ISBN - 9788192362182

Current Updates in
Life Sciences

Chief Editors

Dr. Mrs. P. P. Umale Dr. D. K. Koche

Professor & Head, Dept. of Botany, Professor, Department of Botany
Shri Shivaji College of Arts, Shri Shivaji College of Arts,
Commerce & Science , Akola (M.S.) Commerce & Science , Akola (M.S.)
 ISBN - 9788192362182

Current Updates in
Life Sciences

Chief Editors

Dr. Mrs. P. P. Umale Dr. D. K. Koche

Professor & Head, Dept. of Botany, Professor, Department of Botany
Shri Shivaji College of Arts, Shri Shivaji College of Arts,
Commerce & Science , Akola (M.S.) Commerce & Science , Akola (M.S.)
Copyright@2020, Departments of Life Sciences,
Shri Shivaji College of Arts, Commerce and Science, Akola (MS) India

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 EVALUATION OF CYTOTOXICITY OF COMMON VEGETABLES
MOMORDICA CHARANTIA AND LAGENARIA SICERARIA BY
ALLIUM TEST SYSTEM

Aniruddha S. Deshpande, K. D. Aswar and S. N. Malode

Department of Botany, Government Vidarbha Institute of Science and Humanities,
Amravati – 444 604, (MS) India.
Corresponding author: asdeshpande1212@gmail.com

ABSTRACT:
Plants are the source of phytochemical constituents which are effective against
various diseases and hence hold great medicinal and economic importance. Common
vegetables like Momordica charantia and Lagenaria siceraria are consumed by many rural,
tribal and nowadays, even urban people in raw form. There must be adequate dose
determination for consumption of these medicinally important vegetables in order to maintain
their positive impact on health. In present study, the Allium test system has been used to
evaluate the genotoxic, cytotoxic potential of these plants. Plant juices were found to be
cytotoxic even at lowest concentrations i.e. 0.5%, which in turn suggest that daily uptake of
these vegetables in raw form, must be very much lower than 0.5% in order to avoid any
cytotoxic effect on human health instead of being beneficial.
Keywords: Momordica charantia, Lagenaria siceraria, Allium test, Plant juice, cytotoxic
effect.

Introduction:
Most developing countries are benefited with vast resources of medicinal and
aromatic plants. These plant resources have been used over the millennia for welfare of
human and are still in use as traditional medicines by 80% of the population (De Silva, 1997).
From ancient times, many Indian medical practitioners made use of common medicinal plants
or vegetable for curing many devastating diseases. India is rich in food diversity. Diversity
among the food and their preparation procedures is due to availability of indefinite vegetable
resources; some are domesticated while some are wild and consumed by limited people.
Two vegetables used for this study are Momordica charantia and Lagenaria
siceraria. Both of these vegetables belong to family cucurbitaceae and are monoecious
climbers. Fruits of these plants are commonly used as vegetable and mostly consumed in raw

