Received: 10 December 2022 Revised: 12 April 2023
DOI: 10.1111/1750-3841.16597
ORIGINAL ARTICLE
Fo o d C h e m i s t r y
Chemical composition, antioxidant, and antimicrobial
activities of P. roxburghii oleoresin essential oils extracted
by steam distillation, superheated steam, and supercritical
fluid CO2 extraction
Muhammad Adnan Ayub1
Mazhar Abbas4 Qurat Ul Ain1
1 Department of Chemistry, University of
Sahiwal, Sahiwal, Pakistan
2 Plant
Biotechnology Research Center,
Kermanshah Branch, Islamic Azad
University, Kermanshah, Iran
3 Department of Chemistry, University of
Agriculture Faisalabad, Faisalabad,
Pakistan
4 Department of Biochemistry, University
of Veterinary and Animal Sciences,
Lahore (Jhang-Campus), Lahore,
Pakistan
5 Department of Allied Health Sciences,
University of Sargodha, Sargodha,
Pakistan
6 Department
of Food Science and
Technology, Toyserkan Faculty of
Engineering and Natural Resources,
Bu-Ali Sina University, Hamedan, Iran
Correspondence
Muhammad Adnan Ayub, Department of
Chemistry, University of Sahiwal,
Sahiwal, Pakistan.
Email: adnanayub@uosahiwal.edu.pk
Nasrin Choobkar, Plant Biotechnology
Research Center, Kermanshah Branch,
Islamic Azad University, Kermanshah,
Iran.
Email: Nchoobkar20@gmail.com
J. Food Sci. 2023;1–14.
Accepted: 14 April 2023
Nasrin Choobkar2 Muhammad Asif Hanif3
Muhammad Riaz5 Amir Daraei Garmakhany6
Abstract
Pinus roxburghii is a rich source of high-quality oleoresin that is composed of
resin acids and essential oil (EO). The present research work was planned to
study and compare the yield, biological activities, and chemical profiling of P. rox-
burghii oleoresin EOs extracted through various green extraction methods. Steam
distillation (SD), supercritical fluid extraction, and superheated SD (SHSD) at
different temperatures (120, 140, and 160◦ C) were employed to extract EOs from
P. roxburghii oleoresin. Antioxidant potential of EOs was determined by total
antioxidant content/ferric-reducing antioxidant power (FRAP), 2,2-diphenyl-1-
picrylhydrazyl (DPPH)-free radical scavenging activity (DPPH-FRSA), hydrogen
peroxide scavenging assays, and percentage inhibition in linoleic acid. Antimi-
crobial activity of EOs was determined by resazurin microtiter-plate, disc
diffusion, and micro-dilution broth susceptibility assays. Gas chromatography–
mass spectrometry was used to determine the chemical composition of EOs. It
was observed that extraction methods significantly affected the yield, biological
activities, and chemical composition of EOs. The maximum yield (19.92%) was
found in EO extracted by SHSD at 160◦ C. EO extracted by SHSD at 120◦ C showed
the highest DPPH-FRSA (63.33% ± 0.47%), linoleic acid oxidation inhibition
(96.55% ± 1.71%), hydrogen peroxide scavenging activity (59.42% ± 0.32%), and
total antioxidant contents/FRAP (134.49% ± 1.34 mg/L of gallic acid equivalent).
The antimicrobial activity results showed that superheated steam–extracted EO
of 120◦ C revealed the highest antifungal and antibacterial activity. It is concluded
that SHSD is an alternative and effective technique for the extraction of oleo-
resins EO that improves the EO yield and biological activities. Further research
on optimization and experimental parameters for the extraction of P. roxburghii
oleoresin EO by SHSD is required.
wileyonlinelibrary.com/journal/jfds © 2023 Institute of Food Technologists. 1
2 P. ROXBURGHII OLEORESIN ESSENTIAL OILS
Funding information
Higher Education Commission (HEC), KEYWORDS
Islamabad, Pakistan, Grant/Award antimicrobial activity, antioxidant activity, essential oil, GC–MS, oleoresin, P. roxburghii,
Number: 20-15988/NRPU/R&D/HEC/2021 superheated steam distillation
1 INTRODUCTION
Foodborne illness is a major health concern of people
in both developed and developing countries (Abunna
et al., 2016). According to World Health Organization, the
infectious diseases that caused by consuming contami-
nated water or food are called foodborne diseases (Abebe
et al., 2020). Particularly foodborne bacteria, such as
Salmonella enterica, Staphylococcus aureus, Campylobac-
ter jejuni, Escherichia coli, and Listeria monocytogenes, can
spread foodborne illnesses (Zhao et al., 2014). Oxidation
reactions that occur during production, distribution, and
storage of food products can lower their quality (Pateiro
et al., 2018). Synthetic preservatives, which are used as
antimicrobial and antioxidants in food products, may
lead to allergies, intoxication, and degenerative diseases
(Laranjo et al., 2017). So, scientists are searching for antimi-
crobial agents and antioxidants of natural origin that may
help attenuate oxidative damage. Essential oils (EOs) may
be used as an alternative to synthetic preservatives due to
their pathogenic resistance (Myszka et al., 2019).
The compounds that neutralize free radicals and reac-
tive oxygen species in the cells are called antioxidants.
The antioxidants protect organisms from free radicals that
are responsible for chronic diseases, such as cardiovascu-
lar disease, cancer, aging, and anemia (Riaz et al., 2016;
Zehiroglu & Ozturk Sarikaya, 2019). Free radicals are pro-
duced by an uncoupled flow of electrons during metabolic
processes. They may be harmful to lipids, nucleic acids,
proteins, and carbohydrates (Gulcin, 2020). Antioxidants
reduce the oxidative stress-related diseases by counteract-
ing the deleterious effects of free radicals (Neha et al.,
2019; Riaz et al., 2017). Using EOs as natural antioxidants
help avoid degenerative diseases (Bhalla et al., 2013). In
a study 1% (v/w) EO of Thymus capitata to minced beef
significantly reduced the population of L. monocytogenes
(El Abed et al., 2014). Therefore, EOs can be used for
the preservation of meat products to increase the shelf
life against L. monocytogenes. EOs are volatile, secondary
metabolites of medicinal plants. They are aromatic oily liq-
uids obtained from different parts of plants, for example,
roots, leaves, herbs, flowers, barks, and buds (Burt, 2004).
EOs possess antimicrobial and anti-inflammatory proper-
ties so they are used as analgesics, sedatives, anesthetics,
and spasmolytic materials (Bakkali et al., 2008).
