Indonesian propolis: Chemical composition, biological activity and botanical origin

Article abstract of DOI:10.1080/14786419.2010.488235

From a biologically active extract of Indonesian propolis from East Java, 11 compounds were isolated and identified: four alk(en)ylresorcinols (obtained as an inseparable mixture) (1-4) were isolated for the first time from propolis, along with four prenylflavanones (6-9) and three cycloartane-type triterpenes (5, 10 and 11). The structures of the components were elucidated based on their spectral properties. All prenylflavanones demonstrated significant radical scavenging activity against diphenylpicrylhydrazyl radicals, and compound 6 showed significant antibacterial activity against Staphylococcus aureus. For the first time Macaranga tanarius L. and Mangifera indica L. are shown as plant sources of Indonesian propolis.

Full text of DOI:10.1080/14786419.2010.488235

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Indonesian propolis: chemical
composition, biological activity and
botanical origin
Boryana Trusheva a , Milena Popova a , Eko Budi Koendhori b , Iva
Tsvetkova c , Christo Naydenski c & Vassya Bankova a
a Institute of Organic Chemistry with the Centre of
Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria
b Department of Microbiology, School of Medicine, Airlangga
University, Surabaya, Indonesia
c Institute of Microbiology, Bulgarian Academy of Sciences, Sofia,
Bulgaria
Version of record first published: 14 Mar 2011.
To cite this article: Boryana Trusheva , Milena Popova , Eko Budi Koendhori , Iva Tsvetkova , Christo
Naydenski & Vassya Bankova (2011): Indonesian propolis: chemical composition, biological activity
and botanical origin, Natural Product Research: Formerly Natural Product Letters, 25:6, 606-613
To link to this article: http://dx.doi.org/10.1080/14786419.2010.488235
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Natural Product Research
Vol. 25, No. 6, March 2011, 606–613
Indonesian propolis: chemical composition, biological activity and
botanical origin
a
a
b
c
*
Boryana Trusheva , Milena Popova , Eko Budi Koendhori , Iva Tsvetkova ,
Christo Naydenskic and Vassya Bankovaa
aInstitute of Organic Chemistry with the Centre of Phytochemistry, Bulgarian Academy of
Sciences, Sofia, Bulgaria; Department of Microbiology, School of Medicine, Airlangga
b
University, Surabaya, Indonesia; cInstitute of Microbiology, Bulgarian Academy of Sciences,
Sofia, Bulgaria
(Received 15 February 2010; final version received 18 April 2010)
From a biologically active extract of Indonesian propolis from East Java,
11 compounds were isolated and identified: four alk(en)ylresorcinols
(obtained as an inseparable mixture) (1–4) were isolated for the first time
from propolis, along with four prenylflavanones (6–9) and three
cycloartane-type triterpenes (5, 10 and 11). The structures of the compo-
nents were elucidated based on their spectral properties. All prenylflava-
nones demonstrated significant radical scavenging activity against
diphenylpicrylhydrazyl radicals, and compound 6 showed significant
antibacterial activity against Staphylococcus aureus. For the first time
Macaranga tanarius L. and Mangifera indica L. are shown as plant sources
of Indonesian propolis.
Keywords: propolis; alk(en)ylresorcinols; prenylflavanones; cycloartane
triterpenes; Macaranga tanarius; Mangifera indica
1. Introduction
Propolis (bee glue) is a sticky, dark-coloured material that honeybees collect from
living plants. They mix it with wax and use it in the construction and adaptation
of their nests, mainly to fill out cracks in the beehive and for defence against
pathogenic microorganisms. It has been reported that bee glue possesses valuable
biological activities such as antibacterial, antifungal, antiviral, antioxidant, anti-
inflammatory and anticancer activities, mainly due to its aromatic acids, phenolic
compounds and flavonoids (Awale et al., 2008; Gekker, Hu, Spivak, Lokensgard, &
Peterson, 2005; Kujumgiev et al., 1999; Lustosa, Galindo, Nunes, Randau, & Rolim
Neto, 2008; Scazzocchio, D’Auria, Alessandrini, & Pantanella, 2006). Owing to its
valuable qualities, propolis is extensively used in complementary and alternative
medicine, in food and beverages to improve health, and to prevent diseases such as
inflammation, heart disease, diabetes and cancer (Bankova, de Castro, & Marcucci,
2000).
*Corresponding author. Email: bobi_tru@orgchm.bas.bg
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2011 Taylor & Francis
DOI: 10.1080/14786419.2010.488235
http://www.informaworld.com

