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The Pacific Journal of Science and Technology –85–
https://www.akamai.university/pacific-journal-of-science-and-technology.html Volume 23. Number 1. May 2022 (Spring)
Chemical Composition, Antioxidant, and Antibacterial Activity of the Essential Oil
from the Leaves of Pinus sylvestris
Isaac S. Njoku1,2,3*; Nisar-Ur Rahman3; Ahsan Mohammed Khan3;
Idowu Otunomo4; Olayinka T. Asekun2; Oluwole B. Familoni2; and Chibuko Ngozi5
1Department of Chemistry, Faculty of Basic Medical and Applied Sciences, Trinity University, Yaba,
Lagos, Nigeria.
2Department of Chemistry, Faculty of Science, University of Lagos, Akoka-Yaba, Lagos, Nigeria.
3Department of Pharmacy, COMSATS University, Islamabad, Abbottabad Campus, Pakistan.
4Department of Chemistry, Faculty of Science, University of Nigeria, Nsuka, Enugu State, Nigeria.
5Department of Fisheries, Faculty of Science, University of Lagos, Akoka-Yaba, Lagos, Nigeria
E-mail: isaac.njoku@trinityuniversity.edu.ng*
ABSTRACT
The essential oil of air-dried leaves of Pinus
sylvestris obtained through hydro-distillation was
characterized by gas chromatography-flame
ionization detection (GC-FID) and gas
chromatography-mass spectrometry analyses
(GC-MS). The oil yielded 0.78% per dry weight of
sample. The oil was composed majorly of mono-
and sesquiterpenoids. The major components of
the essential oil of P. sylvestris were humulene
(13.24%), α-guaiene (11.57%), allo-ocimene
(9.40%), terpinolene (8.84%), caryophyllene
(8.84%), and terpineol (5.02%). The air-dried leaf
oil showed strong activity against Pseudomonas
aeruginosa, Escherichia coli, and Klebsiella
pneumoniae, as well as a promising antioxidant
potency. To our knowledge, this is the first report
concerning chemical composition and
antimicrobial activities of the essential oil from
Pinus sylvestris.
(Keywords: essential oil, Pinus sylvestris, Scots pine,
Scotch pine, Baltic pine, GC-MS, GC-FID, antibacterial,
humulene, antioxidants)
INTRODUCTION
Reactive oxygen species (ROS) are formed as a
natural byproduct of the normal metabolism
of oxygen and environmental stress can increase
their level dramatically resulting in significant
damage to cell structures1. This is cumulatively
known as oxidative stress. They are chemically
reactive chemical species containing oxygen.
Examples include peroxides, superoxide, hydroxyl
radical, and singlet oxygen2.
Molecular oxygen (O2) is the premier biological
electron acceptor that serves vital roles in
fundamental cellular functions3. However, its
reduction in the cells produces superoxide, which
is the precursor of most other reactive oxygen
species4. The damaging effect of these oxygen
species include apostasies, cardiovascular
diseases, oxidative damage, cellular ageing, and
cancer 5,6,7,8,9.
The damaging effects of these reactive oxygen
species can be mitigated through the positive
effects of antioxidants, both natural and synthetic.
Natural antioxidants are the secondary
metabolites of phytochemicals and are preferred
over synthetic antioxidants, which are found to
impose side effects10.
Pinus sylvestris is of the genus pinus belonging
to the family Pinaceae, is a species widely
distributed from Europe, including Britain, from
Scandanavia South and East to Spain, Albania,
temperate Asia, and tropical locations11. The
medicinal properties of various parts of P.
sylvestris have been studied by many
researchers. It has been used as an antiseptic
agent and is known to have beneficial effect on
the respiratory system12.
It is a valuable remedy in the treatment of kidney,
bladder, and rheumatic affections, and also in
diseases of the mucous membranes and the
treatment of respiratory complaints. Externally it
is used in the form of liniment plasters and
inhalers13. The leaves and young shoots are
antiseptic, diuretic, and expectorant. They are
harvested in the spring and dried for later use.
They can be added to the bath water for treating
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fatigue, nervous exhaustion, sleeplessness, and
skin irritations. They can also be used as an
inhalant in the treatment of various chest
complaints14. The essential oil from the leaves is
used in the treatment of asthma, bronchitis, and
other respiratory infections, and also for digestive
disorders such as excessive flatulence. An
essential oil obtained from the seed has diuretic
and respiratory-stimulant properties12.
Although some earlier studies revealed the
medicinal attributes of P. sylvestris, very little work
has been done on the antioxidant and
antimicrobial activities of the essential oil of this
plant. It is on this backdrop that this research was
undertaken.
