Industrial Crops & Products 107 (2017) 90–96

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Industrial Crops & Products

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Chemical composition and anticancer activity of Elsholtzia ciliata essential MARK

oils and extracts prepared by different methods
Lauryna Pudziuvelytea, Mantas Stankeviciusb, Audrius Maruskab, Vilma Petrikaited,e,
⁎
Ona Ragazinskienec, Gailute Draksienea, Jurga Bernatonienea,
a
Department of Drug Technology and Social Pharmacy, Lithuanian University of Health Sciences, Eivenių str. 4, LT-50161, Kaunas, Lithuania
b
Department of Biology, Vytautas Magnus University, Vileikos str. 8, LT-44404, Kaunas, Lithuania
c
Kaunas Botanical Garden of Vytautas Magnus University, Kaunas, Lithuania
d
Department of Drug Chemistry, Faculty of Pharmacy, Lithuanian University of Health Sciences, LT-44307, Kaunas, Lithuania
e
Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, LT-10222, Vilnius, Lithuania

A R T I C L E I N F O A B S T R A C T

Keywords: E. ciliata (Lamiaceae) is very interesting and promising herb mainly for chemical composition and pharmaco-
Elsholtzia ciliata logical activities. The aim of this study was to determine chemical composition of the essential oils of fresh,
Anticancer activity frozen and dried herbal materials of E. ciliata and compare different extraction methods This is the first study on
Essential oil composition of E. ciliata volatile compounds from fresh, frozen and dried herbal samples. The samples were
Ethanolic extracts
prepared by hydrodistillation (HD), extraction with polar solvent-ethanol (ESE) and dynamic headspace solid-
Hydrodistillation
GC–MS
phase micro extraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC–MS) method. A
total of 48 compounds were identified by GC–MS. Dehydroelsholtzia ketone, elsholtzia ketone, sesquiterpenes β-
bourbonene, caryophyllene, α-caryophyllene, germacrene D and α-farnesene were identified and found to be
predominant compounds in SPME composition of the fresh, frozen and dried herbal samples. The major amounts
of ketones (dehydroelsholtzia and elsholtzia) were determined in dried herbal samples where they made up
24.94% (p < 0.05) and 71.34% (p < 0.05) of the SPME composition. Artemisia ketone was determined only in
fresh herb. No previous report exists regarding this ketone in E. ciliata fresh, frozen and dried herbal materials or
essential oil. There were 26 components identified in the essential oil obtained by HD. The main compounds of
this essential oil were dehydroelsholtzia ketone (78.28%) and elsholtzia ketone (14.58%).
Essential oil showed antiproliferative activity on three tested cancer cell lines (human glioblastoma (U87),
pancreatic cancer (Panc-1) and triple negative breast cancer (MDA-MB231)) in vitro. EC50 (half maximal
effective concentration) values of essential oil against those cells were in the range of 0.017–0.021%. The
viability of human normal fibroblasts exposed to the same concentrations of the essential oil was statistically
significantly higher compared to the viability of cancer cells (p < 0.05). The extracts did not show any effect on
U87 cells, and only slightly decreased MDA-MB231 cell viability (up to 76.4%) at the highest concentration of
10 mg/mL.
The impact that different extraction methods have on the yield of the essential oil from the tested herbal
materials requires a further research. It would enable production of pharmaceutical forms enriched by these
essential oils, which are notable for their anticancer activity.

1. Introduction are usually obtained by HD, steam distillation or solvent extraction

Essential oils are considered to be one of the most important
substances in plants and to possess antimicrobial, antiviral, antifungal,
antioxidant and anti-inflammatory activities (Bey-Ould Si Said et al.,
2016; Raut and Karuppayil, 2014; Teixeira et al., 2013; Buchbauer,
2010). Essential oils are complex mixtures of various terpenes and their
oxygenated derivatives such as alcohols, phenols, ketones. Essential oils
(Bakkali et al., 2008; Longaray Delamare et al., 2007; Raut and
Karuppayil, 2014). For the experiments herbal materials of E.ciliata
grown in Lithuania (Europe) were chosen. A flowering plant Elsholtzia
ciliata (Thunb.) Hylander of the family Lamiaceae is native to Asia and
is also found in Europe, Africa, North America, India (Guo et al., 2012;
Korolyuk et al., 2002). Lamiaceae family plants are well known for their
antiviral, antimutagenic, chemotherapeutic, antimicrobial, antioxidant

⁎
Corresponding author.