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A single propolis sample can contain more than 300 components, as the plant
source and respectively the chemistry of the bee glue depend on the specificity of the
local flora at the site of collection, which differs at different geographic locations
(Bankova, Trusheva, & Popova, 2008). This causes problems in the medicinal use
and standardisation of propolis, and generates the necessity of more thorough
investigations on the chemical composition, plant origin and biological activity of
propolis from different regions of the world, especially from places yet to be
investigated (Bankova, 2005). For this reason, tropical bee glue, in particular from
regions of Southeast Asia, has become the subject of increasing interest, concerning
its relatively unknown and highly varied chemistry and biological activity.
Indonesian propolis has received little attention to date and no data have been
published on its detailed chemical composition and botanical origin (Syamsudin,
Wiryowidagdo, Simanjuntak, & Heffen, 2009). In this article, we report our results
on the constituents and plant sources of Indonesian propolis from Java. The
antibacterial and radical scavenging activities of extracts and isolated compounds
were determined.
2. Results and discussion
The chloroform 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 PTLC one inseparable
mixture, one complex mixture and four individual compounds were obtained
(Figure 1). All of them are among the main sample components.
Figure 1. Components identified in Indonesian propolis.

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B. Trusheva et al.
The inseparable mixture was analysed by 1D and 2D NMR and GC–MS.
It contained the following alk(en)ylresorcinols: 5-pentadecylresorcinol (1), 5-(80Z,
110Z-heptadecadienyl)-resorcinol (2), 5-(110Z-heptadecenyl)-resorcinol (3) and
5-heptadecylresorcinol (4). 5-(80Z,110Z-Heptadecadienyl)-resorcinol (2) was the
most abundant. Other than the substances mentioned above, the mixture contained
some minor C15-, C17- and C19- substituted alk(en)ylresorcinols with different
degrees of unsaturation. Because they were present in a very small amounts they
remained unidentified. Isolated alk(en)ylresorcinols have recently been found
in mango (Mangifera indica L.) peel and proved to have antioxidant and very
strong cyclooxygenase (COX-1 and COX-2) inhibitory activity (Knoedler, Berardini,
Kammerer, Carle, & Schieber, 2007; Knoedler et al., 2008). Compounds 1–4 were
found for the first time in propolis.
The complex mixture was comprised of prenylflavanones, the flavonoid
compounds with geranyl side chains, which were identified by means of GC–MS
after silylation and comparison with authentic samples to be propolin C (7), propolin
F (8) and propolin G (9). The most abundant among them was propolin C (7). One
of the isolated pure compounds was also a prenylflavanone: propolin D (6).
Compounds 6–9 were found for the first time in Indonesian propolis, but these
constituents are very characteristic of Macaranga (pacific)-type propolis (Taiwanese
and Okinawan), originating from the resinous surface material on the fruits of the
tropical tree Macaranga tanarius L. (Chen et al., 2004; Kumazawa et al., 2004, 2008).
Prenylated flavanones are known to possess antibacterial, antioxidant and
antitumour activities (Shirataki et al., 2001).
Three individual cycloartane-type triterpenes were isolated and identified, based
on comparison of their spectral properties with literature data: mangiferolic acid (5),
isomangiferolic acid (10) and 27-hydroxyisomangiferolic acid (11). To date, the
cycloartane triterpenes have been isolated from Brazilian and Myanmar propolis
(Li et al., 2009; Silva et al., 2005).
Compounds 5, 10 and 11 and alk(en)ylresorcinols 1–4 are characteristic
components of M. indica and prenylflavanones of the fruits of M. tanarius
(Anjaneyulu et al., 1989; Knoedler et al., 2007, 2008; Kumazawa et al., 2008).
Moreover, both M. indica and M. tanarius are widely distributed in East Java, where
the propolis used in this study was collected. Thus, the plant sources of Indonesian
propolis from East Java are M. indica and M. tanarius, which distinguish it from the
already known pacific-type propolis (Macaranga type), containing only prenyl-
flavonoids (Popova, C. Chen, P. Chen, Huang, & Bankova, 2009).
The ethanol and chloroform extracts and isolated compounds were tested for their
antimicrobial and radical scavenging activities. The results are presented in Table 1.
No activity was found against C. albicans or E. coli (data not shown), similar to most
propolis samples of different origin and constituents (Kujumgiev et al., 1999). The
chloroform extract possessed good antibacterial activity against S. aureus, commen-
surable to that of the standard, due to the presence of alk(en)ylresorcinols 1–4 and
mainly to propolin D (6), which showed the highest antibacterial activity.
The ethanol and chloroform extracts demonstrated low RSA against DPPH
radicals (5.0% and 9.2%, respectively; Table 1), compared to the well-known
antioxidant caffeic acid (75.5%), used as a positive control, but the isolated
prenylflavanones 6–9 showed significant activity (more than 50% inhibition). Thus,
the main biologically active components of Indonesian propolis are prenylflavanones.

