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Article
Evaluation of Anticancer Activity of Satureja montana
Supercritical and Spray-Dried Extracts on Ehrlich’s
Ascites Carcinoma Bearing Mice
Jelena Vladi´c 1, Tatjana ´
Cebovi´c 2, *, Senka Vidovi´c 1and Stela Joki´c 3,*
1
Department of Biotechnology and Pharmaceutical Engineering, Faculty of Technology, University of Novi Sad,
Bulevar cara Lazara 1, 21000 Novi Sad, Serbia; vladicjelena@gmail.com or vladicj@uns.ac.rs (J.V.);
senka.vidovic@uns.ac.rs (S.V.)
2Department of Biochemistry, Faculty of Medicine, University of Novi Sad, Hajduk Veljkova 3,
21000 Novi Sad, Serbia
3Faculty of Food Technology Osijek, Josip Juraj Strossmayer University of Osijek, Franje Kuhaˇca 20,
31000 Osijek, Croatia
*
Correspondence: cebovictatjana@gmail.com or tatjana.cebovic@mf.uns.ac.rs (T. ´
C.); stela.jokic@ptfos.hr (S.J.)
Received: 1 October 2020; Accepted: 5 November 2020; Published: 10 November 2020
Abstract:
Satureja montana herbal species belongs to aromatic medicinal plants with a significant
place in traditional medicine. However, products produced with conventional procedures do not
meet the requirements of the modern market which include environmentally-safe processes that
provide quality, safe, and standardized products. In this study, the antiproliferative activity of
S. montana extracts obtained by supercritical carbon dioxide and solid–liquid extraction followed by
spray drying was investigated using the
in vivo
model of Ehrlich ascites carcinoma (EAC) in mice.
The impact of two concentrations of extracts on the growth of tumor and the redox status of malignant
cells was monitored. It was determined that the extracts induced oxidative stress in the malignant
cells which was confirmed by the changes in activity of biochemical indicators of oxidative stress.
The posttreatment was not an efficient approach, while the extracts applied as pretreatment and
treatment resulted in an increase in the xanthine oxidase (XOD) activity, a decrease in catalase (CAT)
activity, and an increase in the intensity of lipid peroxidation (LPx). Furthermore, a decrease in the
values of reduced glutathione (GSH) and an increase in glutathione reductase (GR) and glutathione
peroxidase (GSHPx) in EAC cells were recorded.
Keywords:
Satureja montana; winter savory; cytotoxic activity; Ehrlich ascites tumor; supercritical
carbon dioxide; spray drying; carvacrol
1. Introduction
Winter savory (Satureja montana L.) represents an aromatic plant from the Lamiaceae family.
The benefits of S. montana have long been recognized in traditional medicine which has established
the plant as a widespread folk remedy. Furthermore, winter savory is used as a spice. S. montana
is rich in essential oil with oxygen-containing phenolic monoterpenes carvacrol and thymol as its
most dominant components [
1
]. In addition to lipophilic fraction, winter savory possesses significant
bioactive components in its content which are hydrophilic in character, such as phenolic acids [2].
It was determined that the most dominant components of winter savory, isomers carvacrol and
thymol, exhibit numerous beneficial biological activities, such as antioxidative, antimicrobial, diuretic,
anti-HIV-1, antidiarrheal, and antiproliferative activities [
3
–
8
]. Very often, the biological activity of
herbal products is attributed to its most dominant components. However, the effect of the herbal
products due to the synergy of all the components present in the plant can be more significant than the
Plants 2020,9, 1532; doi:10.3390/plants9111532 www.mdpi.com/journal/plants
Plants 2020,9, 1532 2 of 14
activity of an individual component [
9
]. Moreover, the body’s response to the extract is not the same as
the response to individual components.
The induction of apoptosis of cancer cells without significant side effects is a characteristic of a
good chemoprotective agent [
10
]. Therefore, herbal extracts represent excellent candidates for research
in this area as potential non-toxic agents. Herbal polyphenols demonstrated to be significant potential
chemopreventive and chemotherapeutic agents considering that they can lead to the suppression
of malignant proliferation with different mechanisms. Additionally, they can have an effect on the
reduction of consequences of oxidative stress. For certain herbal species that belong to the Satureja genus,
it was determined that they exhibit antiproliferative activity. Examples of those species are: Satureja
thymbra and Satureja parnassica [
11
], Satureja bakhtiarica [
12
], and Satureja khuzistanica [
13
]. Furthermore,
the antitumor activity of carvacrol was demonstrated in numerous studies [
14
–
18
]. Additionally, it was
found that S. montana extracts exhibit antiproliferative activity on cancer cells [
19
,
20
]. With regard
to investigating the activity of S. montana extracts on an
in vivo
model however, no study has yet
been conducted.
