Food and Bioproducts Processing 1 2 0 ( 2 0 2 0 ) 158–165

Contents lists available at ScienceDirect

Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

Microwave-assisted extraction of O. vulgare L. spp.

hirtum essential oil: Comparison with conventional
hydro-distillation

Zorica Drinić ∗ , Dejan Pljevljakušić, Jelena Živković, Dubravka Bigović,

Katarina Šavikin
Institute for Medicinal Plant Research “Dr. Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: Microwave-assisted hydro-distillation (MAHD) at different power levels has been applied for
Received 8 May 2019 the extraction of essential oil from O. vulgare L. ssp. hirtum. Hydrolats and residual extracts,
Received in revised form 2 which present by-products, were also collected. A comparison was done with conventional
December 2019 hydro-distillation (HD) in terms of extraction time, extraction yield, the chemical composi-
Accepted 23 January 2020 tion of essential oils and environmental impact. MAHD proved to be a technique with many
Available online 30 January 2020 advantages compared to conventional HD. MAHD had shorter extraction time (24–45 min)
compared to HD (136 min), better yields of essential oil (2.55–7.10 % for MAHD compared to
Keywords: 5.81 % for HD), higher content of oxygenated compounds (78.89–85.15 % for MAHD compared
Microwave-assisted to 76.82 for HD), and a more environmentally friendly approach (electrical consumption was
hydro-distillation from 0.135–0.240 kW h for MAHD compared to 1.360 kW h for HD). The main component in
Oregano all essential oils, as well as in hydrolats, was carvacrol. MAHD at the power level of 600 W
Essential oil may be considered as the most promising among the tested power levels with respect to
Hydrolat all investigated parameters. Residual extracts after HD and MAHD showed to be valuable
by-products rich in phenolic compounds, with rosmarinic acid as dominant.
© 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction Plants belonging to the genus Origanum are widely distributed in