EXPERIMENTAL
Plant Material and Essential Oil Extraction
Technique
The healthy leaves of Pinus sylvestris were
collected from the botanical garden of the
University of Lagos, Akoka, Yaba Area of Lagos
State, Nigeria in August 2020. The botanical
identification and authentication was done in the
Herbarium of the Department of Botany,
University of Lagos, Nigeria. The fresh leaves of
P. sylvestris were air-dried for a period of one
week and pulverized using a mechanical grinder
prior to extraction. The essential oils from the air-
dried leaves were obtained by hydro-distillation of
300g each of the plant material using the modified
Clevenger-type apparatus15. The oil was dried
over anhydrous sodium sulphate and stored in a
refrigerator prior to analysis.
GC–FID and GC/MS Analyses of Volatile Oils
The essential oil samples were analyzed using a
Varian CP-3800 gas chromatograph fitted with a
flame ionization detector (FID) and
dimethylpolysiloxane (100%) column (CP Sil-5
CB: 50 m length × 0.25 mm i.d. × 0.4 μm film
thickness) (Varian, Netherlands). Nitrogen was
the carrier gas with a 16-psi inlet pressure.
Samples (0.2 μL) were injected in split mode with
a ratio of 1:100. The column was initially held at
60°C for 5 minutes then heated to 220°C at a
5°C/minute ramp rate and was held for 3 minutes
at that temperature. The temperature was further
raised to 250°C at a 5°C/minute ramp rate and
was held at this temperature for 4 minutes. The
injector and detector temperatures were
maintained at 250° and 300°C, respectively.
The gas chromatography/mass spectrometry
(GC/MS) analyses performed on a Perkin Elmer
Turbo mass Clarus 600 Instrument at 70 eV
ionization energy with a mass range of 40–500
amu, employing an Elite-5 column (5 % phenyl
and 95 % dimethylpolysiloxane) of 30 m length,
0.25 mm internal diameter and 0.25 μm film
thickness (PerkinElmer, USA). Helium (1 mL/min)
was used as a carrier gas. The initial temperature
was 60 °C (1 min), this was increased to 240 °C
at rate of 6 °C/min, and remained at 240 °C for 6
min, and then continued to increase to 250 °C at
rate of 10 °C/min, with a final stage of 10 min at
250 °C. The oven temperature was programmed
from 50 °C to 250 °C at a 5 °C/min dynamic rate
and remained for 15 min at 250 °C. Sample (0.1
uL) were injected with a split less mode.
Identification of Volatile Oil Constituents
Component identification was accomplished by
comparison of the retention indices (RI) of the
GC peaks with those obtained using saturated n-
alkanes (C8–C30) (Aldrich, USA), those reported
in the literature16,17,18,19 and by comparison of the
mass spectra of the peaks with those reported in
the literature20,21 and stored in the NIST library.
Peak area percentages were calculated from
GC–FID response without employing correction
factors. RI values were calculated for all
components using a homologous series of n-
alkane. Mixtures (C7-C30) injected under
conditions similar to those of the samples and
computer matched with the NIST libraries.
Antioxidant Assay
DPPH Radical Scavenging Assay: The free
radical scavenging capacity of the compounds
was measured by 1,1-diphenyl-2-picrylhydrazyl
(DPPH) method22,23 with modifications. The
essential oil was allowed to react with stable free
radical, DPPH for half an hour at 37 OC. The
concentration of DPPH was 1 mM. The oils (10,
20, 30, 40 and 50 µL) were mixed with DPPH
prepared in methanol. Ascorbic acid (4 mg/mL in
methanol) was used as positive control. DPPH
solutions at the same concentration without the
tested oil was used as negative control. Each
sample, as well as each control was analyzed in
triplicate. The end volume for each sample was
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100 µL in each well of the 96 well plate. After
incubation, decrease in absorbance was
measured at 517 nm using microplate reader
(BMG Labtech Fluostar Omega UV-VIS
microplate reader Instrument, Inc., USA).
Percentage radical scavenging activity was
calculated using the formula:
AC = Absorbance of control.
AS = Absorbance of Sample.
In order to calculate the IC50, the essential oil was
prepared in a series of concentrations of 1, 10, 20,
40, 60, 80, 200, 400, 800, and 2000 μg/mL. The
test was repeated as described above for all
concentration of each oil in triplicates. Inhibition %
was plotted against concentration and the IC50
was calculated graphically.
