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Chemical Composition, Antimicrobial andAntioxidant Activity of Birch (Betula pendula Roth.) Buds Essential Oil

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  • University of Niš, Faculty of technology, Bulevar Oslobodjenja 124, Leskovac

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The essential oil from birch (Betula pendula Roth.) buds was obtained by Clevenger-type hydrodistillation. Its qualitative and quantitative composition was determined by gas chromatography in combination with mass spectrometry (GC/MS) and flame ionization detection (GC/FID), respectively. Twenty-seven compounds, mainly sesquiterpene hydrocarbons (78.7 %) and oxygenated sesquiterpenes (14.8 %), were identified comprising 93.5 % of total identified components in the essential oil. The most abundant compounds were germacrene D (21.7 %) and δ-cadinene (17.0 %). Antimicrobial activity of isolated essential oil against Gram-positive and Gram-negative bacteria as well as fungus Candida albicans was determined using the disc-diffusion method. The isolated essential oil has shown antimicrobial activity against the following microorganisms: Proteus vulgaris, Bacillus luteus and Klebsiella pneumoniae. Antioxidative activity of the essential oil was determined using the DPPH assay, after adding DPPH radical and after 20 min, 30 min and 60 min incubation with radical. Essential oil showed antioxidant activity.
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Journal of Essential Oil Bearing Plants
ISSN: 0972-060X (Print) 0976-5026 (Online) Journal homepage: https://www.tandfonline.com/loi/teop20
Chemical Composition, Antimicrobial
andAntioxidant Activity of Birch (Betula pendula
Roth.) Buds Essential Oil
Marijana S. Vladimirov, Vesna D. Nikolic, Ljiljana P. Stanojevic, Jelena S.
Stanojevic, Ljubisa B. Nikolic, Bojana R. Danilovic & Valentina D. Marinkovic
To cite this article: Marijana S. Vladimirov, Vesna D. Nikolic, Ljiljana P. Stanojevic, Jelena S.
Stanojevic, Ljubisa B. Nikolic, Bojana R. Danilovic & Valentina D. Marinkovic (2019) Chemical
Composition, Antimicrobial andAntioxidant Activity of Birch (Betula�pendula Roth.) Buds Essential
Oil, Journal of Essential Oil Bearing Plants, 22:1, 120-130, DOI: 10.1080/0972060X.2019.1602084
To link to this article: https://doi.org/10.1080/0972060X.2019.1602084
Published online: 17 Apr 2019.
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Chemical Composition, Antimicrobial and Antioxidant
Activity of Birch (Betula pendula Roth.) Buds Essential Oil
Marijana S. Vladimirov 1*, Vesna D. Nikolic 1, Ljiljana P. Stanojevic 1, Jelena S.
Stanojevic 1, Ljubisa B. Nikolic 1, Bojana R. Danilovic 1, Valentina D. Marinkovic 2
1 University of Nis, Faculty of Technology, Bulevar Oslobodjenja 124, 16000 Leskovac, Serbia
2 University of Belgrade, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia
Abstract: The essential oil from birch (Betula pendula Roth.) buds was obtained by Clevenger-type
hydrodistillation. Its qualitative and quantitative composition was determined by gas chromatography in
combination with mass spectrometry (GC/MS) and flame ionization detection (GC/FID), respectively. Twenty-
seven compounds, mainly sesquiterpene hydrocarbons (78.7 %) and oxygenated sesquiterpenes (14.8 %), were
identified comprising 93.5 % of total identified components in the essential oil. The most abundant compounds
were germacrene D (21.7 %) and δ-cadinene (17.0 %). Antimicrobial activity of isolated essential oil against
Gram-positive and Gram-negative bacteria as well as fungus Candida albicans was determined using the disc-
diffusion method. The isolated essential oil has shown antimicrobial activity against the following microorganisms:
Proteus vulgaris, Bacillus luteus and Klebsiella pneumoniae. Antioxidative activity of the essential oil was
determined using the DPPH assay, after adding DPPH radical and after 20 min, 30 min and 60 min incubation with
radical. Essential oil showed antioxidant activity.
