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Inula viscosa (L.) Aiton Ethanolic Extract Inhibits the Growth of
Human AGS and A549 Cancer Cell Lines
Habiba Rechek,a, b, c Ammar Haouat,d, e Kaouther Hamaidia,*a, f Diana C. G. A. Pinto,*c
Tarek Boudiar,gMónica S. G. A. Válega,cDavid M. Pereira,hRenato B. Pereira,hand
Artur M. S. Silva*c
a
Faculty of Sciences of Nature and Life, Mohamed Cherif Messaadia University, Souk Ahras, 41000 Souk-Ahras,
Algeria, e-mail: k.hemaidia@univ-soukahras.dz
b
Department of Biology of Organisms, Faculty of Sciences of Nature and Life, University of Batna 2, Mostefa Ben
Boulaid, 05078 Batna, Algeria
c
LAQV-REQUIMTE & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal,
e-mail: diana@ua.pt; artur.silva@ua.pt
d
Unité de Valorisation des Ressources Naturelles, Molécules Bioactives et Analyse Physicochimiques et Biolo-
giques (VARENBIOMOL), Université des Frères Mentouri, 25000 Constantine, Algeria
e
Department of Biology, Faculty of Sciences of Nature and Life, University of Oued Souf, 39 000, Oued Souf,
Algeria
f
Laboratory of Applied Animal Biology, Badji Mokhtar University, 23000 Annaba, Algeria
g
Center de Recherche en Biotechnologie, Ali Mendjli Nouvelle Ville UV 03, BP E73 Constantine, Algeria
h
REQUIMTE/LAQV, Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Uni-
versidade do Porto, R. Jorge Viterbo Ferreira, n°228, 4050-313 Porto, Portugal
© 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AG. This is an open access article under the terms of the
Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the
original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
The present study shows the chemical profile and cytotoxic properties of the ethanolic extracts of Inula
viscosa from Northeast Algeria. The extract was obtained by maceration using ethanol. Its phenolic profile was
determined using ultra-high-performance liquid chromatography coupled with a diode array detector and an
electrospray mass spectrometer (UHPLC-DAD-ESI/MS), which allowed the identification and quantification of 17
compounds, 1,5-O-caffeoylquinic acid being the most abundant. The cytotoxic activity was assessed against
human gastric cancer (AGS) and human non-small-cell lung cancer (A549) cell lines, whereas ethanolic extract
elicited nearly 60% and 40% viability loss toward AGS and A549 cancer cells, respectively. Results also showed
that cell death is caspase-independent and confirmed the involvement of RIPK1 and the necroptosis pathway in
the toxicity induced by the I. viscosa extract. In addition, the ethanolic extract would not provoke morphological
traits in the cancer cells. These findings suggest that I. viscosa can be a source of new antiproliferative drugs or
used in preparation plant-derived pharmaceuticals.
Keywords: LC/MS analysis, cytotoxic activity, AGS and A549 cell lines, RIPK1, caspases, necroptosis.
Introduction
Treating cancer is considered one of the most
challenging problems in medicine. In addition to
conventional cancer treatments, patients increasingly
resort to alternative and/or complementary treatments
focusing in particular on the use of medicinal
plants.[1–3] This practice is widespread in developing
countries due to its low cost, easy access to these
plants, and, above all, the concern about the harmful
effects of synthesized drugs.
Supporting information for this article is available on the
WWW under https://doi.org/10.1002/cbdv.202200890
doi.org/10.1002/cbdv.202200890 RESEARCH ARTICLE
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Previous studies have shown that the consumption
of certain plants can promote chemopreventive and
antineoplastic actions.[3–6] To improve cancer patients’
survival and quality of life, research aiming at identify-
ing new substances with anti-cancer properties has
been steadily increasing in recent years.
Medicinal plants contain compounds that can be
used either for therapeutic purposes or as precursors
for pharmaceutical chemical synthesis.[7–9] Inula visco-
sa (L.) Aiton (currently accepted name Dittrichia viscosa
subsp. viscosa (L.) Greuter)[10] belongs to the Aster-
aceae family found in the Mediterranean, Africa, Asia,
and Europe.[11] It is known for its traditional use in
treating several diseases, such as anti-inflammatory,
astringent, vulnerary, antipyretic, antiseptic, and antie-
metic properties. It is also used to treat gastrointestinal
disorders and skin diseases.[12–17] Previous works
reported some interesting activities displayed by I.
viscosa extracts that can corroborate its traditional use.