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form. Momordica charantia (Bitter gourd or Karla) is a vigorously growing herbaceous
climber with deeply incised leaves. Edible fruits are berries which are cylindric to fusiform
and green at young age. Lagenaria siceraria (L. leucantha or Bottle gourd or Dudhi bhopla)
is an extensive climber with strong stem creeping on ground and rooting at nodes. Leaves are
ovate and fruits are bottle shaped or cylindric-oblong berries (Dhore, 2002).
Momordica charantia fruit possess many medicinal properties. It has antidiabetic,
anticancer, anti-inflammatory, antiviral, antioxidant properties. Fruits are even use to treat
digestive disorders, hyperglycemia, microbial infections and menstrual problems and known
to have cholesterol lowering effects (Joseph and Jini, 2013). Even though, bitter guard have
potential to lower blood glucose level; its more consumption may lead to diarrhea, headache
and low blood sugar. It is known to have severe hypoglycemic effect due to daily
consumption. Children have been reported to become ill from eating bitter gourd and even
show hypoglycemic coma and convulsions after drinking bitter gourd tea (Krawinkel et. al.,
2006).
Lagenaria siceraria fruit pulp is used both as an emetic and purgative, and for its
cooling, diuretic, antibilious, and pectoral properties. Pulp boiled in oil is used to treat
rheumatism and insomnia (Prajapati et. al., 2010). Sharma et. al. (2012) found that
consumption of fresh juice causes many side effects like severe abdominal pain accompanied
by vomiting with or without blood, diarrhea, and excessive ulceration seen in distal
oesophagus, stomach and duodenum with some lethal consequences. These toxic effects may
be attributed to presence of tetracyclic triterpenoid compounds called cucurbitacins which is a
responsible for the bitter taste. These vegetables, distinction must be made between health
promoting and toxic doses. Allium test system is the most effective tool to determine toxicity
of certain phytochemicals present in plant extracts. Allium test involves the direct treatment
of onion bulbs with plant extract or such substances suspected of being capable of causing
cytological defects. Such defects are damages to DNA. It is therefore, believed that the
defects occurring on treated onion bulbs can also be expressed in human cells since both
Allium and human genomes are living systems containing DNA in the cells. Positive results
in the Allium test should be considered as a warning and also as an indication that the tested
chemical may be a risk to human health and to our environment (Fiskesjo, 1985).
Present study aimed to investigate the effects of fresh juices of Momordica charantia
and Lagenaria siceraria fruits in Allium test system. Secondly to study, effect of different
concentrations of fresh juices on Allium root tip cells to evaluate cytotoxicity assay for

Current Updates in Life Sciences 41

appropriate dose determination for safety purpose and healthcare to avoid harmful side effect
of juice consumption.

Materials and Methods:

a. Vegetables - Momordica charantia and Lagenaria siceraria were purchased from local
market of Amravati.
b. Fruits were washed with tap water; air dried and cut down. Extracts were prepared by
crushing 5gm, 10gm and 15gm of fruits in mortar and pestle and about 1 liter of tap water
added to each sample to squeeze out the extract. By this extraction, the three different
concentrations of the extract obtained are 0.5, 1.0 and 1.5 % (Figure 1).
c. Allium cepa assay:
For experiment, dried and good bulbs were used. 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 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 of plant
material of three different concentrations viz. 0.5%, 1.0%, and 1.5% 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 (Figure 1). About six onion bulbs
were also treated with distilled water which served as control. The root tips were cut after the
treatment duration and fixed in Carnoy‘s fixative (Glacial Acetic Acid: Absolute Alcohol in
1:3 proportions).
d. Cytological study:
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
chromosome aberrations was calculated by expressing the percentage of number of aberrant
cells. Mitotic index was calculated by following formula for each of the treatments and the
control (Figure 2 and Figure 3).
Mitotic Index = Number of dividing cells × 100 / Total number of cells
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

Current Updates in Life Sciences 42

the different cell division stages, total dividing cells, non-dividing cells and number of cells
showing chromosomal aberrations. The per cent chromosomal aberration (Abnormality %)
was calculated for each treatment and the control using the following formula
Percent of chromosomal aberration = Number of aberrant cells × 100 /Total number of dividing cells

Results and Discussions:

This study determines the cytotoxic and genotoxic effects of raw aqueous extracts of
Momordica charantia and Lagenaria siceraria fruits on mitotic index and chromosomal
aberrations in growing root tip cells of A. cepa. The number of normal dividing phases and
chromosomal aberrations observed in the cells of the treated onion roots with tested aqueous
extracts and the control are shown in Tables 1 and 2 (Figure 4 and Figure 5). No
chromosomal abnormalities were reported in control. Chromosomal abnormalities were
mainly observed in the form of chromosomal bridges, clumped phases (prophase and
metaphase), lagging chromosomes, precocious movements, and sticky chromosomes.
Table 1 represent effect of different concentrations of Momordica charantia fruit
extract on mitotic index and chromosomal abnormalities in Allium cepa root tips. Linear
decrease in mitotic index was observed with increase in the concentration over the control
(20.13%) except 0.5% (20.40%) concentration. 1.5% shows the least value of mitotic index
(12.53%). While contrasting results were obtained in case of abnormality percent. Linear
increase in abnormality percent was observed with increase in concentration of extract.
Highest abnormality percent (15.15%) was recorded in 1.5% concentration while 0.5%
concentration shows least chromosomal abnormalities (4.39%).
Table 2 represent effect of different concentrations of Lagenaria siceraria fruit
extract on mitotic index and chromosomal abnormalities in Allium cepa root tips. Similar
trend of linear decrease in mitotic index and linear increase in chromosomal abnormalities
found to be coupled with increase in the concentration of fruit extract was observed. Control
shows highest mitotic index (20.09%) while least value (15.73%) of mitotic index was
observed in highest concentration i.e. 1.5%. Abnormality percent shows linear increase with
increase in concentration of extract. Highest abnormality percent (13.70%) was recorded in
1.5% concentration while 0.5% concentration shows least chromosomal abnormalities
(7.05%).

