Industrial Crops & Products 139 (2019) 111505

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Industrial Crops & Products

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Chemical composition, kinetic study and antimicrobial activity of essential T

oils from Cymbopogon schoenanthus L. Spreng extracted by conventional and
microwave-assisted techniques using cryogenic grinding
⁎
Fatima-Zohra Bellika, Farid Benkaci-Alia, , Zouheir Alsafrab, Gauthier Eppeb, Samira Tatac,
Nasserdine Sabaouc, Racim Zidanid
a
Laboratory of Functional Organic Analysis, Department of Organic Chemistry, Faculty of Chemistry, University of Sciences and Technology Houari Boumediene (U.S.T.H.
B), El Alia, BP 32, Bab Ezzouar, 16111, Algiers, Algeria
b
Laboratoire de Spectrometrie de Masse L.S.M, University of Liege, Allée du 6 Août, Bât B6c, 4000, Liège, Sart-Tilman, Belgium
c
Department of Biology, Laboratory of Biology of the Microbial Systems, Ecole Normale Supérieure El Bachir El Ibrahimi (E.N.S), Kouba-Alger, BP 92, Algeria
d
SYMAO, 15 rte de fay 01660 mezeriat, France

A R T I C LE I N FO A B S T R A C T

Keywords: In this present work, volatile oils from Cymbopogon schoenanthus L. Spreng’s (CS) leaves extracted by microwave
Volatile oil assisted hydrodistillation (MAHD) and microwave assisted steam distillation (MASD) were studied according to
Microwave - assisted distillation simple (SG) and cryogenic grinding (CG) and compared with conventional hydrodistillation (HD).
Cryogenic grinding Extraction time, kinetics, energy consumption, CO2 rejected, physical properties, chemical composition and
GC–MS
antimicrobial activity were investigated for the first time with this specie using microwave extraction and
Antimicrobial activity
Cymbopogon schoenanthus L. Spreng (Poaceae)
showed that MAD is a promising and innovative technique for extraction of volatile oils; also, cryogenic grinding
allowed a high extraction yield compared to the classical grinding (MAHD-CG: 1.76% - MAHD-SG: 1.25%) and
(MASD-CG: 1.5% - MASD-SG: 1.11%). GC and GC–MS analysis showed variability in composition according to
the technique used especially in major constituents and in chemical classes.
Qualitative and quantitative kinetic study demonstrated a significative effect of extraction technique and
grinding mode on aromatic profile.
Evaluation of antimicrobial activity showed that the efficiency of volatiles from CS varied according to ex-
traction technique used where MASD-SG volatile was the most effective one.

1. Introduction
Cymbopogon schoenanthus L. Spreng (CS) commonly named as Camel
grass or Lemongrass, known in Algeria, as ‘Lemmad’. Is a sub-sponta-
neous (Naima et al., 2016), perennial (Khadri et al., 2010), aromatic/
medicinal plant that belongs to the Poaceae family (Cafferty et al.,
2000). Cymbopogon genus includes 56 species widely spread through
the Mediterranean area (Lino et al., 2010). Native to tropical Asia, this
plant is now distributed in Asia and in north and central Africa (Al-
snafi, 2016). Growing in dry stony places (Hashim et al., 2016). Cym-
bopogon schoenanthus L. Spreng was traditionally used to treat kidney
and urinary diseases, digestive diseases, rheumatisms, fever, food poi-
soning (Ramdane et al., 2015), intestinal troubles and for bringing back
the appetite (Khadri et al., 2008). In Menia (Algeria) where the plant
was collected for this experimental study, herb decoction is used as
mouthwash to relief dental pain, and to diminish cramping pain for
pregnant women before delivery. Essential oil isolated from this plant is
used in perfumery, cosmetics and pharmaceutics (Khadri et al.,
2010).Volatile oils can be isolated from aromatic plants using several
techniques including hydrodistillation (HD) which has been the tradi-
tional technique for essential oil extraction. However, this method is
high consuming time (causing thermal degradation) and energy as well
as releasing considerably carbon dioxide in the atmosphere. While,
many other techniques based on microwave heating constitutes a good
alternative for conventional processes (Benkaci-Ali et al., 2007), be-
cause of their low extraction time and energy consumption. Hence, the
extraction cost without adverse effecting essential oil quality as well as
enhancing extraction efficiency (Benkaci-Ali et al., 2006; Mandal et al.,
2007).
In recent years, microwave assisted extraction (MAE) has proven it

⁎
Corresponding author.
E-mail address: benkaciaf@yahoo.fr (F. Benkaci-Ali).

