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.
Keywords: propolis – plant origin – bioactive compounds – composition/activity relationship

Introduction

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.
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.

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 Table 1: 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 Compounds responsible for the biological activity of different propolis types
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

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.

Important Trends and Developments in Recent Propolis Research

Biological Studies Performed with Chemically Characterized Samples
More and more publications are appearing which combine antimicrobial and other biological studies with chemical analyses of the tested propolis samples. The most often used techniques for chemical analyses are gas chromatography– mass spectrometry (GC–MS) (18–24) and high-performance liquid chromatography (HPLC) (25–27). In a recent work, qualitative chemical characterization of the samples tested for antibacterial activity was combined with quantification of the major groups of biologically active substances of the corresponding samples (28). The use of chemically characterized propolis samples for biological experiments is the only meaningful way to study the biological and pharmacological activities of bee glue at the beginning of the third millennium.
Bioassay-guided Studies of Active Principles
Studies based on bioassay-guided chemical analysis represent a very promising trend in propolis research. Using this approach, Chen et al. isolated two new cytotoxic prenylflavones from Taiwanese propolis (29). Both compounds demonstrated cytotoxic properties on three cancer cell lines and also were potential radical scavengers – radicals of 1, 1 diphenyl-2-picrylhydrazyl (DPPH). Banskota et al. (30) isolated the active components from Netherlands propolis with antiproliferative activity in cancer cell lines: caffeic acid phenethyl ester (CAPE) and several analogs, including two new glyceryl esters of substituted cinnamic acids. The same compounds were found by Nagaoka et al. (31) to be responsible for the nitric oxide-inhibiting activity of Netherlands propolis. Usia et al. (32) isolated from Chinese propolis a number of compounds with antiproliferative activity. Most of them were known ‘poplar propolis’ constituents, but among them were two new flavonoids: 2-methylbutyrouylpinopbanskin and 6-cinnamylchrysin. From Greek propolis, the new flavanone derivative 7-prenylpinocembrin has been isolated, together with totarol and 7-prenylistrobopinin, as important antibacterial principles (33).
Banskota et al. (34) studied Brazilian propolis in order to identify the substances with hepatoprotective activity and those active against Helicobacter pylori. They found that these activities were due mainly to phenolic components, but diterpenic acids also contributed to hepatoprotective activity. Several anti-HIV compounds, derivatives of moronic acid, and a new triterpenoid called melliferon were isolated from Brazilian bee glue (35). The major component of Cuban red propolis, the prenylated benzophenone nemorosone, was found to possess cytotoxic activity against several tumor cell lines and to have radical scavenging action (13).
Comparative Biological Studies of Propolis of Different Origin and Chemical Composition
Perhaps the most interesting trend in recent propolis research is the comparative study of biological properties of propolis from different geographic locations and different chemical composition. The number of this type of studies is as yet limited. Kujumgiev et al. (36) compared the antimicrobial (antibacterial, antifungal and antiviral) activity and chemical composition of propolis from diverse geographic origins. The results presented unambiguous proof that in spite of the great differences in the chemical composition of propolis from different geographic locations, all samples exhibit significant antibacterial and antifungal (and most of them antiviral) activity. This study clearly demonstrated that in different samples, different combinations of substances are essential for the biological activity of bee glue. Trying to develop this comparative approach, Popova et al. (37) searched for a statistically significant correlation between biological activity and geographic origin of propolis samples. Analysis of variance (ANOVA) was used to compare the antibacterial action of three groups of bee glue: European, Brazilian and Central American. The results showed that propolis from Europe and Brazil had similar activity despite the drastic differences in chemical composition. Their antibacterial activity was significantly higher than that of Central American propolis. The ANOVA was also applied to compare the toxicity of the same three propolis groups with Artemia salina (Crustaceae). In this case, there was no significant correlation between geographic origin and potential cytotoxicity. This demonstrates that the search for new promising cytotoxic compounds in new propolis sources is reasonable.
The cytotoxic, hepatoprotective and free radical scavenging activity of propolis from Brazil, Peru, The Netherlands and China was compared by Banksota et al. (38). They found that propolis from The Netherlands and China possessed the strongest cytotoxic activity; while almost all samples possessed significant hepatoprotective activity. The scavenging activity against DPPH free radicals of all samples was similar; only the Peruvian sample showed weaker scavenging activity.
Salomao et al. (39) compared the microbicidal activity of Brazilian and Bulgarian propolis and analyzed their chemical composition by High Temperature – High Resolution Gas Chromotography – Mass Spectrometry (HT-HRGC-MS), and found that although they were of totally distinct compositions, they were active against Trypanozoma cruzi and some pathogenic fungi.
The work of Kumazawa et al. (40) is an excellent example of this approach. The authors compared the antioxidant activity of propolis of various geographic origins (Argentina, Austria, Brazil, Bulgaria, Chile, China, Hungary, New Zealand, South Africa, Thailand, Ukraine, Uruguay, the USA and Uzbekistan) and combined these data with chemical analyses. Major constituents of the samples tested were identified by HPLC with photo-diode array and mass spectrometric detection. Seventeen phenolic compounds in 16 kinds of propolis were identified and quantified by HPLC. Propolis with strong antioxidant activity contained antioxidative compounds such as kaempferol and phenethyl caffeate. In the same way, antioxidant activities and chemical constituents of propolis from different regions of Japan were compared by the same research group (41). They concluded that strong antioxidant activity correlated with a high concentration of caffeic acid and phenethyl caffeate. In addition, propolis from Okinawa was found to have antioxidants not seen in propolis from other areas.
Following a similar model, Chen et al. (42) compared the radical scavenging activity, cytotoxic effects and apoptosis induction in human melanoma cells of Taiwanese propolis from different locations. Propolins (C-prenylated flavanones) in the samples were detected by HPLC, and the total phenolic content was determined by spectrophotometry. The high concentration of propolins was found to be essential for the apoptosis induction in human melanoma cells and for the antiradical properties.

