Assessment of microwave assisted and hydrodistllation extraction on Echinops persicus essential oils chemical composition and evaluation of its biological activity

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TMR | September 2019 | vol. 4 | no. 5 |246
Submit a manuscript: https://www.tmrjournals.com/tmr
doi: 10.12032/TMR20190826132
Persian Medicine
Assessment of microwave assisted and hydrodistllation extraction on
Echinops persicus essential oils chemical composition and evaluation
of its biological activity
Maryam Soori1
, Hossein Abbaspour1
, Hamid Hashemi-Moghaddam2
*
1
Department of Biology, Damghan Branch, Islamic Azad University, Damghan 3671639998, Iran. 2
Department of
Chemistry, Damghan Branch, Islamic Azad University, Damghan 3671639998, Iran.
*Corresponding to: Hamid Hashemi-Moghaddam, Department of Chemistry, Basic Science Faculty, Damghan
Branch, Islamic Azad University, Cheshme ali BLVD, Damghan 3671639998, Iran. Email:
h.hashemimoghadam@damghaniau.ac.ir, hashemimoghaddam@yahoo.com.
Highlights
Microwave assisted hydrodistillation method can extract more compounds and yield of essential oils from
Echinops persicus than conventional hydrothermal method as well as further confirms that the methanol
extract of E. persicus plant exhibits considerable antioxidant and antimicrobial properties.
Traditionality
E. persicus is well-known as Shakarook in local Persian botany and it has been known since the time of
Avicenna and was prescribed for cough and respiratory system as an effective drug. In Persian folk
medicine, this plant has been widely prescribed as a flavoring agent and an effective remedy to treat
influenza, cough, fever, throat dryness, etc. E. echinatus is also extensively utilized in Indian traditional
folklore and Pakistan ethno-veterinary medicine.

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TMR | September 2019 | vol. 4 | no. 5 |247
Submit a manuscript: https://www.tmrjournals.com/tmr
doi: 10.12032/TMR20190826132
Abstract
Background: E. persicus which is well-known as Shakarook in local Persian botany and is extensively utilized in
different parts of in Iran. Materials and methods: Essential oils from the aerial parts of Echinops persicus were
isolated using hydrodistillation (HD) and microwave assisted hydrodistillation (MAHD) methods and the
respective chemical profiles were analyzed by means of GC-MS technique. The in vitro antioxidant and
antimicrobial activities of methanol extracts of E. persicus were investigated via using
2,2'-diphenyl-1-picrylhydrazyl (DPPH) assay as well as agar well-diffusion methods. The minimun inhibitory
concentrations (MICs) of the methanol extracts of E. persicus against the test microorganisms were determined by
the broth microdilution method. Results: GC-MS essential oils analysis shows 29 and 36 compounds constituting
91.9% and 98.2% of the total oils using HD and MAHD methods, respectively. Furthermore, the methanol extracts
of E. persicus exhibited higher DPPH radical scavenging activity than vitamin C with an IC50 value of 0.42 ± 0.16
µg/mL. Moreover, the prepared methanol extracts preliminarily showed promising antimicrobial activities against S.
aureus with the MIC value of 6.2 mg/mL. Conclusion: This study confirms that the methanol extract of E. persicus
plant exhibits considerable antioxidant and antimicrobial properties in vitro.
Keywords: Echinops persicus, Essential oil, Methanol extracts, Antioxidation, Antimicrobial.
Acknowledgments:
Financial and technical supports from the office for research affair of Islamic Azad University, Damghan
Branch are gratefully acknowledged.
Abbreviations:
HD, Hydrodistillation; MAHD, Microwave assisted hydrodistillation; MIC, Minimun inhibitory concentration;
DPPH, 2,2'-Diphenyl-1-picrylhydrazyl; NFT, Nitrofurantoin; NA, Nalidixic acid; MHA, Mueller-Hinton agar;
FID, Flame ionization detector; CLSI, Clinical and Laboratory Standards Institute; OM, Oxygenated
monoterpenes; SH, Sesquiterpene hydrocarbons; OS, Oxygenated sesquiterpenes; NH, Non-terpene
hydrocarbons, DH: Diterpene hydrocarbons; OD: Oxygenated diterpenes; MH: Monoterpene hydrocarbon;
RE: Rutin equivalent.