FRAP - Ferric Reducing Antioxidant Power
Assay: Ferric ion reducing capacity of the
essential oil of P. sylvestris was conducted using
the method described by24. The ability of the
essential oil to reduce ferric tripyridyltriazine
(Fe(III)-TPTZ) complex to its ferrous colored form
(Fe(II)-TPTZ) at low pH was determined using a
spectrophotometer. 1.5 mL of FRAP reagent (2.5
mL of 10 mM TPTZ solution in 40 mM HCl, 2.5 mL
of 20 mM FeCl3 and 25 mL of 0.3 M acetate
buffer, pH 3.6) was added to 50 µL of each
sample (100 µg/mL). After incubation at 37 °C for
10 min, the absorbance was measured at 593 nm.
FRAP reagent without the sample was as blank
and the experiment was performed in triplicate.
Different concentration of aqueous solution of
FeSO4.7H2O (in a range of 125-1000 μmol/L) was
used for calibration curve. The relative antioxidant
activities of samples were reported as mmole
Fe2+/100 g of fractions.
β-Carotene Bleaching Test: The β-carotene
bleaching capacity of the essential oil of P.
sylvestris was conducted using the method of
Kelvin et al25 with slight modification; 10 mg of β-
carotene was dissolved in 10 mL of chloroform.
The carotene-chloroform solution, 0.2 mL, was
pipetted into a boiling flask containing 20 mg
linoleic acid and 200 mg Tween 40. Chloroform
was removed using a rotary evaporator and 50
mL of distilled water were added slowly with
vigorous agitation to the residue, to form an
emulsion. 5 mL of the emulsion were added to a
tube containing 2 mg of essential oils and the
absorbance was immediately measured at 470
nm against a blank, consisting of an emulsion
without β-carotene. The tubes were placed in a
water bath at 50 oC and the oxidation of the
emulsion was monitored spectrophotometrically
by measuring absorbance at 470 nm over a 60
min period. Control samples contained 10 μL of
water instead of essential oils. Butylated hydroxy
anisole (BHA) was used as a reference.
The antioxidant activity was expressed as
inhibition percentage with reference to the control
after a 60 min incubation using the following
equation:
AA = 100(DRC − DRS)/DRC
where AA is the antioxidant activity, DRC is the
degradation rate of the control = [log (a/b)/60],
DRS is the degradation rate in presence of the
sample = [log (a/b)/60]; a is the absorbance at
time 0; b is the absorbance at 60 min.
Antibacterial Assay
The essential oil of P. sylvestris was tested on
three different bacterial strains. The strains were
maintained at 4 °C and they are, P. aeruginosa
ATCC 21234, K. pneumoniae ATCC 15522 and
E. coli ATCC 25922. The bacterial strains were
cultured in a Thermo Scientific Oxoid Nutrient
agar (NA) at 37 °C for 24 hours. The disc
diffusion method26 was used to determine the
antimicrobial activities of the essential oils. Petri
plates were prepared by pouring 20 mL Thermo
Scientific Oxoid Nutrient agar (NA) and the
solution was allowed to solidify. The plates were
then dried, and 0.1 mL of the standardized
inoculum containing 106-107 colony-forming
units/mL of the bacterial suspension was poured,
uniformly spread, and allowed to dry for 5
minutes.
The essential oil was prepared in dimethyl
sulfoxide (DMSO) at a concentration of 1 mg/mL.
100 μL was taken from this stock solution and
was added to respective wells. The control well
received only 100 μL DMSO. Gentamycin
(positive control) was used as the reference
antibiotics. The plates were left at room
temperature to allow diffusion and then incubated
at 37 °C for 24 hours for bacterial growth. The
antimicrobial activity was evaluated by measuring
the diameter of the zones of inhibition against the
test organisms. The experiments were repeated
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in triplicate and the results are expressed as
average values.
The minimum inhibitory concentration (MIC) was
determined using the broth microdilution method
using 96-well microplates. The inoculum of the
microbial strains was prepared from 24 to 48
hours broth cultures and suspensions were
adjusted to 0.5 McFarland standard turbidity.
Serial concentrations (500, 250, 125, 62.5, 31.3,
15.6, 7.81, 3.9, 1.95, 0.98, and 0.49 μg/mL) of
essential oil were prepared. 100 μL from culture
broth was mixed with 100 μL of different
concentration of the essential oils of P. sylvestris
in the corresponding well and plates were
incubated either at 37 °C for 24 hours for
antibacterial activity. The lowest concentration of
the tested oil showing no microbial growth was
defined as the MIC.
Minimum bactericidal concentration (MBC) values
were determined by taking a part of the liquid from
each well that showed no growth and incubating
on agar plates at 37 °C for another 24 hours. The
lowest concentration that disclosed no visible
growth of bacteria or fungi was confirmed as
MBC.