Key words: Birch (Betula pendula Roth.); essential oil; GC/MS analysis; antimicrobial activity;
antioxidative activity.
Introduction
Family Betulaceae is widespread around the
world, mainly in the north and cold temperature
zones, especially in the mountain regions of North
hemisphere, from the East Europe and North
America to China and Japan. The use of this plant
is known from the antic 1-3. White birch (Betula
pendula Roth.) is one of the most abundant and
best-known species from Betulaceae family. It is
known as silver birch and European white birch.
White birch is a tree up to 30 m high, with silver
white bark, smooth in the higher parts, black and
cracked in the lower parts. Betula pendula ex-
erts large morphological variability. It could be
divided in 3 subspecies, with ssp. pendula espe-
cially and sufficiently abundant in Europe 1,2,4-6.
Almost every part of white birch (buds, bark,
leaves, juice and resin) found its application in the
folk medicine. This medicinal plant exerts differ-
ent pharmacological effects due to the synergis-
tic effect of biologically active compounds present
therein. White birch isolates could be applied in
different pathological conditions such as inflam-
mation, infections, urinary tract disturbances, skin
diseases and rheumatism 1,2,7,8. European medi-
cal agency (EMEA) cites birch leaves as tradi-
tional plant medicine used, due to its diuretic ef-
fect, as the auxiliary agent in the therapy of mild
urinary disorders 9. Russian traditional medicine
cites that birch buds could be used as diuretics 10.
ISSN Print: 0972-060X
ISSN Online: 0976-5026
Received 10 March 2019; accepted in revised form 27 March 2019
*Corresponding author (Marijana S. Vladimirov)
E-mail: < marijana89@hotmail.co.uk > © 2019, Har Krishan Bhalla & Sons
TEOP 22 (1) 2019 pp 120 - 130 120
In addition, they could be used as expectorants,
cholagogues, diaphoretics and analgetics. It is also
highlighted that birch buds could be used as anti-
inflammatory agents and antiseptics for wound
healing, washing and removing stains from hu-
man skin. Infusions and decocts of birch buds are
also used in dentistry and otolaryngology due to
their antiinflammatory activity, as well as for the
treatment of stomatitis, gingivitis, periodontitis,
glossitis, throat pain, chronic tonsillitis and acute
respiratory diseases 10.
The essential oil, often used as an ingredient in
hair care products, could be obtained from the
birch buds, leaves and young branches. It could
be applied in the skin disease treatment, aroma-
therapy as well as in the treatment of various res-
piratory disorders 3,8.
The essential oil content is the highest in the
birch buds, up to 3.8 %. Determination of birch
essential oil chemical composition was the sub-
ject of numerous studies 1-3,6,7,10-12. The qualita-
tive and quantitative composition of essential oil
mainly depends on the isolation method applied
and the area where the plant material was col-
lected. The most described methods in the litera-
ture used for essential oil isolation are Clevenger-
type hydrodistillation, micro distillation and Lik-
ens-Nickerson distillation 1,7.
Plant extracts are complex mixtures of differ-
ent compounds, and exhibit powerful biological ef-
fects as a result of synergistic effect. Numerous
studies have shown that essential oils exhibit strong
antimicrobial and antioxidant activity and thus they
can be used in the food, pharmaceutical and cos-
metic industries 13.
There is no data regarding the chemical com-
position analysis of birch essential oil from Serbia
in the available literature. Therefore, the aim of
this study was to isolate the essential oil from dried
birch buds by Clevenger-type hydrodistillation, to
determine qualitative and quantitative composition
of the isolated oil as well as to determine its anti-
microbial and antioxidant activity.
Materials and methods
Plant material
White birch (Betula pendula Roth.) buds (300
g) were collected from the locality Vrnjacka
Banja (GPS-coordinates: 43°37'30.266" N 20°53'
39.077" E) in March 2018. In order to maintain its
natural color and medicinal properties, plant ma-
terial was dried in a shaded, drafty place, for 7
days, and then used for hydrodistillation.