Competing interests:
The authors declare that there is no conflict of interest.
Citation:
Maryam Soori, Hossein Abbaspour, Hamid Hashemi-Moghaddam. Assessment of microwave assisted and
hydrodistllation extraction on Echinops persicus essential oils chemical composition and evaluation of its
biological activity. Traditional Medicine Research 2019, 4 (5): 246-256.
Executive Editor: Nuo-Xi Pi.
Submitted: 17 May 2019, Accepted: 26 August 2019, Online: 5 September 2019.

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Submit a manuscript: https://www.tmrjournals.com/tmr TMR | September 2019 | vol. 4 | no. 5 |248
doi: 10.12032/TMR20190826132
Background
Echinops is a famous genus from the Asteraceae
family consisting of about 120 species of flowering
plants [1]. It is usually known as globe thistles. The
plants belonging to this genus frequently appear as
spiny foliage leading to spherical flower heads in
nature [2].
E. persicus which is well-known as Shakarook in
local Persian botany and it has been known since the
time of Avicenna and was prescribed for cough and
respiratory system as an effective drug [3]. In Persian
folk medicine, this plant has been widely prescribed as
a flavoring agent and an effective remedy to treat
influenza, lung disease, asthma, constipation,
depression, overweight, bellyache, cough, fever, as
well as throat dryness [4-8]. It also possesses strong
laxative, expectorant and anti-cancer impacts [9-10].
Moreover, a balancing role in the digestive system of
the human body has been attributed to this medicinal
species [5]. In Indian traditional folklore usage, E.
echinatus is extensively utilized as a potent drug to
cure sexual debility [11]. In Pakistan ethno-veterinary
medicine, E. echinatus has been recommended as a
tonic for camels [12].
Diverse pharmacological, biological, and
phytochemical reports on different Echinops species
have been reported in the literature involving
antihelmentic [13], antimalarial [14],
anti-inflammatory [15-17], larvicidal [18], diuretic [11],
insecticidal [19], analgesic [11], reproductive [11],
antiproliferative [20], antifungal [21-25], antioxidant
[26], hepatoprotective [11], cytotoxic [27-29],
anti-ulcer [30], antifeedant [29], immunomodulatory
[4], antiplasmodial [31], antibacterial [25, 32, 33],
toxicological [31], anti-irritant [34], antipyretic [11,
35], anti-worm [25], wound healing [36] and
antimicrobial [37-39] activities.
As there are several activities on Echinops genus,
developing more effective extraction techniques are
required which was secure and agreeable to automation.
Today, microwave heating there has been a growing
interest because of its speed, also it is an environment
friendly technology for the extraction of a wide array
of samples [40]. The combination of
microwave-assisted extraction and hydrodistillation
(HD) has been effectively applied to extraction
essential oils from different plants [41-46]. More
effective and selective heating, time efficiency, faster
response to process heating control are advantages
microwave assisted hydrodistillation (MAHD)
extraction compared with conventional extraction
methods [40, 42].
In this work, essential oil was extracted from the
aerial parts of E. persicus by using HD and MAHD
methods for the first time. Separation and
identification of the constituents of the chemical
profiles have been made using GC and GC-MS
approaches. Furthermore, antioxidant activity of the
plant methanol extract was assessed using the
2,2'-diphenyl-1-picrylhydrazyl (DPPH) assay while its
antimicrobial properties were examined using broth
microdilution and agar well diffusion methods.
Experimental
Chemicals
2,6-Ditertbutyl-4-methylphenol (butylated
hydroxytoluene) standard antioxidant agent,
nitrofurantoin (NFT) antibiotic, nalidixic acid (NA)
antibiotic and DPPH were all purchased from
Sigma-Aldrich GmbH (Munich, Germany). The
Mueller-Hinton agar (MHA) and MHB (Muller Hinton
broth) culture media were purchased from Merck
Company, Germany. Standard strains: Staphylococcus
aureus (ATCC 25923) and Escherichia coli (ATCC
25922) were provided by Iran Medical Sciences
University, Tehran, Iran.
