versão impressa ISSN 0103-5053
J. Braz. Chem. Soc. v.11 n.5 São Paulo set./out. 2000
Comb and Propolis Waxes from Brazil (States of São Paulo and Paraná)
Giuseppina Negria, Maria Cristina Marcuccia, Antonio Salatinob* and Maria Luiza F. Salatinob
aUniversidade Bandeirante de São Paulo, Rua Maria Cândida, 1813, 02071-013, São Paulo - SP, Brazil
bInstituto de Biociências, Universidade de São Paulo, CP 11461, 05422-970, São Paulo - SP, Brazil
Amostras de ceras do ninho e da própolis de Apis mellifera foram analisadas. Observou-se a predominância de ésteres, seguidos de hidrocarbonetos. Os constituintes foram identificados por cromatografia a gás/espectrometria de massas. Amplas variações foram observadas nos padrões de hidrocarbonetos e dos ácidos e álcoois de ésteres. As cadeias carbônicas dos hidrocarbonetos abrangem a faixa C23 - C35, com o predomínio de C27 e C31. O principal ácido carboxílico foi C16:0, seguido de C18:0 e C18:1. Os principais álcoois constituintes de ésteres foram homólogos saturados normais, na faixa C24 - C32, C30 sendo o mais abundante, seguido de C24. Não foram observadas diferenças que permitam distinção, o que sugere uma origem comum para ambas as fontes de cera.
Samples of propolis and comb waxes of Apis mellifera were analyzed. Monoesters predominated, followed by hydrocarbons. The constituents were identified by gas chromatography/mass spectrometry. Wide variations in the patterns of hydrocarbons, acids and alcohols of the esters were found. Hydrocarbon chains cover the range C23 - C35, C27 and C31 alkanes predominating. The main carboxylic acid was C16:0, followed by C18:0 and C18:1. The alcohols were predominantly saturated n-homologues, ranging from C24 to C32, C30 being the most abundant, followed by C24. No differences were found to allow a distinction, suggesting a common origin for both wax sources.
Keywords: propolis wax, beeswax, Apis mellifera
Propolis is a complex mixture of waxes, resins and other organic and inorganic compounds used by bees as a general sealer, draught excluder and antibiotic1-3. Bees use propolis to prevent decomposition of creatures such as beetles and mice which they have killed after invasion of the hive4. Propolis derived products are widely used in folk medicine and reputedly have antibacterial, antimycotic, anti-inflammatory and other pharmacological properties1.
The term "waxes" is used to designate mixtures of long-chain non-polar compounds commonly found mainly on the surfaces of plants and animals5. Commercially, beeswax is the most important natural wax. It is obtained chiefly from the domesticated European honey bee Apis mellifera, although other important taxa exist, such as the Asiatic A. dorsata, A. florea and A. indica and the African A. mellifera adansonii. Aliphatic saturated and monounsaturated compounds are major comb wax constituents6,7. The composition of comb wax is dependent on the genetics of the insects8. European and African bees produce waxes with different hydrocarbon patterns9,10 and the process of bee africanization may be detected by analysis of the hydrocarbons encountered in bee products11. Recent analysis of comb wax using two-stage resolution of mixtures of heterogenous compounds by supercritical fluid chromatography12 revealed hydrocarbons, esters of higher alcohols and fatty acids and free higher fatty acids among the wax constituents.
Samples of propolis contain a whitish material which can be extracted by treatment with hot chloroform. This substance has a composition similar to comb wax13 and is apparently secreted by the bees. In comparison with comb wax, much less is known about the composition of propolis wax. Negri et al.13 observed that monoesters and hydrocarbons are the predominant constituents of propolis wax. Alkanes, alkenes, alkadienes, diesters, ketones and fatty acids, previously reported as propolis constituents14,15, are classes of compounds commonly found as natural wax substances.