There are various techniques used for the extraction
of EOs from plants. The hydrodistillation (HD) process,
devised by Avicenna, is the oldest method of EO extraction
(Madani et al., 2023). HD is the simplest and oldest method
used for the extraction of EOs from plant materials. How-
ever, this technique has some disadvantages, such as low
extraction efficiency, high fuel and water consumption,
hydrolysis of compounds, prolonged extraction time, and
decomposition of heat-sensitive compounds. Steam distil-
lation (SD) is simple, convenient, more reproducible, and
has high energy efficiency (Akdağ & Öztürk, 2019). SD has
disadvantages, just like HD, such as prolonged extraction
time, degradation of ester or unsaturated compounds, and
low extraction efficiency (Reyes-Jurado et al., 2015).
Supercritical fluid (SCF)–CO2 involves the use of recy-
cling fluid in periodic steps of compression or decompres-
sion. Carbon dioxide is mainly used to extract EOs by
SCFE (Fornari et al., 2012). This process provides various
properties, such as lower density, lower viscosity, and high
diffusivity. These properties enable a fast extraction of EO
at low temperatures. It is an environment-friendly, cost-
effective, and simple process (El Asbahani et al., 2015). This
method requires less time as compared to conventional
techniques. The major demerit of this technique lies in the
nonpolar nature of CO2 . Due to the nonpolar nature of
CO2 , the total content of bioactive phenolic compounds in
EOs cannot be extracted (Tyśkiewicz et al., 2018).
Superheated SD (SHSD) is an alternative extraction
technique in which superheated steam is used for the
extraction of EO. Superheated steam has a temperature
higher than the saturation temperature at normal pressure.
In the form of latent heat of vaporization, it carries a lot of
thermodynamic energy. It has a high thermal conductivity,
extraction capacity, and low oxygen conditions, prevent-
ing the oxidation of extracted components (Shuaib et al.,
2018). Ayub et al. (2018) used the SHSD technique for the
extraction of EO from oleogum resin of Boswellia serrata.
Pinus roxburghii, referred to as Chir Pine, belongs to
the Pinaceae family of gymnosperms. It is located in the
Himalayas range of Pakistan, China, Afghanistan, India,
and Nepal (da Silva Rodrigues-Corrêa et al., 2013). P.
roxburghii is the rich source of high-quality oleoresin. Ole-
oresin, on distillation, produces an EO generally known
as turpentine oil and rosin is left behind as a non-
volatile solid residue (Khajenoori et al., 2015). Diterpene
resin acids are major constituents of rosin. Oleoresin is
also composed of terpenes that are complex mixtures
of monoterpenes, diterpenes, sesquiterpenes, oxygenated
monoterpenes, oxygenated diterpenes, and oxygenated
P. ROXBURGHII OLEORESIN ESSENTIAL OILS
sesquiterpenes (Mottahedin et al., 2017). Turpentine oil is
a complex mixture of monoterpenes and sesquiterpenes.
Pinus oleoresins have a role in pharmaceuticals and cos-
metics (Mottahedin et al., 2017). It is also used for scorpion
and snakebite stings (Badrkhani et al., 2013). Turpentine
oil is used in disinfectants and denaturants (Labib et al.,
2017).
A thorough evaluation of previously published research
studies manifested that SHSD is still not practiced for the
extraction of EO from P. roxburghii oleoresin. Therefore,
the research work was performed to extract EO from P. rox-
burghii oleoresin by SD, SCF–CO2 , and SHSD at different
temperatures. Moreover, the effect of extraction methods
and extraction temperatures on the yield, antioxidant and
antimicrobial activities, and chemical composition of EOs
was evaluated.
2 MATERIALS AND METHODS
The P. roxburghii oleoresin was collected by tapping
method from fully grown plants of Abbottabad, Pakistan in
July–August 2021. Plant material was identified (voucher
number ABT 004) and verified by Dr. Fahim Arshad,
Department of Botany, University of Okara, Pakistan.
2.1 Extraction of essential oil
P. roxburghii oleoresin EO was extracted by SD, supercrit-
ical fluid extraction–CO2 (SCFE–CO2 ), and SHSD (at 120,
140, and 160◦ C).
2.1.1 Steam distillation
SD apparatus consisted of a round bottom flask, a biomass
flask, condenser, a heating mantle, thermometer, and
collecting vessel. The oleoresin sample (100 g oleoresin
soaked in 100 g cotton wool) was placed in a biomass flask
that was adjusted on the top of round bottom flask. Steam
was generated by boiling water using heating mantle. The
steam was allowed to enter biomass flask where it inter-
acted with oleoresin sample. The EO vapors along with
water vapors were passed through a condenser that cooled
them to liquid droplets. The EO and water layers were sep-
arated on the basis of density in collecting vessel. The EO
layer was separated and collected in amber glass vial. SD
was carried out for 3 h. The EO was dried using anhyd.
Na2 SO4 , followed by filtration using a micro filter, and was
stored at +4◦ C until further processing. The extraction pro-
cedure was repeated five times to ensure that results were
reproducible.
3
2.1.2 Supercritical fluid CO2 extraction
EO was extracted by supercritical fluid CO2 extractor
(NPX-10 Extractor, Supercritical Fluid Technologies Pvt.
Ltd., Mumbai, India). Liquid CO2 chilled to 10◦ C was
charged into the extraction tank (which was already kept
at 45◦ C) using a high-pressure pump. The pressure was
adjusted to an accuracy of 1% over the measuring range.
To enable the easy effusion of supercritical CO2 through
extraction vessel, it was filled with raw materials and glass
beads. After passing through a heated micrometer valve,
the SCF–CO2 with dissolved compounds was inflated to
ambient pressure. The extract was precipitated in a collec-
tion vessel at room temperature and pressure. At a known
temperature and pressure, the total amount of CO2 was
measured using a calibrated wettest meter. For each extrac-
tion test, the extractor was stuffed with about 500 g of
oleoresin soaked in 500 g cotton wool. Flow rate of CO2
was 2 L/min. An analytical balance was used to measure
the weight of oil (Sarker et al., 2007).
2.1.3 Superheated steam distillation
SHSD was carried out in a custom-sized distillation
apparatus (SHSD-001, PAMICO Technologies, Faisalabad,
Pakistan) consisting of a stainless steel extraction ves-
sel connected to an electrical steam boiler, super heater,
condensers, and extraction vessel to collect oil. Tem-
perature of superheated steam was continuously moni-
tored by thermocouples attached at the entry and exit
of stainless steel extraction vessel. The oleoresin (100 g
soaked in 100 g cotton wool) was packed in the extrac-
tion chamber and subjected to superheated steam at
120, 140, and 160◦ C for 1 h. Moisture from EOs (10 g)
was removed by adding 0.25–0.50 g of anhyd. Na2 SO4 ,
filtered using a micro filter and was stored in amber
glass vials till further analysis. The extraction proce-
dure was repeated five times to ensure that results were
reproducible.