Furthermore, the demand for natural products which is, along with market competition, constantly
on the rise, dictates the demand for introducing new, modern, efficient, and green procedures which are
to provide a stable, quality, and safe product. Modern production processes have to meet the following
requirements of the modern market: production without the use of toxic solvents, extended stability
of products, optimized chemical composition with efficient use of natural resources, reduced use
of energy, higher process safety, and reduction of waste. The attainment of winter savory extracts
was investigated by applying different modern green techniques and alternative solvents including
supercritical and subcritical fluids [
21
–
25
], eutectic solvents [
26
], microwave-assisted extraction,
and hydrodistillation [27,28].
For the aforementioned reasons, this study investigated the antiproliferative activities of S. montana
extracts obtained by applying environmentally-friendly techniques and using the model of induced
Ehrlich ascites carcinoma (EAC) in mice. Considering that winter savory possesses significant bioactive
components that are polar and non-polar in character, lipophilic extracts obtained by supercritical
carbon-dioxide extraction (Sc-CO
2
) and extracts with a more polar character obtained by solid–liquid
extraction with ethanol followed by the spray drying process were investigated. To determine the
potential of extracts, parameters of tumor growth and the antioxidant status of malignant cells were
monitored. In addition, the impact of extracts before, at the time of, and after the implantation of EAC
cells was monitored.
2. Results
2.1. Impact of S. montana Extracts on the Volume of Ascites, Viability, and Number of Cells
In order to determine the activity of S. montana extracts on the parameters of tumor growth,
the changes in cell parameters were monitored—ascites volume, number of tumor cells, and tumor
cells variability (Figure 1; Tables A1–A3). The impact of spray-dried (SD) extract and extract obtained
by Sc-CO
2
(SC extract) was investigated in two concentrations (1% (SD1 and SC1) and 5% (SD5 and
SC5)) and different impacts of the extracts on the volume of ascites were recorded (Figure 1a; Table A1).
After the application of extracts in the pretreatment (both extracts and both concentrations), there was
a significant decrease in the volume of ascites compared to the EAC control group. Moreover, the most
significant decrease was recorded in the group that was pretreated with SC5. During treatment,
there was also a significant decrease in the volume of the ascites when all extracts were applied.
In addition, the most efficient decrease among the treated groups was recorded in a group in which
SD5 was applied. In the posttreatment, a reduced volume was only recorded in a group in which SD1
was applied, while in all other groups an increase in the volume of ascites compared to the EAC group
was recorded.
Plants 2020,9, 1532 3 of 14
Figure 1.
Impact of S. montana extracts on (
a
) Ehrlich ascites carcinoma (EAC) ascites volume, (
b
) EAC
cells viability, and (c) EAC cells number (SD—spray-dried extract; SC—extract obtained by Sc-CO2).
The groups which were pretreatedand post-treated with extracts, a significant change in variability
was not recorded (Figure 1b; Table A2). In the groups of animals that were treated, a statistically
significant decrease of cell variability was recorded only after the treatment with the SD1 extract,
while in all other treated groups of animals, there was a statistically insignificant decrease in variability.
The impact on the number of cells compared to the EAC control group was statistically significant
only in groups of animals that were pretreated with SD and an increase in the number of cells was
determined. In the treatment with SD1, the same effect was recorded—a significant increase in
Plants 2020,9, 1532 4 of 14
the number of cells, while the administration of SC extracts led to a mild reduction (statistically
insignificant) in the number of malignant cells. In the post-treated groups, there was an increase
recorded after the application of all extracts (Figure 1c; Table A3).
2.2. Impact of S. montana Extracts on the Antioxidant Status Malign Cells of EAC
The changes in the antioxidative status of EAC cells were monitored by measuring the activity of
antioxidative enzymes (XOD, CAT, Px, GSHPx, and GR) and the quantity of GSH and the intensity of
LPx in EAC cells. The obtained results were compared with the control group, that is, with the results
of measurements of the same parameters in EAC cells (Table 1).
Table 1. Impact of S. montana extracts on biochemical parameters in EAC cells.