RESULTS AND DISCUSSION
The yield of leaf essential oil obtained by hydro-
distillation was 0.65 % (w/w relative to dry material
weight). The analysis by gas chromatography-
flame ionization detection (GC-FID) and gas
chromatography-mass spectrometry (GC-MS)
identified 35 volatile compounds, accounting for
98.86 % of the total extracted oil, which were
identified by matching retention times of available
authentic standards, retention indices (RIs), and
mass spectra in the NIST 17 database (Table 1).
The essential oil was mainly composed of 13
monoterpene hydrocarbon (37.14%), 2
oxygenated monoterpenes (5.71%), 13
sesquiterpene hydrocarbon (37.14%), 5
oxygenated sesquiterpenes (17.14%) and 1
diterpenes (2.85%). As shown in Table 1, the
major compounds are humulene (13.24%), α-
guaiene (11.57%), allo ocimene (9.42%),
terpinolene (8.84%), caryophyllene (8.27%), γ-
terpinene and terpineol (5.02%).
The essential oil P. sylvestris from Turkey
contained α-pinene, camphene, and β-pinene as
major constituents27 while those from Lithuania
had significantly higher concentration of γ-
Terpinene, Caryophyllene oxide, δ-3-Carene, α-
Terpinene, γ-Terpinene and Terpinolene.
Sabinene + β-Pinene, 1-epi-Cubenol, Camphene,
Sabinene + β-Pinene, Myrcene, α-Cadinene and
1-epi-Cubenol28. In another research, the
essential oil of from the twigs of P.sylvestris was
composed 49·2% α-pinene, 30·1% sabinene,
14·9 % β-pinene and 7·9 % limonene while the
needles had 69·5 % α-pinene, 14·9 %
camphene, 9·1 % β-pinene, 3·6 % sabinene and
2·8 % limonene29.
The essential oil from this research (Nigeria) had
some components common to those from
Lithuania, Turkey and Spain. These components
include: α-terpinene, γ-terpinene and terpinolene.
However, this research reports the presence of
some major components not found in or present
in very low composition in the essential oil P.
sylvestris earlier reported.
These components include humulene, allo
ocimene, α-guaiene, caryophyllene,
Isoaromadendrene epoxide and longiverbinone.
Worthy of note is the presence of diterpene
hydrocarbon, cembrene in the essential oil from
this research, which has never been reported in
earlier reports. These variations observed in the
chemical composition of the essential oil may be
due to geographical location. A different
chemotype is suggested.
Antimicrobial Activity
The in vitro antimicrobial activities of essential oil
against 3 pathogenic microorganisms (Klebsialla
pneumoniae, Escherichia coli, and Pseudomonas
aeruginosa) were evaluated using the disc
diffusion method. The disc diameters of the zone
of inhibition and the MIC of the essential oil for
the tested microorganisms are shown in Table 2.
The essential oil was effective against K.
pneumoniae, E. coli and P. aeruginosa with
inhibition zones of 21.50, 18.90, and 20.20 mm
respectively. In the broth microdilution assay, the
essential oil showed the highest sensitivity to K.
pneumoniae with MIC and MBC values of 65.5
and 135.6 μg/mL, followed by E. coli (MIC and
MBC values of 105.2 and 215.50 μg/mL), and P.
aeruginosa (MIC and MBC values of 78.3 and
156.70 μg/mL).
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Table 1: Chemical Composition of the Essential Oil from Pinus sylvestris.
Compounds
aRI
bRI
%
α-Pinene
931
932
1.25
Fenchene
946
945
0.38
Camphene
942
946
1.47
Sabinene
961
967
0.24
β-Pinene
980
974
1.66
2,4,6-Octatriene, 3,4-dimethyl-
998
993
0.78
3-Carene
1014
1008
0.05
Benzene, 1-methyl-3-propyl-
1038
1042
1.85
γ-Terpinene
1048
1054
7.87
Terpinolene
1086
1086
8.84
Cycloheptene, 5-ethylidene-1-methyl-
1090
1095
1.55
Allo-Ocimene
1122
1128
9.42
Terpinen-4-ol
1165
1174
0.55
Terpineol
1174
1186
5.02
α-Terpinyl acetate
1342
1346
0.92
Bicyclo[5.2.0]nonane, 4-methylene-2,8,8-trimethyl-2-vinyl-
1411
1407
0.43
Isocaryophyllene
1416
1409
2.83
Cedrene
1405
1410
0.81
Caryophyllene
1416
1417
8.27
γ-Elemene
1440
1434
0.12
α-Guaiene
1437
1437
11.57
cis-β-Farnesene
1444
1440
0.52
Humulene
1453
1452
13.24
cis-Muurola-4(15),5-diene
1384
1465
3.23
Cadina-1(10),4-diene
1467
1469
3.44
α-Muurolene
1504
1500
1.72
γ-Cadinene
1514
1513
0.49
(Z)-γ-Bisabolene
1511
1514
0.09
Caryophyllene oxide
1583
1582
0.48
trans-Z-α-Bisabolene epoxide
1590
1586
0.10
Humulene epoxide 2
1603
1608
0.14
Longiverbenone
1637
1632
4.75
Isoaromadendrene epoxide
1645
1639
4.77
11,11-Dimethyl-4,8-dimethylenebicyclo[7.2.0]undecan-3-ol
1641
1646
0.18
Cembrene
1938
1937
0.93
aRI: Retention index determined relative to n-alkanes (C7-C30) on the HP-5ms column.
