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Article
Antioxidant, Antigenotoxic and Cytotoxic Activity of Essential
Oils and Methanol Extracts of Hyssopus officinalis L. Subsp.
aristatus (Godr.) Nyman (Lamiaceae)
Tijana Mićović 1 , Dijana Topalović 2 , Lada Živković 2 , Biljana Spremo-Potparević 2 , Vladimir Jakovljević 3,4 ,
Sanja Matić 5 , Suzana Popović 6 , Dejan Baskić 6,7 , Danijela Stešević 8 , Stevan Samardžić 9 , Danilo Stojanović 10
and Zoran Maksimović 9, *
1 Institute for Medicines and Medical Devices of Montenegro, Bulevar Ivana Crnojevića 64a,
81000 Podgorica, Montenegro; tijana.micovic@calims.me
2 Department of Pathobiology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450,
11000 Belgrade, Serbia; dijana.topalovic@pharmacy.bg.ac.rs (D.T.); lada.zivkovic@pharmacy.bg.ac.rs (L.Ž.);
biljana.potparevic@pharmacy.bg.ac.rs (B.S.-P.)
3 Department of Physiology, Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovića 69,
34000 Kragujevac, Serbia; drvladakgbg@yahoo.com
4 Department of Human Pathology, First Moscow State Medical University I. M. Sechenov, Trubetskaya Street
8, Str. 2, 119991 Moscow, Russia
5 Department of Pharmacy, Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovića 69,
34000 Kragujevac, Serbia; sanjad.matic@gmail.com
6
Department of Microbiology and Immunology, Center for Molecular Medicine and Stem Cell Research,
Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovića 69, 34000 Kragujevac, Serbia;
Citation: Mićović, T.; Topalović, D.; popovic007@yahoo.com (S.P.); dejan.baskic@gmail.com (D.B.)
Živković, L.; Spremo-Potparević, B.; 7 Public Health Institute, Nikole Pašića 1, 34000 Kragujevac, Serbia
Jakovljević, V.; Matić, S.; Popović, S.; 8 Faculty of Natural Sciences and Mathematics, University of Montenegro, Džordža Vašingtona bb,
Baskić, D.; Stešević, D.; Samardžić, S.; 81000 Podgorica, Montenegro; danijela.stesevic@ucg.ac.me
9 Department of Pharmacognosy, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450,
et al. Antioxidant, Antigenotoxic and
Cytotoxic Activity of Essential Oils 11000 Belgrade, Serbia; stevan.samardzic@pharmacy.bg.ac.rs
10 Department of Botany, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450,
and Methanol Extracts of Hyssopus
11000 Belgrade, Serbia; dancho@pharmacy.bg.ac.rs
officinalis L. Subsp. aristatus (Godr.)
* Correspondence: zmaksim1@pharmacy.bg.ac.rs
Nyman (Lamiaceae). Plants 2021, 10,
711. https://doi.org/10.3390/
Abstract: Hyssopus officinalis L. is a well-known aromatic plant used in traditional medicine and the food
plants10040711
and cosmetics industry. The aim of this study is to assess the antioxidant, genotoxic, antigenotoxic and
cytotoxic properties of characterized hyssop essential oils and methanol extracts. Chemical composition
Academic Editor: Suresh Awale
was analyzed by gas chromatography - mass spectrometry (GC-MS) and liquid chromatography with diode
Received: 25 February 2021
array detection and mass spectrometry (LC-DAD-MS), respectively. Antioxidant activity was examined
Accepted: 30 March 2021 by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) tests; genotoxic
Published: 7 April 2021 and antigenotoxic activity were examined by the comet assay, while cytotoxicity was evaluated by the 3-
(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide dye (MTT) test against tumor cell lines (SW480,
Publisher’s Note: MDPI stays neutral MDA-MB 231, HeLa) and non-transformed human lung fibroblast cell lines (MRC-5). The essential oils
with regard to jurisdictional claims in were rich in monoterpene hydrocarbons (e.g., limonene; 7.99–23.81%), oxygenated monoterpenes (1,8-
published maps and institutional affil- cineole; 38.19–67.1%) and phenylpropanoids (methyl eugenol; 0.00–28.33%). In methanol extracts, the most
iations. abundant phenolics were chlorogenic and rosmarinic acid (23.35–33.46 and 3.53–17.98 mg/g, respectively).
Methanol extracts expressed moderate to weak antioxidant activity (DPPH IC50 = 56.04–199.89 µg/mL,
FRAP = 0.667–0.959 mmol Fe2+/g). Hyssop preparations significantly reduced DNA damage in human
whole blood cells, induced by pretreatment with hydrogen peroxide. Methanol extracts exhibited selective
Copyright: © 2021 by the authors. and potent dose- and time-dependent activity against the HeLa cell line. Results of the current study
Licensee MDPI, Basel, Switzerland. demonstrated notable H. officinalis medicinal potential, which calls for further investigation.
This article is an open access article
distributed under the terms and Keywords: Hyssopus officinalis; antioxidant activity; antigenotoxic activity; comet assay; cytotoxic
conditions of the Creative Commons
activity; HeLa cell line; essential oil; methanol extract; GC-MS; LC-DAD-MS
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Plants 2021, 10, 711. https://doi.org/10.3390/plants10040711 https://www.mdpi.com/journal/plants

Plants 2021, 10, 711 2 of 21
1. Introduction
Hyssop, Hyssopus officinalis L. (Lamiaceae), is a shrubby perennial herbaceous plant,
distributed mostly in the Mediterranean area [1–3]. In Montenegro and Serbia, Hyssopus
officinalis L. subsp. aristatus (Godr.) Nyman (syn. H. officinalis L. subsp. pilifer (Gris. ex
Pant.) Murb.) can be found in plant communities of rocky pastures [2].
Hyssop herb (Hyssopi herba) and its pharmaceutical preparations (infusions, syrups,
tinctures, extracts) have been used in traditional medicine since ancient times as antiseptic,
carminative, diaphoretic, emenagogue, expectorant, muscle relaxant, stomachic and tonic
agents. As an aromatic plant, it is also used in the food and cosmetics industry [4–6].
Essential oil is the most important and the most frequently investigated product of hyssop.
Available literature data on wild and cultivated plants indicate that its herb yields 0.3–1% of
essential oil with isopinocamphone as the dominant compound, along with pinocamphone,
β-pinene, 1,8-cineole, pinocarvone, linalool, sabinene and methyl eugenol [7–9]. Beside the
essential oil, hyssop herb contains flavonoids and phenolic acids, tannins, diterpene lactones
(marrubiin) and triterpenoid compounds such as ursolic and oleanolic acid [5,7,10].
Antimicrobial activity is one of the most commonly examined pharmacological effects of
various hyssop preparations. Extensive experimental evidence also speak in favor of antioxi-
dant [10], antiviral [11], sedative and anxiolytic [12,13], spasmolytic [14], anti-inflammatory [15],
antiulcer [16], anti-asthmatic [17] and antidiabetic activities of the hyssop herb [18]. However,
despite the diversity in scientific information on pharmacological activities of Hyssopi herba and
its preparations, genotoxicity, antigenotoxicity and cytotoxic activity of the essential oils and
polar extracts of this herbal drug are still insufficiently investigated.
In attempt to better understand medicinal potentials of H. officinalis herb in this field,
we designed and performed a set of chemical and physiological investigations on the
plant material collected from five wild-growing populations in Montenegro and on one
commercial sample available in herbal apothecaries in Serbia, and manufactured by a local
enterprise from wild-growing sources.
Hyssopi herba, as an herbal medicinal substance and a raw material for pharmaceutical
and related industries, is neither official in pharmacopoeias, nor listed in the other well-
established documents. Therefore, the question of its pharmaceutical quality still remains
open. Having in mind that growing (for plants harvested from the wild) and/or cultivation
conditions can significantly affect the composition and activity of a given plant, it would
explain the need for sampling at different sites, as well as the use of a commercial sample-
that is something that a person can find, if needed.
Consequently, the main objectives of the present work were to quantify the levels of
in vitro antioxidant activity by commonly used 2,2-diphenyl-1-picrylhydrazyl (DPPH) and
ferric reducing/antioxidant power (FRAP) tests, to assess potential genotoxicity/antigenoto
xicity by comet assay, and to determine the overall cytotoxic, cytostatic and cytocidal effects
against human tumor and non-transformed human lung fibroblast cell lines of investigated
hyssop essential oils and/or extracts, with respect to their chemical composition.
2. Results and Discussion

2.1. Essential Oils Compositions
Produced oils were pale yellowish-green liquids with characteristic pleasant odors.
The yields ranged from 0.4% to 0.79% (v/w) for samples collected from the wild in Mon-
tenegro. However, the highest yield was obtained from the commercial sample (Serbia),
amounting to 1% (v/w). The results of the gas chromatography coupled with mass spec-
trometry (GC-MS) analysis of the essential oils (1EO–6EO) obtained from tested samples of
H. officinalis subsp. aristatus (1–6, Table 1) are presented in Table 2. Overall, 12 to 16 com-
pounds were identified depending on the sample, which is more than 98% of the total oil on
average, with one exception (sample 2EO), where the percentage of identified compounds
was lower (86.84%).
Plants 2021, 10, 711 3 of 21
Table 1. Plant material: origin, collection data and yields of essential oils and extracts. a Values are the means of five consecutive determinations.
Essential Oil MeOH Extract