Medicinal plants are a valuable source of bioactive compounds. World
Health Organization reported that medicinal plants have been used
in every country in the world. Interest for use plant-based natural
products increases over years (Al-Kalaldeh et al., 2010; Chishti et al.,
2013; Ahmad Khan and Ahmad, 2019). Medicinal plants are used in the
pharmaceutical, food and cosmetic industry. The major compounds
of plants that humans have used due to their beneficial role are ter-
penoids, alkaloids, and phenolic compounds (Harborne, 1999). The
many aromatic and medicinal plants have become attractive as natu-
ral sources of bioactive compounds. Aromatic plants are naturally rich
sources of essential oils and their components have a potential multi-
purpose functional use in different fields such as medicine, pharmacy,
cosmetic and food (Mohammadhosseini et al., 2017).
∗
Corresponding author. Moreover, Koukoulitsa et al. (2006) reported that the inhibitory activity
E-mail address: zdrinic@mocbilja.rs (Z. Drinić).
https://doi.org/10.1016/j.fbp.2020.01.011
0960-3085/© 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Eurasia and North Africa and have been used for centuries as a spice,
as well as in ethnomedicine (Ietswaart,1980; Fleisher and Fleisher,
1988). Various biological activities have been reported for Origanum
species such as antifungal, antimicrobial, antioxidant, antispasmodic,
antitumoral, expectorant, antiparasitic, antihelminthic (Kokkini, 1997;
Hummer et al., 1999; Elgayyar et al., 2001; Baser, 2002; Puertas et al.,
2002; Sokovic et al., 2002; Baydar et al., 2004; Kulisic et al., 2004; Sari
et al., 2006; Bakkali et al., 2008; Dundar et al., 2008). Still, oregano is
mainly used as a spice for a variety of dishes in Mediterranean cui-
sine, while for medicinal purposes, its essential oil is used due to its
antimicrobial properties.
Monoterpenoid phenolic isomers carvacrol and thymol are the
main components of the essential oil of all O. vulgare subspecies
(D’Antuono et al., 2000; Kulisic et al., 2004; Menaker et al., 2004) and
those two compounds are primarily responsible for diverse biological
activities (Aeschbach et al., 1994; Yanishlieva et al., 1999; Mastelic et al.,
2008). Besides its essential oil, oregano is also rich in various biologi-
cally active phenolic compounds such as flavonoids and phenolic acids.
 Food and Bioproducts Processing 1 2 0 ( 2 0 2 0 ) 158–165
of polar extracts of O. vulgare L. ssp. hirtum has a promising potential in
the prevention of diabetes complications in long-lasting treatments.
Essential oils can be isolated using several classical and advanced
approaches. Some of the conventional and most commonly used
methods are hydro-distillation, steam distillation, and organic solvent
extraction. The essential oil yield, losses of some volatile compounds
and the content of bioactive components depend on the method used
(Ferhat et al., 2007). Nevertheless, all of these conventional techniques
have some shortcomings. Some components of essential oils are sen-
sitive to steam distillation conditions (Lo Presti et al., 2005; Karakaya
et al., 2011), while, on the other hand, organic solvents such as n-
hexane and methylene chloride provide good recovery of oil and lipid
compounds, but they are toxic (Mamidipally and Liu, 2004). Moreover,
conventional techniques are usually time- and cost-consuming (Pavlić
et al., 2015).
Nowadays, alternative techniques for the isolation of essential
oils such as subcritical water extraction, supercritical carbon dioxide
extraction and microwave-assisted hydro-distillation have been devel-
oped (Karakaya et al., 2011; Pavlić et al., 2015). For these new extraction
techniques, it is expected to be economically and environmentally
friendly, sustainable, highly efficient and to provide good product
quality. Microwave energy with a frequency of 2.45 GHz has attracted
attention in analytical chemistry due to reduced analysis time, simpli-
fied manipulation and work-up, and higher purity of the final product
(Kingston and Jassie, 1988; Zlotorzynski, 1995; Kingston and Haswell,
1999). Many authors reported the main advantages of microwave-
assisted hydro-distillation of essential oils regarding shorter extraction
time, better yields, a higher amount of oxygenated compounds, envi-
ronmentally friendly character and lower cost (Ferhat et al., 2006;
Golmakani and Rezaei, 2008; Abdellatif and Hassani, 2015; Hashemi-
Moghaddam et al., 2018).
In the present study, conventional hydro-distillation (HD) and alter-
native microwave-assisted hydro-distillation (MAHD) techniques were
applied for the isolation of essential oil from O. vulgare L. subspecies hir-
tum and to obtain hydrolats. The qualitative and quantitative profiles
of essential oils and hydrolats were analyzed using GC/MS. Moreover,
water extracts remaining after HD and MAHD were collected and ana-
lyzed for total phenolics content as well as for individual phenolic
compounds.
2. Material and methods
2.1. Standards and reagents
Ethanol, distilled water, and n-hexane were purchased from
Zorka Pharma, Šabac (Serbia). Gallic acid, sodium carbonate
anhydrous (Na2 CO3 ), sodium sulfate anhydrous (Na2 SO4 ) and
Folin–Ciocalteu phenol reagent were purchased from Sigma
Chemicals Co. (USA). The phenolic compounds standards
were from Merck (Germany). All chemicals used in the experi-
mental procedure were of analytical grade purity. For the HPLC
analysis solvents and formic acid were of HPLC grade.
2.2. Plant material
The aerial parts of O. vulgare L. subspecies hirtum were
collected in July 2018 from the collection of the Institute
for Medicinal Plant Research “Dr. Josif Pančić “(20◦ 42 4.55 E,
44◦ 52 20.54 N, altitude 74 m, Belgrade. Voucher samples are
stored in the Herbarium of the Institute of Botany and Botan-
ical Garden “Jevremovac”, Faculty of Biology, University of
Belgrade; voucher No. 181014.
2.3. Essential oils, hydrolats and residual extracts
Isolation of essential oils was performed using HD and
MAHD approaches. HD was performed using a Clevenger type
159
apparatus according to the procedure I of the Yugoslavian
Pharmacopoeia IV (1984). MAHD was performed using the spe-
cially created system, consisting of the microwave oven (HMT
72M450, Bosch, Gerlingen, Germany) connected to the Cle-
venger type apparatus. The water/plant ratio was the same
for HD and MAHD, 20:1 (w/w). MAHD was performed at three
different power levels (180, 360, and 600 W) until no more
essential oil was obtained (ca. 20 min). The time required to
reach the final amount of essential oil will be referred to’ total
extraction time’ in further discussion. The essential oils were
collected, and dried over anhydrous sodium sulphate. The
essential oil yield, expressed as a percentage, was calculated