Plant material
E. persicus was harvested in the late of April 2016 in
the agricultural research farm, Hamadan Province,
Hamadan, Iran. The plant material was first
characterized by Dr Bostan Rodi botanist in the
Damghan Branch, Islamic Azad University and for
further authentication the whole plant material was
deposited at the Research Institutes of Forest
Herbarium and Rangelands (No: 4123451). For oil
extraction, the aerial parts were separated and final
step it dried in a clean place at shadow to avoid extra
damaging and to lower unpleasant
cross-contamination.
HD of essential oil
Commercially available clevenger apparatus
(Ashk shishe Co.Tehran Iran) was used for HD. In a
preliminary step, the E. persicus sample was dried for
one week at shade and then it was precisely weighed.
In order to hydrate its external layers, 50 g of plant was
immersed in 500 mL water, then volatile oils were
extracted at successive times. Each experiment was
carried out twice. The optimized distillation time was 3
h. Finally, the oil was dehydrated with anhydrous
sodium sulfate, it was separated and capped under
nitrogen and kept in refrigerator at -4 °C.
MAHD of essential oil
The microwave oven (Samsung, South Korea) used for
performance of the MAHD was operated at a safe and
commonly used frequency of 2450 MHz. This oven
had an interior cavity with the general dimensions of
29 cm × 37 cm × 40 cm which was capable of
providing of maximum powers equal to 1000 W within

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doi: 10.12032/TMR20190826132
the experiments. In our proposed MAHD methodology,
a proper hole was first drilled on the top of the
microwave. Immediately after, a flat-bottom flask
(1000 mL) was situated in the cavity of the microwave
assemble and directly connected to the circulatory
Clevenger via the provided hole [47-49]. To avoid
scattering of microwave beams in the laboratory
environment, around the hole was completely closed
with a layer of an aluminum foil. To perform the
MAHD approach, fifty g portions of the plant were
first immersed in 0.5 L of distilled water at 25 °C
under the atmospheric pressure for 1 h. Immediately
after, the excess water was drained off. The required
time for the soaking step was determined through our
primary evaluations. In the next step, the moistened
plant material was placed in a flat-bottom flask of
the system assembly. Throughout the extraction, the
vapor constantly passed through the condenser. Time
of extraction was optimized, as in long times the
extraction yield was reduced. For each case,
experiments were replicated twice. The storage
procedure for the essential oil obtained by the MAHD
approach was the same as described for the
water-distilled oil.
Preparation of the methanol extracts of E. persicus
In the first step, 50 g the aerial parts of plant (E.
persicus) were cut up and crushed in a coffee mill
(Moulinex Corporation, France). Obtained powder was
transferred to a darkcolored flasks, mixing with 85%
(v/v) methanol at a ratio of 15:100 (m/v plant material
to-solvent) and heating at 50 °C for 35 min.
Subsequently, the slurry was filtered through Whatman
No. 1 filter paper and the resulting deposit was
extracted twice and supernatants were combined and
evaporated under vacuum using a rotary vapor (IKA,
Germany) at 40 °C. Finally, the extracts were stored in
refrigerator at 4 °C.
GC and GC-MS Analysis
Quantitative and qualitative assessments of the
extracted oils were carried out using GC and GC-MS
instruments. A gas chromatograph (Varian 3800,
Australia) was used for quantitative analyses;
experimental conditions were chosen as follow:
injector temperature 290 °C with the split ratio 1:10
and a FID (flame ionization detector) temperature
250 °C. N2 at flow rate of 0.8 mL/min was used as
carrier gas. Capillary column was CP-Sil 5 CB (30 m ×
0.25 mm × 0.25 μm film thickness). The oven
temperature was held at 50 °C for 5 min and heated to
240 °C at a rate of 3 °C/min followed by an extra rise
to 300 °C with a programmed 5 °C/min ramp. Three
min final hold was applied for a clean-up of column.
Quantitative data were obtained through the system
area percentage. GC-MS determinations were
performed on an HP-6890 GC system coupled with a
5973-network mass selective detector, and equipped
with an HP-5MS capillary fused silica column (30 m ×
0.25 mm I.D. × 0.32 μm film thickness). The working
conditions were the same as described above but the
carrier gas was He. Mass spectra were taken at an
ionization voltage of 70 eV and were recorded over the
m/z range 20-500 amu. Chromatographic
measurements repeated three times, and the mean of
the retention indices as well as the mean percentage of
each component were considered. Duplicate times
were not taken into consideration if they had
differences higher than 1 s.
