Phytotoxicity and cytogenetic action mechanism of leaf extracts of Psidium cattleyanum Sabine in plant bioassays

Abstract

The search for more environmental friendly herbicides, aiming at the control of agricultural pests, combinated with less harmfulness to human health and the environment has grown. An alternative used by researchers is the application of products of secondary plant metabolism, which are investigated due to their potential bioactivities. Thus, species belonging to the Myrtaceae family are potential in these studies, since this family is recognized for having high biological activity. A species belonging to this genus is Psidium cattleyanum, which has a medicinal effect and its fruits are used in human food. Thus, the objective of this research was to evaluate and compare the phyto-cyto-genotoxicity of aqueous and ethanolic leaf extracts of the specie P. cattleyanum, from plant bioassays, as well as to identify the main classes of compounds present in the extracts. For this, the extracts were prepared, characterized and biological tests were carried out by evaluating, in seeds and seedlings of lettuce and sorghum, the variables: percentage of germination, germination speed index, root growth and aerial growth; and in meristematic lettuce cells the variables: mitotic phases, mitotic index, nuclear alterations and chromosomal alterations. Flavones, flavonones, flavonols, flavononols, flavonoids, alkaloids, resins, xanthones and anthraquinone glycoside were characterized in the ethanolic extract. Both evaluated extracts, in the highest concentration, inhibited the initial plant development. All treatments caused alterations in the mitotic phases and inhibited mitotic index. In addition, the treatments promoted an increase in nuclear and chromosomal alterations. The mechanism of action presented was aneugenic, clastogenic and determined in epigenetic alterations. The ethanolic extract was more cytotoxic, since it had a more expressive effect at a lower concentration. Despite the cytotoxicity of the extracts under study, they promoted alterations at lower levels than the glyphosate positive control.

Full text

1/10Brazilian Journal of Biology, 2024, vol. 84, e260985 | https://doi.org/10.1590/1519-6984.260985
Original Article
THE INTERNATIONAL JOURNAL ON NEOTROPICAL BIOLOGY
THE INTERNATIONAL JOURNAL ON GLOBAL BIODIVERSITY AND ENVIRONMENT
ISSN 1519-6984 (Print)
ISSN 1678-4375 (Online)
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
The search for more environmental friendly herbicides, aiming at the control of agricultural pests, combinated with less
harmfulness to human health and the environment has grown. An alternative used by researchers is the application
of products of secondary plant metabolism, which are investigated due to their potential bioactivities. Thus, species
belonging to the Myrtaceae family are potential in these studies, since this family is recognized for having high biological
activity. A species belonging to this genus is Psidium cattleyanum, which has a medicinal effect and its fruits are used in
human food. Thus, the objective of this research was to evaluate and compare the phyto-cyto-genotoxicity of aqueous
and ethanolic leaf extracts of the specie P. cattleyanum, from plant bioassays, as well as to identify the main classes of
compounds present in the extracts. For this, the extracts were prepared, characterized and biological tests were carried
out by evaluating, in seeds and seedlings of lettuce and sorghum, the variables: percentage of germination, germination
speed index, root growth and aerial growth; and in meristematic lettuce cells the variables: mitotic phases, mitotic index,
nuclear alterations and chromosomal alterations. Flavones, flavonones, flavonols, flavononols, flavonoids, alkaloids,
resins, xanthones and anthraquinone glycoside were characterized in the ethanolic extract. Both evaluated extracts,
in the highest concentration, inhibited the initial plant development. All treatments caused alterations in the mitotic
phases and inhibited mitotic index. In addition, the treatments promoted an increase in nuclear and chromosomal
alterations. The mechanism of action presented was aneugenic, clastogenic and determined in epigenetic alterations.
The ethanolic extract was more cytotoxic, since it had a more expressive effect at a lower concentration. Despite the
cytotoxicity of the extracts under study, they promoted alterations at lower levels than the glyphosate positive control.
Keywords: aqueous extract, citotoxicity, ethanolic extract, genotoxicity, Lactuca sativa.
