BioRisk | 7 | 79— | 90 (2022) Apeer-reviewed open-access journal

doi: 10.3897/biorisk.17.77313 RESEARCH ARTICLE & Ke Ke) Re IS k

https://biorisk.pensoft.net

On the mode of action of Origanum vulgare spp.
hirtum methanolic extract and essential oil
on Chlamydomonas reinhardtii

Maria D. Todorova', Petya N. Parvanova', Teodora I. Todorova',
Georgi D. Dronchev', Milena T: Nikolova’, Strahil H. Berkov’, Stephka G. Chankova'

| Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Str., 1113 Sofia,
Bulgaria 2. Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Acad. G. Bonchev
Str, Bl. 23, 1113 Sofia, Bulgaria

Corresponding author: Stephka Georgieva Chankova (stephanie.chankova@yahoo.com)

Academic editor: K. Danova | Received 29 October 2021 | Accepted 14 December 2021 | Published 21 April 2022

Citation: Todorova MD, Parvanova PN, Todorova TI, Dronchev GD, Nikolova MT, Berkov SH, Chankova SG
(2022) On the mode of action of Origanum vulgare spp. hirtum methanolic extract and essential oil on Chlamydomonas
reinhardtii. In: Chankova S, Peneva V, Metcheva R, Beltcheva M, Vassilev K, Radeva G, Danova K (Eds) Current trends
of ecology. BioRisk 17: 179-190. https://doi.org/10.3897/biorisk.17.77313

Abstract

Aim: To reveal whether methanolic extract and essential oil from Origanum vulgare subsp. hirtum in doses causing
even low levels of mortality in aphids, would have harmful effects on plants-genotoxic, mutagenic and/or DNA
damaging. Materials and methods: Aerial parts of Origanum vulgare ssp. hirtum from the ex-situ collection of
IBER, BAS during flowering were collected. Extraction and isolation procedures, as well as GC/MS analysis of
essential oil and methanolic extract were performed by standard protocols. The components were identified by
comparing their relative retention times to the retention times of authentic standards, and with mass spectra
with the NIST. Test system: Chlamydomonas reinhardtii strain 137 C+ (WT). Endpoints: “clonal” assay, the
test of “visible mutations”, constant field gel electrophoresis. Statistics: GraphPad Prism version 6.04 (San
Diego, USA) and One-way Analysis of Variance ANOVA with multiple comparisons using the Tukey method.
Results: A good correlation was observed between chemical composition of essential oil and methanolic extract,
and their mode of action. Our genotoxic and double strand breaks results demonstrated mild genotoxic and
statistically non-significant DNA damaging potential of methanolic extract and concentration-dependent well
- expressed genotoxic and DNA damaging potential of essential oil. A good relationship between increased
double strand breaks levels and decreased survival might be related to one of the main constituents of essential
oil, suspected to be carvacrol. No mutagenic effect for ME and EO was found. Conclusion: Well-expressed
toxic/genotoxic capacity of essential oil, as well as its capacity to damage DNA inducing double strand breaks,
but the absence of mutagenic potential, could be considered as a good reason to recommend Origanum vulgare

subsp. irtum essential oil as a promising candidate for purposes of “green” technologies.

Copyright Maria Dimitrova Todorova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

180 Maria Dimitrova Todorova et al. / BioRisk 17: 179-190 (2022)

Keywords
Cell survival, Chlamydomonas reinhardtii, DSBs, mutations, Origanum vulgare spp. hirtum methanolic

extract, Origanum vulgare spp. hirtum essential oil

Introduction

For decades, the application of chemical / synthetic pesticides has been the most
effective and common tool for weed, and pests control in agriculture. Unfortunately,
their long-term use negatively affects the environment and biota, including human
health (Gill and Garg 2014; Chu and Karr 2017; Bocker et al. 2019) due to the
low biodegradability of most of them, and their ability to accumulate in the basic
environmental matrices (Ali et al. 2019). On the other hand, many target organisms
have increased their resistance to certain groups of pesticides that has provoked the
development and release of new groups of chemical compounds (Heap 2021). As
of 28 October 2021, the International Herbicide Resistant Weed website reported
that 266 weed species (153 dicots and 113 monocots) were resistant to 164 different
herbicides (Heap 2021). These alarming data “force” the scientific community to look
for an environmentally-friendly solution for successful control of weed populations
(Gnanavel 2015; Stankovic et al. 2020), and increased target specificity, as well as rapid
degradation of the active substance (Cordeau et al. 2016).