Conclusion

Such comparative studies are extremely valuable with respect to propolis standardization and practical applications in therapy. It is our hope that in the near future their number is going to grow significantly. They will allow scientists to connect a particular chemical propolis type to a specific type of biological activity and formulate recommendations for the practitioners. This could help the general public to make more efficient use of the beneficial properties of propolis with respect to CAM.

References

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Received September 30, 2004; Revision received December 8, 2004. accepted December 24, 2004

Bioactive Constituents of Brazilian Red Propolis

Boryana Trusheva1, Milena Popova1, Vassya Bankova1, Svetlana Simova1, Maria Cristina Marcucci2, Patricia Laguna Miorin2, Flavia da Rocha Pasin2 and Iva Tsvetkova3
1 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences 1113 Sofia, Bulgaria, 2 Faculdade de Farmacia, Universidade Bandeirante de Sao Paulo Rua Maria Candida, 1813, Vila Guilherme, Campus MC, 02071-013 Sao Paulo, SP Brazil, and 3 Institute of Microbiology, Bulgarian Academy of Sciences Acad. G. Bonchev Street, Building 26, 1113 Sofia, Bulgaria

Abstract

In a new propolis type, red Brazilian propolis, 14 compounds were identified (six of them new for propolis), among them simple phenolics, triterepenoids, isoflavonoids, prenylated benzophenones and a naphthoquinone epoxide (isolated for the first time from a natural source). Three of the major components demonstrated significant antimicrobial activity, and two (obtained as inseparable mixture) possessed radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH).
Keywords: antibacterial activity – chemical constituents – propolis – radical scavenging activity

Introduction

Propolis (bee glue) has a long history of being used as a remedy, dating back to the times of ancient Greece and Rome. Nowadays, it is still used for the treatment of various diseases, and in products like ‘health foods’, ‘biocosmetics’, etc., because of its versatile biological activities (1). Tropical propolis samples, and especially Brazilian ones, have shown significant differences in their chemical composition to propolis from temperate zone (2,3). For this reason, Brazilian bee glue has recently become a subject of increasing interest for scientists (4–7). It was found that propolis from different regions of Brazil display different chemical composition, depending on the local flora at the site of collection. Park et al. (8) have specified 12 types of Brazilian propolis according to its geographical origin, chemical composition and source plant. The most popular and well studied Brazilian propolis is the so-called green or Alecrim propolis, which originates from Baccharis dracunculifolia (Asteraceae) (9–12). Till now, no chemical data have been published on red propolis from Brazil. In Brazil, red propolis is collected in the North regions. Red colored propolis is reported to be typical for Cuba, where its plant source was identified as Clusia nemorosa (Clusiaceae) (13), and for Venezuela, where bees collect it from Clusia scrobiculata (14). In this study, we report our results on antibacterial and antioxidant activity of chemical constituents of red Brazilian propolis.

