Omani propolis: chemical profiling, antibacterial activity and new propolis plant sources
Chemistry Central Journal
Omani propolis: chemical profiling, antibacterial activity and new propolis plant sources
Milena Popova 0
Rosa Dimitrova 0
Hassan Talib Al-Lawati 2
Iva Tsvetkova 1
Hristo Najdenski 1
Vassya Bankova 0
0 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences , Sofia 1113 , Bulgaria
1 Institute of Microbiology “Stefan Angelov”, Bulgarian Academy of Sciences , Sofia 1113 , Bulgaria
2 Honey Bee Research Lab, Directorate General of Agriculture and Livestock Research, Ministry of Agriculture and Fisheries , Muscat, Sultanate of Oman
Background: Propolis (bee glue) is a resinous honeybee product having a long history of application in many countries as a traditional remedy for treating wounds, burns, soar throat, stomach disorders, etc. It has been proved to possess beneficial biological effects, including antimicrobial, antioxidant, anti-inflammatory, cytotoxic, antiulcer, and many others. Bees gather propolis from diverse resinous plant parts and in different phytogeographic regions its chemical composition might vary significantly. In this article we report the results of the first study on the chemical profiles of propolis from Oman, its plant origin and antibacterial activity. Results: The chemical profiles of Omani propolis extracts were obtained by GC-MS analysis after silylation. Over 50 individual compounds were identified in the samples, belonging to different compound types: sugars, polyols, hydroxy acids, fatty acids, cardanols and cardols, anacardic acids, flavan derivatives, triterpenes, prenylated flavanones and chalcones. The profiles were dissimilar from other known propolis types. They demonstrate that although Oman is not a large country, the plant sources of propolis vary significantly, even in the same apiary and the same season. Based on chemical profiles, and isolation and identification of major marker compounds (new propolis constituents), new plant sources of propolis were found: Azadiracta indica (neem tree) and Acacia spp. (most probably A. nilotica). The ethanol extracts of the studied propolis samples demonstrated activity against S. aureus (MIC < 100 μg. mL-1) and E. coli (MIC < 380 μg. mL-1). Conclusion: Omani propolis is different form the known propolis types and demonstrates significant chemical diversity. Its most important plant source is the resin of Azadirachta indica, and as a result its typical components are С5-prenyl flavanones. Other plant sources have been identified, too, playing some role in resin collection by bees in Oman: Acacia spp. (most probably A. nilotica) and Mangifera indica. The results demonstrate also the potential of Omani propolis as antimicrobial.
Propolis; Chemical profiling; Propolis plant sources; Azadiracta indica; Acacia; Antibacterial activity
Propolis (bee glue) is a resinous honeybee product collected
by bees from plants and used as a building material and as
a defensive antimicrobial substance in their hives [
]. It has
a long history of application in many countries as a
traditional remedy for treating wounds, burns, soar throat,
stomach disorders, etc [
]. In the last decades, the
medicinal properties of propolis attracted the attention of
scientists and nowadays a lot of data exist concerning its
chemical composition and diverse biological effects,
including antimicrobial, antioxidant, immunomodulating,
antiinflammatory, cytotoxic, antiulcer, and many other activities
]. It is a popular ingredient of food supplements, health
foods and beverages, cosmetics .
Bees gather propolis from diverse resinous parts of living
plants and in different phytogeographic regions its chemical
composition might vary significantly due to the specificity
of the local flora and the choices it offers to bees. At
present, several propolis types are known, according to
their chemical profiles and source plants: poplar type
(European) propolis, Brazilian green propolis (Baccharis
type), red South American propolis (Dalbergia type),
Mediterranean propolis (rich in diterpenes from Cupressus spp.)
], etc. Numerous studies have revealed that in different
environments honeybees are capable of finding appropriate
propolis floral sources with significant antimicrobial
]. Because of this, new propolis types from
unexplored regions of the world have the potential to provide
valuable leads to secondary metabolites with important
bioactivities. In this article we report the results of the first
study on the chemical profiles of propolis from Oman, its
plant origin and antibacterial activity.