Resumo
A busca por herbicidas mais amigáveis ao meio ambiente, visando o controle de pragas agrícolas, aliado a uma
menor nocividade à saúde humana e ao meio ambiente tem crescido. Uma alternativa utilizada pelos pesquisadores
é a aplicação de produtos do metabolismo secundário de plantas, que são investigados em virtude do seu potencial
bioativo. Assim, espécies pertencentes à família Myrtaceae são potenciais nestes estudos, uma vez que esta família
é reconhecida por possuir alta atividade biológica. Uma espécie pertencente a este gênero é Psidium cattleyanum,
que possui efeito medicinal e seus frutos são utilizados na alimentação humana. Assim, o objetivo desta pesquisa foi
avaliar e comparar a fitocitogenotoxicidade de extratos foliares aquosos e etanólicos da espécie P. cattleyanum, a partir
de bioensaios vegetais, bem como identificar as principais classes de compostos presentes nos extratos. Para isso, os
extratos foram preparados e caracterizados e foram realizados testes biológicos avaliando, em sementes e plântulas de
alface e sorgo, as variáveis: porcentagem de germinação, índice de velocidade de germinação, crescimento radicular e
crescimento aéreo; em células meristemáticas de alface foram avaliadas as variáveis: fases mitóticas, índice mitótico,
alterações nucleares e alterações cromossômicas. Flavonas, flavononas, flavonóis, flavononóis, flavonóides, alcalóides,
resinas, xantonas e glicosídeo de antraquinona foram caracterizados no extrato etanólico. Ambos os extratos avaliados,
na maior concentração, inibiram o desenvolvimento inicial da planta. Todos os tratamentos causaram alterações nas
fases mitóticas e inibiram o índice mitótico. Além disso, os tratamentos promoveram aumento de alterações nucleares
e cromossômicas. O mecanismo de ação apresentado foi aneugênico, clastogênico e epigenético. O extrato etanólico
foi mais citotóxico, pois teve efeito mais expressivo em menor concentração. Apesar da citotoxicidade dos extratos
em estudo, eles promoveram alterações em níveis inferiores ao controle positivo glifosato.
Palavras-chave: extrato aquoso, citotoxicidade, extrato etanólico, genotoxicidade, Lactuca sativa.
Phytotoxicity and cytogenetic action mechanism of leaf
extracts of
Psidium cattleyanum
Sabine in plant bioassays
Fitotoxicidade e mecanismo de ação citogenético de extratos foliares de
Psidium cattleyanum Sabine em bioensaios vegetais
T. A. Alvesa , M. S. Spadetoa , L. C. Vasconcelosa , J. R. C. L. Souzab , L. Meninib , M. F. S. Ferreirac 
and M. M. Praça-Fontesa* 
aUniversidade Federal do Espírito Santo – UFES, Centro de Ciências Exatas, Naturais e da Saúde, Departamento de Biologia, Programa de
Pós-graduação em Genética e Melhoramento, Laboratório de Citogenética e Cultura de Tecidos Vegetais, Grupo de Pesquisa Agroquímicos e
Análise de Toxicidade em Bioensaios, Alegre, ES, Brasil
bInstituto Federal de Educação, Ciência e Tecnologia do Espírito Santo – IFES, Laboratório de Química Aplicada, Alegre, ES, Brasil
cUniversidade Federal do Espírito Santo – UFES, Centro de Ciências Agrárias e Engenharias, Departamento de Agronomia, Programa de Pós-
graduação em Genética e Melhoramento, Laboratório de Genética Vegetal, Grupo de Pesquisa Agroquímicos e Análise de Toxicidade em
Bioensaios, Alegre, ES, Brasil
*e-mail: milene.fontes@ufes.br
Received: February 11, 2022 – Accepted: May 19, 2022

Brazilian Journal of Biology, 2024, vol. 84, e2609852/10
Alves, T. A. et al.
large amount of small-sized seeds (Aragão et al., 2015), in
addition to germinating within 24 hours (Alves et al., 2018).
The Myrtaceae family agroup taxon with high biological
activity. Among the species of this family with potential
phytochemical content is the araça Psidium cattleyanum
Sabine (Vinholes et al., 2018), which presents fruits used
in human food and has bioactive compounds previously
proven in studies that investigated its medicinal activity
(Pereira et al., 2018; McCook-Russell et al., 2012;
Vinholes et al., 2018).
Given the above, the objective of the present study
was to evaluate and compare the phyto-cyto-genotoxicity
of aqueous and ethanolic leaf extracts of the species P.
cattleyanum, from bioassays with model plants, as well
as to identify the main classes of compounds present in
the extracts.
2. Material e Methods
2.1. Material vegetal
Young leaves were collected from adult individuals of
Psidium cattleyanum at a height of 1.30 m, in the month
of February (summer), in the morning period, at the
experimental field of the Center of Agricultural Sciences
and Engineering (CCAE) (altitude 254 m, coordinates 20°45’
41°31’) at the Federal University of Espírito Santo (UFES),
and used as test agents. The voucher was collected, dried,
and deposited at the MBML-Herbario (48718). The number
of SISGEN authorization is AGF0DB3.
