Journal of Scientific Research in Science 2023, 40, (2): 1-25

Genotoxic impacts of Teucrium apollinis medicinal plant via HGPRT gene

and DNA damage

Nagat S. Elhaddad*, Hoda A. Khatab, Eman M. Belkasem and Nagah S.A. ABubaker.

Department of Botany, University of Omer Almukhtar, Al-Baida, Libya

Abstract

We report the applicability of the Hypoxanthine Guanine Phosphoribosyl Transferase

(HGPRT) gene to test the potential mutagenic activity of Teucrium apollinis (TA).
Genotoxicity of low dose (LD) 50µl and high dose (HD) 250µl of fixed oil extracted from TA
were examined using asexual cell (oidia) of Coprinopsis cinerea. Surprisingly, survival of wild
type cells was significantly decreased to 45.8 and 29.3% when oidia incubated with LD and
HD respectively illustrating its cytotoxic effect. The mutation rate decreased to 0.33 and 0.4
with selected doses of the plant extract correspondingly which means that both were mutagenic.
DNA damage of Coprinopsis cinerea was also quantified by spectrophotometer. There were
significant differences (LSD= 0.05) between negative control and treatments, since the highest
value was 94.33±2.41µg/ml in control, and the lowest amount of DNA (32.53±3.40) was
obtained when hypha was treated with the positive control (MSG). Unsurprisingly, LD and HD
reduced DNA values to 33.84±3.26 and 31.56±2.70 respectively, indicating the genotoxicity
of TA concentrations. Ten components were identified by GC-MS of the oil extract involving
Camphor and Furanone. Thus cytotoxic and mutagenic of medicinal plants must be assessed
to ensure a relatively save use for our important population.
Keywords: HGPRT gene, mutation, Teucrium apollinis, DNA damage, genotoxicity.

1. Introduction

The Hypoxanthine Guanine Phosphoribosyl-Transferase HGPRT gene mutation assay is a

remarkable tool for testing genotoxic chemicals and allows the isolation and screening of
mutation in different living organisms such as humans, hamsters, bacteria, and fungi. In the
current project, the HGPRT assay for gene mutation at the HGPRT locus of Coprinopsis
cinerea was used to test the mutagenicity of the Teucrium apollinis medicinal plant. The genus
Jaada (Teucrium L.) is considered one of the most important plants of the Lamiaceae family in
Libya. Plants of this genus are stunted shrubs and shrubs, with plant heights ranging from 10
to 100 cm based on [1]. Some plants of this genus are popularly used in Libya to treat stomach
and intestinal pain reduce blood sugar levels blood and cure colds, and is used to treat kidney
*Corresponding author: Nagat S. Elhaddad, Department of Botany, University of Omer
Almukhtar, Al-Baida, Libya.
E-mail: [email protected].
(Received 04 September 2023, revised 26 October 2023, accepted 29 October 2023)
https://doi.org/10.21608/JSRS.2023.234096.1117
Nagat S. Elhaddad et al. J. Sci. Res. Sci., 2023, 40, (2): 1-25
stones and blood pressure [1]. Many studies demonstrated that the extractions of a range of
medicinal plants have antioxidant, antibacterial, antimicrobial, anti-parasitic, anti-mutagenic,
antifungal, and anti-carcinogenic properties. In recent years, using herbal drugs to treat
different diseases has renewed interest, and the World Health Organization (WHO) has
recommended an evaluation of the efficiency of medical plants in conditions that require or
lack safe modern drugs [2]. Because of the high cost and the range of side effects that may have
been caused by taking synthetic drugs, alternative medicine became necessary. Traditional
medicinal plants are used over long periods for the above reasons, however, this is not always
the situation [3, 4]. Acute toxicity due to the use of extracts from medicinal plants is more
common than is often thought. It was estimated that annual deaths were between 8000 and
20,000 because of inappropriate use of medicinal plants (1). In addition, research has shown
that many plants that are either used as food additives or in traditional medicine not only can
be toxic drugs [4], but they can also be mutagenic [4, 5, 6, 7] or even carcinogenic [4, 7].
Although plant extracts have been used in the treatment of diseases according to knowledge
accumulated over centuries, scientific research has shown some substances present in these
medicinal plants to be potentially toxic and carcinogenic [7]. Many plant species commonly
considered medicinal can contain potentially dangerous substances [8]. Recent research studies
conducted in vitro and in vivo assays have revealed that many plants used in traditional
medicine have cytotoxic and genotoxic effects [9, 10, 11, 12]. However, according to [13], rats
were injected with different Concentrations of Teucrium polium L. or normal saline, following
28 days of T. Polium consumption, kidney damage was not increased in comparison with a
control group. However, following 28 days, kidney damage including degeneration,
destruction, and vacuolization, appeared and they concluded that T. polium should not be used
or should be consumed with great caution [14]. The aqueous extract of Teucrium polium has
strong hypoglycemic properties in experimental animals consequently it was concluded that it
is not suitable for use in humans as an antidiabetic agent [15]. Moreover, [16] suggested that
female rats are more sensitive to higher doses of Teucrium and that the liver could serve as a
target organ toxicity of this extraction. Moreover, [17] reported the first case of hepatotoxicity
was probably caused by T. viscidumone of Teucrium species indicating that using T. viscidum
may pose similar to the other Teucrium species. Similarly, Lavandula stoechas was used to
treat various diseases around the world but [18] reported cytotoxic and genotoxic effects of
aqueous extracts from L. stoechas on Allium cepa root tip meristems. Their results showed that
aqueous extracts significantly reduced mitotic index, induced chromosome aberrations, and
mitotic aberrations in comparison with control [18]. Traditional plants, even though natural,

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can be harmful. There is a rare of published data on the safety profile of these herbals. It was
recommended to enhance the investigations of the safety of medicinal herbs and the adverse
effects of their products and also consuming them in clinical practice [17].

