activity Antitumor activity Hepatoprotective activity Antioxidant activity Allergenic action European (poplar type) Flavanones, flavones, phenolic acids and their esters (14) Flavanones, flavones, phenolic acids and their esters (15) Caffeic acid phenethyl ester (16) Caffeic acid, ferulic acids acid, caffeic acid their esters (15) Flavonoids, phenolic and their esters (15) 3,3-Dimethylallyl caffeate (14) Brazilian (Baccharis type) Prenylated p-coumaric acis, labdane diterpenes (15) Unidentified (15) Prenylated p-coumaric acids, clerodane diterpenes, benzofuranes (15) Prenylated p-coumaric acis, flavonoids, lignans, caffeoyl quinic acids (15) Prenylated p-coumaric acis, flavonoids (15) Not tested Cuban Prenylated benzophenones (17) Not tested Prenylated benzophenones (13) Unidentified (15) Prenylated benzophenones (13) Not tested Taiwanese Not tested Not tested Prenylated flavanones (42) Not tested Prenylated flavanones (42) Not tested Open in a separate window
The only exception seems to be the allergenic property of European (poplar type) propolis. This problem needs detailed investigations. Until now, no studies have been performed to find out if other propolis types have allergenic properties. It is very tempting to search for propolis that causes no contact allergy or causes it much less often.
The fact that different chemistry leads to the same type of activity and in some cases even to activity of the same order of magnitude is amazing. Nonetheless, it is important to have detailed and reliable comparative data on every type of biological activity, combined with chemical data, in order to decide if some specific areas of application of a particular propolis type can be formulated as preferable. The biological tests have to be performed with chemically well characterized and, if possible, chemically standardized propolis. Such detailed comparative investigations are a challenge to propolis researchers. The most important recent developments in propolis research are those which are aimed at meeting this particular challenge.
Chemical characterization and biological activity of six different extracts of propolis through conventional methods and supercritical extraction
The variation found between the samples studied and with those from other studies, including the significant differences between the samples for the humidity, water activity and lipids, can be explained by the type of propolis, the flora of the region and the period of collection [ 50 ].
The results for the lipid analysis showed that the red variety of propolis had 15.61%, which was 47.54 and 29.27% more lipid compared to the same analysis of the brown and green varieties, respectively. These values proved to be below the values found by Machado et al [ 5 ], for red propolis (65.74%) originating from Sergipe.
The protein content values found in the samples showed no significant difference ( Table 1 ). According to Bogdanov et al. [ 52 ], the content of protein in the composition that determines quality of the sample is above 0.7%. Therefore, compared with results from this work, the propolis samples were considered quality according to the literature.
The importance of the determination of total ash in propolis material were due to the possibility of commercialization in a powder form, where this analysis can identify any adulteration [ 50 ]. The samples agree with the limit established by Brazilian legislation (a maximum 5%) [ 51 ].
Concerning the results of total ash, the green and red propolis showed similar values compared with the brown propolis. The values found for the analysis of ash proved to be slightly lower that the values found by Machado et al. [ 5 ] for brown propolis from the state of Santa Catarina (1.73%).
In relation to the water activity, the samples demonstrated a value of 0.765% for red, 0.803% for green and 0.876% for brown propolis. The values are in agreement with the results of humidity, where the samples with higher humidity showed higher water activity . The water activity and the humidity are the parameters that permit the determination of conservation, microbial propagation and the occurrence of chemical reactions of the products [ 49 ].
The brown variety showed a humidity value of 8.03%, which was slightly out of the required standards for the humidity (a maximum of 8%) [ 26 ]. The green and red propolis were demonstrated to be within the standard required.
Analyzing the physicochemical composition of propolis is important for determining the quality of this material when it is considered for use in industrial areas, such as the food, cosmetics and pharmaceutics industries. The results of the physicochemical characterization of the three different raw propolis extracts analyzed in this study are found in Table 1 . Significant differences were found in the analyses of humidity, water activity and lipids.
Determination of content for phenolic compounds, flavonoids and antioxidant activity of ethanolic and supercritical extraction
The results for the phenolic, flavonoid analysis and antioxidant capacity of the extracts from different samples of propolis obtained through conventional (ethanolic) methods and supercritical extraction are found in Table 2. The results showed significant differences (p>0.05) for the extracts analyzed (Table 2) when comparing the extraction method for the same sample, as well as for the extracts obtained by the same method and samples of different types.
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TIFF original image Download: Table 2. Determination of the content of total phenolics (mg EAG/g), flavonoids (mg EQ/g) and the antioxidant activity by DPPH (IC 50 ) of extracts of three different samples obtained by ethanolic (EtOH) and supercritical (SFE) extraction.
The variations identified among the samples were already expected, considering that propolis of different types exhibit very different chemical profiles [51–52]. Furthermore, the method of extraction and solvent can change the chemical composition of propolis extract [53]. The results found in this study confirm the influence of the type and origin of the raw material [54], as well as the extraction method [55], in the composition and characteristics of the extracts. Serra Bonvehí and Ventura [56] investigated fifteen propolis samples from various botanic and geographic origins, verifying significant differences in their contents of polyphenols, flavonoids and active components.