Essential oil isolation
The essential oil from dried birch buds was ob-
tained by Clevenger-type hydrodistillation.
Hydrodistillation time was 4 h and hydromodule
1:5 m/V. A hundred grams (100 g) of buds and
500 ml of water were used for hydrodistillation.
Round bottom flask containing water and plant
material was connected to Clevenger-type appa-
ratus and tap water was opened for running the
flow through the condenser. The heating mantle
was heated to the temperature high enough to
reach the boiling point of water. The essential oil
and water vapors were come into the graduated
distillate receiving tube and the excess water (hy-
drosol) was returned back into the flask. After 4
h, the hydrosol was removed from the distillate
receiving tube, and the oil was dried over anhy-
drous Na2SO4. The hydrodistillation was repeated
3 times to establish the reproducibility of results.
The essential oil yield was 1 ± 0.044 %. The oil
was kept in a dark bottle, at +4°C until the analy-
sis.
GC/MS and GC/FID analysis
GC/MS analysis of the essential oil obtained
from birch buds was performed on Agilent Tech-
nologies 7890B gas chromatograph, coupled with
inert, selective 5977A mass detector of the same
company. The essential oil obtained was dissolved
in diethyl ether to concentration of 1000 ppm. 1 μl
of the prepared solution was injected in split/
splitless inlet set at 250°C in 10:1 split mode. Com-
ponents were separated on weakly polar, silica
capillary column, HP-5MS (5 % diphenyl- and 95
% dimethyl-polysiloxane, 30 m × 0.25 mm, 0.25
μm film thickness; Agilent Technologies, USA).
Helium was used as the carrier gas, at a constant
flow rate of 1 ml/min. The oven temperature was
programmed from 50°C for 2.25 minutes and then
increased to 290°C at the rate of 4°C/min. Sepa-
rated components were further analyzed by mass
spectrometer. Temperatures of the MSD trans-
Marijana S. Vladimirov et al., / TEOP 22 (1) 2019 120 - 130 121
fer line, ion source and quadruple mass analyzer
were set at 300°C, 230°C and 150°C, respectively.
The ionization voltage was 70 eV and mass de-
tection was done in the Scan mode, in m/z range
from 35 to 650.
Quantitative results were obtained on the gas
chromatograph used for the GC/MS analysis
equipped with the flame-ionization detector (FID)
of the same company. The GC program was the
same as previously described for GC/MS analy-
sis. The flows of the carrier gas (He), make up
gas (N2), fuel gas (H2) and oxidizing gas (Air)
were 1 ml/min; 25 ml/min; 30 ml/min and 400 ml/
min, respectively. The temperature of the FID
detector was set at 300°C.
Data processing was performed using MSD
ChemStation (revision F.01.00.1903) in combina-
tion with AMDIS (revision 2.70) and NIST MS
Search (version 2.0g) softwares (Agilent Tech-
nologies, USA). Retention indices of the compo-
nents from the sample were experimentally de-
termined using a homologous series of n-alkanes
from C8-C20 (Alkane standard solution C8-C20,
analytical standard, contains C8-C20, ~40 mg/l
each in hexane, Sigma-Aldrich, USA) as stan-
dards. 20 μl of alkanes solution were diluted with
200 μl of hexane and the solution prepared was
analyzed under identical GC/MS and GC/FID
conditions used for the sample. The retention in-
dices of sample compounds represent their re-
tention times normalized to the retention times of
the adjacent eluting n-alkanes. The purpose of
retention indices determination is to compare the
retention of compounds analyzed under different
conditions (different column dimensions, carrier
gas flows and oven temperature programs) as long
as the stationary phase is the same. The identifi-
cation of compounds was based on the compari-
son of experimentally obtained retention indices
(RIexp-Table 1) with those found in the literature
14 (RIlit-Table 1) as well as on the comparison of
EI mass spectra of essential oil components with
data from Willey 6, NIST11 and RTLPEST 3 mass
spectra libraries. The percentage composition of
particular component in the essential oil was de-
termined on the basis of area percent report
(uncalibrated calculation procedure) generated by
Agilent ChemStation software.