Plant Material Site of Geographic Altitude Collection Date Voucher
Sample Habitat (EO) Yield (E) Yield (%
Origin Collection Coordinates (m) (dd/mm/yyyy) Specimen
(mL/100 g) a w/w)
Commercial Southeastern
1 N/A N/A N/A N/A N/A 1.00 12.02
sample (Serbia) Serbia
Wild-growing N 42◦ 310 5500
2 Kuči 870 rocky 13/09/2018 1420263 0.40 9.48
(Montenegro) E 19◦ 240 0700
3 Šavnik 880 rocky pasture 19/09/2018 1420261 0.54 10.24
4 Piva 750 rocky 14/09/2018 1420162 0.65 9.05
5 Piperi 800 rocky pasture 07/09/2018 1420259 0.79 10.21
(Montenegro) E 19◦ 160 0.800
6 Cuce 820 rocky 12/09/2018 1420260 0.48 9.64
Plants 2021, 10, 711 4 of 21
Table 2. Essential oil composition of Hyssopus officinalis subsp. aristatus. * Arithmetic retention index.
Amount (%)
tR [min] AI * Compound 1EO 2EO 3EO 4EO 5EO 6EO
5.517 925 α-Thujene 0.00 0.51 0.00 1.05 1.44 0.00
5.706 932 α-Pinene 2.08 4.13 1.12 0.53 0.79 1.03
6.762 972 Sabinene 1.86 1.24 0.57 0.47 0.54 0.56
6.872 976 β-Pinene 6.73 9.13 16.33 15.79 9.69 5.48
7.238 990 β-Myrcene 0.93 0.46 0.46 0.00 0.43 0.36
8.343 1024 p-Cymene 0.27 1.92 0.00 0.00 0.00 0.28
8.482 1028 Limonene 7.99 7.99 16.11 23.81 21.77 15.43
8.569 1030 1,8-Cineole 67.10 42.07 9.77 1.42 38.19 56.08
8.765 1036 Z-β-Ocimene 3.57 2.94 2.06 1.88 3.11 3.06
9.142 1046 E-β-Ocimene 0.27 0.00 0.00 0.00 0.00 0.00
9.531 1057 γ-Terpinene 0.31 0.58 0.00 0.00 0.00 0.00
12.592 1138 trans-Pinocarveol 0.23 2.26 0.83 0.54 0.00 0.61
13.463 1159 trans-Pinocamphone 0.00 1.84 3.34 8.34 4.72 0.00
13.556 1162 Pinocarvone 0.00 1.20 3.99 1.67 0.00 0.41
14.027 1173 cis-Pinocamphone 1.15 5.61 22.75 14.72 14.54 0.00
14.961 1196 Myrtenal 0.32 3.71 1.02 0.66 0.69 0.80
20.403 1325 Myrtenyl acetate 0.00 1.25 0.00 0.00 0.00 0.00
22.856 1384 β-Bourbonene 0.00 0.00 0.00 0.00 0.00 0.31
23.758 1406 Methyl eugenol 5.43 0.00 19.24 28.33 3.52 13.70
24.265 1418 E-β-Caryophyllene 0.47 0.00 0.00 0.00 0.00 0.00
26.771 1480 Germacrene D 0.40 0.00 0.00 0.00 0.00 0.36
Monoterpene hydrocarbons 24.01 28.9 36.65 43.53 37.77 26.2
Oxygenated monoterpenes 68.8 57.94 41.7 27.35 58.14 57.9
Sesquiterpene hydrocarbons 0.87 0.00 0.00 0.00 0.00 0.67
Phenylpropanoids 5.43 0.00 19.24 28.33 3.52 13.70
Total identified 99.11 86.84 97.59 99.21 99.43 98.47
The dominant group of identified volatiles was monoterpenes (70.88–95.91%). Their

oxygenated derivatives were the most abundant (41.7–68.8%), among which 1,8-cineole
and cis-pinocamphone were found in the highest contents. Only in the sample 4EO, the
content of monoterpene hydrocarbons (43.53%) was higher than the content of oxygenated
monoterpene derivatives. With regard to the monoterpene hydrocarbons, the dominant
constituents were β-pinene and limonene. Methyl eugenol (up to 28.33%), the only com-
pound belonging to the phenylpropanoid group, and sesquiterpene hydrocarbons (up to
0.87%) were present in a lower percentage. Monoterpene hydrocarbons, such as β-pinene,
limonene, Z-β-ocimene, α-pinene, sabinene and the oxygenated monoterpene derivative
myrtenal, were present in all the investigated samples.
The principal component analysis (PCA) confirmed the existence of significant chem-
ical variation in the investigated essential oils. Performed on the entire dataset, PCA
detected five principal components (PCs), with the first three accounting together for more
than 86% of total variance (Figure 1). The constituents of the oils which contribute the most
to the corresponding PCs are listed in Table S1 (Supplementary Material), along with their
loadings and scores. Along the first PC axis, the highest number of significant characters
of separation (factor loadings higher than ±0.7) was detected. The second and third PCs
further underscored the chemical variations between samples.
The cluster analysis of entire dataset revealed the similarity in the composition of
essential oils from the commercial sample of H. officinalis and plants collected from the
locality Cuce in Montenegro (Cluster 1), plants collected from localities Kuči and Piperi
(Cluster 2) and plants collected from localities Šavnik and Piva (Cluster 3), as shown in
Figure 2. The results indicated that the classification proposed by the PCA and hierarchical
cluster analysis (HCA) are in good agreement.
tected five principal components (PCs), with the first three accounting together for more
than 86% of total variance (Figure 1). The constituents of the oils which contribute the
most to the corresponding PCs are listed in Table S1 (Supplementary Material), along with
their loadings and scores. Along the first PC axis, the highest number of significant char-
Plants 2021, 10, of
acters 711separation (factor loadings higher than ±0.7) was detected. The second and third 5 of 21
Plants 2021, 10, x FOR PEER REVIEW
PCs further underscored the chemical variations between samples.
Figure 1. Plots of principal component analysis (PCA) scores (a) and loadings (b) along the
two principal components (PCs) extracted from the dataset of Hyssopus officinalis essential
Figure 1. Plots of principal component analysis (PCA) scores (a) and loadings (b) along the first two principal components
from six mutually independent sources,
Figure 1. Plots as listed
of principal in Table
component 2. (PCA) scores (a) and loadings (b) a
analysis
(PCs) extracted from the dataset of Hyssopus officinalis essential oils from six mutually independent sources, as listed
two principal components (PCs) extracted from the dataset of Hyssopus officinalis es
in Table 2.
from six mutually independent sources, as listed in Table 2.
The cluster analysis of entire dataset revealed the similarity in the compos
Considering the previously reported literature data, numerous compounds have
essentialbeen
formerly oilsidentified
from the commercial
in essential
The ofsample
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cluster hyssop of several
of and H.dataset
entire officinalis and the
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described. essential oils(Cluster
from the 1), plants collected
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officinalis Kuči coll
and plants an
Differences in oil composition
locality Cuce (deriving
in from
Montenegro climatic conditions,
(Cluster 1), plants the origin
collected
(Cluster 2) and plants collected from localities Šavnik and Piva (Cluster 3), as sh of plant
from localities K
material, subspecies or(Cluster
variety, developmental stages, soil type, cultivation technologies,
Figure 2. The results indicated that the classification proposed by the PCA and3
2) and plants collected from localities Šavnik and Piva (Cluster
extraction methods, etc.) determine
Figure its organoleptic
2. The results indicatedandthatphysiological properties,
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chicalits
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possibilities of (HCA)
application are in good
[1,5,7,19–23]. agreement.
chical cluster analysis (HCA) are in good agreement.
Figure 2. Dendrogram representing the similarity relations of the chemical compos

