Journal of Ethnopharmacology

In vivo antioxidant activity of Clinopodium vulgare L. leaves extract correlates with

amelioration of zeocin-induced Ty1 retrotransposition, reverse mutations in the ilv1-92
allele and mitotic crossingover in ade2 locus on Saccharomyces cerevisiae
--Manuscript Draft--

Manuscript Number:

Article Type: Research Paper

Keywords: Clinopodium vulgare; Saccharomyces cerevisiae; antioxidant; mutagenesis;

carcinogenicity

Corresponding Author: Teodora Todorova

Bulgarian Academy of Sciences
Sofia, BULGARIA

First Author: Teodora Todorova

Order of Authors: Teodora Todorova

Krum Bardarov

Stephka Chankova

Abstract: Ethnopharmacological relevance

Antibacterial, anti-inflammatory, DNA protective and anticancer activity of wild basil (

Clinopodium vulgare L.), widely applied in folk medicine for treatment of cancer,
hemorrhagic disease, gastric ulcers , diabetes, mastitis, prostatitis, skin irritation and
swelling in the Balkan Peninsula has been reported. Little is currently known about the
genotoxicological mode of action.

Aims of the study

to enlarge the present state of knowledge concerning 1) in vivo pro-

oxidant/antioxidant as well as genotoxic, mutagenic and carcinogenic potential of
Clinopodium vulgare L. leaves extract; 2) the relationship between antioxidant
capacity of Clinopodium vulgare L. leaves extract and its antigenotoxic, antimutagenic
and anticarcinogenic potential against the standard radiomimetic zeocin.

Material and methods

Three Clinopodium vulgare L. leaves extract concentrations (10, 100 and 1000 µg/ml)
are tested on Saccharomyces cerevisiae as a human cell model. In vivo antioxidant
activity (antiROS test), antimutagenic (Zimmermann test) and anticarcinogenic (Ty1
transposition test) effects were tested. Zeocin was used as a positive control.

Results

No pro-oxidative, mutagenic or carcinogenic effect was obtained for concentrations of

10 and 100 µg/ml Clinopodium vulgare extract. The minor pro-oxidative activity of
1000 µg/ml extract measured in vivo by antiROS test did not correspond with increase
of mitotic gene conversion, reversion and mitotic crossing-over. Well-expressed
protective capacity of Clinopodium vulgare L. leaves extract against the radiomimetic
zeocin was determined, decreasing zeocin-induced superoxide anions (6-fold);
revertants (4-fold); total aberrants (2-fold) and transposants (4-fold).

Conclusion

Here, for the first time, in vivo antioxidant activity of Clinopodium vulgare L. leaves
extract has been evaluated. The concentrations in the range of 10-1000 µg/ml do not
possess any genotoxic, mutagenic or carcinogenic capacity on Saccharomyces
cerevisiae as a model for human cell. Experimental evidence is provided that the
extract possesses well-expressed protective effect against the radiomimetic zeocin.

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 Strong correlation between antioxidant activity and amelioration of zeocin-induced Ty1
retrotransposition, reverse mutations in the ilv1-92 allele and mitotic crossingover in
ade2 locus – some of the initial steps of tumorigenesis is defined.

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Cover Letter

To: prof. A.M. Viljoen, PhD.

Editor-in-Chief
Journal of Ethnopharmacology
July 27, 2020

Dear prof. A.M. Viljoen,

My team and I are pleased to submit an original research article entitled “In vivo antioxidant activity of
Clinopodium vulgare L. leaves extract correlates with amelioration of zeocin-induced Ty1 retrotransposition,
reverse mutations in the ilv1-92 allele and mitotic crossingover in ade2 locus on Saccharomyces cerevisiae” by
Teodora Todorova, Krum Bardarov and Stephka Chankova for consideration for publication in the Journal of
Ethnopharmacology.
In the present study, for the first time, in vivo antioxidant activity of Clinopodium vulgare L. leaves extract
has been evaluated. The concentrations in the range of 10-1000 µg/ml do not possess any genotoxic, mutagenic
or carcinogenic capacity on Saccharomyces cerevisiae as a model for human cell. Experimental evidence is
provided that the extract possesses well-expressed protective effect against the radiomimetic zeocin. Strong
correlation between antioxidant activity and amelioration of zeocin-induced Ty1 retrotransposition, reverse
mutations in the ilv1-92 allele and mitotic crossingover in ade2 locus – some of the initial steps of tumorigenesis
is defined.
We believe that our findings would appeal to the readership of Journal of Ethnopharmacology because
Clinopodium vulgare L. is a worldwide distributed plant, successfully applied in alternative medicine as a remedy
for cancer, gastric ulcers, diabetes, treatment of skin irritation and swelling, as well as relieving symptoms
associated with mastitis and prostatitis.The manuscript provides evidence for the genotoxicological mode of action
of this herb and throw more light into the molecular events regarding the initial steps of tumorigenesis. Such
research could be considered as a scientific evidence concerning the long-term use of wild basil as an anticancer
drug in traditional medicine. The model system in our study - Saccharomyces cerevisiae could be successfully
applied as an alternative to the use of animals in mutagenicity/carcinogenicity testing because of the similarities in
the main stress response pathways with the mammalian cells as well as the large number of homologous proteins,
and oncogenes in yeasts and humans.
Our manuscript may contribute in the long-run to improved health care and for the possible application of
Clinopodium vulgare leaves extract as plant-derived drugs. It also creates a paradigm for future in vivo and clinical
studies and for better understanding of some mechanisms such chromatin rearrengements.
This manuscript has not been published and is not under consideration for publication elsewhere in English
or any other languages. We have no conflicts of interest to disclose. If you feel that the manuscript is appropriate for
your journal, we suggest the following reviewers:
1. Bektas Tepe - e-mail: [email protected]
2. Stefania Frassinetti - e-mail: [email protected]
3. Luigi Del Giudice - e-mail: [email protected]