In Brazil, there has been extensive hybridization between the European bees A. mellifera mellifera and A. mellifera ligustrica with the African bee A. mellifera adansonii (=A. m. scutellata) after the introduction of the latter in the 1950's16. Contemporarily, all honey bees found in Brazil are said to be Africanized. The present work presents data of propolis wax from Brazilian localities not included in reference 13. In addition, it includes information about comb wax, for the purpose of comparison between the composition of the waxes from both natural sources.
Results and Discussion
The contents of wax in the collected samples of comb and propolis are presented in Table 1. The values for the samples of propolis range from 4.8% to 19.3%. Apparently, there is no correlation between the percentages and sites of collection. The yields are relatively small compared with those of Bonvehí et al.17, who found values close to 30% for samples from China. The contents of wax in samples of comb collected in two cities of the state of São Paulo are much lower (1.5% and 3.0%) than the contents found in samples of propolis. Table 1 also presents the percentages of the constituent hydrocarbons and monoesters of the samples of comb and propolis waxes. In both sources, monoesters are clearly the predominant class of constituents, followed by hydrocarbons. Similar results were obtained by Negri et al.13 for samples of propolis waxes. There is no homogeneity in the results and no correlation with locality. The compositions of comb and propolis waxes are probably more dependent on the genetic characteristics of the bees than on the site of collection. In fact, different degrees of hybridization have been found to occur between European and African bees in Brazil16,18,19,20.
Table 2 shows the distribution of the hydrocarbon fraction of comb and propolis waxes. A wide variation in the hydrocarbon patterns among the samples is visible. Most samples of comb (1c, 2c and 4c), as well as propolis (2p, 5p, 6p, 7p, 8p and 9p), presented heptacosane as the main component. This alkane has been referred to as the main hydrocarbon of both comb wax9 and propolis wax13. The comb wax sample 3c and that of propolis 1p presented the alkene C33 as the main hydrocarbon. Other authors9,14,21 have reported the predominance of alkenes in comb wax. The main hydrocarbon of propolis samples 3p, 4p and 10p was C31. Occasionally, branched alkanes (iso-alkanes) were found in low amounts and exclusively in propolis waxes (samples 1p, 6p and 7p, Table 2). No correlation is apparent between hydrocarbon patterns and localities, contrary to what is known about the composition of the constituents of propolis resin, which is dependent on the local flora1. A possible explanation for differences in hydrocarbon patterns between colonies may lie in genetic factors8, 11, particularly in bee populations from Brazil, which are the result of different levels of hybridization (see Introduction). However, high levels of consistency within and among families of bees has been found by means of correlation analysis8, indicating structural constancy in comb wax. Analyses of surface hydrocarbons also indicated that a significant proportion of the variation among bees may be attributable to genetic factors22. Some insect surface compounds may also be important constituents of comb wax, as are the cases of the hydrocarbons C27 and C29 (but not C31) and the carboxylic acid C14. It is interesting to note that the latter acid has been found neither in samples of wax of Brazilian propolis analyzed by Negri et al.13 nor in the samples of the present work.
The need to hydrolyze the esters for identification of the constituent acid and alcohol residues is a shortcoming in the analysis of natural waxes, because the outcome is only a partial analysis of the product. Under suitable conditions it is possible to analyze intact high molecular weight esters9, 23, 24, 25. Novel techniques involving high temperature gas chromatography have enabled the direct analyses of seed triglycerides26 and propolis extracts27,28 without derivatization. In the present work the analysis of the ester fraction followed the conventional procedure of hydrolysis and derivatization prior to GC/MS analysis.
The distribution of the alcohols of monoesters covered the range C24-C32 in both sources of waxes 3) presented C24 as the main compound. For some samples (1c, 2c, 1p and 6p, Table 3) C32 was an important component. In most samples, however, it was a minor constituent of the ester fraction and, in some samples (2p, 7p and 10p), C32 was not detected.