2.2 Antioxidant activity
2.2.1 Ferric reducing antioxidant power
(FRAP) assay
The FRAP assay was used to determine total antioxidant
content (Wayne, 2008). A volume of 1 mL of each essen-
tial oil extracted by different extraction methods was mixed
with 2.5 mL of phosphate buffer (pH 6.6, 0.2 M) and
2.5 mL of 1 w/v% potassium ferricyanide solution. The
test tubes were incubated in water at 50◦ C for 25 min. A
4 P. ROXBURGHII OLEORESIN ESSENTIAL OILS
volume of 2.5 mL of 10 w/v% solution of trichloroacetic
acid was added to each test tube. A volume of 2.5-mL
reaction mixture from each test tube was transferred to a
separate test tube and diluted with 2.5 mL of deionized
water. A volume of 500 µL of 0.1 w/v% solution of fer-
ric chloride was added to each test tube. Test tubes were
incubated at room temperature for 30 min. A UV–Visible
spectrophotometer (UV-2600i, Shimadzu, Kyoto, Japan)
was used to measure the absorbance of each solution at
710 nm. Total antioxidant activity is expressed in terms
of mg/mL of gallic acid equivalents. Total antioxidant
activity was calculated by the gallic acid calibration curve
(0–100 mg/mL).
2.2.2 DPPH-free radical scavenging activity
(DPPH-FRSA)
DPPH assay was carried out in accordance with the liter-
ature (Hussain et al., 2013). A volume of 1 mL of DPPH
solution (0.09 mM) was added to 2.5 mL of EO, and the
final volume was made up to 4 mL using MeOH (95%).
A spectrophotometer (UV-2600i) was used to measure the
absorbance of the solution after 1 h at room temperature in
the dark at 515 nm. A positive control was BHT at a con-
centration of 100 ppm. The following equation was used
to calculate the percentage of free radical inhibition by
DPPH:
𝐴𝑏𝑙𝑎𝑛𝑘 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒
%𝐼 = × 100 (1)
𝐴𝑏𝑙𝑎𝑛𝑘
2.2.3 Percentage inhibition in a linoleic acid
system
The percentage inhibition in linoleic acid peroxide forma-
tion was estimated based on Singh et al. (2006). A volume
of 50 µL EOs dissolved in 1 mL EtOH was mixed with 1 mL
of 2.5 v/v% solutions of linoleic acid, 4 mL of 99.5% EtOH,
and 4 mL of 0.05 M sodium phosphate buffer (pH 7). The
solution was incubated at 40◦ C for 175 h. The colorimetric
method was employed to estimate the extent of oxidation
based on the peroxide value.62 To 0.2 mL sample, 10 mL
of 75% of EtOH, 0.2 mL of 30% aqueous solution of ammo-
nium thiocyanate, and 0.2 mL of ferrous chloride solution
(20 mM in 3.5% HCl) were sequentially added. The solu-
tion was stirred for 3 min. The absorbance was measured
at 500 nm using a spectrophotometer (UV-2600i). A spec-
trophotometer was used to measure absorbance at 500 nm.
The same procedure was used to run a control group. The
positive control was BHT. The following equation gives the
percent inhibition of linoleic acid.
%𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑙𝑖𝑛𝑜𝑙𝑒𝑖𝑐 𝑎𝑐𝑖𝑑 𝑜𝑥𝑖𝑑𝑎𝑡𝑖𝑜𝑛 = 100
[( ) ]
𝐴𝑏𝑠. 𝑖𝑛𝑐𝑟𝑒𝑎𝑠𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑡 175 h
− × 100 (2)
𝐴𝑏𝑠.𝑖 𝑛𝑐𝑟𝑒𝑎𝑠𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑎𝑡 175 h
2.2.4 Hydrogen peroxide scavenging activity
The spectrophotometric estimation of hydrogen peroxide
scavenging activity was described by (Bakkali et al., 2008).
In phosphate buffer (pH 7.4), 2 mM solution of hydrogen
peroxide was prepared. A volume of 600 µL EO was mixed
with 600 µL of hydrogen peroxide (2 mM). The resulting
mixture was incubated at room temperature for 10 min.
The absorbance was measured at 230 nm. Same procedure
was carried out for ascorbic acid (100 mg/L). The following
equation was used to compute the percentage of hydrogen
peroxide scavenging activity:
( )
𝐴0 − 𝐴1
%𝐻𝑦𝑑𝑟𝑜𝑔𝑒𝑛 𝑝𝑒𝑟𝑜𝑥𝑖𝑑𝑒 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 =
𝐴0
× 100 (3)
2.3 Antimicrobial activity
2.3.1 Microbial strains
EOs extracted by different extraction techniques were
tested against E. coli, Aspergillus flavus, S. aureus, A. alter-
nata, Bacillus subtilis, F. solani, Pasteurella multocida, and
Aspergillus niger collected from Institute of Microbiology,
University of Agriculture Faisalabad, Pakistan.
2.3.2 Agar well diffusion method
Agar well diffusion was used to test the antibacterial activ-
ity of P. roxburghii oleoresin EO.63 The overnight cultures
of indicator strains were transferred to flasks containing
25 mL nutrient agar. The mixture of flasks was trans-
ferred to 6-in. petri dishes to solidify at room temperature.
A sterilized cork borer was used to create wells. These
wells were filled with 10 µL EOs and standard drug, that
is, ampicillin (1 mg/mL) for the evaluation of antibacte-
rial activity. The petri plates were incubated at 37◦ C for
24 h. The width of zones of inhibition (mm) was measured
after incubation in order to determine the antimicrobial
activity.
P. ROXBURGHII OLEORESIN ESSENTIAL OILS
2.3.3 Resazurin microtiter-plate assay
A modified resazurin microtiter-plate test was used to
determine the MIC of EOs against various bacterial strains,
as mentioned in the literature.64 A volume of 10-mL EO
was dissolved in 1 mL of 10% DMSO to make the sam-
ple solution. An amount of 27-mg resazurin was dissolved
in 4 mL of sterilized deionized water to make resazurin
indicator solution. In the first row of 96 well plates, about
100 𝜇L of sample solution and standard antibiotic ampi-
cillin (1 mg/mL in 10% DMSO) were pipetted. After that,
with the exception of first row; 50 mL nutrient broth was
added in all wells and twofold serial dilutions were con-
ducted until each well had 50 𝜇L of the sample mixture. In
each well, 30 𝜇L of 3.3× strength isosensitized broth, 10 𝜇L
of resazurin solution, and 10 𝜇L (5 × 105 colony forming
units per mL) microbial culture were added. Plates were
incubated at 37◦ C for 24 h. After incubation, MIC values
were measured visually. The bacterial growth was shown
by the color change from purple to colorless or pink, and
the MIC value was determined by the lowest concentration
at which the color change occurred.