E-mail addresses: jurgabernatoniene@yahoo.com, jurga.bernatoniene@lsmuni.lt (J. Bernatoniene).

http://dx.doi.org/10.1016/j.indcrop.2017.05.040
Received 11 January 2017; Received in revised form 19 May 2017; Accepted 21 May 2017
Available online 26 May 2017
0926-6690/ © 2017 Elsevier B.V. All rights reserved.
L. Pudziuvelyte et al.
and anti-inflammatory activities (Raut and Karuppayil, 2014). E. ciliata
is widely used in folk medicine for antibacterial, anticancer and anti-
inflammatory properties (Tian, 2013). In traditional Chinese medicine
the herb has been used to treat the common cold, headaches,
pharyngitis, fever, edema, diarrhea, rheumatic arthritis, digestion
disorders, nephritises (Guo et al., 2012; Sung et al., 2011). The
pharmacological activities, such as antibacterial, antiviral, antioxidant,
anti-inflammatory, diuretic and anti-obese, of extracts and pure com-
pounds from E. ciliata are under investigation (Guo et al., 2012; Sung
et al., 2011). The major chemical constituents in Elsholtzia are
flavonoids, phenylpropanoids, phytosterols, cyanogenic glycosides
and triterpenes (Guo et al., 2012; Liu et al., 2012).
Essential oils accumulating herbal materials are used to produce
various pharmaceutical forms such as tablets, capsules, microcapsules,
ointments, creams or gels. Usually materials used in production are air
dried; however, using fresh or frozen materials could also be suitable.
There has not been a research done yet comparing chemical composi-
tion of essential oils from freshly collected and from frozen herbal
materials, and it is not known what effect the material preparation has
on the volatile composition. The aim of this study was to investigate the
effects of different methods of material preparation and extraction on
chemical composition of volatile compounds from E. ciliata. Also, one of
the objectives was to evaluate the toxicity and anticancer activity in
vitro of the plant extracts as well as the essential oil.
2. Materials and methods
2.1. Plant material
E. ciliata aerial parts were collected in Vilnius, Lithuania, in July
2016 and were purchased as fresh and dried herbs from “Žolynų namai”
(Vilnius, Lithuania). The herbs were identified by Dr. Prof. Nijole
Savickiene, Medical Academy, Lithuania University of Health Sciences,
Kaunas, Lithuania. A voucher specimen (L 17710) was placed for
storage at the Herbarium of the Department of Drug Technology and
Social Pharmacy, Lithuanian University of Health Sciences, Lithuania.
Fresh and dried materials were mechanically ground in a laboratory
mill to a homogenous powder or paste. A sample of fresh herb was
frozen in a freezer (−18 °C) until preparation of extracts and SPME by
GC–MS method.
2.2. Isolation of the essential oil
30 g dried E. ciliata sample was mixed with 500 mL bi-distilled
water and submitted to HD for 4 h using a Clevenger-type apparatus
(European pharmacopoeia). A yellow colored oil with specific aroma
was obtained. The essential oil was collected with water and stored in a
refrigerator at +4 °C until needed.
2.3. Preparation of ethanolic extracts
Fresh, frozen and dried powdered materials (Fig. 1) of plant’s areal
parts (0.5 g each) were extracted for 24 h with 20 mL 96% ethanol in
TiterTek shaker (Germany). The extracts were filtered through a paper
filter and 0.22 μm pore size PVDF membrane filter and stored at +4 °C
in a refrigerator until GC–MS analysis.
2.4. Dynamic headspace SPME
Samples for gas chromatography analysis were prepared using
SPME. Extraction of E. ciliata volatiles was performed on 65 μm
PDMS/DVB (polydimethylsiloxane/divinylbenzene) Stable Flex fibre
(Supelco, Bellefonte, USA). 10 mg of fresh, frozen and dried samples
were added into 10 mL vials and placed in the AOC-5000 autosampler.
