Advance Access Publication 7 July 2007 eCAM 20085(4)435441 [628727]

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
Propolis contains resinous substances collected by honey bees from various plant sources and
has been used as a traditional folk medicine since ca 300 BC. Nowadays, the use of evidence-
based complementary and alternative medicine (CAM) is increasing rapidly and so is the use ofpropolis in order to treat or support the treatment of various diseases. Much attention has beenfocused on propolis from Populus sp. (Salicaceae) and Baccharis dracunculifolia (Asteracea), but
scientific information about the numerous other types of propolis is still sparse. We gathered sixsamples of red propolis in five states of Northeastern Brazil. The beehives were located nearwoody perennial shrubs along the sea and river shores. The bees were observed to collect redresinous exudates on Dalbergia ecastophyllum (L) Taub . (Leguminosae) to make propolis.
The flavonoids of propolis and red resinous exudates were investigated using reversed-phase
high-performance liquid chromatography and reversed-phase high-performance thin-layerchromatography. We conclude that the botanical origin of the reddish propolis is D.
ecastophyllum . In areas where this source ( D. ecastophyllum ) was scarce or missing, bees were
collecting resinous material from other plants. Propolis, which contained the chemicalconstituents from the main botanical origin, showed higher antimicrobial activity.
Keywords: Apis mellifera – botanical origin – Dalbergia ecastophyllum – flavonoids – propolis
Introduction
Propolis is a resinous mixture of substances collected by
honey bees ( Apis mellifera ) from various plant sources.
It is used by the bees e.g. to seal holes in their
honeycombs and protect the hive entrance (1,2). Due to
its large variety of biological activities, it has beensuccessfully used in balsam and ointments to treat battlewounds (3). It has been used as a traditional folk
medicine since ca 300 BC. Numerous biological proper-
ties have been reported including cytotoxic (4), antiherpes(5), antitumor (6), radical scavenging (7), antimicrobial(8), antifungal (9), anti-HIV (10) and suppressive effects
of dioxin toxicity (11). As a result of this wide range ofbiological 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 physiochem-
ical characteristics, five in southern Brazil, one in
southeastern Brazil and six in northeastern Brazil.It was also reported that the main botanical origin ofpropolis group 3 was the bud resin of Populus
(Salicaceae). The botanical origin of propolis group 6and 12 were resinous coatings from young leaves of
Hyptis divaricata (Lamiaceae) and Baccharis dracunculi-
folia (Asteracea), respectively (13).
Propolis normally is a dark yellow or brownish
resinous material. Recently, we found reddish propolisin beehives located along the sea and river shores innortheastern Brazil.
Previously, Trusheva et al . (14) reported bioactive
constituents of Brazilian red propolis, but they did not
For reprints and all correspondence: Yong K. Park, Department of
Food Science, College of Food Engineering, State University ofCampinas, PO Box 6177, Campinas, SP, Brazil. Tel.: ț55-19-3521-2157;
Fax:ț55-19-3521-2153; E-mail: ykpark@fea.unicamp.br
/C2232007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.or g/
licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original wor ki s
properly cited.

describe the botanical origin of red propolis. We
observed that bees kept in that area were collecting thereddish exudates on the surface of Dalbergia ecastophyl-
lum (L) Taub . (15,16), it was assumed that this was the
botanical origin of the reddish propolis. We thereforeanalyzed comparatively samples of the plant exudates aswell 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 perennialshrubs along the sea and river shores in the states ofBahia, Sergipe, Alagoas, Pernambuco, and Paraı ´ba in
northeastern Brazil. The red resinous exudates secretedfrom a hole in a branch of D. ecastophyllum that was
made by tree-boring-insects as shown in Fig. 1A. It wasobserved that the bees visited mainly D. ecastophyllum to
collect the resinous exudates on its surface and fromholes in its branches (Fig. 1B). The resin issuedfrom these holes was collected and then passed tothe hind leg (Fig. 1C). Samples of the red exudates(Fig. 1A and B) were collected for analysis and comparedwith samples of propolis collected from a beehivethat was located in the same area. The red resinousexudates were dissolved in 80% ethanol. In the caseof propolis, approximately 50 g of the red propolis werecollected from one beehive that was located in the samearea. We collected six same samples of red resinousexudates from the botanical origin and six samples of redpropolis from respective states to examine the qualityof the propolis.
Preparation of Ethanolic Extracts of Red Resinous
Exudates and Propolis
500 mg of red resinous exudates were mixed with 5 ml of
80% ethanol and the mixtures were shaken for 10 minat 70
/C14C. After centrifugation, the supernatant was used
for analysis. Propolis samples ( /C2450 g) were frozen in a
freezer and then immediately grounded to a fine powderwith a blender. Then, 2 g of the powder were mixed with25 ml of 80% ethanol and shaken at 70
/C14C for 30 min.
After extraction, the mixtures were centrifuged and thesupernatants used for analysis.
Reversed-Phase High-Performance Thin-Layer
Chromatography (RPHPTLC)
Portions of 3 ml of the ethanolic extracts of propolis and
resinous exudates solutions were plated on pre-coatedplates of silica gel RP-18F
254S for RPHPTLC purchased
from Merck Co. and were chromatographed usingethanol/water (55:45, v/v) as solvent. The detection of
flavonoids was carried out using UV-visualizationat 366 nm.
Reversed-Phase High-Performance Liquid
Chromatography (RPHPLC)
Analysis of flavonoids and other phenolic compounds
from ethanolic extracts of propolis and red resinous
Figure 1. Collection of propolis from reddish resinous exudates of
D. ecastophyllum by africanized Apis mellifera .(A)Secrete reddish
exudates from a hole in a branch of the tree. (B)Bee is collecting the
reddish exudates. (C)The collected exudates passed to the hind leg to
make propolis.436 Brazilian Red Propolis