Investigation of traditionally medicinal plants is thus valuable on two levels: firstly, as a source
of potential chemotherapeutic drugs, and secondly, as a measure of safety for the continued use
of medicinal plants [19]. Mutagenicity studies of traditional medicinal plants are important for
risk assessment when applied as a medication against different diseases. Since mutagens are
involved in the initiation and/or promotion of cancer, research would focus on the identification
of a novel of traditional plants that may act as mutagenesis. HGPRT gene mutation assay is
one of the strategies that is quite accessible to test mutagenicity. The objective of this study is
to examine the mutagenicity/cytotoxicity of Teucrium apollinis extract using HGPRT gene
mutation assay and the analysis of DNA damage of Coprinopsis cinerea.

2. Materials and Methods

2.1 The fungi

The wild strain of Coprinopsis cinerea used in this study is H1(allelic gene pairs of A and B).
It has undergone five name changes in the last 30 years, most recently from Coprinus
cinereus to Coprinopsis cinerea [20].

2.1.1 Strain and culture conditions

Strain AmutBmut is a self-compatible homokaryon of Coprinopsis cinerea which due to

mutations in the A and B mating type genes, produces fruiting bodies without mating to another
compatible monokaryon [21]. The hypha was isolated from horse dung and cultivated on YMG
agar plates (4 g yeast extract, 10 g malt extract, 4 g glucose, and 10 g agar per l) for 5 days at
28°C until the mycelium fully covered the substrate [22]. Oidial suspensions were prepared by
harvesting the hypha that were cultured on about 50 Petri dishes [21].

2.1.2 Complete media

The medium consists of (4 g yeast extract, 10 g malt extract, 4 g glucose, and 15 g agar per L)
and it is named Yeast malt-glucose (YMG) medium. A complete medium is used to grow and
sustain the wild- types of monokaryons [23].

2.1.3 Selective media

This medium is mainly used for genetic experiments such as isolating spontaneous and induced
mutations and for measuring fungal growth rates. The medium contains the following: 4 g yeast

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extract, 10 g malt extract, 4 g glucose, and 15 g agar per L, and then 20µl/ml 6-thioguanine
was added to 100 ml medium. A 6-thioguanine (6- TG, 20µl/ml) solution was prepared by
dissolving 120µg of 6-thioguanine in the freshly prepared 0.1M NaOH solution that made up
to 60ml with distilled water and then stored at -20 C° until use (Sigma instructions).

2.1.4 Isolation and identification of fungi

Coprinopsis cinerea was isolated from the horse's dung in sterilized conditions. It was planted
on the YMG media and incubated at 25 °C for 2-4 days, where the C. cinerea appeared to clear
growth several seedlings using a needle sterile vaccination and this process was repeated until
pure and good growth of C. cinerea was obtained.

The morphology of Coprinopsis cinerea was studied and the colony was first grown on the
YMG medium and using the standard cover-slip technique with lacto phenol cotton blue
staining procedure. The cover of the slide was inserted in the petri plate itself and the culture
was allowed to grow for fourteen days. Every two days with the help of forceps, the coverslip
was taken and inverted on a slide containing a drop of stain and visualized under a microscope
(Lica microsystem 1000 Led) at 10X, 40X, and 100X magnification App. The fungi were
identified based on mycelia and spore characteristics.

2.2 Hypoxanthine-guanine phosphoribosyl transferase locus (HGPRT) assay

The HGPRT assay was performed according to [24] Experiment was divided into four groups;
the first group is the negative control in which the asexual (oidia) cells were incubated with
Distilled Water (DW). Cells in the next two groups were treated with selected concentrations
of oil extract of Teucrium apollinis (50 and 250µg/l) for one hour at 37°C. The fourth group,
Oidia were treated with 7g/ml of mono-sodium glutamate as appositive control. Three replicate
tubes were used for each group. After the incubation, cells were washed twice with DW and
centrifuged at 1500 rpm. Treated oidia were then divided and cultured on two different culture
mediums. Non-selective medium (YMG agar plates) to detect the wild-type phenotype,
whereas, the selective medium (YMG agar plates containing 6-TG) to detect the mutant
phenotype cells. Plates were incubated at 28°C for 48 hrs. Colonies on both non-selective and
selective medium were counted. Three separate experiments (n=3) were carried out for each
treatment. Viability was expressed as the number of colonies on the non-selective medium.
Mutation frequency was determined phenotypically as the number of mutant colonies in the
selective medium and the number of colonies in the non-selective medium.