2.3.4 Micro-dilution broth susceptibility
assay
The micro-dilution broth susceptibility assay was used to
determine the MIC of EOs extracted by different extraction
techniques against various fungal strains, as previously
described in the literature.65 An amount of 10 mg of EO
and 1 mL of standard antibiotic in 1 mL of 10% DMSO solu-
tion were mixed to prepare sample solution of EOs and
standard antibiotic (Fluconazole). Into the first row of the
96 well plates, about 100 𝜇L of samples and standard solu-
tions were pipetted. After that, with the exception of the
first row, 50 𝜇L of Sabouraud dextrose broth was added
in all wells and twofold serial dilutions were conducted
until each well had 50 𝜇L of the sample mixture. Then,
130 𝜇L Sabouraud dextrose broth and 20 𝜇L of microorgan-
ism suspension (5 × 105 colony forming units per mL) were
added in all wells, and well plate was incubated at 30◦ C
for 48 h. The MIC value was visually determined. It is the
lowest concentration at which fungal growth is completely
inhibited.
2.4 Gas chromatography/mass
spectrometry (GC–MS) analysis
Gas chromatography–mass spectrometry (GC–MS) (Shi-
madzu gas chromatograph GC-2010 system, Kyoto, Japan)
5
equipped with QP-2010 plus mass detector and DB-5 cap-
illary column (50 m × 0.25 mm, film thickness of 0.25 µm)
was used to quantify the volatile components of EOs
extracted by different extraction techniques. Temperature
of injector was set at 200◦ C. An injection volume of 1 µL EO
was injected in to injection port using an injection syringe.
Column was heated at 60◦ C for 3 min and then gradually
its temperature was increased up to 240◦ C at the heating
rate of 24◦ C/min and kept constant for the next 10 min.
Carrier gas was nitrogen gas that was flown through the
system at the flow rate of 1.5 mL/min. The temperature
of MS transfer line was kept to 240◦ C. For MS detection,
an electron ionization mode (70 eV) was used (Shuaib
et al., 2013). By running n-alkanes (C9 –C24 ) standards
under same conditions, retention indices of identified
compounds were determined. These retention indices and
mass spectrum data were also compared to previously pub-
lished data, the NIST mass spectral database library, and
pherobase mass spectral database library (Shuaib et al.,
2013).66
2.5 Statistical analysis
All the experiments were carried out in triplicate and
data was statistically analyzed using analysis of variance
(ANOVA) with post hoc Tukey HSD test using STA-
TISTICA 5.5 (StatSoft Inc., Tulsa, OK, USA) software.
A probability value at p ≤ 0.05 was considered statis-
tically significant. The results are presented as mean
values ± standard deviation calculated from triplicate
determinations.
3 RESULTS AND DISCUSSION
3.1 Yield of EOs
P. roxburghii oleoresin EO was extracted by using SD, SCF–
CO2 , and SHSD (carried out at 120, 140, and 160◦ C), and
results are shown in Figure 1. It was inferred from the
results that extraction methods affected the yield of EO.
The highest EO yield was observed by SHSD (19.92%) at
160◦ C. The higher temperature of SHSD may increase
the yield of EO. A high temperature of SHSD causes
more vaporization of EO from oleoresin which leads to
a high yield. These results are similar to the previous
study in which the EO yield of thyme leaves and black
pepper extracted by SHSD was higher than HD (Zafar
et al., 2010). It was observed that EO yield was increased
with the increasing temperature of superheated steam.
A much similar pattern was reported in the previous
6 P. ROXBURGHII OLEORESIN ESSENTIAL OILS
F I G U R E 1 Yield (g/100 g) of Pinus roxburghii oleoresin essential oils extracted by steam distillation, supercritical fluid CO2 extraction,
and superheated steam distillation.
literature in which EO yield of Boswellia serrata oleo-
gum resin was increased with increasing temperature of
superheated steam (8.50%–10.75%) (Shuaib et al., 2018).
Similarly, it was reported that Citrus hystrix leaves EO
yield was increased by increasing the extraction temper-
ature of subcritical water extraction (Khan et al., 2012).
It was also mentioned that high temperature decreased
the hydrogen bonding, dielectric constant, polarity, viscos-
ity, density, and surface tension of water molecules and
increased the diffusing power, mass transfer rate, and EO
yield (Khan et al., 2012). Extraction techniques compari-
son showed that SHSD revealed higher EO yield followed
by SD and SCF–CO2 , respectively. EO yield of P. roxburghii
oleoresin was much higher than other parts of its plant
extracted by HD, that is, bark 0.022% (Salem et al., 2014),
needles 0.11% (Tillah et al., 2016), wood 0.5% (Karapand-
zova et al., 2019), flowers 0.08% (Wang et al., 2018), and
stem 0.24% (Sinha & Tandon). Similarly, SD of P. roxburghii
needles produced 0.26% EO (da Silva Rodrigues-Corrêa
et al., 2013). Therefore, it can be concluded that SHSD is
the most effective extraction technique, as it yields a higher
EO percentage compared to SD and SCF–CO2 .
3.2 Antioxidant activity of EO
Antioxidant activity of EOs was evaluated using 2,2-
diphenyl-1-picrylhydrazyl–free radical scavenging activity
(DPPH-FRSA), ferric reducing antioxidant power (FRAP)
assay, H2 O2 scavenging activity, and % inhibition in linoleic
acid system. DPPH is a reliable and simple assay used for
evaluating the antioxidant activity of plant extracts. The
EOs antioxidant activity is due to their ability to donate
the hydrogen, resulting in the scavenging of free radi-
cal. This leads the reduction of DPPH and discoloration
of violet color (Tsvetkov et al., 2019). Antioxidant activ-
ity of P. roxburghii oleoresin EO obtained by different
extraction techniques is given in Table 1. The experimental
results showed that EOs exhibited less DPPH-free radi-
cal scavenging activity as compared to standard butylated
hydroxytoluene (BHT). Notably, the extraction techniques
had a significant effect on the antioxidant activity of EOs of
P. roxburghii oleoresin. Among the extraction methods, EO
obtained by the SHSD (120◦ C) demonstrated the highest
DPPH-free radical scavenging activity (63.33%), whereas
SHSD (160◦ C) had the lowest (49.53%). SD exhibited a
DPPH-FRSA activity of (62.40%). The difference in FRSA
may be attributed to the variation in the concentration
of chemical compounds present in EOs. Previous studies
have reported that EOs extracted from fresh fruits of P.
roxburghii showed negligible scavenging activity (Shamim
et al., 2016). In addition, P. roxburghii needles EO showed
lower antioxidant activity (50%) as compared to tannic
acid (80%), whereas bark (85%) and wood (82%) exhib-
ited higher antioxidant activity than tannic acid (Hajdari
et al., 2016). Furthermore, resin extracts and turpentine
oils of Pinus oocarpa, Pinus insularis, Agathis loranthifolia,
and Pinus merkusii exhibited less antioxidant potential as
compared to control (ascorbic acid) (Mercier et al., 2009).
