Antibacterial mono- and sesquiterpene esters of benzoic acids from Iranian propolis
Chemistry Central Journal
Antibacterial mono- and sesquiterpene esters of benzoic acids from Iranian propolis
Boryana Trusheva 0
Ivelin Todorov 0
Mariana Ninova 2
Hristo Najdenski 2
Ali Daneshmand 1
Vassya Bankova 0
0 Institute of Organic Chemistry with the Centre of Phytochemistry, Bulgarian Academy of Sciences , Acad G Bonchev str, bl 9, 1113 Sofia , Bulgaria
1 Department of Animal Science, Faculty of Agriculture University of Kurdistan , 15175, Pasdaran BLVD, Sanandaj, Kurdistan , Iran
2 Institute of Microbiology, Bulgarian Academy of Sciences , 1113 Sofia , Bulgaria
Background: Propolis (bee glue) has been used as a remedy since ancient times. Propolis from unexplored regions attracts the attention of scientists in the search for new bioactive molecules. Results: From Iranian propolis from the Isfahan province, five individual components were isolated: the prenylated coumarin suberosin 1, and four terpene esters: tschimgin (bornyl p-hydroxybenzoate) 2, tschimganin (bornyl vanillate) 3, ferutinin (ferutinol p-hydroxybenzoate) 4, and tefernin (ferutinol vanillate) 5. All of them were found for the first time in propolis. Compounds 2 - 5 demonstrated activity against Staphylococcus aureus. Conclusions: The results of the present study are consistent with the idea that propolis from unexplored regions is a promising source of biologically active compounds.
Propolis (bee glue) has been used as a remedy since
ancient times. At present, propolis is a popular remedy
in the folk medicine of many nations and a row material
for numerous over-the-counter preparations, health
foods and beverages. Propolis has been proved to
possess valuable biological activities: antimicrobial, antiviral,
anti inflammatory, antioxidant, antitumor, etc. [1,2]. Bee
glue is a plant derived product: bees collect it from
resinous plant parts, and its chemical composition strongly
varies in different eco-geographic zones. Despite the
chemical variability however, it always demonstrates
considerable biological activity, especially antimicrobial
activity [3,4]. For this reason, propolis from unexplored
regions attracts the attention of scientists in the search
for new bioactive molecules . In this article we report
on the isolation of antibacterial terpene esters of
phenolic acids, new propolis constituents, from bee glue
originated from the central part of Iran.
Results and Discussion
The total ethanol extract of Iranian propolis and the
light petroleum fraction of this extract demonstrated
significant antibacterial activity against Staphylococcus
aureus, similar to the activity of the poplar type
propolis. For this reason, GC-MS chemical profiling of
this propolis was performed.
The GC-MS analysis of the ethanol extract after
silylation (data not shown) revealed the presence of the
poplar bud metabolites that are characteristic for poplar
type propolis: pinocembrin, pinobanksin acetate,
pentenyl caffeates, caffeic acid phenethyl ester (CAPE) .
However, the GC-MS profile demonstrated also the
peaks of several terpene esters of substituted benzoic
acids among the major components. Such compounds
have never been found in propolis till now and for this
reason, more detailed chemical studies of this propolis
sample were performed.
The light petroleum fraction of the ethanol extract
afforded, after repeated chromatographic procedures,
five pure individual compounds. One of them was
identified as the prenylated coumarine suberosin 1 (Scheme
1), by comparison of its spectral properties (MS, UV,
NMR) with literature data . It was found for the first
time in propolis. This coumarin has been isolated earlier
from numerous plant species belonging to different
genera, e. g. Citrus , Peucedanum , Heracleum , etc.
Thus, it cannot be used as taxonomic marker for the
source plant of this propolis type.
The other four compounds were esters of mono- and
sesquiterpene alcohols with p-hydroxybenzoic and
vanillinic acids (Scheme 1), according to their mass spectra.
Based on the detailed analysis of their mass and NMR
Scheme 1 Constituents of Iranian propolis.
spectra (including 2D NMR) these compounds were
identified as tschimgin (bornyl p-hydroxybenzoate) 2
, tschimganin (bornyl vanillate) 3 , ferutinin
(ferutinol p-hydroxybenzoate) 4  and tefernin (ferutinol
vanillate) 5 , new propolis constituents. These
compounds have been found in different Ferula species. The
presence of sesqui- and diterpene esters of oxygenated
benzoic acids is a characteristic feature of the metabolic
profile of the plants of genus Ferula (Apiaceae) .
Several Ferula species grow in Iran, F. gumosa (an
endemic to Iran) and F. asafetida being the most widespread
[15,16]. They have flowering stems with a number of
schizogenous ducts in the cortex containing the resinous
gum , which is a potential propolis source. Thus, the
source of compounds 2 - 4 in propolis has to be a plant
species of the genus Ferula and the studied sample has
two plant sources: poplar and ferula.
The total extract and the light petroleum fraction
demonstrated low radical scavenging activity against
DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals (5.3 and
2.4%, respectively), compared to the well-known
antioxidant caffeic acid (75.5%), used as a positive control. The
individual compounds showed practically no antiradical
activity (lower than 1%).
The antimicrobial activity of the isolated compounds
was tested and compared to the activities of the total
extracts (Table 1). No activity was found against
Candida albicans and Escherichia coli, similar to most
propolis samples of different origin and their constituents
Table 1 Antimicrobial activities against S. aureus (zones
of inhibition) of propolis extract and the isolated
components (at 400 μg in the cup).