https://doi.org/10.1016/j.indcrop.2019.111505
Received 7 February 2019; Received in revised form 16 June 2019; Accepted 22 June 2019
Available online 11 July 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
F.-Z. Bellik, et al.
efficiency for the extraction compared to traditional techniques being
time and energy consuming, generating thermal and hydrolytique de-
gradations of thermolabile components, devaluing the quality of oils.
Besides, grinding process which precedes extraction is performed in
order to facilitate liberation of aroma by reducing size of particle and
increasing plant material surface (Murthy et al., 1999). However, this
procedure can cause loss of volatiles (Fornari et al., 2012), in addition
to thermal degradation for thermolabile compounds due to heat gen-
eration that accompanies frictional forces (Kaur and Srivastav, 2018).
Cryogenic grinding or cryo-grinding (CG) uses cryogenic liquids in
order to reduce temperature between the two rubbing surfaces pre-
venting the easily oxidative compounds from degradation which pre-
serves the quality of plant material. Liquid nitrogen (N2) supplies re-
frigeration effect at −196 °C and has been used in many studies in
agricultural field (Mékaoui et al., 2016; Singh and Goswami, 2000).
To the best of author’s knowledge, no work has been published on
kinetic extraction, characterization or evaluation of biological activity
of essential oil isolated from cryogrinded Cymbopogon schoenanthus L.
Spreng. Therefore, the objectives of this present work is to investigate
the chemical composition, extraction kinetics and antimicrobial activity
of Cymbopogon schoenanthus L. Spreng essential oil using microwave
assisted hydrodistillation (MAHD) and microwave assisted steam dis-
tillation (MASD) performing two grinding modes as classical and
cryogenic grinding (N2 at −196 °C) in terms of comparison. A quali-
tative and quantitative comparison was made with conventional hy-
drodistillation. Our objective is to make a convenient comparison in
terms of extraction time, energy consumption, environmental impact,
yield, physical properties, chemical composition and antimicrobial ac-
tivity.
2. Experimental
2.1. Plant material
Cymbopogon schoenanthus L. Spreng (CS) was collected from Oued
Sbaa, El-Menia, ALGERIA (Latitude: 30° 48′ N; Longitude: 2°50′ E;
Altitude 420 m) in March 2017. The Identification of the investigated
specie was confirmed in the herbarium of Botany Department, National
superior School of Agronomy (ENSA), Algiers. The plant material was
air-dried in the shade, sifted to remove sand residues, then, leaves were
separated from roots using pruning shears, leaves were stored at room
temperature in a moisture-free atmosphere until extraction. The mass of
CS’s leaves were 100, 50 and 40 g for HD, MAHD and MASD respec-
tively.
2.2. Chemicals
Alkane standard solutions C6-C28 (40 mg/ml) and the internal
standard C12, were purchased from Sigma-Aldrich (Steinheim,
Germany), Mueller-Hinton agar (MHA) and Sabouraud dextrose agar
(SDA) were bought from Merck (Darmstadt, Germany), Dimethyl sulf-
oxide (DMSO) was purchased from Carlo Erba (Val de Reuil, France).
All other solvents were of analytical grade purchased from Fluka and
Sigma-Aldrich.
2.3. Simple and cryogenic grinding
Rotor mill (Model Pulverisette 14, Fritsch, Germany) was worn for
simple (SG) and cryogenic grinding (CG) with a peripheral rotors speed
of 70 (15,000 RPM) m/ second. Plant material was grinded to high
particle size particle size (dp1) and low particle size (dp2) for SG and
CG respectively. The cryo-grinding was performed by pouring liquid
nitrogen at −196 °C to the vegetable matrix.
2
Industrial Crops & Products 139 (2019) 111505
2.4. Extraction procedure
Extraction of the essential oils from CS leaves was performed using
five different methods HD, MAHD-SG, MAHD-CG, MASD-SG and
MASD-CG. Extraction temperature is equal to water boiling tempera-
ture at atmospheric pressure (100 °C).
2.4.1. Hydrodistillation (HD) apparatus and procedure
100 g of grinded CS leaves were submitted to conventional hydro-
distillation using a Clevenger-type apparatus (Pharmacopée
Européenne, 1996), immersed with 1 L of distilled water. The collected
essential oil was dried under anhydrous sulphate and conserved at 4 °C
in a sealed amber vial until used for analyses. Each extraction was
realized three times.
2.4.2. Microwave assisted hydrodistillation (MAHD) apparatus and
procedure
A mass of 50 g of grinded CS leaves were soaked with 375 ml of
solvent (distilled water) in a 1 liter capacity round flask. The MAHD has
been performed using microwave laboratory as described in reference
(Mékaoui et al., 2016) at atmospheric pressure using a power of 800 W.
Ratio (mass of plant material /volume of water) and microwave power
were optimized in a preliminary study. In fact, four microwave powers
were tested: 500 W, 600 W, 800 W and 1000 W. It was shown that for
MAHD, increasing the power accelerated the time of extraction and
allowed to raise the yield of essential oil, but at 1000 W, a light smell of
burning has been noted; thus, the power value was fixed at 800 W. The
volatile oil isolated was dried under anhydrous sulphate and preserved
at 4 °C in a sealed amber vial until used for analysis. Each extraction
was achieved three times.
2.4.3. Microwave assisted Steam distillation (MASD) apparatus and
procedure
40 g of grinded leaves were submitted to MASD using a simple
column (Height =40 cm, Diameter =3 cm) coupled to a Clevenger-type
apparatus and treated with 300 ml of water. The power used was 600 W
in order to avoid the hazards of overpressure in the extraction appa-
ratus. The plant was exposed to steam without been immersed into
water. After condensation, the collected essential oil was dried under
anhydrous sulphate and stored at a temperature of 4 °C in a sealed
amber vial until used for analysis. Each extraction was carried out in
triplicate.
2.4.4. Extraction kinetics
For the kinetic experiments, the same conditions were applied for all
extraction methods; each sample of volatile oil was collected at dif-
ferent times (the sampling times differed from method to another).
Each fraction was weighted to measure the yield, and stored at 4 °C
until used for chromatographic analyses. The operation was carried out
in duplicate.
2.5. Physical constants
Specific gravity and refractive index of essential oils from
Cymbopogon schoenanthus L. Spreng extracted by the five methods have
been determined as reported by standard methods (Agence française de
Normalisation, 2007): specific gravity corresponds to the ratio of the
weight of the essential oil to the weight of an equal volume of water at
the same temperature. Refractive index was measured using an ATAGO
refractometer at 20° ± 1 °C.
2.6. GC-FID analysis
Gas chromatography analysis was performed using a 6890 series
instrument (Agilent Technologies, Palo Alto, CA, USA) gas chromato-
graph equipped with flame ionization detector (FID), the conditions
F.-Z. Bellik, et al.
followed were: fused-silica capillary column HP5-MS (60 m, 0.25 mm
i.d, 0.25 μm film thickness, 5% biphenyl, 95% dimethyl polysiloxane).
Carrier gas Helium (0.03 MPa, flow rate 0.5 ml/min), injector and de-
tector temperature were regulated at 280 and 300 °C. 1 μl of each
sample (oil diluted in hexane at 1%) was injected in the splitless mode,
oven temperature progress was from 60 °C for 10 min, then increased
from 60 to 250 °C at 4 °C/min and held at 250 °C for 10 min.
2.7. GC–MS analysis
Gas Chromatography-mass spectrometry (GC–MS) analysis was
achieved using same gas chromatograph and column (HP5-MS capillary
column, 60 m, 0.25 mm i.d, 0.25 μm film thickness) coupled to a MSD
5973 mass spectrometer. Oven temperature was programmed at 60 °C
for 8 min, then, increased from 60 to 250 °C at 4 °C/min and held at
250 °C during 10 min. in a splitless mode and injection volume of 1 μl
(samples were diluted in hexane at 1%). Helium was used as carrier gas
(flow rate of 1.2 ml/min). Injector and detector temperatures were kept
at 250 and 280 °C respectively. Scan time: 1.5 s, splitless time 0.75 min.
For MS detection, The MS source temperature was 230 °C; MS quad-
rapole temperature at 150 °C; mass scan, 30–550 amu and interface
temperature at 290 °C. ChemStation software was used to handle
chromatograms and mass spectra.
The volatile components were identified comparing their GC re-
tention indices (RI) on a HP5-MS column calculated according to Van
den Dool and Kratz (Van Den Dool and Dec. Kratz, 1963) determined
with reference to an homologous series of C6-C28 n-alkanes with those
of authentic standards (authors laboratory), and by comparing their
mass spectral fragmentation patterns with literature (Adams, 2007) and
wiley / NBS library as data bank. Quantification was done using re-
tention factors relative to dodecane (internal standard). Percentages of
components were calculated as average values of three analyses.
2.8. Antimicrobial activity
Antimicrobial activity of Cymbopogon schoenanthus L. Spreng essen-
tial oils isolated by the five methods was evaluated using two tests: Agar
disk diffusion method and determination of Minimum Inhibitory
Concentration (MIC).
2.8.1. Microbial strains
Antimicrobial activity of CS essential oils was evaluated against two
Gram-positive bacteria: Staphylococcus aureus MRSA 639cand Listeria
monocytogenes ATCC 13932, three Gram-negative bacteria: Klebsiella
pneumoniae E40, Salmonella Typhi ATCC 14028 and Escherichia coli
ATCC 8739, three fungi: Aspergillus westerdijkiae ATCC 3174, Fusarium
culmorum ATCC 36017 and Aspergillus flavus NRRL 3251) and one yeast:
Candida albicansM3.
Regarding experiments, microorganisms were cultured in Mueller-
Hinton Agar broth (MHA) for bacteria (at 30 °C for 24 h) and Sabouraud
dextrose Agar broth (SDA) for filamentous fungi (at 30 °C for 48 h).
2.8.2. Antimicrobial activity by Agar disc diffusion method
Paper disk diffusion method was used for the determination of an-
timicrobial activity as described by Djouahri et al. (2017) with slight
modifications: microbial suspensions were prepared by mixing and
vortexing fresh active cultures with physiological water; optical den-
sities were adjusted to 1 × 106 CFU/ml for bacterial strains and to
1 × 106 spore/ml for fungal strains. Then, spread using a sterile cotton
swab in post-autoclaving MHA medium for bacterial strains and SDA
medium for fungal strains (100 μl suspension /100 ml medium) in
90 mm Φ Petri dishes. 6 mm Φ filter paper discs were sterilized under
UV hood for 45 min, afterward, individually impregnated with 10 μl of
essential oil, then, placed at the surface of agar Petri dishes using
sterilized tweezers. Subsequently, these Petri dishes were kept at 4 °C
for 1 h for essential oil diffusion in the medium, then, incubated at 30 °C
3
Industrial Crops & Products 139 (2019) 111505
during 24 h for bacterial strains and at 30 °C during 48 h for fungal
strains. Antimicrobial activity was estimated by measuring diameter of
inhibition zones (mm). Amoxicillin was used as positive control. The
antimicrobial activity is expressed as the zone of inhibition (in milli-
meters) surrounding the discs (including diameter of disc). Anti-
microbial activity potential is estimated according the size of inhibition
zone, it is considered to be great (inhibition zone ≥ 20 mm), moderate
(inhibition zone 15–19 mm), weak to moderate (inhibition zone
9–15 mm) or weak (inhibition zone < 9 mm). All essays were per-
formed three times.
2.8.3. Determination of minimum inhibitory concentration (MIC)
Minimum inhibitory concentrations of different essential oils were
performed using broth micro-dilution assay according to National
Committee for Clinical Standards Guidelines (Jorgensen, 1993) with
slight modifications. For each sample of tested essential oils, an initial
solution of 1500 μg/ml was prepared in Dimethyl Sulfoxide (DMSO),
then, serial dilutions were realized with the same solvent to obtain
concentrations from 1 to 1500 μg/ml. Afterwards, each dilution was
added to Agar medium 10:90 (v/v) (MHA for bacterial strains and SDA
for fungal strains) in order to obtain concentrations from 0.1 to 150 μg/
ml. those resulted mixtures were immediately poured into Petri dishes.
Fresh cultures of each strain tested in agar disc diffusion method
(1 × 106 CFU/ ml for bacterial strains and 1 × 106 spore/ ml for fungal
strains) were prepared and inoculated in those Petri dishes, then, in-
cubated at 30 °C during 24 h for bacterial strains and at 30 °C during
48 h for fungal strains. Standard antibiotic Amoxicillin was tested for
comparison using the same concentrations. DMSO was used as negative
control. MIC corresponds to the lowest concentration that inhibits
growth of microbial strains. All experiments were performed in tripli-
cate.
2.9. Statistical analysis
Experimental results were recorded as means ± standard deviation
of three values (n = 3) using Microsoft Excel statistical analysis pro-
gram. K-Means clustering and Principal Component Analysis (PCA)
were performed by R programming language using RStudio software
(RStudio, Inc., Boston, Massachusetts, USA).
3. Results and discussion
3.1. Effect of extraction technique and time on yield of essential oil
To assess the influence of extraction time on the essential oil yield,
the overall extraction curves were constructed using the cumulated
mass of volatile oil collected at different time intervals. Variation of
extraction yield (%) according to time (min) for the different methods
used is represented by (Fig. 1) observing three phases. The first step is
Fig. 1. Variation of global (accumulated) yield (%) according to the extraction
time (min) for the different methods.
F.-Z. Bellik, et al. Industrial Crops & Products 139 (2019) 111505