Results and discussion
The chemical profiling of propolis extracts was performed
by GC-MS analysis. Over 50 individual compounds were
identified in the samples (see Additional file 1: Table S1),
belonging to different compound types: sugars, polyols,
hydroxy acids, fatty acids, cardanols and cardols, anacardic
acids, flavan derivatives, triterpenes, prenylated flavanones
and chalcones. A representation of the chemical profiles by
groups of compounds is shown in Figure 1. It is obvious
that the sample profiles display significant chemical
differences, both qualitative and quantitative. The profiles
demonstrate that although Oman is not a large country, the
plant sources of propolis vary significantly, even in the same
apiary and the same season. In addition, they are
completely dissimilar from poplar type (European) propolis, and
from the recently described Mediterranean propolis. Omani
samples do not contain phenolic acids and their esters
found in poplar propolis [
]. The typical poplar flavonoids
pinocembrin, pinobanksin 3-O-acetate, galangin, and
] are not present, either. Further, the samples do
not contain any diterpenes, which are characteristic for
Mediterranean type propolis [
In order to analyze the large amount of analytical data we
applied chemometric approach: the Principal Component
Analysis (PCA). The central idea of PCA is to reduce the
dimensionality of a data set in which there are a large
number of correlated variables, while retaining as much as
possible the total information. We used for PCA analysis the
relative amounts of individual constituents of propolis from
the GC-MS analysis (data from Additional file 1: Table S1).
The obtained two-dimensional plot (Figure 2) covers 78%
of the total variation. One well defined group is formed by
samples OM-1, OM-2, OM-4, OM-6 and OM-7. These
samples (excluding OM-1) are characterized by relatively
high concentration of prenylated flavanones and chalcones.
Concerning the presence of chalcones, it is important
to note that flavanones can easily turn to chalcones
under the conditions of silylation for GC-MS analysis
]. In order to check whether the chalcones are native
constituents of Omani propolis, we examined the
1HNMR spectra of the extracts from the latter samples,
looking for the typical doublets (J ~ 16 Hz) of the α- and
β-protons of the chalcone skeleton in the range δ 6.7 –
7.4 and δ 7.3 – 8.0 [
]. We were unable to detect any
such signals in the 1H-NMR spectra, which lead to the
conclusion that chalcones registered in GC-MS are
artifacts produced from the corresponding flavanones and
are not present in the native propolis.
Till now, several prenylated flavanones (propolins)
have been found in propolis from Pacific islands: Taiwan
], Okinawa [
], Indonesia [
]; the plant sources of
these compounds were identified as Macaranga species.
All of them contain C10 prenyl moieties in their
molecules, while the flavanones identified in Omani samples
contained C5 prenyl side chains. Obviously, the floral
source of the latter flavanones has to be some plant
different from Macaranga. In order to identify this floral
source, the complete structure elucidation of the major
Cardanols and cardols
Prenylated flavanones and chalcones
Figure 1 Chemical profiles of Omani propolis extracts.
Projection of the cases on the factor-plane ( 1 x 2)
Cases with sum of cosine square >= 0,00
prenylated flavanones was necessary. Mass spectral data
of silylated flavanones (See Additional file 1: Table S2)
allow the identification of the type and number of
substituents (OH-, OMe-, prenyl- groups) and their location
in rings A or B of the flavanone frame. The exact
position of the prenyl groups, however, cannot be detected
from the mass spectrum. For this reason, isolation of
important constituents of sample OM-6 was performed. Its
ethanol extract afforded, after repeated chromatographic
separations, two pure individual compounds. By
comparison of their spectral properties (NMR spectra,
MS) with literature data [
], these compounds were
identified as 7-O-methyl-8-prenylnaringenin 1 and
3’,8diprenylnaringenin 2 (Figure 3). Both compounds are
new propolis constituents and have been isolated
previously from the neem tree Azadiracta indica [
this reason we consider this tree as a likely major plant
source of the samples OM-2, OM-6 and OM-7.
The adaptation of A. indica to hot and dry climates
has made it one of the most commonly planted species
in arid and semi-arid areas [
], and it is widespread in
]. The material collected by bees seems to be
the resin, produced by resin glands on the leafy shoots
of neem trees [
]. The chemical composition of this resin
is largely unknown; only one prenylated flavanone
8-prenyl-5,7-dihydroxy-3’-(3-hydroxy-3-methylbutyl)4’-methoxyflavanone 3 has been identified in it so far. This
flavanone has been isolated from neem resin glands as a
new natural product [
] and no other source of 3 has yet
been reported, thus it is a good taxonomic marker for A.
indica resin. Compound 3 was detected in samples OM-2,
OM-6 and OM-7. It was tentatively identified in the
GCMS profiles of the samples on the basis of its characteristic
mass spectral fragmentation. The spectrum of the silylated
compound is characterized by a low intensity molecular ion
peak at m/z 656, corresponding to a molecular formula
C26H32O6 and base peak at m/z 641 [M-15]+, This
molecular formula corresponds to a flavanone with one prenyl
group, one hydroxylated pentyl group, two OH and one
OCH3 groups. The mass spectrum displays the typical
fragments of a flavanone bearing 5 and 7 hydroxyl groups and
one prenyl group in ring A: ions at m/z 349 [A1 – CH3]+,
321 [A1 – CH3 - CO]+, 309 (98%) [A1 – C4H7]+ [
the other hand, the presence of a non-aromatic silyloxy
group is indicated by the ion m/z 552 [M-CH3-OTMS]+.