on a moisture-free basis. Oil samples (20 ␮L) were dissolved in
EtOH 96 % (2 mL).
The hydrolats are defined as the distilled water from the
production of essential oils obtained by steam water distilla-
tion from aromatic plants (flowers, leaves, stems, roots), so
the main components are water-soluble volatile compounds
(Rajeswara Rao, 2013). Hydrolats, which presented water from
the production of essential oils obtained by steam distilla-
tion or hydro-distillation from aromatic plants, were collected
from the burette after HD and MAHD, extracted with n-hexane
(5 mL of hydrolats with 3 × 1 mL n-hexane), hexane fractions
were collected and combined. Residual extracts after HD and
MAHD were also collected. Prepared samples of essential oil,
hydrolats and residual extracts were stored in the freezer until
further analysis.
2.4. Chemical analyses of essential oil and hydrolats
The chemical composition of the essential oil was analyzed
using GC/MS technique. GC/MS analyses were performed on a
Shimadzu GCMS-QP2010 ultra mass spectrometer fitted with
a flame ionic detector and coupled with a GC2010 gas chro-
matograph. The InertCap5 capillary column (60.0 m × 0.25
mm × 0.25 ␮m) was used for separation. Helium (He), at a split
ratio of 1:5 and a linear velocity of 35.2 cm/s was used as car-
rier gas. Initially, the oven temperature was 60 ◦ C, which was
held for 4 min, then increased to 280 ◦ C at a rate of 4 ◦ C/min,
and held for 10 min. The injector and detector temperatures
were adjusted at 250 ◦ C and 300 ◦ C, respectively. The ion source
temperature was 200 ◦ C. The identification of the constituents
was performed by comparing their mass spectra and reten-
tion indices (RIs) with those obtained from authentic samples
and/or listed in the NIST/Wiley mass-spectra libraries, using
different types of search (PBM/NIST/AMDIS) and available lit-
erature data (Hochmuth, 2006; Adams, 2007).
2.5. Determination of total phenolic contents of
residual extracts
The total phenolic content of residual extracts was deter-
mined by Folin-Ciocalteu spectrophotometric procedure with
slight modification (Harris, 2000). Two hundred microliters of
extracts were added to 1 mL of 1:10 diluted Folin-Ciocalteu
reagent. After 4 min, 800 ␮L of sodium carbonate (75 g/L) was
added. After 2 h of incubation at room temperature, the
absorbance was measured at 765 nm. Gallic acid (0–100 mg/L)
was used for the calibration of a standard curve. The results
were expressed as milligrams of gallic acid equivalents per g
of plant dry weight (mg GAE/g dw).
160 Food and Bioproducts Processing 1 2 0 ( 2 0 2 0 ) 158–165
2.6. HPLC analysis of residual extracts
The HPLC-DAD analysis of phenolic compounds was per-
formed using the Agilent 1100 Series (Agilent Technologies,
Palo Alto, CA, USA) comparing the retention times and
absorption spectra (200−400 nm) of unknown peaks with the
reference standards. The column was Lichrospher® 100 RP 18e
(5 ␮m, 250 × 4 mm). Mobile phase A was formic acid in water
(0.17 %), while mobile phase B was acetonitrile. The injection
volume was 10 ␮L, and the flow rate 0.8 mL/min with gradient
program (0–53 min 0–100 % B). The stop time of the analy-
sis was 55 min. The investigated samples were analyzed in
triplicate.
2.7. Environmental impact of applied techniques
Regarding environmental impact, electrical consumption and
CO2 emission were calculated. The electrical consumption (A)
of different methods was calculated as the electrical power for
a time, using the following equation:
A = P×t,
where A is electrical consumption (kWh), P is electrical power
(kW) and t is time (h).
According to Ferhat et al. (2006) to obtain 1 kW h from coal
or fuel, 800 g of CO2 will be rejected in the atmosphere during
the combustion of fossil fuel. CO2 emission can be described
by the equation:
ECO2 = (A × 800)/1000
where ECO2 is CO2 emission (kg) and A is electric consumption
(kWh).
2.8. Statistical analysis
All experiments were made in triplicate and the obtained
results are expressed as mean values. Regarding phenolic
compounds, a null hypothesis was tested by one-way ANOVA
and differences among mean values subsequently estimated
by post hoc Duncan’s MR test. Statistical analysis was per-
formed using the MS Office Excel v. 2010, while charts were
produced in R Base Plot environment (CRAN).
3. Resultion and discussion
3.1. Extraction time
From the results obtained after comparison of HD and MAHD
at different power levels, it can be concluded that the total
extraction time differed among applied techniques and con-
ditions: total extraction time was 24, 29, and 45 min for MAHD
at 600, 360, and 180 W, respectively, while for HD it was 136 min
(Fig. 1). The time required to start the extraction was 4, 9, and
25 min for MAHD at 600, 360, and 180 W, respectively, while for
HD it was 16 min. These differences can be explained by a dif-
ferent mechanism of extraction with microwaves compared
to hydro-distillation or in other words by the mechanism of
heat penetration into the plant material. Veggi et al. (2013)
highlighted that heating during HD is achieved by gradual
heat transfer from the heating medium to the interior of the
samples, while microwave heating is volumetrically dispersed
within the radiation. Furthermore, microwave heating is based
on two phenomena: ionic conduction and dipole rotation, and
Fig. 1 – Total extraction time for HD and MAHD at different
power levels.
it is directly connected with the dielectric constant of the used
medium (Chemat et al., 2009). Water is a solvent with a high
dielectric constant which leads to rapid heating in the MAHD
process. Differences in the MAHD extraction time at different
power levels are correlated to the density of the waves.
The reduction in the extraction time of essential oil by
microwaves was significantly higher. Even at the level of the
lowest microwave power (180 W), the total extraction time was
significantly shorter than in hydro-distillation (Fig. 1). Similar
to our results, many authors reported that MAHD significantly
reduces extraction time in comparison to conventional HD
(Ferhat et al., 2007; Golmakani and Rezaei, 2008; Rezvanpanah
et al., 2008).
3.2. Essential oils yield
Essential oil yield was 7.10, 5.67, and 2.55 % for MAHD at
600, 360 and 180 W, respectively, while for HD it was 5.81 %
(Fig. 2). The highest yield of the essential oil was obtained
after MAHD at 600 W, while the yield using MAHD at 360 W
was similar to HD. The lowest oil yield was obtained by MAHD
at 180 W. Higher yield of extracted essential oil at higher
microwave power has been previously explained by Chemat
et al. (2009) as synergistic effect of two transport phenom-
ena, such as temperature gradient and mass gradient, acting
in the same direction. In contrast, at lower levels of microwave