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2.3 Preparation of plant extractions
2.3.1 Plant material
In April aerial parts of Teucrium apolinis were collected from natural populations in the regions
of Sousa in the AlJabel Alakhdar area. The specimens of T. apolinis were confirmed and
deposited in the sylphium Herbarium at the Department of Botany, Faculty of Science,
University of Omer-Almukhtar. The collected plant material was kept in sterilized plastic bags
and immediately transferred to the laboratory.

2.3.1.1 Fixed oil extraction

Plant material was transferred to tightly dark-colored bottles and was soaked in 250ml of
acetone and then stored at room temperature. After 48 h, the infusions were filtered through
Whatman No. 1 filter paper. After that, the combined supernatants were evaporated under
vacuum at 40 °C using a Rotary evaporator according to [25]. The obtained oil extracts were
kept in a sterile bottle and stored in a refrigerator at 4°C until needed.

2.3.1.2 Determination of antioxidant

The antioxidant content of the Teucrium apolinis plant was determined following the Prussian
blue method [26]. One gram of powder was defatted twice with petroleum ether, the defatted
powder was then extracted sequentially by stirring with 10ml methanol twice, then with 10 ml
1% hydrochloric acid: methanol (v/v). The three combined extracts were evaporated under
vacuum and the residue was dissolved in 10ml methanol. Half ml of the solution was diluted
with 3 ml distilled water, 3 ml 0.008 M K3Fe(CN)6 was added, 3 ml 0.1M HCl, and 1 ml 1%
FeCl3. The blue color was allowed to develop for 5 min and the absorbance was measured at
720nm against the blank.

2.3.1.3 Determination of total phenol

Aliquots of the extracts were taken in a 10 ml flask and made up to a volume of 3 ml with
distilled water. Then 0.5 ml folin ciocalteau reagent (1:1 with water) and 2 ml Na2Co3(20%)
were added. The test solutions were warmed for 1 minute, cooled and absorbance was
measured at 650 nm against the reagent used as a blank [27].

2.4 Gas Chromatography-Mass Spectrum Analysis

The extraction was kept in a dark bottle and transferred to (the central lab of the faculty of
science at Alexandra University-Moharam Bey) to commence to Gas Chromatography- Mass
Spectrum (GC-MS) examination.

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2.5 Plant extract applications

In this experiment, oidia were treated with monosodium glutamate MSG (7g/l, published data
at http://www.scholarsresearchlibrary.com) as a positive control and two different
concentrations of the fixed oil (low and high) and then incubated for two hours which were
determined based on the preliminary experiments. The experiment of the interaction between
MSG and plant extracts was divided into two sup-experiments as follows: in the first treatment:
three sterile test tubes containing 2mL of oidial suspension were allocated for all of the
negative, and positive control groups and treatment groups.

In the treatment group: 7g/l of MSG was added, cells were incubated for two hours, washed,
and then the fixed oil (50 and 250 µl) was added to the oidia. The tube was incubated for
another two hours at 37°C, subjected to centrifugation, and then the supernatant was discarded.
The washing step was repeated twice using distilled water and then the oidia were re-suspended
with 2ml of distilled water to be ready for cultivating. The non-treated oidia tubes represent the
negative control group. The rest of the tubes represent a putative mutagenicity of the oil extract.
In the second treatment: the steps mentioned above in the first treatment were repeated but this
time the oidia were incubated for two hours with oil extract, then the MSG was added and
incubated for two more hours. Here the first group represents negative untreated oidia, while
the rest of the tubes represent the putative enhancing mutagenicity role of the fixed oil were
incubated for two hours with the plant extraction before adding the food enhancer MSG for
more two hours.

2.6 Fungal genomic DNA extraction

The genomic DNA was extracted from five to seven day old fungal cultures grown in culture
plates. The fungal mass of 10-20 mg fungal mass (20-40 mm2mycellial tissue) from the culture
plate was collected with the help of a fine spatula. The fungal mass obtained from the culture
plate placed in a 2ml tube containing a500µl extraction buffer (1M Kcl, 100 mM Tris-Hcl, 10
mM EDTA). Homogenization of fungal mass was done using sterilized sand and vortexed for
30 seconds, 50μl freshly made proteinase K (10mg/ml PBS) was added. The resulting fungal
tissue homogenate was centrifuged at 5,000 rpm for 10 min and the supernatant was transferred
to an Eppendorf tube containing 300µl of isopropanol and mixed by inverting tube several
times, centrifuged at 12,000 rpm for 10 min, and the supernatant discard as much as possible,
then dissolved the DNA pellet in 1X TE with vortex at the low speed [28]

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2.6.1 Quantitative estimation of genomic DNA:

DNA quantitation of different treatments by Spectrophotometer. The A260/A280 of each

sample was read against distilled water as a blank using an Eppendorf Spectrophotometer,
fitted with 1000 µl of DNA samples, demonstrating genomic DNA by spectrophotometric
analysis.

2.7 Statistical Analysis

One-way ANOVA was used to compare the viability and rate of mutation values of both
controls and treated samples. For each treatment, three tubes were included and then from each
tube, six plates were divided into two groups and cultured on complete and selective media
(n=27). Data were collected using CoStat statistical computing software, and results with The
Least Significant Difference (LSD˂0. 05) were considered to be statistically significant.
Results were presented as mean ± standard deviation (SD). In the estimation of DNA quantities,
differences in average values between control and treated samples (n=3) were analyzed using
one-way ANOVA. Again, results with (LSD˂0.05) were considered to be statistically
significant.