Materials and Methods

Nuclear magnetic resonance (NMR) spectra were measured on a Bruker AVANCE 250 MNR spectrometer; mass-spectra were measured on a Hewlett Packard 5972 mass spectrometer system.
Propolis. Propolis was collected near Maceio city, Alagoas State, Brazil.
Extraction of propolis. Propolis (61 g) was cut into small pieces and extracted with 70% ethanol (1 : 10, w/v) at room temperature for 24 h. The ethanol extract was concentrated in vacuo and extracted successively with petrol ether (40–60°C) three times. The petrol ether extract was evaporated to give 5 g dry residue after evaporation.
Isolation of compounds. The petrol ether extract was subjected to column chromatography on silica gel (300 g) with an n-hexane/acetone gradient (1 : 0.05/1 : 0.4) to produce 20 fractions (I–XX).
Fraction I (300 mg) was rechromatographed on a silica gel column eluted with n-hexane/diethyl ether gradient (1 : 0.01/1 : 1). The first fraction of this column, I.1 (12 mg), was subjected to gas chromatography-mass spectrometry (GC-MS) analysis with a Hewlett Packard Gas Chromatograph 5890 Series II Plus linked to Hewlett Packard 5972 mass spectrometer system equipped with a 23 m long, 0.25 mm id, 0.5 µm film thickness HP5-MS capillary column. The temperature was programmed from 100 to 310°C at a rate of 5°C.min–1. Helium was used as a carrier gas, flow rate 0.7 ml min–1, split ratio 1 : 80, injector temperature 280°C, ionization voltage 70 eV. Using computer searches on a NIST98 MS data library, the following compounds were identified in the mixture: trans-anethol 1 (13%), methyl eugenol 2 (14%), trans-methyl isoeugenol 3 (18%), elemicin 4 (26%) and trans-isoelemicin 5 (11%).
The second fraction I.2 (37 mg), after additional separation by preparative thin layer chromatography (TLC) (silica gel, n-hexane/ethyl methyl ketone 1 : 0.06) yielded 20(29)-lupen-3-one 6 (6 mg) and 2,3-epoxy-2-(3-methyl-2-butenyl)-1,4-naphthalenedione 7 (8.6 mg).
Fraction II (300 mg) was rechromatographed on a silica gel column eluted with n-hexane/diethyl ether gradient (1 : 0.01/1 : 1). After further purification by preparative TLC (silica gel, n-hexane/ethyl methyl ketone 1 : 0.06), a mixture of triterpenic alcohols (36 mg) (1H-NMR) was obtained. This mixture was analyzed after silylation, using the above mentioned GC-MS apparatus and the same analysis conditions as with fraction I.1. Using computer searches on a NIST98 MS data library, -amyrin 8, ß-amyrin 9 (identity also confirmed by comparison with an authentic sample), cycloartenol 10 and lupeol 11 were identified.
Fraction VIII (241 mg) was rechromatographed on a silica gel column, mobile phase n-hexane/acetone gradient (1 : 0.05/1 : 0.8). Additional purification by preparative TLC (silica gel, toluene/acetone 1 : 0.2) yielded isosativan 12 (40.8 mg).
Fraction X (454 mg) was rechromatographed on a silica gel column with mobile phase n-hexane/acetone gradient (1 : 0.05/1 : 1). Further purification by preparative TLC (n-hexane/ethyl methyl ketone 1 : 0.1) led to the isolation of 11.8 mg medicarpin 13.
Fraction XIII (620 mg) was rechromatographed on a silica gel column with mobile phase n-hexane/acetone gradient (n-hexane/acetone 1 : 0.01/1 : 1) to yield 20.5 mg of an inseparable mixture of guttiferone E 14 and xanthochymol 15.
20(29)-Lupen-3-one 6 was identified based on comparison of its EIMS, 1H- and 13C-NMR spectra and optical rotation with literature data (15).
2,3-Epoxy-2-(3-methyl-2-butenyl)-1,4-naphthalenedione 7, colorless oil, [ ]D 0 (c 0.2, acetone). MS (EI, 70 eV), m/z (rel. int. %): 242, M+. (28), 227 (M-15)+ (85), 213 (M-29)+(100), 196 (64), 171 (81), 105 (35%), 89 (36), 69 (30). HRMS (EI) m/z: 242.09553 (Calc. for C15H14O3 : 242.09430). For 1H-and 13C-NMR, see Table 1.

Isosativan 12, colorless crystals. UV, EIMS, 1H- and 13C-NMR spectra identical with literature data (16), [ ]D 0 (c 0.27, chloroform).
(6aS,11aS)-Medicarpin 13. UV, EIMS, 1H- and 13C-NMR spectra identical with literature data (17), [ ]D +184 (c 0.51, acetone).
Guttiferone E 14 and xanthochymol 15. The components of this inseparable mixture were identified by comparison of the spectral data for the same mixture published by Gustafson et al. (18) 1H- and 13C-NMR, MS (Fig. 1).

Antimicrobial tests. For the investigation of the antibacterial and antifungal activity, the agar cup method (19) was used with test strains Staphylococcus aureus 209 (obtained from the Bulgarian Type Culture Collection, Institute for State Control of Drugs, Sofia), Escherichia coli WF+ (obtained from the Collection of ZIMET, Central Institute of Microbiology and Experimental Therapy, Jena, Germany) and Candida albicans 562 (obtained from the Bulgarian Type Culture Collection, Institute for State Control of Drugs, Sofia). An inhibitory zone with a diameter <10 mm corresponds to lack of activity (10 mm is the diameter of the cup). The test solution (0.1 ml) containing 0.4 mg of each substance in ethanol was applied to every cup (concentration of the test solution 4 mg ml–1). Control experiments with solvents showed that solvents do not have any activity.
DPPH free radical scavenging activity. DPPH free radical scavenging activity was measured according to the procedure described in the literature (20). The decrease of the absorption at 516 nm of the DPPH solution after addition of the tested solution was measured. An aliquot (2960 µl) of 0.1 mM ethanolic DPPH solution was mixed with 40 µl of a 3.6 mM solution of the tested substance. The radical scavenging activity was expressed as percentage decrease with respect to control values. Caffeic acid was used as positive control.