power, the density of microwaves is also lower, leading to
lower heating ability and consequently to lower extraction
yield. In a nutshell, the extraction of active substances from
plant material using microwave extraction occurs as a result
of a change in cell structure caused by electromagnetic waves
(Veggi et al., 2013). At higher power levels, water can absorb
more microwave beams in that way disrupting cell structure
which leads to increased release of the essential oil (Chen and
Spiro, 1995). Higher power levels can lead to an increase in
system temperature and extraction yield are directly depen-
dent on the extraction temperature (Veggi et al., 2013). The
extraction efficiency increases with increasing temperature to
the optimum temperature, which depends on the stability of
the target compounds. Therefore, it is important to select the
appropriate microwave power because the high microwave
 Food and Bioproducts Processing 1 2 0 ( 2 0 2 0 ) 158–165
Fig. 2 – Total essential oil yield for HD and MAHD at
different power levels.
power can cause degradation of temperature sensitive com-
pounds. Raner et al. (1993) reported that variation of power
level from 500 to 1000 W had no significant effect on the yield
of flavonoids, while Mohamadia et al. (2013) reported that
powers greater than 600 W had no significant effect on the
extraction efficiency of essential oil.
3.3. Composition of essential oils and hydrolats
The chemical constituents of obtained essential oils by both
applied methods are presented in Table 1. A total of 42
compounds have been identified, representing more than
99.97 % of the oil. The dominant compound in all samples
was carvacrol followed by ␥-terpinene, p-cymene, trans-
caryophyllene, ␣-terpinene, and myrcene. In respect to this
order, the chemical composition of the essential oils obtained
by HD and MAHD at 600 W were similar, while essential oils
obtained by MAHD at 360 and 180 W showed slight differences.
The content of carvacrol in essential oils was higher when
MAHD was applied (76.02, 86.13, and 82.65 % for power lev-
els 600, 360, and 180 W, respectively), while in HD it was 71.71
%. Carvacrol was also dominant compounds in O. vulgare L.
ssp. hirtum essential oil analyzed by other authors (Kokkini
et al., 1991; Adam et al., 1998). Essential oils obtained by MAHD
had a higher content of oxygenated monoterpenes and lower
content of monoterpenes hydrocarbons. The amounts of oxy-
genated monoterpenes in essential oils obtained by MAHD at
600, 360, and 180 W was 78.89, 87.83, and 85.15 %, respectively,
while the content in HD obtained essential oil was 76.02 %.
Since hydrocarbon monoterpenes are the product of thermal
and hydrolytic decomposition of oxygenated monoterpenes,
higher content of oxygenated monoterpenes in essential oils
obtained by MAHD is probably due to shorter time need for
MAHD compared to HD. This is in agreement with previously
reported studies (Filly et al., 2014; Ferhat et al., 2006). Also, the
different chemical composition in obtained essential oil, in
addition to hydrolytic processes, can be related to the possible
degradation of products by oxidation and trans-esterification
due to different extraction time (Benkaci–Ali et al., 2007).
Chemical constituents in the hydrolats obtained by HD and
MAHD at different power levels are given in Table 2. Same
161
as in the essential oils, the main component in all hydrolats
was carvacrol and its content represented 99.00, 99.58, 99.40,
and 94.71 % of all identified components by HD, MAHD at 600,
360, and 180 W, respectively. Some authors reported that the
main component in essential oil was also the main compo-
nent of corresponding hydrolats (Paolini et al., 2008; Shahani
et al., 2011). Since, carvacrol was reported as a key compo-
nent for the biological activity of Origanum species essential
oil, the high content of carvacrol in obtained hydrolats indi-
cates the possibility for their use in the pharmaceutical and
cosmetic industry. The number of identified compounds in
hydrolat obtained by MAHD at 180 W power level was signifi-
cantly higher than in other hydrolats. This discrepancy is most
probably the result of prolonged time of water extraction of the
plant material before medium boiling and evaporation of the
essential oil.
3.4. Total phenolic content and composition of residual
extracts
Residual extracts after isolation of essential oils are usually
recognized as waste. On the other hand, such extracts have
a remarkable potential as valuable by-products in the dis-
tillation process. Total phenols content in obtained extracts
ranged from 9.34 to 17.74 ␮g GAE/g dw. The most abundant
phenolic compound in all obtained extracts was rosmarinic
acid (Table 3). Some authors reported that rosmarinic acid is
the dominant phenolic acid of family Lamiaceae in general
(Wang et al., 2004; Lee, 2010; Embuscado, 2015). The highest
rosmarinic acid content was found in the extract obtained by
MAHD at 600 W. Other phenolic components determined using
HPLC were chlorogenic acid (0.34–1.50 ␮g/g dw), caffeic acid
(1.61–5.37 ␮g/g dw) and luteolin-7-O-glucoside (0.57–1.51 ␮g/g
dw). The highest phenolic content was noticed in the extract
obtained by MAHD at 600 W which is in accordance with
previously mentioned fact that extraction with microwaves
achieves a higher yield than the classic HD method. Among
all tested distillation models, MAHD at 600 W yielded signifi-
cantly higher amounts of individual phenolic compounds. To
the best of our knowledge, there are no previously published
results on the phenolic compounds of the O. vulgare L. ssp.
hirtum residual extract after distillation.
3.5. Environmental impact
Electrical consumption and CO2 emission were smaller for
MAHD compared to HD (Fig. 3). HD electrical consumption was
1.360 kW h, while for MAHD at levels 600 W, 360 W, and 180 W
it was 0.240, 0.174, and 0.135 kW h, respectively. CO2 emission
for HD was 1.09 kg, while for MAHD at 600 W, 360 W, and 180 W
it was 0.19, 0.14, and 0.11 kg, respectively. Since the yield of
essential oils obtained by HD and MAHD at different power
levels was not the same, in order to compare environmen-
tal impact, the total electrical consumption and CO2 emission
were converted to electrical consumption and CO2 emission
for the yield of 1 % of essential oil. Electrical consumption
for yield of 1 % was 0.2341, 0.0338, 0.0307, 0.0529 kW h, while
CO2 emission was 0.1873, 0.0270, 0.0246, and 0.0424 kg for
HD, MAHD at 600 W, 360 W and 180 W, respectively. Using the
MAHD method energy saving was on average about 75 % com-
pared to the hydro-distillation, and the emission CO2 was
reduced in the same percentage. Electrical consumption and
CO2 emission are related to the extraction time. Since MAHD
162 Food and Bioproducts Processing 1 2 0 ( 2 0 2 0 ) 158–165