3. Results

3.1 Identification of fungi

3.1.1 Cultural characteristics

The fungi were isolated from horse dung and grown on Yeast malt-glucose (YMG) medium,
colonies attaining a diameter of 7-8cm after 5 days at 28C, edges and aerial mycelium were
white (Figure 1).

Figure 1: Coprinopsis cinerea grown on YGM medium

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3.1.2 Microscopic examination

Figure 2 Developmental structures of Coprinopsis cinerea.(a-g) a: Mycelium, b:

Chlamedospores, c: Oidia with oidiophore, d: Oidia, e: ( A.Carpophore. B, Basidiocarp), f:
Fruit body (A, young fruit body), g: ( A, Basidia. B, Oidia. C: Cheilocystidia. D:
Pilealcuticular elements).

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Oidia with 44.95µm in diameter (Figure 2), small unicellular haploid asexual spores are
produced in abundant numbers in the aerial mycelium of monokaryons by splitting short oidial
hyphae produced at the tips of specialized conidiophores, terminal chlamydospores,
odioiphores caring oidia, basidiocarp (young fruit body) on the tip of carpophore 68.13µm in
height. Septa hyphal foot cells within the conidiophore were found, heteromorphous showing
cheilocystidia (27.72µm in diameter), basidia and pilealcuticular elements, and Fused clamp
cells at hyphal septa

3.2 The consequence of plant extract applied to the wild-type cells

A fluctuation of the viability percentages was noticed when the interaction between two
selected doses of plant extract and the positive control (highest mutagenic concentration of
MSG, previously published data at (http://www.scholarsresearchlibrary.com) where applied on
Coprinopsis cinerea (Figure 3). The lowest viability (27.5%) was remarked when the oidia
were treated with 7g/l of mono-sodium glutamate while the control oidia reached the highest
viability. Surprisingly, the number of wild type cells decreased to 45.8 and 29.3% after
applying low dose (LD) 50 µl and high dose (HD) 250 µl of plant extract respectively. There
were four altered treatments of the interactions; a low dose of plant extraction and then 7g/l of
MSG (LD-7) was the first, in the second treatment MSG was added to the cell, and then a low
dose of plant extraction (7-LD). The addition of a high dose of plant extract before adding 7g/l
of MSG (HD-7) was the third treatment and lastly, the reverse (7-HD) was the fourth
application. The interaction between plant extract (LD and HD) and the positive control (7g/l)
of MSG illustrates an improvement in viability. 59.5% was the highest reached viability when
cells were treated with a low dose of plant extraction then 7g/l of mono-sodium glutamate (LD-
7). However, the viability decreased to the lowest percentage 35.6% following the application
of MSG and then a high dose of plant extraction (7-HD). There was a significant difference at
(p< 0.05) when comparison was made between the mean of each treatment and the mean of
oidia that treated with only MSG. Whereas, there was no significant difference between oidia
treated only with a high dose (HD, 29.3%) of plant extraction and the oidia treated only with
MSG (7g/l, 27.5%).

3.3 The consequence of the plant extract applied to the mutant cells

In this experiment, cells were treated in the same manner as described above in the 3.2 section;
negative control group, both of low and high doses of the plant extract, 7g/l of MSG as a
positive control, and the interactions. Figure 4 demonstrates that there were no mutations in the

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control group whereas the mutation rate reached 0.33 and 0.4 of both selected doses of the plant
extract correspondingly which means that both were mutagenic. The highest mutagenic rate
attained was 1.68 when cells were treated with 7g/l of mono-sodium glutamate. The mean
number of the mutant colonies on the 6-thioguanine plates for the interactions was significantly
(p< 0.05) less than the means obtained in the MSG group. It was not expected to develop a
higher mutagenic rate when cells incubated with plant extract (LD-7 and HD-7) before the
MSG than the reverse (7-LD and 7-HD) incubation which were 0.51, 0.55,0.47, and 0.36
respectively.

120
a
100
Viability %

80
60 cd c
d
e
40 f f
20
0
C LD HD 7G/L LD-7 7-LD HD-7 7-HD
Treatment

Figure 3. The consequences of the interaction between two selected doses of the plant extract
and the highest mutagenic concentration of MSG (7g/l) on the viability. Values plotted are
means of 3 readings from 27 plates. Error bars represent the standard deviation. Values were
statistically tested using one-way ANOVA (small letters: significant differences at p< 0.05).
C= control, LD=low dose, HD= high dose.