sopus officinalis essential oils. For this analysis, an entire chromatographic dataset w
consideration. Amalgamation rule: single-linkage. Distance metric is Euclidean dis
Figure 2. Dendrogram standardized).
representing the similarity relations ofrelations
the chemical composition of Hyssopus
Figure 2. Dendrogram representing the similarity of the chemical composition of
officinalis essential oils.
sopus officinalis For thisoils.
essential analysis,
For an entire
this chromatographic
analysis, an entire dataset was taken intodataset
chromatographic considera-
was take
Considering
tion. Amalgamation rule: single-linkage. the previously
Distance reported
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consideration. Amalgamation
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standardized). have and
The most characteristic beenimportant
described.components of so far investigated H. officinalis
essential oils are 1,8-cineoleDifferences in oil composition trans-pinocamphone
[1,21,22], cis-pinocamphone, (deriving from climaticand conditions,
their the
Considering
precursor the previously
material,
β-pinene [1,19]. Among the otherreported
subspecies or literature
variety,
principal data,
developmental
constituents, numeroussabinene,
stages,
pinocarvone,soil compounds h
type, cultivation
germacrene D, germacren D-4-ol, 4-carvomenthenol,
merly been identified in essential oils of hyssop and several chromatographic
α-, β-phellandrene, thymol, carvacrol,
have been described.
Differences in oil composition (deriving from climatic conditions, the origin
material, subspecies or variety, developmental stages, soil type, cultivation techn
Plants 2021, 10, 711 6 of 21
elemol, limonene, linalool, α-terpinene, myrtenol, myrtenyl acetate and methyl eugenol
were also reported [7].
With regard to the hyssop growing in Serbia, Mitić et al. (2000) identified cis-pinocamp-
hone (44.7%) as the most abundant constituent of its essential oil, followed by trans-
pinocamphone (14.1%), germacren-D-11-ol (5.7%) and elemol (5.6%) [19]. Gorunović et al.
(1995) examined hyssop from the territory of Montenegro. The main constituents were
methyl eugenol (38.30%), limonene (37.40%) and β-pinene (9.6%) [20].
Hajdari A. et al. (2018) investigated the composition of the essential oil of wild-
growing H. officinalis subsp. aristatus (aerial parts) from five different localities in Kosovo,
and found that in four out of five samples, the dominant compound was cis-pinocamphone,
with the content ranging between 30.44% and 57.73%. In a sample from one of the localities,
the dominant compound was 1,8-cineole (45.27%). The same authors found that the content
of trans-pinocamphone (14.76%) was significant in one of the samples, as well as that of
β-pinene (23.31%) and caryophyllene oxide (12.66%) [21].
The essential oils obtained from wild-growing H. officinalis L. subsp. aristatus in
Bulgaria in two stages of development (during the flower bud formation and in the full
bloom) were similar in composition, with 1,8-cineole (48.2% and 39.6%), isopinocamphone
(16.3% and 29.2%) and β-pinene (11.4% and 39.6%) as the major constituents. The essential
oil obtained from cultivated H. officinalis contained larger amounts of isopinocamphone
(40.2%), pinocamphone (10.3%) and β-pinene (14.2%), but no traces of 1,8-cineole [22].
In the essential oil of wild-growing H. officinalis subsp. aristatus (aerial parts) native to
Italy, the main compound was linalool (35.3–51.2%), whereas methyl eugenol (7.3–22.7%),
limonene (3.7–4.4%), germacrene D (1.9–4.1%), (Z)-β-ocimene (5.1–5.8%) and (E)-β-ocimene
(2.1–5.3%) were reported as well [5].
Our results revealed three chromatographic profiles in investigated essential oils of
wild-growing plants from Montenegro. The essential oil obtained from plants collected
from the locality Cuce in Montenegro (sample 6EO) was similar with the essential oil of the
commercial sample from southeastern Serbia (sample 1EO), being high in 1,8-cineole and
relatively rich in β-pinene, but low in cis-pinocamphone. On the other hand, the essential
oils of plants collected from the localities Šavnik and Piva in Montenegro (samples 3EO
and 4EO, respectively) stood out for being high in β-pinene, limonene, cis-pinocamphone
and methyl eugenol, but relatively low in 1,8-cineol at the same time. Finally, the essential
oils obtained from the plants collected from the localities Kuči and Piperi in Montenegro
(samples 2EO and 5EO, respectively) appeared to be relatively rich in 1,8-cineole, limonene,
β-pinene and cis-pinocamphone.
2.2. Methanol Extract Compositions and Contents of Total Polyphenols

The results of the liquid chromatography with diode array and mass spectrometry
(LC-DAD-MS) analysis of methanol extracts (1E–6E) obtained from the tested samples of H.
officinalis subsp. aristatus (1–6, Table 1) are presented in Tables 3 and 4. LC-DAD-MS analysis
of methanol extracts of hyssop flowering aerial parts revealed the presence of phenolic
compounds, specifically benzoic acid derivative (syringic acid), hydroxycinnamic acid
derivatives (chlorogenic, feruloylquinic and rosmarinic acids, as well as caffeoyl pentoside)
and flavonoids (derivatives of quercetin and diosmetin). The identified compounds, their
spectral characteristics and their retention times are given in Table 3. The comparative
view of chromatograms of 1E–6E recorded at 320 nm is given in Supplementary Material
Figure S1. It showed that chlorogenic and rosmarinic acids can be considered quantitatively
dominant compounds based on their relative peak areas (%).
Plants 2021, 10, 711 7 of 21
Table 3. Assignment, retention times, UV and MS spectral data of phenolic compounds in methanol extracts of Hyssopus
officinalis subsp. aristatus. a Identification by comparing with commercial reference compounds. b Tentative identification by
comparing acquired UV and MS spectral data with literature data.
Peak No. tr (min) UV λmax (nm) ESI-MS Data (m/z) Assignment

1 4.255 280 395.1 [2M-H]− , 197 [M-H]− , 153.1 Syringic acid b
Chlorogenic acid
2 9.107 218, 240, 298 sh, 326 707.1 [2M-H]− , 353.1 [M-H]− , 191
(5-O-caffeoylquinic acid) a
3 10.954 218, 238, 298 sh, 328 623.1 [2M-H]− , 311.1 [M-H]− , 134.1 Caffeoyl pentoside b
4 12.087 218, 238, 296 sh, 326 735.2 [2M-H]− , 367.1 [M-H]− , 173.1 Feruloylquinic acid b
5 15.339 256, 266 sh, 356 463.1 [M-H]− , 300.1 Quercetin O-hexoside b
Diosmetin
6 17.215 252, 266, 348 607.2 [M-H]− , 299.1, 284
O-deoxyhexosyl-hexoside b
7 18.461 286 sh, 328 719.1 [2M-H]− , 359 [M-H]− , 197, 161.1 Rosmarinic acid a
Table 4. Content of total phenols, chlorogenic acid and rosmarinic acid in methanolic extracts of Hyssopus officinalis subsp.
aristatus. Different letters in the superscript indicate statistically different values at p < 0.05.
Sample Total Phenols (mg GAE/g) Chlorogenic Acid (mg/g) Rosmarinic Acid (mg/g)
c a
1E 74.7 ± 0.8 23.35 ± 0.2 13.71 ± 0.19 d
2E 68.2 ± 0.8 b 30.94 ± 0.11 d 5.35 ± 0.02 b
3E 64.1 ± 1.3 a 24.12 ± 0.11 b 3.53 ± 0.03 a
4E 112.0 ± 1.6 e 33.46 ± 0.08 e 17.98 ± 0.25 e
5E 81.8 ± 0.8 d 33.17 ± 0.1 e 4.97 ± 0.12 b
6E 69.0 ± 0.3 b 30.19 ± 0.1 c 8.13 ± 0.04 c
Identified phenolics were present in all extracts, regardless of the site of the plant
material collection. Variability was reflected through relatively small differences in the
concentrations of individual constituents. The content of chlorogenic acid was in the range
between 23.35 and 33.46 mg/g, whereas rosmarinic acid was present in lower amounts
(3.53–17.98 mg/g) (Table 4). Among the analyzed preparations, sample 4E was the richest
in chlorogenic and rosmarinic acids.
The results are in good agreement with the literature data. Previous studies of ethanol
and deodorized aqueous extracts of the aerial parts of wild-growing H. officinalis subsp.
aristatus (originating from central Italy and eastern Serbia) showed the presence of chloro-
genic acid, rosmarinic acid, 4-O-feruloylquinic acid and syringic acid [1,5]. Flavonoids,
isoquercitrin (quercetin 3-O-glucoside) and diosmin (diosmetin 7-O-rutinoside), were also
previously detected in extracts of hyssop herb [10,24]. In a study conducted by Borrelli
et al. (2019), ethanol macerate of the hyssop aerial parts was chemically analyzed and
the occurrence of caffeoyl pentoside, a hydroxycinnamate derivative, was confirmed [25].
In addition, a phenylethanoid glycoside martynoside was reported as a constituent of H.
seravshanicus [26]. The findings of other authors regarding the quantitative composition
of different extracts of hyssop herb are consistent with the presented results. Namely,
Venditti et al. (2015), as well as Hatipoğlu et al. (2013), demonstrated that the content of
chlorogenic acid is the highest among the quantities of phenolics [5,27]. Detailed analysis
indicated that the contents of chlorogenic acid in the examined samples were 4–5 times
higher than the corresponding values formerly reported, whereas the rosmarinic acid
contents were closer to the literature values. However, certain variations can be expected
and explained by a number of factors, e.g., differences in the extraction solvent used, the
extraction methodology, the origin of the plant material and/or the developmental stage of
the plant during collection.
The contents of total polyphenols (TPC) in tested samples ranged between 64.1 and
112.0 mg GAE/g (Table 4). The highest TPC was determined in sample 4E (112 mg GAE/g),
Plants 2021, 10, 711 8 of 21
whereas the lowest one was obtained in sample 3E (64.1 mg GAE/g). The sample richest
in chlorogenic and rosmarinic acids was also the richest in total polyphenols. The order of
the remaining extracts, by the decreasing TPCs, was: 5E > 1E > 6E > 2E.
Previous studies have yielded variable results, which is expected given that TPC can
be affected by numerous factors. Namely, reported values for TPC in several different
preparations of H. officinalis aerial parts were in a wide range, between 2.69 and 497.6 mg
GAE/g [1,10,21,25,28].
2.3. Antioxidant Activity

Dry methanol extracts (1E–6E) of hyssop herb exhibited notable antioxidant activity
in DPPH and FRAP assays (Table 5).
Table 5. Total antioxidant activity and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
activity in methanolic extracts of Hyssopus officinalis subsp. aristatus. Different letters in the superscript
indicate statistically different values at p < 0.05.
Sample DPPH-IC50 (µg/mL) FRAP mmol Fe2+ /g