Identification and quantification of essential oil
ingredients
Characterization and recognition of the constituent
components of the essential oils from the aerial parts of
E. persicus were made by: (1) exhaustive comparison
of the mass spectral fragmentation pattern with respect
to the authentic samples and the Kovat's retention
indices (RI) relative to normal n-alkanes (C9-C25)
with the authentic data tabulated in the literature [50]
and a homemade software based upon our previously
published papers in the literature; (2) the mass spectral
archive available in the library (Wiley 275) of the
GC-MS instrument; (3) matching of the fragmentation
pattern of each constituent in each characterized profile
with those available in National Institute of Standards
and Technology Mass Spectral Library package with a
resemblance percentage above 90%.
The relative content of each constituent was
evaluated regarding the related peak area through
summing of all of the peaks (%). In this regard, no
correction factor was used in the calculation process.
In the last step, distinct volumes of the essential oils
were injected onto the injection port of the GC-MS
column, separately.
Determination of total phenolic contents
A proposed method that based on using the
Folin-Ciocalteu reagent is utilized to assess the amount
of total phenolic [51]. One hundred μL portions of
methanol extracts from the aerial parts of E. persicus
were transferred into the test tubes, followed with 0.75
mL of Folin-Ciocalteu reagent, which was previously
diluted 10 times with deionized water. This solution
was mixed gently and allowed to stand at 25 °C for 5
min. Then 0.75 mL of sodium carbonate (6.0% w/v)
was added to the mixture and stirred with other
reactants. Ninety min later, the absorbance was
determined at 725 nm using the double beam UV-Vis
spectrophotometer (Varian Cary 50, Australia). All of
the determinations were performed in triplicate.
Determination of total flavonoid contents
The contents of total flavonoids in the methanol
extracts from the aerial parts of E. persicus were
assessed with a spectrophotometric method reported by
Zhishen et al. [52] with some modification. Rutin was

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doi: 10.12032/TMR20190826132
used as the standard. One mL of the prepared extract
was diluted with an aqueous-ethanol solution (40.0%
v/v) to the final volume of 5 mL, followed by 0.3 mL
of an aqueous solution of NaNO2 (1:20, w/v), and then
after 5 minutes followed by addition of 0.3 mL
Al(NO3)3 (1:10, w/v). After being mixed and standing
for 6 min, the resulting solution was added with 2.0
mL of NaOH (1.0 M) and then brought up to 10 mL by
an aqueous ethanol (40% v/v). The mixed solution was
incubated at 25 °C for 10 min. A spectrophotometer
(UV2550, Shimadzu, Japan) using quartz cuvettes (10
×10 mm) were used to measure its absorbance at 510
nm. The solution containing all the reagents except for
the plant extract in an aqueous ethanol medium was
used as the blank solution. All of the determinations
were performed in triplicate.
Determination of antioxidant activity by the DPPH
method
Based on the method of Onder et al. [50] with some
modifications, the capability to scavenge DDPH free
radicals was used to evaluate the in vitro antioxidant
activities of methanol extracts from the aerial parts of
E. persicus. The methanolic extracts and vitamin C,
both at the concentration of 0.2 mg/L, were prepared
and were separately to react with 2 mL of a methanolic
solution of DPPH (0.004 g in 100 mL of methanol),
vitamin C was used as positive control while DPPH
without test samples was used as blank. Each
experiment was performed 3 times. Then the plate was
incubated in dark at 25 °C for 60 min and the
absorbance was subsequently measured at 517 nm
using a microplate reader (Biotek Epoch, USA). The
ability to scavenge the DPPH free radical was
calculated using the following equation [53]. DPPH
scavenging effect (%) = (Ao-A1)/Ao × 100. Where Ao
and A1 respectively imply the absorbance of the
control (blank) and the absorbance in the presence of
sample (E. persicus extract). Furthermore, the
antioxidant activities of the plant extract was evaluated
using the ascorbic acid standard curve and its
corresponding activities were expressed as IC50
(mg/L).
