Cytogenotoxicity evaluation of Carica papaya Linn.

using Allium
cepa assay

Meca Angela Baliva, Joan M. Pangyan, Charlene Tampos

Master of Arts in Education
Institute of Graduate Studies
Southern Philippines Agri-Business and Marine and Aquatic School of
Technology

INTRODUCTION

Natural products have long been recognized since ancient times as

potential sources of human drugs. Recently, phytotherapy, or the use of
medicinal plants in healing ailments, has received increased attention
since it is effective and safe (Responte et al., 2015). Natural plant extracts
are widely used in traditional medicine and modern pharmacology due to
their therapeutic properties. However, their safety profiles must be
thoroughly evaluated to ensure they do not pose cytogenotoxic risks
(WHO, 2015).
Carica papaya Linn., commonly known as papaya, is a tropical fruit
renowned for its nutritional and medicinal benefits, including anti-
inflammatory, antioxidant, and anticancer properties. Different parts of
papaya plant viz. fruit, bark, roots, seeds, peel, pulp, and leaf have many
known therapeutic uses around the world (Prashar, 2015). However,
leaves have been emerged as one of the most useful parts with plethora
of health-promoting compounds and activities. Despite these benefits, it is
essential to assess whether papaya extracts can cause cytogenotoxic
effects (Eur and Orafa, 2019).
The research aims to solve this gap by investigating the
cytogenotoxicity of Carica papaya using the Allium cepa assay.
This assay is a valuable tool for assessing the mitotic, cytotoxic,
and genotoxic effects of various medicinal plants (Bailar & Gornick, 1997).
Understanding the cytogenotoxicity of Carica papaya is crucial in
evaluating its safety and potential impact on genetic material.
 This study highlights Carica papaya's cytogenetic performance,
usage, advantages, and chromosomal aberrations. The researchers will
assess the cell cycle phases of the Allium cepa plant as a model assay for
the Carica papaya, providing valuable insights into its impact on genetic
material. Understanding the cytological effects of the leaves of Carica
papaya is essential for ensuring its safe and effective use in medicinal
and traditional settings.

Objectives of the Study

This study assessed the cytogenotoxicity of Carica papaya using the

Allium cepa assay. Specifically, this study was to determine the following:
1. Determine the diameter of root growth of the Allium cepa tip as
exposed to the following concentration of Carica papaya based on
the following:
a. Negative control (water)
b. 50 ppm
c. 125 ppm
d. 250 ppm
e. 500 ppm
2. Determine the mitotic index of Allium cepa based on the following
concentration of the Carica papaya:
a. Negative control (water)
b. 50 ppm
c. 125 ppm
d. 250 ppm
e. 500 ppm
3. Determine the % aberrant cells of Allium cepa exposed to the
following concentrations:
a. Negative control (water)
b. 50 ppm
c. 125 ppm
d. 250 ppm
e. 500 ppm
4. Determine the significant difference of root growth diameter of
Allium cepa tip after exposure to the different treatments of Carica
papaya.
 5. Determine the significant difference of % mitotic index of Allium
cepa cells after exposure to the different treatments of Carica
papaya.
6. Determine the significant difference of % aberrant cells of Allium
cepa after exposure to the different treatments of Carica papaya.

MATERIALS AND METHODS

Research Design
The study employs an experimental research design whereby the
different treatments of Carica papaya leaves were tested for
cytogenotoxicity using the allium cepa assay. To assess the
cytogenotoxicity, root growth, mitotic index, and aberrant cells were used
as an indicator.

Research Locale

The study was conducted at Malita, Davao Occidental, particularly

in barangay Poblacion. This study was conducted at the Research and
Laboratory Services Center in SPAMAST Malita, particularly focused on the
evaluation of cytogenotoxicity specifically to the medicinal plant Carica
papaya using the model test of the Allium cepa assay. Research and
Laboratory Services Center is situated at intersection roads of Ipil Street
and Relocation Street. The institution approximately lies between 6.41803
north longitude, 125.608348 east latitude.