an increasing line AB’, and the curvilinear B’B defining the first quan-
tities of volatile oil located in exogenous sites of the plant, this last is
marked by a decline in extraction yield which diminish gradually until
reaching the point B. During the first AB step the amount of EO ex-
tracted by the different techniques HD, MAHD-SG, MAHD-CG, MASD-
SG and MASD-CG are respectively 53.25, 62.07, 39, 92.4 and 85.74%.
The second step is represented by another curvilinear branch BC
characterizing the penetration of solvent (water) into the endogenous
sites of the plant causing the diffusion of essential oil from the midst of
particles via external environment. This stage took from 20 to 210 min
depending on the technique used and over of 75% of the remaining
amount of volatile oil has been extracted.
The third phase consists of a horizontal line marking the end of
extraction. Speed of extraction differs from a technique to another
noting that MASD is most rapid process than MAHD (40–50 min com-
pared to 120–150 min respectively) and HD (240 min), which leads to
conclude that microwave hydrodistillation (MAHD) is better than
conventional hydrodistillation, in terms of short extraction time,
cleanliness (using water as a green and an alternative solvent for ex-
traction) (Filly et al., 2016) and energy saving as confirmed by many
studies (Akloul et al., 2014; Benkaci-Ali et al., 2007; Bouchachia et al.,
2017; Chemat et al., 2006).
Table 1 shows variation of essential oil’s yield according to extrac-
tion technique and time. The HD technique provided, at 180 min of
extraction, a yield of 1.92 ± 0.01% while MAHD-SG at 45 min and
MASD-SG at 40 min allowed extracting 1.25 ± 0.14 and
1.11 ± 0.07% respectively. We point out that the total extraction time
was chosen to be lower than the one recorded in the kinetic study, in
order to prevent any thermal degradation reactions of thermolabile
compounds and allowing accurate evaluation of the chemical compo-
sition of the volatile oils isolated.
Cryogenic grinding process increased the recovered EO’s yields in
the same extraction time MAHD-CG at 45 min (1.76 ± 0.09%) and
MASD-CG at 40 min (1.5 ± 0.03%) compared to simple grinding
techniques. The cryogenic grinding (N2 at −196 °C) helps to maintain
low temperature by absorbing the generated heat during grinding
process (Akloul et al., 2014), this heat represents almost 99% of the
generated energy, whereas only 1% is used for real grinding (Tischer
et al., 2017).
In addition we noted a significative variation of essential oil yield
according the grinding mode and particle size (PS). The CG essential oil
recovery (low particle size) was higher than those of classical or simple
grinding (high particle size). This may be explained by the fact that
when the particles become finer, the solid-liquid contact surface of the
plant material increases, allowing a better solvent permeation and
improved extraction. In addition to keeping the temperature lower and
reducing the size of particles, the vapors from the liquid nitrogen create
an inert and dry environment giving supplementary protection of plant
quality (Saxena et al., 2018).
In agreement with our study, authors (Sharma et al., 2016) showed
that cryogenic grinding technology had significantly raised the amount
of extracted essential oil with high quality.
Extraction times and stages (kinetic) of highlighted techniques are
listed in Table 1. For economical (time and energy saving) and ecolo-
gical (reducing the quantity of CO2 released in the atmosphere) pur-
poses, it is better to choose an optimal extraction time permitting the
extraction of 80% of total yield, and according to that, the chosen times
for highlighted techniques are approximately: 120 min (HD), 50 min
(MAHD-SG), 30 min (MAHD-CG), 15 min (MASD-SG) and 15 min
(MASD-CG). In addition, it is preferable to limit the time at these values
for the reason of preventing the thermal degradation reactions (hy-
drolytic and oxidative) of thermolabile compounds. In the whole, per-
forming CG as well as increasing the applied energy (600 and 800 W)
accelerated extraction time, but raising the power can cause thermal
and hydrolytic degradations. Indeed, when we attempted to investigate
the extraction process at 1000 W, a light smell of burning has been
noticed; as a consequence, we avoided working above 800 W.
3.2. Cost, energy consumption and environmental impact
In term of extraction cost, MASD technique showed a clear ad-
vantage over MAHD and conventional HD. Regarding the energy con-
sumption, HD was the most high consuming energy technique (1.548
KWh) recorded in our study. In addition, MASD-SG (0.510 KWh) and
MASD-CG (0.517 KWh) techniques were low consuming energy com-
pared the MAHD-SG (0.854 KWh) and MAHD-CG (0.868 KWh). As
shown in Table 1, HD required before extraction a heating time of
26.58 min raising the temperature of water (1000 ml) and the plant
material (100 g) up to 100 °C, while MAHD took 19–20.30 min, how-
ever MASD needed only 11.92–12.53 minutes with a slight augmenta-
tion when the extraction is performed by cryogenic grinding. The last,
needs more time to heat the frozen plant material treated by liquid
nitrogen. From where, microwave assisted techniques reduces sig-
nificatively energy consumption compared to conventional hydro-
distillation as also confirmed by many authors (Asghari et al., 2012;
Bouchachia et al., 2017; Ferhat et al., 2007).
For environmental impact, the carbon dioxide released in the at-
mosphere was calculated according to the fact that 1 KWh generated by
the combustion of coal or other fossil fuel, releases 800 g of CO2 into the
atmosphere (Bernard, 2001). The higher amount of CO2 rejected were
recorded in HD technique (1238 g) followed by MAHD (SG: 683 – CG:
694.14 g) and MASD (SG: 408.24 – CG: 413.34 g).

Table 1
Effect of different parameters and extraction technique on yield, energy consumption, CO2 released in the atmosphere and extraction stages of CS essential oil
recorded by HD, MAHD-SG, MAHD-CG, MASD-SG and MASD-CG.
HD MAHD-SG MAHD-CG MASD-SG MASD-CG

Mass of plant material (g) 100 100 100 40 40

Volume of water (ml) 1000 750 750 300 300
Heating time (min) 26.58 19 20.13 11.92 12.53
Extraction time after heating (min) 180 45 45 40 40
Total extraction time (min) 206.58 64 65.13 51.92 52.53
Yield (% w/w) 1.92 ± 0.01 1.25 ± 0.14 1.76 ± 0.09 1.11 ± 0.07 1.5 ± 0.03
Electric consumption (kWh) 1.548 0.854 0.868 0.510 0.517
CO2 released (g) 1238.016 683 694.144 408.24 413.336
Extraction steps
AB' (min) 10 20 2 5 5
B'B (min) 20 10 3 15 15
BC (min) 210 120 115 20 30
CD (min) 30 50 60 10 10

AB: first step of extraction (AB’: linear profile representing the extraction of the essential oil situated in the external solid surface, B’B: curvilinear, characterized by a
reduction in extraction rate); BC: second step of extraction; CD: third step of extraction.