Current Updates in Life Sciences 43

 Table- 1: Mitotic activities and chromosomal abnormalities observed in root tip cells of A.
cepa treated with different concentrations of Momordica charantia fruit extract.

Conc. of
Abnormalities
M. Total
Total Mitotic
charantia No. of Abnormality
cells Index
fruit dividing Percent (%)
counted (%) CB CP L PM SC
extract cells
(Percent)
Control 174 864 20.13% 00 00 00 00 00 00%
0.5% 182 892 20.40% 3 1 - - 4 4.39%
1.0% 121 800 15.12% 4 - 1 - 6 9.09%
1.5% 99 790 12.53% 5 2 - 2 6 15.15%

Table- 2: Mitotic activities and chromosomal abnormalities observed in root tip cells of A. cepa
treated with different concentrations of Lagenaria siceraria fruit extract.
Conc. of
Abnormalities
L. Total
Total Mitotic Abnormality
siceraria No. of
cells Index Percent (%)
fruit dividing
counted (%) CB CM L PM SC
extract cells
(Percent)
Control 171 851 20.09% 00 00 00 00 00 00%
0.5% 156 846 18.43% 4 - - 2 5 7.05%
1.0% 142 796 17.83% 7 1 - 1 4 9.15%
1.5% 124 788 15.73% 8 - 3 - 6 13.70%
CB – Chromosomal Bridge, CM – Clumped phases, L – Lagging chromosomes,
PM – Precocious movements, SC - Sticky chromosome

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Current Updates in Life Sciences 45
 Lowered mitotic index in onion root tip cells treated with various concentration of
plant juices implies that these extract lower down normal rate of cell divisions i.e. they cause
mito-depressive effect. Mito-depressive effects may be caused due to ability of these extracts
to block the synthesis of DNA and nuclear proteins or even they may not allow the initiation
of their biosynthesis. Such action occurring in the interphase nucleus apart from influencing
the ultimate structure of the chromosome during cell division could also cause reduction of
number of other stages as earlier proposed by many coworkers (Akinboro and Bakare, 2007).

Since mitotic index is the quantitative estimation of the mitotic activities in an

organism, it is a good indicator to evaluate the cytotoxicity level of certain plant based and
chemical compounds. Decreasing mitotic index with increase in concentration of plant