Parameter Group EAC SD1 SD5 SC1 SC5
XOD
Pretreatment
0.156 ±0.009
0.142 ±0.004 b0.151 ±0.005 b0.807 ±0.138 a,* 0.822 ±0.057 *,a
Treatment 0.104 ±0.011 c,*0.836 ±0.069 b,*1.035 ±0.064 a,* 1.012 ±0.014 a,*
Posttreatment 0.220 ±0.014 a,*0.102 ±0.005 b,* 0.097 ±0.012 b,* 0.101 ±0.004 b, *
CAT
Pretreatment
0.503 ±0.013
0.585 ±0.019 a,*0.429 ±0.002 b,*0.233 ±0.016 c,*0.136 ±0.002 d*
Treatment 0.636 ±0.027 a,* 0.138 ±0.002 c,*0.194 ±0.006 b,* 0.191 ±0.003 b,*
Post-treatment 0.316 ±0.008 b,*0.698 ±0.052 a,* 0.713 ±0.002 a,* 0.742 ±0.041 a,*
Px
Pretreatment
0.325 ±0.023
0.302 ±0.016 c0.432 ±0.010 c,*0.618 ±0.003 b,*0.881 ±0.019 a,*
Treatment 0.120 ±0.006 c,* 0.875 ±0.035 a,*0.715 ±0.004 b, * 0.710 ±0.002 b,*
Post-treatment 0.474 ±0.035 a,*0.100 ±0.008 b,* 0.114 ±0.023 b,* 0.118 ±0.007 b, *
GR
Pretreatment
2.187 ±0.107
2.115 ±0.165 b3.012 ±0.105 b,*4.957 ±0.135 a,* 5.707 ±0.667 a,*
Treatment 1.202 ±0.095 b,*5.627 ±0.363 a, * 5.172 ±0.111 a, * 5.195 ±0.024 a,*
Post-treatment 3.930 ±0.217 a,*1.195 ±0.002 b,* 1.254 ±0.028 b,* 1.284 ±0.185 b, *
GSHPx
Pretreatment
0.779 ±0.048
0.796 ±0.024 d0.933 ±0.022 c,*1.302 ±0.049 b,*1.697 ±0.038 a,*
Treatment 0.474 ±0.034 b,*1.91 ±0.345 a, * 2.043 ±0.020 a,* 2.007 ±0.031 a,*
Post-treatment 1.131 ±0.151 a,*0.395 ±0.065 b,* 0.401 ±0.068 b,* 0.421 ±0.005 b, *
GSH
Pretreatment
1.603 ±0.110
1.417 ±0.056 a,* 1.358 ±0.029 a,*0.871 ±0.033 b,* 0.884 ±0.043 b,*
Treatment 1.513 ±0.058 a,*0.926 ±0.069 b,*0.621 ±0.011 c, * 0.635 ±0.005 c,*
Post-treatment 1.105 ±0.063 b,*1.801 ±0.045 a,* 1.799 ±0.029 a,* 1.842 ±0.003 a,*
LPx
Pretreatment
0.032 ±0.008
0.030 ±0.009 c0.041 ±0.003 c0.100 ±0.007 a,*0.080 ±0.005 b,*
Treatment 0.022 ±0.005 c0.042 ±0.521 b0.077 ±0.005 a0.083 ±0.005 a
Post-treatment 0.056 ±0.018 a,*0.011 ±0.006 b,* 0.019 ±0.006 b,* 0.014 ±0.001 b, *
Results are presented as a mean value
±
SD from six mice. *—statistically significant difference compared to the
EAC control group; different letters within a row indicate a significant difference between the samples in the same
group at p<0.05.
In the control group, the XOD activity in EAC cells was low, and by applying the SC extracts
in pretreatments, there was a significant increase in the XOD activity in malignant cells, without a
difference in their impact on this parameter (SC1 and SC5). On the other hand, the application of
SD extracts did not cause any significant changes in the intensity of XOD. In the groups treated with
extracts, significant changes in the activity of enzymes were determined where SC extracts intensified
the XOD activity in EAC cells, as well as in the treatment with SD5. Furthermore, the treatment with a
lower concentration of SD significantly decreased the activity of XOD. The application of extracts in
post-treatments caused significant changes in all groups (except in the posttreatment with SD1)—a
decrease in XOD activity compared to the EAC group, while the post-treatment with SD1 caused an
increase in the XOD activity.
Compared to the activity of CAT in the EAC control group, the application of extracts resulted in
a different impact on the activity of this enzyme. In the groups pretreated with extracts, the activity of
CAT in EAC cells was significantly increased with the application of SD1, while it was significantly
decreased in other pretreated groups. Moreover, the highest decrease was achieved when SC5 was
applied in the pretreatment. The treatment with extracts in all animal groups caused significant
changes in the CAT activity. In the pretreatment, the application of SD1 increased the CAT activity,
while all other extracts caused a decrease in the activity of CAT of malignant cells. The application
Plants 2020,9, 1532 5 of 14
of extracts in post-treatments in all groups caused significant changes compared to the EAC control
group in terms of decreasing the CAT activity with posttreatment with SD1, and an increase in the
remaining three groups of post-treated mice.
The activity of Px in malignant cells compared to the control EAC group increased after the
application of SC extracts and SD5 in pretreatments. The treatment with SD1 resulted in a decrease
in the activity of Px, and an increase was determined in the treatments with SD5, SC1, and SC5.
In post-treated groups, the SD1 extract caused changes in the intensity through the increase in the Px
activity, while SD5 and SC decreased the activity of Px in EAC cells.
The activity of the GR enzyme in tumor cells was significantly different after the application
of extracts. SC extracts and SD5 in the pretreated groups caused an increase in the activity of GR.