Data Gathering Procedure

Preparation of Treatments
In this study, three distinct groups were employed to ensure a
comprehensive evaluation of the cytogenotoxicity effects of Carica papaya
leaves. The control group consisted of onion bulb exposed exclusively to
distilled water, serving as the negative control. This group is essential for
establishing a baseline mitotic index and assessing chromosomal stability.
The Carica papaya treatment group was the focal point of the study,
subjecting onion root tips to varying concentrations of the Carica papaya
leaf extract, precisely prescribed at 50 ppm (7 g), 125 ppm (12.5 g), 250
ppm (25 g) , and 500 ppm (50 g).
Test Organism
Healthy and equal-sized bulbs were chosen, and the series of
onions was grown in each test chemical. Onions showing green leaves and
mold-attacked bulbs were discarded. To start, the dried scales of onion
and old roots were removed. Thus, 15 onions were included in the series,
and the best onion roots grown were selected and treated.

Application of Treatment
Day 0 (24hrs), onion (Allium cepa) was submerged in distilled
water at room temperature 25ºC with no exposure media, the experiment
was performed at a relatively constant temperature and avoided direct
sunlight. When the onion bulb root tips were grown about the length of
0.3 – 3 mm, the root tips were then immersed in the exposure media,
with various concentrations. Root tips will be put on test per the
concentrations (50 ppm, 125 ppm, 250 ppm, 500 ppm), different for
onion bulbs with distilled water only (control). Root growth was monitored
daily, and measurements were taken at the end of each day (24-hour
intervals) for macroscopic evaluation. Additionally, root tips were collected
at 24, 48, and 72 hours from all treatments and placed in fixatives for
further processing.
In the processing for the evaluation of the Carica papaya leaves for
finer quality, approximately 3 mm of root tips were removed per
treatment group. These root tips were transferred to the aceto-alcohol
fixative (1:3 ratio), which had just been prepared, and stored for 24
hours. Afterward, undergoes hydrolyzation with 1 Normality of HCl at
60ºC for 1 minute. Subsequently, the root tips were washed with water
for 1 and a half minutes, equivalent to about three rinses. Petri dishes and
watch glasses was utilized for placing the root tips both before and after
fixation, followed by a heating process lasting 10-20 seconds and a
subsequent resting period of approximately 5-12 minutes.
Next, the root tips undergo staining using a safranin solution,
allowing it to penetrate for 10 minutes. Placed on glass slides, a sharp
blade or scalpel was used to remove about a millimeter of the root tip,
discarding the rest. With three replicates per concentration (one root tip
on each slide), the slides were covered by slips to avoid air bubbles.
Gently squashing and applying slight pressure with a rod will follow,
removing excess stain before sealing. The prepared slides were ready for
observation under a light microscope. The effects of the Carica papaya
leaf extract on A. cepa root tip cells were determined, and the results or
findings were subsequently analyzed.

Observation
To assess the cytogenotoxicity of the medicinal plant Carica
papaya, the slides were viewed at 400x magnification. The potency of the
medicinal plant Carica papaya and its impact on the tips of onion roots
were only determined after several hours of observation for each
treatment. A light microscope fitted with a digital camera was used to
record chromosomal abnormalities and mitotic activity to get better-
quality photos. Photomicrographs were useful in determining A's
chromosomal abnormalities and mitotic activity. root tip cells of A. cepa.
For each Carica papaya concentration, a minimum of 500 cells per slide
was observed for analysis.

For Microscopic Evaluation

A total of 5 root tips were collected for each treatment to assess
the mitotic index (MI). The MI represents the ratio of the total number of
dividing cells in the cell cycle (prophase, anaphase, metaphase, telophase)
to the total number of cells. To evaluate the rate of cellular division based
on microscopic criteria, the mitotic index was calculated. Different
concentrations were examined by scrutinizing categories through the
computation of cells exposed to various concentrations.

For Aberrant Cell Evaluation

For chromosomal aberrations (CA), are monitored under various
conditions of cell division (prophase, metaphase, anaphase, and
telophase). CA perhaps distinguishes the chromosome breaks, laggards,
vagrants, stickiness, bridges, and polar deviation. The MI and CA were
computed based on the number of aberrant cells per scored total cells at
various concentrations exposed in each sample.