The main chemical classes present in propolis are flavonoids, phenolics, and aromatic compounds [57]. The content of phenolic compounds varied from 113.41±0.01 (Brown SCO 2 ) to 481.59±0.02 mg EAG/g (Red EtOH), whereas the content of flavonoids varied from 29.67±0.01 (Brown EtOH) to 186.96±0.01 mg EQ/g (Red EtOH) among other samples, and the antioxidant capacity varied from 371.12±0.01 (Brown SCO 2 ) to 89.90±0.02 (Red EtOH) (IC 50 ).
For the major procedures analyzed, the ethanolic extraction yielded the best results. The ethanolic extraction of red propolis showed 48% more phenolic compounds compared to the brown propolis and 23.89% more than the green variety. Comparing the supercritical extractions with regards to phenolic compounds, the green propolis yielded 1.7% more compared to the red propolis and 34.9% more than the brown propolis.
The results found by Tei et al. [58], for five green propolis samples from Paraiba (Brazil) and five samples from Minas Gerais (Brazil) had 70.9% less phenolic compounds compared with the results found in this study.
Frozza et al. [59] demonstrated 68.53% less phenolic compounds in red propolis and Machado et al. [5], showed 13.61% less for brown propolis from Paraná (Brazil) extracted using the supercritical fluid extraction method.
The values identified in this study for the red and green samples appeared to be higher than the values found in the literature. These results are justified by the fact that the samples were from different origins [60].
Regarding the flavonoid analysis, the red propolis extracted by the ethanolic method indicated a difference of 84.13% more compared with the brown propolis extracted by the same method, while for the green propolis, the difference was 29.56%. Among the supercritical extracts, the total flavonoid content ranged from 1.24% (brown)– 6.23% (green) to red propolis.
The green sample tested in this present study had 64.46% more flavonoids compared with the results identify by Machado et al. [5] for green propolis originating from Minas Gerais extracted by the same method (Ethanolic extraction) and 74.17% more flavonoid compounds compared to the same sample extracted by supercritical extraction. Alencar et al. [61] also found lower values of flavonoid content for ethanolic extracts of red propolis from Sergipe.
Lower IC 50 values indicated a higher radical scavenging activity; the brown and green propolis extracted by conventional methods demonstrated 77.68% and 48.22% less antioxidant activity when compared with the red propolis, respectively. In respect to the supercritical method, the green propolis showed 86.10% less antioxidant activity in relation to the red type. Frozza et al [59] found an IC 50 value of 270.13 for red propolis from the northeast of Brazil, showing that the red propolis studied required less mass to inhibit 50% of DPPH radical formation.
Comparing the results presented in Table 2 in relation to the extraction method, it is possible to notice a significant difference (p>0.05) between the values for the phenolic, flavonoid and antioxidant activity (DPPH), where the ethanol extraction presented the best results between the samples and in the samples of different types. These results demonstrate the importance of the extraction method in the composition of the extract.
Similar results were observed by Zordi et al. [62], who determined that the highest concentrations of antioxidant compounds from ethanolic extracts of Italian propolis were obtained, when compared with the extracts obtained by the SFE process under different conditions and using SCO 2 . Machado et al., [5] and Silva et al. [40] also found higher values of total phenols and flavonoids in ethanolic extracts of Brazilian propolis, in relation to the supercritical extracts.
Considering the different types of processes used around the world to obtain propolis extracts, ethanol is the first choice of solvent, especially due to the affinity of its chemical characteristics with the matrix. Other solvents such as ethylic ether, water, methanol and chloroform can also be used for the extraction of specific classes of propolis constituents [62–63]. According to Biscaia et al. [64], low concentrations of flavonoid, phenolic, and antioxidant activity were shown in the extracts obtained by SFE (SO 2 ) and can be explained by the fact that unwanted substances such as resin, wax and other materials that are present in propolis in high concentrations can interfere with the biological potential of the extracts. The wax and other organic wastes are removed during the process of ethanolic extraction [65].
SFE extraction is currently an alternative to conventional processes, presenting numerous advantages. Although some studies show advantages in the use of SFE to obtain ecologically clean extracts and with greater biotechnological potential [17,66,67], in this study the conventional extraction was more efficient. Most polar phenolic compounds are practically insoluble in pure CO 2 , but are sufficiently soluble in a CO 2 +ethanol mixture or in a CO 2 +ethanol+water mixture, allowing for their separation on the basis of molecular weights and polarity. Monroy et al. [55] used green propolis from southeastern Brazil to obtain extracts concentrated in phenolic compounds using supercritical carbon dioxide as an anti-solvent to selectively fractionate ethanolic and hydroalcoholic extracts of green propolis by precipitation in four separators in series.
In general, red propolis presented the best levels of antioxidant compounds, regardless of the extraction method used. Red propolis has been classified as a separate type based on its unique chemical composition, particularly rich in isoflavonoids [68]. Furthermore, ethanolic extraction was more efficient to obtain extracts with higher antioxidant capacity. Extraction with ethanol is particularly suitable to obtain dewaxed propolis extracts rich in polyphenol components [14,39].
Hatano et al. [69] also studied the red propolis (from Shandong–China). Extracts obtained by ethanolic extraction showed strong antioxidant activity. The total polyphenol content, the flavonoid content, DPPH radical scavenging activity values were 433.8 mg.g-1 of extract, 129.6 mg.g-1 of extract, and 98.8%, respectively.