Table 1 shows the percentage inhibition of linoleic
acid oxidation by P. roxburghii oleoresin EOs. The EOs
exhibited lower linoleic acid oxidation inhibition than the
control BHT. This finding is consistent with Karapandzova
et al. (Ju et al., 2019) reported on Pinus mugo needles EO.
EO extracted by SHSD at 120◦ C showed highest linoleic
acid oxidation inhibition (96.55%), whereas EO extracted
by SHSD at 160◦ C SD showed lowest linoleic acid oxidation
inhibition (80.46%). The results of hydrogen peroxide–free
P. ROXBURGHII OLEORESIN ESSENTIAL OILS
T A B L E 1 Antioxidant potential of Pinus roxburghii oleoresin essential oils extracted by steam distillation, supercritical fluid CO2
extraction, and superheated steam distillation.
Inhibition in linoleic
Extraction method acid system (%)
Steam distillation 80.46 ± 1.42f
Supercritical fluid 88.37 ± 1.92 e
extraction (40◦ C,
80 bar)
Superheated steam 92.18 ± 1.33d
(120◦ C)
Superheated steam 93.16 ± 1.53c
(140◦ C)
Superheated steam 96.55 ± 1.71b
(160◦ C)
BHT 98.85 ± 1.89a
Ascorbic acid – –
Note: Values are mean ± standard deviations of three separate determinations. Different letters in superscript represent significant differences among P. roxburghii
oleoresin essential oils extracted by different extraction methods.
Abbreviations: BHT, butylated hydroxytoluene; DPPH, 2,2-diphenyl-1-picrylhydrazyl; FRAP, ferric reducing antioxidant power.
a
Total antioxidant contents/FRAP (mg/L of essential oil, measured as gallic acid equivalent).
radical scavenging activity are also shown in Table 1. The
EO extracted by SHSD at 120◦ C had highest hydrogen per-
oxide scavenging activity (59.42%), whereas EO extracted
by supercritical fluid extracted EO had lowest hydrogen
peroxide scavenging activity (50.43%). Total antioxidant
contents, represented as mg/L of GAE, are given in Table 1.
Total antioxidant contents in EO ranged from 67.44 to
134.49 mg/L. The EO extracted by SHSD at 120◦ C con-
tained the highest amount of total antioxidant contents,
whereas EO extracted by SHSD at 160◦ C contained the low-
est. These finding are similar to a published study in which
scavenging activity of Citrus pomace extract processed by
superheated water decreased with extraction temperature
(Mohamed et al., 2020).
EOs are a complex mixture of variety of compounds,
making it difficult to assess specific compound responsi-
ble for their antioxidant activity (Shuaib et al., 2013). It
has been reported that scavenging activity of P. roxburghii,
Pinus wallichiana, and Pinus gerardiana extracts might be
due to the presence of phenolic compounds, which can
donate electrons to H2 O2 converting it to H2 O (Tsvetkov
et al., 2019). According to Tsvetkov et al. (Gündüz et al.,
2012), the antioxidant activity of crude acetone extracts of
P. roxburghii knot wood compounds was less than that of
gallic acid. Moreover, it was also mentioned that antioxi-
dant activity was related to the number of hydroxyl groups
present on benzene ring, the degree of shielding of the
hydroxyl groups by other functional groups, and the nature
of other substituents present at the benzene ring, capable
of increasing and decreasing the electron density of the
ring and thus modifying the oxidizability of the hydroxyl
groups (Gündüz et al., 2012).
7
DPPH-free radical Hydrogen peroxide Total antioxidant
scavenging activity (%) scavenging (%) contents/FRAPa
62.4 ± 0.41c 55.32 ± 0.29c 67.44 ± 0.95e
49.53 ± 0.11 f
52.49 ± 0.26 e
99.92 ± 1.06d
54.67 ± 0.38e 53.76 ± 0.16d 102.07 ± 1.12c
60.68 ± 0.34d 50.43 ± 0.19f 127.12 ± 1.21b
63.33 ± 0.47b 59.42 ± 0.32b 134.49 ± 1.34a
97.92 ± 1.82a – –
85.57 ± 1.14a
–
3.3 Antimicrobial activity of EOs
Antimicrobial activity of EOs of P. roxburghii oleoresin
extracted by different extraction techniques against bac-
teria and fungi was evaluated by well diffusion assay,
micro-dilution broth assay, and resazurin microtiter plate
assays, and results are given in Table 2. The inhibition
zone and minimum inhibitory concentration (MIC) val-
ues for antibacterial activity of EOs extracted by various
extraction techniques were in the range of 10.02 ± 0.03–
24.74 ± 0.08 mm and 54.27 ± 0.46–281.47 ± 1.76 µg/mL,
respectively. The values for positive control (Amoxicillin)
were 21.11 ± 0.08–28.53 ± 0.06 mm and 2.92 ± 0.04–
13.33 ± 0.07 µg/mL, respectively. The lowest antibacterial
activity was shown by SHSD EO of 160◦ C with inhibi-
tion zone (10.02 ± 0.03–14.22 ± 0.07 mm) and MIC values
(242.22 ± 0.74–281.47 ± 1.76 µg/mL). The highest antibacte-
rial activity was shown by EO extracted by SHSD at 120◦ C
with inhibition zones (14.77 ± 0.08–24.74 ± 0.08 mm) and
MIC values (54.27 ± 0.46–112.58 ± 1.18 µg/mL). Varia-
tion in chemical constituents of EOs might be responsible
for differences in antimicrobial activity. The P. roxburghii
oleoresin EO was found to be more effective against S.
aureus, which is a Gram-positive strain of bacteria. This
result is consistent with published studies (Hajdari et al.,
2016; Sinha & Tandon; Tillah et al., 2016, Wang et al.,
2018) that reported EO of P. roxburghii extracted from
stems, needles, wood, and bark was effective against S.
aureus. According to another study (da Silva Rodrigues-
Corrêa et al., 2013), the methanolic resin extract of P.
roxburghii showed better activity against Gram-positive
bacteria than Gram-negative bacterial strains. In another
 8

TA B L E 2 Antimicrobial activity of Pinus roxburghii oleoresin essential oils extracted by steam distillation, supercritical fluid CO2 extraction, and superheated steam distillation.