Zone of inhibition (mm)
. The total ethanol extract, the light petroleum
fraction and its constituents 2 - 5 were active against S.
aureus. The most active compounds were 2 and 4, the
esters of p-hydroxybenzoic acid. Till now, antibacterial
activity has been documented for 4 and5 only , but
for the bornyl esters 2 and 3 this is the first report on
antibacterial activity. The studied propolis type of mixed
origin (poplar & ferula) demonstrated slightly higher
antibacterial activity compared to propolis from poplar
origin (the Bulgarian sample) but the difference was not
The results of the present study are consistent with the
idea that propolis from unexplored regions is a
promising source of biologically active compounds.
UV-VIS: Helios Gamma spectrophotometer, spectra
were taken in methanol. IR: Bruker IFS 113v, spectra
were taken in KBr pellets 1H NMR (600 MHz) and 13C
NMR (150 MHz), Bruker AV 600; spectra were taken in
CDCl3 (deuterated chloroform). MS: Hewlett Packard
5972 mass spectrometer, ionization voltage 70 eV.
Column and flash chromatography were performed on
Silica gel 60 (Merck, 63-200 μm), normal phase.
Analytical thin-layer chromatography (TLC) was performed on
Silica gel 60 F254 plates (Merck). Preparative thin-layer
chromatography (PTLC) was performed on Silica gel 60
F254 glass plates (Merck, 20 × 20 cm, 0.25 mm).
Detection of the spots was achieved under UV light (254 and
366 nm) and by spraying with vanillin- sulfuric acid in
methanol (5:95 w/v vanillin:methanol solution, freshly
mixed with a 5:95 v/v sulfuric acid:methanol solution),
followed by heating at 100°C.
Extraction and isolation
Propolis (31 g) was cooled in a refrigerator, ground and
extracted twice with 70% ethanol (1:10, w:v) at room
temperature for 24 h. A small part of this extract (10
ml) was evaporated to dryness and subjected to GC-MS
analysis after silylation (5 mg of the residue was mixed
with 50 μL of dry pyridine and 75 μL of BSTFA
(bis(trimethylsilyl)-trifluoroacteamide) and heated at 80°C
for 20 min). The gas-chromatography - mass
spectrometry (GC-MS) analysis was performed with a Hewlett
Packard Gas Chromatograph 5890 Series II Plus linked
to Hewlett Packard 5972 mass spectrometer system
equipped with a 30 m long, 0.25 mm id and 0.5 μm film
thickness HP5-MS capillary column; temperature
program from 100 to 300°C at a rate of 5°C/min. carrier
gas helium, flow rate 0.7 mL/min. Split ratio 1:20,
injector temperature 280°C, interface temperature 300°C,
ionization voltage 70 eV.
The ethanol extract was concentrated in vacuo and
extracted successively with light petroleum (40 - 60°C) 3
times. The light petroleum extract was evaporated in
vacuo to dryness to give 4.3 g dry residue.
The dry light petroleum extract was subjected to flash
chromatography on silica gel, mobile phase light
petroleum - EtOAc (ethyl acetate) (100% light petroleum to
100% EtOAc) and 10 fractions (A - J) were obtained.
Fractions D and E (1.131 g, eluted with light petroleum
EtOAc 70:30- and 60:40) were re-chromatographed on a
silica gel column eluted with light petroleum - EtOAc
(90:10 to 100% EtOAc) and 27 fractions were obtained.
Fraction 4 (55.7 mg, eluted with light petroleum - EtOAc
80:20) was subjected to PTLC (silica gel, mobile phase
methylene chloride - EtOAc 99:1) to yield 16.5 mg 2
(tschimganin). Fraction 6 (66 mg, eluted with light
petroleum - EtOAc 70:30) was subjected to PTLC (silica gel,
mobile phase methylene chloride - EtOAc 99:1) and gave
two pure compounds: 2.8 mg of 1 (suberosin) and 2.7 mg
of 3 (tschimgin). Fraction 15 (11.3 mg, eluted with light
petroleum - EtOAc 45:55) was further purified by PTLC
(silica gel, mobile phase methylene chloride - EtOAc
20:1, two-fold development) to afford 5.8 mg ferutinin 4
and 2.1 mg teferin 5.
For the investigation of the antibacterial activity, the
agar cup method  was used 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).
An inhibitory zone with a diameter less than 10 mm
corresponds to lack of activity (10 mm is the diameter
of the cup). 0.1 ml of test solution containing 0.4 mg of
each extract and substance in ethanol was applied to
every cup (concentration of the test solution 4 mg/ml).
Control experiments with solvent showed that it does
not have any activity. Streptomycin and nystatin were
used as positive controls.
DPPH free radical scavenging activity
DPPH free radical scavenging activity was measured
according to the procedure described in . In brief,
the extracts and tested compounds were dissolved in
ethanol (0.36 mg/ml), and to 2 ml DPPH solution(0.1
mM in ethanol) 30 μl of the analyzed solution was
added. The resulting solution was thoroughly mixed and
absorbance was measured at 516 nm after 20 min. The
scavenging activity was determined by comparison of
the absorbance with blank (100%), containing only
DPPH and solvent. Caffeic acid was used as positive
BT participated in the separation and identification of the compounds and
performed DPPH tests. IT performed performed the extractions and some of
the chromatographic separations. MN and HN performed the antimicrobial
tests. AD performed sample collection. 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.
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