For example, antioxidant,[18– 21] antifungal,[22]
antibacterial,[21] anti-inflammatory,[23]
hypoglycemic,[19,21] and cytotoxic[21,24– 28] effects can
be highlighted. The chemical composition for some I.
viscosa extracts was reported before, namely the
ethanol, methanol, water, and ethyl acetate
fractions.[19– 22,29,30]
Although I. viscosa has been the subject of some
research works, there is still some missing information
concerning the species growing in Algeria and its
effect on some prevalent human cancer cells. So, in
the present article, we aimed to investigate the
chemical composition and cytotoxic properties of the
I. viscosa extract against human gastric cancer (AGS)
and human non-small-cell lung cancer (A549) cell lines
due to the high prevalence of cancer from which they
originate. The mechanisms of cell death associated
were also assessed.
Results and Discussion
Total Bioactive Content
Total phenolic content (TPC) of I. viscosa extract were
estimated using the Folin-Ciocalteu method,[31] while
the total flavonoid content (TFC) was determined
spectrophotometrically based on the AlCl3method.[32]
The ethanolic extract showed contents of 145.3�
4.4 mg gallic acid equivalents/g of extract and 22.1�
0.6 mg quercetin equivalents/g of extract in terms of
TPC and TFC contents, respectively.
The results herein discussed showed that ethanolic
extract TPC is different from those previously cited for
the same species and ranging from 75.3 �1.3 and
299.1�34.5 mg GAE/g of extract.[22 –24,29,30] These dif-
ferences between our findings and those reported in
the literature could be related to the solvents used but
also to several other factors such as the geographical
location, collection year, and extraction procedures.
LC/MS characterization of I. viscosa extracts
Inula viscosa ethanolic extract was further charac-
terized by UHPLC-UV-MS/MS. The chromatogram was
recorded at 280 nm (Figure 1), and the compounds
identification was achieved by comparing the reten-
tion time and the MS data from samples and reference
standards injected under the same chromatographic
conditions, or by comparing their UV/VIS, MS and MS/
MS spectra data with those reported in the literature.
The peak characteristics and the assigned identifica-
tion of compounds present in the ethanolic extract of
I. viscosa are presented in Table 1, respecting their
elution order.
The phenolic profile of I. viscosa ethanolic extract
shows the presence of 17 compounds, 16 identified,
and dominated by hydroxycinnamic acid and flavonol
derivatives (Table 1). The hydroxycinnamic acid deriva-
tives comprised, caffeoyl derivatives, mainly chloro-
genic acid derivatives. One monocaffeoylquinic acid,
compound 2, was identified comparing the obtained
data with previously reported one, mainly its pseudo-
molecular ion at m/z 353 and its typical base peak at
m/z 191, which is correspondent to quinic acid
fragment.[33] Naturally, the standard used to confirm
the identification of caffeic acid also helped establish
some fragment ions.
The other chlorogenic acids, compounds 5,6,7,
and 8, are dicaffeoyl quinic acid derivatives, with the
typical pseudomolecular ion at m/z 515 and exhibiting
a base peak at m/z 353 due to the loss of one caffeoyl
moiety (Table 1). Considering the retention time and
their spectra data, these compounds were assigned to
1,4-O-diCQA, 1,5-O-diCQA, 4,5-O-diCQA.[33– 36] Cyto-
toxic properties of caffeoylquinic acid and derivatives
are well reported in the literature; for example, Ha and
Park (2018) previously reported that six dicaffeoyl-
quinic acids isomers, including 1,5-O-diCQA and 4,5-O-
diCQA, showed an inhibitory effect on melanogenesis
in B16F1 murine melanoma cells.[37] Additionally, 4,5-
O-diCQA displayed therapeutic potential against pros-
tate cancer by inactivating Bcl-2.[38] Using a modified
TUNEL assay, Ooi et al. (2011) found that 3,4-O-diCQA
can inhibit the growth of human lung adenocarcinoma
cell lines (NCIH23) by inducing apoptosis.[39] On the
other hand, Teoh et al. (2016), reported that 3,5-O-
diCQA displayed cytotoxic effects against colon cancer
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cells with minimal cytotoxic effects against normal
colon cells.[40]
One of the central nuclei of these metabolites,
caffeic acid, also occurs naturally in plants and is
known to inhibited the growth of Ht-29 cell lines via
induction of apoptosis[41,42] and has an antitumor
effect against the human cutaneous melanoma cell
lines (SK-Mel-28).[43]
The last caffeoyl derivative, compound 9, showing
a pseudomolecular ion at m/z 857, was assigned to
tetracaffeoyl hexaric acid.[44] Not only it presents the
four fragment ions typical of cleavage of the caffeoyl
moieties, m/z 695 [MC9H7O3(caffeoyl moiety)], m/z
533 [MC18H13O6(2xcaffeoyl moiety)], m/z 371
[MC27H19O9(3xcaffeoyl moiety)], and m/z 209
[MC36H25O12 (4xcaffeoyl moiety)], but also the UV/VIS
Figure 1. UHPLC chromatogram of I. viscosa ethanolic extract, recorded at 280 nm.