The samples were thermostated for 10 min at 40 °C and the fiber was
exposed in the headspace.
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Industrial Crops & Products 107 (2017) 90–96
2.5. GC–MS analysis
The analysis was carried out using a GCMS-QP2010 system
(Shimadzu, Tokyo, Japan). For separation of volatiles a low polarity
RTX-5MS (Restec, USA) (30 m × 0.25 mm i.d. × 0.25 μm film thick-
ness) GC column was used. The oven temperature gradient started at
60 °C and was raised to 150 °C at 5 °C/min, and then raised to 280 °C at
20 °C/min and was held at it for 3 min. The carrier gas helium
(99.999%, AGA Lithuania) was used at a flow rate of 1.2 mL/min.
The injector temperature was kept at 230 °C in a split mode (1:20). The
mass detector electron ionization was 70 eV. The ion source and
interface temperatures were set at 220 °C and 260 °C correspondingly.
The compounds preliminary were identified by mass spectra library
search (NIST, v1.7) and by comparing with the mass spectral data from
literature (Adams, 1995). The repeatability of solid-phase micro
extraction – gas chromatography mass spectrometry method was
evaluated by injecting the same sample three times. The relative
standard deviation for the peak area was 5.15%.
2.6. Cell lines
Anticancer activity was tested on three selected cancer cell lines:
human glioblastoma (U87), pancreatic ductal adenocarcinoma (Panc-1)
and triple-negative mammary gland adenocarcinoma (MDA-MB231).
Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
high glucose (Gibco) supplemented with 10% fetal bovine serum (FBS)
(Gibco) and 1% antibiotics (10,000 units/mL penicillin and 10 mg/mL
streptomycin) (Gibco) at 37 °C in a humidified atmosphere containing
5% CO2. Human fibroblasts were grown in a Medium 106 with Low
Serum Growth Supplement (LSGS) (Gibco) supplemented with 1%
antibiotics (10,000 units/mL penicillin and 10 mg/mL streptomycin)
(Gibco) at 37 °C in a humidified atmosphere containing 5% CO2. All cell
lines were grown to 70% confluence and trypsinized with 0.125%
TrypLE™ Express solution (Gibco) before passage. They were used until
passage 20.
2.7. Determination of cell viability
Cell viability was studied using the method of 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma). 100 μL of
cells were seeded in 96-well plates in triplicates (5000 cells/well for
U87, MDA-MB231 and human fibroblasts cells, and 3000 cells/well for
Panc-1 cells) and incubated at 37 °C for 24 h. Then serial dilutions of
different extracts and the essential oil were made in microplates. The
essential oil before the experiment was mixed with 10% of Tween 80 to
enhance its solubilization in culture medium. In order to avoid the
essential oil evaporation, 0.05% of methylcellulose (Sigma) was added
into the medium. Cells treated only with medium containing the same
concentration of ethanol (in the case of extracts) or Tween 80 (in the
case of essential oil) served as a negative control. Only medium without
cells was used as a positive control. After 72 h incubating at 37 °C,
10 μL of MTT was added in each well. After 3 h the liquid was aspirated
from the wells and discarded. Formazan crystals were dissolved in
100 μL of DMSO, and absorbance was measured at a test wavelength of
490 nm and a reference wavelength of 630 nm using a multidetection
microplate reader. The experiments were repeated three times inde-
pendently.
2.8. Statistical analysis
Data are presented as mean ± SEM. Statistical analysis was
performed by using Student’s t-test. A value of p < 0.05 was taken
as the level of significance.
L. Pudziuvelyte et al.
Fig. 1. Fresh (A.), frozen (B.) and dried (C.) herbal materials of E. ciliata areal parts.
3. Results
In the first experiments the chemical compositions of essential oils
in the fresh, frozen and dried E. ciliata herbal materials were deter-
mined.
To our knowledge, this is the first study of E. ciliata volatile
compounds extracted from fresh, frozen and dried herbal samples.