1E 88.42 ± 3.48 d 0.815 ± 0.012 b
2E 175.41 ± 2.92 e 0.781 ± 0.012 a,b
3E 199.89 ± 0.60 f 0.667 ± 0.004 a
4E 56.04 ± 0.19 b 0.959 ± 0.003 c
5E 79.37 ± 1.51 c 0.877 ± 0.007 b,c
6E 87.90 ± 0.67 d 0.736 ± 0.023 a,b
Rutin 4.67 ± 1.41 a 4.111 ± 0.0253 d
Ascorbic acid - 8.181 ± 0.136 e
The lowest IC50 value, i.e., the best ability to neutralize DPPH radicals, was shown for
the extract 4E (56.04 µg/mL), followed by 5E (79.37 µg/mL), whereas the lowest activity
was observed in the case of 3E (199.89 µg/mL).
These results correlate well with the values of total antioxidant activity estimated
by the FRAP assay. Namely, the highest FRAP value was obtained for the 4E extract
(0.959 mmol Fe2+ /g), followed by the 5E extract (0.877 mmol Fe2+ /g), whereas the lowest
value was demonstrated for the 3E extract (0.667 mmol Fe2+ /g).
The data obtained in antioxidant assays correlate well with the contents of total
polyphenols, which are known as constituents that contribute to the antioxidant activity of
the plants. With regard to extracts 1E, 2E and 6E, there was no such strong link between
the antioxidant activity and total polyphenol contents as there was with the aforemen-
tioned extracts. Compared to standard substances (rutin and ascorbic acid), tested hyssop
preparations were less effective in DPPH radical scavenging and in the reduction of ferrous
ion-2,4,6-tri(2-pyridyl)-s-triazine complex (Table 5).
Taking into account the presented results, it can be concluded that moderate antioxi-
dant efficacy (IC50 < 100 µg/mL) was demonstrated for four of the six analyzed samples,
with the best activity shown for sample 4E.
Literature reports on hyssop aerial parts preparations indicate considerable variability
in IC50 values (25–2970 µg/mL) obtained in the DPPH test [1,10,25,28], which could be
expected as the geographical origin of plant material, extraction procedures and antioxidant
activity test protocols differ. With regard to the total antioxidant activity, Stanković et al.
(2016) examined methanol extract of vegetative parts of H. officinalis from southeastern
Serbia and found its FRAP value to be 0.73 mmol Fe2+ /g [29]. The current study FRAP
value is in good agreement with this reported value, as it ranged from 0.667 mmol Fe2+ /g
to 0.959 mmol Fe2+ /g. The chemical composition may help explain the documented
antioxidant activity. Namely, earlier published papers provide evidence that the dominant
compounds of the tested extracts (chlorogenic and rosmarinic acids) exhibit significant
efficacy in neutralizing DPPH radicals and reducing the ferrous ion complex [30–34].
ern Serbia and found its FRAP value to be 0.73 mmol Fe2+/g [29]. The current study FRAP
value is in good agreement with this reported value, as it ranged from 0.667 mmol Fe2+/g
to 0.959 mmol Fe2+/g. The chemical composition may help explain the documented anti-
oxidant activity. Namely, earlier published papers provide evidence that the dominant
Plants 2021, 10, 711 compounds of the tested extracts (chlorogenic and rosmarinic acids) exhibit significant
9 of 21
efficacy in neutralizing DPPH radicals and reducing the ferrous ion complex [30–34].
2.4. Genotoxic and Antigenotoxic Activity

Potential genotoxicity and antigenotoxicity of methanol extracts and essential oils of
hyssopPotential
herb weregenotoxicity and antigenotoxicity
assessed using the Comet assay.of methanol extracts and essential oils of
hyssop herb were assessed using the Comet assay.
2.4.1. Genotoxic Activity
2.4.1. Genotoxic Activity
Hyssop herb extracts did not exhibit a genotoxic effect at concentrations 100, 200 and
Hyssop herb extracts did not exhibit a genotoxic effect at concentrations 100, 200 and
400 µ g/mL (data not shown). With regard to the essential oils, the genotoxic effect did not
400 µg/mL (data not shown). With regard to the essential oils, the genotoxic effect did not
manifest at the lowest tested concentration (2.5 µ g/mL). These results were used to select
manifest at the lowest tested concentration (2.5 µg/mL). These results were used to select
concentrations for antigenotoxic activity testing.
concentrations for antigenotoxic activity testing.
2.4.2.
2.4.2.Antigenotoxic
AntigenotoxicActivity
Activity
AtAt 400 µg/mL, alltested
400 µ g/mL, all testedH.H.officinalis
officinalisextracts
extractssignificantly
significantly(p(p<<0.0001)
0.0001)reduced
reducedDNA
DNA
damage
damageininhuman
humanperipheral
peripheral blood
blood leukocytes, inducedby
leukocytes, induced bythe
thepretreatment
pretreatmentwithwithhydrogen
hydro-
gen peroxide
peroxide (Figure
(Figure 3). 3).
Figure 3. Antigenotoxic properties of methanol extracts of H. officinalis subsp. aristatus (1E–6E)

against
Figure 3. DNA damage inproperties
Antigenotoxic human peripheral blood
of methanol leukocytes,
extracts induced by
of H. officinalis hydrogen
subsp. peroxide
aristatus (1E–6E)(H2 O2 )
against DNA damage
in post-treatment in human
protocol. peripheral
Bars representblood leukocytes,
the mean value ofinduced
cells withby hydrogen
DNA damage peroxide
± standard
(H 2O2) of
error in the
post-treatment
mean (SEM) protocol.
versus theBars represent
control treatedthe mean
with value
H2 O2 (n =of cells
3). PBS:with DNA damage
phosphate-buffered ± saline
standard
solution.error
*** pof the mean (SEM) versus the control treated with H2O2 (n = 3). PBS: phosphate-
< 0.0001.
buffered saline solution. *** p < 0.0001.
A decrease in the mean number of cells with DNA damage was the most pronounced
for A decrease
extracts 2Ein the4E;
and mean numberthere
however, of cells withno
were DNAmajordamage was the
differences inmost pronounced
the antigenotoxic
for extracts
activity 2E and
among the4E; however,
tested there
extracts. were no
Similarly, majoretdifferences
Borrelli in the antigenotoxic
al. (2019) indicated ac-
that the ethanol
tivity among
extracts the tested
of aerial partsextracts. Similarly,H.
of wild-growing Borrelli et al.
officinalis (2019)aristatus,
subsp. indicated that the
native ethanol
to southern
extracts of not
Italy, did aerial partsgenotoxicity,
display of wild-growing H. officinalis DNA
but counteracted subsp.damage
aristatus,
in native
Caco-2to southern
cells caused
Italy, did not display genotoxicity, but counteracted DNA damage in Caco-2
by hydrogen peroxide [25]. The notable antigenotoxic activity of the tested extracts can cells caused
bybehydrogen
attributed peroxide
at least [25]. Theto
in part notable antigenotoxic
the significant activity
content of the testedand
of polyphenols extracts
theircan be
ability
attributed at least
to neutralize freeinradicals.
part to the significant and
Chlorogenic content of polyphenols
rosmarinic acids, asand their ability
dominant to neu-
compounds
tralize
in the free radicals.
examined Chlorogeniccould
preparations, and be
rosmarinic
important acids, as observed
for the dominantactivity,
compounds in the
considering
that earlier published data have shown that these compounds are effective in the Comet
test [35,36].
The statistically significant antigenotoxic activity of the essential oils of hyssop aerial
parts, applied at the concentration of 2.5 µg/mL, was revealed in the post-treatment. The
best activity was shown for the essential oil 4EO (p < 0.0001), followed by commercial
sample 1EO (p < 0.001), whereas other samples (2EO, 3EO, 5EO and 6EO) exhibited weaker,
but statistically significant activity (p < 0.01) (Figure 4).
[35,36].
The statistically significant antigenotoxic activity of the essential oils of hyssop aerial
parts, applied at the concentration of 2.5 µ g/mL, was revealed in the post-treatment. The
best activity was shown for the essential oil 4EO (p < 0.0001), followed by commercial
Plants 2021, 10, 711 sample 1EO (p < 0.001), whereas other samples (2EO, 3EO, 5EO and 6EO) exhibited 10 of 21
weaker, but statistically significant activity (p < 0.01) (Figure 4).
Figure 4. Antigenotoxic properties of essential oils of H. officinalis subsp. aristatus (1EO–6EO) against
Antigenotoxic
DNA4.damage
Figure in human properties of essential
peripheral oils of H. induced
blood leukocytes, officinalisby
subsp.
H2 O2aristatus (1EO–6EO)protocol.
in post-treatment
against DNA damage in human peripheral blood leukocytes, induced by
Bars represent the mean value of cells with DNA damage ± standard error of the meanH 2 O 2 in post-treatment
(SEM) versus
protocol. Bars represent the mean value of cells with DNA damage ± standard
control treated with H2 O2 (n = 3). PBS: phosphate-buffered saline solution. * p < 0.01, error of the**mean
p < 0.001,
(SEM) versus control
*** p < 0.0001. treated with H 2O2 (n = 3). PBS: phosphate-buffered saline solution. * p < 0.01,
** p < 0.001, *** p < 0.0001.

