eCAM 2007;Page 1 of 4
doi:10.1093/ecam/nem059
Original Article
Chemical Composition and Botanical Origin of Red Propolis,a New Type of Brazilian Propolis
Bruno B. Silva
1
, Pedro L. Rosalen
1
, Jaime A. Cury
1
, Masaharu Ikegaki
2
, Vinı´ cius C. Souza
3
, Alessandro Esteves
4
and Severino M. Alencar
3
1
Piracicaba Dentistry School (FOP/UNICAMP), Department of Physiologic Science, C.P. 52; Zip-code: 13414-903,Piracicaba, SP,
2
Federal University of Alfenas; Zip-code 37130-000, Alfenas, MG,
3
College of Agriculture ‘Luiz deQueiroz’ (ESALQ/USP), C.P. 9; ZIP-CODE: 13418-900, Piracicaba, SP and
4
Apia´ rios Almar e Essenciale LTDA,Maceio´ , AL, Brazil
Red propolis is a new type of Brazilian propolis. This material, as well as the secretions of 20plant species that are often mentioned as its probable botanical source, have been investigatedby RP-HPTLC. Phytochemical evidence based on UV-VIS spectra, RP-HPLC and GC-MS,showed
Dalbergia ecastophyllum
(L.) Taub. to be the main source of red propolis in Alagoasstate. The propolis and plant resin showed high relative percentages of the isoflavonoids 3-Hydroxy-8,9-dimethoxypterocarpan and medicarpin. To our knowledge this is the first reportof the secretion of a leguminous species being the source of propolis.
Keywords:
Dalbergia ecastophyllum
–isoflavonoids–plant resin–red propolis
Introduction
Brazilian propolis is a non-toxic resinous substance,collected from plant buds or exudates by
Apis mellifera
bees, which was classified into 12 types accordingto physicochemical properties and related to geographiclocations; however, the botanical origin of only three typeswere identified (1). The main botanical originof the Brazilian propolis types 3 (Southern), 6 (North-eastern) and 12 (Southeastern), has been reported to beresins from
Populus
sp.,
Hyptis divaricata
and
Baccharisdracunculifolia
, respectively (1). Brazilian propolis isquite diverse in chemical composition, due to Brazil’srich biodiversity, which needs to be investigated as asource of new bioactive substances, such as cinnamicacid derivatives (2), chiefly artepillin C (3), flavonoids (4)and others with pharmacological or functional properties.A new type of propolis, named Brazilian red propolis(BRP) because of its color, as yet not classified, wasfound in Maceio City (Alagoas state, Northeastern Brazil)and has attracted the attention of international business. Sofar, this unique propolis has not been found elsewhere inBrazil.The best approach to finding a plant source of propolis would be by an investigation to compare itschemical composition with that of the supposed plantsource and by demonstrating that the source of thispropolis is a plant resin, as in the case of red Brazilianpropolis.Thus, the main objective of this study was to identifythe plant source and chemical composition of the newBrazilian red propolis, by exploratory phytochemicalanalysis of the microflora of resins produced in themangrove area.
Material and Methods
Plant Propolis and Resin
Apis mellifera
bees from the mangrove region inMarechal Deodoro (a city in the vicinity of Maceio,capital of Alagoas State, in Northeastern Brazil, wet
For reprints and all correspondence: Pedro L. Rosalen, PiracicabaDentistry School (FOP/UNICAMP), Department of PhysiologicScience, C.P. 52; Zip-code: 13414-903, Piracicaba, SP, Brazil. Tel:
þ
55-19-2106-5313; Fax:
þ
55-19-2106-5250; E-mail: rosalen@fop.unicamp.br
ß
2007 The Author(s).
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 work isproperly cited.
eCAM Advance Access published July 7, 2007
tropical climate, SL 09.40 and WL 35.41) were visuallymonitored and photographically recorded to observetheir behavior and vegetational preference in order todetermine which plant was visited to collect resin forpropolis production. Twenty plant samples (
Acrostichumaureum, Anacardium occidentale, Avicennia Germinans,Avicennia schaueriana
,
Borreria verticillata L. Byrsonimacrassifolia, Byrsonima verbascifolia, Canavalia obtusifolia,Cnidosculus urens L. Conocarpus erectus, Cyperus liguralisL. Dalbergia ecastophyllum
,
Hibiscus pernambucencis,Hibiscus titiaceus
,
Ipomoea prescapae, Lagunculariaracemosa, Paspalum vaginatum, Passiflora subrotundaMart. Remirea maritima, Rhizophora mangle)
werecollected after monitoring the bee visits. Propolis producedin the mangrove area was sampled from a collector insidea hive, and samples from all parts of vegetation visitedby bees were collected. Plant material taxonomy of thebotanical source of propolis was performed in the depart-ment of Biological Science of College of Agriculture ‘‘Luizde Queiroz’’ (University of Sa ˜o Paulo) and a voucherspecimen (reference number ESA 96543) was deposited inthe herbarium of Biological Science of College of Agriculture ‘Luiz de Queiroz’ (University of Sao Paulo),Piracicaba, SP, Brazil.