Sincerely yours,
assist. prof. Teodora Todorova, PhD
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences,
2 Gagarin str., 1113, Sofia, Bulgaria
e-mail: [email protected]
Tel: (+359)886 96 5713; Fax: (+359) 2 870 54 98
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Manuscript File Click here to view linked References

In vivo antioxidant activity of Clinopodium vulgare L. leaves extract correlates with amelioration

of zeocin-induced Ty1 retrotransposition, reverse mutations in the ilv1-92 allele and mitotic

crossingover in ade2 locus on Saccharomyces cerevisiae

Teodora Todorova1, Krum Bardarov2,3,4, and Stephka Chankova1

Affiliations:
1
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin str.,

1113, Sofia, Bulgaria

2
Sofia University “St. Kliment Ohridski”, Faculty of Physics, 5 James Bourchier Blvd., 1164,
Sofia, Bulgaria
3
Chromana Ltd, 12 Rojak Str., 1225, Sofia, Bulgaria
4
InoBioTech Ltd, 78 Samokov Str., 1113, Sofia, Bulgaria

Corresponding author:

Teodora Todorova

Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences,

2 Gagarin str., 1113, Sofia, Bulgaria

e-mail: [email protected]

Tel: (+359)886 96 5713; Fax: (+359) 2 870 54 98

Abstract

Ethnopharmacological relevance: Antibacterial, anti-inflammatory, DNA protective and

anticancer activity of wild basil (Clinopodium vulgare L.), widely applied in folk medicine for

treatment of cancer, hemorrhagic disease, gastric ulcers, diabetes, mastitis, prostatitis, skin

irritation and swelling in the Balkan Peninsula has been reported. Little is currently known about

the genotoxicological mode of action.

Aims of the study: to enlarge the present state of knowledge concerning 1) in vivo pro-

oxidant/antioxidant as well as genotoxic, mutagenic and carcinogenic potential of

Clinopodium vulgare L. leaves extract; 2) the relationship between antioxidant capacity of

Clinopodium vulgare L. leaves extract and its antigenotoxic, antimutagenic and anticarcinogenic

potential against the standard radiomimetic zeocin.

Material and methods: Three Clinopodium vulgare L. leaves extract concentrations (10,

100 and 1000 µg/ml) are tested on Saccharomyces cerevisiae as a human cell model. In vivo

antioxidant activity (antiROS test), antimutagenic (Zimmermann test) and anticarcinogenic (Ty1

transposition test) effects were tested. Zeocin was used as a positive control.

Results: No pro-oxidative, mutagenic or carcinogenic effect was obtained for

concentrations of 10 and 100 µg/ml Clinopodium vulgare extract. The minor pro-oxidative

activity of 1000 µg/ml extract measured in vivo by antiROS test did not correspond with

increase of mitotic gene conversion, reversion and mitotic crossing-over. Well-expressed

protective capacity of Clinopodium vulgare L. leaves extract against the radiomimetic zeocin was

determined, decreasing zeocin-induced superoxide anions (6-fold); revertants (4-fold); total

aberrants (2-fold) and transposants (4-fold).

Conclusion: Here, for the first time, in vivo antioxidant activity of Clinopodium vulgare L.

leaves extract has been evaluated. The concentrations in the range of 10-1000 µg/ml do not

possess any genotoxic, mutagenic or carcinogenic capacity on Saccharomyces cerevisiae as a

model for human cell. Experimental evidence is provided that the extract possesses well-

expressed protective effect against the radiomimetic zeocin. Strong correlation between

antioxidant activity and amelioration of zeocin-induced Ty1 retrotransposition, reverse mutations

in the ilv1-92 allele and mitotic crossingover in ade2 locus – some of the initial steps of

tumorigenesis is defined.