4
F.-Z. Bellik, et al.
Table 2
Physical properties of Cymbopogon schoenanthus L.Spreng essential oils isolated
by different extraction techniques.
HD MAHD-SG MAHD-CG MASD-SG MASD-CG
Refractive index at 1.4950 1.4900 1.4910 1.4885 1.4890
20 °C
Relative density at 0.8890 0.8780 0.8730 0.8700 0.8690
20 °C
Indeed, microwave can be selected as an alternative for the reason
of protecting thermolabile compounds, diminishing the amount of CO2
rejected in addition to energy and time economization (Asghari et al.,
2012; Bouchachia et al., 2017; Ferhat et al., 2007).
3.3. Physical constants
Evaluation of physical properties (refractive index and specific
gravity) of essential oil of Cymbopogon schoenanthus L. Spreng extracted
by HD, MAHD-SG, MAHD-CG, MASD-SG and MASD-CG are displayed in
Table 2. Both properties fall within the ranges 1.478–1.500 (at 20 °C)
for refractive index and 0.869- 0.904 (at 20 °C) for specific gravity (Al-
snafi, 2016) with a slight difference noted between oils extracted by the
microwave techniques (MAHD and MASD) compared to HD volatile oil,
due to their chemical composition changing depending on the tech-
nique used.
3.4. Effect of extraction technique on chemical composition
Chemical composition of Cymbopogon schoenanthus L. Spreng es-
sential oils isolated using the different methods HD, MAHD-SG, MAHD-
CG, MASD-SG and MASD-CG are displayed in Table 3.
In total, 109 components were identified in the volatile oils isolated
by all methods studied constituting 99.33% to 99.73% of the essential
oil isolated.
The major component for all techniques studied was α-eudesmol at
different concentration (HD: 17.89%; MAHD-SG: 14.18%; MAHD-CG:
15.54%; MASD-SG: 15.91% and MASD-CG: 11.51%). Based on previous
works, it was mentioned that α-eudesmol has antimicrobial activity
(Costa et al., 2008), antitrypanosomal activity (Otoguro et al., 2012)
and useful for the treatment of neurogenic inflammation and brain
injury (Asakura et al., 2000), this compound was also identified in the
CS essential oil of Saudi Arabia (11.5%) (Hashim et al., 2016), Benin
(2.1%–3.5%) (Bossou et al., 2015), Tunisia (0.4%–5.1%) (Khadri et al.,
2008) and Illizi from Algeria (1.8%) (Naima et al., 2016) with piper-
itone as major constituent, except from Tunisia CS with limonene as
major compound. This shows clearly the variability of the chemical
composition of Cymbopogon schoenanthus L. Spreng’s essential oil related
to geographical factors.
Other major components were also identified (Fig. 2) such as trans-
dauca-4(11),7-diene (10.39–12.58%); α-muurolol (3,77–6,55%); hi-
nesol (3.72–6.1%); allo-ocimene (5.02–7.71%) and δ-2-carene
(2.78–5.19%).
Thus, as observed in Table 3, α-eudesmol and hinesol (oxygenated
sesquiterpenes) were present in high amount in the HD essential com-
pared to allo-ocimene and isopulegol which were better extracted from
microwave techniques. Regarding, δ-2-carene and sylvestrene (mono-
terpene hydrocarbons) the highest percentages were recorded with
MASD-CG compared to trans-dauca-4 (11),7-diene with MASD-SG.
As shown in Table 3, we noted a predominance of oxygenated ses-
quiterpenes (30.28–50.37%) followed by sesquiterpenes hydrocarbons
(23.49–29.63%), monoterpene hydrocarbons (14.91–26.66%) and
oxygenated monoterpenes (8.47–13.62%) as well as traces of di-
terpenes (totarene, carissone, Z,E-geranyl linalool and manool). This
confirms that essential oil from Cymbopogon schoenanthus L. Spreng is a
5
Industrial Crops & Products 139 (2019) 111505
complex mixture of secondary metabolites and rich in oxygenated
sesquiterpenes, generally, the oxygenated compounds are strongly fra-
grant, making them much valuable (Akloul et al., 2014).
Additionally, it is important to note that extraction technique has
significant effect on qualitative and quantitative composition of CS’s
essential oil. Indeed, Table 3 reveals that oxygenated sesquiterpenes
were present in larger amount in HD essential oil (50.37%) compared to
microwave techniques MAHD and MASD. This is may be explained by
the fact that HD, long heating time based, allowed the penetration of
water (polar solvent) into the internal sites of the plant extracting a
greater amount of oxygenated compounds with higher molecular
weight. However this technique gives a low fraction of oxygenated
monoterpenes (8.47%) and monoterpene hydrocarbons (14.9%).
On other hand, using cryogenic grinding permitted rapid extraction
with considerable monoterpene hydrocarbons yields (MAHD-CG:
20.86%, MAHD-SG: 20.2% and MASD-CG: 26.66%, MASD-SG: 14.96%)
due to the loss of this compounds during the classical grinding because
of their lower boiling point (Tischer et al., 2017). Hence, liquid nitrogen
at −196 °C allowed decreasing the loss in monoterpene volatiles by
cooling the plant material during the grinding as well as enhancing the
extraction of monoterpenes compared to conventional hydrodistillation
by reducing the time of heating processes causing their thermal de-
gradation.
A non negligible compounds (in traces) were also isolated only
specifically according the technique achieved such as verbenene
(MASD-CG), neoiso-dihydro carveol acetate (MASD-SG), guaiol (MASD-
SG), aristolone (HD), 14-hydroxy-δ-cadinene (HD) and cryptomeridiol
(HD and MASD-SG). Besides, traces of three diterpenes (carissone, Z,E-
geranyl linalool and manool) were exclusively extracted by HD. In
addition, seven other components were totally absent in the EOs as Z-β-
ocimene (MASD-SG), trans-carveol (HD and MASD-CG), cis-thujopsa-
diene (MASD-SG and MASD-CG), nootkatene (MAHD-CG), eudesma-
4(15),7-dien-1β-ol (MASD-CG), iso-longifololacetate (MASD-SG) and
totarene (MASD-CG).
In agreement with previous works (Bossou et al., 2015; Hashim
et al., 2016; Yagi et al., 2016; Yentema et al., 2007), among the most
representative components of Cymbopogon schoenanthus L. Spreng oil
were eudesmane derivatives. In our study, seven eudesmane-type ses-
quiterpens were identified representing from 14.1 to 25.3% of total
chemical composition : 5-epi-7-epi-α-eudesmol (1.11–2.16%), γ-eu-
desmol (0.1–2.44%), α-eudesmol (11.64–17.89%), 7-epi-α-eudesmol
(0.61–1.2%), eudesma-4(15),7-dien-1β-ol (0.03–0.07%), eudesm-
7(11)-en-4-ol (0.61–2.34%) and eudesm-7(11)-en-4-ol, acetate
(0.02–0.05%).
Eudesmols belong to sesquiterpenoid alcohols having several phar-
macological effects (Britto et al., 2012). Previous studies realized by
Britto et al. (2012) showed that antitumor effect of essential oil from
Guatteria friesiana is related to its major constituents (α-, β-, and γ-
eudesmol). Other research showed that eudesmols possess a strong
antifungal activity (Mori et al., 2000; SeonHong et al., 2016).
3.5. Effect of extraction technique on chemical variability of essential oil
(statistical analysis)
In order to better evaluate the chemical variability of Cymbopogon
schoenanthus L. Spreng’s essential oils according to extraction technique,
data analysis with K-means clustering and Principal Component
Analysis (PCA) based on a scaled and centered matrix linking percen-
tages of components to extraction techniques (HD, MAHD-SG, MAHD-
CG, MASD-SG and MASD-CG) were performed using R programming
language.
Clustering classification permitted to distinguish three groups of
essential oils, the group 1 including HD and MASD-SG essential oils;
Class 2 combining MAHD-SG with MAHD-CG essential oils and class 3
corresponding to MASD-CG essential oil. This shows that composition is
much related to the technique and grinding mode used, from where a
F.-Z. Bellik, et al. Industrial Crops & Products 139 (2019) 111505

Table 3
Chemical composition of Cymbopogon schoenanthus L. Spreng extracted by the different Methods.
N° compound RI RI (lit.) HD MAHD-SG MAHD-CG MASD-SG MASD-CG