Essential oils extracted by different extraction methods
Supercritical fluid Superheated steam Superheated steam Superheated steam
Steam distillation extraction (120◦ C) (140◦ C) (160◦ C)
Microorganism Inhibition zone (mm) Positive controla
Escherichia coli 13.90 ± 0.09 d 14.44 ± 0.04c 14.77 ± 0.08b 13.12 ± 0.05e 12.86 ± 0.06f 21.11 ± 0.08a
d c b e f
Staphylococcus aureus 21.12 ± 0.05 22.00 ± 0.07 24.74 ± 0.08 16.30 ± 0.06 14.22 ± 0.03 25.38 ± 0.09a
Pasteurella multocida 16.12 ± 0.04c 15.90 ± 0.05d 16.80 ± 0.07b 10.14 ± 0.05e 10.02 ± 0.03f 28.53 ± 0.06a
d c b e f
Bacillus subtilis 14.15 ± 0.04 14.70 ± 0.03 15.45 ± 0.06 13.20 ± 0.08 10.44 ± 0.07 22.32 ± 0.07a
Fusarium solani 12.13 ± 0.04d 12.42 ± 0.05c 14.03 ± 0.06b 10.50 ± 0.06e 9.00 ± 0.07f 24.36 ± 0.08a
e c b d f
Aspergillus niger 9.00 ± 0.04 10.58 ± 0.03 17.84 ± 0.08 10.02 ± 0.03 7.00 ± 0.05 22.61 ± 0.06a
Alternaria alternata 14.50 ± 0.06d 15.02 ± 0.05c 22 ± 0.09b 11.00 ± 0.05e 10.00 ± 0.04f 24.82 ± 0.05a
d c b e f
Aspergillus flavus 11.00 ± 0.06 11.46 ± 0.05 19.80 ± 0.04 8.97 ± 0.03 8.06 ± 0.03 23.32 ± 0.07a
Minimum inhibition concentration (µg/mL)
E. coli 126.64 ± 1.28c 119.60 ± 1.08d 112.58 ± 1.18 e 140.73 ± 1.22b 168.88 ± 1.52a 5.88 ± 0.06f
b d e c a
S. aureus 168.88 ± 1.24 56.29 ± 0.88 54.27 ± 0.46 140.73 ± 1.16 242.22 ± 0.74 2.92 ± 0.04f
P. multocida 168.88 ± 1.34c 140.73 ± 1.20d 112.58 ± 1.02e 274.43 ± 1.86b 281.47 ± 1.76a 8.33 ± 0.08f
c d e b a
B. subtilis 140.73 ± 1.44 70.36 ± 0.48 56.29 ± 0.96 168.88 ± 1.32 281.47 ± 1.72 13.33 ± 0.07f
F. solani 245.22 ± 1.72c 337.76 ± 1.56d 168.88 ± 1.24e 253.32 ± 1.18b 506.65 ± 2.56a 5.88 ± 0.05f
c d e b a
A. niger 337.76 ± 1.86 329.66 ± 1.86 112.58 ± 1.06 478.49 ± 2.42 675.54 ± 3.42 8.42 ± 0.06f
A. alternata 168.88 ± 1.28c 270.36 ± 0.82d 160.78 ± 1.4e 281.46 ± 1.14b 337.76 ± 1.56a 5.12 ± 0.03f
c d e b a
A. flavus 281.46 ± 1.22 273.34 ± 1.86 84.44 ± 1.04 422.80 ± 2.24 562.94 ± 2.56 6.66 ± 0.03f
Note: Values are mean ± standard deviations of three separate determinations. Different superscript letters represent significant difference among P. roxburghii oleoresin essential oils extracted by different extraction
methods.
a
Positive control for bacteria and fungi was Amoxicillin and Fluconazole (25 µg/disc), respectively.
P. ROXBURGHII OLEORESIN ESSENTIAL OILS
P. ROXBURGHII OLEORESIN ESSENTIAL OILS
study, it was reported that n-hexane resin extract of P.
oocarpa was most effective against S. aureus (Mercier
et al., 2009). The results of antifungal activity are given
in Table 2. The inhibition zone and MIC values for anti-
fungal activity of EO extracted by different extraction
techniques were in the range of 7 ± 0.05–22 ± 0.09 mm
and 70.36 ± 0.82–675.54 ± 3.42 µg/mL, respectively, and the
values for positive control (Fluconazole) were 22.61 ± 0.06–
24.82 ± 0.05 mm and 5.12 ± 0.03–8.42 ± 0.06 µg/mL,
respectively. The highest antifungal activity was exhib-
ited by EO extracted by superheated steam at 120◦ C with
larger inhibition zone 17.84 ± 0.08–22 ± 0.09 mm and
lower MIC values 84.44 ± 1.04–168.88 ± 1.24 µg/mL, respec-
tively. The lowest antifungal activity was exhibited by EO
extracted by SHSD at 160◦ C with lowest inhibition zone
7.00 ± 0.05–10.00 ± 0.04 mm and highest MIC values
337.76 ± 1.56–675.54 ± 3.42 µg/mL. It was noted that P. rox-
burghii oleoresin EO was more effective against Fusarium
solani and Alternaria alternata with maximum inhibition
zone 9.00 ± 0.07–14.03 ± 0.06, 10.00 ± 0.04–22 ± 0.09 mm
and minimum MIC values of 168.88 ± 1.24–506.65 ± 2.56,
70.36 ± 0.82–337.76 ± 1.56 µg/mL, respectively. Similar
observations were made by Shamim et al. (Bhagat et al.,
2018) who reported that chloroform extract of P. rox-
burghii female cones showed a maximum inhibition zone
22 mm against A. alternata, whereas methanol extract of
P. roxburghii female cones showed a maximum inhibition
zone 20 mm against F. solani. Similarly, chloroform and
methanol extract of P. roxburghii needles showed maxi-
mum inhibition zone 9 mm against A. alternata, whereas
distilled water extract of P. roxburghii needles showed
maximum inhibition zone 12.33 mm against F. solani.