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2 a

Mutation rate X 105 1.75

1.5
1.25
1 b
0.75 b c
d e de
0.5
0.25
f
0
-0.25 C LD HD 7G/L LD-7 7-LD HD-7 7-HD
Treatment

Figure 4. The consequences of the interaction between two selected doses of the plant extract
and the highest mutagenic concentration of MSG (7g/l) on mutation rate. Values plotted are
means of 3 readings from 27 plates. Error bars represent the standard deviation. Values were
statistically tested using one-way ANOVA (small letters: significant differences at p< 0.05).
C= control, LD=low dose, HD= high dose.
3.4 The analysis of DNA damage

The genomic DNA was extracted from the hypha of Coprinopsis cinerea and divided as the
following; negative control, mono-sodium glutamate as a positive control, Teucriuma pollinis
extract, and interaction between plant extract and MSG. This experiment was carried out to
determine the in vitro DNA damage that may be caused by exposure to low and high doses of
Teucrium apollinis.

The DNA concentration for untreated and treated samples was measured by spectrophotometer,
three replicates were recorded at 260/230nm of each treatment (Table 1). Unsurprisingly, the
negative control had the highest concentration (94.33µg/ml) whereas the concentrations of
DNA after MSG (7g/l) application were significantly (P>0.05) decreased to 32.33µg/ml. The
significant decline in the amount of DNA was also noted when samples were treated with low
(LD; 50µg/ml) and high (HD; 250µg/ml) doses of plant extract. The DNA quantity obtained
following the LD treatment (33.84µg/ml) was slightly higher than the DNA quantity that was
recorded after HD application (31.56µg/ml). The interaction between two doses of the fixed
oil and 7g/l of MSG was also assessed. Unexpected quantities of DNA were achieved
especially when hypha incubated with the plant extract before adding MSG. DNA quantities
were 30.83, 32.53, 30.73, and 28.03 µg/ml following the treatments of 7g/l of MSG then LD

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of plant fixed oil, the reverse application, 7g/l of MSG then HD of plant extract, and the last
treatment of the fixed oil first then MSG correspondingly.

Table 1: DNA quantitation of different treatments recorded by Spectrophotometer

Amount of
Treatments
DNA (µg/ml)±SE

Control 94.33±2.41a

7g/l(MSG) 32.53±3.40b

LD 33.84±3.26b

HD 31.56±2.70b

LD then7g/l 23.53±2.84b

7g/l then LD 30.83±4.11b

HDthen7g/l 30.73±2.96b

7g/l then HD 28.03±3.99b

MSG: mono-sodium glutamate, LD: low dose of plant extract, HD: high dose of plant extract.
Small letters: significate differences at (P>0.05)

3.5 Analysis of plant extract:

3.5.1 Determination of antioxidant and phenolic compounds

The total phenolic compounds and the antioxidant content of T.apollinis were measured by
spectrophotometer and the values were 11.312%±0.46 and 1.334%±0.005 correspondingly per
1g of dry weight of plant material

3.5.2 Analysis of plant fixed oil by GC–MS technique

The oil extracted from fresh aerial parts of Teucrium apollinis was analyzed by gas
chromatography–mass spectrometry (GC–MS). Fixed oil components were identified based on
their retention index values and also by comparing their mass spectra with known compounds
existing in the data system libraries. Table 2 summarizes the composition of T.apollinis leaf
fixed oils. In total, 10 compounds were identified (Figure 5). The active principles with their
retention time (RT), molecular formula, molecular weight, and peak area (%) are presented,
and Figure 6 shows: a qualitative analysis of the chemical composition of Teucrium apollinis.

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Table 2: Chemical composition of the fixed oil of Teucrium apollinis fresh leaves
identified by (GC–MS) technique

Peak Compound molecular MW RT Peak

formula area
(%)

1 Propanoic acid, 2-methyl-, C19H26O6 350 15.244 100

(dodecahydro-6a-hydroxy-9a-methyl-
3-methylene-2,9-dioxoazuleno[4,5-
b]furan-6 –yl)methyl ester, [3aS-
(3aα,6β,6aα,9aβ,9bα)]-

2 Eucalyptol C10H18O 154 5.394 42.5

3 1,5,5-Trimethyl-6-methylene- C10H16 136 8.026 28.96

cyclohexene

4 10,12-Tricosadiynoic acid, methyl C24H40O2 360 12.557 25.51

ester

5 6,9-Octadecadiynoic acid, methyl ester C19H30O2 290 17.163 16.85

6 Spiro[13ricycle[4.4.0.0(5,9)]decane- C15H24O3 252 14.92

10,2’-oxirane], 1-methyl-4-isopropyl- 16.548
7,8-dihydroxy-, (8S)-

7 9,10-Secocholesta-5,7,10(19)-triene- C27H44O3 416 16.308 12.89

3,25,26-triol, (3β,5Z,7E)-

8 Androstan-17-one, 3-ethyl-3-hydroxy-, C21H34O2 318 16.062 11.05

(5α)

9 2(3H)-Furanone, dihydro-4,4- C9H14O3 170 6.679 7.75

dimethyl-5-(2-oxopropyl)

10 Camphor C10H16O 152 7.479 6.94

MW=Molecular weight, RT= Retention time

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Retention Time

Figure 5. Qualitative analysis of fixed oil of Teucrium apollinis fresh leaves by Gas
chromatography/Mass spectrometry (GC/MS)
4. Discussion

Fungi display several characteristics that make them suitable objects for a great variety of
studies: although they are eukaryotic and, therefore, are related to higher organisms, they are
modest regarding cultivation requirements, and the genetics of many strains are well
documented. In the current study, we use Coprinopsis cinerea fungi for the assessment of
genotoxicity of TA extract.