Plants-based bioactive compounds with pesticide and/or herbicidal potential have
been the focus of scientists for at least three decades (Balandrin and Klocke 1988;
Gerwick and Sparks 2014; Fouad et al. 2015; Bona et al. 2016; Della Pepa et al. 2019;
Elshafie et al. 2019; Grulova et al. 2020) due to their chemical composition (Araniti et
al. 2018; Jankowska et al. 2018; Lins et al. 2019).

Till now, the question of whether and how plants’ essential oils or/and extracts
could be effectively used for the purposes of “green agro chemistry” is ongoing. New
information has been gathered about their insecticidal and inhibitory activity on seed
germination and weed seedling growth (Matkovié et al. 2018; Nikolova and Berkov
2018; Stankovic et al. 2020), but information about their mutagenic, and/or DNA
damaging effects are scarce (Karpouhtsis et al. 1998; Llana-Ruiz-Cabello et al. 2018).

This investigation was based on our previous finding that Origanum vulgare ssp.
hirtum extracts and essential oil negatively affect Myzus persicae survival (Parvanova et
al. 2020). Here, using C. reinhardtii as a plant test system, we aimed to reveal whether
methanolic extract and essential oil of Origanum vulgare subsp. hirtum in doses causing
even low levels of aphid’s mortality would have harmful effects on plants — genotoxic,
mutagenic, and/or DNA damaging.

C. reinhardtii was chosen because it is a robust model test-system in environmental
mutagenesis (Chankova et al. 2006, 2013; Dimitrova et al. 2007, 2009, 2014; Chank-
ova and Yurina 2012; Chen et al. 2012; Kopaskova et al. 2012; Jamers et al. 2013;
Melegari et al. 2013; Aksmann et al. 2014; Angelova et al. 2014; Boenigk et al. 2014;
Chalifour et al. 2014; Almeida et al. 2019; Xu et al. 2019; Todorova et al. 2020).

Effect of Origanum vulgare ME and EO on Chlamydomonas reinhardtii 181

Materials and methods

Plant materials. Aerial parts of Origanum vulgare ssp. hirtum were collected during
the flowering stage from the ex-situ collection of the Institute of Biodiversity and Eco-
system Research (IBER), http://www. iber.bas.bg/sites/default/files/projects/plantscol-
lection/index.html.

Preparation of methanolic extract (ME). Air-dried, ground aerial parts of the
species were extracted with methanol by classical maceration for 24 h. After filtration,
the organic solvent was evaporated and the resulting crude extract was subjected to
further analysis.

Isolation of essential oil (EO). ‘The essential oil was extracted on a Clevenger appara-
tus by water distillation from 50 g dry plant material in a flask with 500 ml water for 2 h.

GC/MS analysis of EO and ME. For GC/MS analysis, 50 mg of methanolic extract
was silylated with 50 ul of N, o-bis- (trimethylsilyl) trifluoroacetamide (BSTFA) in 50
ul of pyridine for 2 h at 50 °C. The spectra were recorded on a Thermo Scientific Focus
GC combined with a Thermo Scientific DSQ mass detector as described previously
(Berkov et al. 2021). The chromatographic conditions for EO analysis were described
by Traykova et al. (2019). The quantities of the compounds have been expressed as
the percentage area of the total peaks’ area of the chromatogram. The components
were identified by comparing their mass spectra and retention indices (RI) to known
compounds from the literature, National Institute of Standards and Technology
(NIST) and home-made MS databases.

Toxicity/Genotoxicity — a “clonal” assay, based on colony forming ability, was
performed (Dimitrova et al. 2007). C. reinhardtii WT137C was cultivated at standard
conditions — light of 70 umol-m™-s! and t = 25 + 3 °C to the end of the exponential
and the beginning of the stationary phase. Concentrations of ME and EO — 250,
500, 750, 1000 ppm, as well as exposure time, were defined previously. Two negative
controls — Sager-Granick liquid medium (SG) and 1000 ppm DMSO as a solvent, and
one positive control — Nurelle D at the commercially- recommended concentration of
500 ppm for insects, were used. Both survival fraction (SF) (Bryant 1968) and three
levels of lethality were calculated (Lidanski 1988).

Mutagenicity — test of “visible mutations”, based on the changes in size,
morphology, and pigmentation of surviving colonies, was applied. The method and
calculations of a percentage of induced mutant colonies and index of mutagenicity
(IM) were described by Dimitrova et al. (2007). When IM < 2.5 — no mutagenic effect
is identified, when IM is in the range of 2.5 to 10, the mutagenic effect is mild, and
when IM > 10, the mutagenic effect is moderate to strong.

DNA -— the damaging potential of both ME and EO was evaluated by CFGE
(Constant Field Gel Electrophoresis) (Chankova and Bryant 2002). The advantage
of this method is well described by (Chankova et al. 2007). After electrophoresis
ended, the gel was visualised in UV light and captured with a digital camera and the
GeneSnap programme (SynGene). The images were analysed with GeneTools software
(SynGene). The fraction of DNA released (FDR) from the wells was calculated
according to Chankova et al. (2009).

