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Volume 9 | Issue 5 | Print ISSN: 2395-6011 | Online ISSN: 2395-602X (www.ijsrst.com)

Cytotoxicity Assay of Azadirachta indica and Carica papaya Leaf Extracts by

Allium Test System
U. A. More1, A. S. Deshpande2, K. D. Aswar2, S. N. Malode2*
1Department of Botany, Late Rajkamlji Bharti Arts, Commerce and Smt. Sushilabai R. Bharti Science College,
Arni - 445103, Maharashtra, India
2 Department of Botany, Govt. Vidarbha Institute of Science and Humanities, Amravati, Maharashtra, India

ABSTRACT

Azadirachta indica and Carica papaya are two well-known medicinal plants used for their antibacterial,
antiviral and nutritive properties. Most often they are consumed raw for their different health benefits by the
people worldwide. Even though they are effective against various diseases, certain side effects occur due to
their consumption impeding their popularity as traditional medicines. There must be evaluation of adequate
dose of these medicinal plants to prevent their negative effect on life systems. In present study, the Allium
test has been used to evaluate the genotoxic, cytotoxic potential of these plants. Effects of three different
concentrations of these plant extracts on mitotic chromosomes have been considered as reliable major for
cytotoxicity assay. Mitotic abnormalities (MA%) observed were chromosomal bridges, clumped metaphases
and ghost cells. Although mitotic abnormalities were noted in all concentrations but percentage of
abnormalities are very low. Highest percentage of abnormalities for Azadirachta indica (1.58%) and Carica
papaya (1.97%) noted in 1.5% concentration. Present study showed that extracts of Azadirachta indica and
Carica papaya are safer to use in low concentrations.

Keywords: Azadirachta indica, Carica papaya, Allium test, cytotoxic effect, MA% etc.

I. INTRODUCTION information available (Zink and Chaffin, 1998,

Since the millennia, plants having medicinal and
health promoting properties have been used in
many folk medicines and herbal preparations. In
recent times, purely scientific study on the effect of
such herbal preparations on different health
parameters has been flourished worldwide. But
some of these herbal preparations found to have
side effects. Moreover, there is little or no
scientific
Basaran et al., 1996).
Azadirachta indica and Carica papaya are the most
commonly used and well-known medicinal plant in
traditional medicine. Both of these plants possess
wide array of functions in large number of medicinal,
cosmetics, toiletries. Neem leaves is used to treat
certain diseases like - leprosy, eye disorders, bloody
nose, intestinal worms, stomach upset, loss of appetite
(Vanna, 1976; Schmutterer, 1995); while papaya leaf
extract used to treat dengue fever and found to have

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 Volume 9 - Issue 5 - Published :
strong medicinal properties such as antibacterial,
antiviral, antitumor, hypoglycemic and anti-
inflammatory activity (Singh et al., 2020). Although
these plants possess medicinal application, they also
found to exert negative impact on human health.
Neem leaves found to cause mortality in pregnant
woman and renal failures while papaya leaf extracts
have certain side effects like stomach trouble,
purgative effects and abortion (Raj, 2014; Patil et al.,
2014).
To overcome these limitations, there is an urgent
need to differentiate between therapeutic and toxic
doses of these plant extracts. Allium test serves as an
effective tool for evaluation of toxic effects of such
herbal and chemical extracts. This test based on the
toxic effects of such extracts on somatic chromosomes
of Allium cepa. It is considered that any toxic effects
occur to somatic chromosomes in Allium cepa maybe
induced in human cell on consumption (Fiskesjo,
1985).
II. MATERIALS AND METHODS
 Fresh leaves of Neem (Azadirachta indica) and
Papaya (Carica papaya) were collected from
campusof Govt. Vidarbha Institute of Science and
Humanities, Amravati (Plate I).
 Leaves of collected plants were washed with tap
water; air dried and cut down. Extracts were
prepared by crushing 5gm, 10gm and 15gm of
collected leaves in mortar and pestle and about 1
liter of total volume was made up with tap water.
By this extraction, the three different
concentrations of the extract obtained are 0.5%,
1.0% and 1.5 %.
 The loose outer scale of bulbs and old roots were
removed with the help of sharp and pointed
forceps or blade so as to expose the root
primordia. A series of bulbs were then placed on
coupling jars containing water at room
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September 13, 2021 Page No : 181-186
temperature for three days. After three days the
new root tips were immersed out.
 Onion with new root tips were placed on
coupling jars containing the extracts plant
material of three different concentrations such
that the growing root tips were immersed in the
extracts. The duration of the treatments for each
extract was three hours. The experimental set up
had three replicates. Three onion bulbs were also
treated with distilled water which served as
control (Plate I).
 The root tips were cut after the treatment and
fixed in Carnoy’s fixative (Glacial acetic acid:
Absolute alcohol in 1:3 proportion) for about
24Hrs. and then in 70% alcohol. For the
preparation of slides, the root tips were
hydrolyzed in 1N HCL for 10 minutes. The
squashing was done over 2% Aceto-orcein stain.
The slides were then studied under the high
magnification compound microscope.
 The numbers of dividing and non-dividing cells
were recorded. Incidence of chromosomal
abnormalities was calculated by expressing the
number of aberrant cells as a percentage of total
dividing cells for each treatment. Mitotic index
was calculated by following formula for each of
the treatments and the control.
Data on cytological parameters were taken at 40X
magnification of compound microscope from at least
20 slides for each treatment and control. The cells
were scored for the different cell division stage, total
dividing cells, non-dividing cells and number of cells
showing chromosomal aberrations (Plate I). The per
cent chromosomal aberration (Mitotic Abnormality %)
was calculated for each treatment and the control
using the following formula
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III. RESULTS AND DISCUSSIONS Table 1. Mitotic activities and chromosomal