Table 1. Chemical composition of I. viscosa extracts by UHPLC-DAD-ESI/MS. Retention time (Rt), not quantified (NQ), caffeoylquinic
acid (CQA).
Peak Rt (min) λmax [MH]MS2(m/z) Identified compound Quantification
(μg/mg extract)
17.73 324, 239, 221 189 127, 115, 99 Unknown NQ
27.99 325, 239, 218 353 191, 179, 173 1-O-CQA 7.397�0.329
38.75 322, 239, 221 179 135 Caffeic acid 0.305�0.069
411.82 352, 256, 203 477 301 Quercetin-O-glucuronide 1.192�0.560
512.43 326, 241 515 353, 317, 299, 255, 235, 203 1,4-O-diCQA 4.674 �0.010
612.84 341, 311, 289, 245 515 353, 335, 191 1,5-O-diCQA 52.422 �4.035
713.51 326, 242, 220 515 353, 299, 203 3,4-O-diCQA 5.517�0.067
813.91 326, 242 515 353, 335, 299, 255, 203 4,5-O-diCQA 4.213 �0.134
915.52 328, 244, 221 857 695, 533, 371, 353, 209 Tetracaffeoyl hexaric acid 5.486�0.512
10 15.88 351, 267, 254 315 300, 287, 271 Methylquercetin isomer 1 2.315�0.141
11 16.61 355, 255, 205 315 300, 287, 271 Methylquercetin isomer 2 3.962�0.615
12 16.80 289, 222, 204 317 299, 289, 193 Myricetin 13.311�0.876
13 17.70 334, 273, 217 299 284 Chrysoeriol 8.785�0.497
14 18.67 340, 291, 269, 230 299 284 Diosmetin 2.847�0.264
15 18.92 355, 254, 205 329 314, 301 Dimethylquercetin 3.174�0.319
16 19.87 367, 256 315 300, 287, 193, 164 Isorhamnetin 2.945�0.667
17 20.17 290, 229 359 341, 317, 299, 193 Trimethylmyricetin 26.682�2.441
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data identical to the one reported by Dudek et al.
(2016).[45]
It is worth mentioning that Trendafilova et al.