The volatile compounds found in tested samples are listed in Table 1,
their relative percent contents were calculated from the peak areas. E.
ciliata grown in Lithuania is not rich in quantity and variety of the
volatile compounds. From all SPME samples there were 16 different
compounds identified (Table 1). Dehydroelsholtzia ketone, elsholtzia
ketone, sesquiterpenes β-bourbonene, caryophyllene, α-caryophyllene,
germacrene D and α-farnesene were identified and found to be
predominant compounds in fresh, frozen and dried herbal samples.
The major amounts of ketones (dehydroelsholtzia and elsholtzia) were
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Industrial Crops & Products 107 (2017) 90–96
found in dried herbal samples and made up 21.94% and 71.34% of
SPME composition accordingly (p < 0.05 vs fresh and frozen material)
(Table 1). Artemisia ketone was identified only in fresh herbal sample
and made up 1.83% of SPME composition.
Sesquiterpenes were obtained from dried (3.3%), frozen (1.73%)
and fresh (1.95%) E. ciliata samples (Table 1). Predominant sesquiter-
penes were α-caryophyllene and β-bourbonene in fresh (1.04% and
0.53%), frozen (0.84% and 0.49%) and dried (1.6% and 0.97%) herbal
materials. Three alcohols were identified in frozen samples only and
one alcohol ((S)-3,4-dimethylpentanol (0.08%)) in dried samples
(Table 1). The predominant compound in fresh herb was eucalyptol
(0.38%). The tests showed that 2-propenoic acid and 5-methyl-furan-2-
carboxylic acid (1H-[1,2,4]-triazol-3-yl)-amide were not present in air-
dried herbal material SPME composition. However, they were identi-
fied in fresh and frozen material SPME composition. 2,3-Dimethyl-5-
(2,6,10-trimethylundecyl)-furan was present only in dried material. The
L. Pudziuvelyte et al. Industrial Crops & Products 107 (2017) 90–96

Table 1 Table 2
SPME composition of fresh, frozen and dried E. ciliata herbal materials. Chemical composition of essential oil obtained by hydrodistillation from E. ciliata dried
herb.
Compounds Fresh (%) Frozen (%) Dried (%) Retention
index Compounds Retention Composition (%)
index
3-Methyl-3- – 0.22 – 935
oxetanemethanol Eucalyptol 963 0.05
3-Octanol – 0.11 – 944 Cyclohexene, 2-ethenyl-1,3,3-trimethyl 1011 0.15
Eucalyptol – 0.38 – 963 2-propenoic acid, 2-methyl-, ethenyl ester 1053 0.06
2-Propenoic acid, 2-methyl-, 0.12 0.16 – 1052 Elsholtzia ketone 1066 14.58
ethenyl ester Furane-2-carboxaldehyde, 5- 1079 0.43
Elsholtzia ketone 23.09 33.64 24.94 1067 (nitrophenoxymethyl)-
2,3-Dimethyl-5-(2,6,10- – – 0.26 1082 (−)-1R-8-Hydroxy-p-menth-4-en-3-one 1110 0.08
trimethylundecyl) furan Dehydroelsholtzia ketone 1117 78.28
5-Methyl-furan-2-carboxylic 0.28 0.23 – 1083 Beta-Bourbonene 1152 0.57
acid (1H-[1,2,4]triazol- Isocaryophyllene 1166 0.57
3-yl)-amide Beta-Cubebene 1170 0.06
Dehydroelsholtzia ketone 72.64 63.31 71.34 1118 Ledene 1174 0.05
Artemizia ketone 1.83 – – 1123 Alpha-Caryophyllene 1180 1.84
Beta-Bourbonene 0.53 0.49 0.97 1152 Alpha-Cubebene 1186 0.02
Caryophyllene 0.23 0.23 0.42 1167 Naphthalene 1190 0.13
Alpha-Caryophyllene 1.04 0.84 1.60 1181 Germacrene D 1192 0.24
Germacrene D 0.07 0.09 0.14 1193 Trans-alpha-Bergamotene 1197 0.55
Alpha-Farnesene 0.08 0.08 0.17 1199 Alpha-Farnesene 1202 0.66
1,3,6,10-Dodecatetraene, 0.09 – – 1205 Gamma-Cadinene 1205 0.15