Keywords: Clinopodium vulgare; Saccharomyces cerevisiae; antioxidant; mutagenesis;

carcinogenicity

Abrreviations: EURL ECVAM, European Union Reference Laboratory for alternatives to animal

testing; XTT, 2,3-bis (2-methoxy-nitro-5-sulphophenyl)-5-[(phenylamino)-arbonyl]-2H-

tetrazolium hydroxide; PBS, phosphate-buffered saline; NS, nonsignificant; SD, standard

deviation; SEM, standard error of mean; ROS, reactive oxygen species; ORAC, oxygen radical

absorbance capacity; NQO1, NAD(P)H: quinone oxidoreductase 1; NQO, NAD(P)H−quinone

oxidoreductase; Nrf2, NF-E2-related factor 2; HR, homologous recombination;

1. Introduction:

Wild basil (Clinopodium vulgare L.) is a worldwide-spread plant, widely used in folk

medicine in Balkan Peninsula for treatment of cancer, hemorrhagic disease, gastric ulcers,

diabetes, mastitis, prostatitis, skin irritation and swelling (Badisa et al., 2003; Burk et al.,

2009; Batsalova et al., 2017). Based on the ethnopharmacological use, several pharmaceutical

products have been developed (Burk et al., 2009). Last two decades in vitro antioxidant,
antibacterial, anti-inflammatory, plasmid DNA protective and anticancer activity of wild basil

has been revealed (Dzhambazov et al., 2002; Yanchev, 2007; Tepe et al., 2007; Burk et al., 2009;

Kratchanova et al., 2010; Stefanovic et al., 2011; Todorova et al., 2016; Batsalova et al., 2017).

Although the application of herbal medicine is growing rapidly all over the world

(discussed in Thomford et al., 2018), herbal medications could also possess some harmful

activities (De Smet, 2004; Jordan et al., 2010). Newly provided information concerning

modulation capacity of Clinopodium vulgare L. extract and its constituents - catechin, caffeic

and chlorogenic acids on the cyclooxygenase-2 expression in neutrophils (Amirova et al. 2019)

is available. Further, low toxicity of Clinopodium vulgare L. extract in acute and sub-acute oral

administration in mice and rats is reported by Zheleva-Dimitrova et al. (2019). These findings

provoke us to look inside the genotoxicity mode of action of leaves extract of Clinopodium

vulgare L and its safety use.

Why the bioactivity of leaves extract has been chosen by us? In our previous paper

(Todorova et al., 2016) comparing the bioactivity of Clinopodium vulgare L. extracts from

different plant organs both the most pronounced radical scavenging activity and DNA protective

activity of leaves extract was shown. Although, the chemical methods for antioxidant activity are

widely applied, they do not provide information concerning the fate of the extract inside the

cells.

In the present study, we hypothesized that Clinopodium vulgare L. extract with well-

expressed antioxidant activity could ameliorate zeocin-induced mutagenic, recombinogenic and

carcinogenic effects on Saccharomyces cerevisiae model organism.

Here we aimed to enlarge the present state of knowledge concerning: 1) in vivo pro-

oxidant/antioxidant as well as genotoxic, mutagenic and carcinogenic potential of

Clinopodium vulgare L. leaves extract in Saccharomyces cerevisiae as a model for human cell;

2) the relationship between antioxidant capacity of Clinopodium vulgare L. leaves extract and its

antigenotoxic, antimutagenic and anticarcinogenic potential.

Saccharomyces cerevisiae has been chosen as a model system for human cell due to

the similarities in main stress response pathways (Todorova et al., 2015a). Moreover,

experiments on yeasts could be a valuable tool when taking into consideration the Directive

2010/63/EU. This directive is aiming to anchor firmly the principle of the “Three Rs, to Replace,

Reduce and Refine” the use of animals for experimental and scientific purpose in the EU

Member States. According to the Annex (47) there is a need to develop new methods alternative

to animal testing and proposed to validation in the European Union Reference Laboratory for

alternatives to animal testing (EURL ECVAM) (http://ihcp.jrc.ec.europa.eu/our_labs/eurl-

ecvam).

2. Materials and methods:

2.1. Plant material

Aerial parts of the herb Clinopodium vulgare L. were collected in Lozenska Mountain (Sofia

Region, Bulgaria), near Monastery “Saint Spas”, open grass area with shrubs, 850 m a.s.l.,

Bulgaria. Date: 01/07/2014, Leg.: Krum Bardarov, Det. Anely Nedelcheva. A voucher specimen

is deposited in Herbarium of Sofia University “St. Kliment Ohridski” (SO 107606). The plants

were air-dried in a clean, dark and airy room, and stored in ventilated paper boxes, before

preparation of the materials for investigation. Plant leaves were gently separated and

milled/homogenized in a grinder-mill. Sixty grams of powdered plant material were incubated in

a glass beaker with 1000 mL 95oC HPLC-grade water, and gently agitated, until cooled at room

temperature (approximately 2 hours). The aqueous extracts were centrifuged, filtered, frozen at t
= -50 oC, and then lyophilized. The leaves lyophilizates were kept at t = -20 oC until the

preparation of experimental solutions. Prior to each experiment stock solution dissolved in sterile

distilled water was prepared.

2.2. Chemicals

Zeocin, purchased from Invitrogen was used as a positive control in all experiments; 2,3-

bis (2-methoxy-nitro-5-sulphophenyl)-5-[(phenylamino)-arbonyl]-2H-tetrazolium hydroxide

(XTT) - from BioShop Canada Inc. Nutritional components for yeast YEPD media preparation

were from Difco Chem. Co. (USA). Chemicals and reagents were of analytical grade.