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Table 1. Antibacterial and RSA of extracts and isolated compounds.
Antibacterial activity against S. aureus
DPPH RSA
RSA (%)a
Sample
Inhibitory zone (mm)a
Ethanol extract
Chloroform extract
14 ꢃ 0
18 ꢃ 1
5.0 ꢃ 0.4
9.2 ꢃ 0.1
2.7 ꢃ 0.1
2.4 ꢃ 0.1
52.5 ꢃ 1.5
56.7 ꢃ 1.1
2.4 ꢃ 0.2
75.5 ꢃ 1.1
Not tested
(1) þ (2) þ (3) þ (4)
17 ꢃ 1
5
6
Not tested
21 ꢃ 1
7 þ 8 þ 9
Not tested
0
11
Caffeic acidb
Not tested
20 ꢃ 1
Bulgarian propolis extractb
a
b
Notes: Tests were done in triplicate, values are mean ꢃ standard deviation (SD). Bulgarian
propolis extract and caffeic acid are designated only as standards for antibacterial and DPPH
RSA, respectively.
Once again, the results demonstrate the fact that propolis, independently of its
plant source and chemical composition, always possesses antimicrobial and antiox-
idant activity. The presence of biologically active constituents, like the alk(en)ylre-
sorcinols and prenylflavonoids in the propolis from East Java, Indonesia is a
confirmation of its potential for application in complementary and alternative
medicine.
3. Experimental
3.1. General experimental procedure
NMR spectra were recorded on a Bruker AV 600 spectrometer (600 MHz for 1H and
150 MHz for 13C) in CDCl3. The GC–MS analysis was performed after silylation of
the samples with a Hewlett Packard gas chromatograph 5890 Series II Plus linked to
a Hewlett Packard 5972 mass spectrometer system, equipped with a 23 m long,
0.25 mm i.d. and 0.5 mm film thickness HP-5 capillary column. The temperature was
programmed from 100ꢀC to 310ꢀC at a rate of 5ꢀC minꢁ1. Helium was used as a
carrier gas, at flow rate 0.7 mL minꢁ1, split ratio 1 : 80, injector temperature 280ꢀC
and ionisation voltage 70 eV. For silylation, about 5 mg of the sample was mixed
with 75 mL bis(trimethylsilyl)trifluoroacetamide and 50 mL of dry pyridine, heated at
80ꢀC for 20 min and analysed by GC–MS. Column chromatography was performed
on silica gel 60 (Merck, 63–200 mm), normal phase. Analytical TLC was performed
on silica gel 60 F254 plates (Merck). Preparative TLC (PTLC) was performed on
silica gel 60 F254 glass plates (Merck, 20 ꢂ 20 cm and 0.25 mm). Detection of the
spots was achieved under UV light (254 and 366 nm) and by spraying with vanillin–
sulphuric acid in methanol (5 : 95 w/v vanillin : methanol solution, freshly mixed with
a 5 : 95 v/v sulphuric acid : methanol solution), followed by heating at 100ꢀC.
3.2. Propolis sample
Propolis was produced by Apis mellifera subsp. mellifera and was collected near
Batu City, East Java Province, Indonesia, by scraping from the bee container.