1 Verbenene 963 961 – – – – 0.02 ± 0

2 Cis-meta-mentha-2,8-diene 985 983 0.3 ± 0.02 0.3 ± 0.02 0.36 ± 0.03 0.19 ± 0.01 0.57 ± 0.06
3 Dehydro-1,8-Cineole 990 988 0.02 ± 0 0.02 ± 0 0.03 ± 0 0.02 ± 0 0.03 ± 0
4 δ-2- Carene 1003 1001 4.16 ± 0.44 4.32 ± 0.39 5.19 ± 0.47 2.78 ± 0.23 7.42 ± 0.68
5 δ-3-Carene 1009 1008 0.57 ± 0.03 1.05 ± 0.05 0.94 ± 0.06 0.62 ± 0.04 1.34 ± 0.11
6 α-Terpinene 1017 1014 0.27 ± 0.03 0.56 ± 0.05 0.48 ± 0.04 0.31 ± 0.03 0.68 ± 0.07
7 p-Cymene 1023 1020 1.1 ± 0.09 1.35 ± 0.18 1.16 ± 0.11 0.74 ± 0.03 1.89 ± 0.07
8 Sylvestrene 1028 1025 3.38 ± 0.16 4.69 ± 0.19 4.83 ± 0.2 2.72 ± 0.12 7.16 ± 0.3
9 Z- β-Ocimene 1035 1032 0.02 ± 0 0.04 ± 0 0.14 ± 0.02 – 0.02 ± 0
10 y-Terpinene 1056 1054 0.02 ± 0 0.05 ± 0 0.05 ± 0 0.02 ± 0 0.04 ± 0
11 Terpinolene 1087 1085 0.03 ± 0 0.07 ± 0.01 0.06 ± 0 0.02 ± 0 0.1 ± 0.02
12 p-Cymenene 1091 1089 0.04 ± 0 0.06 ± 0 0.05 ± 0 0.04 ± 0 0.06 ± 0
13 Exo-Fenchol 1120 1118 0.06 ± 0 0.02 ± 0 0.07 ± 0.01 0.04 ± 0 0.09 ± 0.02
14 Allo-ocimene 1130 1128 5.02 ± 0.34 7.71 ± 0.64 7.6 ± 0.47 7.52 ± 0.32 7.36 ± 0.29
15 Cis-Verbenol 1140 1137 0.05 ± 0.01 0.05 ± 0 0.07 ± 0.01 0.04 ± 0 0.05 ± 0
16 Isopulegol 1147 1145 3.67 ± 0.22 5.58 ± 0.31 5.22 ± 0.37 4.61 ± 0.41 5.27 ± 0.19
17 p-Mentha-1,5-dien-8-ol 1167 1166 0.08 ± 0.02 0.13 ± 0.03 0.12 ± 0.09 0.1 ± 0.07 0.11 ± 0.06
18 Terpinen-4-ol 1177 1174 0.02 ± 0 0.03 ± 0 0.02 ± 0 0.02 ± 0 0.02 ± 0
19 Cryptone 1185 1183 0.1 ± 0.02 0.13 ± 0.03 0.1 ± 0.01 0.08 ± 0 0.09 ± 0.02
20 Cis - Piperitol 1197 1195 2.07 ± 0.15 3.79 ± 0.28 3.51 ± 0.17 2.81 ± 0.21 3.87 ± 0.25
21 p-Cymen-9-ol 1206 1204 0.03 ± 0 0.04 ± 0 0.03 ± 0 0.02 ± 0 0.05 ± 0
22 Trans- Piperitol 1210 1207 1.97 ± 0.09 3.13 ± 0.23 2.78 ± 0.17 2.79 ± 0.20 2.94 ± 0.13
23 Trans-Carveol 1217 1215 – 0.03 ± 0 0.02 ± 0 0.02 ± 0 –
24 Neo iso-dihydro carveol 1229 1226 0.02 ± 0 0.04 ± 0 0.03 ± 0 0.03 ± 0 0.05 ± 0.01
25 Cumin aldehyde 1240 1238 0.03 ± 0 0.05 ± 0 0.04 ± 0 0.03 ± 0 0.04 ± 0
26 Car-3-en-2-one 1247 1244 0.08 ± 0.01 0.18 ± 0.03 0.12 ± 0.05 0.1 ± 0.01 0.14 ± 0.02
27 Piperitone 1251 1249 0.22 ± 0.06 0.35 ± 0.05 0.31 ± 0.02 0.26 ± 0 0.26 ± 0.03
28 p-Menth-1-en-7-al 1275 1273 0.03 ± 0 0.03 ± 0 0.02 ± 0 0.02 ± 0 0.03 ± 0
29 3'-methoxy-Acetophenone 1296 1298 0.05 ± 0.01 0.04 ± 0 0.03 ± 0 0.04 ± 0 0.04 ± 0
30 p-Menth-1-en-9-ol 1396 1294 0.02 ± 0 0.02 ± 0 0.03 ± 0 0.03 ± 0 0.02 ± 0
31 δ-Elemene 1337 1335 0.09 ± 0.02 0.07 ± 0 0.09 ± 0.01 0.14 ± 0.03 0.11 ± 0
32 α-Cubebene 1347 1345 0.08 ± 0.02 0.09 ± 0.01 0.06 ± 0 0.07 ± 0 0.14 ± 0.06
33 Neo iso dihydro carveol acetate 1358 1356 – – – 0.02 ± 0 –
34 α-Ylangene 1373 1373 0.08 ± 0.01 0.04 ± 0 0.06 ± 0.01 0.11 ± 0.03 0.07 ± 0.01
35 α-Copaene 1377 1374 0.12 ± 0.09 0.15 ± 0.07 0.09 ± 0.02 0.1 ± 0.01 0.21 ± 0.02
36 Modheph-2-ene 1385 1382 0.09 ± 0.01 0.08 ± 0 0.07 ± 0 0.11 ± 0 0.1 ± 0.01
37 β-Bourborene 1387 1387 0.02 ± 0 0.02 ± 0 0.02 ± 0 0.02 ± 0 0.02 ± 0
38 β- Elemene 1391 1389 2.56 ± 0.17 2.38 ± 0.22 2.14 ± 0.12 2.92 ± 0.26 3.49 ± 0.31
39 β-Longipinene 1399 1400 0.04 ± 0 0.03 ± 0 0.03 ± 0 0.05 ± 0 0.04 ± 0
40 β-Cedrene 1421 1419 0.35 ± 0.07 0.31 ± 0.05 0.35 ± 0.03 0.55 ± 0.06 0.49 ± 0.05
41 β-Duprezianene 1424 1421 0.16 ± 0.02 0.15 ± 0.02 0.11 ± 0.01 0.21 ± 0.03 0.21 ± 0.04
42 Cis-Thujopsene 1431 1429 0.27 ± 0.03 0.16 ± 0.02 0.29 ± 0.03 0.52 ± 0.04 0.2 ± 0.01
43 β-Gurjunene 1433 1431 0.34 ± 0.04 0.38 ± 0.04 0.35 ± 0.02 0.34 ± 0.01 0.67 ± 0.05
44 α-Guaiene 1439 1437 0.14 ± 0.01 0.08 ± 0 0.11 ± 0 0.2 ± 0.02 0.99 ± 0.03
45 6,9-Guaiadiene 1444 1442 0.06 ± 0 0.04 ± 0 0.05 ± 0 0.1 ± 0.04 0.05 ± 0
46 α-Himachalene 1450 1449 0.06 ± 0 0.07 ± 0.01 0.07 ± 0 0.09 ± 0.02 0.12 ± 0.03
47 α-neo-Clovene 1454 1452 0.12 ± 0.01 0.12 ± 0.02 0.11 ± 0 0.13 ± 0 0.17 ± 0.02
48 α-Humulene 1454 1452 0.09 ± 0.01 0.11 ± 0 0.09 ± 0 0.1 ± 0.01 0.14 ± 0.01
49 Allo-Aromadendrene 1460 1458 0.05 ± 0 0.04 ± 0 0.04 ± 0 0.08 ± 0.01 0.04 ± 0
50 Cis-Muurola-4(14),5-diene 1466 1465 0.08 ± 0 0.06 ± 0 0.08 ± 0.01 0.1 ± 0.01 0.16 ± 0.02
51 Cis-Thujopsadiene 1468 1465 0.12 ± 0 0.11 ± 0.03 0.02 ± 0 – –
52 y-Muurolene 1479 1478 0.71 ± 0.05 0.71 ± 0.03 0.15 ± 0.02 0.15 ± 0.01 0.11 ± 0.03
53 Germacrene D 1484 1484 0.21 ± 0.02 0.1 ± 0 0.47 ± 0.02 0.63 ± 0.04 0.77 ± 0.03
54 Aristolochene 1489 1487 0.54 ± 0.01 0.65 ± 0.02 0.83 ± 0.04 1.42 ± 0.09 0.85 ± 0.08
55 Epi-Cubebol 1496 1493 3.02 ± 0.25 2.65 ± 0.13 1.96 ± 0.11 2.6 ± 0.17 3.29 ± 0.28
56 α-Muurolene 1502 1500 1.9 ± 0.11 1.97 ± 0.1 1.52 ± 0.09 2.5 ± 0.09 2.32 ± 0.1
57 α-Bulnesene 1507 1509 0.2 ± 0.04 0.11 ± 0.01 0.15 ± 0.03 0.17 ± 0.01 0.15 ± 0
58 Modhephene-8-β-ol 1515 1513 0.1 ± 0.02 0.04 ± 0 0.05 ± 0.01 0.05 ± 0.01 0.02 ± 0
59 Nootkatene 1515 1517 0.07 ± 0 0.02 ± 0 – 0.02 ± 0 0.05 ± 0
60 7-epi-α-Selinene 1520 1520 1.75 ± 0.19 1.64 ± 0.1 1.29 ± 0.09 1.86 ± 0.14 2.24 ± 0.2
61 δ- Cadinene 1524 1522 2.4 ± 0.17 2.98 ± 0.17 2.11 ± 0.09 2.3 ± 0.22 3.23 ± 0.14
62 (E)-iso-y-Bisabolene 1530 1528 0.35 ± 0.03 0.3 ± 0.01 0.3 ± 0.02 0.51 ± 0.02 0.48 ± 0.02
63 α-Cadinene 1537 1537 0.79 ± 0.04 0.91 ± 0.02 0.67 ± 0.03 0.75 ± 0.03 0.88 ± 0.04
64 Selina-3,7(11)-diene 1546 1545 0.29 ± 0.04 0.29 ± 0.02 0.21 ± 0 0.27 ± 0.04 0.32 ± 0.02
65 α-Calacorene 1547 1544 0.25 ± 0.04 0.15 ± 0 0.14 ± 0.01 0.17 ± 0.02 0.17 ± 0.01
66 Trans- dauca-4(11),7-diene 1558 1556 10.67 ± 0.51 11.62 ± 0.3 11.1 ± 0.46 12.58 ± 0.54 10.39 ± 0.42
67 β-Calacorene 1566 1564 0.22 ± 0.01 0.18 ± 0.02 0.25 ± 0.01 0.27 ± 0 0.19 ± 0.03
68 1α,10α-epoxy-Amorph-4-ene 1572 1570 0.03 ± 0 0.04 ± 0 0.03 ± 0 0.03 ± 0 0.03 ± 0
69 Germacrene D-4-ol 1576 1574 0.16 ± 0.02 0.34 ± 0.03 0.36 ± 0.03 0.37 ± 0.03 0.29 ± 0.01
70 Caryophyllene oxide 1585 1582 0.45 ± 0.04 0.48 ± 0.02 0.4 ± 0.01 0.47 ± 0 0.28 ± 0.02
71 Cis-dihydro-Mayurone 1597 1595 0.18 ± 0 0.17 ± 0.02 0.14 ± 0.01 0.07 ± 0.01 0.09 ± 0.01
72 Guaiol 1602 1600 – – – 0.08 ± 0.01 –
73 5-epi-7-epi-α -Eudesmol 1610 1607 2.16 ± 0.12 1.8 ± 0.21 1.79 ± 0.08 1.77 ± 0.19 1.11 ± 0.07
74 1,10-di-epi-Cubenol 1620 1618 0.16 ± 0.01 0.16 ± 0.01 0.16 ± 0.02 0.14 ± 0.03 0.24 ± 0
(continued on next page)