The dominant compound in the 4EO was phenylpropanoid methyl eugenol. With
The to
regard dominant
the 1EO,compound
2EO, 5EO in andthe6EO
4EOsamples,
was phenylpropanoid methyl eugenol.
the dominant constituent was an With
oxy-
regard to the 1EO, 2EO, 5EO and 6EO samples, the dominant constituent
genated monoterpene 1,8-cineole, whereas cis-pinocamphone was present in the highest was an oxygen-
ated
amountmonoterpene 1,8-cineole,
among components whereas
of 3EO. cis-pinocamphone
For methyl eugenol [37]was present in [38],
and 1,8-cineole the highest
there are
amount
literatureamong components
data that confirm theirof 3EO. Forto
ability methyl eugenol
neutralize free [37] and which
radicals, 1,8-cineole
could[38], there
contribute
are
to literature data thateffect.
the antigenotoxic confirm Thetheir abilityaction
beneficial to neutralize free radicals,
of essential oils couldwhich
be basedcouldoncon-
their
tribute to the antigenotoxic effect. The beneficial action of essential oils
participation in the direct neutralization of free radicals, but also their contribution to DNA could be based on
their participation
molecule repair. in the direct neutralization of free radicals, but also their contribution
to DNA molecule
These resultsrepair.
show that hyssop extracts and essential oils exhibit statistically significant
These results
antigenotoxic show that
activity. hyssop conduction
Therefore, extracts andof essential
in vivo oils
testsexhibit statistically
is needed signifi-
to estimate the
cant antigenotoxic
potential activity.
of H. officinalis Therefore, conduction
preparations more reliably.of in vivo tests is needed to estimate the
potential of H. officinalis preparations more reliably.
2.5. Cytotoxic Activity
2.5. Cytotoxic Activity
A cytotoxic agent can induce cell death when it leads to cell demise or, on the other
hand, it can cause
A cytotoxic agentreproductive
can inducecell celldeath,
deathinhibiting
when it leadscell growth and proliferation,
to cell demise while
or, on the other
the cell remains alive. This study aimed to determine the overall
hand, it can cause reproductive cell death, inhibiting cell growth and proliferation, while cytotoxic potential,
cytostatic
the and cytocidal
cell remains alive. Thiseffects
studyofaimedextracts (1E–6E) against
to determine human
the overall tumor cell
cytotoxic lines (SW480,
potential, cyto-
MDA-MB 231 and HeLa). Additionally, these effects were examined
static and cytocidal effects of extracts (1E–6E) against human tumor cell lines (SW480, on non-transformed
human lung
MDA-MB 231 fibroblast
and HeLa). cell line (MRC-5).
Additionally, these effects were examined on non-transformed
Cytotoxic effects of these
human lung fibroblast cell line (MRC-5). extracts were examined in a range of seven concentrations
after 24, 48 and
Cytotoxic 72 h of
effects of treatment
these extracts by 3-(4,5-dimethylthiazol-2-yl)-2,5
were examined in a range of seven diphenyltetrazolium
concentrations
bromide dye (MTT) colorimetric assay. MTT assay is a method that
after 24, 48 and 72 h of treatment by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium indirectly determines
cell viability. MTT is a water-soluble yellow-colored crystal
bromide dye (MTT) colorimetric assay. MTT assay is a method that indirectly determines that easily passes through
the cell membrane because of its positive charge. In metabolically
cell viability. MTT is a water-soluble yellow-colored crystal that easily passes through active cells, MTTtheis
reduced to non-soluble purple formazan crystals. Mitochondrial reductase (succinate
dehydrogenase), active only in viable cells, catalyzes this reaction, so the reduction of the
original compound to formazan is directly proportional to the number of viable cells. The
obtained results are represented as dose-response curves (Supplementary Figure S2) and
IC50 , SI (Table 6) parameters.
Plants 2021, 10, 711 11 of 21
Table 6. Concentrations of extracts (µg/mL) that induce inhibition of biological activity in 50% cells (IC50 ), 50% growth
inhibition (GI50 ), total growth inhibition (TGI) and 50% lethality (LC50 ) in the HeLa cell line, expressed as X ± SD. SI:
selectivity index.
HeLa 1E 2E 3E 4E 5E 6E
24 h >100 >100 >100 >100 >100 >100
IC50 48 h 22.72 ± 3.53 16.97 ± 2.10 44.38 ± 1.96 16.74 ± 1.43 25.90 ± 4.60 25.32 ± 7.80
72 h 19.53 ± 1.03 15.15 ± 1.72 33.43 ± 1.36 14.97 ± 0.78 18.73 ± 0.53 20.04 ± 5.10
24 h 0.97 1.52 1.71 1.31 1.10 3.61
SI 48 h 14.19 20.14 12.08 19.61 8.34 11.87
72 h 12.17 13.87 8.30 15.04 11.31 7.82
24 h 6.95 ±0.95 6.00 ± 0.34 98.09 ± 11.08 5.56 ± 0.18 7.67 ± 1.45 7.46 ± 0.99
GI50 48 h 4.91 ± 0.84 3.49 ± 0.46 65.64 ± 3.66 2.54 ± 0.45 5.61 ± 1.22 5.75 ± 0.68
72 h 0.86 ± 0.51 <0.3 59.38 ± 1.85 <0.3 4.97 ± 0.54 4.76 ± 0.52
24 h 16.90 ± 4.96 14.67 ± 0.58 >100 12.91 ± 0.44 47.25 ± 5.66 27.55 ± 1.89
TGI 48 h 13.60 ± 1.75 11.00 ± 0.56 >100 9.18 ± 0.59 19.90 ± 4.38 16.77 ± 1.52
72 h 13.27 ± 1.33 1.69 ± 0.36 >100 <0.3 18.42 ± 3.57 13.63 ± 1.26
24 h >100 >100 >100 >100 >100 >100
LC50 48 h 61.09 ± 16.15 35.65 ± 1.16 >100 27.07 ± 1.56 63.66 ± 2.30 41.15 ± 6.75
72 h 43.19 ± 10.03 26.02 ± 2.88 >100 20.28 ± 1.10 60.65 ± 1.59 31.20 ± 5.93
The data revealed that extracts 1E–6E displayed a statistically significant percentage
of growth inhibition in a dose-dependent manner on all designated cell lines after 48 h and
72 h (p < 0.05); however, that trend was not noticed after 24 h of treatment (p > 0.05). Time-
dependent growth inhibition was present only on the HeLa cell line with high statistical
significance (p < 0.0001), while a significant time-dependent effect was revealed only at
the highest examined concentration on MRC-5 and MDA-MB 231 cells (p < 0.05). On the
other hand, the increase in growth inhibition of the SW480 cell line was independent of the
exposure period.
To evaluate the overall inhibitory potential of the examined extracts, we calculated
IC50 as a parameter of growth inhibition in relation to the control, which did not take
into account the initial cell number at time zero. Examined extracts showed very low
overall inhibitory activity against healthy cell line MRC-5, but also against the SW480 and
MDA-MB 231 tumor cell lines, because their IC50 values exceeded the highest examined
concentration (data not shown). On the other hand, the HeLa cell line was susceptible
to their effect with high overall inhibition indicated by low IC50 values. Extracts 2E and
4E exhibited the strongest overall inhibitory activity after 48 h and 72 h of treatment,
followed by extracts 1E, 5E and 6E, while extract 3E had the highest IC50 . Regardless,
there was no statically significant difference among the tested extracts. Importantly, the
extracts displayed activity highly selective for HeLa cells with selectivity index (SI) values
that ranged between 8 and 20. Antitumor activity of most clinically applied agents is
restricted because of their large spectrum of side effects and general toxicity, including to
some normal cells. Although scientists continue to develop compounds with a targeted
mechanism of action, many of those compounds still lack selectivity for tumor cells [39]. In
that term, natural products are considered as less toxic for normal cells and as a biologically
friendly approach, as evidenced by the large number of extracts and secondary metabolites
in clinical trials [40].
Further, according to National Cancer Institute (NCI) recommendations [41], we
calculated three parameters to disclose whether the examined extracts had cytostatic
(GI50 , TGI) or cytocidal (LC50 ) effects on designated cell lines (Table 6). The calculated
parameters showed low to absent cytostatic or cytocidal activity of tested extracts 1E–
6E against the SW480 and MDA-MB 231 cancer cell lines and, importantly, against the
non-transformed cell line MRC-5 (data not shown). Contrarily, on the HeLa cell line,
all examined extracts acted as very potent inhibitors of net cell growth with very low
Plants 2021, 10, 711 12 of 21
GI50 values, especially extracts 2E and 4E, which exhibited a net cell growth inhibition
for 50% of cells at concentrations lower than the minimum concentration examined after
72 h of treatment (GI50 < 0.3 µg/mL). Extracts 1E, 5E and 6E exhibited a net cell growth
inhibition of 50%, with similar potency as the previous extracts. Compared to the extract of
commercial hyssop herb (1E), only extract 3E had lower cell growth inhibition activity with
a high statistical significance (p < 0.0001). The same trend was present in the perspective of
total growth inhibition and cytocidal activity. Namely, extracts 2E and 4E provoked strong
cytostatic effect after 72 h of treatment with TGI values of 1.69 µg/mL and <0.3 µg/mL,
respectively. Also, LC50 values of these extracts were significantly lower than for other
extracts (p < 0.05), indicating their potent cytocidal nature. Extracts 1E, 5E and 6E followed
the same trend. The tumor grows when the total rate of division of its cells exceeds the
total mortality rate. The ability to grow uncontrollably is gained through the accumulation
of mutations of genes that manage cell proliferation and cell death. Therefore, the agents
that can override these defects, stop uncontrolled cell division and kill cancer cells are
beneficial in cancer treatment. Tested extracts 1E, 2E and 4E–6E showed, along with high
selectivity, a potent ability both to inhibit cell proliferation and to induce cell death in a
human cervical cancer cell line. Therefore, those extracts and their compounds should be
further examined for their possible application in the therapy of this type of cancer.
The dominant compounds in the extracts are, as mentioned above, chlorogenic and
rosmarinic acids, whose cytotoxic potential has been reported earlier [42,43]. The extract
4E had the highest content of chlorogenic and rosmarinic acids, as well as total phenolic
compounds, while the extract 3E, which exhibited the weakest cytotoxic activity compared
to other tested extracts, had the lowest contents of total phenols and rosmarinic acid. On
the other hand, the extract 2E, which also gave very good results in this study, together with
the extract 4E, was distinguished neither by the content of total phenols nor by chlorogenic
or rosmarinic acids. The extracts 2E and 4E also showed antigenotoxic activity in the
comet assay (post-treatment protocol). Therefore, we can conclude that chlorogenic and
rosmarinic acid probably contributed to the overall cytotoxic potential of the methanol
extract of hyssop herb. The contribution of individual components of the extract and/or
their synergistic/additive action to selective cytotoxicity against HeLa cells is of particular
interest and should be further investigated in the future.
3. Material and Methods

