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Brazilian Red Propolis
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Monday, 30.05.2011, 02:38pm (GMT+1)
Advance Access Publication 7 July 2007 | eCAM 2008;5(4)435–441 |
doi:10.1093/ecam/nem057 | |
Original Article |
Brazilian Red Propolis—Chemical Composition and Botanical Origin
Andreas Daugsch, Cleber S. Moraes, Patricia Fort and Yong K. Park
Department of Food Science, College of Food Engineering, State University of Campinas, PO Box 6177, Campinas, SP, Brazil
Propoliscontainsresinoussubstancescollectedbyhoneybeesfromvariousplantsourcesandhasbeenusedasatraditionalfolkmedicinesinceca300BC.Nowadays,theuseofevidence-basedcomplementaryandalternativemedicine(CAM)isincreasingrapidlyandsoistheuseofpropolisinordertotreatorsupportthetreatmentofvariousdiseases.MuchattentionhasbeenfocusedonpropolisfromPopulussp.(Salicaceae)andBaccharisdracunculifolia(Asteracea),butscientificinformationaboutthenumerousothertypesofpropolisisstillsparse.WegatheredsixsamplesofredpropolisinfivestatesofNortheasternBrazil.Thebeehiveswerelocatednearwoodyperennialshrubsalongtheseaandrivershores.ThebeeswereobservedtocollectredresinousexudatesonDalbergiaecastophyllum(L)Taub.(Leguminosae)tomakepropolis.Theflavonoidsofpropolisandredresinousexudateswereinvestigatedusingreversed-phasehigh-performanceliquidchromatographyandreversed-phasehigh-performancethin-layerchromatography.WeconcludethatthebotanicaloriginofthereddishpropolisisD.ecastophyllum.Inareaswherethissource(D.ecastophyllum)wasscarceormissing,beeswerecollectingresinousmaterialfromotherplants.Propolis,whichcontainedthechemicalconstituentsfromthemainbotanicalorigin,showedhigherantimicrobialactivity.
Keywords: Apis mellifera – botanical origin – Dalbergia ecastophyllum –flavonoids–propolis
Introduction
Propolisisaresinousmixtureofsubstancescollectedbyhoneybees(Apismellifera)fromvariousplantsources.Itisusedbythebeese.g.tosealholesintheirhoneycombsandprotectthehiveentrance(1,2).Duetoitslargevarietyofbiologicalactivities,ithasbeensuccessfullyusedinbalsamandointmentstotreatbattlewounds(3).Ithasbeenusedasatraditionalfolkmedicinesinceca300BC.Numerousbiologicalproper-tieshavebeenreportedincludingcytotoxic(4),antiherpes(5),antitumor(6),radicalscavenging(7),antimicrobial(8),antifungal(9),anti-HIV(10)andsuppressiveeffectsofdioxintoxicity(11).Asaresultofthiswiderangeof
For reprints and all correspondence: Yong K. Park, Department of Food Science, College of Food Engineering, State University of Campinas, PO Box 6177, Campinas, SP, Brazil. Tel.: þ55-19-3521-2157; Fax: þ55-19-3521-2153; E-mail: ykpark@fea.unicamp.br
� 2007 The Author(s).
biological activities, propolis is now increasingly being used as a health food supplement and in beverages (12).
Previously, we reported on the classification of Brazilian propolis into 12 groups, based on physiochemical characteristics, five in southern Brazil, one in southeastern Brazil and six in northeastern Brazil. It was also reported that the main botanical origin of propolis group 3 was the bud resin of Populus (Salicaceae). The botanical origin of propolis group 6 and 12 were resinous coatings from young leaves of Hyptis divaricata (Lamiaceae) and Baccharis dracunculifolia (Asteracea), respectively (13).
Propolis normally is a dark yellow or brownish resinous material. Recently, we found reddish propolis in beehives located along the sea and river shores in northeastern Brazil.
Previously, Trusheva et al. (14) reported bioactive constituents of Brazilian red propolis, but they did not
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original workis properly cited.
Brazilian Red Propolis
describe the botanical origin of red propolis. We observed that bees kept in that area were collecting the reddish exudates on the surface of Dalbergia ecastophyllum (L) Taub. (15,16), it was assumed that this was the botanical origin of the reddish propolis. We therefore analyzed comparatively samples of the plant exudates as well as of this special propolis.