Antimicrobial activity
The following microorganisms were selected to
determine the antimicrobial activity of birch buds
essential oil: Staphylococcus aureus (ATCC
25923), Bacillus cereus (ATCC 11778), Bacil-
lus luteus (clinical isolate), Listeria mono-
cytogenes (ATCC 19166), Pseudomonas
aeruginosa (ATCC 2785), Proteus vulgaris
(ATCC 8427), Klebsiella pneumoniae (ATCC
700603), Escherichia coli (ATCC 25922) and
Candida albicans (ATCC 10259). Mediums
used for the growth of the microorganisms: nutri-
ent agar (Merck, Germany) for bacterial growth
and Sabouraud maltose agar (Torlak, Belgrade)
for fungus. Microorganisms are from the collec-
tion of the Microbiology Laboratory, Faculty of
Technology, Leskovac.
The agar disc-diffusion method was used for
testing the antimicrobial activity of birch buds es-
sential oil. The mediums were sterilized for 15
minutes in an autoclave at 121°C under 110 kPa.
An inoculum of 0.1 cm3 of culture was added to
10 cm3 of the medium and poured into petri dishes.
Sterilised filter paper disks (12.7 mm dia.,
Schleicher & Schuell) were placed on the sur-
face of inoculated mediums and impregnated with
60 μl of the essential oil (the ratio of essential oil
and dimethylsulfoxide (DMSO) was 1:7 v/v in
DMSO). The plates were incubated for 24 hours
at 37°C for bacteria, and 48 hours at 25°C for
fungus.
After incubation, the inhibition zone diameters
were measured and expressed in mm (the ex-
periments were carried out in three replications
and the results represent the mean value ± stan-
dard deviation). The presence of the inhibition
zone indicates the activity of the tested samples
against bacteria or fungus. Standardized discs of
Ampicilin (10 μg/disc), Bactrim (25 μg/disc),
Cefalexin (30 μg/disc) (Bio Rad) and Nystatin
(100 U/disc) (Bioanalyse) diameter 6 mm were
used as reference standards. DMSO was used
as negative control. Minimal inhibitory concen-
tration (MIC) was determined against the isolates
which were sensitive to birch buds essential oil.
Determination of MIC was performed according
to Clinical and Laboratory Standard Institute
(2012) protocols 15.
Marijana S. Vladimirov et al., / TEOP 22 (1) 2019 120 - 130 122
Antioxidative activity
DPPH assay
The ability of the essential oil to scavenge free
DPPH radicals was determined using the DPPH
assay. Essential oil was dissolved in the ethanol
and a series of different concentrations was pre-
pared. Ethanol solution of DPPH radical (1 ml,
150 μmol solution (1.5x10-4 mol/l)) was added to
2.5 ml of the prepared essential oil solutions. Ab-
sorption was measured at 517 nm after 20, 30
and 60 minutes incubation with radical. Absorp-
tion at 517 nm was determined for the ethanolic
solution of DPPH radical as well, which was di-
luted in the aforementioned ratio (1 ml of the
DPPH radical of the given concentration with 2.5
ml ethanol added). Ethanol was used as a blank.
Free radical scavenging activity was calculated
according to the formula:
DPPH radical scavenging capacity (%) = 100
- [(As - Ab) * 100/Ac]
AS - Absorption of the “sample” at 517 nm.
“Sample” - ethanolic solution of the essential oil
treated with DPPH radical solution
AB - Absorption of the “blank” at 517 nm.
“Blank” - ethanolic solution of the essential oil
which is not treated with DPPH radical solution
AC - Absorption of the “control” at 517 nm.
“Control” - ethanolic solution of the DPPH radi-
cal
All absorptions were measured on UV-VIS
VARIAN-Cary 100 Conc. Spectrophotometer.
Essential oil concentration needed for the neu-
tralization of 50 % of the initial DPPH radical
concentration is called EC50 value. This value was
determined by using linear regression analysis in
the concentration range 1.055-4.22 mg/ml of es-
sential oil added to the reaction mixture.