3.1. Chemicals and Reagents
The acetonitrile and methanol for the chemical analysis and antioxidant activity
testing were from J.T. Baker Chemicals Co. (Phillipsburg, NJ, USA); formic acid, 2,4,6-
tris(2-pyridyl)-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferrous sulphate,
Folin–Ciocalteu (FC) reagent, ferric chloride and gallic acid were purchased from Sigma-
Aldrich (St. Louis, MO, USA); L-ascorbic acid was procured from Acros (Geel, Belgium),
whereas rosmarinic acid (≥99%), chlorogenic acid (>97%) and rutin were supplied by Carl
Roth (Karlsruhe, Germany). Solvents used for the LC-DAD-MS analysis were of LC-MS
grade, whereas the other solvents and reagents were of analytical purity.
For determination of genotoxic and antigenotoxic activity, phosphate-buffered saline
solution (PBS) was purchased from Fisher Scientific (Pittsburgh, PA, USA); hydrogen
peroxide was purchased from Zorka Pharma (Šabac, Serbia), low-melting-point agarose
(LMPA) and normal-melting-point agarose (NMPA) were purchased from Sigma-Aldrich
(St. Louis, MO, USA).
For the investigation of cytotoxic activity, Dulbecco’s modified Eagle medium (DMEM),
heat-inactivated fetal bovine serum (FBS), L-glutamine, non-essential amino acids, dimethyl
sulfoxide (DMSO), penicillin and streptomycin, as well as a trypsin and ethylenediaminete-
traacetic acid (EDTA) combination dissolved in phosphate-buffered saline (PBS) were
purchased from Sigma Aldrich (St. Louis, MO, USA). Finally, 3-(4,5-dimethylthiazol-2-yl)-
2,5 diphenyltetrazolium bromide dye (MTT) was purchased from Sigma Aldrich (St. Louis,
MO, USA).
Plants 2021, 10, 711 13 of 21
3.2. Plant Material

The flowering aerial parts of Hyssopus officinalis subsp. aristatus (Godr.) Nyman were
collected from five localities in the territory of Montenegro (Table 1).
Identification of the plant material was carried out according to the Flora Europaea [44].
Voucher specimens (Table 1) were deposited in the herbarium of the Faculty of Natural
Sciences and Mathematics in Podgorica (Montenegro), Department of Biology (TGU). The
collected plant material was air-dried at room temperature.
In addition, a commercial sample of hyssop herb was purchased from a local produc-
tion enterprise in Serbia. Commercial material was produced from the wild-growing plants
collected from the sites located in southeastern Serbia (Pirot and Nišava Districts).
Prior to hydrodistillation or extraction, dried plant material was ground to a coarse powder.
3.3. Essential Oil Isolation

Essential oils were isolated from the plant material by hydrodistillation in a clevenger-
type apparatus, according to Procedure I of the Pharmacopoea Jugoslavica IV (1984), suitable
for isolation of essential oils lighter than water [45].
For calculating the yield of essential oils (%, based on the dry weight of the plant
material), five consecutive volumetric determinations were performed.
The essential oils were kept at 4 ◦ C in amber glass vials that were tightly closed and
protected from light.
3.4. Extraction Procedure

Methanol extracts were prepared by bimaceration, according to the Pharmacopoea Ju-
goslavica IV (1984) [45]. Obtained methanol extracts were brought to dryness using a rotary
evaporator under reduced pressure and a temperature below 50 ◦ C, and subsequently, by a
stream of nitrogen. Prior to analysis, the extracts were stored at 4 ◦ C in tightly closed glass
jars. The extracts were reconstituted just before the analysis by the addition of methanol up
to the concentration of 5 mg/mL and filtered through a 0.45 µm membrane filter (Captive
Syringe Filters, Agilent, Germany).
3.5. GC-MS Analysis of Essential Oils

Qualitative and semi-quantitative chemical analysis of essential oils was performed by
gas chromatography coupled with mass spectrometry (GC-MS) on an Agilent Technologies
6890 Series gas chromatograph.
A 0.2 µL aliquot of each essential oil solution (10 µL/mL in hexane) was injected
in split mode, with a split ratio of 1:20, at the temperature 220 ◦ C. The components
were separated on a nonpolar poly (tetramethyl-1,4-sulfenylenesiloxane) HP-5ms column
(Agilent Technologies; 30 m × 0.25 mm, layer thickness 0.25 µm). The column was eluted
in the temperature-programmed mode: initial temperature 60 ◦ C, increase 3 ◦ C/min to
246 ◦ C (total analysis time 62 min). High-purity helium (5.0) was used as the carrier gas with
a constant flow of 0.9 mL/min. The effluent was transferred to the Agilent Technologies
5975 series electron ionization mass spectrometer via a transfer line maintained at 280 ◦ C.
The parameters of the mass spectrometer were as follows: electron energy 70 eV, ion source
temperature 230 ◦ C, quadrupole temperature 150 ◦ C. Acquisition scan mode was applied
in the range m/z 35–400 with a solvent delay of 2.30 min. To achieve a better agreement
between the experimental and library spectra, the standard spectra tune was used.
Acquired data were processed using Agilent Technologies MSD ChemStation software
(revision E01.01.335) in combination with NIST MS Search software (ver. 2.0d). The
spectral libraries Wiley Registry of Mass Spectral Data [46], NIST/EPA/NIH Mass Spectral
Library [47] and Adams’ mass spectral library of essential oils (3rd Edition) [48] were used
to identify mass spectra. The identity of the compounds was demonstrated by comparing
the MS spectra and linear retention indices with the literature data. The relative proportion
of compounds was determined using the area standardization method and expressed
as area %.
Plants 2021, 10, 711 14 of 21
3.6. LC-DAD-MS Analysis of Methanol Extracts

Liquid chromatography with diode array and mass spectrometry (LC-DAD-MS) was
carried out on Agilent LC/MS System 1260/6130 (Agilent Technologies, Waldbronn, Ger-
many), equipped with ChemStation software Rev. B.04.03-SP1, a degasser (model G1311B),
a quaternary pump (G1311B/1260), an autosampler (G1329B), a diode array detector (DAD)
(G4212B), a single quadrupole atmospheric pressure ionization - electrospray ionization
(API-ESI) mass selective detector (MSD) (6130) and a reverse-phase column Zorbax SB-Aq
(150 × 3.0 mm; particle diameter 3.5 µm, Agilent Technologies), maintained at an operating
temperature of 25 ◦ C.
The mobile phase consisted of 0.1% aqueous formic acid (phase A) and acetonitrile
(phase B). The following gradient elution program was used: 10% B to 35% B (0–20 min),
35% B to 90% B (20–24 min), 90% B (24–25 min) and 90% B to 10% B (25–30 min), at a total
operating time interval of 30 min, a mobile phase flow rate of 0.35 mL/min and an injection
volume of 3.00 µL. UV spectral data of all peaks were collected in the range 190 to 640 nm,
and chromatograms were recorded at 210, 270, 320 and 350 nm. API-ESI in the negative
polarity and the range m/z 100–1000 was used for analyte ionization. The parameters of
the ion source were as follows: fragmentation voltages 100 and 250 V, drying gas flow
(nitrogen) 10.0 L/min, drying gas temperature 350 ◦ C, nebulization pressure 40 psi and
capillary voltage 3500 V.
The compounds were identified by comparing their UV and MS spectral data, and the
retention times (Rt) with the corresponding data obtained for the standard compounds un-
der the same chromatographic conditions, as well as by comparing the data with previously
published literature data.
The contents of chlorogenic and rosmarinic acids were determined using the external
standard method at 320 nm.
Stock solutions of chlorogenic acid (0.4 mg/mL) and rosmarinic acid (0.5 mg/mL)
were prepared by dissolving the reference substances in methanol and their subsequent
filtration through a syringe filter (0.45 µM, Captiva, Agilent). By further dilution with the
same solvent, calibration standards with a wide range of concentrations were prepared.
This procedure (including the preparation of stock solutions) was repeated three times so
that three measurements were made for each calibration point. Calibration curves and
coefficients of determination were obtained by linear regression analysis. Limit of detection
(LoD) and limit of quantification (LoQ) were calculated according to the International
Conference on Harmonization guidelines [49].
Chlorogenic acid calibration curve (y = 26733x + 70.594, R2 = 0.9998; linearity range
0.02–0.4 mg/mL; LoD = 0.005 mg/mL; LoQ = 0.015 mg/mL) and rosmarinic acid
(y = 18000x + 39.94, R2 = 0.9998; 0.00625–0.5 mg/mL; LoD = 0.003 mg/mL; LoQ =
0.010 mg/mL) were used for contents determination.
3.7. Total Phenols