610
B. Trusheva et al.
3.3. Extraction of propolis
Propolis (1 kg) was cut into small pieces and extracted with 96% ethanol (1 : 10, w/v)
at room temperature for 7 days; after filtration the ethanol extract was concentrated
under reduced pressure to give a crude extract (152 g, yield 15.2%). The crude
ethanol extract (152 g) was dissolved in ethanol–water and extracted successively
with chloroform (three times). The chloroform extract was evaporated to give 68.5 g
dry residue after evaporation.
3.4. Isolation of compounds
The chloroform extract was subjected to column chromatography on silica gel
(350 g) with an n-hexane/ethyl acetate gradient (1 : 0.05/1 : 1) to produce 27 fractions
(I–XXVII).
From fraction VI (95.8 mg), after additional purification by PTLC (silica gel, n-
hexane/ethyl acetate 1 : 0.16, two-fold development), an inseparable mixture of
alk(en)ylresorcinols (32.7 mg) was obtained. This mixture (after silylation) was
analysed by GC–MS under the above-mentioned conditions. The following
compounds were identified in the mixture: 5-pentadecylresorcinol (1) (21%), 5-
(80Z, 110Z-heptadecadienyl)-resorcinol (2) (30%), 5-(110Z-heptadecenyl)-resorcinol
(3) (12%) and 5-heptadecylresorcinol (4) (12%) (Figure 1).
Fraction XIV (778 mg) was rechromatographed on a silica gel column eluted with
n-hexane/acetone, gradient (1 : 0.2/1 : 0.8). Fraction XIV.7 (19.5 mg) after additional
purification with PTLC (n-hexane/acetone 1 : 0.4) yielded mangiferolic acid (5) (3ꢀ-
hydroxycycloart-24-en-26-oic acid; 7.1 mg). From the 11th fraction of this column
XIV.11 (87 mg), propolin D (6) (23 mg) and a mixture of prenylflavanones were
isolated after further separation by PTLC (silica gel, n-hexane/acetone 7 : 3). The
mixture was analysed by GC–MS after silylation and propolin C (7) (42%), propolin
F (8) (40%) and propolin G (9) (17%) were identified.
Fraction XIV.16 (5.2 mg) after further purification by PTLC (silica gel, n-hexane/
acetone 7 : 3) yielded isomangiferolic acid (10) (3ꢁ-hydroxycycloart-24-en-26-oic
acid; 1.5 mg).
Fraction XXIII (200 mg) was rechromatographed by PTLC, mobile phase
chloroform/methanol/water 60 : 15 : 4, to afford 27-hydroxyisomangiferolic acid (11)
(50 mg).
3.5. Identification of compounds
3.5.1. Alk(en)ylresorcinols (1–4)
Pink resin. Alk(en)ylresorcinols (1–4) as an inseparable mixture were identified based
1
on their GC–MS analysis and comparison of their H- and 13C-NMR spectra with
literature data (Gunstone, 1993; Knoedler et al., 2008).
. 5-Pentadecylresorcinol diTMS (1), retention time (RT) 35.3 min, MS (EI,
70 eV), m/z (relative intensity %): 464 Mþ (33), 449 [M–CH3]þ (9), 268
[M–C14H28]þ (100), 73 (13).

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. 5-(80Z,110Z-Heptadecadienyl)-resorcinol diTMS (2), RT 37.6 min, MS
(EI, 70 eV), m/z (relative intensity %): 488 Mþ (24), 473 [M–CH3]þ (6),
268 [M–C16H28]þ (100), 73 (14).
. 5-(110Z-Heptadecenyl)-resorcinol diTMS (3), RT 37.9 min, MS (EI, 70 eV),
m/z (relative intensity %): 490 Mþ (25), 475 [M–CH3]þ (6), 268
[M–C16H30]þ (100), 73 (13).
. 5-Heptadecylresorcinol diTMS (4), RT 38.0 min, MS (EI, 70 eV), m/z
(relative intensity %): 492 Mþ (35), 477 [M–CH3]þ (8), 268 [M–C16H32]þ
(100), 73 (11).
3.5.2. Mangiferolic acid (5), isomangiferolic acid (10) and 27-hydroxyisomangi-
ferolic acid (11)
1
White crystals. These compounds were identified based on their H- and 13C-NMR
spectra, which are identical with literature data (Anjaneyulu, Ravi, Prasad, &
Connolly, 1989; Silva, Cito, Chaves, & Dantas, 2005).
1
3.5.3. Propolin D (6). Yellow resinous substance. Its EI–MS, H- and 13C-NMR
spectra are identical with literature data (Chen, Weng, Wu, & Lin, 2004).
3.5.4. Mixture of propolin C (7), propolin F (8) and propolin G (9)
Yellow resin. The components of this mixture were identified by TLC and GC–MS
comparison with an authentic sample.
3.6. Antimicrobial tests
For the investigation of the antibacterial and antifungal activity, the agar cup
method was used with test strains Staphylococcus aureus 209 (obtained from the
Bulgarian Type Culture Collection, Institute for State Control of Drugs, Sofia),
Escherichia coli WF þ (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; Spooner & Sykes, 1972). An inhibitory zone with a diameter
510 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 mLꢁ1). Control experiments with
solvents showed that the solvent does not exhibit any activity.
3.7. DPPH free radical scavenging activity
The DPPH free radical scavenging activity (RSA) was measured according to a
procedure previously described in the literature (Nenadis & Tsimidou, 2002). The
decrease of the absorption at 516 nm of the DPPH solution after addition of the
tested solution was measured. An aliquot (2000 mL) of 0.1 mmol ethanolic DPPH
solution was mixed with 30 mL of a 2 mmol ethanolic solution of the tested substance.

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The RSA was expressed as percentage decrease with respect to a control value.
Caffeic acid was used as a positive control.
Acknowledgement
The authors thank Ms Daniela Antonova for running GC–MS analyses.
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