exudates were performed by RPHPLC with a chroma-
tograph equipped with an YMC Pack ODS-A column(RP-18, column size 4.6 /C2250 mm; particle size
5mm) and photodiode array detector (SPD-M10A,
Shimadzu Co.). The column was eluted by using a
linear gradient of water (solvent A) and methanol(solvent B), starting with 30% B (0–15 min) andincreasing to 90% B (15–75 min), held at 90% B(75–95 min) and decreasing to 30% B (95–105 min) witha solvent flow rate of 1 ml/min and detection with adiode array detector. Chromatograms were recorded at268 nm. The authentic standards of flavonoids andother chemical compounds were purchased from
Extrasynthese Co. France.Antimicrobial Activity of Ethanolic Extracts of Propolis
and Resinous Exudates
Examination of antimicrobial activity of propolis to
Staphylococcus aureus ATCC 25923 was determined
according to the method described in (17). Actively
growing nutrient broth cultures of S. aureus were
inoculated in nutrient agar plates with sterile swabs,which were dipped in broth culture. On the inoculated
plate, disks with extracts of propolis were placed and
incubated overnight at 37
/C14C. The extracts of propolis and
resinous exudates were prepared by submerging 10 mL
into Whatman filter paper no. 3 disks (5 /C21 mm) and
dried under low vacuum at room temperature overnightand then incubating at 60
/C14C for 4 h.
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 profileas the red resinous exudates (Fig. 2B and C) from the
Figure 2. RPHPTLC of the ethanolic extracts of propolis and reddish
exudates from D. ecastophyllum .3mL of respective solution described in
method were applied. (A)Ethanolic extracts of propolis; (B)Ethanolic
extracts of reddish exudates from sample of Fig. 1A; and (C)Ethanolic
extracts of reddish exudates from sample of Fig. 1B.
Figure 3. RPHPLC of ethanolic extracts of propolis and reddish
resinous exudates from D. ecastophyllum . Respective numbers of peak
represent chemical constituents that were described in Table 1.eCAM 2008;5(4) 437

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 withauthentic 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 chemi-cal 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 thesame 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, fourand five showed nearly identical profiles. But sample oneand six appeared weaker. Finally, Fig. 4A demonstrated
RPHPLC with similar characteristics in sample two,
three, four and five and similar chemical constituents andquantitative amounts (Table 2). It indicates that thebotanical origin of these samples is the same. But samples
one and six demonstrated similar chemical constituents in
extremely lower concentrations than in samples two,Table 1. Flavonoids and other chemical constituents of propolis
andD. ecastophyllum
PropolisyD. ecastophyllumy
PeakRetention
time (min) CompoundContent
(mg/g)Content
(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 6.05 23.84 UV /C21251, 292 nm
zțț
6 24.59 Quercetin 0.5 1.9
7 28.40 Luteolin 1.2 2.1
8 30.46 UV /C21241, 272,
282 nmzțț
9 32.15 Dalbergin 0.4 0.9
10 34.62 Isoliquiritigenin 4.8 12.111 36.97 Formononetin 10.2 19.512 39.28 UV /C21235, 263 nm
zțț
13 40.08 Pinocembrin 3.3 7.1
14 42.30 Pinobanksin-3-acetate 1.7 2.615 46.45 Biochanin A 0.5 1.5
16 55.96 UV /C21238, 260,
269 nm
zțț
17 60.53 UV /C21233, 249,
329 nmzțț
18 63.43 UV /C21233, 256 nmzțț
yQuantity of constituents in mg/g of propolis and D. ecastophyllum .
Symbols: ‘ ț’ means present, but not quantified.
zUnidentified constituents represent only UV spectral absorption
maximum.
Figure 4. RPHPLC and RPHPTLC of the ethanolic extracts of 6
samples of propolis (one sample from one beehive) collected in same
regions.438 Brazilian Red Propolis