182 Maria Dimitrova Todorova et al. / BioRisk 17: 179-190 (2022)

Data analysis — All presented data are averages from at least three independent
experiments. Statistical data processing was performed with GraphPad Prism version
6.04 (San Diego, USA) and One-way Analysis of Variance (ANOVA) with multiple

comparisons, using the Tukey method to compare the results of different treatments.

Results

Chemical composition — carvacrol (74.34%), p-cymene (9.46%), y-terpinene (10.66%),
a-pinene (1.73%), J-pinene (1.34%) and carvacrol methyl ether (1.23%) were identi-
fied as the main components of essential oil. The other components were presented in
quantities of less than 1%.

In the methanolic extract, various primary metabolites as fructose (10.32%),
glucose (11.65%), sucrose (10.51%), organic acids — succinic (0.60%), malic (1.09%)
and linolenic acid (0.78%) were found. Phenolic acids — 4(p)-hydroxybenzoic (0.53%)
and vanillic (0.22%), rosmarinic acid (6.06%), terpenoids — carvacrol (15.67%),
caryophyllene (0.40%), flavonoids — catechin (0.23%) 6-hydroxyflavone glycoside
(1.49%) were identified as the main secondary metabolites.

Toxicity/Genotoxicity — as the first step of our investigation, we had to clarify
two points: whether Nurelle D, chosen as a positive control at the recommended
commercial dose for aphids control, would have a detrimental effect on the model
plant cells and whether DMSO, as a solvent, would affect C. reinhardtii cells
negatively. As seen in Fig. 1, no statistically significant decrease in the cells survival
fraction after the treatment with DMSO and Nurelle D was found, compared to the
negative control SG (P < 0.05).

ns ns

SG DMSO 1000 ppm _ =~=-—s Nurrelle D® 500 ppm

Figure |. Cells survival fraction in negative and positive control samples of C. reinhardtii strain 137C.
Mean values from at least three independent experiments. Error bars represent standard errors of mean
values. The statistical significance of the differences is presented as follows: * P < 0.05; ** P < 0.01;

** P < 0.001; ns — no significant difference.

Effect of Origanum vulgare ME and EO on Chlamydomonas reinhardtii 183

Analysing curves in Fig. 2, statistically significant reduction of cell survival after
the treatment with both highest concentrations of ME 750 ppm (SF = 0.25 + 0.08)
and 1000 ppm (SF = 0.00026 + 0.08) was shown. In the range of 250 ppm and
500 ppm, some plateau was formed. Better expressed dose-effect was obtained for
EO (Fig. 2) at a concentration of 250 ppm or above (SF = 0.51 + 0.09; SF = 0.34
+ 0.11 and SF = 0.09 = 0.02) (*** p < 0.0001).One-way ANOVA analysis reveals
statistically significant differences between: SG vs. 250 ppm EO; SG vs. 500 ppm
EO; SG vs. 750 ppm EO; SG vs. 1000 ppm EO; SG vs. 750 ppm ME; SG vs.
1000 ppm ME (***, p < 0.001).

-—@-methanolic extract -M&essential oil

a

am

2 VR eg
508 7 ~*
5S

E 0.6

20.4

a

= 0.2

=

+h

0 250 500 750 1000
Concentration (ppm)

Figure 2. Cells survival fractions (SF) after the treatment with Origanum vulgare ME and EO. Mean data
are from three independent experiments. Error bars represent standard errors of mean values. Where no

error bars are evident, errors were equal to or smaller than the symbols.

Further, we had to calculate three levels of lethality - LD,,, LD,, and LD,, as a
commonly-used approach for comparing the genotoxic potential of standard mutagens
or other chemical/physical factors. The stronger genotoxic potential of essential oil is
obvious by data shown in Table 1. Approximately two-fold lower EO concentrations
can induce LD,,, LD,, and LD,, comparing with those of ME.

20°

Table I. LD,,, LD,, and LD,, in strain 137C, measured after the treatment with Origanum vulgare spp.
hirtum ME and EO.

Origanum vulgare subsp. hirtum LD LD LD
Methanolic extract [ppm] 523 634 810
Essential oil [ppm] < 250 263 588

184 Maria Dimitrova Todorova et al. / BioRisk 17: 179-190 (2022)

Mutagenicity — test of “visible mutations”

The next step in our investigation was to reveal whether both ME and EO of Origanum
vulgare spp. hirtum possess some mutagenic potential on C. reinhardtii. The level of
spontaneous “visible mutations” was 0.136%. Calculated IM clearly demonstrated an
absence of mutagenic capacity for DMSO and Nurelle D. No mutagenic capacity of
ME and EO was identified (IM < 2).