Moreover, the highest increase was recorded in the group of mice pretreated with SC5. By starting
therapy at the moment of tumor implantation, there were significant changes in the activity of enzymes
in EAC cells. The treatment with SC and SD5 resulted in an increase, while treating mice with SD1
resulted in a decrease in the activity in the GR in EAC cells. After the application of SD1 in post-treated
groups of animals, an increase was recorded, while the post-treatment with other extracts resulted in a
decrease in the activity of GR.
The GSPHx activity in EAC cells when extracts SD5 and SC were applied prior to the implantation
of tumor, increased compared to the control group. The impact of extracts that were applied as a
treatment was also significant. It was determined that there was an increase in this activity in all
treated groups (SC1, SC5, and SD5), except for the group treated with SD1 where the GSHPx activity
was significantly decreased. The post-treatment of animals with SC1, SC5, and SD5 extracts caused a
decrease in the GSPHx activity, and an increase with the application of SD1.
Compared to the EAC control group, the amount of GSH was reduced in all groups that were
pretreated. Moreover, a significant reduction in GSH was recorded in groups in which SC extracts were
applied. Furthermore, animal treatment with extracts also resulted in a decrease in the amount of GSH,
and SC extracts in the treatment also led to a significant reduction in the amount of GSH compared
to SD extracts. In post-treatments, a reduction was recorded only in the case of SD1, while with SC
extracts and SD5, an increase in the amount of GSG was determined.
The intensity of the lipid peroxidation in the group with EAC was low. In the application of
extracts in the pretreatment, only SC extracts led to a significant increase in the intensity of the lipid
peroxidation. The treatment with extracts did not have a statistically significant impact on the changes
in intensity of LPx. By post-treating mice with extracts, the changes in the intensity of LPx were
significant, so the post-treatment with SD1 resulted in an increase, while others caused a decrease in
the intensity of LPx.
3. Discussion
The study investigated the antiproliferative activity of S. montana extracts obtained by different
techniques of extraction. Modern extraction techniques were selected which, once the process is
established, can easily be scaled-up to an industrial level and provide high-quality extracts while
protecting the environment. The Sc-CO
2
extraction represents the method of choice for the isolation of
lipophilic components [
29
]. The spray drying process has proved to be the superior and efficient method
because it is multiple times cheaper compared to freeze drying and provides extracts of extended
stability with the possibility of concealing unpleasant odors and tastes. In addition, the smaller
volume of extracts compared to liquid ones further contributes to lower expenses of storage and
transport. Furthermore, dry extracts are characterized by instantaneous solubility and they can easily
be incorporated in other products like pills and tablets and instant teas [30,31].
In the previously published studies by our research group, the procedures for obtaining
extracts of winter savory with optimal physical–chemical characteristics were established [
32
,
33
].
Sc-CO
2
(
350 bar and 50 ◦C
) was applied to obtain extracts that are lipophilic in character and contained
the highest content of carvacrol [
32
]. In addition, the mixture water/ethanol was used for the extraction
Plants 2020,9, 1532 6 of 14
of predominantly polar components and spray drying was applied further to obtain powder [
33
].
The goal was to evaluate the antitumor activity of extracts of winter savory of polar and non-polar
character to identify the potential mechanism of activity of extracts. Furthermore, to determine whether
carvacrol as the most dominant component in winter savory is responsible for the manifestation of the
activity, essential oil was isolated from powder and its chemical profile was determined [33].
Considering that the Sc-CO
2
is a solvent that extracts only non-polar components while the
mixture water/ethanol shows an affinity toward more polar components, the content of carvacrol in
supercritical extract was expectedly multiple times higher compared to the powder (
SC 60.82 g/100 g
;
SD 902.52 mg/100 g), according to the GC-FID (gas chromatography with flame-ionization detection)
analysis [
32
,
33
]. Furthermore, the presence of other less represented compounds was recorded
in essential oil isolated from the S. montana powder and supercritical extract, such as p-cymene,
trans-caryophyllene, caryophyllene oxide, linalool, and terpinen 4-ol (Table 2). The presence of
lipophilic components in powder was not dominant and a higher presence of polyphenols was
expected. Polyphenolic components that were identified in the extracts of winter savory are caffeic,
syringic, rosmarinic, gallic, ferulic, cinnamic, syringic, gentisic, ferulic, and vanillic acids, as well as
luteolin, rutin, epicatechin, catechin, and quercetin [34,35].
Table 2.
GC/MS analysis of extract obtained by Sc-CO
2
((SC) extract) and essential oil isolated from
spray-dried (SD) extract (relative percentage; %) [32,33].
Compound SC SD
α-Terpinene 0.15 n.i.
p-Cymene 3.96 0.36
γ-Terpinene 0.80 n.i.
α-Terpineol n.i. 0.21
Eucalyptol 0.42 n.i.
Trans-sabinene hydrate 0.33 n.i.
Cis-sabinene hydrate 0.15 n.i.
Linalool 0.29 0.18
Borneol 1.56 n.i.