2.3. Strains

Two strains were chosen in order to evaluate the mutagenic/antimutagenic,

recombinogenic/antirecombinogenic and carcinogenic/anticarcinogenic potential of Clinopodium

vulgare leaves extract:

D7ts1 - diploid strain with genotype MATa/α ade2-119/ade2-40 trp5-27/trp5-12 ilv1-

92/ilv1-92 ts1/ts1 for detection of mutagenic/recombinogenic activity. D7ts1 strain provides

simultaneous detection of mitotic gene conversion at the trp-5 locus, reversion mutations in ilv1

locus and mitotic crossing-over between the centromere and ade2 allele (Freeman and

Hoffmann, 2007). All genetic events linked with the ADE2 locus are classified as total

aberrations (Poli et al., 1999; Terziyska et al., 2000).

551 (DG1141ts1) - haploid strain with genotype MATα ura3-167 his3Δ200:TymHIS3AI

sec53 rho+ (National Bank for Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria, Cat

№ 8719) for detection of carcinogenic activity. This strain is a derivative of DG1141 (Curcio and

Garfinkel, 1991). DG1141 strain is constructed for determination of the Ty1 transposition in the
genome as a whole by insertion of a Ty1 element marked with the indicator gene HIS3AI

(Pesheva et al, 2005).

The yeast strains 551 and D7ts1 have temperature sensitive allele ts1, leading to

increased cellular permeability to different substances, including mutagens/carcinogens.

2.4. Single treatment experiments:

2.4.1. Clinopodium vulgare L. leaves extract treatment

Cell suspensions were cultivated in standard conditions (300C, 200rpm) to the end of

exponential and the beginning of stationary phase with cell density 1x107cells/ml. Cells were

treated with three concentrations of the extract - 10, 100 and 1000 µg/ml for 1 hour at 300C with

aeration (on a rotary shaker, 200 rpm). After incubation, cells were washed twice with YEPD

medium.

2.4.2. Zeocin treatment

Previously it was shown by us that zeocin treatment for 1 min results in pronounced

increase superoxide anions, genotoxic, mutagenic and carcinogenic effects (Todorova et al.,

2015). Based on this, the same treatment was performed: cell suspensions were treated with 100

µg/ml zeocin for 1 min on ice (to prevent DNA repair).

2.5. Pretreatment experiments:

Cell suspensions were pretreated with 10, 100 or 1000 µg/ml Clinopodium vulgare L.

extract, washed twice with YEPD and subsequently treated with 100 µg/ml zeocin for 1 min on

ice.

2.6. Quantitative Assay for Superoxide Anions (antiROS test)

The quantitative measurement of superoxide anions was performed as described in

(Stamenova et al., 2008; Todorova et al., 2015). After the application of single and/or subsequent
treatments, cells were washed in phosphate-buffered saline (PBS) and 125 µM XTT was added.

The aliquots were incubated for 6 hours at t=30 0C on a rotary shaker, spun and the absorbance

of supernatant was measured at 470 nm. The number of living cells was determined as colony

forming units and results were presented as pM O2•–/cell ± standard error of mean (SEM).

2.7. Zimmermann’s test

Zimmermann’s test (Zimmermann, 1984) was performed for detection of

mutagenic/antimutagenic and recombinogenic/antirecombinogenic activity. Zeocin (100 µg/ml)

was used as a positive control.

Single and/or pretreatment experiments were applied; the cell pellet was then

resuspended in PBS. Appropriate dilutions of cell suspensions were plated on solid complete

medium for survival and total aberrants. Gene conversion was detected on selective media

lacking tryptophan. Selective media lacking isoleucine was used for reverse mutations. Five

plates in each category were incubated for 5-7days at t=300C. Yeast media were prepared as

described by Zimmermann et al., 1975.

2.8. Ty1 transposition assay using S. cerevisiae strain 551rho+

Ty1 transposition assay was performed as described by Pesheva et al., 2005 and Dimitrov

et al., 2013. After the treatments, cell suspensions were washed with YEPD medium, and

cultivated at t=20°C (optimal conditions for Ty1 transposition) for 24 hours. Appropriate

dilutions of cells were plated on YEPD to evaluate survival and on selective medium lacking

histidine – for His+ transposants. Zeocin (100 µg/ml) was used as a positive control. Yeast media

were prepared as described by Sherman et al., 2001.

Mean transposition rates were determined and results presented as “fold increase of Ty1

transposition rate” related to control sample, taken as 1.00. A fold increase in treated cultures
equal or higher than 2.00 is considered as positive response of the Ty1 assay (Pesheva et al.,

2005).

2.9. Statistical analysis

At least three experiments with independently grown cultures were performed for each test. One-

way analysis of variances (ANOVA) with Bonferroni’ spot hoc-test was used to calculate

statistically significant differences between tested concentrations and positive controls.

Calculations were done with GraphPad Prism program, version 6.04 (San Diego, USA).

Asterisks provide information for the significance in the differences where ns P > 0.05; *P <

0.05; **P < 0.01; ***P < 0.001.