Seeds of two species were adopted as plant models
(Alves et al., 2018):
a) Lactuca sativa L. ‘Crespa Grand Rapids’ (Isla Pak)
(eudicot), with germination rate of 97%, purity of 100%
and within the validity period indicated by the supplier;
b) Sorghum bicolor L. Moench ‘AL Precioso’ (BR Seeds)
(monocot), with germination rate of 87%, purity of
99.7% and within the validity period indicated by the
supplier.
2.2. Extract preparation
To obtain the test extracts, the collected leaves were
dried in forced air circulation oven at 60°C for 72 h and
subsequently ground in a blender.
a) Aqueous extract
30 g of the dried leaf powder were weighed and
300 mL of distilled water at 100°C were added. After
10 minutes, the infusion was filtered, yielding the extract
at the concentration of 100 mg mL
-1
(Almeida et al., 2006;
Prichoa et al., 2013), from which dilutions were made to
obtain the concentrations of 50, 25 and 12.5 mg mL-1.
b) Ethanolic extract
10 g of the dried leaf powder were weighed and 100 mL
of 70% ethanol were added, being kept on a shaker for
three days. Subsequently, the solution was filtered and
placed in a rotary evaporator, yielding a concentrate of
500 mg mL
-1
(concentration not tested). From this solution,
dilutions were made to obtain the tested concentrations
of 100, 50, 25 and 12.5 mg mL-1 (the same as tested with
the aqueous extract).
1. Introduction
Most of the known organic compounds can be found in
nature, and plants are the collaborators in the formation
and supply of these molecules. The production is through
the natural synthesis of phytochemicals in the plant,
which are commonly known as primary and secondary
metabolites. This classification is carried out considering the
role played by each compound in the plant (Mohan et al.,
2019; Almeida et al., 2021).
The molecules and substances necessary for plant
growth, such as chlorophyll, sugars, proteins, amino
acids, lipids, among others, are considered primary
metabolites. The compounds intended to defend the
individual’s biotic and abiotic stresses, such as essential
oils, alkaloids, tannins, flavonoids, saponins, terpenoids,
phenolic compounds, among others, are the secondary
metabolites (Mohan et al., 2019; Oszmiański et al., 2020).
These phytochemicals products of secondary metabolism
have biological activities, being studied due to this potential
(Alves et al., 2018; Oszmiański et al., 2020).
Studies have been developed seeking to prove the
effects of the different uses and applications of plant
materials. In this way, plant chemical compounds have
been applied in various activities of human interest, such
as in the synthesis of agrochemicals, cosmetics, medicines
and condiments (Braga et al., 2019; Alves et al., 2018;
Oszmiański et al., 2020).
Different ways of extracting and obtaining plant
products are described (Alothman et al., 2009; Ferreira-
Dias et al., 2003; Ozer et al., 2016; Saklani et al., 2017), in
order to optimize your applications. One of the ways uses
water as a solvent, obtaining aqueous extracts as a product.
This type of extract is the most friendly to the environment,
in addition, water is the most accessible and inexpensive
solvent, making it very applicable to the extraction of
bioactive vegetable compounds (Vuong et al., 2013). This
type of extraction determines the greater obtaining and
release of hydrophilic compounds (Kubiliene et al., 2018).
Another way is ethanol extraction, which allows greater
access to lipophilic substances, such as phenolic acids,
aromatic acids, flavonoids and terpenes (Kubiliene et al.,
2018). Thus, research that evaluates the bioactivity of
the products of the different methods of obtaining plant
metabolites is relevant and can provide illuminating results
related to the different applications of these compounds.
Moreover, the understanding of bioactivity, as well as
the differential effect of aqueous and ethanolic extract is
important for the establishment of cultures, the study of
possible bio-chemicals, in addition to allowing knowledge
about the allelopathic activities of organisms.
To investigate the bioactivity of natural products
against plant development, bioassays with model plants
are used (Alves et al., 2018), for elucidating the different
levels of toxicity of the test agent and indicating potential
agrochemicals (Pinheiro et al., 2015). In addition, they
feature fast response and are low cost (Alves et al., 2018).