The fungal genera and species were identified based on mycelium and spore characteristics via
microscopic examination and referring published data. Coprinopsis cinerea (previously called
Coprinus cinereus) is the developmentally best-understood species of the Agaricomycetes that
produces typical fruit bodies with the sexual basidiospores on the dikaryon (Figure 2: e &f).
Genetics proved that the tested model has morphological varieties of the same species [29, 30].
Oidiophores can also differ very much in structure, four main types were distinguished by the
attendance or non-attendance of an oidiophore stipe, by the presence or absence of a septum
that separates oidiophores from their foot cells, by the length of the oidiophores, and by
existence or lack of side branches at the oidiophores. About these four types, our strain matches
the type I of the oidiophore (Figure 2: c).

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4.1 The analysis of HGPRT gene mutation assay

The purpose of the in vitro gene mutation test is to detect gene mutations induced by chemicals.
This test measures forward mutations in the reporter gene, endogenous hypoxanthine guanine
phosphoribosyl-transferase gene (HGPRT). The HGPRT mutation test detects different spectra
of genetic events. Base pair substitutions, frameshifts, small deletions, and insertions are
mutational events that were detected by the HGPRT test. The assessment of mutations on the
HGPRT locus is expected to be an effective tool for various genetics, medical, and agricultural
applications [31,32,33]. Organization for Economic Co-operation and Development (OECD)
recommended many tests for assessing the genotoxicity of chemicals including HGPRT Gene
Mutation [34]. This test is an operative and widely used method in the detection of mutagenicity
and genotoxicity of chemicals in laboratory conditions [35, 36, 37, 38, 39].

4.1.1 The consequences of Teucrium apollinis extract on survival and mutation cell
frequency

Traditional medicine plants are widely worldwide used and have attracted renewed interest in
developed countries over the last decades. Despite the positive observation of herbal medicines,
cytotoxic cases have been reported in different literature [40, 41, 42]. Some herbal medicines
have been shown to contain toxic compounds, these compounds react with cellular
macromolecules, including DNA, causing cellular toxicity and/or genotoxicity [43]. The
HGPRT mutation assay used in the present study for evaluating the cytotoxic and mutagenic
potentials of medicinal plant, Teucrium apollinis. Two different ranges of concentrations (low
and high) were selected and tested in the current study exhibiting significant cytotoxic
influence by decreasing cell viability (Figure 3). The deficiency of chemo-protective effect in
current extraction was also evident from the high mutant phenotype in selective medium and
thus the high mutation frequencies (Figure 4). It was reported that components of plant extracts
can contribute to a variety of effects on cells, and run specified mechanisms of cytotoxicity
[44, 45]. Ten compounds were identified by (GC-MS) analysis of the air aerial parts of
Teucrium apollinis of greenish oil with a strong deterrent odor. Those ten compounds are
represented as the main constituents of the oil (Table 2). The chemical composition of T.
apollinis oil from different parts of the world has been reported by some researchers. The
variations in the composition of the oil are likely the result of plant parts used, harvesting time,
and geographical location [46]. Our results could be related to the chemical composition of
the extract since the GC-MS analysis exhibits Camphor and Furanone compounds that were
previously reported as genotoxic phytochemicals [47, 48, 49, 50]. Camphor, a terpenoid

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Nagat S. Elhaddad et al. J. Sci. Res. Sci., 2023, 40, (2): 1-25
compound, is a common traditional remedy that has been used to cure a wide range of
symptoms [51]. However, the main problems with camphor toxicity in humans are connected
more to the large availability of camphor secondary products and their diffused perception
rather than to the toxicity of camphor [52]. The oral administration of different doses of
camphor to rats and rabbits caused pronounced signs of toxicity. Reduced body weight gain
and food consumption with a reduction of motility were observed in both rats and rabbits [52].
The chemical composition of the extract, as well as their relationship, may alter the cytotoxicity
of plants and could be the reason for their different effects [44]. Following that, the difference
in the effects between the different species of the same genus Teucrium may depend on the
chemical composition of the extracts or can be caused by many ecological factors. Moreover,
plant compounds and selected doses may affect the activity of many cell receptors and enzymes
[53]. In general, both doses extract of Teucrium apollinismay somehow impaired the set of
signaling pathways and metabolic functions that affect the cell cycle and proliferation, protein
synthesis, RNA biosynthesis, DNA replication, repair, and membrane biosynthesis. For this
reason, in mice, the HGPRT gene regulates multiple developmental and metabolic pathways
of embryonic stem cell neuronal differentiation [54, 55, 56].

4.2 The analysis of genomic DNA damage

In this section, the mutagenicity of fixed oil of Teucrium apollinis in comparison to negative
and positive controls was evaluated by assessing the genomic DNA damage based on
spectrophotometer analysis. The quantities of the genomic DNA extracted from treated hypha
are illustrated in Table 1. In the current study, it can be said that MSG and extract of T. apollinis
had a negative effect on genomic DNA, which produced DNA with very low concentrations in
all treatments.