bRI: literature retention indices16-19
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Table 2: Zones of Growth Inhibition (mm), MICs, and MBCs of Essential Oil from Pinus sylvestris Against
the Growth of Microorganisms. a
Microorganisms
Diameters of Zones of Inhibition
MICs
MCBs
Essential oil
Antibiotic
(μg/mL)
(μg/mL)
Klebsiella pneumonia
21.50±0.2
23.00±0.2
65.50
135.60
Escherichia coli
18.90±0.1
23.00±0.3
105.20
215.50
Pseudomonas aeruginosa
20.20±0.1
21.00±0.2
78.30
156.70
Abbreviations: MBC, minimal bactericidal concentration; MIC, minimum inhibitory concentration. Results were mean ± SD of triplicate values.
Antibiotics used was, gentamicin
Table 3: DPPH, FRAP, and β-Carotene Bleaching Antioxidant Assay of the Essential Oil From Pinus
sylvestris.
Antioxidant Assay
Pinus Sylvestris Essential Oil
Ascorbic Acid (Positive control)
DPPH Assay (μg/mL)
45.12 ± 3.48
40.24 ±. 3.22
FRAP Assay (μg/g)
3.70 ± 0.04
5.55 ± 0.04
β-Carotene Bleaching Assay (%)
80.66 ± 4.34
84.82 ± 4.36
Results of antioxidant capability were reported in mean ± SD of triplicate values.
The promising antibacterial activity of the
essential oil could be attributed to the presence of
major chemical components in the oil. The
antimicrobial activity of humulene and
caryophyllene and terpinene has been
reported30,31,32,33,34.
The activity shown by the essential oil of this plant
could also be attributed to the synergistic effect of
some of its minor components. Earlier reports
have shown that components such
isocaryophyllene, which in minor composition
could also elicit good antimicrobial activities35,36,37
Antioxidant Activity
The antioxidant activity of the essential oil of P.
sylvestries was evaluated using three different
methods. These are, the FRAP, DPPH and β-
carotene bleaching assays (Table 3). The
essential oil of P. sylvestris showed good
antioxidant activity with 45.12 μg/mL, 3.70 μg/g
and 80.66 % for DPPH, FRAP and β-carotene
bleaching assays. The antioxidant activity of the
essential oil of this plant could be attributed to the
presence of some major components of its
essential oil.
The antioxidant property of caryophyllene has
been earlier reported38.The antioxidant effect of
β-caryophyllene protects rat liver from carbon
tetrachloride-induced fibrosis by inhibiting hepatic
stellate cell activation39.
The antioxidant activity of the essential oil of
Photinia serrulata and senecio nudicaulis was
attributed to the presence of α-humulene,
caryophyllene oxide, γ-elemene, β-
caryophyllene, epi-α-cadinol, epi-α-muurolol and
δ-elemene40,41. In the 3 Assays carried out, the
essential oil of P. sylvestris showed antioxidant
potency that was almost similar to that of the
standard drug (Ascorbic Acid) used (Table 3).
CONCLUSION
The essential oil of P. sylvestris was composed
mainly of mono and sesquiterpenoids. The
essential oil showed promising antibacterial and
antioxidant capacity, which is suggested to be
due to the presence of the major and minor
components in the oil and their synergistic effect.
The results indicate that the essential oil of Pinus
sylvestris might be suitable for use as a natural
antibacterial and antioxidant agent.
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ACKNOWLEDGMENT
Profound appreciation to the World Academy of
Sciences (TWAS) for the award of the Scholarship
that enabled this research to be conducted and to
Comsats Institute of Information Technology,
Abbottabad, Pakistan for been a worthy host.
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