3,7,11-trimethyl-, (Z,E)- Delta-Cadinene 1208 0.28
(S)-3,4-Dimethylpentanol – – 0.08 1256 Caryophyllene oxide 1224 0.21
Sesquiterpenes 1.95 1.73 3.3 Nonane 1243 0.05
Oxygenated monoterpenes – 0.38 – 3-Tetradecen-5-yne, (Z)- 1268 0.05
Ketones 97.01 96.95 96.28 Palmitic acid 1275 0.16
Other 0.49 0.72 0.34 Phytol 1286 0.06
Total 99.45 99.78 99.92 Methyl (Z)-5,11,14,17-eicosatetraenoate 1289 0.61
2,6-octadiene, 2,7-dimethyl- 1294 0.08
SPME – solid-phase micro extraction. Sesquiterpenes 4.99
Oxygenated monoterpenes 0.05
Oxygenated sesquiterpenes 0.21
results presented in Table 1 show that the methods of herb preparation
Ketones 92.86
affected the chemical composition. Others 1.86
Further tests were aimed to determine the effect of HD method on Total 99.97
the chemical composition of the essential oil. In our study, 26
components of the essential oil obtained by HD were identified
(Table 2). The main compounds in the essential oil were dehydroel- tions of essential oil was statistically significantly higher compared to
sholtzia ketone (78.28%) and elsholtzia ketone (14.58%). These the viability of cancer cells (p < 0.05).
ketones were also predominant in SPME composition of fresh, frozen The extracts were not very active against tested cell lines (Fig. 4).
and dried herbal samples. The second major identified group of volatile They did not show any effect on U87 cells, and only slightly decreased
compounds was sesquiterpenes (5.02%), which included α-caryophyl- the viability of MDA-MB231 cells (up to 76.4%) at the highest
lene (1.87%), α-farnesene (0.66%), β-bourbonene (0.57%), isocaryo- concentration of 10 mg/mL. However, they also did not decrease the
phyllene (0.57%), trans-α-bergamotene (0.55%), δ-cadinene (0.28%), viability of human fibroblasts at the same dilutions.
germacrene D (0.24%), γ-cadinene (0.15%), β-cubebene (0.06%),
ledene (0.05%), and α-cubebene (0.02%). Some chemical compounds, 4. Discussion
not present in the herbal material SPME composition, were identified in
the essential oil: isocaryophyllene, β-cubebene, ledene, α-cubebene, γ- In this study the fresh, frozen and dried herbal samples and different
cadinene and δ-cadinene. Eucalyptol, caryophyllene oxide, palmitic extraction methods were analyzed. Our results show that E. ciliata main
acid were determined only in the essential oil as well. volatile components are dehydroelsholtzia, elsholtzia ketones and
A total of 23 components were identified in E. ciliata ethanolic sesquiterpenes α-caryophyllene, β-bourbonene. We determined the
extracts (Fig. 2). The main compounds in fresh herb extracts were composition of extracts obtained from dried herbal samples using HD
dehydroelsholtzia ketone (45.74%), cis,cis,cis-7,10,13-hexadecatrienal method in percentages of these major components. Other authors
(16.94%), elsholtzia ketone (7.34%), palmitic acid (4.69%), α-caryo- reported that α-caryophyllene demonstrated anti-inflammatory, anti-
phyllene (0.87%). viral activities (Djilani and Dicko, 2012; Fernandes et al., 2007).
Dehydroelsholtzia ketone (58.47%), elsholtzia ketone (11.49%), Caryophyllene oxide (12.45%) rich essential oil of Helichrysum fulgidum
linolenic acid (8.5%), palmitic acid (3.95%), α-caryophyllene (1.3%) showed antibacterial and antifungal activities (Bougatsos et al., 2004).