abnormalities observed in root cells of A. cepa treated
This study determine the cytotoxic effects of aqueous with different concentrations of Azadirachta indica
extracts of Azadirachta indica and Carica papaya leaf leaf extract.
extracts on mitotic index and mitotic abnormalities in
growing root tips of A. cepa. Data on number of Conc.of A.
MA
dividing cells, mitotic index and mitotic abnormality indica leaf MA%
TDC TCO MI%
extract (%) CB CM
percent is shown in Table 1 and Table 2 (Figure 1 and
Figure 2). Control 527 1957 26.92% 0 0 00
Table 1 show that mitotic index (MI) decreases in
linear manner with increase in concentration of A. 0.5% 511 2217 23.04% 2 0 0.39
indica leaf extracts in Allium cepa root tip cells.
Highest value of mitotic index (26.92%) was observed 1.0% 464 2866 16.18% 3 1 0.86%
in control while lowest value (14.00%) was observed
in 1.5% concentration. Similar trend of decrease in 1.5% 316 2265 14.00% 2 3 1.58%
mitotic index over the control was followed in Allium
cepa root tip cells treated with C. papaya leaf extracts.
All the concentrations show decreased value of
mitotic index over the control with least value Table 2. Mitotic activities and chromosomal
(12.77%) in 1.5% concentration. (Table 2). abnormalities observed in root cells of A. cepa treated
with different concentrations of Carica papaya leaf
extract.

Conc.of C.
MA
papaya leaf MA%
TDC
extract (%) TCO MI%
CB CM GC

Control 294 1922 15.29% 0 0 0 00%

0.5% 276 1956 14.08% 2 0 0 0.72%

1.0% 260 1952 13.31% 2 1 0 1.15%

1.5% 253 1980 12.77% 3 1 1 1.97%

TDC – Total number of dividing cells; TCO – Total number of cells

observed; MI% - Mitotic Index percent; MA – Mitotic Abnormalities;
CB – Chromosomal bridges; CM – Clumped metaphases;
GC – Ghost cells; MA% - Mitotic abnormality percent.

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 Volume 9 - Issue 5 - Published : September 13, 2021 Page No : 181-186

Control shows only the preparatory phase (interphase)

and normal phases of mitotic division (viz. prophase,
metaphase, anaphase and telophase). While all the
concentrations shows development of different
chromosomal anomalies along with normal mitotic
phases. These mitotic abnormalities include –
chromosomal bridges, clumped metaphases and ghost
cells. Both the extract shows that there is a dose
dependent increase in abnormality percent i.e. highest
abnormality percent was observed in highest
concentration (1.5%) of extract. Mitotic abnormalities might resulted due to
Lowering of mitotic index with higher concentration mutagenic effect of phytochemical constituents like
of extract implies that phytochemicals in the extracts phenols, flavonoids, alkaloids in plant extract which
affect normal rate of cell division adversely. Mitotic interferes with process of normal mitotic division.
index found to be a reliable index to evaluate These extract might have mito-depressive effects and
comparative toxicity of different concentrations of might interfere with spindle formation, attachment of
plant extracts by many workers. Yekeen et al. (2011) chromosome to spindle causing disorientation,
found that extracts of Solanum aethiopicum, Lactuca chromosomal bridges, stickiness and lagging
taraxacifolia and Talinum triangulare lowers the movements. As most of the herbal preparations are
mitotic index. Similar results were obtained by not processed, cells of consumer might be exposed to
Rajkumar et al. (2009) and Chukwujekwu et al. (2014) such mutagenic activities after consumption of such
when Allium root tip cells treated with extracts of extracts.
Acorus calamus and Distephanus angulifolius Rathnasamy et al. (2013) observed cytotoxic effects of
respectively. The level of cytotoxicity of any extract aqueous and methanolic extracts of three plants -
can be relate to both increase or decrease in mitotic Clinacanthus nutans, Adhatoda vasica and Carica
index value. Comparatively, extract which shows papaya on Allium cepa. Adegbite and Sanyaolu (2009)
lower MI value than control might result due to effect tested aqueous extract of Vernonia amygdalina
of test extract on growth and development of test for cytotoxicity on Allium cepa root tip cells. They
organism while extract showing increased MI value observed chromosomal aberrations such as
can be a potent carcinogen which encourages rapid endopolyploidization, lagging of chromosomes and
cell proliferation and tumor formation (Fernandes et cells with giant chromosomes. They stated that
al., 2007; Hoshina, 2002). abnormal use of Vernonia amygdalina could cause
genetic damage. Similar results were reported by
Yuet et al. (2012) in Euphorbia hirta, Pastori et al.
(2013) in Campomanesia xanthocarpa, Anjana and
Thoppil (2013) in Pogostemon auricularius etc.

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V. REFERENCES

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IV. CONCLUSIONS
Allium cepa root tip assay. International Journal
of Research and Development in Pharmacy and
Root tip cells of Allium cepa found to constitute an
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excellent system for testing toxicity and mutagenicity
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