3. Results:

3.1. Pro-oxidative potential of Zeocin and Clinopodium vulgare leaves extract

Around 15-fold higher levels of superoxide anions (O2•-) were measured after Zeocin

treatment in vivo compared to the control samples. Clinopodium vulgare L. extract at

concentrations 10 and 100 µg/ml does not possess pro-oxidative properties (Figure 1) while after

the treatment with the highest concentration (1000 µg/ml) around 1.5-fold increase in the level of

superoxide anions (P<0.05) was measured.

Figure 1: Potential pro-oxidative properties of Clinopodium vulgare L. leaves extract applied at

different concentrations (10, 100 and 1000 µg/ml). Error bars represent standard error of the

mean from at least three independent experiments. Where no error bars are evident, they are

equal of less than the symbols. The statistical significance of differences is indicated with an

asterisk (ns P > 0.05; * P < 0.05).

3.2. Genotoxic potential after single treatment with Zeocin and Clinopodium vulgare

leaves extract

Further, the potential genotoxic action of zeocin and leaves extract was evaluated based

on the cell survival of two Saccharomyces cerevisiae strains – one diploid (D7ts1) and one

haploid (551).

Zeocin treatment (positive control) resulted in about 30-35% cell survival for both

strains. Data presented on table 1 show no statistically significant decrease of cell survival

depending on the extract concentrations and the genotype (Table 1).

Table 1: Cell survival of Saccharomyces cerevisiae strains D7ts1 and 551 after the

treatment with 10, 100 and 1000 µg/ml Clinopodium vulgare leaves’ extract

Zeocin C. vulgare L. extract

Control
Strain 100 µg/ml 10 µg/ml 100 µg/ml 1000 µg/ml
(%)a
(%)a (%)a (%)a (%)a
551 100 30,19±4,37*** 98,24±3,88 NS 98,17±4,30 NS 100,96±1,37 NS

D7ts1 100 35,61±0,64*** 88,33±8,13 NS 92,61±5,97 NS 87,32±9,14 NS

a
Values are mean ±SD from at least three independent experiments. The significance

of differences between negative control (untreated cells) and single treatments was calculated

by One-way ANOVA with Bonferroni’spot hoc-test (***P<.001). SD, standard deviation

 3.3. Mutagenic and recombinogenic potential after single treatment with Clinopodium

vulgare leaves extract

Single treatment with any of the Clinopodium vulgare extract concentrations did not

result in statistically significant increased levels of gene conversion, reversion and mitotic

crossing-over suggesting lack of mutagenic and recombinogenic effect (Table 2). The

frequency of the genetic events in treated samples was comparable to the spontaneous ones in

untreated cells.

Table 2: Frequency of genetic events in S. cerevisiae D7ts1 cells after the treatment

with three concentrations Clinopodium vulgare L leaves extract.

C. vulgare L.
Zeocin Convertants/ 105 Revertants/ 104 Total aberrants
extract
(µg/ml) survivors survivors (%)
(µg/ml)

0 0 0,46±0,19 0,008±0,001 1,38±0,20

0 100 1,42±0,28** 0,094±0,009*** 8,49±0,49***

10 0 0,007±0,002 NS 1,27±0,11 NS
0,45±0,11 NS

100 0 0,39±0,16 NS 0,006±0,001 NS 1,92±0,56 NS

1000 0 0,49±0,24 NS 0,009±0,003 NS 1,98±0,42 NS

Frequencies are means ± SD from at least three independent experiments. The statistical

significance of differences between positive control (zeocin) and single treatments with C.

vulgare L. extract were calculated by ANOVA with a post-hoc test Dunnett's Multiple

Comparison Test (NS: nonsignificant; **P < 0.01; ***P < 0.001).

3.4. Carcinogenic potential presented as induction of Ty1 retrotransposition after

single treatment with zeocin and Clinopodium vulgare leaves extract

Treatment with zeocin resulted in around 3-4-fold higher levels of Ty1 retrotransposition

(Figure 2). Further, our experimental results show no capacity of Clinopodium extract to

enhance Ty1 retrotransposition rate in comparison with the negative control - untreated cells

(Figure 2).

Figure 2. Potential carcinogenic effect of different concentrations C. vulgare L extract on S.

cerevisiae 551rho+ measured as Fold increase transposition rate. Average values ± SD from at

least 3 independent experiments. The significance in differences between negative control -

untreated cells and treatment with different concentrations of C. vulgare extract was calculated

by ANOVA with post-hoc test- Bonferroni’s Multiple Comparison Test (***P < 0.001). Where

no error bars are evident, they are equal or less than the symbols.

3.5. In vivo antioxidant potential of Clinopodium vulgare L. extract towards Zeocin-

induced oxidative stress

 Around 15-fold reduction of zeocin-induced ROS levels was measured when pretreatment

with different concentrations of the extract (figure 3) and zeocin was performed. The levels were

comparable with those in untreated cells (P<0.0001). The antioxidant activity of the extract was

similar despite the concentration applied.