The species Lactuca sativa L. (lettuce) and Sorghum bicolor
(L.) Moench are applied as a model in bioassays, as they
are easily found agricultural supply stores and have a

Brazilian Journal of Biology, 2024, vol. 84, e260985 3/10
Toxicity of extracts of Psidium cattleyanum
2.3. Chemical characterization of aqueous and ethanolic
extracts
The phytochemical screening to determine the main
classes of secondary metabolites present in aqueous
and ethanolic leaf extracts were performed as described
in the literature for phenols, hydrolyzable tannins,
condensed tannins, anthocyanins, anthocyanidins,
leucoanthocyanidins, flavones, flavonols, flavononols,
flavanones, xanthones, chalcones, aurones, catechins,
steroids, triterpenoids, saponins, strong fixed acids,
resins and alkaloids (Matos, 2009); for anthraquinone
glycosides (Joshi et al., 2013); and for cardiac glycosides
(Ayoola et al., 2008).
2.4. Phytotoxicity assay
The experiment was established following the method
of direct treatment application in completely randomized
design using five repetitions per treatment, with 25 seeds
per repetition
22
. Distilled water was used as negative
control (C-) and the commercially available herbicide
glyphosate (0.1%) as positive control (C+) (Pinheiro et al.,
2015). The following variables were analyzed (Alves et al.,
2018; Silveira et al., 2017):
a) Germination percentage (GP) – number of germinated
seeds after 48 h of exposure to the treatments, calculated
by the ratio between the number of germinated seeds
times 100 divided by the total number of exposed seeds
per repetition.
b) Germination speed index (GSI) – number of germinated
seeds counted every 8 h during the first 48 h of exposure
to the treatments, calculated by the following formula
(Equation 1):
( ) ( ) ( )
( )
( )
1 *1 2 1 *1 / 2 3 2
*1 / 3 ... 1 *1 /
N NN NN
Ny Ny y
+− +−
+ −−
(1)
Where: Ny refers to the number of seeds germinated
within a given period; y: represents the total number of
time intervals (Maguire, 1962).
c) Root growth (RG) – measured (in mm) with the aid of a
digital caliper after 48 h of exposure to the treatments.
d) Aerial growth (AG) – measured (in mm) after 120 h
of exposure to the treatments with the aid of a digital
caliper.
2.5. Cyto-genotoxicity assay
To assess cyto-genotoxicity, tip of roots of lettuce were
fixed in an methanol: acetic acid fixative (3:1/vv-1) after 48h
of exposure to treatments (Alves et al., 2021; Dutra et al.,
2020), and then they were stored at -20ºC. Two fastener
changes were made; the first after ten minutes of fixation
and the second after 24 hours. The roots remained fixed
for at least 24 hours, until the end of the last fixation step.
The roots were washed three times, for ten minutes
each, in distilled water and hydrolyzed in 5N HCl at 25ºC
for 18 minutes. For each slide, semi-permanent and
prepared by the crushing technique, two root meristems
were used, which were cut, stained with 2% acetic orcein
for 15 minutes and sealed with colorless enamel. For each
treatment, five slides were prepared and 1000 cells were
evaluated per slide, totaling 5000 cells per treatment.
The following variables were evaluated:
a) Mitotic index (MI) - refers to the number of cells that are
dividing, calculated by the ratio between the number of
cells in division and the total number of cells observed
(Fiskesjö, 1985).
b) Chromosomal alterations (CA) - refers to the changes
observed at the chromosomal level, calculated by the
ratio between the number of cells with CA and the
total number of cells observed (Fiskesjö, 1985).
c) Nuclear alterations (NA) - refers to the changes observed
at the nuclear level, calculated by the ratio between
the number of cells with NA and the total number of
cells observed (Fiskesjö, 1985).
The CA and NA were assessed separately according
to their categories and their frequencies were measured
individually (Pinheiro et al., 2015).
a) The CA are – c-metaphase, adherence, bridge, lost
chromosome, chromosome not oriented, fragmentation,
polyploidization, multipolarity, the frequency of each
alteration being calculated individually by the ratio of
the number of cells with each CA to the total number
of cells in division.
b) The NA are – micronucleus and condensed nucleus
the frequency of each alteration being calculated
individually by the ratio of the number of cells with
each NA to the total number of cells observed.
2.6. Statistical analysis
The values obtained in each observation were tabulated.
The means were obtained by analysis of variance and
submitted to the Tukey test (p<0.05) using the statistical
program Genes and the graphics were plotted in the
program R, version 3.3.2 (R Development Core Team,
2020; Cruz, 2013). Regression analysis was used to assess
the mitotic index (MI). The polynomial regression models
were adjusted according to the significance of ANOVA F and
the quality of the models was assessed by the coefficient
of determination (R
2
). The analysis were performed using
the R computational environment (R Development Core
Team, 2020).