Results and Discussion

The petrol ether fraction of the ethanol extract of the investigated propolis sample was subjected to column chromatography on silica gel and several fractions were produced. After further purification by repeated column chromatography and preparative TLC, two complex mixtures, one inseparable mixture of two isomers and four pure compounds were obtained.
The most unpolar fraction, isolated by repeated column chromatography, was of complex composition and was analyzed by GC-MS. It turned out to be composed of following phenylpropene derivatives: trans-anethol 1, methyl eugenol 2, trans-methyl isoeugenol 3, elemicin 4 and trans-isoelemicin 5. Elemicin was the most abundant. Of these compounds, methyl eugenol, methyl isoeugenol, elemicin and isoelemicin were found for the first time in propolis. The composition of this fraction also explains the very unusual anis-like odor of this Brazilian red propolis sample.
The second complex mixture (see Materials and Methods) was comprised of triterpenic alcohols, which were identified by means of GC-MS as -amyrin 8, ß-amyrin 9, cycloartenol 10 and lupeol 11. The most abundant among them was ß-amyrin. Triterpenic alcohols are typical for Brazilian propolis (2).
One of the pure compounds isolated was also of triterpenic nature: the ketone 20(29)-lupen-3-one 6 [identified by comparison of spectral information with literature data (13)], found for the first time in propolis. This compound has recently been found to possess antibiotic activity against bacteria and fungi, and antioxidant activity similar to that of tocopherol (15).
Compound 7 deserves special attention. Its structure was determined as 2,3-epoxy-2-(3-methyl-2-butenyl)-1,4-naphthalenedione on the basis of its MS, infrared, 1H- and 13C-NMR spectra. This is the first isolation of 7 from a natural source. Till now, it was known only as a synthetic product (21,22). The mass- and 1H-NMR spectra of our compound were identical with the literature data (no 13C-NMR data have been published). Compound 7, obtained synthetically from a natural product, demonstrated antibacterial, antifungal and cytotoxic properties (22).
Two isoflavonoids were isolated and identified: the isoflavan isosativan 12 and the pterocarpan medicarpin 13, based on comparison of their spectral properties with literature data (including absolute stereochemistry of 13, confirmed by optical rotation measurements; 12 was racemic). This is the first report of isoflavonoids in propolis other than Cuban. Compounds 12 and 13 were till now found only in Cuban propolis (16,17). This fact suggests that Cuban red propolis and Brazilian red propolis might have a common plant source, but a plant that produces isoflavonoids as components of its exudates is not yet known. The presence of isoflavonoids suggests some plant of the Leguminosae family but further studies are needed for confirmation. Especially 13 is of particular interest: it is an important plant phytoalexin well known for its antimicrobial and especially antifungal activity (23).
Compounds 14 and 15 are double bond isomers which occur as an inseparable mixture, but the structures were deduced by comparison of the spectral data of the mixture with the values for 14 and 15 from the literature (22–25). 1H- and 13C-NMR spectra of the mixture were virtually identical to those of 14 and 15 and all HMBC correlations were fully consistent with these structures. Moreover, Gustafson et al. (18) reported the isolation of the same inseparable mixture from Clusia rosea leaves as the active anti-HIV principle. These compounds have been detected as traces in red Cuban propolis (13) originating from C. rosea floral resins. In Cuban propolis nemorosone, another polyisoprenylated benzophenone, is the most important constituent (13). In our case, however, the mixture of 14 and 15 is among the major components of the extract. So the main plant source is most probably some other Clusia species. Moreover, the presence of isoflavonoids in our sample is an indication that another plant source could be involved, as isoflavonoids have never been found in the resins of Clusiaceae plants.
Three of the isolated compounds were tested for their antibacterial and radical scavenging activity against DPPH radicals. The results are represented in Table 2. The results indicated that the isoflavonoids 12 and 13 are important antimicrobial components of red propolis, especially concerning the activity against C. albicans. This is not surprising, taking into consideration that pterocarpans are known for their antifungal activity and play a defensive role in many plants due to this activity (26). The mixture of prenylated benzophenones 14/15 demonstrated good activity against S. aureus. The mixture showed also significant radical scavenging activity against DPPH, obviously it is one of the most important antioxidant components of the extract.

Table 2 Antimicrobial and antiradical activity of isolated compounds
Sample Antimicrobial activity inhibitory zone ± SD, mma
________________________________________ DPPH radical scavenging activity

________________________________________
S. aureus E. coli C. albicans % inhibition
________________________________________
12 14 ± 0 0 15 ± 1 4.5
13 23 ± 1 14 ± 0 26 ± 0 0.7
14/15 19 ± 1 12 ± 0 0 49
Caffeic acid – – – 85.6

aMean of three measurements.