Antimicrobial activity of Pinus species is related to com-
pounds, such as terpenes, alkaloids, and phenols (Satyal
et al., 2013). Similarly, Salem et al. (Hajdari et al., 2016)
reported that terpenes and phenolic compounds of P. rox-
burghii act as antibacterial and antifungal agents. The α-
and β-pinenes of P. roxburghii were the essential com-
pounds of oleoresin EOs due to their capability to induce
toxic effects on membrane structure and function of bac-
teria and fungi. Cytoplasmic membranes of bacteria and
fungi provide a barrier to ions, that is, K+ , H+ , Ca2+ ,
and Na+ . This property of semipermeability of membrane
is essential for many cellular functions. Monoterpenes,
partition from aqueous phase into membrane structures,
due to their lipophilic character. This causes membrane
expansion, enhanced membrane fluidity, and inhibition
of membrane-embedded enzymes (Guan et al., 2007). In
another study, it was reported that the hydrophobic char-
acter of EOs and their constituents are responsible for
their interaction with lipids in the microbial plasma mem-
brane and mitochondria, rendering them more permeable.
This enhanced permeability causes the outflow of ions and
9
other cell components. A certain amount of outflow from
microbial cells can be endured without causing cell death
but a significant loss of cell contents or essential molecules
and ions will result in cell death (Chan et al., 2007).
GC–MS results showed that a high percentage of
monoterpenes (3-carene, α-pinene, and β-pinene) in P.
roxburghii oleoresin EO might be responsible for antimi-
crobial activity. Similar findings were mentioned by Zafar
et al. (Tillah et al., 2016) that terpenes of P. roxburghii nee-
dles EO were highly active against microbes. In another
study, antifungal activity of P. roxburghii wood treated
with P. halepensis branch n-hexane extract and S. tere-
binthifolius branch EO was found to be active against the
growth of Rhizoctonia solani, Fusarium oxysporum, and
Bipolaris oryzae. It was also reported that monoterpenoids,
the most prevalent compounds found in the extract, might
be responsible for this effect (Bozin et al., 2006). Accord-
ing to Singh et al. (2006), the major components present in
EO of plants were monoterpenes, sesquiterpenes, and their
derivatives, which act as antifungal and antibacterial sub-
stances. The most well known of these components were
terpenes and phenolic compounds. It was also reported by
that P. roxburghii needles EO was effective against Gram-
positive bacteria (Yen et al., 2000). It was also mentioned
that it the antimicrobial activity could be attributed to
the higher concentration of α-phellandrene, α-pinene, and
α-terpinolene in P. roxburghii needles EO.
3.4 Chemical composition
The results of GC–MS analysis of P. roxburghii oleo-
resin EOs extracted by different extraction techniques are
given in Table 3. Monoterpene hydrocarbons were the
major compounds present in P. roxburghii oleoresin EO
(98.21%–86.24%). Oxygenated monoterpenes were 0.59%–
0.19%, whereas sesquiterpenes were 0.59%–4.65%. Overall,
17 compounds were quantified with 3-carene as the major
compound (44.31%–34.93%) followed by α-pinene (38.01%–
28.36%) and β-pinene (24.46%–16.32%). These results are
in accordance with published study (da Silva Rodrigues-
Corrêa et al., 2013) in which it was stated that P. roxburghii
oleo-gum-resin EO contained a higher percentage of 3-
carene followed by α- and β-pinene. Similarly, in another
study it was reported that GC–MS analysis of P. roxburghii
leaves EO contained 3-carene as the major component
(Wang et al., 2018). Similar findings were observed by
Sharma et al. (2020) (Tsvetkov et al., 2019) in which P. rox-
burghii bark essential oil contained 3-carene as the major
component. EO of P. mugo needles extracted by SD con-
tained 3-carene as a major compound (Ju et al., 2019).
There is no previous literature available on the chemi-
cal composition of P. roxburghii oleoresin EO extracted
 10

TA B L E 3 GC–MS analysis of Pinus roxburghii oleoresin essential oils extracted by steam distillation, supercritical fluid CO2 extraction, and superheated steam distillation.
% Composition of essential oils
Steam Superheated Superheated Superheated Supercritical fluid Method of
Componentsa RTb RIc distillation steam (120◦ C) steam (140◦ C) steam (160◦ C) extraction (40◦ C, 80 bar) identification
Monoterpene hydrocarbon
α-Thujene 5.09 923 0.11 ± 0.00b 0.09 ± 0.00c 0.11 ± 0.00b 0.09 ± 0.00c 0.41 ± 0.02a a, b
c a d e
α-Pinene 5.33 933 30.91 ± 0.56 38.01 ± 0.84 29.53 ± 0.34 28.36 ± 0.18 33.14 ± 0.16b a, b
b a
Camphene 5.75 952 0.18 ± 0.01 – – – 0.67 ± 0.04 a, b
β-Pinene 6.80 988 19.76 ± 0.04c 24.46 ± 0.02a 16.99 ± 0.02d 16.32 ± 0.08b 19.83 ± 0.05b a, b
b d a c c
3-Carene 7.45 1011 44.31 ± 0.42 34.93 ± 0.29 45.78 ± 0.34 40.09 ± 0.31 40.69 ± 0.37 a, b
m-Cymene 7.97 1023 0.19 ± 0.01b 0.17 ± 0.00c 0.07 ± 0.00e 0.15 ± 0.00d 0.22 ± 0.01a a, b
p-Cymene 8.12 1027 0.42 ± 0.01b 0.42 ± 0.00b 0.08 ± 0.00d 0.63 ± 0.01c 0.93 ± 0.02a a, b
d b a c b
Limonene 8.18 1031 0.04 ± 0.00 0.14 ± 0.01 0.64 ± 0.00 0.34 ± 0.00 0.15 ± 0.00 a, b
α-Terpinolene 10.19 1088 0.51 ± 0.01c – 1.39 ± 0.04b 0.26 ± 0.01d 1.81 ± 0.02a a, b
Oxygenated monoterpene hydrocarbons
Trans-Verbenol 12.53 1144 0.06 ± 0.02c 0.15 ± 0.01a 0.13 ± 0.00b 0.11 ± 0.00c 0.09 ± 0.00d a, b
c b a
Pinocarvone 13.33 1162 0.10 ± 0.00 0.16 ± 0.01 0.24 ± 0.00 0.31 ± 0.02 0.48 ± 0.03 a, b
Myrtenal 14.71 1193 0.01 ± 0.00b 0.01 ± 0.00b 0.03 ± 0.00a 0.01 ± 0.00b 0.01 ± 0.00b a, b
Verbenone 15.16 1204 0.02 ± 0.00a 0.01 ± 0.01 b 0.02 ± 0.00a 0.01 ± 0.00b 0.01 ± 0.00 b a, b
Sesquiterpene hydrocarbons
α-Longipinene 21.67 1351 0.07 ± 0.00c – 0.09 ± 0.00b 0.09 ± 0.00b 0.16 ± 0.01a a, b
Longicyclene 22.64 1373 0.89 ± 0.04a 0.68 ± 0.03b 0.58 ± 0.01c 0.53 ± 0.08c – a, b
Sativene 23.51 1393 0.05 ± 0.00c 0.06 ± 0.01bc 0.07 ± 0.00b 0.09 ± 0.00b 0.11 ± 0.01a a, b
Trans- 24.55 1416 0.17 ± 0.00c – 0.43 ± 0.02b 0.03 ± 0.00d 0.47 ± 0.02a a, b
Caryophyllene
Note: “a” is identification based on retention index, whereas “b” is identification based on comparison of mass spectra.
a
Compound listed in order of elution from a DB-5 capillary column.
b
Retention time.
c
Retention indices on the DB-5 capillary column.