were obtained as the main components from frozen herb extracts. Surprisingly, sesquiterpene trans-α-bergamotene in E. ciliata essential
Predominant compounds from dried herb extracts were dehydroelsholt- oil has not been reported earlier, but was determined for the first time
zia ketone (57.94%), linolenic acid (11.79%), elsholtzia ketone in our study (0.55%). This volatile compound is attractive to insects
(11.40%), palmitic acid (3.19%), α-caryophyllene (1.31%). This ex- (Schnee et al., 2006) and was identified as the main component in the
traction method was better for extraction of organic acids, esters and oil of Cichorium intybus (Rustaiyan et al., 2011). Artemisia ketone was
ketones from herbal samples. determined only in fresh herb SPME composition. No previous report
The essential oil showed antiproliferative activity (Fig. 3) on all exists regarding this ketone in E. ciliata fresh, frozen and dried herbal
tested cancer cell lines (human glioblastoma (U87), pancreatic cancer materials or essential oil. Artemisia ketone is a major constituent of
(Panc-1) and triple negative breast cancer (MDA-MB231)) in vitro. EC50 essential oil from some cultivars of A. annua and may have antimalarial,
values of the essential oil were in the range of 0.017–0.021%. The antibacterial and antifungal activities (Bilia et al., 2014; Weathers et al.,
viability of human normal fibroblasts exposed to the same concentra- 2014).

93
L. Pudziuvelyte et al.
Fig. 2. A. Identified sesquiterpenes in ethanolic extracts from E. ciliata. B. Identified ketones in ethanolic extracts from E. ciliata. C. Identified organic acids in ethanolic extracts from E.
ciliata. D. Identified other compounds in ethanolic extracts from E. ciliata.
*Hexadecahydrocyclopenta[a]phenanthrene-3, 17-diol, 16-(1,3-dimethyl-1H-pyrazol-4-ylmethylene)-10, 13-dimethyl-
There is a considerable similarity reported by different authors
about the main components of E. ciliata essential oil. Dehydroelsholtzia
ketone and elsholtzia ketone have been reported to be the main
ingredients (Kharina et al., 1995; Thappa et al., 1999). Researchers
also identified dehydroelsholtzia (66.1–72.4%) and elsholtzia
(3.3–19.3%) ketones as the main volatile constituents of essential oil
prepared by HD of fresh E. ciliata material from suburb area of
Novosibirsk (Korolyuk et al., 2002). There is some difference between
our data and other researcher’s results about the main identified
compounds. Dung et al. (1996) reported geranial (19.5–26.5%), neral
(15.2–20.5%), and limonene (10.9–14.2%) as the main ingredients of E.
ciliata essential oils from the leaves were β-linalool (12.06%), caryo-
phyllene (11.02%), eugenol (9.67%), and caryophyllene oxide (9.61%),
from the flowers β-linalool (11.52%), β-lonone (11.08%), eugenol
(10.56%), and caryophyllene (9.57%), and from the seeds caryophyl-
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Industrial Crops & Products 107 (2017) 90–96
lene (11.13%), β-linalool (10.86%), eugenol (9.32%), and caryophyl-
lene oxide (8.83%) (Tian, 2013). Ketones have not been identified by
these researchers.
GC–MS analysis showed the influence of different techniques of
extraction on the quantity and quality of volatile components composi-
tion. More volatile compounds were obtained using HD (26 compo-
nents). HD has been earlier reported as a better extraction method for C.
aurantium (58 components) and E. fruticosa (35 components) herbal
samples. HD was more suitable for monoterpene and sesquiterpene
extraction (Jiang et al., 2011; Saini et al., 2010). ESE extracted more
organic acids, esters and other compounds. Similar results were
obtained from the research where herbal material was extracted by
ethyl acetate (Jiang et al., 2011).
Volatile compounds composition differences are related to the
extraction methods and other factors, such as growing habitats,
L. Pudziuvelyte et al.
Fig. 3. The activity of E. ciliata essential oil on human normal fibroblasts and cancer cells.
*p < 0.05. Human fibroblasts (HF), human glioblastoma (U87), pancreatic cancer (Panc-
1) and triple negative breast cancer (MB231).
Fig. 4. The activity of E. ciliata extracts on human glioblastoma (U87) and triple-negative
breast cancer (MB231) cell lines. 1 – extract from frozen fresh herbal material, 2 – extract
from fresh herbal material, 3 – extract from dried herbal material.
seasonal variation, parts of the plant, storage conditions and others.