The identification of new propolis constituents in red Brazilian propolis, most of them having antibacterial, antimycotic and antiradical activities, is a further confirmation of the fact that propolis, independently of its plant source and chemical composition, always possesses antimicrobial and antioxidant activity. This is due to the role that propolis plays in the hive: it is the ‘chemical weapon’ of bees against pathogen microorganisms and the elements of weather. However, in different propolis types, different chemical constituents are responsible for the valuable activities (27). The results obtained demonstrate once again that propolis remains a fascinating subject for further studies and application to CAM.

Footnotes

For reprints and all correspondence: Vassya Bankova, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. Tel: +359-2-9606149; Fax: +359-2-8700225; E-mail: bankova@orgchm.bas.bg

Acknowledgments

The authors wish to thank Mrs Diana Nikolova for running GC-MS analyses.

References

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14. Trusheva B, Popova M, Naydenski H, Tsvetkova I, Rodriguez JG, Bankova V. New polyisoprenylated benzophenones from Venezuelan propolis Fitoterapia 2004; 75: 683–9[CrossRef][ISI][Medline]
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Received November 10, 2005; accepted February 10, 2006

Origin and Chemical Variation of Brazilian Propolis

Antonio Salatino1,*, Érica Weinstein Teixeira2, Giuseppina Negri1 and Dejair Message3
1Department of Botany, Institute of Biosciences, University of São Paulo São Paulo, SP, Brazil, 2APTA (Agência Paulista de Tecnologia dos Agronegócios)/SAA-SP Pindamonhangaba, SP, Brazil, and 3Viçosa Federal University, Department of Animal Biology Viçosa, MG, Brazil
*For reprints and all correspondence: Antonio Salatino, University of São Paulo, Institute of Biosciences, Department of Botany, C. Postal. 11461, 05422-970, São Paulo, SP, Brazil. Tel.: +55 11 3091 7532; Fax: +55 11 3091 7416; E-mail: asalatin@ib.usp.br

Abstract

Propolis is a hive product containing chiefly beeswax and plant-derived substances such as resin and volatile compounds. Propolis has been used as an antiseptic and wound healer since ancient times and interest for the product has increased recently. Probably few plant species contribute as major resin sources. Green propolis derives mainly from vegetative apices of Baccharis dracunculifolia (alecrim plants). However, wide variation detected in the chemical composition suggests contributions from alternative resin plant sources. Predominant components of the resin of green propolis are cinnamic acids, chiefly compounds bearing prenyl groups. Terpenoid compounds, such as sesqui, di and pentacyclic triterpenoids, have been detected in many, but not all, samples investigated. Propolis research has uncovered potentialities of substances previously isolated from plants and has detected constituents of plant origin that would hardly be known otherwise.
Keywords: africanized Apis mellifera – Baccharis dracunculifolia – flavonoids – prenylated phenylpropanoids – propolis – terpenoids

Introduction

Propolis is a complex resinous bee product with a physical appearance that varies widely, depending on many factors. The color may be cream, yellow, green, light or dark brown. Some samples have a friable, hard texture, while other samples may be elastic and gummy. The word propolis is of Greek origin, stemming from pro, in defense of, and polis, city. It thus implies a product involved in the defense of the bee community. Bees use propolis for diverse purposes, among them to seal openings in the hive. In addition to avoiding the entrance of intruders, this contributes to maintaining the hive inner temperature at around 35°C. The walls of the comb hexagonal cells contain a mixture of beeswax and propolis. It is believed that propolis not only hardens the cell walls but also contributes to the attainment of an internal aseptic environment (1). The entrance to the hive is also lined internally with propolis. Evidence that leaves no doubt as to the anti-microbial properties of propolis comes from another use of the product in the hive: bees cover the carcasses of intruders that were killed and are too heavy to be thrown off the hive (e.g., small snakes) with propolis. The process comes close to an embalming effect, because the dead bodies dry out without undergoing putrefaction (1). This is obviously important to protect the hive from a widespread bacterial infection. Recognition of the antiseptic efficacy of propolis is ancient. Aristotle recommended the use of propolis to treat abscesses and wounds. Roman soldiers carried propolis as an emergency medicine for war wounds. A medicine containing vaseline and propolis (propolisin vasogen) was used for wound treatment (2) during the Boer war.
Propolis composition is extremely complex. The main constituents are beeswax, resin and volatiles. The insects secrete beeswax, while the latter two constituents are obtained from plants. But contrary to the well-known habit of visiting flowers for collection of nectar and pollen, bees usually take plant material for propolis from plant secretions or by cutting fragments of vegetative tissues (but see below comments about Venezuelan and Cuban propolis). The biological activity of propolis is assigned to these plant-derived substances. Hence, although propolis is obviously an animal product, a considerable proportion of its components, chiefly those upon which rest its biological activity, are plant derived. The resin contains most of the compounds found in alcohol extracts consumed by people from many countries as food complements or alternative medicine. Propolis contains other constituents, such as pollen and amino acids (3,4). In their reviews, Marcucci (5) and Bankova et al. (6) registered over 300 known substances in propolis. Reports in the last 10 years have added a great deal of other substances [e.g. Banskota et al. (7)].
In modern times, propolis started gaining appreciation as a means for the treatment of health problems in the 1950s and 1960s in the former Soviet Union and countries of Eastern Europe, such as Bulgaria, Czechoslovakia and Poland. Matsuno (2) mentions the use of propolis in these countries in cases of chronic medium and external otitis, pharyngitis, chronic rhinitis, amygdalitis and bronchial asthma, among other ailments, with satisfactory results. In Western European countries, in North and South America and in Japan, propolis did not acquire popularity until the 1980s. In the latter country, the first important announcement of propolis as a promising possibility in pharmacology occurred in 1985 (30th International Apiculture Congress, Nagoya). Up to that time, propolis was regarded by beekeepers as an unwanted hive by-product, since it had no market value and its production meant a decline in the amounts of honey obtained. Starting with a gradual rise in interest from people from several countries in the mid 1980s, propolis ended up as an important product in complementary and alternative medicine. Japan is the leading importer of propolis, with a manifest preference for propolis from Brazil.
Presently, many beekeepers in Brazil have, in propolis, their major product, and develop means to maximize its production. One of them is to leave longitudinal slits on both sides of the wooden box that shelters the bee colony. This method relies on the bee’s behavior of depositing propolis seals on all openings they detect in the hive (Fig. 1).