Palmitic acid (C16) was the dominant homologue of the acyl portion of esters in all samples examined (Table 3), in agreement with previous findings about propolis wax13. Although no data were raised about chain lengths of intact esters in the present investigation, the fact that the main alcohol is in general C30 and the main acid is C16 suggests that triacontil palmitate (C46) predominates among the esters of the samples of propolis and comb wax investigated. Esters ranging from C40 to C50 were found to occur in comb waxes of A. mellifera mellifera and A. mellifera adansonii, the most abundant being C469. As in the cases of hydrocarbons and alcohols commented above, a wide variation of patterns of the acid portion of esters is observed in Table 3. For example, samples 1c, 3c, 3p, 5p and 10p presented exclusively palmitic acid. On the other hand, samples 4c and 7p yielded a long series of homologues ranging from C16 to C28; oleic acid is an important constituent in some samples (2c, 2p, 7p-9p), but a minor or undetected component in other samples.
In spite of the wide variation observed in the distribution of the constituents of all fractions analyzed, there is a remarkable similarity between the composition of propolis wax and comb wax. The resin and the volatile fractions of propolis are presumably largely derived from plant secretions collected by bees1. Since plants produce waxes that coat all aerial cutinized parts29, 30, the hypothesis could be raised that propolis wax might also be derived from plant secretions. But several differences can be pointed out between the composition of beeswax (as here reported) and plant waxes. For example, the latter rarely present alkenes and oleic acid as important hydrocarbon constituents29, 30, and esters may predominate in plant waxes, but not always. In contrast, monoesters always appeared consistently as the predominant class of propolis wax (Table 1).
Samples of propolis and comb waxes were collected from hives growing in the states of São Paulo and Paraná (Southeast and Southern Brazil, respectively) (Table 1).
Extraction of the waxes
Samples of propolis were extracted with chloroform in a Soxhlet extractor13. Amounts of comb ranging from 1.0 to 3.0 g were treated with boiling chloroform and filtered while still hot. The chloroform extracts were evaporated to dryness under reduced pressure and dried in a dessicator to constant weight (Table 1).
Separation and identification of constituent fractions
The fractions of constituents of propolis and comb waxes were separated by CC, using silicagel and a mixture of solvents of increasing polarity13, and TLC, using silicagel impregnated with sodium fluoresceine and developing with a mixture of hexane: chloroform (73:27)13. Functional characterization of the constituent classes was achieved by IR spectroscopy with a Perkin Elmer model FTIR spectrophotometer. The esters were hydrolyzed with methanolic KOH and the resultant acid and alcohol fractions separated by means of extraction with chloroform after neutralization with 10% HCl13. The acids were identified as the corresponding methyl esters and alcohols as the corresponding acetyl esters by GC/electron impact mass spectrometry on an HP model 5890 series II GC interfaced with an HP 5989B ChemStation mass spectrometer using conditions identical to those cited in reference 13.
The authors acknowledge financial support provided by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional do Desenvolvimento Científico e Tecnológico).