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 resultsare shown in Figure 5. Samples two, three, four and fivedemonstrated the highest inhibition of bacterial growth
as compared with samples one and six, which contained
lower concentrations of the chemical constituents ofD. ecastophyllum , but also contained chemical constitu-
ents from other plants.
Discussion
As described in previous publications (13,18), Brazilianpropolis has been classified into 12 groups by physico-
chemical characteristics. Among these 12 groups of
propolis, three (group 3, 6 and 12) were sufficientlyobserved to determine which plant bud and unexpandedleaves the bees visited to collect the resins. Recently, wefound reddish propolis from beehives which were locatedalong the sea and river shores in Northeastern Brazil.We found that Brazilian red propolis contained liquir-
itigenin, daidzein, dalbergin, isoliquiritigenin, formono-netin and biochanin A. Three of them (daidzein,formononetin and biochanin A) are isoflavonoids.
However, previously it was reported that Cuban redTable 2. Flavonoids and other chemical constituents of red propolis
Propolis2yPropolis3yPropolis4yPropolis5y
Peak Retention time (min) Compound Content (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 2.23 20.63 Daidzein 0.3 0.6 0.5 0.2
4 22.35 Pinobanksin 1.7 5.7 4.9 3.3
5 23.84 UV /C21251, 292 nm
zțțțț
6 24.59 Quercetin 0.5 3.0 4.5 2.67 28.40 Luteolin 1.2 2.5 2.4 1.5
8 30.46 UV /C21241, 272, 282 nm
zțțțț
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
12 39.28 UV /C21235, 263 nmzțțțț
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 0.416 55.96 UV /C21238, 260, 269 nm
zțțțț
17 60.53 UV /C21233, 249, 329 nmzțțțț
18 63.43 UV /C21233, 256 nmzțțțț
yQuantity of constituents in mg/g of propolis and D. ecastophyllum .
Symbols: ‘ ț’ means present, but not quantified.
zUnidentified constituents represent only UV spectral absorption maximum.
Figure 5. Growth inhibition of Staphylococcus aureus ATCC 25923 by
extracts of propolis. 10 mL of respective solution described in method
were applied to the disks.eCAM 2008;5(4) 439