A great enthusiasm characterizes present-day propolis research, driven by positive results in pharmacological tests, dealing not only with anti-microbial activity, the first [Lavie (8)] and as yet the most investigated effect in propolis research, but also with a wide diversity of effects, including immune activation and cytotoxicity (9).
Besides pharmacological activity, an important point in propolis research refers to its botanical origin and the consequent variation in chemical composition when samples from different locations, and even from the same locality, are compared. Propolis samples produced in Europe and South America share anti-microbial, anti-viral, wound-healing, immune-stimulating, anti-inflammatory and anesthetic activities. However, similar as these samples might be in their biological activities, they are quite different chemically (10), because different plants in Europe and South America provide resin for propolis production in the two continents. In typical European propolis the major pharmacologically active constituents have long been identified as flavonoids, the most common and abundant being galangin (11). It is now well established that such a chemical profile is a consequence of the fact that in Europe bees collect propolis resin mainly from vegetative poplar (Populus nigra, Salicaceae) buds (12). In the tropics, poplars are seldom cultivated, so alternative plants are sources of propolis resin. For example, the flowers of Clusia minor in Venezuela (13) and of Clusia rosea in Cuba (14) produce resin, which bees collect for propolis production. In both cases, flavonoids are minor propolis constituents, the major compounds being polyprenylated benzophenones.
The present paper intends to put forward general comments about the plant origin of green propolis and its varying chemical composition.