The contents of total phenolic compounds in dry methanol extracts were determined
using Folin–Ciocalteu (FC) reagent according to the method described by Velioglu et al. [50].
The results were expressed as mg of gallic acid equivalents (GAE) per g of dry extract (mg
GAE/g) and represent the mean value of the three consecutive measurements.
3.8. Antioxidant Activity

The antioxidant activity of investigated extracts was assessed by a set of two commonly
used tests: DPPH radical scavenging assay and FRAP assay.
3.8.1. DPPH Assay

The DPPH radical scavenging assay was carried out according to the methodology
described by Kukić et al. [51] with slight adaptations that were necessary for conducting
the assay on microtiter plates.
Plants 2021, 10, 711 15 of 21
The methanol solutions of the tested hyssop extracts were prepared in different con-
centrations, along with the standard methanol solutions of rutin. The test solution consisted
of a mixture of 0.1 mL of the methanol solution of tested extract, 0.1 mL of methanol and
0.05 mL of 0.5 mM methanol solution of DPPH. The mixtures were shaken vigorously and
incubated for 30 min in the dark at room temperature. The absorbances were measured at
492 nm against methanol as a blank test on Biochrom EZ Read 400 microtiter plate reader.
The negative control consisted of 0.2 mL of methanol and 0.05 mL of 0.5 mmol/L DPPH
solution. DPPH inhibition was calculated according to the following formula:
I (%) = (Ac − At)/Ac × 100 (1)
where Ac is the absorbance of the control and At is the absorbance of test solutions.
The results are expressed as half-maximum inhibitory concentration (IC50 values;
µg/mL) values, which denote the concentrations that neutralize 50% of DPPH radicals and
are the mean values of the three consecutive determinations.
3.8.2. FRAP Assay

The total antioxidant activity of dry methanol extracts of hyssop herb and standard
solutions was determined using the ferric reducing/antioxidant power (FRAP) assay,
essentially as described by Pellegrini et al. (2003) [52].
In brief, the test, standard (rutin, 0.1 mg/mL; ascorbic acid, 0.05 mg/mL) or control
solutions were transferred (0.1 mL) into test tubes and 3.0 mL of ex tempore prepared
FRAP reagent (25 mL acetate buffer, 300 mmol/L, pH 3.6 + 2.5 mL 10 mmol/L TPTZ in
40 mmol/L HCl + 2.5 mL 20 mmol/L FeCl3 × 6H2 O) was added. The absorbances were
recorded at 593 nm against a blank containing 0.1 mL of solvent after 30 min incubation at
37 ◦ C. FRAP values were calculated from the calibration curve of FeSO4 × 7H2 O solutions,
covering the concentration range between 100 and 1000 µmol/L, and expressed as mmol
Fe2+ /g dry extract (mmol Fe2+ /g). All measurements were performed in triplicate.

Peripheral blood samples from three volunteer subjects (21–35 years of age), were
collected using a method of finger extraction into heparinized containers and immediately
subjected to the experiment. The subjects were non-smokers and they had taken neither
medications nor alcohol and dietary supplements. They signed their written consent,
in accordance with the regulations of the ethical standards of the Ethics Committee for
Biomedical Investigations at the Faculty of Pharmacy in Belgrade.
Dry methanol extracts of the hyssop herb were dissolved in the phosphate-buffered
saline solution (PBS); the essential oils were dissolved in absolute ethanol and then diluted
with PBS.
3.9.1. Genotoxic Activity Assay

Human whole blood cells (WBC) were incubated with different concentrations of
essential oils (12.5, 5 and 2.5 µg/mL) or methanol extracts of hyssop herb (100, 200 and
400 µg/mL) for 30 min at 37 ◦ C. Tested concentrations were selected on the basis of the
literature data [53,54]. The samples were treated in parallel with a negative control of
PBS for 30 min at 37 ◦ C, as well as with a positive control of hydrogen peroxide (H2 O2 ,
50 µM) for 20 min at 4 ◦ C and PBS for 30 min at 37 ◦ C. A concentration of 50 µM H2 O2
was the lowest one that induced a statistically significant increase of DNA damage in the
tested cells.
3.9.2. Antigenotoxic Activity Assay

The antigenotoxic activity of extracts or essential oils (which did not show activity
in the genotoxicity assay) was analyzed in the post-treatment [55,56]. WBC were treated
with H2 O2 for 20 min at 4 ◦ C and washed with PBS; afterward, they were incubated
for 30 min at 37 ◦ C with tested essential oils/methanol extracts. The concentration of
Plants 2021, 10, 711 16 of 21
400 µg/mL was chosen for further testing, because it was the most effective concentration
in the antigenotoxic assessment of commercial extract. The attenuation of H2 O2 -induced
DNA damage in human peripheral blood leukocytes in the post-treatment with EO was
assessed using the concentration that did not induce a statistically significant increase of
DNA damage in the tested cells in the genotoxicity assessment (2.5 µg/mL).
3.9.3. Comet Assay

The comet assay (single-cell gel electrophoresis), which is normally used to detect
DNA damage, was performed according to the protocol described by Singh et al. (1988),
called the alkaline method, according to which DNA breaks and alkaline labile sites are
detected, while the degree of DNA damage is indicated by the extent of DNA molecule
migration [57].
A quantity of 100 µL of the test suspension, containing 6 µL of peripheral blood
suspended in 0.67% of LMPA, was evenly applied to the prepared microscopic plates
coated with a layer of 1% NMPA. Cover glasses were placed on the plates and then left at
4 ◦ C for 5 min. The cover glasses were slowly removed and the cells were exposed to the
appropriate treatment (as previously described for genotoxic or antigenotoxic activities).
After treatment, the third layer of 100 µL of 0.5% LMPA was applied, cover glasses were
placed and the plates were again left at 4 ◦ C for 5 min. The cover glasses were again
carefully removed, the microscope plates were immersed in lysis solution (2.5 M NaCl,
100 mM EDTA, 10 mM Tris, 1% Triton X100 and 10% DMSO; pH 10 adjusted with NaOH)
and kept at 4 ◦ C overnight. After cell lysis, DNA denaturation was performed in the same
solution that was later used for electrophoresis for 30 min (10 M NaOH, 200 mM EDTA,
pH ≥ 13).
Electrophoresis was performed at 25 V and 300 mA for 30 min. After electrophoresis,
alkali neutralization in gels was performed by rinsing twice with a neutralizing buffer
(0.4 M Tris, pH 7.5) for 10 min, and then with distilled water. After rinsing, the comet was
stained and visualized with fluorescent dye-ethidium bromide solution (20 µg/L). After
staining (15 min), the plates were ready for analysis.
All experiments were done three times, in duplicate.
To determine the degree of DNA damage for each donor and each concentration,
200 nucleoids (comets) were selected and analyzed by random selection, or 100 from each
duplicate microscope plate, using an Olympus BX 50 fluorescent microscope (Olympus
Optical Co., GmbH, Hamburg, Germany) with a mercury short arc lamp (HBO) (50 W,
516–560 nm Carl Zeiss Microscopy, Jena, Germany) at 100 × magnification.
The analysis was performed by determining the length and density of DNA in the
“tail” based on which of the nucleoids (comets) were classified into five groups, as described
by Anderson et al. (1994) [58]: A—without the “tail” (<5% DNA damage), B—low degree of
damage (5%–20%), C—medium degree of damage (20%–40%), D—high degree of damage
(40%–95%) and E—complete DNA damage (>95%). The degree of total DNA damage was
expressed as the sum of all DNA damage/migrations over 5% (B + C + D + E).
3.10. Investigation of Cytotoxic Activity of Hyssop Herb

3.10.1. Cell Lines and Cultures
The potential cytotoxic effect of methanolic extracts was examined on the human
cervix (HeLa), breast (MDA-MB 231) and colon (SW480) cancer cell lines, while their
selectivity was tested on non-transformed human lung fibroblasts (MRC-5). Cell lines were
obtained from American Type Culture Collection (ATCC).
Cells were cultured in DMEM at pH = 7.4, enriched with 10% heat-inactivated FBS,
L-glutamine, non-essential amino acids (0.1 mM), penicillin (100 IU/mL) and streptomycin
(100 µg/mL). Cultivation was performed in T-25 flasks (ThermoFisher Scientific, Waltham,
MA, USA) in an aseptic environment under standard culture conditions (37 ◦ C, absolute
humidified air and 5% CO2 ). The media were changed when necessary and cells were
subcultured every fifth day. Just before in vitro experiments, subconfluent cell monolayers
Plants 2021, 10, 711 17 of 21
(~80%) in the logarithmic growth phase were detached from the bottom of the flask by
short-term treatment with 0.25% trypsin and 0.53 mM EDTA combination dissolved in
phosphate-buffered saline.
3.10.2. Extract Solutions

The stock solutions were prepared by dissolving methanol extracts in DMSO at
50 mg/mL and stored at 4 ◦ C. Preceding the treatment, fresh working solutions of the ex-
tracts at different concentrations were prepared by diluting stock solution in supplemented
DMEM. The final DMSO concentration in working solutions was lower than 0.5% (v/v).
3.10.3. MTT Assay