3. Results and Discussion
3.1. Chemical characterization of the aqueous and
ethanolic extracts
Leucoanthocyanidins, catechins, anthocyanins,
anthocyanidins, aurones, chalcones, condensed tannins
and triterpenoids were not observed in any of the
samples (Table 1). This observation corroborates another
investigation that in which neither the aqueous or the
ethanolic extract of P. cattleyanum presented anthocyanins,
anthocyanidins, aurones and chalcones, confirming this
finding for the specie (Gavilla and Muro, 2018).
Strong fixed acids, steroids, simple phenols, saponins,
hydrolyzable tannins, cardiac glycosides and terpenoids
were identified in both evaluated extracts (Table 1).
Saponins – previously described in the composition of

Brazilian Journal of Biology, 2024, vol. 84, e2609854/10
Alves, T. A. et al.
aqueous and ethanolic extracts of P. cattleyanum – show
insecticide activity, causing destruction of hemolymph
components, leading to alterations in the coagulation,
leakage, and ultimately death of the insect (Tello, 2014).
Alkaloids, resins, xanthones and anthraquinone
glycosides were identified only in the ethanolic extract
(Table 1). Alkaloids, previously described in the composition
of the ethanolic extract of P. cattleyanum, but not in the
aqueous one (Gavilla and Muro, 2018), have been described
as insecticides owing to their detrimental effect on the
nervous system of most insects, acting in the ganglion-
cerebral disorientation, culminating in alteration of the
insects’ perception (Tello, 2014).
Also flavones, flavonones, flavonols, flavonnnols and
flavonoids were only identified in the ethanolic extract
of P. cattleyanum (Table 1). Different types of quercetin
in the chromatographic profile of P. cattleyanum leaves
further demonstrated (Wang et al., 2017). Thus, there are
possibly different types of quercetin among the occurring
flavonoids, which are related to different biological activities
(Díaz-de-Cerio et al., 2017).
Considering the above, several compound classes with
biological activity already described in the literature were
observed in the studied extracts, demonstrating their
potential in investigations of novel biological activities.
3.2. Phytotoxicity assay
The lettuce seeds treated with ethanolic extract at the
concentrations of 100, 50 and 25 mg mL
-1
did not germinate,
being completely inhibited (Figure 1a).
Allelochemical compounds exert direct and indirect
action on the plant metabolism (Maraschin-Silva and Aquila,
2006). The production of metabolites is dependent on the
environmental conditions, such as nutrient availability,
soil biota, chemical characteristics of the soil, interaction
between the different populations, among others. Changes
in these conditions that are caused by the presence of the
allelochemical are considered as indirect action. In turn,
the direct action comprises alterations at the level of cells
and plant metabolism, including changes in physiological
processes (e.g. respiration, photosynthesis) and in cell
functioning (e.g. membrane permeability), among others
(Reigosa et al., 1999; Rice, 1984; Rizvi et al., 1992).
In this way, when such effects of phytotoxic activity
are compared to the phytochemical screening, it is noted
that the treatment with aqueous extract was that allowed
germination of lettuce seeds (Figure 1a). This extract
did not present flavonoid compounds, such as flavones,
flavonols and flavononols (Table 1). Since flavonoids are
allelochemicals known to promote inhibition of plant
development, by direct and indirect action (Carvalho et al.,
2019), it can be concluded that the observed result is related
to the absence/presence of these chemical constituents
in the extracts.
The variable GP remained similar among all treatments
in the sorghum model (Figure 1b), whereas in the lettuce
model it was significantly inhibited by all treatments
compared to C- (Figure 1a). However, greater effectiveness
of the ethanolic extract is observed in lettuce compared to
the aqueous extract, as total inhibition of GP occurred at
its three highest concentrations. This greater effectiveness
of the ethanolic vs. aqueous extract may be associated to
the presence of alkaloids only in the first (Table 1). This
metabolite class has been described to promote rupture of
the cell membrane (Santos et al., 2019), allowing leakage
of electrolytes, thus inhibiting the development of seeds/
plantlets in a direct way.
It is important to highlight that C+ reduced the GP in
lettuce by approximately 20% when compared to C-, with
the reductions caused by the extracts being superior to
that promoted by C+ (Figure 1a). Studying the allelopathic
effect of the aqueous extract of P. cattleyanum, other study
also reported inhibition of the germination of lettuce
seeds treated at the concentration of 75 g L-1, associating
this inhibition to the presence of allelopathic compounds,
which act by interfering with membrane permeability,
cell division and enzyme activation (Hister et al., 2016).