Terpinen 4-ol 0.84 0.42
Carvacrol 78.61 71.82
Trans-caryophyllene 2.40 0.24
Caryophyllene oxide 1.26 1.31
α-Amorphen 0.46 n.i.
β-Bisabolene 0.74 n.i.
γ-Cadinene 0.53 n.i.
δ-Cadinene 0.78 n.i.
β-Cadinene n.i. 0.24
Spatulenol n.i. 0.21
Heptakosane 0.17 n.i.
Nonakosane 0.19 n.i.
n.i. not identified.
The antioxidative activity of extracts was investigated by using the DPPH (2,2-diphenyl-1-
picryl-hydrazyl-hydrate) assay. By comparing the manifested activities, expressed as the IC
50
value;
that is, the concentration with which 50% of free radicals is inhibited, the dry extract showed a stronger
antioxidative activity (SC 17.40
µ
g/mL; SD 5.24
µ
g/mL) [
33
,
36
]. The extract with the more dominant
percentage of carvacrol did not exhibit a stronger antioxidative activity. An explanation for this
can be that carvacrol is not the main carrier of antioxidative activities in the extract. Additionally,
Serrano et al.
[
37
] reported a stronger antioxidant activity of ethanolic and aqueous extracts compared
to essential oil. A reason they state the presence of a higher content of phenolic acids, such as rosmarinic
and others, is that, because of their hydroxyl group, they have a strong capability of capturing free
Plants 2020,9, 1532 7 of 14
radicals. Further, it is suggested that the presence of sesquiterpenes, which generally have a lower
antioxidative capacity, contributes to the lower antioxidative activity of oils [37].
In the studies which in their focus have the investigation of antitumor potential of S. montana
extracts, the precise mechanism responsible for the antitumor activity has not been determined.
The antiproliferative activity of different extracts of S. montana was investigated on HeLa (human cervix
adenocarcinoma) [
19
,
20
], HT-29 (human colon adenocarcinoma) [
20
], and MCF-7 (human breast
adenocarcinoma) [
20
], K562 (human chronic myelogenous leukemia cells [
19
], and MDA-MB-453
(breast cancer cells) [
19
]. A strong antioxidative capacity of S. montana extracts was confirmed along
with their antiproliferative properties that were different depending on the type of extract. The authors
made an assumption that S. montana as a strong antioxidant can impact the redox condition of cells
which leads to decreased cell proliferation. A low level of free oxygen species is necessary to promote
cell proliferation and redox changes have a significant role in the signal traductional pathway, which is
important for the regulation of growth of cells [
20
]. Additionally, good selectivity with respect to
activity was determined, especially towards K562 cells in comparison to normal MRC-5 human
fibroblasts [
19
]. It was suggested that cytotoxicity is not the only thing responsible for the manifestation
of the antitumor activity, but also the possible prenylation of proteins including Ras [
17
], as well as the
antioxidant nature of carvacrol [38].
Furthermore, carvacrol is a confirmed antitumor agent. It was demonstrated that supplementation
with carvacrol exhibits the antitumor effect on liver cancer induced with diethylnitrosamine in Wistar
albino rats, most likely protecting the antioxidant defense system and preventing lipid peroxidation
and damage of liver cells [
18
]. Recent studies demonstrate that the antiproliferative effect of carvacrol
on metastatic cells of breast cancer (MDA-MB 231) is based on the activation of classic responses that
belong to the mitochondrial pathway of apoptosis [
39
]. In addition, it was determined that carvacrol
can induce apoptosis in cell lines of the hepatocellular carcinoma and the results suggest that the
induction of apoptosis can be performed through direct activation of the mitochondrial pathway,
and mitogen-activated protein kinase can have a significant role the antitumor effect of carvacrol [
40
].
Ascites fluid is highly important for the development of tumor considering that it represents
a source of food for its cells; therefore, the increase in volume of ascites is a significant indicator of
proliferation of tumor cells [
41
]. It was determined that the application of extracts leads to a decrease
in volume of ascites in groups of mice, but not in the number of cells. Therefore, it can be assumed that
in the decreased volume of ascites there is a concentrated higher number of cells and that extracts do
not exhibit an impact in that respect; that is, they do not lead to the death of cells nor the inhibition of
cell growth. Hence, they do not exhibit cytotoxic nor cytostatic activity with regard to malignant cells.
The potential oncostatic effect (statistically insignificant) was exhibited only by SC (SC1 and SC5) and
SD5 extracts applied as treatment.
It can be noted that SC extracts applied as pretreatment and treatment exhibited a more dominant
effect on SD. The content of carvacrol in SC extracts is significantly higher compared to SD; hence, that
could be the reason for SC extracts’ higher impact on the antioxidant status of EAC cells. Furthermore,
when they were applied as treatment, SC extracts resulted in the reduction in the volume of ascites
(statistically insignificant) and the decrease in malignant cells. On the other hand, SC extracts applied
as pretreatment did not have an impact on the decrease of number of malignant cells so they cannot be
considered as cytotoxic or cytostatic agents.