Table 1 – Chemical constituents in the O. vulgare L. ssp. hirtum essential oil obtained with hydro-distillation and
microwave-assisted hydro-distillation at different power levels.
Peak Compound RIa Hydro-distillation Microwave assisted hydro-distillation

600 W 360 W 180 W

1. ␣-thujene 923.6 0.91 0.60 0.21 0.26

2. ␣-pinene 931.6 0.39 0.32 0.10 0.13
3. camphene 948.5 0.11 0.09 – 0.02
4. 1-octen-3-ol 973.8 – 0.41 0.18 0.32
5. sabinene 973.9 0.40 – – –
6. ␤-pinene 978.6 0.11 0.09 0.02 0.03
7. 3-octanone 981.3 0.11 0.12 0.06 0.09
8. myrcene 987.5 1.38 1.31 0.59 0.63
9. ␣-phellandrene 1002.7 0.19 0.16 0.05 0.06
10. ␦-3-carene 1008.4 0.06 0.04 – –
11. ␣-terpinene 1013.6 1.67 1.46 0.69 0.78
12. p-cymene 1019.6 4.36 4.08 2.43 3.10
13. limonene 1024.0 0.19 0.19 0.06 0.07
14. ␤-phellandrene 1026.3 0.14 0.12 0.04 0.05
15. cis-␤-ocimene 1028.4 0.19 0.20 0.07 0.08
16. trans-␤-ocimene 1039.1 0.10 0.08 0.02 0.03
17. ␥-terpinene 1053.3 10.14 9.51 6.39 7.16
18. cis-sabinene hydrate 1061.8 0.37 0.48 0.26 0.24
19. terpinolene 1083.3 0.07 0.06 – 0.04
20. linalool 1090.7 0.10 0.07 0.02 0.05
21. trans-sabinene hydrate 1093.8 0.24 0.29 0.10 0.11
22. borneol 1164.5 0.57 0.45 0.15 0.29
23. 4-terpinenol 1175.3 0.50 0.41 0.22 0.57
24. ␣-terpineol 1188.7 0.18 0.05 – 0.06
25. cis-dihydrocarvone 1195.7 0.02 0.05 – –
26. trans-dihydrocarvone 1198.6 0.11 0.01 – 0.03
27. carvacrol methyl ether 1241.9 0.48 0.46 0.25 0.40
28. linalool acetate 1254.6 0.04 – – –
29. carvone 1252.0 – 0.03 – –
30. thymol 1289.6 1.60 – 0.70 0.75
31. carvacrol 1297.4 71.71 76.14 86.13 82.65
32. dihydro-carveol acetate 1307.7 0.04 – – –
33. n.i.b 1319.9 0.03 – – –
34. linalool propanoate 1338.9 – 0.17 – –
35. carvacrol acetate 1358.8 0.05 0.28 – –
36. trans-caryophyllene 1416.4 2.26 1.47 0.94 1.46
37. ␣-humulene 1455.7 0.40 0.23 0.12 0.20
38. ␤-acoradiene 1467.1 – 0.03 – –
39. ␣-curcumene 1470.0 – 0.09 – –
40. germacrene D 1487.0 0.05 0.03 – –
41. bicyclogermacrene 1496.7 – 0.03 – –
42. ␣-farnesene 1497.1 0.06 – – –
43. ␤-bisabolene 1504.6 0.66 0.38 0.20 0.34
Monoterpene hydrocarbons 20.41 18.31 10.67 12.44
Oxygenated monoterpenes 76.02 78.89 87.83 85.15
Sesquiterpene hydrocarbons 3.43 2.26 1.26 2.00
Other 0.11 0.53 0.24 0.41
n.i. 0.03 – – –

a
RI, retention indices as determined on HP-5 column using homologous series of C8 -C30 alkanes.
b
n.i. stands for Not identified.

was faster method for the isolation of the essential oils than
HD, it turns out as more “environmentally friendly” approach.
4. Conclusion
Microwave-assisted hydro-distillation (MAHD) at different
power levels has been compared with conventional hydro-
distillation (HD) method for the extraction of essential oil
from O. vulgare L. ssp. hirtum. MAHD at the power level of
600 W may be considered as the most promising among the
tested power levels with respect to all investigated param-
eters. MAHD at the power level of 600 W proved to be a
technique with many advantages compared to conventional
hydro-distillation (HD). MAHD had shorter extraction time,
24 min compared to 136 min, better yields of essential oil,
7.10 % compared to 5.81 %, higher content of oxygenated
compounds, 78.89 % compared to 76.82 %, and a more envi-
ronmentally friendly approach with electrical consumption
0.240 kW h compared to 1.360 kW h and CO2 emission 0.19 kg
compared to 1.09 kg. The main component in all essential
oils, as well as in obtained hydrolats, was carvacrol. Residual
extracts after HD and MAHD showed to be valuable by-
products, rich in phenolic compounds with rosmarinic acid as
the most abundant compound. The higher amount of pheno-
lic compounds was detected in residual extracts after MAHD.
MAHD at the power level of 600 W may be considered as the
 Food and Bioproducts Processing 1 2 0 ( 2 0 2 0 ) 158–165 163

Table 2 – Chemical constituents in the O. vulgare L. ssp. hirtum hydrolat obtained using hydro-distillation and
microwave-assisted hydro-distillation at different power levels.
Peak Compound RIa Hydro-distillation Microwave assisted hydro-distillation