Antimicrobial activity by the agar well diffusion
method
The methanolic extract of was weighed and dissolved
in phosphate buffer saline (PBS; pH 7.0-7.2) and
DMSO at the concentration of 10 mg/mL, respectively
and filtered through a 0.45 µm membrane filter.
Staphylococcus aureus and Escherichia coli was
suspended in sterile saline at the density of 1 × 106
/mL
and inoculated onto the surface of MHA. The wells
with 8 mm in diameter were cut from the agar and 0.06
mL of methanol extract solution at the concentration of
10 mg/L was then delivered into each well. NFT100
antibiotic and NA30 antibiotic were used as positive
control for Staphylococcus aureus and Escherichia coli
respectively. After 24 h incubation at 37 °C, all plates
were examined for any zones of growth inhibition, and
the diameters of these zones were measured in mm
[54]. All the tests were performed in triplicate.
Determination of minimum inhibitory
concentration
A broth microdilution susceptibility assay
recommended by Clinical and Laboratory Standards
Institute (CLSI), formerly known as NCCLS, was used
to determine the MIC [54-55]. All the tests
supplemented with Tween 80 detergent to a final
concentration of 0.5% (v/v) were performed in Mueller
Hinton Broth (MHB). The bacterial strains were
cultured overnight at 37 ºC in MHA. Test strains were
suspended in MHB at a final density of 5 × 105
/mL.
Geometric dilutions of the methanol extract, with the
range from 0.036 to 72.0 mg/mL, were prepared in a
96-well microtitre plate. MHB + Tween 80 was used as
the growth control, MHB + Tween 80 + test oil was
used as the sterility control. Plates were then incubated
at 37 ºC for 24 h and the bacterial growth was
confirmed.
Statistical analysis
All experiments were done in triplicate, and results
were presented as mean ± SD. Statistical analysis was
performed on the data by SPSS 11.0. Differences in
inhibition zone were statistically analyzed by Student's
t test. Values of P < 0.05 were considered significant.
Results
The yield of essential oils with HD and MAHD
methods
The yield of extraction was 0.25 % and 43% (w/w) for
respectively (weight of the collected oil per g of dried
plant). The comparison of obtained results for yield of
essential oils extraction by these two methods show
apparent increase in yield by MAHD method.
Chemical profiles of the volatile oils of E. persicus
by HD and MAHD
Characterization of extracted essential oils of E.
persicus show that its ingredients mainly consisting of
oxygenated monoterpenes (OM) by the aid of the
described HD and MAHD techniques. Identificated
compounds are several OM, sesquiterpene
hydrocarbons (SH), oxygenated sesquiterpenes (OS),
non-terpene hydrocarbons (NH), diterpene
hydrocarbons (DH) and oxygenated diterpenes (OD).
Notably, no monoterpene hydrocarbon (MH)
contributed to the recognized profiles of the analyzed
essential oils. Chromatographic information of the
volatile oils obtained from the E. persicus by the
above-mentioned methods are tabulated in Table 1.
There are satisfactory agreements between the
calculated numerical values of Kovats retention indices

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and those cited in the literature. In Figure 1, The
component classes found in the aerial parts oils in E.
persicus L. by HD and MAHD methods.