The variable GSI in the sorghum model did not present
significant difference between the treatments, including C+
(Figure 1d). However, the lettuce model displayed several
significant alterations (inductions and inhibitions), also in
Table 1. Classes of secondary metabolites found in the aqueous
and ethanolic extracts of Psidium cattleyanum. The signals (+) and
(-) respectively indicate the presence or absence of the chemical
classes in the analyzed plant material.
Chemical class Aqueous
extract
Ethanolic
extract
Strong fixed acids + +
Alkaloids - +
Catechins - -
Steroids + +
Simple phenols + +
Flavonoids - +
Anthocyanins - -
Anthocyanidins - -
Aurones - -
Chalcones - -
Flavones - +
Flavanones +
Flavonols - +
Flavononols - +
Leucoanthocyanidins - -
Resins - +
Saponins + +
Condensed tannins - -
Hydrolyzable tannins + +
Xanthones - +
Anthraquinone glycosides - +
Cardiac glycosides + +
Triterpenoids - -
Terpenoids + +

Brazilian Journal of Biology, 2024, vol. 84, e260985 5/10
Toxicity of extracts of Psidium cattleyanum
relation to C+ (Figure 1c), demonstrating that the extracts
are more effective in lettuce than in sorghum. Alterations in
the GSI evince the occurrence of changes in the metabolic
processes related to germination (Maraschin-Silva and
Aquila, 2006). In addition, highlighted that allelochemicals
selectively inhibit and alter the growth or development
patterns of the plants (Merino et al., 2018). Thus, such effect
can be related to (1) the different responses of the seeds in
the germination process of monocots and eudicots, and (2)
the greater sensitivity of lettuce to the metabolites produced
by Psidium. Studying the phytotoxicity of essential oils from
different species of this genus, also reported greater sensitivity
of lettuce in comparison to sorghum (Vasconcelos et al., 2019).
For the variable RG, significant inhibition was observed
in both plant models (Figure 2). Comparing both test
agents, the ethanolic extract was more efficient in lettuce
(comparing the highest concentration), whereas the
aqueous extract was more efficient in sorghum (Figure 2).
Some authors have already reported differential response
for monocots and eudicots, relating similar results with the
physiology of the plants (Alves et al., 2018; Pinheiro et al.,
2015; Vasconcelos et al., 2019).
For the lettuce model, inhibition of AG was observed
in the treatment with ethanolic extract only at the
concentration of 12.5 mg mL-1, in 35.4% of the plantlets,
compared to C- (Figure 2c). The concentrations of aqueous
Figure 1. Effect of the aqueous and ethanolic extracts of Psidium cattleyanum on the germination percentage of (a) Lactuca sativa and
(b) Sorghum bicolor and germination speed index (GSI) of (c) L. sativa and (d) S. bicolor. The small letters above the boxplots indicate
significant difference between the treatments by Tukey’s test (p < 0.05).
Figure 2. Effect of the aqueous and ethanolic extracts of Psidium cattleyanum on the root growth of (a) Lactuca sativa and (b) Sorghum bicolor
and aerial growth of (c) L. sativa and (d) S. bicolor. The small letters above the boxplots indicate significant difference between the
treatments by Tukey’s test (p < 0.05).

Brazilian Journal of Biology, 2024, vol. 84, e2609856/10
Alves, T. A. et al.
extract that presented inhibitory effect were 25, 50 and
100 mg mL-1, reaching 31.6%, 43.4% and 63.4% of the
plantlets, respectively, in comparison to C- (Figure 2c).
Several factors are determining for RG and AG, including
nutritional and cellular conditions. In order for the plantlets
to grow, it is necessary that cell multiplication and/or
elongation occur. Moreover, the initial development is
dependent on the formation of the cambium and xylem,
which occurs according to the availability and distribution
of nutrients in the plantlets (Merino et al., 2018).
The AG of sorghum was inhibited by the aqueous
extract at the concentration of 100 mg mL-1 in 54.8% of
the plantlets, compared to C- (Figure 2d). The evaluation
of germination parameters and of the initial growth of the
plantlets is considerably elucidative regarding the toxicity
of compounds, since these are critical stages of the plant
development and subject to high error rates, owing to the
seeds presenting lower tolerance to different conditions
imposed by the environment (Merino et al., 2018).
Overall, higher toxicity of the evaluated extracts
was observed for the variable RG than for AG (Figure 2).
The greater sensitivity of RG was also described by
Cândido et al. (2010). These authors highlighted that, when
comparing the action of phytotoxic agents in roots and
aerial parts of plantlets, the effects are more prominent
in the roots, as they remain in direct contact with the
allelochemical, which increases the possibility of toxic
agents influencing the development of this region.