The position of the methyl group and the hydroxylated
pentyl group in ring B are supported by the presence of the
fragment ions at m/z 293 [B2]+ and 291 [B3]+. The peak at
m/z 497, resulting from the loss of the hydroxylated side
chain, supports the structure
8-prenyl-5,7-dihydroxy-3’-(3hydroxy-3-methylbutyl)-4’-methoxyflavanone 3 (analogous
peak was detected in the spectrum of the unsilylated
]). The occurrence of compound 3 in Omani
propolis thus provides an evidence for the contribution of
A. indica as propolis source.
This finding adds a new species to the list of
propolis source plants. It is important to note that A.
indica is one of the most widely used medicinal
plants in India since ancient times [
]; it possesses
diverse biological activities, including antibacterial,
antiviral and antifungal properties. Antifungal activity
has been demonstrated for some individual neem
constituents, including compound 3 [
this fact, the choice of bees for resin source is not
The prenylated flavanones were detected in all Omani
samples studied excluding OM-1. In the latter sample,
the only secondary metabolites found were triterpenic
compounds (individual compounds could not be
identified), their source remaining unclear.
Sample OM-3 has a profile different form the ones on
the other samples, its major constituents being cardanol
(alkylphenol), cardols [alk(en)ylresorcinols)] 4 and
anacardic acids 5 (Figure 4). These three related
compound types have been found in propolis samples from
], while cardols have been detected in poroplis
from Thailand and Indonesia [
]. They most
probably originate from Mangifera indica fruit bark and are
known antifungal substances [
]. Sample OM-3
contained also significant amounts of C5-prenylated
flavanones (and the resulting chalcones), as well as
triterpenes. Obviously, this particular sample is of at
least dual origin: Mangifera indica and Azadiracta
indica. The contribution of M. indica as a resin source
could be detected also in sample OM-8, which comes
from the same region as sample OM-3 (Saham). In
OM8 however, these compounds are present in much lower
concentration compared to OM-3. Nevertheless, this fact
suggests that the bees in this region have access to both
propolis plant sources.
The sample OM-4 is unique among the studied
samples in that it contains high amounts of flavan
derivatives. The mass spectra of their TMS ethers were similar
to those of catechine derivatives with two OH groups in
ring B [
]. Catechins are known constituents of
Azadirachta indica [
], however, the samples where the
Azadirachta prenylflavanones are major components
contained only minor amounts of these flavans. For this
reason, their positive identification was necessary and
several compounds were isolated from this propolis
sample using consecutive chromatographic procedures. Two
pure substances were obtained and characterized by
mass and NMR spectra and comparison with literature
data as fisetinidol 6 [
] (Figure 5) and a mixture of
two stereoisomers of mollisacacidin: 2,3-trans-3,4-trans
(7a) and 2,3-trans-3,4-cis (7b) mollisacacidin (Figure 5)
]. All three compounds are new propolis
constituents, and flavan-3,4-diols have not been found in
propolis till now.
R = alk(en)yl
R = alk(en)yl
Another feature of this sample is the high concentration
of the cyclitol derivative pinitol 8, also new for propolis.
Indeed, pinitol has been found in several honey types but
always as a minor constituent [
], so it is highly improbable
that it has come into propolis from honey, unlike glucose
and other carbohydrates. Pinitol together with the flavan
derivatives form a combination of phytochemicals that can
point to the plant source of this particular propolis sample:
it is a combination typical for many Acacia species [
Taking into consideration the distribution of Acacia nilotica
in Oman , it is the most probable major source of
OM4. The presence of some amounts of prenylated flavanones/
chalcones proves the importance of the neem tree as a
secondary source for this sample.
It is noteworthy that all samples contain triterpenes but
different samples contain different triterpenes and many of
them could not be identified by GC-MS. The one common
for all samples was cycloartenol. The exact plant origin of
the triterpene compounds is yet unknown, but triterpenes
are widely distributed in plant kingdom and the potential
sources are numerous.