Equation 1:
%Root growth of Treatment =
OVERALL MEAN ROOT LENGTH OF SOLUTION
× 100
OVERALL MEAN ROOT LENGTH OF CONTROL
Equation 2:
NUMBER OF DIVIDING CELLS
%Mitotic index = × 100
TOTAL NUMBER OF CELLS
Equation 3:
NUMBER OF ABERRANT CELLS
% Aberrant cells = ×100
TOTAL NUMBER OF CELLS

Statistical Tools
To gather data, the studies were computed, categorized, and
examined data statistically. This process facilitated a clear discussion and
presentation of the study results, utilizing the following statistical tools:
Percentage
Percentage was employed to determine the fraction of responses
from the sample. Percentage was used in the study to determine whether
the root tip cells in the treatment would have a high percentage in terms
of the various concentrations exposed.
Mean
The means were used to calculate the root length of Allium cepa
exposed from different concentrations. The method was used to
determine the degree of dispersion of meristem cells for cytogenetic
analysis of the leaves of Carica papaya.
ANOVA
One-way ANOVA was used to test the difference in the
concentrations significantly. To show the cytotoxic effects in accordance
with statistically dwindled root growth and mitotic index, furthermore, the
genotoxic effects on statistically piled up the chromosomal aberrations
and/or DNA damage.
Tukey’s test
This statistical method is instrumental in identifying significant
differences among treatment groups, with this it helps discern the specific
impact of varying concentrations and exposure durations of the medicinal
plant Carica papaya leaf extract on chromosomal integrity and mitotic
activity.
 Results and Discussion

Root Growth Analysis

The effects of different concentrations of Carica papaya leaf

extracts on the root growth of Allium cepa were observed and measured
over 72 hours. The concentrations used were 50 ppm, 125 ppm, 250 ppm,
and 500 ppm, along with a negative control (distilled water). The data
collected showed a clear trend in root growth inhibition correlated with
increasing concentrations of the extract.
Table 1. Root length diameter (cm) of the Allium cepa tip after 3 days
exposure to Carica papaya leaf extract.

TREATMENT (ppm) Root Length (cm)

a) Control 1.38
b) 50 0.52
c) 125 0.26
d) 250 0.19
e) 500 0.12

The control setup, devoid of Carica papaya leaf extract, exhibited

the highest root growth, with a length of 1.38 cm. Following this, the root
lengths for the 50, 125, 250, and 500 ppm concentrations were 0.52 cm,
0.26 cm, 0.19 cm, and 0.12 cm, respectively. These results indicate
various degrees of suppression in root growth as the concentration of
Carica papaya leaf extract increased. In contrast to the control group,
which experienced normal root growth due to exposure to distilled water
alone, the treated groups exhibited varying levels of root growth
suppression. This suppression intensified with higher concentrations of
Carica papaya leaf extract.

The root growth inhibition was more pronounced at higher

concentrations, indicating a dose-dependent cytotoxic effect of Carica
papaya leaf extracts on Allium cepa. The statistical analysis using one-way
ANOVA revealed a significant difference (p < 0.05) between the control
and the treatment groups, confirming that the extract inhibited root
growth significantly compared to the control.

Mitotic index of the Allium cepa tip as exposed to different

concentrations of Carica papaya leaf extract.

The data gathered from the mitotic index of Allium cepa tip as
exposed to different concentrations of Carica papaya leaf extract (50 ppm,
125 ppm, 250 ppm, 500 ppm). The mitotic index indicates the proportion
of actively dividing cells in the root tip of Allium cepa. Higher mitotic
indices suggest greater cellular proliferation, while lower indices may
indicate inhibition of cell division or cytotoxic effects.

Table 2. Mitotic index of root tip cells of Allium cepa.

TREATMENT Total cell (500 cells) Total Mitotic
(ppm) Dividing index
cells (%)
Prophase Metaphase Anaphase Telophase

a) Control 88 52 26 26 192 38.4%

b) 50 72 45 22 18 157 31.4%
c) 125 54 34 14 12 114 22.8%
d) 250 38 24 10 6 78 15.6%
e) 500 22 18 10 6 56 11.2%

Table 2 presents the mitotic index for each treatment, with

Treatment A (50 ppm), Treatment B (125 ppm), Treatment C (250 ppm),
Treatment D (500 ppm), and Treatment E (Control) showing mitotic
indices of 38.4%, 31.4%, 22.8%, 15.6%, and 11.2%, respectively. The
mitotic index represents the proportion of actively dividing cells in the root
tip of Allium cepa.