Our experimental data showed that the ethanolic extracts from the
fresh, dried and frozen E. ciliata herbal samples were not very active
against different tested cancerous and non-cancerous cell lines. We
have shown for the first time that the essential oil from E. ciliata shows
an anticancer activity against human glioblastoma, breast and pancrea-
tic cancer. However, the oil was significantly less toxic against human
normal fibroblasts. Similar effects have been obtained with the extract
and essential oil of the plant from the same Lamiaceae family by
Fekrazad et al. (2017). They showed that the essential oil from Thymus
caramanicus Jalas possesses antiproliferative properties. The activity of
this essential oil (IC50 = 0.44 μL/mL) against human oral epidermoid
carcinoma KB cells was also much lower compared to that of the
extract. However, it was about 2–3 times more active compared to the
essential oil from E. ciliata against human breast, pancreatic cancer or
glioblastoma cells. Interestingly, cytotoxic effect of essential oil from
Thymus caramanicus Jalas was greater than the effect on normal
gingival cells, what was seen also in our experiments.
In another experiment Russo et al. (2015) showed that the essential
oil from Salvia verbenaca was also active against human melanoma cell
line M14 and possessed lower activity against normal human non-
immortalised buccal fibroblast cells. The activity of that essential oil
mainly has been explained by the presence of sesquiterpenes and
carvacrol. α-Caryophyllene and caryophyllene, which are found in
the essential oil of E. ciliata as the major constituents, also could
contribute to the anticancer activity against tested cell lines as there is
some data published about its in vitro and in vivo anticancer effect
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Industrial Crops & Products 107 (2017) 90–96
(Loizzo et al., 2007; Kim et al., 2014).
We could not find any data in the official literature sources about
the anticancer effect of dehydroelsholtzia and elsholtzia ketones.
However, there are published data that the essential oil from E. ciliata
did not possess significant toxicity on rat pheochromocytoma (PC12)
cell line at doses of 10, 25, and 50 μg/mL (Choi et al., 2015).
It was found that the water extract of E. ciliata could inhibit cytokine
expression through NF-κB and p38 MAPK pathway (Kim et al., 2011).
p38 MAPK participates in the regulation of cell survival/death, is
important for cell invasion and migration, and its levels are usually
associated with short survival in cancer patients (Koul et al., 2013).
Thus, it is very important to study the mechanism of action of E. ciliata
active components. Anticancer activity may be possessed not only by
volatile compounds but maybe by polyphenols as well, which can be
extracted by different solvents. At this moment it is not easy to answer
the question which constituent is the most important for the anticancer
activity and at the same time is not very toxic. It could be that not one
substance only but the whole complex is responsible for this effect, as
different substances may have different mechanisms of action and they
may act synergistically (Bakkali et al., 2008).
5. Conclusion
Volatile compounds from E. ciliata herbal samples were extracted by
different extraction methods and then analyzed by GC–MS. The data
shows that E. ciliata grown in Lithuania is rich in volatile compounds
ketones. Dehydroelsholtzia ketone and elsholtzia ketone were predo-
minant compounds in all samples, and were obtained by using HD and
SPME. Sesquiterpenes are the second major group of compounds
identified in the essential oil obtained by using HD. The impact that
different extraction methods have on the yield of the essential oil from
the tested herbal materials requires a further research. The essential oil
from E. ciliata could be further investigated for application of its
constituents as anticancer substances.
Conflicts of interest
The authors declare that there are no competing interests regarding
the publication of this paper.
Acknowledgments
The authors would like to thank Open Access Centre for the
Advanced Pharmaceutical and Health Technologies (Lithuanian uni-
versity of Health Sciences) for providing the opportunity to use
infrastructure and perform this research and Instrumental Analysis
Open access research center of Vytautas Magnus University of Natural
Sciences.
Also, the authors want to thank Dr. Manel Esteller (Bellvitge
Biomedical Research Institute (IDIBELL), Spain) for kindly providing
the MDA-MB231, Panc-1 and U87 cell lines; and Dr. Ramunas Valiokas
(Center for Physical Sciences and Technology, Lithuania) for providing
human fibroblasts cell line.
References
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