(2020) found that the extract of Inula ensifolia flowers
with a higher concentration of chlorogenic and
dicaffeoylquinic acids decreased the cell proliferation
of A549 cancer cell lines.[46]
Additionally, to caffeic acid derivatives, the etha-
nolic extract profile shows several methylated flavone
derivatives, from which the quercetin derivatives are
the major ones (Table 1). Still, the flavones isomers,
chrysoeriol, and diosmetin, with pseudomolecular ion
[MH]at m/z 299 and releasing a fragment ion at m/z
284, resulting from the loss of the methyl group, were
also detected.[47] Compound 16 possessing a pseudo-
molecular at m/z 315 and a fragment ion at m/z 164
was tentatively identified as isorhamnetin since this
fragment corresponds to the typical fragment
1,3B+.[48,49] On the other hand, compounds 10 and 11
(Table 1), also methylquercetin derivatives, could not
be totally assigned, but due to their UV/VIS data, they
seemed to be methylated in the A ring. The similarities
in the UV/VIS data also suggest that compound 15
should be quercetin with O-methoxy groups in A ring
and C-3. Finally, it should also be highlighted that a
trimethylmyricetin (compound 17) was also identified
in this extract; it presents similar data to compound
12, which is myricetin.[50–52]
Flavonoids, in general, are known to have impor-
tant biological activities, including cytotoxic proper-
ties. Concerning the ones identified in I. viscosa,
previous studies revealed that chrysoeriol significantly
inhibits the growth of A549 lung cancer cells,[53,54] and
exhibited significant anticancer effects against other
tumor cell lines, including cervical cancer,[54,55] colon
cancer cell lines,[55] HL-60 leukemia cells,[55] and human
stomach cancer AGS cells.[56]
Myricetin is another flavonoid identified in the
ethanolic extract of I. viscosa species, and this com-
pound is endowed with remarkable cytotoxic proper-
ties. In many previous reports, it has been shown that
myricetin-induced cytotoxicity against various types of
cancer cell lines, including glioblastoma cells,[57]
A2780, OVCAR3,[58] SKOV3 ovarian cancer cells,[59] and
human cervical cancer (HeLa) cells.[60]
Quercetin methylated derivatives have also been
found to inhibit the growth of cancer cell lines, namely
human lung adenocarcinoma cell lines (A549 and
HCC-44),[61] JB6 P+cells,[62] MDA-MB-231 cells,[63] HCT-
116 cancer cell lines.[64] Finally, it can be highlighted
diosmetin, which inhibits the growth of human
squamous carcinoma cells obtained from the oral
cavity.[65]
Overall, LC/MS analysis revealed that 1,5-O-diCQA
was the major compound in the phenolic profile of
the I. viscosa ethanolic extract. Our results agree with
previous reports demonstrating that hydroxycinnamic
acids were the major phenolic compounds isolated
from the Asteraceae family.[66,67] Data showed that the
phenolic compounds detected in this study differ from
those reported in the literature by Brahmi-Chendouh
et al. (2019).[30] Still, it should be emphasized that their
phenolic profile was established for an ethyl acetate
extract.
Cytotoxic Activity
Screening of Toxicity Towards Cancer Cells
Aiming the evaluation of the potential cytotoxic
properties of the I. viscosa ethanolic extract, human
gastric cancer (AGS) and human non-small-cell lung
cancer (A549) cell lines were used due to the high
prevalence of the cancer from which they originate.
For the assessment of cell viability, we used a
rezasurin-based assay, which has the advantage of not
requiring cell lysis before reading when compared to
assays like MTT, and additionally it can be measured
by the means of fluorescence, while tetrazolium salts
are read by the means of colorimetry. In the case of
AGS cells, the I. viscosa ethanolic elicited more than
60% viability loss (Figure 2).
A549 cells are known to be a drug-resistant cell
lines, being frequently included in biological screen-
ings for this reason.[68] In these cells, a different toxicity
pattern was achieved, ethanolic extract cause nearly
40% viability loss (Figure 2). According to the above
reported results, the ethanolic extract was subjected
to further mechanistic studies in AGS and A549 cells.
Considering the extract phenolic profile, the significant
cytotoxic effect found in this research could be
attributed to the chemical compounds present in the I.
viscosa ethanolic extract.
I. viscosa ethanolic extract-elicited cell death is cas-
pase-independent
Aiming to assess the impact of the I. viscosa ethanolic
extract upon cell morphology, the treated AGS and
A549 cells were imaged to evaluate the chromatin
status and overall cell morphology. Several techniques
could be used to this end, including SEM and
fluorescence microscopy. While SEM provides much
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higher magnification and resolution, the use of
fluorescence microscopy can provide important in-
sights when using actin- and chromatin-specific
probes, as widely described in the literature.[69,70] In
this work we the chromatin status of the cells
incubated with ethanolic extract was evaluated using
DAPI, while the cytoplasmic morphology was assessed
using phalloidin, which binds to actin. The extract
under study was tested at the concentration of
125 μg/mL and 250 μg/mL against AGS and A549 cells,
respectively (Figure 3), due to the marked effect in the
AGS cell lines at 250 μg/mL, that originate the loss of
the majority of the cells after fixation. In both cases, it
is noticeable that there is a marked decrease in cell
density, however, were unable to find morphological
traits that would be expected in case apoptosis was
taking place, such as increased rate of cells exhibiting
chromatin condensation or fragmentation.[71,72]
To further confirm that the loss of viability caused
by the ethanolic extract was caspase-independent,
cells were exposed to the extract under study in the
presence Z-VAD-FMK, a pharmacological pan-inhibitor
of caspase activity, which is capable of rescuing cell
viability in cases where caspase-dependent pathways
are involved. As shown in Figure 4A, co-incubation
with Z-VAD-FMK did not result in a significant increase
in cell viability, thus ruling out the involvement of
these enzymes.