Materials and Methods
Propolis and its Botanical Origin
As indicated in the introduction, the reddish propolis was collected from beehives located in woody perennial shrubs along the sea and river shores in the states of Bahia, Sergipe, Alagoas, Pernambuco, and Paraı´ ba in northeastern Brazil. The red resinous exudates secreted from a hole in a branch of D. ecastophyllum that was made by tree-boring-insects as shown in Fig. 1A. It was observed that the bees visited mainly D. ecastophyllum to collect the resinous exudates on its surface and from holes in its branches (Fig. 1B). The resin issued from these holes was collected and then passed to the hind leg (Fig. 1C). Samples of the red exudates (Fig. 1A and B) were collected for analysis and compared with samples of propolis collected from a beehive that was located in the same area. The red resinous exudates were dissolved in 80% ethanol. In the case of propolis, approximately 50 g of the red propolis were collected from one beehive that was located in the same area. We collected six same samples of red resinous exudates from the botanical origin and six samples of red propolis from respective states to examine the quality of the propolis.
Preparation of Ethanolic Extracts of Red Resinous Exudates and Propolis
500mgofredresinousexudatesweremixedwith5mlof80%ethanolandthemixtureswereshakenfor10minat70C.Aftercentrifugation,thesupernatantwasusedforanalysis.Propolissamples(50g)werefrozeninafreezerandthenimmediatelygroundedtoafinepowderwithablender.Then,2gofthepowderweremixedwith25mlof80%ethanolandshakenat70Cfor30min.Afterextraction,themixtureswerecentrifugedandthesupernatantsusedforanalysis.
Reversed-Phase High-Performance Thin-Layer Chromatography (RPHPTLC)
Portionsof3mloftheethanolicextractsofpropolisandresinousexudatessolutionswereplatedonpre-coatedplatesofsilicagelRP-18F254SforRPHPTLCpurchasedfromMerckCo.andwerechromatographedusingethanol/water (55:45, v/v) as solvent. The detection of flavonoids was carried out using UV-visualization at 366 nm.
Reversed-Phase High-Performance Liquid Chromatography (RPHPLC)
Analysis of flavonoids and other phenolic compounds from ethanolic extracts of propolis and red resinous
eCAM 2008;5(4)
exudates were performed by RPHPLC with a chromatograph equipped with an YMC Pack ODS-A column (RP-18, column size 4.6 250mm;particlesize5mm)andphotodiodearraydetector(SPD-M10A,ShimadzuCo.).Thecolumnwaselutedbyusingalineargradientofwater(solventA)andmethanol(solventB),startingwith30%B(0–15min)andincreasingto90%B(15–75min),heldat90%B(75–95min)anddecreasingto30%B(95–105min)withasolventflowrateof1ml/minanddetectionwithadiodearraydetector.Chromatogramswererecordedat268nm.TheauthenticstandardsofflavonoidsandotherchemicalcompoundswerepurchasedfromExtrasyntheseCo.France.
Antimicrobial Activity of Ethanolic Extracts of Propolis and Resinous Exudates
ExaminationofantimicrobialactivityofpropolistoStaphylococcusaureusATCC25923wasdeterminedaccordingtothemethoddescribedin(17).ActivelygrowingnutrientbrothculturesofS.aureuswereinoculatedinnutrientagarplateswithsterileswabs,whichweredippedinbrothculture.Ontheinoculatedplate,diskswithextractsofpropoliswereplacedandincubatedovernightat37C.Theextractsofpropolisandresinousexudateswerepreparedbysubmerging10mLintoWhatmanfilterpaperno.3disks(51mm)anddriedunderlowvacuumatroomtemperatureovernightandthenincubatingat60Cfor4h.
Results
Red Propolis and Its Botanical Origin
We observed that bees were collecting the red resinous exudates on surfaces of D. ecastophyllum to produce propolis as shown in Fig. 1. The samples of both propolis and resinous exudates were analyzed by RPHPTLC and RPHPLC.