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F.-Z. Bellik, et al. Industrial Crops & Products 139 (2019) 111505

Table 3 (continued)

N° compound RI RI (lit.) HD MAHD-SG MAHD-CG MASD-SG MASD-CG

75 1-epi-cubenol 1628 1627 0.3 ± 0.02 0.21 ± 0.01 0.16 ± 0.05 0.17 ± 0.04 0.77 ± 0.06
76 γ- Eudesmol 1633 1630 1.97 ± 0.08 1.24 ± 0.01 2.19 ± 0.14 2.44 ± 0.16 0.1 ± 0.04
77 Hinesol 1642 1640 6.1 ± 0.2 5.45 ± 0.17 4.85 ± 0.22 5.21 ± 0.19 3.72 ± 0.31
78 α- Muurolol (=δ- Cadinol) 1646 1644 6.55 ± 0.33 4.76 ± 0.24 5.93 ± 0.41 5.64 ± 0.27 3.77 ± 0.36
79 Cubenol 1647 1645 1.66 ± 0.11 2.35 ± 0.08 2.33 ± 0.09 1.13 ± 0.12 1.15 ± 0.07
80 α- cadinol 1655 1652 2.66 ± 0 1.59 ± 0.09 1.25 ± 0.11 1.91 ± 0.07 1.56 ± 0.1
81 α- Eudesmol 1654 1652 17.89 ± 0.79 14.18 ± 0.36 15.54 ± 0.41 15.92 ± 0.75 11.64 ± 0.5
82 7-epi-α-Eudesmol 1664 1662 1.2 ± 0.09 0.68 ± 0.05 0.64 ± 0.01 0.61 ± 0.03 0.62 ± 0.02
83 Longiborneol acetate 1678 1684 1.21 ± 0.07 0.82 ± 0 1.25 ± 0.09 1.49 ± 0.14 0.4 ± 0.08
84 Cadalene 1685 1675 0.13 ± 0.02 0.07 ± 0.01 0.07 ± 0 0.06 ± 0.01 0.06 ± 0
85 Eudesma-4(15),7-dien-1β-ol 1689 1687 0.07 ± 0.01 0.03 ± 0 0.04 ± 0 0.03 ± 0 –
86 Acorenone 1694 1692 0.11 ± 0.02 0.08 ± 0 0.07 ± 0 0.06 ± 0 0.04 ± 0
87 Amorpha-4,9-diene-2-ol 1701 1700 0.08 ± 0.01 0.04 ± 0 0.05 ± 0 0.05 ± 0 0.02 ± 0
88 Eudesm-7(11)-en-4-ol 1703 1700 2.32 ± 0.19 1.14 ± 0.08 2.34 ± 0.24 2.45 ± 0.3 0.61 ± 0.09
89 10-nor-Calamenen-10-one 1705 1702 0.14 ± 0.02 0.11 ± 0 0.14 ± 0.01 0.12 ± 0.01 0.04 ± 0
90 Mayurone 1712 1709 0.59 ± 0.04 0.38 ± 0.04 0.42 ± 0.02 0.32 ± 0.01 0.15 ± 0
91 iso-Longifolol 1730 1728 0.15 ± 0.04 0.07 ± 0 0.11 ± 0.03 0.09 ± 0.01 0.03 ± 0
92 Curcumenol 1735 1733 0.1 ± 0.03 0.05 ± 0 0.06 ± 0 0.06 ± 0 0.02 ± 0
93 7, 14-anhydro-Amorpha-4,9-diene 1754 1755 0.07 ± 0 0.04 ± 0 0.05 ± 0 0.04 ± 0 0.02 ± 0
94 Cyclocolorenone 1760 1759 0.06 ± 0 0.03 ± 0 0.04 ± 0 0.04 ± 0 0.02 ± 0
95 Aristolone 1764 1762 0.04 ± 0 – – – –
96 14-hydroxy-α-Muurolene 1782 1779 0.1 ± 0.01 0.02 ± 0 0.05 ± 0 0.04 ± 0 0.02 ± 0
97 (E)-Isovalencenol 1791 1793 0.1 ± 0.02 0.04 ± 0 0.05 ± 0 0.06 ± 0 0.03 ± 0
98 14-hydroxy-δ-Cadinene 1806 1803 0.02 ± 0 – – – –
99 iso-LongifoloIacetate 1821 1819 0.08 ± 0.01 0.06 ± 0 0.07 ± 0.01 – 0.03 ± 0
100 Cryptomeridiol 1815 1813 0.08 ± 0.02 – – 0.07 ± 0 –
101 β-Vetivone 1824 1822 0.35 ± 0.09 0.14 ± 0.02 0.19 ± 0.04 0.17 ± 0.02 0.07 ± 0
102 Eudesm-7(11)-en-4-ol, acetate 1841 1839 0.02 ± 0 0.02 ± 0 0.02 ± 0 0.05 ± 0 0.02 0
103 (2E,6E)-Farnesyl acetate 1847 1845 0.05 ± 0 0.02 ± 0 0.02 ± 0 0.04 ± 0 0.03 ± 0
104 α-Chenopodiol 1857 1855 0.04 ± 0 0.05 ± 0 0.04 ± 0 0.07 ± 0.01 0.05 ± 0
105 Cubitene 1878 1878 0.03 ± 0 0.02 ± 0 0.02 ± 0 0.03 ± 0 0.02 ± 0
106 Totarene 1925 1922 0.03 ± 0 0.02 ± 0 0.02 ± 0 0.03 ± 0 –
107 Carissone 1928 1926 0.03 ± 0 – – – –
108 Z,E-Geranyl linalool 2001 1998 0.02 ± 0 – – – –
109 Manool 2058 2056 0.02 ± 0 – – – –

Monoterpene hydrocarbons 14.91 20.2 20.86 14.96 26.66

Oxygenated monoterpenes 8.47 13.62 12.52 11.02 13.06
Sesquiterpene hydrocarbons 25.40 26.19 23.49 29.60 29.63
Oxygenated sesquiterpenes 50.37 39.28 42.79 43.88 30.28
Other components 0.18 0.08 0.07 0.1 0.06
Total 99.33 99.37 99.73 99.56 99.69

RI (lit): retention indices of reference (Adams, 2007). RI: calculated retention indices. Values represent the average of three values; Mean ± SD (n = 3).

significative variability between the EOs extracted by highlighted
methods. The first three PCA axes explained 90.24% of information,
and the representation was made in the two first axes (69.27% of in-
formation) (Fig. 3), PCA confirmed K-Means results revealing three
separate groups. The group formed by MASD-CG essential oil has been
previously characterized from other oils by it richness in monoterpene
hydrocarbons (Table 3), the second group is formed by MAHD-SG and
MAHD-GC essential oils, displaying a relative similarities in chemical
classes percentages, whereas the third HD and MASD-SG regroups EOs
marked the highest content in oxygenated sesquiterpenes.
Surprisingly, cryogenic grinding caused a significative variability in
qualitative and quantitative composition for MASD EOs, hence a non
significative effect was noted for the MAHD EOs. From where, results
suggested that extraction time (low for MASD compared to MAHD) and

Fig. 2. percentage of major constituents of Cymbopogon schoenanthus L. Spreng essential oil extracted by different techniques.