In contrast to the expectation, the control treatment (Treatment A)

actually exhibits the highest mitotic index compared to the other
treatments. This indicates a relatively high proportion of actively dividing
cells in the control group. However, it's notable that the mitotic index
decreases as the concentration of Carica papaya leaf extract increases,
suggesting a potential inhibitory effect on cell division with higher
concentrations. Treatments A and B, with lower concentrations of the
extract, demonstrate higher mitotic indices compared to the control,
indicating a stimulatory effect on cell division at these concentrations.
Conversely, treatments C and D, with higher concentrations of the extract,
show lower mitotic indices than the control, suggesting potential cytotoxic
effects or inhibition of cell division. This observation aligns with previous
findings (Jühlen et al., 2018).

The highest mitotic index values were obtained from Treatment A (50
ppm) after 72 hours of application, with a mean of 38.4%. Conversely,
the mitotic index was notably low at Treatment D (500 ppm) after 72
hours, with a mean of 15.6% compared to other concentrations.
Additionally, all mitotic index results were found to be statistically
significant for all concentrations of Carica papaya leaf extract and at each
exposure time. Thus, while the control treatment unexpectedly exhibits
the highest mitotic index, the overall trend suggests a dose-dependent
effect of Carica papaya leaf extract on the mitotic index of Allium cepa
root tip cells.

The decrease in the mitotic index with increasing concentrations of Carica

papaya leaf extracts indicates a cytotoxic effect, reducing the number of
actively dividing cells. The ANOVA test showed a significant difference (p
< 0.05) in the mitotic index between the control and the treatment
groups, demonstrating the inhibitory effect of the extract on cell division.

Table 3. The percentage occurrence of nuclear abnormalities observed in

the dividing cells of Allium cepa root tips exposed to
Carica papaya leaf extract capsule in different treatments.
TREATMENT Chromosomal aberrations of Total CA / AC
 (ppm) Allium cepa root tip cells AC rate (%)
L&B S PD V CB b-NL
a) Control 2 5 4 0 2 0 13 2.6%
a) 50 5 7 8 3 5 3 31 6.2%
b) 125 7 13 11 5 9 5 50 10%
c) 250 13 24 13 10 15 8 83 16.6%
d) 500 17 32 20 15 19 9 112 22.4%
*L & B- laggards and bridges; S- sticky; PD- polar deviation; V- vagrants;
CB- chromosome breaks; b-NL- bi-nuclei lesions. CA-chromosomal
aberrations/ AC-Aberrant cells.

The Allium cepa chromosome aberration test results, as depicted in

Table 3, provide insights into the effects of different concentrations of
Carica papaya leaf extract on chromosomal abnormalities in root tip cells.
The total mean percentages of various chromosomal aberrations,
including laggards, stickiness, polar deviation, vagrants, breaks, and
nuclear lesions, were calculated for each treatment group. Contrary to the
hypothesis, the control group exhibited an average rate of 2.6%
compared to the 22.4% average rate observed in the 500 ppm treatment
group.

The data indicate a clear trend of increasing chromosomal

aberrations with higher concentrations of Carica papaya leaf extract,
suggesting a dose-dependent effect. Treatments C, D, and E showed
significantly higher frequencies of chromosomal abnormalities compared
to the control (Treatment A), indicating a potential genotoxic effect. This
is supported by the elevated occurrences of chromosome breaks,
laggards, vagrants, stickiness, bridges, and polar deviation in these
treatments. The findings align with previous research suggesting that
exposure to certain substances can lead to increased chromosomal
aberrations, potentially due to impaired DNA repair, direct DNA damage,
and the generation of reactive oxygen species. Prolonged exposure to
such substances can promote genetic instability and the onset of disease.
 The percentage of aberrant cells increased with higher
concentrations of Carica papaya extracts, indicating a dose-dependent
genotoxic effect. The statistical analysis showed a significant difference (p
< 0.05) between the control and all treatment groups, highlighting the
genotoxic potential of the extract.

Significant difference of root growth lengths of Allium cepa tip

after exposure to the different concentrations of Carica papaya
leaf extract

Table 4 shows the study that examines the root growth of Allium
cepa exposed to different concentrations of Carica papaya leaf extract.
The study revealed that there was a significant difference between the
root growth of Allium cepa applied concentrations of Carica papaya leaf
extract. It notes that the mean score of each treatment were 1.38, 0.52,
0.26, 0.19, and 0.12. It later shows that it has a p-value of 0.00
respectively which was lesser than 0.05 in the level of significance. This
implies that the hypothesis was rejected which denotes that there is a
significant difference in each concentration and control group. The result
of the analysis pointed out that it obtained a greater mean in the
concentration than the control group.
The statistics further demonstrate that, although being higher than
those of treatments A, B, and C, the means of treatments D and E are
comparable to one another. The null hypothesis is rejected because
Treatment A has a substantial impact on root growth length. With lower
values, treatments B, C, D, and E may not differ substantially within the
same group.
Additionally, Allium cepa root structure altered in response to
different Carica papaya leaf extract exposure levels. Treatments A and B,
have more roots than treatment C, D, and E, which has fewer roots and a
curled texture. This indicates that the leaf extract can affect the root
structure of Allium cepa, causing stunted or diminished root development.