Results
Qualitative and quantitative composition of
birch essential oil
The average essential oil yield, after 3 repeated
hydrodistillations was 1 %. The qualitative and
quantitative composition of essential oil from birch
buds determined by GC/MS and GC/FID meth-
ods respectively (Table 1).
GC/FID chromatogram of the isolated essen-
tial oil is shown in Figure 1. The main peaks are
marked with numbers from 1-27 according to the
order of their elution from HP-5MS column.
Twenty-seven compounds, comprising 93.5 %
of the total oil composition, were identified by GC/
MS in this study. All identified components were
divided in two groups: sesquiterpene hydrocarbons
and oxygenated sesquiterpenes.
Twenty sesquiterpene hydrocarbons were iden-
tified in this study comprising 78.7 % of the total
oil composition. The most abundant sesquiterpene
hydrocarbons were germacrene D (21.7 %) and
δ-cadinene (17.0 %), followed by α-copaene (7.0
%), γ-cadinene (5.8 %) and (E)-caryophyllene (4.0
%). Their structures are shown in Figure 2.
Oxygenated sesquiterpenes contribute with 14.8
% in the total composition of isolated essential oil.
The most abundant compounds from this group
were sesquiterpene alcohols α-cadinol (5.8 %)
and its isomer α-muurolol (torreyol) (5.0 %). Their
structures are shown in Figure 3.
Antimicrobial activity of birch buds essen-
tial oil
Antimicrobial activity of birch buds essential oil
was determined against nine microorganisms: S.
aureus, B. cereus, B. luteus, L. monocytogenes,
P. aeruginosa, P. vulgaris, K. pneumoniae, E.
coli and C. albicans. The results of antimicrobial
activity are given in Table 2.
The birch essential oil showed antimicrobial ac-
tivity against B. luteus, P. vulgaris and K.
pneumoniae (Table 2). Diameter of the inhibition
zone diameter against B. luteus was 18 mm, while
the MIC value was the lowest observed at 32 μl/
ml. There is no literal data about antimicrobial
activity of birch essential oil.
Highest antimicrobial activity was observed
against P. vulgaris with inhibition zone of 4.15
mm (Table 2). The isolated essential oil showed
more antimicrobial activity than Ampicillin, a com-
mercial broad-spectrum antibiotic (inhibition zone
width of 3.6 mm, Table 2). MIC value was the
highest for these bacteria at 128 μl/ml.
The essential oil isolated from birch buds in this
study has shown antimicrobial activity against K.
pneumoniae almost twice better than commer-
cial cephalosporin antibiotic Cephalexin (the di-
ameter of the inhibition zone width in the case of
Marijana S. Vladimirov et al., / TEOP 22 (1) 2019 120 - 130 123
Table 1. Chemical composition of birch buds essential oil
No. R.T. Compound Molecular RI exp RI lit Method of Composition
min formula identification %
1 24.59 α-Cubebene C15H24 1351 1345 RI, MS 0.5
2 25.33 α-Ylangen C15H24 1375 1373 RI, MS 0.2
3 25.47 α-Copaene C15H24 1379 1374 RI, MS 7.0
4 25.78 β-Bourbonene C15H24 1389 1387 RI, MS 0.4
5 25.92 iso-Longifolene C15H24 1393 1389 RI, MS 0.4
6 26.88 E-Caryophyllene C15H24 1424 1417 RI, MS 4.4
7 27.16 β-Copaene C15H24 1433 1430 RI, MS 1.0