1. Marcucci, M. C. Apidologie 1995, 26, 83. [ Links ]
2. Marcucci, M. C. Quim. Nova 1996, 19, 529. [ Links ]
3. Greenaway, W.; May, J.; Scaysbrook, T.; Whatley, F. R. Z. Naturforsch. C: J. Biosci. 1991, 46, 111. [ Links ]
4. Brumfitt, W.; Hamilton-Miller, J. M. T.; Franklin, I. Microbios 1990, 62, 19. [ Links ]
5. Hamilton, R. J. In Waxes: Chemistry, Molecular Biology and Functions; Hamilton, J. R., Ed.; The Oily Press; Dundee, 1995, p 257. [ Links ]
6. Kolattukudy, P. E. Chemistry and Biochemistry of Natural Waxes; Elsevier; Amsterdam, 1976. [ Links ]
7. Brossard, S.; Lafosse, M.; Dreux, M.; Becart, J.; Tranchant, J. F. Parf. Cosmét. Arômes 1994, 117, 48. [ Links ]
8. Breed, M. D.; Page, R. E.; Hibbard, B. E.; Bjostad, L. B. J. Chem. Ecol. 1995, 21, 1329. [ Links ]
9. Tulloch, A. P. Bee World 1980, 61, 47. [ Links ]
10. Hepburn, H. R. Honey Bees and Wax; Springer-Verlag; Berlin, 1986. [ Links ]
11. Carlson, D. A. In Africanized Honey Bees and Bee Mites; Needham, G. R.; Page, R. E.; Delfinado-Baker, M.; Bowman, C. E., Eds; Ellis Horwood Ltd.; Chichester, 1988, p 264. [ Links ]
12. Takeuchi, M.; Saito, T. J. Chromatogr. 1996, 722, 317. [ Links ]
13. Negri, G.; Marcucci, M. C.; Salatino, A.; Salatino, M. L. F. Apidologie 1998, 29, 305. [ Links ]
14. Seifert, M.; Haslinger, E. Liebigs Ann. Chem. 1989, 1123. [ Links ]
15. Bankova, V.; Christov, R.; Stoev, G.; Popov, S. J. Chromatogr. 1992, 607, 150. [ Links ]
16. Lobo, J. A.; Del Lama, M. A.; Mestriner, M. A. Evolution 1989, 43, 794. [ Links ]
17. Bonvehí, J. S.; Coll, F. V. V.; Jordá, R. E. J. Am. Oil Chem. Soc. 1994, 71, 529. [ Links ]
18. Lobo, J. A.; Krieger, H. Heredity 1992, 68, 441. [ Links ]
19. Diniz-Filho, J. A. F.; Malaspina, O. Evolution 1995, 49, 1172. [ Links ]
20. Diniz-Filho, J. A. F. J. Apic. Res. 1996, 35, 104. [ Links ]
21. Seifert, M.; Haslinger, E. Liebigs Ann. Chem. 1991, 93. [ Links ]
22. Page, R. E.; Metcalf, R. A.; Metcalf, R. L.; Erickson, E. H.; Lampman, R. L. J. Chem. Ecol. 1991, 17, 745. [ Links ]
23. Gülz, P. G.; Müller, E. Z. Naturforsch. 1992, 47, 800. [ Links ]
24. Summchen, P.; Markstadter, C.; Wienhaus, O. Phytochemistry 1995, 40, 599. [ Links ]
25. Shepherd, T.; Robertson, G. W.; Griffiths, D. W.; Birch, A. N. E. Phytochemistry 1997, 46, 83. [ Links ]
26. Reiter, B.; Lechner, M.; Lorbeer, E.; Aichholz, R. J. High Resolut. Chromatogr. 1999, 22, 514. [ Links ]
27. Pereira, A. S.; Pinto, A. C.; Cardoso, J. N.; Aquino Neto, F. R. ; Ramos, M. F. S.; Dellamora-Ortiz, G. M.; Santos, E. P. J. High Resolut. Chromatogr. 1998, 21, 396. [ Links ]
28. Pereira, A. S.; Ramos, M. F. S.; Poças, E. S. C.; Dias, P. C. M.; Santos, E. P.; Silva, J. F.; Cardoso, J. M.; Aquino Neto, F. R. Z. Naturforsch. 1999, 54, 395. [ Links ]
29. Baker, E. A. In The Plant Cuticle; Cutler, D. F.; Alvin, K. L.; Price, C. E., Eds; Academic Press; New York, 1982, p 139. [ Links ]
30. Bianchi, G. In Waxes: Chemistry, Molecular Biology andFunctions; Hamilton, R. J., Ed.; The Oily Press; Dundee, 1995, p 175. [ Links ]
Received: April 08, 1999
Published on the web: August 31, 2000
FAPESP helped in meeting the publication costs of this article.