600 W 360 W 180 W

1 ␣-thujene 923.9 – – – 0.05

2 1-octen-3-ol 973.8 – – – 0.09
3 myrcene 987.7 – – – 0.15
4 ␣-terpinene 1013.8 – – – 0.18
5 p-cymene 1019.6 0.11 – – 0.83
6 ␥-terpinene 1052.9 0.06 – – 1.97
7 cis-sabinene hydrate 1061.9 – – – 0.03
8 camphor 1143.1 0.04 – – –
10 4-terpinenol 1175.6 – – – 0.31
11 carvacrol methyl ether 1242.2 – – – 0.10
12 p-8-cymenol 1179.8 0.07 – – –
13 thymol 1289.1 0.72 0.42 0.60 0.88
14 carvacrol 1296.7 99.00 99.58 99.40 98.22
15 trans-caryophyllene 1416.5 – – – 0.71
16 ␣-humulene 1455.9 – – – 0.06
17 ␤-bisabolene 1504.7 – – – 0.13

a
RI, retention indices as determined on HP-5 column using homologous series of C8 -C30 alkanes.

Table 3 – Chemical constituents in the O. vulgare L. ssp. hirtum extracts obtained after hydro-distillation and
microwave-assisted hydro-distillation at different power levels.
Total phenols contentb Chlorogenic acid Caffeic acid Rosmarinic acid Luteolin-7-O-glucoside
[␮g GAE/g dw] [␮g/g dw] [␮g/g dw] [␮g/g dw] [␮g/g dw]

Hydro-distillation 10.59 0.92 ± 0.03 b 3.30 ± 0.08 b 31.76 ± 1.10 b 1.10 ± 0.04 b
MAHD 600 Wa 17.74 1.50 ± 0.04 a 5.37 ± 0.12 a 56.86 ± 1.42 a 1.51 ± 0.04 a
MAHD 360 W 9.34 0.74 ± 0.02 c 2.43 ± 0.07 c 26.59 ± 1.00 c 0.77 ± 0.03 c
MAHD 180 W 9.58 0.34 ± 0.02 d 1.61 ± 0.05 d 19.14 ± 0.40 d 0.57 ± 0.02 d

a
MAHD stands for Microwave Assisted Hydro-Distillation.
b
Means followed by different letters are significantly different according to the post hoc Duncan’s test al level P < 0.05.

Fig. 3 – Environmental impact: A) Electrical consumption for yield of 1 % (kWh), and B) CO2 emission (kg).