Figure 3. Antioxidant activity of Clinopodium vulgare L. measured as levels of superoxide

anions. Error bars represent standard error of the mean from at least three independent

experiments. Where no error bars are evident, they are equal of less than the symbols. Statistical

significance of differences is indicated with an asterisk (*** P < 0.001).

3.6. Antimutagenic and antirecombinogenic potential of Clinopodium vulgare L.

extract towards zeocin

The revertant frequency was used as an endpoint for mutagenicity. Treatment with 100

µg/ml zeocin led to around 12-fold higher levels of revertants in comparison with the

spontaneous levels in untreated cells suggesting strong mutagenic activity of zeocin.

The pretreatment with different concentrations Clinopodium vulgare L. resulted in 4-

 fold amelioration of the zeocin-induced reverse mutations (Table 3).

Table 3. Pretreatment with C. vulgare L can reduce genetic events induced by zeocin

C. vulgare
L. Zeocin Convertants/ Revertants/ 104 Total
Survival (%)
extract (µg/ml) 105 survivors survivors aberrants (%)
(µg/ml)

0 0 100 0,46±0,19 0,008±0,001 1,38±0,20

0 100 35,61±0,64*** 1,42±0,28** 0,094±0,009*** 8,49±0,49***

Pretreatment experiments

10 100 69,47±13,35*** 2,4±0,44 NS 0,018±0,003*** 6,22±0,27 NS

100 100 73,40±12,27*** 2,16±0,26 NS 0,026±0,008*** 6,07±0,89 *

1000 100 84,13±15,97*** 1,49±0,27 NS 0,021±0,002*** 4,83±0,58***

1
Values are mean ± SD from at least three independent experiments. The significance of

differences between positive control (zeocin) and pretreatment treatments – Clinopodium vulgare

extract and zeocin were calculated by One-way ANOVA with Bonferroni’s post hoc test (ns P >

0.05; *P < 0.05; **P < 0.01; ***P < 0.001).

The antirecombinogenic activity was evaluated based on the frequency of mitotic gene

conversion and mitotic crossing-over (Table 3).

Single treatment with Zeocin resulted in 3-fold higher frequency of convertants and 6-

fold higher rates of total aberrants.

Effect of the pretreatment was calculated only for the mitotic crossing-over. Pretreatment

with the extract concentrations 10 and 100 µg/ml led to around 1.5-fold decrease in the

percentage of zeocin-induced total aberrant while pretreatment with 1000 µg/ml extract resulted

in around 2-fold decrease. Concerning the mitotic gene conversion, no statistically significant
difference was calculated among the levels measured in the pretreatment and zeocin-treated

samples (Table 3).

3.7. Anticarcinogenic potential of Clinopodium vulgare L. extract towards Zeocin

After single treatment with zeocin the retrotransposition rates was around 4 fold higher

comparing with that in control samples. Based on this, strong carcinogenic capacity of zeocin

could be suggested (Table 4).

In the pretreatment experiments, dose-dependent decrease in transposition rate is

detected with increasing the extract concentration. Around 4-fold lower transposition rate was

obtained, after the pretreatment with the highest tested C. vulgare L. concentration (1000

µg/ml), suggesting anticarcinogenic activity of this extract in our test system.

Table 4. Ty1 transposition test for potential anticarcinogenic effect of different

concentrations C. vulgare L extract on S. cerevisiae 551rho+.

C. vulgare L. extract Zeocin

Survival (%)a Fold increase transposition rateb
(µg/ml) (µg/ml)

0 0 100 1

0 100 30,19±4,37*** 4,32±0,27***

Pretreatment experiments

10 100 48,16±2,91*** 3,65±0,39 NS

100 100 46,13±0,95*** 2,11±0,08***

1000 100 47,69±2,10*** 1,17±0,25 ***

a
Average values ± SD from at least three independent experiments.
b
The significance in differences between positive control - single zeocin treatment and split

treatment with different concentrations of C. vulgare extract and zeocin was calculated by

ANOVA with post-hoc test- Bonferroni’s Multiple Comparison Test (***P < 0.001).

4. Discussion:

In the present study, the mutagenic and recombinogenic potential of zeocin has been

determined analysing the frequencies of three types of genetic events in nuclear DNA: reverse

mutation in the ilv1-92 allele- 12-fold; mitotic gene conversion in the trp5 locus - 3-fold; and

total aberrants in the ade2 locus - 6-fold. An enhancement of the Ty transposition rate – marker

for carcinogenic effect, was also observed.

Some data illustrate the relationship among bleomycin-induced higher levels of total

aberrants, changes in chromatin structure and an increased transcription (Freeman and

Hoffmann, 2007). Previously, we have shown that zeocin can induce high levels of Ty1

retrotransposition (Todorova et al., 2015) similarly to the action of ionizing radiation (Sacerdot

et al., 2005) as a consequence of a modification of the chromatin structure. Thus, high levels of

Ty1 transposition and carcinogenic potential respectively could be explained by the elevation

of Ty1 transcription due to a misregulation of the histone degradation resulting in oncogenic

translocations (Hauer et al., 2017). Both, well-expressed pro-oxidative and carcinogenic potential

of zeocin described in the current study have confirmed the present state of knowledge that the

only inducers of Ty1 transposition are carcinogens overproducing ROS (Stamenova et al., 2008;

Stoycheva et al., 2010; Dimitrov et al., 2011).