Origin of Green Propolis

Among the propolis types produced in Brazil, green propolis has gained preference in the world propolis market. Typical green propolis is hard and friable, easily made into powder by mechanical milling. It exhales a pleasant resinous odor and the color ranges from greenish-yellow to deep green. Lack of records on propolis characteristics means that a comparison between the contemporary product and samples produced before 1960 in Brazil cannot be made. Such data could reveal possible influences of the genetic change that took place after the accidental escape of African Apis mellifera scutellata queen bees (15) from a laboratory in the State of São Paulo (southeast Brazil). All honeybees in Brazil are now africanized and presumably more productive than European bees with regard to propolis. The degree of introgression of the African genes varies according to geography, probably because of a gradual loss of European genes due to a better fitness of the African gene pool to the neotropical environment (16). Chemical affinities based on comb and propolis waxes in Brazil (17) probably reflect genetic differences linked to a higher or lower incidence of the African genes.
Suggestions have been made that probable sources of Brazilian propolis are Araucaria heterophylla, Clusia major, Clusia minor and species of Baccharis (7). Other possible sources of Brazilian propolis that have been suggested are Araucaria angustifolia, Baccharis dracunculifolia and Eucalyptus citriodora (18). Chemical evidence has suggested that some Baccharis species are resin sources for propolis from Botucatu (São Paulo State, southeast Brazil) (6).
Analyses of pollen and plant structures in propolis samples from cerrado (a savanna Brazilian ecosystem in central and southeastern Brazil) suggest that bees visit species of Baccharis, Vernonia, Diclenia, Hyptis, Myrcia, Schinus and Weinmania (19) for resin collection. While remains of vegetative structures may be given credit as consistent evidence for propolis origin, pollen analysis is dangerously misleading, because it is impossible to know with certainty whether a detected pollen is the result of a visit aimed at collecting resin or just a contamination of material obtained by bees for other purposes. Secretory hairs belonging to Baccharis dracunculifolia (called alecrim in some parts of Brazil) have been found in high quantities in both dry and rainy seasons (20). Alecrim is used in traditional medicine and corresponds to a dioecious shrubby member of the Asteraceae, widely distributed in open field ecosystems of southeast and south Brazil, spreading into Uruguay, Argentina, Paraguay and Bolivia (21). Identical prenylated cinnamic acids have been found in its leaves and in Brazilian propolis (22). A similar composition has also been observed between alecrim material and propolis from the state of São Paulo. Direct evidence for such a relationship has been provided (22), through observations of bee behavior and chemical analyses of alecrim leaves and propolis.
An attempt to classify propolis produced in Brazil according to botanical origin and chemical composition (23) recognized 12 types. Five of them correspond to southern, one to southeastern and six to northeastern regions of the country. It was suggested that Hyptis divaricata is the resin source of northeastern propolis, Baccharis dracunculifolia of southeastern propolis and poplar (Populus nigra) of southern propolis. This study by Park et al. (23) is indicative that just stating that a certain sample corresponds to ‘Brazilian propolis’ hardly means anything indicative of physical, chemical and biological characteristics, because a wide diversity of propolis types exist in a country as huge as Brazil, housing a wide plant diversity and a complex honeybee genetic variation. Most studies on ‘Brazilian propolis’ published in recent years have, in fact, dealt with the southeastern type, classified as ‘group 12’ (green propolis), such selectivity by propolis researchers reflecting the above mentioned international market preference. Given the widespread occurrence of B.dracunculifolia not only in the southeast but also in the south of Brazil, claims that the origin of the southern propolis are poplar trees (23) may be an oversimplification of the real range of propolis types in that region. For example, chemical evidence led Bankova et al. (24) to suggest that Araucaria may be a resin source for samples of propolis from southern Brazil, based on the finding of labdane-type diterpenes in propolis from that region and the fact that natural populations of Araucaria angustifolia characterize Brazilian southern flora; however, those compounds were shown to be present also in leaves of B.dracunculifolia (25).
The characteristic green color of alecrim-propolis is a consequence of its botanical origin, because bees collect young chlorophyll-containing tissues, namely vegetative buds and unexpanded leaves of B.dracunculifolia. Such young leaves contain secretory hairs, probably with volatile and aromatic oils inside, hence the resinous aroma of the typical green propolis. Volatile oils have been studied from Baccharis species (26,27). A volatile oil is obtained from the leaves of B.dracunculifolia and is commercially marketed under the name of vassoura oil (28).
Not so long ago beekeepers and many researchers shared a view that bees in Brazil collected material from practically any abundant plant source in the neighborhood of the hive, be it eucalyptus, pine, sugar cane, cashew nut or orange trees. It was usually said that this or that product was a ‘eucalyptus propolis’ or ‘pine propolis’. Such an idea probably derived from the common observation that bees visit a wide diversity of plants seeking nectar and pollen. The accumulated experimental data on propolis origin have not given support to such assumptions. It seems that certain plants are in fact major providers of propolis resin, while many others do not fit this role. It is, however, premature to conclude that in Brazil a certain plant species is the sole resin source for a certain propolis type. Indeed the probability is higher that, depending on the availability of representatives in the field, plants of a certain species prevail to a higher or lower extent as resin sources, while other species may also contribute (sometimes substantially) material for propolis production. The latter possibility is coherent with observations of more or less strong deviations from an expected chemical profile for a propolis type, a circumstance frequently pervading studies of propolis chemistry.

Chemical Composition

This is not a full account of what has been done on the chemistry of green propolis, rather it is a general idea of the diversity of classes of secondary metabolites and the extent of variation that has been noted in green propolis composition. Since most analyses carried out with European propolis until the mid 1990s revealed that flavonoids predominated as resin constituents, researchers assumed that a similar picture was apt to prevail regarding Brazilian propolis, especially taking into consideration that such propolis samples were pharmacologically similar to the European counterparts. However, contrary to those expectations, the first detailed chemical analyses revealed quite distinct profiles for samples of Brazilian propolis. Prenylated phenylpropanoids were shown to be very common and abundant constituents in propolis from Brazil, mainly from the southeastern region. For example Banskota et al. (29) identified in a sample of ‘Brazilian propolis’ pharmacologically active prenylated cinnamic acid-derived compounds, four of them bearing a prenyl group making up a heterocyclic ring that gives rise to chromenes (Fig. 2A). Although no information was given as to the provenance of the propolis sample, investigation carried out in Japan with Brazilian propolis deal practically only with green propolis. Other cinnamic acid-derived compounds common in green propolis have one or two prenyl groups not involved with ring formation. Prenylated cinnamic acids turned out to be a salient chemical feature of green propolis. Among the non-chromene prenylated cinnamic acids of green propolis, artepillin C (Fig. 2B) has attracted great attention, not only for its antimicrobial (30) but also for its toxicity to tumor cells (31). Prenylated cinnamic acids may be present as esters, such as 3-prenyl-cinnamic acid allyl ester (32) (Fig. 2C). Despite not being major constituents, flavonoids do occur in green propolis, one frequent example being kaempferide (Fig. 2D), a compound shown to possess antioxidant activities (33). Interesting benzofurans (Fig. 2E), to our knowledge never detected in B.dracunculifolia, were obtained from Brazilian propolis and shown to possess cytotoxic properties (34).