8 27.93 α-Humulene C15H24 1458 1452 RI, MS 3.7
9 28.16 allo-Aromadendrene C15H24 1466 1458 RI, MS 3.6
10 28.59 γ-Muurolene C15H24 1480 1478 RI, MS 3.8
11 28.77 Germacrene D C15H24 1486 1484 RI, MS 21.7
12 29.13 γ-Amorphene C15H24 1497 1495 RI, MS 1.8
13 29.28 α-Muurolene C15H24 1503 1500 RI, MS 4.0
14 29.50 δ-Amorphene C15H24 1511 1511 RI, MS 0.6
15 29.72 γ-Cadinene C15H24 1518 1513 RI, MS 5.8
16 29.98 δ-Cadinene C15H24 1527 1522 RI, MS 17.0
17 30.06 Zonarene C15H24 1530 1528 RI, MS 1.1
18 30.25 trans-Cadina-1,4-diene C15H24 1536 1533 RI, MS 0.4
19 30.39 α-Cadinene C15H24 1541 1537 RI, MS 1.1
20 30.56 α-Calacorene C15H20 1547 1544 RI, MS 0.2
21 32.08 Salvial-4(14)-en-1-one C15H24O 1600 1594 RI, MS 0.3
22 32.78 Junenol C15H26O 1625 1618 RI, MS 1.1
23 32.99 1-epi Cubenol C 15H26O 1633 1627 RI, MS 0.8
24 33.34 α-Muurolol (Torrejol) C15H26O 1645 1644 RI, MS 5.0
25 33.47 Cubenol C15H26O 1650 1645 RI, MS 1.1
26 33.71 α-Cadinol C15H26O 1659 1652 RI, MS 5.8
27 34.58 Eudesma-4(15), C15H24O 1691 1687 RI, MS 0.7
7-dien-1β-ol
Total identified 93.5
Grouped components (%)
Sesquiterpene hydrocarbons (1-20) 78.7
Oxygenated sesquiterpenes (21-27) 14.8
essential oil is 6.65 mm in comparison to 3.5 mm
in the case of antibiotics, Table 2) while deter-
mined MIC value was 64 μl/ml.
Antioxidative activity of birch essential oil
Essential oil concentrations needed for the neu-
tralization of 50 % of the initial DPPH radical
concentration (EC50 value) were 3.31 mg/ml (af-
ter 20 minutes); 2.67 mg/ml (after 30 minutes)
and 1.70 mg/ml (after 60 minutes incubation with
radical) (Figure 4). The synthetic antioxidant BHT
showed EC50 value of 0.021 mg/ml after 20 min-
utes incubation with DPPH radical 16 .
DPPH radical neutralization capacity depends
on the applied oil concentration, as well as on the
incubation time - it increases with the concentra-
tion and time increase. The essential oil showed
the best antioxidant activity (with the degree of
DPPH radical neutralization of %) after 90 min-
utes of incubation.
Marijana S. Vladimirov et al., / TEOP 22 (1) 2019 120 - 130 124
Figure 1. GC-FID chromatogram of essential oil from birch buds
Marijana S. Vladimirov et al., / TEOP 22 (1) 2019 120 - 130 125
Figure 2. Structures of the most abundant sesquiterpene hydrocarbons in birch buds essential oil
Figure 3. Structures of the most abundant sesquiterpene alcohols in birch buds essential oil
Figure 4. Antioxidant activity of birch buds essential oil
Marijana S. Vladimirov et al., / TEOP 22 (1) 2019 120 - 130 126
Table 2. Antimicrobial activity of selected antibiotics and birch buds essential oil
Inhibition zone diameter (mm)
Microorganism MIC Essential Antibiotic
(μμ
μμ
μl/ml) oil A B C N
Staphylococcus aureus - - 36.7 42.1 26.0 n.t.
Bacillus cereus - - n.t. n.t. n.t. n.t.
Bacillus luteus 32 18.0±0.15 n.t. n.t. n.t. n.t.
Listeria monocytogenes - - n.t. n.t. 34.0 n.t.
Pseudomonas aeruginosa - - n.t. n.t. n.t. n.t.
Proteus vulgaris 128 21.0±0.10 13.2 22.9 n.t. n.t.
Klebsiella pneumoniae 64 26.0±0.15 n.t. n.t. 13.0 n.t.
Escherichia coli - - n.a. 15.0 26.0 n.t.