Toxicity of I. Viscosa Ethanolic Extract is
RIPK1-Dependent
Resistance to apoptosis is often responsible for both
tumorigenesis and drug resistance, resulting in chemo-
therapy failure.[73] There is an increasing number of
pathways for programmed cell death. Necroptosis
emerging as a novel approach to eliminate tumor
cells, multiple therapeutic agents being reported as
inducers or manipulators of this cell death
pathway.[74,38] In contrast to apoptosis, necroptosis is a
regulated necrotic cell death modality in a caspase-
independent fashion and is mainly mediated by
Receptor-Interacting Protein 1 (RIP1), RIP3, and Mixed
Lineage Kinase Domain-Like (MLKL).[73] In order to
evaluate the possible involvement of RIP1/RIP3 path-
way to the mechanism of action of the I. viscosa
ethanolic extract, cells were co-incubated with necros-
tatin-1 (nec-1), a serine/threonine kinase RIP1 inhibitor.
As seen in Figure 4B, the inhibition of the RIP1/RIP3 (EE
+nec-1) significantly rescued the cell death caused by
the ethanolic extract treatment in A549 cells (47.5 %
vs. 57.4%, p=0.0103), thus pointing to the involve-
ment of RIPK1 and necroptosis pathway to the toxicity
previously detected. In the case of AGS cells no
significant differences have been noticed (48.0 % vs.
55.7%, p=0.0965).
The marked reduction in cell density shown in
Figure 3 could also suggest that an antiproliferative
effect is taking place. Considering the quantification
presented in Table 1, the major compounds are 1,5-O-
diCQA, a myricetin derivative (trimethylmyricetin) and
myricetin itself. Together, they account for nearly 60 %
of the total amount of phytochemicals identified and
quantified in the extract. Several di-caffeoylquinic
acids have been described for their antiproliferative
effects in cancers cells, specifically in colon cancer and
promyelocytic cells, with results showing only minor
changes in activity according to the isomer
involved.[75] Another study showed the antiprolifera-
tive of di-caffeoylquinic acids towards breast cancer
cells via the IL6/JAK2/PI3 K pathway.[76]
In the case of myricetin, its antiproliferative effect
has been described against several cancer cell lines
ranging from esophageal carcinoma[77] to liver and
bladder.[78] The mechanism of action involved in this
effect seems to be cell type-dependent[79] and includes
binding to p90 ribosomal S6 kinase (RSK2),[77] phos-
phorylation of the p38 MAPK[78] and binding to
PI3 K.[80] Considering the significant amounts in which
these molecules occur in the extract, it is highly
Figure 2. Viability of AGS and A549 cells exposed to the I.
viscosa extract (250 μg/mL), or medium (control). EE: ethanolic
extract. Cells were incubated for 24 h, after which viability was
evaluated. Data represents the mean �SD, of at least three
independent experiments performed in triplicate. *** p<0.001
compared to the respective control (Student’s t-test).
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Figure 3. Morphology of AGS and A549 cells exposed to the I. viscosa ethanolic extract (EE) (AGS: 125 μg/mL; A549: 250 μg/mL)
after 24 h of incubation (S Plan Fluor ELWD 20 ×DIC N1 objective). Overall cell morphology was evaluated using phalloidin (actin)
and DAPI (chromatin status).
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probably that can explain, at least in part, the bio-
logical effect we describe here.