Mono and sesquiterpenes are frequently detected in green propolis, accounting for its characteristic resinous odor and probably contributing to the antimicrobial activity. For example, farnesol (Fig. 3D) has been shown to affect the accumulation and polysaccharide content of Streptococcus mutans biofilms (35). Labdane-type diterpenes, several with hepatoprotective activity (25), such as isocupressic (Fig. 3A) and agathic acid, have been found in green propolis. Clerodane diterpenoids with anticancer activity have been isolated, such as 13-symphyoreticulic acid (36) (Fig. 3B). Non-volatile sesquiterpenes, such as dehydrocostus lactone, a compound shown to inhibit the killing function of cytotoxic T-lymphocytes (37), may also occur in green propolis (38). Triterpenoids, some with wide occurrence in plants, have been found in green propolis (38–40). Two novel esters of long chain fatty acids and the pentacyclic triterpenoid lupeol (procrims a and b, Fig. 3C) have also been obtained from green propolis (41). Notwithstanding botanical origin being a major factor affecting propolis chemistry, other aspects also seem to influence the product composition. Notable differences are often found between propolis samples, not only from distant but also from nearby, or even the same, locations. This holds either for European (42,43) or for green propolis (32,38,39), even restricting the analysis to samples of ‘typical’ green propolis. For example, 3-prenylcinnamic acid allyl ester (Fig. 2C) was only recently reported to be a major constituent of green propolis (32), despite the considerable number of previous analyses. Among samples of green propolis there seems to be a gradual variation in the proportion of mevalonate-derived substances (terpenoids, including sesqui, di and triterpenoids) and the typical shikimate-derived (phenolics, prenylated or not) compounds. For example, analyses of green propolis have detected pentacyclic triterpenoids in some samples (38,39) but not in others (32). The relative amount of triterpenoids may sometimes be evaluated by the physical appearance of the sample, which loses the hardness and depth of green, turning increasingly cream and pulverulent with increasing levels of triterpenoids and reduction in the amounts of the typical shikimate derivatives. A relatively high content of dehydrocostus lactone (a pharmacologically active sesquiterpenoid (37,44)] was found in a sample from the state of São Paulo, together with 14 pentacyclic triterpenoids, four of them unreported for propolis (39). Bauer-7-en-3-yl acetate (a triterpenoid) was found in this sample in relatively low amounts, but we found it to represent 7% of the weight of another sample in a region where green propolis predominates (unpublished results). Are such inconsistencies accounted for by behavioral shifts to alternative resin plant sources? Baccharis is a large genus, with many representative species in the Brazilian flora. Species of the genus other than B.dracunculifolia are likely providers of propolis resin, not to mention species of other plant groups, including Clusiaceae, which are known to be predominant resin sources in Venezuela and Cuba, as noted above.

Park et al. (20) verified that very young leaves of B.dracunculifolia had chemical composition similar to the vegetative buds of the same plant, but the composition of successively more expanded leaves showed an increasing deviation from the bud pattern. Could this developmental factor account for some of the differences in chemical composition between propolis samples of the same region?
It very likely that phenological differences in plant chemistry may be another important effect accounting for divergences in propolis composition. Another point that may be put forward is the behavioral factors linked to the random location of a different source. It is well known that a bee can sometimes find a suitable source of plant material and then return to the hive, where it performs a ‘dance’ by means of which their sister laborers are informed about the location of the new source.
Finally, what could be said about genetic factors? How strong is the likelihood that the degree of africanization introgression may affect preference of honeybees for B.dracunculifolia?

Concluding Remarks

A matter of great concern regarding the production and use of propolis is the variation of its chemical composition, which has motivated proposals for quality chemical control (45,46). It has been claimed that the inconsistency of chemical composition, provided the product houses active antimicrobial substances, is a favorable characteristic, from the point of view that it precludes the development of resistance by microrganisms. Hence, propolis should be viewed more appropriately as a complex natural resource for the control of microorganisms rather than as a source of potent antimicrobials (10). Such a view from the scientific community meets the expectation of a large proportion of the population of many western and oriental countries, who seek alternative, natural (and hence complex) means for nutrition and health care, while placing great distrust in potent and technologically developed pharmaceuticals containing pure compounds. However, propolis has been the subject of research aiming at the isolation of compounds and opening the possibility of development of sophisticated pharmaceutical products (9). One of the salient examples is artepillin C, a component of B.dracunculifolia and green propolis with strong anti-bacterial and anti-tumor activities. The next step in this line of research seems to be the laboratorial synthesis of the substances, starting from abundant and low-cost raw material, thus making their use economically feasible in pharmacy and medicine.
A quick glance at the wide diversity of propolis composition revealed in the last 10–15 years foretells much further research work and a distant horizon for the completion of the evaluation of the full potentiality of propolis chemistry and pharmacology. Propolis research has uncovered pharmacological potentialities of substances previously known as plant constituents but never evaluated before. On the other hand, were it not for the labor of so many insects, each collecting minute portions of tissues and incorporating them into propolis, many valuable plant substances would hardly be uncovered.

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