Propolis Extraction
Propolis was ground to a fine powder and 2g (dry weight)were mixed with 25ml of 80% (v/v) ethanol and shakenat 70
C for 30min. After extraction, the mixture wascentrifuged and the supernatant was evaporated under lowpressure to produce the ethanolic extract of propolis (EEP),which was prepared at 1% (p/v) with ethanol 80% (v/v).
Resin Extraction
Resin was extracted immediately after it was removed fromthe plant shoot surface with a knife. A 10mg of sample (dryweight) of the resin was mixed with 1ml of 80% (v/v)ethanol to prepare the ethanolic extract of resin (EER).Both EEP and EER were used for chemical analysis.
UV-VIS Spectra
UV-VIS spectra of the EEP and EER samples wererecorded from mixture of 25
m
l of each extract plus 30mlof96%ethanol.Themixturewasscannedat200–500nmbyUV-spectrophotometer (UVMini 1240, Shimadzu Co.).
Reversed Phase–High Performance Thin LayerChromatography (RP–HPTLC)
Precoated silica gel plates RP-18 F
254
S were purchasedfrom Merck Co. Six
m
L of EEP and EER were applied tothe lower edge of the plate, and ascendingchromatography was run using a mobile phase of ethanol:water (55:45, by v/v). After development,the chromatograms were observed under UV lightat 366nm.
Reversed-Phase High Performance LiquidChromatography (RP–HPLC)
EEP and EER were performed by RP-HPLC using achromatograph equipped with a Shimadzu ODS-Acolumn (RP-18, column size 4.6
Â
250mm; particle size,5
m
m) and photodiode array detector (SPD-M10AVp,Shimadzu Co.). EEP and EER were filtered with 0.22
m
mfilter (Millipore) prior to 20
m
l injected into the HPLCsystem. The column was eluted by using a linear gradientof water (solvent A) and methanol (solvent B), startingwith 40% B and increasing to 60% B (45min), held at90% B (45–75min), and decreasing to 30% B (75–85min)with a solvent flow rate of 1ml/min and detection with adiode array detector. Chromatograms were recorded at260nm as described by Park
et al
. (5). The followingauthentic standards of phenolic acids and flavonoids(Extrasynthese Co.) were examined:
p
-coumaric, ferulicacid, cinnamic acid, gallic acid, quercetin, kaempferol,kaempferide, apigenin, isorhamnetin, rhamnetin, sakur-anetin, isosakuranetin, hesperidin, hesperetin, pinocem-brin, chrysin, acacetin, galangin, myricetin, tectochrysinand artepillin C.
Gas Chromatography–Mass Spectrometry (GC-MS)
The EEP and EER samples were chemically analyzedafter methylation of the extracts, as described byMarkham
et al
. (6). Samples of the methylated solutionswere analyzed by GC–MS by using a CP-Sil 8CB fused-silica capillary column (30m
Â
0.25mm; 0.25
m
m filmthickness) installed in a GC (Varian Saturn 2100D)instrument, interfaced to a Varian EM-AI mass selectivedetector, operated in scanning mode (
m/z
40–400). Thetemperature program employed was 50
C (0.3)–285
C(15min) at a rate of 6
C/min, with injection and detectortemperatures maintained at 280 and 290
C, respectively.The split ratio was 20:1 with 0.5
m
l of sample injected(EEP and EER). Carrier gas (He) was 1.0ml/min. TheGC–MS peaks were identified by comparison with datafrom literature (7) and the profiles from the Nist 98library.
Results
Botanical Origin of Red Propolis
Twenty samples of plants were checked for the possibilityof finding botanical origin and tested in our laboratoryto compare the chemical profiles of propolis (EEP)and plant resin (EER) in order to select an identical
2 of 4
Composition and botanical origin of Red Propolis
or similar profile. At first, only one plant showed anEER with profile similar to that of red propolis whencompared with RP-HPTLC plate. The plant, locally andpopularly identified as rabo-de-bugio (
monkey tail
), waslater identified as
Dalbergia ecastophyllum
(L.) Taub.Family: Fabaceae (Leguminosae).