Candida albicans - - n.t. n.t. n.t. 17.0
MIC - minimal inhibitory concentration
A-Ampicilin; B-Bactrim; C-Cefalexin
N-Nystatin; n.t. - not treated; n.a. - no activity
Discussion
Qualitative and quantitative composition of
birch essential oil
Generally, essential oils are complex mixtures
of volatile organic compounds. The most abun-
dant components in the essential oils are hydro-
carbon terpenes (monoterpenes and sesquiterpe-
nes) and their oxygenated derivatives (terpenoids),
phenolic compounds and their derivatives, other
oxygenated molecules (alcohols, ethers, acids,
etc.) as well as phenylpropanoids and heterocy-
clic compounds, containing nitrogen and sulfur
atoms 17. Birch buds represented significant
source of sesquiterpenes, especially germacrene
D and δ-cadinene.
The sesquiterpenes are of special interest in the
natural products chemistry 1,3,6-8,10-12,18-22. More
than thousand sesquiterpene structures are known
nowadays. Therefore, they represent the most
numerous class of terpenes derived from farnesyl
pyrophosphate and composed of three isoprene
units, i.e. 15 carbon atoms. In comparison to
monoterpenes, molecules composed of two iso-
prene units; sesquiterpenes have less volatility and
therefore make a lesser contribution to the scent
of essential oils. On the other side, antioxidant and
antimicrobial activity of essential oils are often
ascribed to the presence of sesquiterpenes 17.
The results obtained in this study are in agree-
ment with results of some previous studies inves-
tigating birch essential oil 1,7. They have also
showed that in the essential oil of fresh white birch
buds obtained by Clevenger hydrodistillation, col-
lected in Russia, in the period of plant’s physi-
ological hibernation (dormancy) - October 1997,
March and November 1998 and February 1999,
the most abundant sesquiterpenes (contributing
with 67.6 % ± 4.9 % in the total composition)
were germacrene D (14.8 %) and δ-cadinene
(13.5 %).
Completely different composition of essential
oil from birch buds was obtained in the study of
Demirci and Baser 11. The most dominant com-
ponents in the oil obtained from dried birch buds,
collected from the Turkey in May, were α-betulenol
(25.3 %) and 14-hydroxy-4,5-dihydro-β-caryo-
phyllene (17.2 %) which were not identified in
our study. In addition, the content of δ-cadinene,
one of the most abundant compounds in the es-
sential oil isolated in our study (17.0 %), was sig-
nificantly less (0.1 %) in the oil studied earlier 11.
On the other side, the presence of germacrene
D, the most abundant compound in the oil isolated
in our study (21.7 %) was not confirmed in previ-
ous study 11.
The different qualitative and quantitative com-
position of the essential oil isolated in our study in
comparison to other studies is most probably due
Marijana S. Vladimirov et al., / TEOP 22 (1) 2019 120 - 130 127
to the different climatic and vegetation conditions,
the collection period of plant material, the way of
storage and processing of plant material etc.
Therefore, further research on this topic is nec-
essary in order to obtain as much as possible data
about the possible use of B. pendula buds as a
source of sesquiterpenes.
Antimicrobial activity of birch buds essen-
tial oil
The results obtained from antimicrobial activity
of isolated essential oil showed that birch buds
could be used as a source of natural antimicrobial
compounds as a safer alternative to the synthetic
antimicrobial agents. Among the tested microor-
ganisms, K. pneumoniae was the most sensitive
to the effect of isolated essential oil.
Germacrene D exhibits antibacterial, antioxi-
dative and cytotoxic effects on certain human tu-
mor cells. Also, it is the precursor of different ses-
quiterpenes (cadinene, selinene) 19,20,23,24. Biologi-
cal role of germacrene D in plants is still not clear
but it is assumed that it repels herbivores, acts as
an insecticide against mosquitoes and as repel-
lent against louses and ticks 17.
δ-Cadinene was identified as the most abun-
dant component in the essential oil isolated from
K. longipedunculata (contributing with 21.8 %
of the total oil composition). It showed antibacte-
rial activity against Gram-positive cocci S.
agalactiae and S. pyogenes 25 . δ-Cadinene also
showed anti-inflammatory and antiproliferative
activity 21,22. α-Copaene exerted antibacterial
activity against some Gram-positive (B. subtilis,
S. aureus 26 and Gram-negative bacteria. It also
showed dose dependent cytotoxic effect, without
genotoxic and mutagenic effects 27,28. (E)-
Caryophyllene exhibited cytotoxic activity against
certain human tumor cells 19. Antibacterial activ-
ity of essential oil isolated from Teucrium
divaricatum Sieb. Ssp. villosum (Celak.) Rech.