In this study, eight propolis samples were studied. Six of
them were collected at the same apiary (region of Rumai)
in different months of 2011, and only two samples (OM-3
and OM-8) originated from another location, Saham.
Surprisingly, samples collected at virtually the same time of
year from the same apiary from neighboring hives
demonstrated different chemical profiles, for example OM-4
(September 10th) and OM-5 (September 11th). On the
other hand, samples from different seasons were similar:
OM-2 (June) and OM-6 (September). This difference
indicates that propolis collection could be directed by
some random factors.
The two samples from Saham (OM-3 and OM-8) have
in common the presence of cardols and anacardic acids
which sets them apart from the other samples, but they
also display considerable quantitative differences
The alcohol extracts of the studied Omani propolis
samples were tested for their activity against Staphylococcus
aureus, Escherichia coli and Candida albicans.
Somewhat surprisingly, no antifungal activity against C.
albicans could be detected (MIC > 1000 μg. mL-1). The
results of the antibacterial tests are presented in Table 1.
Al samples demonstrated good antibacterial activity,
taking into consideration that natural products which
produce minimum inhibitory concentrations (MIC) in
the range 100–1000 mg mL-1 in in vitro susceptibility
tests can be classified as antimicrobials [
]. The MIC
for all tested samples against E. coli were higher (lower
activity) than against S. aureus. Most Omani samples
were more active than a typical poplar type propolis
sample from Bulgaria, both against S. aureus and E. coli.
There was a statistically significant correlation
(r = 0,9646; p < 0.01) between the MIC against S. aureus
and E. coli. The most active samples against both
microorganisms were OM-6 and OM-7, the samples richest in
prenylated flavonoids. Prenylated flavonoids are known
to possess antimicrobial activity [
] and that may be
the explanation of this observation.
The results obtained in the present study lead to the
conclusion that Omani propolis is different form the
known propolis types and demonstrates significant
chemical diversity. Its most important plant source turns
out to be the resin of Azadirachta indica, and as a result
its typical components are С5-prenyl flavanones. Other
plant sources have been identified, too, playing some
role in resin collection by bees: Acacia spp. (most
probably A. nilotica) and Mangifera indica. Further studies of
propolis from the region of the Persian Gulf should
clarify the importance of neem and Acacia species as
The results demonstrate also the potential of Omani
propolis as antimicrobial. The presence of biologically
active phenolic constituents (prenylflavanones, cardols,
anacardic acids, etc.) is an indication for its potential for
application in complementary and alternative medicine,
cosmetics and health foods.
The samples were collected in 2011 (with one exception
collected in 2012) at two locations. The exact time and
site of collection are given in Table 2.
Extraction and sample preparation
Propolis was cooled in a refrigerator, grinned and
extracted twice with 70% ethanol (1:10, w:v) at room
temperature for 24 h. A part of the ethanol extract
(5 mL) was evaporated to dryness. The balsam yield was
determined by two parallel measurements. (Balsam
content could not be determind only in sample OM-9
because propolis couls not be separated quantitatively
from the textile fabric). The dry extract (combined from
the two parallel measurments) was further analyzed by
36.4 ± 0.4
49 ± 1
GC-MS after silylation, as well as for its antimicrobial
activity. About 5 mg of the extract were mixed with
50 μL of dry (water-free) pyridine and 75 μL of bis
(trimethylsilyl)-trifluoroacetamide (BSTFA) and heated
at 80°C for 20 min. The silylated extracts and reference
compounds were analysed by GC– MS.
The GC–MS analysis was performed with a Hewlett–
Packard gas chromatograph 5890 series II Plus linked to a
Hewlett–Packard 5972 mass spectrometer system equipped
with a 30 m long, 0.25 mm i.d., and 0.5 μm film thickness
HP5-MS capillary column. The temperature was
programmed from 60 to 300°C at a rate of 5°C/min, and a
10 min hold at 300°C. Helium was used as a carrier gas at a
flow rate of 0.8 mL/min. The split ratio was 1:10, the
injector temperature 280°C, the interface temperature 300°C,
and the ionization voltage 70 eV. Every extract was analyzed
Identification and quantification of compounds
The identification of individual compounds was performed
using computer searches on commercial libraries,
comparison with spectra of authentic samples and literature data. If
no reference spectra were available, identification was
performed based on the mass-spectral fragmentation, in
such cases for some compounds only tentative structures
were proposed. Some constituents remained unidentified
because of the lack of relevant references and information
(none of them major constituent). The quantification of
individual constituents is based on internal normalization.