7
F.-Z. Bellik, et al.
Fig. 3. PCA for CS essential oils extracted by the five methods according to
chemical composition.
the contact solid-liquid (no contact plant material-boiling water) can be
responsible of this distinguished essential oils.
3.6. Effect of time (kinetic extraction) on chemical composition of
Cymbopogon schoenanthus L. Spreng essential oil isolated by different
methods
In order to spot the influence of extraction time on chemical com-
position and behavior of CS L. Spreng essential oil during the extraction
process, each volatile oil sample collected corresponding to each point
of the curves presented in Fig. 1, was injected and analyzed by GC-FID
and GC–MS analysis in the same conditions as the previous essential oils
extracted. The accumulated percentages of ten major constituents of
essential oils extracted by HD, MAHD-SG, MAHD-CG, MASD-SG and
MASD-CG according to different times are presented in Figs. 4, 5 and 6.
Regarding Fig. 4a α-eudesmol was present during all time of ex-
traction varying according extraction technique (HD: 8.68–15.91%;
MAHD-SG: 4.26–18.13%; MAHD-CG: 4.65–13.14%; MASD-SG:
4.31–12.21% and MASD-CG: 2.84–8.45%), showing a continuous in-
creasing of this compound during the extraction process in the five
highlighted methods especially in the first step of extraction (AB). Other
major constituents such as trans-dauca-4(11),7-diene (Fig. 4b), allo-
ocimene (Fig. 4c), α-muurolol (Fig. 4d), Hinesol (Fig. 5a), δ-2-carene
(Fig. 5c), sylvestrene (Fig. 5d)and β-elemene (Fig. 6b), all of them were
also present in high yield during the first time of extraction process,
Fig. 4. Accumulated percentages of major constituents according to extraction time: (a) α-eudesmol, (b) trans-dauca-4(11),7-diene, (c) allo-ocimene, (d) α-muurolol.
8
Industrial Crops & Products 139 (2019) 111505
varying according the method used (except for isopulegol (Fig. 5b) and
cis-piperitol (Fig. 6a)).
In general, for low molecular weight compounds like cis-piperitol
(Fig. 6a), allo-ocimene (Fig. 4c) and isopulegol (Fig. 5b) we observed an
increase in percentages to reach a maximum value in the first step of
extraction as cis-piperitol (HD: 4.54% at 10 min; MAHD-SG: 6.41% at
2 min; MAHD-CG: 6.42% at 2 min; MASD-SG: 6.36% at 5 min and
MASD-CG: 6.80% at 5 min); allo-ocimene (HD: 15.50% at 10 min;
MAHD-SG: 19.66% at 2 min; MAHD-CG: 16.03% at 2 min; MASD-SG:
15.48% at 5 min and MASD-CG: 13.56% at 5 min) and isopulegol (HD:
11.37% at 10 min; MAHD-SG: 11.99% at 2 min; MAHD-CG: 10.05% at
2 min; MASD-SG: 9.96% at 5 min and MASD-CG: 8.52% at 5 min). This
step is followed by a decreasing curvilinear line (second step) to reach
the end of extraction (last step); this decreasing step is probably due
simultaneously to the decrease of the relative percentage and the ab-
solute value during the extraction time. This phenomenon can be at-
tributed to many factors such as the low molecular weight of mono-
terpenes as well as the content of these components in exogenous and/
or endogenous sites. Other previous studies are in agreement with our
findings (Benyoussef et al., 2005; Fornari et al., 2012; Tigrine-Kordjani
et al., 2012).
Whilst, for the compounds with high molecular weight such as trans-
dauca-4(11),7-diene, Hinesol and α-muurolol (sesquiterpene hydro-
carbons and oxygenated sesquiterpens), the yield increased during ex-
traction time. Conversely, δ-2-carene, sylvestrene and β-elemene
showed a decreasing profile for microwave techniques compared to HD,
their percentage increased significatively during the first minutes of
extraction then tending to a horizontal line indicating the end of the
extraction process.
Chemically, and as indicated in Figs. 4, 5 and 6, the essential oils
collected in each time by MAHD-CG and MASD-CG were different to
those obtained by MAHD-SG and MASD-SG respectively, this variation
is probably caused by change in the structure inside the grinded plant in
contact with water or water vapors during the extraction process.
Indeed, physical structure of the plant affects the diffusion of oil from
this matrix.
Globally qualitative kinetic study proved that microwave extraction
processes (internal heating) were fast compared to conventional hy-
drodistillation (external heating).Yet, the oxygenated compounds iso-
lated in less amounts with microwave methods specially MASD-CG
compared to long time extraction technique as HD do not signify a
lower quality of microwave volatiles. Moreover, some of the oxyge-
nated compounds (HD oils) can be degradation products due to thermal
F.-Z. Bellik, et al.
Fig. 5. Accumulated percentages of major constituents according to extraction time: (a) Hinesol, (b) Isopulegol, (c) 2-δ-carene, (d) sylvestrene.
reactions. In addition, the essential oils present in the plants can contain
more monoterpene and sesquiterpene hydrocarbons than those ob-
tained during the heating process (Akloul et al., 2014; Benkaci-Ali et al.,
2006).
3.7. Effect of extraction technique on antimicrobial activity
The antimicrobial activity of Cymbopogon schoenanthus L. Spreng
essential oils against tested microorganisms was qualitatively and
quantitatively estimated by inhibition zone diameter and MIC values
respectively.
3.7.1. Agar disc diffusion test
Results show that inhibitory effect was detected on eight pathogens
(Table 4). Essential oil extracted by HD showed a high activity against
Fusarium culmorum (26.33 ± 0.58 mm) and Listeria monocytogenes
(21.67 ± 0.58 mm), moderate activity against Staphylococcus aureus
(15.33 ± 0.58 mm), weak to moderate activity against Klebsiella pneu-
moniae (11.67 ± 0.29 mm), Aspergillus flavus (10.33 ± 0.58 mm) and
Salmonella Typhi (09.00 ± 0.50 mm) and week activity against Candida
albicans (07.33 ± 0.58 mm) and Aspergillus westerdijkiae
(06.50 ± 0.50 mm). Whereas, Essential oil extracted by MAHD-SG
presented a high activity against Fusarium culmorum
(20.33 ± 0.58 mm), moderate activity against Listeria monocytogenes
(19.00 ± 1.00 mm), weak to moderate activity against Staphylococcus
aureus (13.17 ± 0.29 mm), Aspergillus flavus (13.17 ± 0.29 mm),
Klebsiella pneumoniae (10.50 ± 0.50 mm) and weak activity against
Salmonella Typhi (08.67 ± 1.15 mm), Aspergillus westerdijkiae
Fig. 6. Accumulated percentages of major constituents according to extraction time: (a) cis-Piperitol, (b) β-Elemene.
9
Industrial Crops & Products 139 (2019) 111505
(08.67 ± 0.58 mm) and Candida albicans (08.67 ± 0.58 mm).
In addition, essential oil isolated from MAHD-CG exhibited a high
activity against Fusarium culmorum (27.00 ± 1.00 mm), moderate ac-
tivity against Listeria monocytogenes (17.33 ± 0.29 mm) and Aspergillus
westerdijkiae (15.33 ± 0.29 mm), weak to moderate activity against
Aspergillus flavus (12.50 ± 0.29 mm), Staphylococcus aureus
(11.50 ± 0.50 mm) and Candida albicans (10.00 ± 0.00 mm) and
weak activity against Salmonella Typhi (08.00 ± 0.50 mm) and
Klebsiella pneumoniae (07.67 ± 0.29 mm).
For MASD-SG essential oil, results revealed a great activity against
Fusarium culmorum (45.67 ± 1.15 mm), Listeria monocytogenes
(26.17 ± 0.29 mm) and Aspergillus westerdijkiae (20.67 ± 1.15 mm),
moderate activity against Aspergillus flavus (15.33 ± 0.58 mm) and
weak to moderate activity against Staphylococcus aureus
(14.50 ± 0.50 mm), Klebsiella pneumoniae (14.17 ± 0.29 mm),
Candida albicans (10.67 ± 0.58 mm) and Salmonella Typhi
(10.33 ± 0.29 mm).
Finally, essential oil extracted by MASD-CG presented great activity
against Fusarium culmorum (25.83 ± 0.29 mm) and Listeria mono-
cytogenes (20.67 ± 0.29 mm), weak to moderate activity against
Aspergillus flavus (11.17 ± 0.76 mm), Staphylococcus aureus
(10.33 ± 0.29 mm), Aspergillus westerdijkiae (9.33 ± 0.58 mm) and
Klebsiella pneumoniae (09.17 ± 0.29 mm) and weak activity against
Salmonella Typhi (08.17 ± 0.29 mm) and Candida albicans
(07.33 ± 0.58 mm).
Table 4 indicates clearly that filamentous fungus Fusarium culmorum
as well Listeria monocytogenes bacteria were strongly inhibited by C.
schoenanthus L. Spreng essential oils specially MASD oil with classical
F.-Z. Bellik, et al. Industrial Crops & Products 139 (2019) 111505

Table 4
Disk diffusion (mm) of Cymbopogon schoenanthus L. Spreng essential oils (10 μl) isolated by the five extraction methods.
Microorganisms Inhibition zone (mm)

HD MAHD-SG MAHD-CG MASD-SG MASD-CG Amoxicillin (30 μg/disc)