Table 1 The essential oils chemical compositions from aerial parts of Echinops persicus obtained using the
HD and MAHD methods
Number Compound Class KI (Cal.) KI (Lit.) RT
HD
RT
MAHD
Characterization
criteria
1 n-Undecane NH 1100.1 1100 1.2 0.5 RI, MS, LRR
2 Borneol OM 1167.1 1165 0.7 0.9 RI, MS
3 Isomenthol OM 1181.2 1182 0.6 1.1 RI, MS
4 ND - 1190.7 - - 0.6 RI, MS, LRR
5 trans-4-Caranone OM 1195.5 1196 1.4 0.9 RI, MS, LRR
6 Methyl
α-Cyclogeranate
OM 1199.1 1197 1.8 1.3 RI, MS, LRR
7 Dodecane NH 1200.4 1200 4.0 4.4 RI, MS, LRR
8 cis-4-Caranone OM 1201.2 1200 1.3 1.2 RI, MS, LRR
9 Fenchyl acetate OM 1221.3 1221 7.0 4.8 RI, MS, LRR
10 β-Cyclocitral OM 1220.6 1219 2.5 1.9 RI, MS, LRR
11 Isobornyl formate OM 1238.8 1239 5.6 4.1 RI, MS, LRR
12 ND - 1389.4 - 1.6 0.4 RI, MS, LRR
13 n-Tetradecane NH 1398.5 1400 - 0.9 RI, MS
14 trans-Caryophyllene SH 1421.3 1417 2.0 2.8 RI, MS, LRR
15 Aromadendrene SH 1440.2 1439 - 1.2 RI, MS, LRR
16 Germacrene D SH 1485.1 1484 0.7 1.0 RI, MS, LRR
17 β-Dihydro agarofuran OS 1504 1503 2.0 1.4 RI, MS, LRR
18 β-Bisabolene SH 1507.3 1505 1.3 2.2 RI, MS, LRR
19 γ-Cadinene SH 1516.5 1513 1.4 2.4 RI, MS
20 δ-Cadinene SH 1527.5 1522 1.3 2.9 RI, MS, LRR
21 Elemol OS 1524.5 1548 0.6 0.7 RI, MS, LRR
22 Caryophyllene oxide OS 1550.1 1582 1.0 1.7 RI, MS, LRR
23 Hexadecane NH 1599.4 1600 6.0 1.6 RI, MS, LRR
24 Guaiol OS 1601.3 1600 - 4.6 RI, MS, LRR
25 γ-Eudesmol OS 1629.4 1630 16.7 12.1 RI, MS, LRR
26 7-epi-α-Eudesmol OS 1658.1 1662 1.4 0.9 RI, MS, LRR
27 Hinesol OS 1638 1640 5.7 7.0 RI, MS, LRR
28 Valerianol OS 1657.1 1656 9.5 5.9 RI, MS, LRR
Pogostol OS 1651.2 1651 8.6 0.9 RI, MS, LRR
30 Heptadecane NH 1699.9 1700 - 1.5 RI, MS, LRR
31 n-Octadecane NH 1800.4 1800 1.8 1.5 RI, MS, LRR
32 Beyerene DH 1933.1 1931 0.7 2.2 RI, MS, LRR
33 ND - 1804.1 - - 0.7 RI, MS
34 n-Eicosane NH 2001.2 2000 1.0 1.4 RI, MS, LRR
35 ND - 1947.1 - - 0.6 RI, MS, LRR
36 Incensole acetate OD 2184.3 2184 - 1.2 RI, MS, LRR
37 ND - 2198.3 - 1.3 0.6 RI, MS, LRR
38 ND - 2201.1 - - 0.5 RI, MS,

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Table 1 The essential oils chemical compositions from aerial parts of Echinops persicus obtained using the
HD and MAHD methods (Continued)
39 n-Docosane NH 2219.9 2200 1.2 0.7 RI, MS, LRR
40 n-Tricosane NH 2302.1 2300 - 0.7 RI, MS, LRR
41 Libocedrol NH 2343.1 2344 - 3.2 RI, MS, LRR
42 ND - - - - 0.6 RI, MS
43 n-Tetracosane NH 2398.7 2400 - 10.5 RI, MS, LRR
Total 91.9 98.2
KI (Cal.), Kovats retention index calculated with respect to n-alkenes on a HP-5MS capillary column; KI (Lit.),
Kovats retention index given in literature; RT, Retention time; HD, Hydrodistillation; MAHD, Microwave assisted
hydrodistillation; ND: Not detected; NH: Non-terpene hydrocarbons; OM: Oxygenated monoterpens; SH:
Sesquiterpene hydrocarbons; OS: Oxygenated sesquiterpens; DH: Diterpene hydrocarbons; OD: Oxygenated
diterpenes; RI: Retention index, MS: Mass spectra, LRR: Laboratory resemblance report ( > 90%).
Figure 1 The component classes in the aerial parts of the E. persicus L. oils by HD and MAHD methods
HD, Hydrodistillation; MAHD, Microwave assisted hydrodistillation; MH: Monoterpene hydrocarbons; OM:
Oxygenated monoterpens; SH: Sesquiterpene hydrocarbons; OS: Oxygenated sesquiterpens; NH: Non-terpene
compounds; DH: Diterpene hydrocarbons; OD: Oxygenated diterpenes; ND: Not detected.