4.2.1 Teucrium apollinis extract showed DNA damage effect

Much research effort has focused on the identification of phytochemicals in traditional plants,
fruits, and vegetables that utilize beneficial effects. However, Assessment of the potential
genotoxicity of traditional medicines is indeed an important issue as damage to the genetic
material may lead to critical mutations and therefore also to an increased risk of cancer and
other diseases. In the current project, we are evaluating the damaging effects of Teucrium
apollinis plant extract on genomic DNA. Numerous studies provide evidence for
mutagenic/antimutagenic or prooxidant /antioxidant activities largely depending on the
concentration used of medicinal plants [57, 58, 59]. That is why two different doses (50 and

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Nagat S. Elhaddad et al. J. Sci. Res. Sci., 2023, 40, (2): 1-25
250µg/ml) of current oil extract were applied. Data in Table 1 reflect the DNA damage when
mycelia were treated with low and high doses of plant extract and also when combined with
7g/l of MSG. This damage might have resulted from direct interaction between a DNA-reactive
component in Teucrium apollinis extract, and DNA since such kind of interaction is one of
several pathways that may lead to primary DNA damage [60, 61]. Specific components in the
composition of plant extracts can contribute to a variety of effects on cells, and run specified
mechanisms of cytotoxicity [62, 47]. Our data may reflect the changes in the redox status of
the DNA by increasing the level of superoxide anion radicals following T.apollinis oil
application [44]. In addition, T. apollinis extraction in present research could alter the
production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can
modify signaling pathways in the cell resulting in damage to DNA [63. The total phenolic
compounds (11.312%±0.46) and the antioxidant content (1.334%±0.005) per 1g of the dry
weight of plant material of T. apollinis were measured, both values were relatively high as
represented in the 3.5.1 section. The situation of the fairly high content of phenols and
antioxidant content is in agreement with Tepe and his colleagues in 2011[64], who examined
antioxidant and DNA damage protection activities of Teucrium and reported that it was rich in
phenolic and flavonoid contents and can be used as an alternative to a synthetic antioxidant
source [64]. In contrast, the present study reflects that the oil of T. apollinis induced significant
DNA damage suggesting that components in our extract might interact directly with the DNA.
In plant extract, major components have been identified and evaluated in the present study
(Table 2), but the potential genotoxicity of these constituents remains rather unclear. However,
there is little data regarding furanone (which was detected in our extract components) which
was responsible for DNA-breaking activity [65]. also, the furanone/transition metal–mediated
generation of reactive oxygen species was held responsible for DNA strand breaks and the
formation of 8-hydroxy-2′- deoxyguanosine [66]. Moreover, the furanone, which are known
pro-oxidants in foods [67], can produce superoxide radicals through, the reduction of cupric
ions to cuprous ions, resulting in the conversion to hydrogen peroxide and hydroxyl radicals
[68]. It is therefore fair to assume that the DNA damaging effect observed at 50 and 250 µg/ml
might be induced by at least one or more of those components. The reason for this is not known,
but it is not an uncommon phenomenon especially if the tested plant was previously situated in
great concern. It was reported that an aqueous extract of Teucrium polium has some hepatotoxic
effects and it is not suitable for use in humans as an antidiabetic agent [69]. Moreover, a
significant increase was seen in both alanine aminotransferase and aspartate aminotransferase
enzyme activities in female rats, also a significant increase in liver weight of male rats after

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Nagat S. Elhaddad et al. J. Sci. Res. Sci., 2023, 40, (2): 1-25
administration of Teucrium polium extract [70]. There are few reports about the possible
toxicological actions of T. polium. Acute and chronic toxicity of Teucrium stocksianum in rats
has been considered [71]. There is also a report about the hepatotoxic effects of Teucrium
polium in rats [72]. Surprisingly, the essential oil of T. Polium has been effectively used as a
bio-insecticide demonstrating its toxic content [73]. Much progress has been made in our
understanding of the medicinal plant mechanisms that underlie cytotoxic, genotoxic, mutagenic, and
carcinogenic actions. However, difficult challenges remain to be addressed to improve our
understanding of their molecular mechanism.In summary, this research paper suggest
mutagenic impacts of Teucrium apollinis.

Genotoxic
TA/ MSG

Cell membrane

TA / MSG

Oxidative stress

+ROS, - AO

+ LPO, - GSH, +
SOD,+GST Mutations (HPRT),
DNA damage

±cellular metabolism Low DNA damage High DNA damage

DNA
repair
Viable cell/no mutation Viable cell/mutation
- Functional -Loss of function
-Cancer
Cell damage
Cell damage

Cell membrane

Figure 6. Proposed diagram of cytogenic/mutagenic of Teucrium apollinis (TA) and

monosodium glutamate (MSG). LPO: lipid peroxidation, GSH: Glutathione, SOD: superoxide
dismutase, GST: Glutathione-s-transferase, +: induction, -: reduction, ±: disruption, dotted
arrows: unknown steps.

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Nagat S. Elhaddad et al. J. Sci. Res. Sci., 2023, 40, (2): 1-25
Figure 6. reflects possible genotoxic mechanisms of Teucrium apollinis. Current results
revealed that TA has cytotoxic and mutagenic impacts on wild type in a similar way to MSG
which is used as a positive control, mutant cell, and also on the genomic DNA in the tested
model fungi. By understanding previous research and comparing their explanation with
present data, proposed diagram 6 was generated assuming that TA and MSG have similar
mutagenic impacts. At the molecular level, TA and MSG increase oxidative stress and reduce
antioxidants leading to cell damage after multiple molecular processes involving mutation in
the HGPRT gene and DNA damage.