In the groups of animals that were post-treated with SC1, SC5, and SD5, it is possible that there
was an occurrence of a higher level of development of the implanted tumor before the application of
extract. Based on the results of the activity of enzymes (XOD, CAT, Px, GSHPx, and GR), the amount of
GSH, and intensity of LPx, as well as the results of the volume of ascites and the number of cells in the
mentioned groups of animals, it can be concluded that post-treatment of animals is not an adequate
therapeutic approach for EAC. Therefore, the time of the application of extracts is of high importance.
During the development of tumor, the cells become resistant due to constant exposure to oxidative
stress and they develop strong mechanisms of antioxidative protection [
42
,
43
]. The intensity of LPx,
Plants 2020,9, 1532 8 of 14
as well as the XOD and CAT activities in EAC cells was low. However, after the application of certain
extracts, it was observed that there was an increase in the XOD activity, decrease in the CAT activity,
and an increase in the intensity of LPx. Moreover, it was recorded that there was a decrease in the
values of GSH in EAC cells in all groups of examined animals (except for groups post-treated with
SC1, SC5, and SD5), as well as an increase in GR and GSHPx in malignant cells after the application of
S. montana extract. These enzymes represent markers of oxidative stress and their increased activity
suggests that due to the application of certain extracts, malignant cells were exposed to oxidative stress.
As a result, it was determined that extracts of S. montana induce the production of reactive oxygen
species in malignant EAC cells. However, the exact mechanism of activity was not clarified and further
research is necessary.
Additionally, Table 3contains values of biochemical parameters of oxidative stress measured in
the control group that was treated with distillated water and in groups that were treated only with SC1
and SD1 extracts [
36
]. By comparing these values with the parameters of oxidative stress determined
in this study, it can be concluded that extracts exhibit selectivity towards tumor cells by inducing them
into oxidative stress, while not manifesting any side-effects on the healthy cells.
Table 3.
Biochemical parameters ofcontrol group and the groups treated withthe SD1 and SC1 extracts [
36
].
Group XOD CAT Px GR GSH-Px GSH LPx
Control 1.93 ±0.02 a9.56 ±0.37 b10.68 ±1.38 b6.13 ±0.07 a8.70 ±0.38 a4.98 ±0.16 a3.04 ±0.07 a
SC 1.87 ±0.03 ab 13.4 ±0.59 a14.88 ±0.38 a7.03 ±0.46 a7.54 ±2.23 a4.99 ±0.17 a2.62 ±0.54 a
SD 1.86 ±0.03 b12.37 ±0.18 a14.57 ±0.80 a6.53 ±1.61 a6.67 ±1.06 a4.92 ±0.39 a2.21 ±0.24 a
Values are expressed as mean
±
standard deviation for six mice. Activities of xanthine oxidase (XOD), catalase
(CAT), peroxidase (Px), glutathione reductase (GR) and glutathione peroxidase (GSHPx) are expressed in nmol/mg
of protein min-1. Content of hepatic reduced glutathione (GSH) is expressed in nmolGSH/mg of protein. Intensity
of lipid peroxidation (LPx) is expressed in nmol/MDA/mg of protein; MDA, malonyldialdehyde. Different letters
within a column indicate a significant difference between the samples at p<0.05.
4. Materials and Methods
4.1. Plant Material
Aerial parts of winter savory (Satureja montana) were collected at the Institute of Field and
Vegetable Crops, Backi Petrovac, Republic of Serbia, in July 2012. The collected plant material was
naturally dried and then stored in paper bags at room temperature.
4.2. Preparation of Extracts
The detailed procedure of obtaining S. montana extract (SC) by Sc-CO
2
extraction was described by
Vladi´c et al. [
32
]. Briefly, the extraction of S. montana herbal material was conducted using Sc-CO
2
at a
pressure of 350 bar and a temperature of 50
◦
C. The extraction time was 4.5 h. The separator conditions
were 15 bar and 23 ◦C [32].
The detailed procedure of obtaining S. montana extracts via spray drying is described by
Vidovi´c et al.
[
33
]. Spray-dried extract (SD) is produced by drying liquid extracts via spray drying
(inlet temperature 120
◦
C, outlet temperature 80
◦
C). S. montana liquid extracts were obtained using a
50% ethanol mixture as an extraction solvent. The extraction was carried out for five days at room
temperature in a dark place. Maltodextrin (DE16) in the percentage of 10% (calculated on extract dry
weight) was used as a carrier material [33].