Chemical Assays
The results of UV-VIS spectroscopy, HPLC (Fig. 1) andGC-MS (Table 1) demonstrated that the chemical profileof
Dalbergia ecastophyllum
was similar to the chemicalprofile of red propolis. According to GC-MS, the mainconstituents of both extracts are the isoflavononoidsmedicarpin and 3-Hydroxy-8,9-dimethoxypterocarpan,the latter representing more than 60% of the compositionin both extracts (Table 1).
Discussion
The chemical composition of propolis is variable depend-ing on the biodiversity and the geographical origin of thisnatural substance (5,8).In this study, a new type of Brazilian propolis, namedred propolis, collected from Northeastern Brazil ispresented. It has an intense red color and its chemicalcomposition differs from that of the 12 types of Brazilianpropolis classified by Park
et al
. (5).From the results of UV–VIS spectroscopy, HPLC(Fig. 1) and GC-MS (Table 1), we could observe thatnot only did EEP and EER present a similar spectralpattern in the region between 200–500nm, but also thesame wavelength of maximum absorption at 282nm. TheEEP and EER analysis using HPLC also demonstratedan identical chemical profile. In addition, we found thatthe two extracts had at least 12 chemical substances incommon, and in similar proportions (Fig. 1). Only twoflavonoids (quercetin and chrysin) and one phenolic acid(ferulic acid), identified as standard in the 12 types of Brazilian propolis classified by Park
et al
. (5), were foundin Brazilian red propolis. Absorption spectra obtainedwith a photodiode detector were used to compare anddistinguish peaks. According to the results of thechemical profile obtained with HPLC, it was possible tostate that this material proved to be a new type of Brazilian propolis.The presence of the isoflavononoids medicarpin and3-Hydroxy-8,9-dimethoxypterocarpan in both extracts isin agreement with Trusheva
et al
. (9) who also observedthe presence of isoflavonoids, such as isosativan andmedicarpin, in samples of Brazilian red propolis.The isoflavonoids are compounds typical of theleguminosae family. Thus, these compounds may beuseful as chemical markers of this new type of Brazilianpropolis. Several isoflavonoids have already been foundin
Dalbergia ecastophyllum
, among which are medicarpin
0 10 20 30 40 50 60 70025050075010000250500750100012501500124563789101211 13020040060080010001200020040060080010001200124578910136113
EEPEER
1212501500
m A U m A U
Minutes0 10 20 30 40 50 60 70Minutes
m A U m A U
Figure 1.
HPLC chromatograms of ethanolic extracts of red propolis (EEP) and
Dalbergia ecastophyllum
resin (EER).
1
, Fereulic acid;
2
, UV
238,276nm;
3
, Quercetin;
4
, UV
239, 246nm;
5
, UV
237, 279nm;
6
, UV
281, 284nm;
7
, UV
237, 279nm;
8
, UV
243, 320nm;
9
, UV
232,261nm;
10
, UV
246, 265nm;
11
, Chrysin;
12
, UV
246, 324nm;
13
, UV
246, 265nm.
Table 1.
Relative percentages of compounds, determined by GC/MS,from ethanolic extracts of red propolis and
Dalbergia ecastophyllum
resin; compounds are referred to by their respective names or massspectra data (molecular ion and base peak)RetentiontimeCompounds Relative%Plant Resin Propolis20.24 208 [M
þ
], 193 [M-15]
þ
0.89 2.4731.62 254 [M
þ
], 223 [M-31]
þ
0.71 0.8035.64 284 [M
þ
], 253 [M-31]
þ
1.91 6.6236.10 Medicarpin 22.20 6.1237.33 3-Hydroxy-8,9-dimethoxypterocarpan 61.58 67.5937.80 330 [M
þ
], 300 [M-30]
þ
1.55 2.2538.00 330 [M
þ
], 300 [M-30]
þ
isomer 0.68 0.8138.32 330 [M
þ
], 298 [M-32]
þ
4.01 7.9338.53 330 [M
þ
], 298 [M-32]
þ
isomer 1.03 0.83
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Red Bee Propolis - Brazilian Red Propolis - The Botanical Origin
This is the botanical orgin of Red Bee Propolis, discovery by Alessandro Esteves Founder COO of NaturaNectar™ and Apicola Almar Ltda
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