The cytotoxic potential of the extracts against HeLa, MDA-MB 231, SW480 and MRC-
5 was evaluated in vitro by MTT assay as a common colorimetric technique for cell viability
determination [59].
The cells were seeded in 96-well flat-bottom microtiter plates (ThermoFisher Scientific,
Waltham, MA, USA) at a density of 5 × 103 cells per well and incubated overnight to
adhere. After 24 h, the supernatant was replaced with extract solutions at seven different
concentrations (0.3, 1, 3, 10, 30, 100 and 300 µg/mL). In the control wells, cells grew in the
presence of supplemented nutrient media only. The cells were incubated for 24, 48 and 72 h.
The MTT solution, at a final concentration 0.5 mg/mL in the unsupplemented medium,
was added to each well at time zero (after overnight incubation) and the end of different
incubation periods. Following 2 h of incubation, the MTT solution was discarded and
formazan crystals were solubilized with 150 µL of DMSO. The plates were shaken for 5 min
and the absorbance was measured at 550 nm with a multiplate reader (Zenyth 3100, Anthos
Labtec Instruments GmbH, Wals-Salzburg, Austria).
All experiments were repeated at least three times in triplicate.
3.10.4. Cytotoxicity Parameters

The results of the MTT assay are presented as the percentage of the values for control
cells that was arbitrarily set to 100%. Cell growth inhibition was calculated according to
the expression:
(A0 − A) × 100/A0 (2)
where A0 is absorbance from control wells and A is absorbance from wells exposed to the
tested extracts.
The measure of the overall inhibitory activity of the agent was evaluated through IC50 ,
which is defined as the concentration of the agent that inhibits the biological activity of the
target cells by 50%.
The selectivity index (SI) was calculated as the quotient of IC50 values for the treated
non-transformed cell line and the IC50 values for the tested extracts on malignant cells.
SI < 2 indicates general toxicity of a compound, SI ≥ 2 indicates selective toxicity and
SI ≥ 3 indicates highly selective toxicity [60].
Following the NCI recommendations [41], GI50 , TGI and LC50 parameters were calcu-
lated for each extract:
• The GI50 value is the concentration where 100 × (T − T0)/(C − T0) equals 50 and
measures the growth inhibitory power of the examined extracts;
• The TGI value is the concentration of the tested extract where 100 × (T − T0 )/(C − T0 )
equals 0 and measures the cytostatic effect;
• The LC50 value is the concentration of the drug where 100 × (T − T0 )/T0 equals
50 and measures the cytotoxic effect of extracts.
In these formulas, T0 is the absorbance at time zero (when the compound is added),
Tis the absorbance of the test well after 24, 48 or 72 h of exposure to the test compound and
C is the optical density of the control wells (cells incubated for 48 h with no additives). If
Plants 2021, 10, 711 18 of 21
the effect was not reached or was exceeded, the value for that parameter was expressed as
greater or less than the maximum or minimum concentration tested.
3.11. Statistics
The results of the experiments are represented as mean ± standard deviation.
Principal component analysis (PCA) and hierarchical cluster analysis (HCA) using
Statistica® v.8.0 (www.statsoft.com, accessed on 1 April 2021) and StatistiXL® Version
2.0 add-in for MS Excel® (www.statistixl.com, accessed on 1 April 2021) were applied to
examine the interrelationships between the chemical compositions of the essential oils.
The results of LC-MS quantitative analysis and assays on antioxidant activity were
analyzed by SPSS software (version 20.0) using one-way ANOVA and post hoc Tukey’s test.
Differences between the mean values were considered statistically significant if p < 0.05.
The IC50 , GI50 , TGI and LC50 parameters were calculated using MS Office Excel® free
add-in ED50 plus v1.0 software (www.sciencegateway.org/protocols/cellbio/drug/data/,
accessed on 1 April 2021). SPSS software version 20 was used for statistical data analysis.
The Shapiro–Wilk test was used to test the normality of data distribution. Depending
on the results normality test, for the comparison of groups, one-way analysis of variance
(ANOVA) or its non-parametric equivalent Kruskal–Wallis test was used.
For the genotoxic and antigenotoxic activity assays, the results are expressed as the
mean value (n = 3) ± standard error of the mean (SEM). Statistical analysis of the comet
assay results was performed using one-way analysis of variance (ANOVA) with Tukey’s
post hoc test for comparisons of different treatments vs. the respective controls. GraphPad
Prism 6.0 software was used. A regression was used to determine the effect of the bioactive
substance concentration on the outcome. The values of the obtained data were considered
statistically significant if p < 0.05, and statistically highly significant if p < 0.001.
4. Conclusions
This study evaluated the antioxidant activity and genotoxicity/antigenotoxicity, as
well as cytotoxic, cytostatic and cytocidal effects against human tumor and non-transformed
human lung fibroblast cell lines of the investigated Hyssopus officinalis essential oils and/or
extracts, with respect to their chemical composition. Our results revealed high variability
in the composition of essential oils, as three chromatographic profiles of the investigated
essential oils of wild-growing plants from Montenegro could be distinguished: oils rich
in 1,8-cineole and relatively rich in β-pinene, but low in cis-pinocamphone; oils rich in
β-pinene, limonene, cis-pinocamphone and methyl eugenol, but relatively low in 1,8-cineol;
and oils relatively rich in 1,8-cineole, limonene, β-pinene and cis-pinocamphone. The
essential oil from the commercial plant material from Serbia, being rich in 1,8-cineole
and β-pinene, but low in cis-pinocamphone, appeared similar to only one of the samples
obtained from wild-growing plants from Montenegro. Both the extracts and the essential
oils significantly reduced in vitro DNA damage. In addition, potent and selective cytotoxic
action of the hyssop methanol extracts on the HeLa cell line was observed. These findings
deserve closer attention and our further investigations, which will be performed in the
prospective future, and should be directed to the panel of cancer cell lines derived from the
most sensitive tissue (cervix), along with a detailed mechanism of antitumor effect and the
isolation/chemical characterization of the constituents that are presumably responsible for
observed activity.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/plants10040711/s1, Figure S1: A comparative view of LC-DAD chromatograms of methanol
extracts (1E–6E) of H. officinalis recorded at 320 nm, Figure S2: Dose-response curves in MTT assay
after 24, 48 and 72 h treatment of MRC-5 (a–f), SW480 (g–l), MDA-MB 231 (m–r) and HeLa (s–x) cell
lines with extracts 1E–6E, Table S1: Component loadings and score coefficients for constituents of
Hyssopus officinalis essential oils.
Plants 2021, 10, 711 19 of 21
Author Contributions: T.M. participated in all the experiments (as a part of her PhD work) and
wrote the manuscript draft. D.T. performed investigation on genotoxicity and antigenotoxicity, the vi-
sualization of the data and contributed to writing the original draft. L.Ž. performed investigation and
provided resources for the tests on genotoxicity and antigenotoxicity. B.S.-P. performed investigation,
provided methodology for the tests of genotoxicity and antigenotoxicity, and conducted supervi-
sion. V.J. provided resources, methodology and supervision for investigations on cytotoxic activity.
S.M., S.P. and D.B. performed investigations on cytotoxic activity, their validation, visualization and
writing of the original draft. D.S. (Danijela Stešević) and D.S. (Danilo Stojanović) performed field
investigations, identified plant material and performed the writing, review and editing process. S.S.
performed chromatographic analyses and the interpretation of collected data, and also contributed
to writing of the original draft and the review and editing processes. Z.M. was responsible for the
conceptualization, resources, supervision, and the writing, review and editing process. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study on genotoxic and antigenotoxic activity was
conducted according to the guidelines of the Declaration of Helsinki, and approved by Ethics
Committee for biomedical investigations at the Faculty of Pharmacy in Belgrade (protocol code
1121/2; date of approval: 31 August 2020).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study on genotoxic and antigenotoxic activity.
Data Availability Statement: Data available upon request.
Acknowledgments: Supported by the Ministry of Education, Science and Technological Develop-
ment of the Republic of Serbia; contract No. 451-03-9/2021-14/200161.
Conflicts of Interest: The authors declare no conflict of interest.
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Plants 2021, 10, 711. https://doi.org/10.3390/plants10040711 https://www.mdpi.com/journal/plants

2. Results and Discussion

Essential Oil MeOH Extract

The dominant group of identified volatiles was monoterpenes (70.88–95.91%). Their

Figure 2. Dendrogram representing the similarity relations of the chemical compos

2.2. Methanol Extract Compositions and Contents of Total Polyphenols

Peak No. tr (min) UV λmax (nm) ESI-MS Data (m/z) Assignment

2.3. Antioxidant Activity

Sample DPPH-IC50 (µg/mL) FRAP mmol Fe2+ /g

2.4. Genotoxic and Antigenotoxic Activity

Figure 3. Antigenotoxic properties of methanol extracts of H. officinalis subsp. aristatus (1E–6E)

** p < 0.001, *** p < 0.0001.

3. Material and Methods

3.2. Plant Material

3.3. Essential Oil Isolation

3.4. Extraction Procedure

3.5. GC-MS Analysis of Essential Oils

3.6. LC-DAD-MS Analysis of Methanol Extracts

3.7. Total Phenols

3.8. Antioxidant Activity

3.8.1. DPPH Assay

I (%) = (Ac − At)/Ac × 100 (1)

3.8.2. FRAP Assay

3.9. Genotoxic and Antigenotoxic Activity

3.9.1. Genotoxic Activity Assay

3.9.2. Antigenotoxic Activity Assay

3.9.3. Comet Assay

3.10. Investigation of Cytotoxic Activity of Hyssop Herb

3.10.2. Extract Solutions

3.10.3. MTT Assay

3.10.4. Cytotoxicity Parameters

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

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p < 0.001, * p < 0.0001.