Table 2 Methanolic extracts antimicrobial activity of the E. persicus L
Note: a: according to CLSI 2013 (Clinical and Laboratory Standards Institute. Performance Standards for
Antimicrobial Susceptibility Testing; Twentieth Informational Supplement, Document M100-S20. Wayne, PA:
Clinical and Laboratory Standards Institute 2013; 22-50 [51].). b: Compared with NA30, P < 0.05. NFT:
Nitrofurantoin；NA, Nalidixic acid.
Bacteria
Diameter of inhibition zone (mm)
Positive control Methanolic extracts
Escherichia coli 25 ±1.7 (NA30, standdrad range: 23-28 mm [51]) 18 ± 1.5 b
Staphylococcus aureus 21 ±1.1 (NFT100, standdrad range: 20-25 mm [51]) 22 ± 0.8

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In the HD extraction method (Table 1), 29
components could be identified in the oil from the
aerial parts: eight OM (20.9%), five SH (6.7%), eight
OS (45.5%), six NH (15.2%), one diterpene
hydrocarbon (0.7%), and one oxygenated diterpene
(1.3%). Specifically, in the hydrodistilled oil from the
plant aerial parts γ-eudesmol (16.7%) was found to be
the major constituent followed by valerianol (9.5%),
pogostol (8.6%), fenchyl acetate (7.0%), hexadecane
(6.0%), hinesol (5.7%) and isobornyl formate (5.6%).
On the other hand, in the essential oil was extracted
by MAHD method, 36 volatile components were
identified in the aerial parts oils using. The main
components were γ-eudesmol (12.1%), hinesol (7.0%),
valerianol (5.9%), fenchyl acetate (4.8%), guaiol (4.6%)
and dodecane (4.4%). In terms of general categories,
eight OM (16.2%), six SH (12.5%), nine OS (35.2%),
eleven NH (26.9%), one diterpene hydrocarbon (2.2%)
and one oxygenated diterpene (1.2%) were identified
in the essential oil of the plant sample (E. persicus)
obtained by the MAHD method.
Total phenolic and flavonoids contents
To assess the total phenolic levels in the organic
extract of the plant material, a standard calibration
curve (0.01-0.05 mg mL-1
) was plotted using gallic
acid (r2
= 0.99). The total phenolic content was finally
calculated as gallic acid equivalents in milligram per
100 g of plant extract. Accordingly, the contents of
total phenolic were found to be 99.22 (mg gallic acid
equivalents/g dry leaves).
On the other hand, in the determination of the
flavonoid contents in the methanol extract of E.
persicus, a standard curve equation obtained having an
equation of A = 1.971 × C, R2
= 0.9986 where A was
the absorbance and C was the rutin equivalent (RE)
mg/mL concentration. The total flavonoids content in
the extracts was calculated and expressed as mg RE
per g dry weight. In our study, total flavonoids
contents in the methanol extracts from the aerial parts
of E. persicus were found to be 17.5 ± 0.50 mg RE/g
dry weight.
Antioxidant activity results
A methanolic solution of the stable free radical, DPPH
was used for the radical-scavenging activity of the
prepared plant extracts. The obtained results showed
that IC50 value for the methanolic extracts from the of
E. persicus and vitamin C were 0.42 ± 0.16 and 0.55 ±
1.8 µg/mL, respectively. This implies that the extracts
of this plant possess a very strong antioxidant activity
even more than vitamin C as a powerful antioxidant
agent.
Antimicrobial activity
In this study, methanolic extract from of the E.
persicus was evaluated for exploration of possible
antimicrobial activity against certain bacterial strains,
which are frequently regarded as important human
pathogens. Susceptibility of the plant extracts was
evaluated by the agar well diffusion method. In our
experiments, the methanolic extract of the E. persicus
showed strong bactericidal effect against all the tested
microorganisms involving both Gram positive and
Gram-negative bacterial strains (Staphylococcus
aureus and Escherichia coli) (Table 2). According to
the result of broth microdilution susceptibility assay,
the extract isolated from the aerial parts of E. persicus
was found to be sensitive against Staphylococcus
aureus microorganism with a minimum inhibitory
concentration (MIC) value of 6.2 mg/mL. However,
MIC of this extract against Escherichia coli was not
detected and further studies need to be undertaken to
elucidate its exact antimicrobial activities.