Thus, in the present study we hypothesized that Clinopodium vulgare L. extract with

well-expressed antioxidant activity could ameliorate the zeocin-induced mutagenic,

recombinogenic and carcinogenic effects on Saccharomyces cerevisiae model organism.

 The antioxidant activity of aqueous Clinopodium vulgare L. leaves extract has already

been revealed based on several chemical methods: oxygen radical absorbance capacity (ORAC)

method (Kratchanova et al., 2010); DPPH assay; determination of total phenolic/flavonoid

content (Todorova et al., 2016). Additionally to chemical methods, in vivo experiments should be

done in order to clarify the contribution of cellular penetration (Dimitrov et al., 2013),

bioavailability or metabolism (Stinco et al., 2015) of the studied antioxidants. Saccharomyces

cerevisiae is considered as an attractive alternative to mammalian cell lines for studying pro-

oxidant/antioxidant activity in vivo (de Oliveira et al., 2018; Islam et al., 2016) due to presence

of P450 system for metabolic activation and detoxification. Our antiROS data provides

information concerning the ability of Clinopodium vulgare L. leaves extract to penetrate into

the cell wall and to neutralize physiologically active oxidative radicals inside the living cells.

No dose dependent antioxidant activity against zeocin was measured using the antiROS

test despite the fact that single treatment with the highest concentration (1000 µg/ml) resulted in

a slight pro-oxidant effect. It is known that plant extracts or/and some constituents might be

referred to antioxidants via different modes of action (Procházková et al., 2011). The antioxidant

properties of the highest tested concentration Clinopodium vulgare L. extract applied before the

zeocin treatment could be explained by the activation of Nrf2 by the polyphenols presented such

as rosmarinic acid, quercetin, kaempferol etc. (Cai et al., 2019; Li et al., 2019; Birringer, 2011).

As discussed in Birringer (2011) the activation of Nrf2 results in induction of phase II enzymes

such as glutathione S-transferases (GST), antioxidant enzymes like superoxide dismutase (SOD).

Such activation is related to cellular redox regulation genes such as NAD(P)H: quinone

oxidoreductase 1 (NQO1). Thus, the amelioration of zeocin-induced oxidative stress could be

related to the activation of enzymes such as NAD(P)H−quinone oxidoreductase (NQO) and

glutathione S-transferases by the rosmarinic acid as a major constituent. It is known that

glutathione S-transferases play an important role not only in defense against ROS, but also

indirectly in the elimination of toxic substances (Todorova et al., 2007). Data regarding NQO

report not only its role in antioxidant defense mechanisms, but also in the stabilization of p53

protein in response to DNA-damaging agents (Nioi and Hayes, 2004).

Previously, it was reported by us that the chemical analysis of the extract show high

amount of phenolic and acylquinic acids, flavonoids and triterpenoid saponins (Bardarov et al.,

2016) and also that the total phenolic content in the extract is 47.03 mg gallic acid/g extract

(Todorova et al., 2016). The preliminary screening revealed the presence of twenty-five plant

secondary metabolites (Bardarov et al., 2016). Additionally to these data, fifty-two compounds,

including 7 flavonoids, 41 caffeic acids derivatives and 5 saponins in the extract have been

identified (Zheleva-Dimitrova et al., 2019). The rosmarinic acid has been reported as a major

component (Bardarov et al., 2016; Nasar-Eddin et al., 2019; Zheleva-Dimitrova et al., 2019).

Around 43% saponins were quantified in the extract after an application of the vanillin-

perchloric acid assay (Bardarov et al., 2016). According to Amić et al. (2007) and Galati et al.

(2002), flavonoids could increase the levels of superoxide anions inside the cells. Data already

exist that the pro-oxidant properties of flavonoids above certain concentrations are directly

proportional to the total number of hydroxyl groups in a molecule. Nevertheless, penetrated

into the cell polyphenols undergo metabolism by at least three forms: oxidative metabolism,

P450-related metabolism, or conjugation with thiols (Procházková et al., 2011). Certain

conditions such as high pH, high concentration of the phenolic compounds and/or the presence

of redox-active transition metals (particularly Fe and Cu), promote the pro-oxidant activity

(Procházková et al., 2011; Park and M Pezzuto, 2012). Data exist that polyphenols can induce
ROS, deplete glutathione (GSH), and activate NF-E2-related factor 2 (Nrf2), which could be

explained by the conversion of diphenols to quinones (Birringer, 2011).

Concerning the antimutagenic and antirecombinogenic potential, three genetics events

were examined – point reverse mutations in the ilv1-92 allele, mitotic gene conversion in the trp5

locus and mitotic crossing-over in the ade2 locus. The decrease of zeocin-induced reverse

mutations demonstrates antimutagenic activity of the extract which could be explained by the

phytochemical composition (Bardarov et al., 2016; Zheleva-Dimitrova et al., 2019) and

prevalence of rosmarinic acid. Our suggestion is in agreement with data of other authors

reporting antimutagenic activity against ethyl methanesulfonate and acridine (Gulluce et al.,

2012), radio-protective, antimutagenic activity against y-rays (Sánchez-Campillo et al., 2009) of

rosmarinic acid.