RPHPTLC (Fig. 2) revealed that chromatographic profiles of propolis (Fig. 2A) showed the same profile as the red resinous exudates (Fig. 2B and C) from the
Brazilian Red Propolis
Table 1. Flavonoids and other chemical constituents of propolis and D. ecastophyllum
PropolisyD.ecastophyllumy | ||||
---|---|---|---|---|
Retention | Content | Content | ||
Peak | time (min) | Compound | (mg/g) | (mg/g) |
- 1 13.42 Rutin 0.7 1.3
- 2 16.99 Liquiritigenin 1.8 7.1
- 3 20.63 Daidzein 0.3 4.3
- 4 22.35 Pinobanksin 1.7 6.0
- 523.84UV�251,292nmz
þþ
- 6 24.59 Quercetin 0.5 1.9
- 7 28.40 Luteolin 1.2 2.1
- 8 30.46 UV � 241, 272,
þþ
282nmz
9 32.15 Dalbergin 0.4 0.9
10 34.62 Isoliquiritigenin 4.8 12.1
11 36.97 Formononetin 10.2 19.5
1239.28UV�235,263nmz
þþ
13 40.08 Pinocembrin 3.3 7.1
14 42.30 Pinobanksin-3-acetate 1.7 2.6
15 46.45 Biochanin A 0.5 1.5
16 55.96 UV � 238, 260,
þþ
269nmz
17 60.53 UV � 233, 249,
þþ
329nmz
1863.43UV�233,256nmz
þþ
yQuantityofconstituentsinmg/gofpropolisandD.ecastophyllum.Symbols:‘þ’meanspresent,butnotquantified.zUnidentifiedconstituentsrepresentonlyUVspectralabsorptionmaximum.
surface of D. ecastophyllum. These results suggested that
D. ecastophyllum is the botanical origin of the red propolis. Furthermore, these results were confirmed by RPHPLC as shown in Fig. 3.
Figure 3 showed profiles of qualitative and quantitative comparisons of the flavonoids and other chemical constituents in propolis and resinous exudates from
D. ecastophyllum. The chemical constituents were quantified by RPHPLC. Identification of the chemical compounds was carried out by direct comparison with authentic standards and was based on retention time, co-chromatography and on the identity of the absorption spectra. The profiles of Fig. 3 and Table 1 indicated that the chromatographic profiles of propolis were exactly the same as those of D. ecastophyllum. These results clearly indicated the botanical origin of the propolis (see quantitative comparisons of flavonoids and other chemical constituents in Table 1). We also collected six further samples of red propolis in respective states. All states showed the similar results and here we demonstrate the results of Alagoas state in Fig. 4. Figure 4C showed the same redness of ethanolic extracts. But the degree of redness is variable, for instance the redness of samples one and six showed a weaker redness than the others. According to Fig. 4B propolis samples two, three, four and five showed nearly identical profiles. But sample one and six appeared weaker. Finally, Fig. 4A demonstrated RPHPLC with similar characteristics in sample two, three, four and five and similar chemical constituents and quantitative amounts (Table 2). It indicates that the botanical origin of these samples is the same. But samples one and six demonstrated similar chemical constituents in extremely lower concentrations than in samples two,
eCAM 2008;5(4)
Table 2. Flavonoids and other chemical constituents of red propolis
Propolis2yPropolis3yPropolis4yPropolis5yPeakRetentiontime(min)CompoundContent(mg/g)Content(mg/g)Content(mg/g)Content(mg/g)
- 1 13.42 Rutin 0.7 1.1 0.9 0.6
- 2 16.99 Liquiritigenin 1.8 5.7 5.0 2.2
- 3 20.63 Daidzein 0.3 0.6 0.5 0.2
- 4 22.35 Pinobanksin 1.7 5.7 4.9 3.3
- 523.84UV�251,292nmz
þþþþ
- 6 24.59 Quercetin 0.5 3.0 4.5 2.6
- 7 28.40 Luteolin 1.2 2.5 2.4 1.5
- 830.46UV�241,272,282nmz
þþþþ
9 32.15 Dalbergin 0.4 2.4 2.5 1.4
10 34.62 Isoliquiritigenin 4.8 9.9 8.3 5.5
11 36.97 Formononetin 10.2 10.7 10.9 10.5
1239.28UV�235,263nmz
þþþþ
13 40.08 Pinocembrin 3.3 9.8 8.3 7.3
14 42.30 Pinobanksin-3-acetate 1.7 3.6 2.4 2.3
15 46.45 Biochanin A 0.5 2.1 1.7 0.4
1655.96UV�238,260,269nmz
þþþþ
1760.53UV�233,249,329nmz
þþþþ
1863.43UV�233,256nmz
þþþþ
yQuantityofconstituentsinmg/gofpropolisandD.ecastophyllum.Symbols:‘þ’meanspresent,butnotquantified.zUnidentifiedconstituentsrepresentonlyUVspectralabsorptionmaximum.
three, four and five. On the other hand other chemical compounds were shown (Retention time 80–100 min). These are probably from other botanical origins.