most promising among the tested power levels with respect Conflict of interest
to all investigated parameters. Therefore, MAHD at the power
level of 600 W can be good alternative technique for the extrac- None.
tion of essential oil from O. vulgare ssp. hirtum.
164 Food and Bioproducts Processing 1 2 0 ( 2 0 2 0 ) 158–165
Acknowledgements
This work was supported by the Ministry of Education, Sci-
ence and Technological Development of the Republic of Serbia,
project number III 46013.
Appendix A. Supplementary data
Supplementary material related to this article can be
found, in the online version, at doi:https://doi.org/10.1016/j.
fbp.2020.01.011.
References
Abdellatif, F., Hassani, A., 2015. Chemical composition of the
essential oils from leaves of Melissa officinalis extracted by
hydrodistillation, steam distillation, organic solvent and
microwave hydrodistillation. J. Mater. Environ. Sci. 6 (1),
207–213.
Adam, K., Sivropoulou, A., Kokkini, S., Lanaras, T., Arsenakis, M.,
1998. Antifungal activities of Origanum vulgare subsp. hirtum,
Mentha spicata, Lavandula angustifolia, and Salvia fruticosa
essential oils against human pathogenic fungi. J. Agric. Food
Chem. 465, 1739–1745.
Adams, R.P., 2007. Identification of Essential Oil Components by
Gas Chromatography/Mass Spectrometry. Allured Publishing
Corporation, Google-Books-ID: 9ut3PQAACAAJ.
Aeschbach, R., Loliger, J., Scott, C., Murcia, A., Butler, J., Halliwell,
B., Aruoma, O.I., 1994. Antitoxidant actions of thymol,
carvacrol, 6–gingerol, zingerone and hydroxytyrosol. Food
Chem. Toxicol. 32, 31–36.
Ahmad Khan, M.S., Ahmad, I., 2019. Herbal medicine: current
trends and future prospects. In: Ahmad Khan, M.S., Ahmad, I.,
Chattopadhyay, D. (Eds.), New look to Phytomedicine:
Advancements in Herbal Products as Novel Drug Leads.
Academic Press, pp. 3–13.
Al-Kalaldeh, J.Z., Abu-Dahab, R., Afifi, F.U., 2010. Volatile oil
composition and antiproliferative activity of Laurus nobilis,
Origanum syriacum, Origanum vulgare, and Salvia triloba against
human breast adenocarcinoma cells. Nutr. Res. 30, 271–278.
Bakkali, F., Averbbeck, S., Averbeck, D., Idaomar, M., 2008.
Biological effects of essential oils—a review. Food Chem.
Toxicol. 46, 446–475.
Baser, K.H.C., 2002. The Turkish Origanum species. Oregano, the
Genera Origanum and Lippia. In: Kintzios, S.E. (Ed.),
Oregano—The Genera Origanum and Lippia. Taylor and
Francis, London and New York, pp. 109–126.
Baydar, H., Osman, S., Ozkan, G., Karadoa, T., 2004. Antibacterial
activity and composition of essential oils from Origanum,
Thymbra and Satureja species with commercial importance in
Turkey. Food Control 15, 169–172.
Benkaci–Ali, F., Baaliouamer, A., Meklati, B.Y., Chemat, F., 2007.
Chemical composition of seed essential oils from Algerian
Nigella sativa extracted by microwave and hydrodistillation.
Flavour Fragr. J. 22, 148–153.
Chemat, F., Abert-Vian, M., Zill-e-Huma, Y.J., 2009. Microwave
assisted separations: green chemistry in action. In: Pearlman,
J.T. (Ed.), Green Chemistry Research Trends. Nova Science
Publishers, New York, pp. 33–62.
Chen, S.S., Spiro, M., 1995. Kinetics of microwave extraction of
rosemary leaves in hexane, ethanol and a hexane + ethanol
mixture. Flavour Fragr. J. 10, 101–112.
Chishti, S., Kaloo, Z.A., Sultan, P., 2013. Medicinal importance of
genus Origanum: a review. J. Pharmacogn. Phytother. 5 (10),
170–177.
D’Antuono, L.F., Galletti, C.G., Bocchini, P., 2000. Variability of
essential oil content and composition of Origanum vulgare L.
populations from a North Mediterranean area (Liguria region,
Northern Italy). Ann. Bot. 8, 471–478.
Dundar, E., Olgun, E.G., Isiksoya, S., Kukcuoglu, M., Baser, K.H.C.,
Bal, C., 2008. The effects of intra-rectal and intra-peritoneal
application of Origanum onites L. essential oil on
2,4,6-trinitrobenzene sulfonic acid induced colitis in the rat.
Exp. Toxicol. Pathol. 59, 399–408.
Elgayyar, M., Draughon, F.A., Golden, D.A., Mount, J.R., 2001.
Antimicrobial activity of essential oils from plants against
selected pathogenic and saprophytic microorganisms. J. Food
Prot. 64, 1019–1024.
Embuscado, M.E., 2015. Spices and herbs: natural sources of
antioxidants—a mini review. J. Funct. Foods 18, 811–819.
Ferhat, M.A., Chemat, F., Meklati, B.Y., Smadja, J., 2006. An
improved microwave clevenger apparatus for distillation of
essential oils from orange peel. J. Chromatogr. A 1112,
121–126.
Ferhat, M.A., Tigrine-Kordjani, N., Chemat, S., Meklati, B.Y.,
Chemat, F., 2007. Rapid extraction of volatile compounds
using a new simultaneous microwave distillation: solvent
extraction device. Chromatographia 65, 217–222.
Filly, A., Fernandez, X., Minuti, M., Visinoni, F., Cravotto, G.,
Chemat, F., 2014. Solvent-free microwave extraction of
essential oil from aromatic herbs: from laboratory to pilot and
industrial scale. Food Chem. 150, 193–198.
Fleisher, A., Fleisher, Z., 1988. Identification of biblical hyssop and
origin of the traditional use of oregano-group herbs in the
Mediterranean region. Econ. Bot. 42, 232–241.
Golmakani, M.T., Rezaei, K., 2008. Comparison of
microwave-assisted hydrodistillation with the traditional
hydrodistillation method in the extraction of essential oils
from Thymus vulgaris L. Food Chem. 109, 925–930.
Harborne, J.B., 1999. Classes and functions of secondary products
from plants. In: Walton, N.J., Brown, D.E. (Eds.), Chemicals
from Plants: Perspectives on Plant Secondary Products. World
Scientific, pp. 1–25.
Harris, D.C., 2000. Quantitative Chemical Analysis, fifth ed. W.H.
Freeman & Company, New York.
Hashemi-Moghaddam, H., Mohammadhosseini, M., Azizi, Z.,
2018. Impact of amine- and phenyl-functionalized magnetic
nanoparticles impacts on microwave-assisted extraction of
essential oils from root of Berberis integerrima Bunge. J. Appl.
Res. Med. Aromat. Plants 10, 1–8.
Hochmuth, D., Hamburg, Germany 2006. Massfinder 3: Software