5. Conclusion

Taken together, after assessing the genotoxicity of Teucrium apollinis extract on the selected
experimental model, we conclude that the HGPRT gene mutation test is an operative method
in the detection of the genotoxicity of chemicals. Also, the efficiency of studying the
degradation of genomic DNA by using a spectrophotometer to investigate the induced damage
caused by different applications. Finally, the findings of the present work support a genotoxic
effect of TA in both of HGPRT mutation assay and DNA damage analysis. It is recommended
to increase health education programs about medicinal plant usage.

Acknowledgment

The authors thank Prof. Mohamed A. M. Adam for occupying all of the fungi images and for
his assistance in measuring DNA content in his laboratory. Also, we would to thank Kamla
Boagila Blash for her helpful advice on statistical analysis. We are grateful for Enas, M.
Ibrahim AL- alwania’s aid in the arrangement of the list of references.

Conflict of interest

The trial is completely devoid of conflict of interest.

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‫‪Nagat S. Elhaddad et al.‬‬ ‫‪J. Sci. Res. Sci., 2023, 40, (2): 1-25‬‬
‫الملخص العربى‬

‫التأثير السمي الوراثي لنبات الجعدة باستخدام جين ‪ HGPRT‬وتلف الـ ‪DNA‬‬

‫نجاة سعد الحداد‪ ،‬هدى أحمد خطاب‪ ،‬إيمان محمد بلقاسم‪ ،‬نجاح سليمان أبوبكر‬

‫قسم النبات – كلية العلوم – جامعة عمر المختار – البيضاء ‪ -‬ليبيا‬

‫الملخص‬

‫في هذه الدراسة‪ ،‬نوثق نجاح تطبيق جين ‪Transferase (HGPRT) Hypoxanthine Guanine Phosphoribosyl -‬‬

‫الختبار القدرة التطفيرية نبات الجعدة ‪ . Teucrium apollinis‬تم فحص التأثير السمي الوراثي باستخدام تركيزين هما‬
‫التركيز المنخفض و المرتفع ‪ 50‬و‪ 250‬ميكروغرام ‪ /‬مل من الزيت الثابت المستخلص من ‪Teucrium apollinis‬‬
‫(‪.) TA‬تهدف الدراسة الحالية للكشف عن الضرر الوراثي الذي قد تسببه الجرعات التي تم اختيارها على الخلية الالجنسية‬
‫(‪ )oidia‬من فطر‪.Coprinopsis cinerea‬‬

‫أثبتت النتائج‪ ،‬طفرة الجين ‪ ،HGPRT‬أن كال التركيزين لهما تأثيرات سامة للخاليا‪/‬مطفرة من خالل تقليل الحيوية وزيادة‬
‫معدل طفرة جين ‪ HGPRT‬في الفطر المدروس‪ .‬والمثير للدهشة أن نسبة الحيوية انخفض بشكل ملحوظ إلى ‪ 45.8‬و‪٪29.3‬‬
‫عندما حضنت األويديا بجرعة منخفضة ‪50‬ميكرولتر وجرعة عالية ‪250‬ميكرولتر من مستخلص النبات على التوالي مما‬
‫يوضح تأثيرها السام للخاليا‪ .‬أيضا وصل معدل الطفرات إلى ‪ 0.33‬و‪ 0.4‬من كلتا الجرعتين المختارتين من الزيت الثابت‬
‫مما يعني أن كالهما كان مطفراً‪ .‬تم الكشف عن تلف الحمض النووي الجيني لـفطر‪ Coprinopsis cinerea‬عن طريق‬
‫القياس الكمي باستخدام التحليل الطيفي‪ .‬كانت هناك فروق ذات داللة إحصائية (‪ )LSD = 0.05‬بين المعالجات الضابطة‬
‫وجميع المعامالت المذكورة أعاله‪ ،‬حيث كانت أعلى قيمة ‪94.33 ± 2.41‬ميكروغرام ‪ /‬مل لمجموعة السيطرة‪ ،‬وأدنى‬
‫كمية من الحمض النووي (‪ )32.53 ± 3.40‬تم الحصول عليها عند معالجة االويديا بـ ‪ 7‬جم ‪ /‬لتر من جلوتامات احاديه‬
‫الصوديوم الذي استخدم كسيطرة موجبة‪ .‬بالتالي فان المستخلص النباتي بالجرعتين المنخفضة والعالية أدى الى خفض تركيز‬
‫الحمض النووي إلى ‪ 33.84 ± 3.26‬و ‪31.56 ± 2.70‬على التوالي‪ ،‬مما يشير إلى السمية الوراثية لنبات الجعدة‪.‬‬

‫تم التعرف على عشرة مركبات كيميائية لزيت نبات الجعدة باستخدام مطياف الكتلة المرتبط بالكروماتوجرافي الغازي ‪GC-‬‬
‫‪ MS‬كان من بينها كال من حمض البروبانويك والكافور‪.‬‬

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