Antimicrobial activity
Antimicrobial activities of six samples of propolis to Staphylococcus aureus ATCC 25923 were measured according to the method described above and the results are shown in Figure 5. Samples two, three, four and five demonstrated the highest inhibition of bacterial growth as compared with samples one and six, which contained lower concentrations of the chemical constituents of
D. ecastophyllum, but also contained chemical constituents from other plants.
Discussion
As described in previous publications (13,18), Brazilian propolis has been classified into 12 groups by physicochemical characteristics. Among these 12 groups of propolis, three (group 3, 6 and 12) were sufficiently We found that Brazilian red propolis contained liquirobserved to determine which plant bud and unexpanded itigenin, daidzein, dalbergin, isoliquiritigenin, formonoleaves the bees visited to collect the resins. Recently, we netin and biochanin A. Three of them (daidzein, found reddish propolis from beehives which were located formononetin and biochanin A) are isoflavonoids. along the sea and river shores in Northeastern Brazil. However, previously it was reported that Cuban red
Brazilian Red Propolis
propolis also contained isoflavonoids (19). The isoflavonoids are a very restricted distribution in the plant kingdom and occur almost exclusively in Leguminosae family such as soybeans, chickpeas and lentils (19). It is interesting that the presence of the isoflavonoids in D. ecastophyllum was found by Donnelly et al. (15). It is well known that dietary consumption of food and food additives containing isoflavonoids has been associated with a variety of health benefits including relief of symptoms of menopause e.g. osteoporosis, hormonal cancer and prostate cancer.
It was already reported that the extracts of South American D. ecastophyllum (Leguminosae), contained liquiritigenin, daidzein, dalbergin, isoliquiritigenin, formononetin and biochanin A (15). Moreover isoliquiritigenin inhibits the growth of prostate cancer (20), whereas liquiritigenin and isoliquiritigenin inhibit xanthine oxidase. Inhibition of xanthine oxidase has been suggested for the treatment of hepatitis and brain tumor because it increased the serum xanthine oxidase levels (21).
Samples one and six also showed the presence of these compounds, but in quantitatively lower concentrations and showed some unidentified peaks that were not found in D. ecastophyllum exudates. We observed that the samples of propolis one and six were collected from beehives, which were located in areas where
D. ecastophyllum was scarce, so that the bees collected from other plants. Therefore, we intend to investigate further the botanical origin of the red propolis which rarely demonstrated unknown constituents in the next project.
Conclusions
Majority samples of red propolis, which were collected from beehives located near woody perennial shrubs along the sea and river shores in Northeastern Brazil (six samples from each State), were analyzed. We observed that bees were collecting the red resinous exudates from surface of D. ecastophyllum to produce propolis. All samples of propolis and red resinous exudates showed very similar profiles of RPHPTLC and RPHPLC.
Therefore, the main botanical origin of the propolis is
D. ecastophyllum. But samples of propolis collected from beehives in areas where D. ecastophyllum was scarce, showed lower concentrations of the chemical constituents found in D. ecastophyllum, instead other chemical compounds appeared (retention time 80–100 min) that were not found in D. ecastophyllum. Consequently, the propolis demonstrated a lower antimicrobial activity. This means that the bees collected resins from different plants to produce propolis. Therefore, the botanical origin and its abundance are essential for the production of this type of propolis.
Acknowledgements
WethankMrEdivaldoPacheco(Apia´rioEdimel,Joa˜oPessoa,Paraı´ba,Brazil)andMrJose´AlexandreAbreu(PharmanectarLtd,BeloHorizonte,MinasGerais,Brazil)forthecollectionofDalbergiaecastophyllumandMrI.B.LimafortheidentificationofDalbergiaecastophyllum.AvoucherofD.ecastophyllum(JPB34951)iskeptintheherbariumofUFPB(FederalUniversityofParaı´ba,Brazil).WealsothankDr
E. Wollenweber of the Institut fu¨ r Botanik, Technische Hochschule Darmstadt, Germany for providing authentic standards. This research was supported by CNPq and CAPES, Brazilian government.
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Received October 17, 2006; accepted December 7, 2006
AC
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