Selectivity of I. Viscosa Ethanolic Extract Towards Cancer
Cells
Therapeutic selectivity is one of the most important
considerations in cancer chemotherapy. The design of
therapeutic strategies to preferentially kill malignant
cells minimizing the harmful effects to non-cancer cells
is essential. For this reason and based on the cytotoxic
results reported above towards cancer cells, the effect
of the I. viscosa ethanolic extract in a human non-
cancer cell lines, specifically HaCaT keratinocytes was
evaluated. When compared with the HaCaT cell lines
at the same concentration, I. viscosa ethanolic extract
displayed 2-fold higher toxicity to AGS cancer cells
which is a promising result (Figure 2 and 4C). Con-
versely, similar toxicity was displayed by this extract in
A549 cells when compared with HaCaT cells (Figure 2
and 4C), suggesting a differential selectivity pattern
according to the cancer cell lines tested. These results
highlight the importance of monitoring the toxicolog-
ical effect of new drug candidates against distinct cell
lines, as they may encompass different selectivity
profiles and even mechanisms of action.
Inula viscosa extracts’cytotoxic and anticancer
effects have recently been shown in cervical
cancer,[81,82] breast cancer,[83] Burkitt lymphoma cell
lines,[84] and colorectal cancer cell lines.[85]
Although the cytotoxic effects of I. viscosa extracts
have been the topic of previous research, some
information is still missing. The chemical composition
of the extracts and the mechanism of action are often
not studied. Furthermore, the activity against typical
human cancer cells, gastric and pulmonary cancers
was not studied. Additionally, the species growing in
Algeria and used in traditional medicine was not
studied. As a result, our research provides new insights
about the cytotoxic effect of I. viscosa species, confirms
its traditional use as an anticancer remedy, and further
highlights it as a potential source of anticancer
compounds.
Conclusions
The present study assessed the phenolic profile of the
I. viscosa ethanolic extract and its cytotoxicity against
AGS and A549 human cell lines. LC/MS/MS allowed the
identification of 17 compounds with 1,5-O-dicaffeoyl
quinic acid as the major component. Results also
showed that I. viscosa elicited nearly 60 % and 40 %
viability loss toward AGS and A549 cancer cells,
respectively. Further, a mechanistic study revealed the
involvement of RIPK1 in the toxicity induced by the I.
viscosa extract and discarded the fact that cell death
was mediated by apoptosis. The results of this study
suggest that the I. viscosa species could be considered
a good candidate for developing new antiproliferative
drugs. However, more studies are required to isolate
and assess the main compounds responsible for this
activity.
Figure 4. (A) Influence of Z-VAD-FMK (25 μM), a pan-caspase inhibitor, on the toxicity elicited by the I. viscosa ethanolic extract (EE)
in AGS and A549 cells after 24 h of incubation. (B) Influence of nec-1 (9 μM), a serine/threonine kinase RIP1 inhibitor, on the toxicity
elicited by I. viscosa ethanolic extract (EE) in AGS and A549 cells after 24 h of incubation. (C) Viability of HaCaT cells exposed to I.
viscosa ethanolic extract (EE), or medium (control). Cells were incubated for 24 h, after which viability was evaluated. Data
represents the mean�SD, of at least three independent experiments performed in triplicate. * p<0.05, *** p<0.001 compared to
the respective control (Student’s t-test).
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Experimental Section
Chemicals
Standards used for the elucidation of the phenolic
compounds and for the elaboration of the calibration
curves were obtained from EXTRASYNTHESE (Genay-
Cedex, France). Acetonitrile HPLC-grade and formic
acid were purchased from Panreac (Barcelona, Spain).
All other chemicals were of analytical grade. Dulbec-
co’s Modified Eagle Medium (DMEM), fetal bovine
serum (FBS), penicillin/streptomycin solution (penicillin
10000 Units/mL and streptomycin 10000 μg/mL), Tryp-
sin-EDTA (0.25%) and PrestoBlueTM were obtained
from Invitrogen (Grand Island, NE, USA). Z-VAD-FMK
and phalloidin (CF543) were provided by Santa Cruz
(Heidelberg, Germany) and Biotium (CA, USA), respec-
tively.
Extract Preparation
Aerial parts of Inula viscosa (L.) Aiton were collected in
Jijel (Northeast of Algeria; 36°46’17’’ N, 5°46’34’’ E) and
identified by Prof. Sarri Djamel (University of M’sila,
Algeria). A specimen (AIV 11/18) was deposited in the
Herbarium of the VARENBIOMOL research unit, Frères
Mentouri University 1, Constantine, Algeria. The leaves
were shade drying at ambient temperature (25 °C), and
then were ground into a fine powder. Ethanol extract
was obtained by maceration at room temperature for
48 h, followed by solvent replacement for 2 additional
times. The solvent was then removed under reduced
pressure.