P. ROXBURGHII OLEORESIN ESSENTIAL OILS
P. ROXBURGHII OLEORESIN ESSENTIAL OILS
by SHSD. So, the results of current study were com-
pared to EO extracted using other extraction methods.
A published study (Rashid et al., 2013) reported that P.
roxburghii cone EO contained caryophyllene, terpinen-4-
ol, δ-3-carene, and α-humulene, whereas needle EO of
P. roxburghii contained caryophyllene, terpinen-4-ol, α-
humulene, and α-terpineol as main compounds. Similarly,
another study (Tillah et al., 2016) reported that Pinus
roxburghii needles EO contained α-pinene, β-myrcene,
3-carene, α-terpineol, and caryophyllene as major com-
pounds. In another study (Sinha & Tandon), it was
reported that stem EO of P. roxburghii contained α-
pinene, 3-carene, limonene, and caryophyllene as major
compounds. The compounds that varied with extraction
techniques were β-pinene, trans-verbenol, pinocarvone,
myrtenal, verbenone, and longicyclene (highest percent-
age composition in EO extracted by SHSD: 24.46%,
0.15%, 0.48%, 0.03%, 0.02%, and 0.89%, respectively); 3-
carene (highest percentage composition in EO extracted
by SD: 44.31%); α-thujene, camphene, m-cymene, p-
cymene, α-terpinolene, α-longipinene, sativene, and trans-
caryophyllene (highest percentage composition in EO
extracted by SCF–CO2 : 0.41%, 0.67%, 0.22%, 0.93%, 1.81%,
0.16%, 0.11%, and 0.47%, respectively).
The variation in the chemical composition of EOs might
be due to differences in extraction power of different
extraction techniques. It has been reported that the chem-
ical composition of Boswellia serrate oleogum resin EO
changes with changing the extraction technique (Ayub
et al., 2022). Similarly, another report (Ayub et al., 2023)
studied the effect of extraction techniques on the chemi-
cal composition of Syzygium aromaticum L. seeds EO. It
was reported that the extraction techniques and condi-
tions significantly affected the chemical composition of
EO. Moreover, cinnamaldehyde, β-elemene, β-cubebene,
isosativene, and β-bisabolol were only detected in super-
heated steam-extracted EO of S. aromaticum L. at the con-
centrations of 4.56%, 1.15%, 0.36%, 0.31%, 0.17%, 0.57%, and
0.29%, respectively. Furthermore, it was reported that the
higher extraction power of superheated steam compared
to normal steam may have contributed to the extraction of
more compounds in SHSD (Ayub et al., 2023). In another
study, it has been reported that the chemical composi-
tion of leaves EO of Tetraclinis articulata (Vahl) Masters
was affected by extraction techniques; hydrocarbon com-
pounds were higher in concentration in EO extracted
by HD, whereas oxygenated compounds were higher in
concentration in EO extracted by microwave-assisted HD
(MAHD) (Djouahri et al., 2013). Similarly, in another study,
the chemical composition of Tunisian Thymus vulgaris
leaves EO was affected by different extraction methods,
that is, solvent-free microwave extraction (SFME), MAHD,
SD, and HD. A total of 17 compounds were extracted
11
by SFME, 11 compounds by both MAHD and SD, and 8
compounds were isolated by HD (Benmoussa et al., 2016).
4 CONCLUSION
In this study, P. roxburghii oleoresin essential oil has been
extracted by various extraction techniques. The effects of
extraction methods on yield, antioxidant potential, antimi-
crobial potential, and chemical composition have also
been studied. According to the results obtained from
this research, SHSD had a significant impact on the
yield, chemical composition, antioxidant, and antimicro-
bial activities of P. roxburghii oleo resin EO. EO extracted
by SHSD at 160◦ C yielded the highest EO yield. The
EO extracted by SHSD at 120◦ C demonstrated the great-
est antioxidant, antibacterial, and antifungal activity. The
major chemical components of P. roxburghii oleoresin
EO according to GC–MS results were 3-carene, α-pinene,
and β-pinene, which may be responsible for their antimi-
crobial activity. In future, more work is required on
optimization and experimental conditions for extraction
of EO from P. roxburghii oleoresin by SHSD and their
applications.
AU T H O R CO N T R I B U T I O N S
Muhammad Adnan Ayub: Investigation; Writing–
review & editing; Project administration; Supervision;
Writing–original draft. Nasrin Choobkar: Formal analy-
sis; Visualization; Conceptualization. Muhammad Asif
Hanif: Formal analysis; Resources. Mazhar Abbas:
Writing–review & editing; Software. Qurat Ul Ain:
Investigation; Writing–review & editing. Muhammad
Riaz: Visualization; Funding acquisition; Validation.
Amir Daraei Garmakhany: Supervision; Resources;
Visualization; Software; Methodology.
AC K N OW L E D G M E N T S
This work is a part of research project funded (grant
number 20-15988/NRPU/R&D/HEC/2021) by the
Higher Education Commission (HEC), Islamabad,
Pakistan.
C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.
D A T A AVA I L A B I L I T Y S T A T E M E N T
The data will be available from corresponding authors
upon reasonable request.
ORCID
Nasrin Choobkar https://orcid.org/0000-0002-8828-
4727
12
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How to cite this article: Ayub, M. A., Choobkar,
N., Hanif, M. A., Abbas, M., Ain, Q. U., Riaz, M., &
Garmakhani, A. D. (2023). Chemical composition,
antioxidant, and antimicrobial activities of P.
roxburghii oleoresin essential oils extracted by
steam distillation, superheated steam, and
supercritical fluid CO2 extraction. Journal of Food
Science, 1–14. https://doi.org/10.1111/1750-3841.16597