Table 4. Significant Difference in the root lengths of Allium cepa applied

to each treatment of Carica papaya leaf extract

TREATMENT MEAN p-VAL DECISION

(ppm)
A) control 1.38 0.00 Reject Hypothesis
B) 50 0.52
C) 125 0.26
D) 250 0.19
E) 500 0.12

Significant difference of mitotic index of Allium cepa cells after

exposure to the different concentrations of Carica papaya leaf
extract
Table 5 presents an evaluation of the Allium cepa mitotic index in
response to varying Carica papaya leaf extract concentrations. The study
found that the concentrations of Carica papaya leaf extract applied had a
substantial impact on the root growth of Allium cepa. The average scores
for each treatment are listed as follows: 38.4%, 31.4%, 22.8%, 15.6%,
and 11.2%. Subsequent analysis reveals that the p-value is 0.006, which
is below the significance level of 0.05. Since the p-value was smaller than
the significance level of 0.05, the hypotheses was rejected, indicating that
there was a significant difference between the scores during the mitotic
index examination. The analysis conclusion indicated that the control
group had a higher mean percentage than the concentration means.
All the treatments’ p-values are 0.006, indicating a marginal level of
a statistical significance. After being exposed to various concentrations of
Carica papaya leaf extract, Allium cepa cells exhibit considerable variations
in their mitotic index, which suggests that the cytotoxic effects of the drug
can differ and showed a dose-dependent association, whereby greater
concentrations resulted in decreased mitotic indices, indicating a potential
suppressive effect on cell division because of chromosome segregation
and DNA replication problems. Furthermore, Onwuamah et al. (2014)
supported these findings by showing that the mitotic index was lowered in
Allium cepa roots with increasing Carica papaya leaf extract
concentrations. The aforementioned studies highlight the sensitivity of the
Allium cepa assay in identifying the cytotoxic effects of Carica papaya leaf
extract on cell division. This emphasizes the significance of
comprehending the dose-response relationship when evaluating the
genotoxic potential of the Carica papaya leaf extract in medical and
environmental contexts.

Table 5. Significant Difference of Allium cepa Mitotic indexes exposed

from different concentrations of Carica papaya leaf extract

TREATMENT MEAN p-VAL DECISION

A 38.4% 0.0063 Reject Hypothesis

B 31.4%
C 22.8%
D 15.6%
E 11.2%

Significant difference in chromosomal aberration of Allium

cepa after exposure to the different treatments of Carica papaya
leaf extract

The study examining the chromosomal aberration of Allium cepa

subjected to varying Carica papaya leaf extract concentrations is displayed
in Table 6. The investigation showed that the aberrant cells of Allium cepa
varied significantly depending on the Carica papaya leaf extract
concentration that was applied. It shows that each treatment's mean
score is 2.6%, 6.2%, 10%, 16.6, and 22.4%. Later analysis reveals that
the p-value was 0.02, which is less than 0.05 at the significance level. This
suggests that the hypotheses were not accepted, indicating a significant
difference between each concentration group and the control group. Its
mean concentration was higher than that of the control group, according
to the analysis's findings.
According to the available information, the treatment designated as
A produced a mean of 2.6%. This indicates that, in comparison to the
control or other treatments, exposure to Carica papaya leaf extract at the
concentration corresponding to treatment A caused a considerable
increase in chromosome aberrations in Allium cepa. The overall result
score for the other treatments, designated B, C, D, and E, is 0.02,
suggesting a statistically significant outcome. Their disparate means
suggest that the impact of Carica papaya leaf extract at various
concentrations on Allium cepa chromosomal abnormalities may vary.

TREATMENT MEAN p-VAL DECISION

A 2.6% 0.02 Reject Hypothesis

B 6.2%
C 10%
D 16.6%
E 22.4%

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