for GC/MS Interpretation and Presentation, Mass Spectral
Library Administration.
Hummer, K.A., Caraon, C.F., Rilet, T.V., 1999. Antimicrobial
activity of essential oils and other plant extract. J. Appl.
Microbiol. 86, 985–990.
Ietswaart, J.H., 1980. A Taxonomic Revision of the Genus Origanum
(Labiatae). Leiden University Press, Hague, Boston, London.
Karakaya, S., Nehir, E.S., Karagozlu, N., Sxahin, S., 2011.
Antioxidant and antimicrobial activities of essential oils
obtained from Oregano (Origanum vulgare ssp. hirtum) by using
different extraction methods. J. Med. Food 14, 645–652.
Kingston, H.M., Haswell, S.J., 1999. Microwave-enhanced
Chemistry: Fundamentals, Sample Preparation and
Applications. American Chemical Society, Washington.
Kingston, H.M., Jassie, L.B., 1988. Introduction to Microwave
Sample Preparation. American Chemical Society, Washington.
Kokkini, S., 1997. Taxonomy, diversity and distribution of
Origanum species. In: Padulosi, S. (Ed.), Proceedings of the
IPGRI International Workshop on Oregano, CIHEAM.
Valenzano, Bari, Italy, pp. 2–12.
Kokkini, S., Vokou, D., Karousou, R., 1991. Morphological and
chemical variation of Origanum vulgare L. in Greece. Bot.
Chron. 10, 337–346.
Koukoulitsa, C., Zika, C., Hadjipavlou-Litina, D., Demopoulos, V.J.,
Skaltsa, H., 2006. Inhibitory effect of polar oregano extracts on
aldose reductase and soybean lipoxygenase in vitro.
Phytother. Res. 20, 605–606.
Kulisic, T., Radoni, A., Katalinic, V., Milos, M., 2004. Use of
different methods for testing antioxidative activity of oregano
essential oil. Food Chem. 85, 633–640.
 Food and Bioproducts Processing 1 2 0 ( 2 0 2 0 ) 158–165
Lee, J., 2010. Caffeic acid derivatives in dried Lamiaceae and
Echinacea purpurea products. J. Funct. Foods 2,
158–162.
Lo Presti, M., Ragusa, S., Trozzi, A., Dugo, P., Visinoni, F., Fazio, A.,
Dugo, G., Mondello, L., 2005. A comparison between different
techniques for the isolation of rosemary essential oil. J. Sep.
Sci. 28, 273–280.
Mamidipally, P.K., Liu, S.X., 2004. First approach on rice bran oil
extraction using limonene. Eur. J. Lipid Sci. Technol. 106,
122–125.
Mastelic, J., Jerkovic, I., Blazevic, I., Poljak-Blazi, M., Borovic, S.,
Ivancic-Bace, I., Smrecki, V., Zarkovic, N., Brcic-Kostic, K.,
Vikic–Topic, D., Muller, N., 2008. Comparative study on the
antioxidant and biological activities of carvacrol, thymol and
eugenol derivatives. J. Agric. Food Chem. 56, 3989–3996.
Menaker, A., Kravets, M., Koel, M., Orav, A., 2004. Identification
and characterization of supercritical fluid extracts from herbs.
CR Chim. 7, 629–633.
Mohamadia, M., Shamspura, T., Mostafavia, A., 2013. Comparison
of microwave-assisted distillation and conventional
hydrodistillation in the essential oil extraction of flowers Rosa
damascena Mill. J. Essent. Oil Res. 25, 55–61.
Mohammadhosseini, M., Akbarzadeh, A., Flamini, G., 2017.
Profiling of compositions of essential oils and volatiles of
Salvia limbata using traditional and advanced techniques and
evaluation for biological activities of their extracts. Chem.
Biodivers., 14.
Paolini, J., Leandrib, C., Desjobert, J., Barbonia, T., Costa, J., 2008.
Comparison of liquid–liquid extraction with headspace
methods for the characterization of volatile fractions of
commercial hydrolats from typically Mediterranean species. J.
Chromatogr. A 1193, 37–49.
Pavlić, B., Vidović, S., Vladić, J., Radosavljević, R., Zeković, Z., 2015.
Isolation of coriander (Coriandrum sativum L.) essential oil by
green extractions versus traditional techniques. J. Supercrit.
Fluid. 99, 23–28.
Puertas, M.M., Hille, B.S., Stashenko, E., Winterhalter, P., 2002.
In vitro radical scavenging activity of essential oils from
Columbian plants and fractions from oregano (Origanum
vulagre L.) essential oil. Flavor. Frag. J. 17, 380–384.
165
Rajeswara Rao, B.R., 2013. Hydrosols and water-soluble essential
oils: their medicinal and biological properties. In: Govil, J.N.,
Bhattacharya, S. (Eds.), Recent Progress in Medicinal Plants,
36. Studium Press LLC, U.S.A, pp. 119–140.
Raner, K.D., Strauss, C.R., Vyskoc, F., Mokbel, L., 1993. A
comparison of reaction kinetics observed under microwave
irradiation and conventional heating. J. Org. Chem. 58,
950–995.
Rezvanpanah, S., Rezaei, K., Razavi, S.H., Moini, S., 2008. Use of
Microwave-assisted hydrodistillation to extract the essential
oils from Satureja hortensis and Satureja montana. Food Sci.
Technol. Res. 14, 311–314.
Sari, M., Biondi, D.M., Kaabeche, M., Mandalari, G., Darrigo, M.,
Bisignano, G., Saija, A., Daquino, C., Ruberto, G., 2006.
Chemical composition, antimicrobial and antioxidant
activities of the essential oil of several populations of Algerian
Origanum glandulosum Desf. Flavour. Frag. J. 21, 890–898.
Shahani, S., Monsef-Esfahani, H.R., Hajiaghaee, R., Gohari, A.R.,
2011. Chemical composition of essential oil and hydrolat of
Geum iranicum. Khatamaz. J. Essent. Oil Res. 23, 29–33.
Sokovic, M., Tzakou, O., Pitarokili, D., Couladis, M., 2002.
Antifungal activities of selected aromatic plants growing wild
in Greece. Nahrung 46 (317-), 320.
Veggi, P.C., Martinez, J., Meireles, M.A., 2013. Fundamentals of
microwave extraction. In: Chemat, F., Cravotto, G. (Eds.),
Microwave-Assisted Extraction for Bioactive Compounds:
Theory and Practice. Springer, Boston, MA, pp. 15–52.
Wang, H., Provan, G.J., Helliwell, K., 2004. Determination of
rosmarinic acid and caffeic acid in aromatic herbs by HPLC.
Food Chem. 87 (307-), 311.
Yanishlieva, N.V., Marinova, E.M., Gordon, M.H., 1999. Antioxidant
activity and mechanism of action of thymol and carvacrol in
two lipid systems. Food Chem. 64, 59–66.
Yugoslavian Pharmacopoeia, 1984. Pharmacopoea Jugoslavica
editio quarta. Ph. Jug. IV. National institute for Health
Protection, Belgrade.
Zlotorzynski, A., 1995. Crit. Rev. Anal. Chem. 25, 43.