The percentage figures in the tables refer to percent of the
Total Ion Current, TIC, and are semi-quantitative.
Isolation of individual compounds
1H NMR (600 MHz) and 13C NMR (150 MHz), Bruker
AV 600; spectra were taken in CDCl3 (deuterated
chloroform) for compounds 1 and 2, in CD3OD
(deuterated methanol) for compounds 6, 7a + 7b.
Individual compounds were isolated from the extract
of sample OM-6 (7.1 g). The ethanol extract was
concentrated in vacuo and extracted successively with
petrol ether (3 x) and CHCl3 mg (3 x). The chloroform
extract was evaporated to yield 2 g dry extract, which
was subjected to column chromatography on a silica gel
column (30 mm d. – 600 mm h; 150 g. silica gel) eluted
with light petroleum — EtOAc (95:5 to 100% EtOAc)
and 30 fractions were obtained. Fraction 4 yielded 2 mg
of 7-O-methyl-8-prenylnaringenin 1, and fractions 9 –
11 yielded 67 mg of 3’,8-diprenylnaringenin 2.
Further individual compounds were isolated from the
ethanol extract of sample OM-4 (3 g). The ethanol extract
was concentrated in vacuo and extracted successively with
petrol ether (3 x), CHCl3 mg (3 x) and EtOAc (3x). The
petrol ether fraction contained mainly triterpenoids and
was not further analyzed. The ethyl acetate extract was
evaporated to dryness (200 mg) and subjected to
preparative thin layer chromatography (PTLC) (silica gel 60 F254
glass plates Merck, 20×20 cm, 0.25 mm, mobile
phaseCHCl3 – MeOH 8: 2; detection under UV light 254
and 366 nm), and two substances were isolated: 1.2 mg of
fisetinidol 6 and 3 mg of a mixture of 2,3-trans-3,4-trans
mollisacacidin 7a and 2,3-trans-3,4-cis mollisacacidin 7b.
1H NMR (600 MHz) and 13C NMR (150 MHz), Bruker
AV 600; spectra were taken in CDCl3 (deuterated
chloroform) for compounds 1 and 2, in CD3OD
(deuterated methanol) for compounds 6, 7a + 7b.
The MIC of propolis extracts were determined using broth
microdilution method with test strains Staphylococcus
aureus 209, Escherichia coli WF + and Candida albicans 562
(obtained from the Bulgarian Type Culture Collection,
institute for State Control of Drugs, Sofia). Stock solution of
propolis extracts was prepared, as follows: 4–5 mg (exact
weight) dry extract were dissolved in 1 mL 70% ethanol.
This stock solution was used for serial dilution in a
96-wells microtiter microplate from 400 – 500 μg/mL to
200–250, 100–125, 50–62.5, 25–31.25, 12.25 -15.62, 6.12
7.81 μg/mL. For the broth microdilution test, 50 μL of
bacterial suspension in exponential phase of the growth
was added to the wells of a sterile 96-well microtiter plate
already containing 50 μL of twofold serially diluted propolis
extracts in growth medium. Control wells were prepared
with culture medium and bacterial suspension only. Three
wells of the microtitre plate were used for each
concentration of tested propolis extracts as well as for control sample.
Incubation of the microplate was done for 24 h in the
cultivation conditions described above. The MIC is the
concentration in the last well in the row where no development of
the microorganism is detected.
Multivariate analysis of propolis chemical profiles was
performed by principal component analysis (PCA), using
the GC-MS data for the identified compounds expressed
as a percentage of the Total Ion Current, respectively.
Statistica Version 8.0 was used for the analyses.
Additional file 1: Table S1. Chemical profiles of Omani propolis
ethanol extracts by GC-MS. Table S2. Important ions in the mass spectra
of silylated compounds in Omani propolis samples (GC-MS).
The authors declare that they have no competing interests.
MP participated in the chromatographic and the identification of individual
compounds, RD performed the GC-MS analysis, HTAL collected the propolis
samples and contributed to drafting the manuscript, IT and HN performed
antimicrobial tests, VB conceived of the study, participated in its design and
coordination and contributed to drafting the manuscript. All authors read
and approved the final manuscript.
Partial financial support from the Bulgarian National Science Fund -Project
DRNF-02-13/2009 is acknowledged. The authors thank Ms. N. Kostova for
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