Total Bioactive Content
Total Phenolic Content
The total phenolic content was determined using the
Folin–Ciocalteu method described by Singleton et al.
(1999).[31] 15 μL of plant extract (1 mg/mL) was mixed
with 15 μL of Folin’s reagent and 60 μL of water. After
5 min of incubation, 150 μL of Na2CO3solution (20 %
w/v) was added. The mixture was once again
incubated in the darkness for 60 min, and absorbance
was recorded at 760 nm. The total phenolic content
was measured as mg equiv. of gallic acid per g of
extract (mg GAE/g of extract).
Total Flavonoid Content
The total flavonoid content was determined according
to the method described by Türkoglu et al. (2007).[32]
with a slight modification. An aliquot of 100 μL of each
plant extract was mixed with 100 mL of AlCl3(2 %)
solution. After incubation at room temperature for
10 min, the absorbance was measured at 415 nm. The
total phenolic content was measured as mg equiv. of
quercetin per g of extract (mg QE/g of extract).
UHPLC/MS Characterization of I. Viscosa Extract
UHPLC-DAD-ESI-MSnprofiling of the I. viscosa etha-
nolic extract was carried out using an Ultimate 3000
(Dionex Co., San Jose, CA, USA) apparatus equipped
with a binary pump, an automatic sampler and a diode
array detector (Dionex Co., San Jose, CA, USA). The MS
analysis was conducted using a Thermo LTQ XL mass
spectrometer (Thermo Scientific, San Jose, CA, USA)
outfitted with an electrospray ionization interface (ESI).
The separation was performed at room temperature
25°C with a Hypersil Gold (Thermo Scientific, USA) C18
column (100 mm length; 2.1 mm i.d.; 1.9 μm particle
diameter, end-capped). Formic acid in water (A) and
acetonitrile (B) were used as analyte solvents of the
extract (1 mg/mL), with an injection volume of 10 μL
and flow rate of 2 mL/min. UV/VIS spectral data were
gathered in a range of 200 to 700 nm and chromato-
graphic profiles documented at 280, 350, 470, 655 nm.
MS and MS/MS data were processed using the Thermo
XcaliburQual Browser data system (Thermo Scientific,
USA). Spectra were recorded in negative-ion mode
with electrospray ionization source of 5.00 kV and ESI
capillarity temperature of 275 °C. The full scan covered
a mass range of 50–2000 m/z. Collision-induced
dissociation MS/MS experiments were simultaneously
acquired for precursor ions. The identification of
individual phenolic compounds by UHPLC/MS was
achieved by comparing their retention times, UV/VIS
spectra, and MSnspectra data available on the
literature. And also, with the data of reference stand-
ards or of the closest available standards, injected
under the same UHPLC/MS conditions. The quantifica-
tion of the individual phenolic compounds in the plant
extract was performed by peak integration at 280 nm,
through the external standard method, using the
closest reference compounds available. The detection
and quantification limits (LOD and LOQ, respectively)
were determined from the parameters of the calibra-
tion curves (LOD=3 standard deviation/slope and
LOQ=10 standard deviation/slope). The calibration
curves were obtained by injection of five known
concentrations with variable ranges and chosen in
order to guarantee the quantification of each com-
pound in the samples by intrapolation in the calibra-
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tion curve. Values of correlation coefficients (>0.99)
confirmed linearity of the calibration plots (6 points; 3
assays). The results were expressed in μg of com-
pound/mg of dried extract, as mean values �standard
deviation (MV �SD) of four independent analyses.
Cytotoxic Activity
Cell Culture
Three human cell lines were used in this research:
gastric carcinoma (AGS; Sigma–Aldrich), lung carcino-
ma (A549; ECACC, Salisbury, UK) and human keratino-
cytes (HaCaT; ATCC, Rockville, MD, USA). Cells were
cultured as monolayer at 37°C in a humidified
incubator with 5% CO2. All cell lines were grown in
glutamine-enriched DMEM (Gibco) supplemented with
1% streptomycin/penicillin and 10% FBS (Gibco). For
subculture, cells were washed with HBSS, treated with
0.25% trypsin-EDTA solution (Sigma–Aldrich) for
3 min at 37°C, resuspended in 5 mL of culture medium
and centrifuged at 1300 rpm for 3 min. The super-
natant was removed, and the cell pellet was resus-
pended in culture medium. Cell passages were kept
low for all cell lines, with a maximum of 12 passages.
