Bioactive Constituents of Brazilian Red Propolis

Evidence-Based Complementary and Alternative Medicine, Sep 2018

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).

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Bioactive Constituents of Brazilian Red Propolis

Bioactive Constituents of Brazilian Red Propolis Boryana Trusheva 2 Milena Popova 2 Vassya Bankova 2 Svetlana Simova 2 Maria Cristina Marcucci 1 Patricia Laguna Miorin 1 Flavia da Rocha Pasin 1 Iva Tsvetkova 0 0 Institute of Microbiology, Bulgarian Academy of Sciences , Acad. G. Bonchev Street, Building 26, 1113 Sofia , Bulgaria 1 Faculdade de Farmacia, Universidade Bandeirante de Sao Paulo , Rua Maria Candida, 1813, Vila Guilherme, Campus MC, 02071-013 Sao Paulo, SP Brazil 2 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences , 1113 Sofia , Bulgaria 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). The Author (2006). Published by Oxford University Press. All rights reserved. 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 mm 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: transanethol 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-2butenyl)-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, a-amyrin 8, b-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, [a]D 0 (c 0.2, acetone). MS (EI, 70 eV), m/z (rel. int. %): 242, Mþ. (28), 227 (M-15)þ (85), 213 (M29)þ(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 ), [a]D 0 (c 0.27, chloroform). (6aS,11aS)-Medicarpin 13. UV, EIMS, 1H- and 13C-NMR spectra identical with literature data ( 17 ), [a]D þ184 (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 WFþ (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 ml) of 0.1 mM ethanolic DPPH solution was mixed with 40 ml 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 a-amyrin 8, b-amyrin 9, cycloartenol 10 and lupeol 11. The most abundant among them was b-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)-1,4naphthalenedione 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. 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. Acknowledgments The authors wish to thank Mrs Diana Nikolova for running GC-MS analyses. 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Boryana Trusheva, Milena Popova, Vassya Bankova, Svetlana Simova, Maria Cristina Marcucci, Patricia Laguna Miorin, Flavia da Rocha Pasin, Iva Tsvetkova. Bioactive Constituents of Brazilian Red Propolis, Evidence-Based Complementary and Alternative Medicine, DOI: 10.1093/ecam/nel006