Antirecombinogenic activity was evaluated by two genetic events – mitotic crossing-over

and mitotic gene conversion. Usually, mitotic crossing-over has a very low frequency and it can

be observed only after the treatment with DNA damaging agents such as ionizing radiation and

radiomimetics, etc. Around 2-fold decrease in the frequency of mitotic crossing-over could be

explained with changes in chromatin structure or transcription rate rather than involvement of

homologous recombination (HR) repair (Freeman and Hoffmann, 2007). This speculation is

dictated by the fact that the extract did not affect the frequency of zeocin-induced mitotic

gene conversion. It is well known that gene conversion is related to HR repair (Freeman and

Hoffmann, 2007). New data elucidate the capacity of polyphenols to modulate the

chromatin structure (discussed in Russo et al., 2017).

Ty1 transposition rate was used as an endpoint for confirmation of the anticarcinogenic

activity of Clinopodium vulgare leaves extract. Our results are in agreement with previous
 study for its antitumor activity (Batsalova et al., 2017). The authors speculated that the

antitumor activity of Clinopodium vulgare extract could be contributed to the most abundant

constituents – rosmarinic acid, caffeic acid-containing glucosides, ursolic acid, gentriacontan

and clinosaponins (Batsalova et al., 2017).

Experimental evidence was provided in confirmation of the reported already relationship

between antioxidant properties of plant extract and its anticarcinogenic potential measured as

transposition rate (Dimitrov et al., 2013).

Based on this fact, the observed anticarcinogenic effect measured as decrease in Ty1

transposition, could be proportionally related to the antioxidant activity of Clinopodium

vulgare L. extract. As discussed above, the elevated levels of Ty1 transposition by Zeocin could

be related to histone imbalance and some chromatin modifications. Data exist about the ability

of some polyphenols, such as quercetin and kaempferol, at certain concentrations to inhibit

histone demethylase LSD1, enzyme studied as a potential target in anticancer drug discovery

(Abdulla et al., 2013; Højfeldt et al., 2013; Stepanić et al., 2014). The potential involvement of

the extract and its polyphenols in chromatin modification needs to be further studied.

5. Conclusion:

Here, for the first time, in vivo antioxidant activity of Clinopodium vulgare L. leaves extract

has been evaluated. The concentrations in the range of 10-1000 µg/ml do not possess any

genotoxic, mutagenic or carcinogenic capacity on Saccharomyces cerevisiae as a model for

human cell. Experimental evidence is provided that the extract possesses well-expressed

protective effect against the radiomimetic zeocin. Strong correlation between antioxidant

activity and amelioration of zeocin-induced Ty1 retrotransposition, reverse mutations in the

ilv1-92 allele and mitotic crossingover in ade2 locus – some of the initial steps of
tumorigenesis is defined.

Authors’ contributions:

Teodora Todorova: Responsible for planning and conducting the experiments; analysing the

results contributed with the discussion of the results, writing the manuscript as well as final

revision of the article; Krum Bardarov: responsible for collection and preparation of

Clinopodium vulgare L. leaves’ extract; Stephka Chankova: responsible for collaboration with

the discussion of the results and final revision before submission.

Acknowledgements: This work was supported by a grant from the National Science Fund,

Ministry of Education and Science, Project No. DH11/10 and “Ecological and genetic

assessment of the environmental - management and strategies for overcoming the risk“–

Bulgarian Academy of Sciences.

Conflict of Interests: Authors declare no conflict of interests.

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Figure Captions

Figure captions
Figure 1: Potential pro-oxidative properties of Clinopodium vulgare L. leaves extract applied at

different concentrations (10, 100 and 1000 µg/ml). Error bars represent standard error of the

mean from at least three independent experiments. Where no error bars are evident, they are

equal of less than the symbols. The statistical significance of differences is indicated with an

asterisk (ns P > 0.05; * P < 0.05).

Figure 2. Potential carcinogenic effect of different concentrations C. vulgare L extract on S.

cerevisiae 551rho+ measured as Fold increase transposition rate. Average values ± SD from at

least 3 independent experiments. The significance in differences between negative control -

untreated cells and treatment with different concentrations of C. vulgare extract was calculated

by ANOVA with post-hoc test- Bonferroni’s Multiple Comparison Test (***P < 0.001). Where

no error bars are evident, they are equal or less than the symbols.

Figure 3. Antioxidant activity of Clinopodium vulgare L. measured as levels of superoxide

anions. Error bars represent standard error of the mean from at least three independent

experiments. Where no error bars are evident, they are equal of less than the symbols. Statistical

significance of differences is indicated with an asterisk (*** P < 0.001).

Figure 1 Click here to access/download;Figure;Todorova_Figure_1.jpg
Figure 2 Click here to access/download;Figure;Todorova_Figure_2.jpg
Figure 3 Click here to access/download;Figure;Todorova_Figure_3.jpg