4.3. Chemical Analysis
Gas chromatography–mass spectrometry (GC/MS) and gas chromatography with flame-ionization
detection (GC/FID) analyses were performed according to the procedures described in previous
studies
[32,33]
. GC analysis was performed on an Agilent GC6890N system coupled with a mass
spectrometer model Agilent MS 5795. An HP-5MS column (30 m length, 0.25 mm inner diameter,
and 0.25
µ
m film thickness) was used. Essential oil was isolated from SD powder using the
Plants 2020,9, 1532 9 of 14
hydrodistillation procedure according to the European Pharmacopoeia (Clevenger-type apparatus)
and its chemical profile was determined. The injected volume of the sample solution (SD in methylene
chloride and SC in methanol) was 5
µ
L with a split ratio of 30:1. Aromatic compounds were identified
using the NIST 05 and the Wiley 7n mass database. The GC/MS operating conditions were as follows:
injector temperature 250
◦
C, temperature program 60–150
◦
C (4
◦
C/min), carrier gas He with flow
rate 2 mL/min. Quantification of carvacrol was performed with an FID detector and the calibration
curve for carvacrol. The GC/FID operating conditions were: injector temperature 250
◦
C, temperature
program 60–150 ◦C (4 ◦C/min), and detector temperature 300 ◦C.
4.4. Antioxidant Activity
The antioxidant activity of extracts was analyzed using the DPPH assay [
44
]. Different volumes
of extracts were mixed with 95% and 90
µ
M DPPH solution. After the 60-min incubation at room
temperature, absorption was measured at a wavelength of 515 nm. The antioxidant activity was
expressed as IC
50
value which represents the concentration of the extract which inhibits 50% DPPH
radicals. All the measurements were performed in triplicate.
4.5. Animals and Treatments
Animal care and all experimental procedures were conducted in accordance with the Guide
for the Care and Use of Laboratory Animal Resources edited by the Commission of Life Sciences,
National Research Council. All procedures performed in the studies involving animals were in
accordance with the ethical standards of the institution (University of Novi Sad; EK: II-2013-03;
01-160-5). For the purposes of this research, female mice of the Hannover National Medicinal Institute
(Hann: NMRI) strain were used, aged 6–8 weeks, weighing 25 g
±
10%. Experimental animals were
obtained from the laboratory of the Independent Department for Biochemistry, Laboratory Medicine
Center, Clinical Center of Vojvodina (Novi Sad, Serbia). The animals were kept under strictly monitored
conditions (temperature 25
◦
C, air humidity 30–50%, 12 h light/day cycles) in adequate cages and
without limitations to access to food (LM2 with 19% protein, Veterinary Institute Subotica, Serbia)
and water.
Extract SC was dissolved in olive oil in a concentration of 1% (SC1) and 5% (SC5). The SD
was dissolved under sonication in water (1% (SD1) and 5% (SD5)), and filtered through A 0.45 mm
membrane filter. The resulting solutions were kept refrigerated at a temperature of 4
◦
C. The extracts
were administered intraperitoneally (i.p.).
The variability in the volume of administered doses was managed by adjusting the concentration
to ensure a constant volume (2 mL/kg body weight).
During experimental work, mice were divided into groups of 6 mice through random selection
and were treated in accordance with the following protocol:
•
Group EAC—animals with implanted Ehrlich ascites carcinoma (EAC) cells treated with 2 mL/kg
of saline, i.p. (n =6).
•
Group PRETREATMENT—animals pretreated with 2 mL/kg of the investigated extract, i.p. during
seven days (n =6).
•
Group TREATMENT—animals were treated with 2 mL/kg of the investigated extract, i.p. seven
days after the implantation of EAC (n =6).
•
Group POSTTREATMENT—animals post-treated with 2 mL/kg of the investigated extract,
i.p. during seven days, seven days after implantation (n =6).
•
After 14 days from the day of implementation of EAC, all animals were sacrificed and the ascites
was collected for further biochemical analyses.
Plants 2020,9, 1532 10 of 14
4.6. Determination of Ascites Volume, Tumor Cell Number, and Cell Viability
The ascites from the abdomen was transferred to Krebs–Ringer phosphate buffer solution
(
0◦C, pH 7.4
) and subjected to sequential centrifuging at 4500 rpm (MSE HIGH SPEED, 4
◦
C) and
12,000 rpm (Eppendorf 3200, 2.5 min) to obtain a dense cell suspension (1:1). The cell number was
counted in a Neubauer chamber and expressed as number of cells/mm
3
. Cell viability was determined
by the Trypan blue exclusion method and expressed as percentage of damaged cells.
4.7. Biochemical Assays
The samples were diluted with Krebs–Ringer phosphate buffer and the activities of antioxidant
enzymes were determined in EAC cells by standard laboratory protocols. The activity of xanthine
oxidase (XOD) was determined using the Bergmayer method [
45
], catalase (CAT) according to Beers and
Sizer [
46
], peroxidase (Px) according to Simon et al. [
47
], glutathione peroxidase (GSHPx) according to
Beutler et al. [
48
], and glutathione reductase (GR) according to Goldberg and Spooner [
49
]. The amount
of reduced glutathione (GSH) was determined according to Beutler [
50
], as well as the intensity of lipid
peroxidation (LPx) using the Buege and Aust protocol [
51
]. All the measurements were performed
in triplicate.