Bacterial strains
Staphylococcus aureus 15.33 ± 0.58 13.17 ± 0.29 11.50 ± 0.50 14.50 ± 0.50 10.33 ± 0.29 17.83 ± 0.76
Listeria monocytogenes 21.67 ± 0.58 19.00 ± 1.00 17.33 ± 0.29 26.17 ± 0.29 20.67 ± 0.58 30.33 ± 1.04
Klebsiella pneumoniae 11.67 ± 0.29 10.50 ± 0.50 07.67 ± 0.29 14.17 ± 0.29 09.17 ± 0.29 40.17 ± 0.29
Salmonella Typhi 09.00 ± 0.50 08.67 ± 1.15 08.00 ± 0.50 10.33 ± 0.29 08.17 ± 0.29 19.50 ± 0.58
Escherichia coli nd nd nd nd nd 29.50 ± 0.50
Fungal strains
Aspergillus westerdijkiae 06.50 ± 0.50 08.67 ± 0.58 15.33 ± 0.29 20.67 ± 1.15 09.33 ± 0.58 nd
Fusarium culmorum 26.33 ± 0.58 20.33 ± 1.15 27.00 ± 1.00 45.67 ± 1.15 25.83 ± 0.29 nd
Aspergillus flavus 10.33 ± 0.58 13.17 ± 0.29 12.50 ± 0.50 15.33 ± 0.58 11.17 ± 0.76 nd
Yeast
Candida albicans 7.33 ± 0.58 8.67 ± 0.58 10.00 ± 0.00 10.67 ± 0.58 7.33 ± 0.58 nd

grinding probably due to it specific composition (Table 3). However,
Escherichia coli was the most resistant strain where no inhibition zone
was observed may be because the qualitative composition of the vola-
tiles not containing specific compounds against this strain.
Results also showed that Gram-positive bacteria were more sensitive
to the action of investigated essential oils than Gram-negative bacteria.
The intricacy of EOs mechanism of action on microorganisms gen-
erally depends on their hydrophilic or lipophilic character, the structure
of microorganism’s cell as well as the arrangement of it external
membrane (Kalemba and Kunicka, 2003). Generally, Gram-positive
organisms are more week to the action of antibacterials (Vaara, 1992)
due to the hydrophilic character of their exterior membrane (Nikaido,
1996) Antibacterials inhibit bacteria by destroying their cell walls in
addition to their cytoplasmic membrane, causing the coagulation and
leakage of microorganism’s cytoplasm. Researchers (Kalemba and
Kunicka, 2003) affirmed that the action EOs on bacterial and fungal
cells is related to the reticence of DNA, RNA, proteins and poly-
saccharides synthesis.
In comparison with previous research (Hashim et al., 2016), it was
mentioned that essential oil from Cymbopogon schoenanthus L. Spreng
exhibited antibacterial activity against Escherichia coli, Staphylococcus
aureus and Klebsiella pneumoniae. The effectiveness against Escherichia
coli is probably be due to composition of essential oil dependent on the
soil, climate, environmental and geographic conditions, harvest time
and extraction method.
3.7.2. Determination of minimum inhibitory concentration (MIC)
From the Table 5 corresponding to minimum inhibitory con-
centrations (MIC) values of each EO and in agreement with disc diffu-
sion results, CS essential oils displayed a great efficacy against Fusarium
culmorum (0.25–2 μg/ml) and Listeria monocytogenes (1–20 μg/ml), and
moderate inhibition against Staphylococcus aureus (1–30 μg/ml), Kleb-
siella pneumoniae (50–60 μg/ml), Aspergillus flavus (10–50 μg/ml),
Aspergillus westerdijkiae (10–100 μg/ml) and Candida albicans
(20–100 μg/ml). However, Escherichia coli and Salmonella Typhi were
the most resistant bacteria against the oils tested. A variability of MIC
values were noted according the technique achieved where HD, MAHD-
SG and MASD-CG essential oil was more efficient against Staphylococcus
aureus, Candida albicans, Klebsiella pneumonia respectively.
Nevertheless, MASD-SG EO was the most effective against the ma-
jority of tested microorganisms. This is may be due to the richness in
some sesquiterpenes hydrocarbons (Trans- dauca-4(11),7-diene) and
oxygenated (eudesmanes) previously mentioned.
In fact, antimicrobial activity is highly dependent on the composi-
tion, thus, the suggestion of Bouchra et al. (2003) that oxygenated
monoterpenes are particularly active against microbial cells, are in
agreement with our results (Table 3) indicating a significant amount of
oxygenated monoterpenes (8.47%–13.62%) consisted essentially of
isopulegol (3.67–5.58%), cis-piperitol (2.07–3.87%) and trans-piperitol
(1.97–3.13%). On the other hand, the presence of p-cymene
(0.74–1.89%) can be responsible for the activity of CS essential oil
(Oussalah et al., 2006). Although these compounds are considered to be
minor components, they might contribute to an increase in activity
(Lopes-Lutz et al., 2008; Mourey and Canillac, 2002; Romagnoli et al.,
2005) in addition to the synergetic and antagonistic effects that must be
taken in consideration (Saka et al., 2017).
In order to highlight the variability of antimicrobial activity of
Cymbopogon schoenanthus L. Spreng’s essential oils according to extrac-
tion technique, data analysis with K-means clustering and Principal
Component Analysis (PCA) was also performed using R programming
language, based on a scaled and centered matrix linking inhibition
zones to extraction techniques (HD, MAHD-SG, MAHD-CG, MASD-SG
and MASD-CG).
Clustering classification permitted to distinguish two separate
classes of essential oils; the class 1 including HD, MAHD-SG, MAHD-CG
and MASD-CG essential oils and class 2 corresponding to MASD-SG

Table 5
Minimum inhibitory concentration (μg/ml) of Cymbopogon Schoenanthus L. Spreng volatile oils extracted by different methods.
Microorganisms HD MAHD-SG MAHD-CG MASD-SG MASD-CG Amoxicillin (μg/ml)

Bacterial strains
Staphylococcus aureus 1 5 30 5 2 1.25
Listeria monocytogenes 10 2 20 1 2 0.25
Klebsiella pneumoniae 20 50 60 5 30 0.1
Salmonella Typhi > 150 > 150 > 150 100 > 150 0.5
Escherichia coli > 150 > 150 > 150 > 150 > 150 0.25
Fungal strains
Aspergillus westerdijkiae 100 30 30 10 30 nd
Fusarium culmorum 1 2 0,5 0,25 1 nd
Aspergillus flavus 50 30 30 50 10 nd
Yeast
Candida albicans 100 20 30 60 100 nd

10
F.-Z. Bellik, et al.
Fig. 7. PCA for antimicrobial activity of Cymbopogon schoenanthus L. Spreng
essential oil based on matrix, linking the extraction methods to inhibition
zones.
essential oil. Indeed, EO isolated by MASD-SG has shown a particularity
in antimicrobial activity to be most effective essential oil on tested
microorganisms. PCA (Fig. 7) confirmed K-Means results showing the
two separate groups.
Globally, our data about antimicrobial activity evaluated by disk
diffusion method and determination of minimum inhibitory con-
centration revealed that essential oils from Cymbopogon schoenanthus L.
Spreng extracted by the studied methods presented different behavior
against tested microorganisms, noting that each strain was particularly
more susceptible to one essential oil as to others.
4. Conclusion
The microwave procedures (MAHD and MASD) offer important
advantages over traditional hydrodistillation, namely: shorter extrac-
tion times, substantial savings of energy, and a reduced environmental
burden. All these advantages make MAHD and MASD a good alternative
for the extraction of essential oils from aromatic plants preserving their
quality (refractive index and specific gravity).
Cryogenic grinding (N2at −196 °C) allowed to increase yield
MAHD-CG (1.76%) and MASD-CG (1.5%) compared to MAHD-SG
(1.25%) and MASD-SG (1.11%), because the reduction of particle size
and increasing surface for better solvent permeation. It has also pre-
served volatile compounds by eliminating their loss during classic
grinding.
Qualitatively, differences in composition according the technique
used were confirmed. HD volatiles showed a predominance of oxyge-
nated sesquiterpens while MASD-GC oil was rich of monoterpene hy-
drocarbons as confirmed by data analysis where K-means clustering and
PCA permitted to distinguish between three separate classes of essential
oils isolated from the different methods.
The kinetic study showed that the qualitative and quantitative
profile of EOs differed significatively according the technique and the
grinding mode used. The cryogenic grinding affects the internal struc-
ture of the plant, hence, the mass transfer and the diffusion of essential
oil toward the external medium allowing the acceleration of the ex-
traction process as well as a qualitative and quantitative selective ex-
traction of compounds from the exogenous and endogenous sides of the
plant. This fluctuation in composition is directly correlated with anti-
bacterial and antifungal properties closely related to the technique
extraction and the kind of the grinding used.
View the high activity of CS volatiles against Fusarium Culmorum,
this plant can used to protect cereals or other seedlings threatened by
this pathogen (Lawrence, 1978).
11
Industrial Crops & Products 139 (2019) 111505
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