propolis also contained isoflavonoids (19). The isoflavo-
noids are a very restricted distribution in the plant
kingdom and occur almost exclusively in Leguminosae
family such as soybeans, chickpeas and lentils (19). It isinteresting 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 foodadditives containing isoflavonoids has been associatedwith 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, for-mononetin and biochanin A (15). Moreover isoliquiriti-genin inhibits the growth of prostate cancer (20), whereasliquiritigenin and isoliquiritigenin inhibit xanthine oxi-dase. 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 concentrationsand showed some unidentified peaks that were notfound in D. ecastophyllum exudates. We observed
that the samples of propolis one and six were collectedfrom beehives, which were located in areas whereD. ecastophyllum was scarce, so that the bees collected
from other plants. Therefore, we intend to investigate
further the botanical origin of the red propolis whichrarely demonstrated unknown constituents in the nextproject.
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 (sixsamples from each State), were analyzed. We observedthat bees were collecting the red resinous exudates fromsurface of D. ecastophyllum to produce propolis.
All samples of propolis and red resinous exudatesshowed 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 constituentsfound in D. ecastophyllum , instead other chemical
compounds appeared (retention time 80–100 min) thatwere not found in D. ecastophyllum . Consequently, the
propolis demonstrated a lower antimicrobial activity.
This means that the bees collected resins from differentplants to produce propolis. Therefore, the botanical
origin and its abundance are essential for the productionof this type of propolis.
Acknowledgements
We thank Mr Edivaldo Pacheco (Apia ´rio Edimel, Joa ˜o
Pessoa, Paraı ´ba, Brazil) and Mr Jose ´Alexandre Abreu
(Pharmanectar Ltd, Belo Horizonte, Minas Gerais,Brazil) for the collection of Dalbergia ecastophyllum and
Mr I. B. Lima for the identification of Dalbergia
ecastophyllum . A voucher of D. ecastophyllum
(JPB34951) is kept in the herbarium of UFPB (FederalUniversity of Paraı ´ba, Brazil). We also thank Dr
E. Wollenweber of the Institut fu ¨r Botanik, Technische
Hochschule Darmstadt, Germany for providing authenticstandards. This research was supported by CNPq andCAPES, Brazilian government.
References
1. Greenaway W, Scaysbrook T, Whatley FR. The composition and
plant origins of propolis. Bee World 1990;71:107–18.
2. Ghisalberti EL. Propolis: a review. Bee World 1979;60:59–84.
3. Thomsom WM. Propolis. Med J Aust 1990;143:654.
4. Matsuno T, Matsumoto Y, Saito N, Morikawa J. Isolation and
characterization of cytotoxic diterpenoid isomers from propolis.
Z Naturforsch 1997;52c:702–4.
5. Vynograd N, Vynograd I, Sosnowski Z. A comparative multicenter
study of the efficacy of propolis, acyclovir and placebo in thetreatment of genital herpes (HSV). Phytomedicine 2000;7:1–6.
6. Kimoto T, Arai S, Kohguchi M, Nomura Y, Micallef MJ,
Kurimoto M, et al. Apoptosis and suppression of tumor growth
by artepillin C extracted from Brazilian propolis. Cancer Detect
Prev 1998;22:506–15.
7. Basnet P, Matsuno T, Neidlein RZ. Potent free radical scavenging
activity of Propol isolated from Brazilian propolis. Z Naturforsch
1997;52c:828–33.
8. Park YK, Koo MH, Abreu JAS, Ikegaki M, Cury JA, Rosalen PL.
Antimicrobial activity of propolis on oral microorganisms. Curr
Microbiol 1998;36:24–8.
9. Ota C, Unterkircher C, Fantinato V, Shimizu MT. Antifungal
activity of propolis on different species of Candida .Mycoses
2001;44:375–8.
10. Ito J, Chang FR, Wang HK, Park YK, Ikegaki M, Kilgore N, et al.
Anti-AIDS agents. 48. Anti-HIV activity of moronic acid deriva-tives and the new mellifore-related triterpenoid isolated from
Brazilian propolis. J Nat Prod 2001;64:1278–81.
11. Park YK, Fukuda I, Ashida H, Nishiumi S, Yoshida K,
Daugsch A, et al. Suppressive effects of ethanolic extracts frompropolis and its main botanical origin on dioxin toxicity. J Agric
Food Chem 2005;53:10306–9.
12. Burdock GA. Review of the biological properties and toxicity of bee
propolis. Food Chem Toxicol. 1998;36:347–63.
13. Park YK, Alencar SM, Aguiar CL. Botanical origin and
chemical composition of Brazilian propolis. J Agric Food Chem
2002;50:2502–6.
14. Trusheva B, Popova M, Bankova V, Simova S, Marcucci MC,
Miorin PL, et al. Bioactive constituents of Brazilian Red Propolis.
ECAM 2006;3:249–54.
15. Donnelly DMX, Keenan PJ, Prendergast JP. Isoflavonoids of
Dalbergia ecastophyllum .Phytochemistry 1973;12:1157–61.
16. Matos FJA, Gottlieb OR, Andrade CHS. Flavonoids from
D. ecastophyllum .Phytochemistry 1975;14:825–6.440 Brazilian Red Propolis

17. Isla MI, Paredes-Guzman JF, Nieva-Moreno MI, Koo H, Park YK.
Some chemical composition and biological activity of NorthernArgentine propolis. J Agric Food Chem 2005;53:1166–72.
18. Park YK, Paredes-Guzman JF, Aguiar CL, Alencar SM,
Fujiwara FY. Chemical constituents in Baccharis dracunculifolia as
the main botanical origin of Southeastern Brazilian propolis.J Agric Food Chem 2004;52:1100–03.
19. Piccinelli AL, Fernandes MC, Cuesto-Rubio O, Herna ´ndez IM,
de Simone F, Rastrelli L. Isoflavonoids Isolated from CubanPropolis. J Agric Food Chem 2005;53:9010–16.20. Kanazawa M, Satomi Y, Mizutani Y, Ukimura O, Kawauchi A,
Sakai T, et al. Isoliquiritigenin inhibits the growth of prostate
cancer. European Urology 2003;43:580–6.
21. Kong LD, Zhang Y, Pan X, Tan RX, Cheng CHK.
Inhibition of xanthine oxidase by liquiritigenin and isoliquiritigenin
isolated from Sinofranchetia chinensis .Cell Mol Life Sci
2000;57:500–5.
Received October 17, 2006; accepted December 7, 2006eCAM 2008;5(4) 441

Submit your manuscripts at
http://www.hindawi.com
Stem Cells
International
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014M ED IATO R S
INFLAM MATIONof
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014Behavioural
Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014Disease Markers
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014BioMed
Research International
OncologyJournal of
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014Oxidative Medicine and
Cellular Longevity
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014PPAR Research
The Scientific
World Journal
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Immunology Research
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014Journal of
ObesityJournal of
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014 Computational and
Mathematical Methods
in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014Research and TreatmentAIDS
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014Gastroenterology
Research and Practice
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014Parkinson’s
Disease
Evidence-Based
Complementary and
Alternative Medicine
Volume 2014Hindawi Publishing Corporation
http://www.hindawi.com