4.8. Statistical Analysis
The activities are expressed as mean
±
standard deviation. Mean values between the groups
in the biochemical analyses were considered significantly different at the p<0.05 confidence level,
after performing a one-way single factor ANOVA, followed by Tukey and Bonferroni multiple
comparison post hoc tests.
5. Conclusions
This study investigated the antiproliferative activity of supercritical and spray dried extracts of
S. montana by using
in vivo
mice model. The impact of extracts on malignant cells was observed by
applying the extracts as pretreatment, treatment, and posttreatment. It was determined that the time of
application of extracts represents a significant parameter in the treatment of EAC cells. The assumption
is that there was a significant development of tumors in the groups in which extracts were applied
as posttreatment and that the application of extracts did not demonstrate to be an efficient approach.
On the other hand, the extracts that were applied as pretreatment and treatment exhibited a far more
significant effect on malign cells. The changes in the activities of biochemical parameters that represent
markers of oxidative stress indicate that the malignant were exposed to oxidative stress when extracts
were applied. Therefore, the results suggest that the extracts resulted in the generation of reactive
oxygen species in malignant EAC cells.
The achieved results indicate the potential of S. montana in the prevention and treatment of
malignant diseases. Furthermore, the results point to the necessity of the application of adequate
environmentally-friendly procedures for the attainment of extracts that exhibit optimal effects and
are safe at the same time. It is necessary to direct further investigation towards determining a precise
mechanism of activity of extracts and their pharmacokinetic properties.
Author Contributions:
Conceptualization, J.V. and S.V.; methodology, T.´
C.; software, J.V.; validation, T. ´
C., S.J.,
and J.V.; formal analysis, T. ´
C. and J.V.; investigation, J.V. and S.V.; resources, T.´
C. and S.V.; data curation, S.J.;
writing—original draft preparation, J.V.; writing—review and editing, J.V., T. ´
C., S.J.; visualization, S.V.; supervision,
T. ´
C.; project administration, S.V.; funding acquisition, S.J. and S.V. All authors have read and agreed to the
published version of the manuscript.
Funding:
This research was funded by Serbian Ministry of Education and Science; Project
No. 451-03-68/2020-14/200134.
Acknowledgments: The authors would like to thank Ana Jovanoski for her technical and editorial support.
Conflicts of Interest: The authors declare no conflict of interest.
Plants 2020,9, 1532 11 of 14
Appendix A
Table A1. Impact of S. montana extracts on the volume of ascites.
Group EAC Ascites Volume (mL)
EAC Control 7.117 ±0.458
Extract SD1 SD5 SC1 SC5
Pretreatment 2.267 ±1.178 a,* 3.283 ±1.301 a,* 2.267 ±0.674 a,* 1.000 ±0.469 a,*
Treatment 2.167 ±0.288 ab,* 0.817 ±0.147 b,*2.933 ±1.147 a,*2.267 ±1.065 ab,*
Post-treatment 3.083 ±0.382 b,*9.657 ±0.359 a,* 9.851 ±0.932 a,* 9.622 ±0.954 a,*
Results are presented as a mean value
±
SD from six mice. *—statistically significant difference compared to the
EAC control group; different letters within a row indicate a significant difference between the samples in the same
group at p<0.05.
Table A2. Impact of S. montana extracts on the viability of EAC cells.
Group EAC Cells Viability (%)
EAC Control 7.243 ±1.425
Extract SD1 SD5 SC1 SC5
Pretreatment 7.183 ±1.705 a6.267 ±0.965 a5.967 ±0.339 a6.250 ±1.205 a
Treatment 4.817 ±1.042 a,* 6.550 ±0.509 a6.733 ±0.857 a6.200 ±1.131 a
Post-treatment 6.533 ±0.476 a7.650 ±0.874 a7.499 ±0.356 a7.586 ±1.023 a
Results are presented as a mean value
±
SD from six mice. *—statistically significant difference compared to the
EAC control group; different letters within a row indicate a significant difference between the samples in the same
group at p<0.05.
Table A3. Impact of S. montana extracts on the number of EAC cells.
Group EAC Cells Number/mm3
EAC Control 119 583.333 ±6 755.862
Extract SD1 SD5 SC1 SC5
Pretreatment 196,583.333
±17,133.058 a,*
158,833.333
±9739.952 ab,*
147,000.000
±22,858.259 b125,583.333
±21,731.122 b
Treatment 247,000.000
±44,340.730 a,*
114,333.300
±1505.545 b112,166.700
±16,898.720 b108,000.000
±17,378.150 b
Post-treatment 141,833.333
±24,717.740 b,*
346,569.58
±34,156.81 a,*
374,511.22
±11,568.13 a,*
361,581.47
±8455.36 a,*
Results are presented as a mean value
±
SD from six mice. *—statistically significant difference compared to the
EAC control group; different letters within a row indicate a significant difference between the samples in the same
group at p<0.05.
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