Fil. (Lamiacae) against Gram-positive bacteria B.
subtilis, S. epidermidis, S. aureus and S.
faecalis was ascribed to the high content (30.1
%) of (E)-caryophyllene 29. α-Cadinol and α-
Muurol are sesquiterpenes alcohols having anti-
parasitic activity on certain Dermatophago-
phagoides subspecies (home mites) 30. α-Cadinol
also exerts antimicrobial and strong antifungal
effect 30,31.
Antimicrobial activity of the essential oil isolated
in our study is most probably due to the synergis-
tic action of sesquiterpene compounds, first of all
the most abundant, germacrene D and δ-cadinene.
Antioxidative activity of birch essential oil
Most of actually used antioxidans are of syn-
thetic origin and there is an evidence about their
pernicious and toxic effects on human health. In-
terest in natural antioxidants is increasing in re-
cent years, especially plant origin antioxidant com-
pounds. Plant - derivate antioxidants are safer
alternative of synthetic antioxidants 13. Isolated
essential oil displayed significant antioxidant ac-
tivity in the DPPH radical-scavenging assay, and
could be a safer alternative to the synthetic anti-
oxidant agents.
A previous investigation determined the antioxi-
dant activity of two different birch species B.
pubescens ssp. pubescens and B. pubescens ssp.
czerepanovii by measuring their ability to scav-
enge DPPH radicals 7. The test was performed
on the samples at concentrations of 0.5 and 1.0
mg/mL but scavenging activity of the radicals was
not determined.
However, no data of the antioxidative activity
of the birch essential oil can be found in the litera-
ture. Our results showed that Betula pendula
buds essential oil could represent an accessible
source of natural antioxidants for use in pharma-
ceutical, cosmetic and food industry.
Conclusions
According to the results obtained in this study,
the essential oil isolated from birch buds grown
on the territory of Vrnjacka banja (Central Serbia)
could be potential source of pharmacologically
active compounds significant for the application
in medicine, pharmacy and food industry. How-
ever, it is necessary to perform further research
that will give closer data about birch buds as a
source of pharmacologically active compounds
and their use in the prevention or therapy of dif-
ferent infections. We can conclude that birch es-
sential oil could be a safer alternative to the syn-
thetic antioxidant and antimicrobial agents with
potential application in pharmaceutical and food
products.
Marijana S. Vladimirov et al., / TEOP 22 (1) 2019 120 - 130 128
Acknowledgment
This work is a part of the research project “Plant
and synthetic bioactive products of new genera-
tion” no. TR 34012, financed by the Ministry of
Education, Science and Technological Develop-
ment of the Republic of Serbia.
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The in vitro and in vivo antioxidant activities of ethanolic extracts of the vegetative organs of Betula pendula Roth. were studied by evaluating the ability to scavenge the DPPH radical, ABTS cation radical or hydrogen peroxide or by the metal chelation activity or reducing power. The phospholipid oxidation processes were studied using the acute hypoxia model. The changing proportion between acid and neutral phospholipids was used for the estimation of the influence of the birch extract on brain tissue. As an index of the pathological state the information entropy parameters were calculated. It has been found that the rats which had experienced ischemia have a decreased amount of acid PL in the homogenates. Preventive injection of birch extracts brings about normalization of PL level. This fact points out a possibility of applying extracts of Betula Pendula Roth in order to regulate the PL level of experimental rats as well as an estimation of the antioxidant effect of exogenous substances. Key words: Betula vegetative organs, Antioxidant activity, Lipid peroxidation, Phosholipids Mashentseva et al. Comparison of the Antioxidant Activity of the Different Betula pendula Roth. Extracts from Northern Kazakhstan. J Phytol 3/1 (2011) 18-25.
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