Discussion
In this project, the essential oils from the aerial parts of
E. persicus were successfully isolated by using HD and
MAHD methods and were subsequently characterized
using the GC-MS instrumentation for the first time.
Comparative assessment of various classes of the oil
ingredient show that in extracted oils by different
methods, OS established the main ingredient and the
second major class are non-terpene and OM when
using the proposed HD and MAHD methodologies.
Regarding our characterization, γ-eudesmol was the
most frequent natural compound occurring in the two
identified profiles.
Weyerstahl et al. have analyzed strong
patchouli-like and woody smelling essential oil of the
rhizomes of E. giganteus var. Ielyi C. D. Adams
(Compositae) and reported that all the chemical profile
consisted of sesquiterpenes, which were mainly due to
triquinane compounds [56]. In this attempt, three new
tricyclic sesquiterpene skeletons, namely cameroonane,
prenopsane and nopsane naturally occurring have been
identified for the first time. Furthermore, a biogenetic
pathway from presilphiperfolane cation C to the
cameroonane K, prenopsane L and nopsane M cations
has been shown. In accordance with this work,
cameroonanol and prenopsanol were recognized as the
main contributors to the fragrance of the total oil.
Also, in the work of Liu et al. on the essential oil
from the aerial parts of E. latifolius Tausch, high
prevalence of monoterpenoids (78.45%) was reviled by
GC-MC analysis [19]. In accordance with this study,
the respective chemical profile was characterized by
high quantities of 1,8-cineole (19.6%), (Z)-β-ocimene
(18.4%), β-pinene (15.6%), β-myrcene (4.8%) and
carvone (4.4%). Finally, Radulovic and Denic have
analyzed the essential oils from the roots of E.
bannaticus Rochel ex Schrad. and E. sphaerocephalus
L. (Asteraceae) from Serbia [57]. In this report, the oils
were characterized by high contents of two relatively
rare groups of compounds: S-containing polyacetylene

ARTICLE
doi: 10.12032/TMR20190826132
compounds (65.5 and 64.1%, respectively) and
triquinane sesquiterpenoids (12.7 and 20.9%,
respectively). The findings given in this work
highlighted that several compounds belonging to
S-containing polyacetylene and triquinane
sesquiterpenoids represent metabolites which were
novel for the analyzed Echinops species or even the
whole genus.
A simple comparison on the obtained results using
the utilized approaches (HD and MAHD) reveals that
considering the most important class of natural
compounds constituting that concerned chemical
profiles, there are some similarities between our
findings and the previously published papers [58-59].
However, there are apparent differences between the
recognized chemical profiles in the present report and
some of the other previous attempts dealing with the
characterization of the chemical composition of the
essential oils from other species of the Echinops genus
[19, 56, 57, 59, 60].
The plant extracts as antioxidant and antimicrobial
agents always show two main features: natural origin
is the first which earnings more safety to the public
and the environment, lower risk for resistance
development through pathogenic microorganisms is
the second characteristic. About of its biological
activity, in the literature, a variety of studies have been
performed on the extracts of various plants to screen
the concerned antimicrobial activity and/or to explore
novel antimicrobial compounds. Therefore, medicinal
plants are finding their way into pharmaceuticals,
nutraceuticals and food supplements disciplines
[61-63]. In short, based on our analysis, the extract
from the aerial parts of E. persicus was observed to
against Gram-positive microorganism
Staphylococcus aureus with a MIC value of 6.2
mg/mL. However, MIC of this extract against
Escherichia coli as a Gram-negative microorganism
was not detected and further studies should be
undertaken to elucidate its exact antimicrobial
activities.
Conclusion
A total of 29-36 compounds were identified in the
related profiles covering 91.9-98.2% of their
compositions in the separated essential oils from the
aerial parts of E. persicus using the HD and MAHD
approches, respectively. In addition, the methanolic
extract of E. persicus was observed to have the definite
antimicrobial activities against S. aureus
microorganism, to be suggested as a new potential
source of natural antioxidants and antimicrobial agents
for pharmaceutical and food industries.
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Assessment of microwave assisted and hydrodistllation extraction on Echinops persicus essential oils chemical composition and evaluation of its biological activity