DNA-damaging potential of ME and EO

Our CFGE results show no DNA damaging capacity of ME. The levels of DSBs,
measured after treatment with concentrations in the range of 250 — 1000 ppm, were
approximately similar to the levels of spontaneously arisen DSB in the control sample
(Fig. 3). One-way ANOVA analysis reveals statistically significant differences between:
SG vs. 250 ppm EO; SG vs. 500 ppm EO; SG vs. 750 ppm EO; SG vs. 1000 ppm EO
(**, p < 0.001); SG vs. 500 ppm ME (*, p < 0.05).

A quite different curve was drawn from the DSB levels measured after treatment
with EO (Fig. 3). Around two-fold higher levels of DSBs were calculated compared
to those in the negative control and the samples treated with methanolic extract. All
differences, showing higher values compared to the negative control, were statistically
significant. These results demonstrate a stronger DNA-damaging potential of oregano
EO than that of ME under our experimental conditions. A well evident correlation
was found between the potential of oregano EO to induce DSBs and lower SF meas-
ured as a colony-forming ability.

-@essential oil -M-methanolic extract

0 250 500 750 1000 1250
Concentration (ppm)

Figure 3. DSBs measured after treatment with different concentrations of ME and EO of Origanum
vulgare spp. hirtum. Mean data are from three independent experiments. Error bars represent standard
errors of mean values. Where no error bars are evident, errors are equal to or smaller than the symbols.
The statistical significance of the differences is presented as follows: * p < 0.05; ** p < 0.01; *** p < 0.001;

ns — no significant difference.

Effect of Origanum vulgare ME and EO on Chlamydomonas reinhardtii 185

Discussion

Previously, it was found by us that Origanum vulgare ssp. hirtum extracts and essential
oil can cause Myzus persicae mortality depending on the concentrations applied. Here,
an attempt was made to clarify whether ME and EO of Origanum vulgare subsp.
hirtum, in doses causing even low levels of mortality in aphids, would have harmful
effects — toxic/genotoxic, mutagenic and/or DNA damaging on C. reinhardtii, used as
a plant test-system.

The better-pronounced capacity of EO vs. ME to decrease C. reinhardtii cell
survival was demonstrated by comparing the concentrations inducing three levels of
lethality - LD,,, LD,, and LD,,. It was calculated that EO is about 1.4—2-fold more
genotoxic for algae cells than ME. Till now, a large spectrum of effects of oregano
EO has been described — phytotoxic (Ibafiez and Blazquez 2017, 2020; Grul’ova et
al. 2020; Abd-ElGawad et al. 2021), antimicrobial (Karaday et al. 2020; Simirgiotis
et al. 2020), antifungal (Puskarova et al. 2017; Saghrouchni et al. 2021); insecticidal
(Alkan 2020), anti-plant pathogens (Raveau et al. 2020) etc. The data reported by us
have further expanded this spectrum.

Information concerning DNA damaging or mutagenic potential of ME and EO
are scarce. Llana-Ruiz-Cabello et al. (2018), using both MN test and comet assay
(standard and enzyme-modified), have reported no increased levels of MN and DNA
damage. Contrary to this study, our experiments revealed, for the first time, the DNA
damaging capacity of oregano EO. It was clarified that the level of DSBs depends on
the concentration applied. The good relationship between increased DSBs levels and
decreased survival, described by us, might be linked to one of the main constituents of
EO, likely to be carvacrol.

Both oregano ME and EO were shown to possess no mutagenic activity in
C. reinhardtii test-system. Similar findings have been described previously by others
(Adam et al. 1998; Karpouhtsis et al. 1998). No mutagenic effect on Aims test and no
mutagenic or recombinogenic activity for ME and EO were found, using the Wing
Somatic Mutation and Recombination Test (SMART) on D. melanogaster.

Conclusion

In this study, mild toxic/genotoxic and statistically non-significant DNA damaging
potential of ME and concentration-dependent effects of EO were identified. The
differences in the mode of action of EO and ME could be related to differences in
their chemical composition. Further experiments are required in order to clarify
the effect of main and minor constituents. Well-expressed toxic/genotoxic capacity
of EO, as well as its capacity to damage DNA inducing DSBs, but the absence
of mutagenic potential, could be considered as a good reason to recommend
Origanum vulgare subsp. hirtum EO as a promising candidate for purposes of
“green” technologies.

186 Maria Dimitrova Todorova et al. / BioRisk 17: 179-190 (2022)

Acknowledgements

This research was funded by The Bulgarian National Science Fund, contract number
16722 Ohl 220173

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