Considering all variables of the assay, it was
demonstrated that the ethanolic extract was the most
toxic agent for lettuce, whereas the aqueous extract was
the most efficient in the sorghum model.
3.3. Cyto-genotoxicity assay
The three largest concentrations (100, 50 and 25 mg
mL
-1
) of ethanolic extract completely inhibited the emission
of roots in the model plant (lettuce). Thus, they could not
be evaluated for cytotoxic parameters.
The mitotic index (MI) of all evaluated treatments,
suffered a significant reduction, less than 50%, when
compared to the C- (Figure 3). According Fiskesjö (1985),
an effectively cytotoxic agent has MI inhibition greater than
50%, as is the case with the C+ used in the study, which
showed a 73.7% reduction when compared to C- (Figure 3).
The most inducing treatment for chromosomal
alterations (CA) was C+, although all other treatments
promoted more CA than C- (Figure 3), showing that the
treatments are less toxic to chromosomes/DNA than
glyphosate. This result is important, since, it is sought,
compounds with high biological activity and that promote
lesser impacts to the environment (Pinheiro et al., 2015;
Aragão et al., 2015; Alves et al., 2018; Aragão et al., 2017).
The treatments with aqueous extract did not promote
an increase in nuclear alterations (NA), with the opposite
being observed with the ethanolic extract (Figure 3). NA are
those that alter the cell nucleus of the cell metabolically
or morphologically or are also related to the appearance
of DNA in a compartmentalized way, as if new nuclei were
being formed, as is the case of micronuclei (Alves et al.,
2018; Aragão et al., 2015; Santos et al., 2019; Andrade-
Vieira et al., 2011). These changes reflect “mistakes” that
are occurring during the split. In some cases, such as
micronuclei (MNC), the goal is to reestablish the DNA
content inside the nucleus; in others, as in the formation
of condensed nuclei (Figure 4b), the damage is large
enough to activate the cell death process, the latter being
considered cytological evidence of the occurrence of cell
death (Andrade-Vieira et al., 2011; Costa et al., 2017).
The types of chromosomal alterations observed in plant
bioassays allow classify the cellular action mechanism of
the evaluated test agent. Thus, test agents that promote
chromosomal changes resulting from changes in the
formation of the mitotic spindle are considered aneugenic
(Fernandes et al., 2009; Pinheiro et al., 2015; Alves et al.,
2018), since they will change the chromosomal number
of the daughter cells, but they will not affect their
DNA sequences. Whereas, test agents that determine
chromosomal changes that alter the DNA sequence
of daughter cells, such as bridges and chromosomal
fragments, are considered clastogenic (Bernardes et al.,
2015; Vasconcelos et al., 2019). Test agents that cause
chromosomal alterations that modify chromosomal
signaling, such as adherence, are considered as epigenetic
action mechanisms because it is triggered by changes
in the phosphorylation pattern of serine 10 in histone
3 (Freitas et al., 2016; Alves et al., 2018). It is important
to note that test agents can have more than one action
mechanism simultaneously.
Both NA MNC (Figure 4a) and CA bridge (Figure 4e)
showed an increase in treatments: ethanolic extract
and aqueous extract (100 mg mL-1) (Figures 3 and 5).
Figure 3. Percentage of observed alterations in the cell cycle
of meristematic cells from the tip of the Lactuca sativa root
exposed to (a) ethanolic extract and (b) aqueous extract from
Psidium cattleyanum leaves. The small letters above the bars indicate
significant differences between the treatments by the Tukey test
(p<0.05). MI = mitotic index; CA = chromosomal alterations; NA
= nuclear alterations, MNC = micronuclei.

Brazilian Journal of Biology, 2024, vol. 84, e260985 7/10
Toxicity of extracts of Psidium cattleyanum
The increase in these alterations are related, since
the chromosome bridge is associated with the break-
fusion-break cycle. In this case, telomeres are lost by
fragmentation leaving cohesive ends of the chromosomes
exposed. Thus, ends of different chromosomes connect,
and at the time of chromosomal segregation, the
formation / visualization of chromosomal bridges occurs.
These linked chromosomes, which are being pulled to
opposite poles, undergo “traction” by depolymerizing the
microtubules, so that a new break occurs giving continuity
to the break-fusion-break cycle (Silveira et al., 2017;
Santos et al., 2019; Costa et al., 2020). These fragments
formed in the cycle are organized in MNC, after the end
of the division, to be exported from inside the cells (Leme
and Marin-Morales, 2009; Andrade-Vieira et al., 2011;
Silveira et al., 2017), resulting in the formation of MNC.