Unless otherwise stated, all assays were carried in 96-
well plates.
Viability Assessment
I. viscosa ethanolic extract was solubilized in DMSO, in
stocks of 50 mg/mL. For the assessment of viability, a
resazurin-based method was used.[70] AGS and HaCaT
cells were plated at a density of 1.5 ×104cells/well,
while A549 cell lines was seeded at a density of 1.0×
104cells/well. The cells were incubated for 24 h being
then exposed to the extract under study (at 250 μg/
mL; maximum DMSO concentration: 0.5%) for another
24 h. After this period, a commercial solution of
resazurin was added (1:10, final volume: 200 μL) and
the plate incubated for 30 min, the fluorescence
increase being monitored at 560/590 nm (excitation/
emission wavelength) in a microplate reader (Cyta-
tion™3, BioTek, Winooski, VT, US). At least three
independent experiments were performed in triplicate.
Caspase Pharmacological Inhibition Assay
AGS and A549 cells were seeded in 96-well plates at
the same density used for viability experiments. After
attachment, cells were pre-incubated with the pan-
caspase inhibitor Z-VAD-FMK at 25 μM for 1 h, as
previously described.[72,86] Then, plant extract was
added, and cells were co-incubated for 24 h. Cell
viability was determined using a resazurin-based
method as described above.
Involvement of the RIP1 Kinase
AGS and A549 cells were pre-incubated with 9 μM
necrostatin-1 (nec-1) for 1 h, as described before.[68,86]
Then, the plant extract was added, followed by co-
incubation for 24 h and subsequent evaluation of
viability by a resazurin-based method as described
above.
Morphological Assessment
For morphological studies, AGS and A549 cells were
cultured in 96-well plates at the same density used for
viability experiments, in the presence of the plant
extract under study. After incubation, cells were
washed with HBSS and fixed in 10 % formalin solution
for 30 min, at room temperature. CF543 phalloidin
(5 U/mL) and DAPI (0.25 μg/mL) were added, and cells
were stained for 25 min at room temperature and
washed with HBSS.[70,87] Images were acquired in an
inverted Eclipse Ts2R-FL (Nikon) equipped with a
Retiga R1 camera and a S Plan Fluor ELWD 20x DIC N1
objective. Images were analyzed with Fiji.[88]
Statistical Analysis
Data were recorded as the mean �standard deviation
(m�SD) from three independent assays. For the
cytotoxicity assays, the Shapiro-wilks normality test
was performed in the data to ensure that it followed a
normal distribution. Comparison between the means
of two groups (controls vs. each experimental con-
dition) was performed using Student’s t-test. Outliers
were identified by the Grubbs’ test. The analyses were
performed using GraphPad Prism 7.0 or ggplot/R
software and values were considered statistically
significant with a p<0.05.
Acknowledgements
Thanks are due to the University of Aveiro and FCT
(Fundação para a Ciência e a Tecnologia), the Euro-
pean Union, QREN, FEDER, and COMPETE for funding
the LAQV-REQUIMTE (UIDB/50006/2020 and UIDP/
50006/2020) and PRFU Project to K. Hamaidia (No:
D01N01UN410120210001). Thanks, are also due to the
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Algerian Higher Ministry and Scientific Research via
PNE (Programme National Exceptionnel) for financial
support, namely the H.R. and A.H. displacements.
Conflict of Interest
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are
available from the corresponding author upon reason-
able request.
Author Contribution Statement
H.R.: Conceptualization, investigation, data curation,
and writing-original draft preparation; A.H.: investiga-
tion, data curation and writing-original draft prepara-
tion; K.H.: conceptualization, supervision, validation,
and writing-original draft preparation; D.M.P. and
R.B.P.: investigation, data curation and writing; T.B.:
investigation; D.C.G.A.P.: conceptualization, supervi-
sion, validation, software, and writing-reviewing the
original draft; M.S.G.A.V.: software, methodology;
A.M.S. S.: conceptualization, validation, resources,
supervision, and writing-reviewing the original draft.
All authors have read and agreed to the published
version of the manuscript.
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Received September 27, 2022
Accepted January 23, 2023
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