The frequency of c-metaphases (Figure 4c) increased in
all treatments evaluated when compared to C- (Figure 5).
This alteration derives from the total dysfunction of the
mitotic spindle, showing that microtubule polymerization
is not occurring, consequently, the chromosomes are not
organized in the cell’s equatorial plane (Silveira et al.,
2017; Santos et al., 2019; Costa et al., 2020).
The lost chromosomes (Figure 4f) were observed,
significantly, in the cells treated with the evaluated
concentration of the ethanolic extract and in the
concentrations of 25 and 100 mg mL
-1
of the aqueous
extract (Figure 5). The lost chromosomes, as well as the
c-metaphases, refer to the bad organization of the spindle.
However, the lost chromosomes are due to a partial change,
while the c-metaphases of a total change in the formation
of spindle fibers (Fernandes et al., 2009; Santos et al., 2019).
Chromosomal adherence (Figure 4d) was observed,
significantly, in the cells treated with all evaluated extracts
(Figure 5). This alteration refers to a series of changes that
occur. This alteration indicates changes in the functioning
of the mitotic machinery, alteration in the chromosomal
constitution, as well as alteration in the phosphorylation
of amino acids that constitute the chromosomes, thus
modifying its signaling (Freitas et al., 2016; Silveira et al.,
2017; Alves et al., 2018; Santos et al., 2019).
Figure 4. Alterations observed in meristematic cells of Lactuca sativa treated with aqueous and ethanolic extracts of Psidium cattleyanum.
Where it is illustrated: (a) micronucleus; (b) condensed nucleus; (c) c-metaphase; (d) adherent; (e) bridge in telophase; (f) anaphase
bridge with lost chromosome. Bar = 10µm.
Figure 5. Distribution of observed chromosomal alterations in the
cell cycle of meristematic cells from the tip of the Lactuca sativa
root exposed to (a) ethanolic extract and (b) aqueous extract of
Psidium cattleyanum.

Brazilian Journal of Biology, 2024, vol. 84, e2609858/10
Alves, T. A. et al.
The observed CAs elucidated the action mechanism of
the treatments. Thus, all treatments were aneugenic, as well
as having an epigenetic effect, acting on mitotic machinery
and chromosomal signaling. In addition, ethanolic extract
in the investigated concentration and aqueous extract in
the concentration of 100 mg mL
-1
were clastogenic, altering
the DNA sequence of the cells.
The knowledge of biological activity in the cell cycle,
as well as the cellular action mechanism of plant extracts
is important for directing studies and understanding
relational dynamics between organisms. In addition,
it helps to elucidate possible adverse health effects,
assisting in the indication and restriction of the daily use
of plants, whether for nutrition, body care and / or as a
herbal medicine.
4. Conclusion
Elucidating the differential effects of extracts according
to the applied extraction method and the target organism
is important for the varied applications of natural products.
This work contributed in the context of showing that the
choice of extraction method should be in accordance with
the aimed purpose; accordingly, the ethanolic extract
favors the extraction of flavones, flavonones, flavonols,
flavonnnols and flavonoids, alkaloids, resins, xanthones
and anthraquinone glycosides.
With regard to phytotoxic potential, the ethanolic
extract was more effective in lettuce, whereas the aqueous
extract was more effective in inhibiting the sorghum model.
Thus, differential toxicity was observed between the two
used extraction methods and for the both model species.
The evaluated extracts were shown to be cytotoxic,
with mechanisms of aneugenic action and promoters
of epigenetic alterations, against the lettuce cell cycle.
The ethanolic extract and the higher concentration of
the aqueous extract promoted a significant increase in
nuclear alterations, when compared to water. In addition,
these treatments proved to be clastogenic. These results
demonstrate the bioactivity of these extracts, as well as
their toxic potential for biological applications.
Acknowledgements
We thank the National Council for Scientific and
Technological Development (CNPq, Brasília — DF, Brazil),
and the Research and Innovation Support Foundation
of Espírito Santo (FAPES / VALE, Vitória — ES, Brazil;
Scholarship 75516586/16; FAPES / UNIVERSAL, Vitória -
ES, Brazil; grant 484/2021 and 2021-ZK8PJ; Cooperação
CAPES/FAPES – PDPG). This study was partially funded by
the Coordination for the Improvement of Higher Education
Personnel - Brazil (CAPES) - Financial Code 001.
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