Current Updates in Life Sciences 46

extracts suggested that the extracts have suppressive effects of mitotic activities in A. cepa
root tip cells. Akinboro and Bakare (2007) reported similar trend of decrease in mitotic index
when onion root tip cells were used to evaluate cytotoxic effects of aqueous extracts of five
medicinal plants Azadirachta indica, Morinda lucida, Cymbopogon citratus, Mangifera
indica and Carica papaya. All these extract shows lowering of mitotic index value over
control wit least mitotic index (2%). Even at higher concentrations cell division process is
completely retarded.
Celik and Sultan in 2010 studied the cytotoxic effects of three different concentrations
of Inula viscosa leaf extracts i.e. 2.5mg/ml, 5mg/ml and 10mg/ml in comparison to tap water
(negative control) and 2×10-2M EMS (positive control). They found that all the extracts and
positive control shows lower value of mitotic index as compared to tap water and even
highest concentration of I. viscosa leaf extract (10mg/ml) was more mutagenic than positive
control showing least value of mitotic index (0.088%).
The mitotic abnormalities like clumped phases, chromosomal bridges, precocious
movements, laggards may results due to various types of phytochemical compounds like
alkaloids, flavonoids, terpenoids, tannins, carcinogens etc. present in the plants. Since most
herbal preparations are not refined and are mostly consumed raw, this suggests the possible
exposure of the cells of the consumers to such genetic defects, as occurred in the
chromosome of A. cepa genome.
Rathnasamy et al. (2013) studied cytotoxic effects of aqueous & methanolic extracts
of three medicinal plants i.e. Clinacanthus nutans, Adhatoda vasica, and Carica papaya on
growing root tips of Allium cepa. Similarly many workers earlier used Allium test for
evaluation of cytotoxic effects of various plant extracts like Anjana and Thoppil for
Pogostemon auricularius (2013), Ilbas et al. for Aloe vera gel (2012), Adegbite and Sanyaolu
for Vernonia amygdalina (2009), Rajkumar et al. for Acorus calamus rhizome (2009),
Oyeyemi and Bakare for Spondias mombin, Nymphea lotus and Luffa cylindrica (2013),
Sultan and Celik for Capparis spinose (2009) etc.

Conclusions:
Cytogenetic tests are desirable for identifying the damaging effects of various
substances and their influence on living organisms. The Allium test is adequately sensitive to
detect numerous substances that cause chromosomal aberrations. Mitotic index is an
acceptable measure of cytotoxicity in all living organisms. The cytotoxicity level can be

Current Updates in Life Sciences 47

determined by the decreased rate of mitotic index. Mitotic index was found to be decreasing
with increasing extract concentration. Mitotic index observed in the treated root tip meristem
were significantly reduced as compared to the control. The decreased mitotic index values in
the treated onion roots may be an indication of the presence of cytotoxic substances in the
aqueous fruit extracts of tested plants samples, which causes inhibition of mitotic activities,
while the aberrant cell in the treated onion root tip meristems indicates genotoxic effects of
the fruit extract. Reduction in the mitotic activities could be due to inhibition of DNA
synthesis or a blocking in the G2 phase of cell cycle, preventing the cell from entering
mitosis.
Chromosomal aberrations were observed in all stages of mitosis. The abnormalities of
chromosome could be due to the blockage of DNA synthesis or inhibition of spindle
formation. The cells of A. cepa root tips after treated with extracts of M. charantia and L.
siceraria fruit showed increased abnormality percent with increased concentration of extract.
Study found to be important for determination of dose of daily intake of these
medicinally important plants. Study suggest that even at lowest concentrations i.e. 0.5%,
plant juices were found to be cytotoxic which in turn suggest that daily raw intake of these
plants must be lower than 0.5% in order to avoid any cytotoxic effect on human health
instead of being beneficial. Further study needs for confirmation after animal experimentation
and cell lines for precise dose determination.

References:
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(Vernonia amygdalina Del.) using the Allium cepa chromosome aberration assay, Scientific
Research and Essay, (11): 1311-1314.
Akinboro, A. and Bakare, A. A. (2007) Cytotoxic and genotoxic effects of aqueous extracts
of five medicinal plants on Allium cepa Linn. Journal of Ethnopharmacology, pp. 470-475.
Anjana, S. and Thoppil, J. (2013) Analysis of cytotoxic potential of the aqueous leaf extracts
of Pogostemon auricularius (L.) Hassk. using Allium cepa root tip assay. International
Journal of Research and Development in Pharmacy and Life Sciences, 2 (5):562-566.
Celik, T. A. and Aslanturk, O. S. (2010) Evaluation of Cytotoxicity and Genotoxicity of Inula
viscosa Leaf Extracts with Allium Test. Journal of Biomedicine and Biotechnology, 1-8.

Current Updates in Life Sciences 48

De Silva, T. (1997) Industrial utilization of medicinal plants in developing countries. Non-
wood forest products. In Medicinal plant for forest conservation and healthcare. FAB, Rome,
Italy.
Dhore, M. A. (2002) Flora of Amravati District with special reference to the distribution of
Tree species, Ph. D. Thesis, submitted to the Faculty of Science, Nagpur University, Nagpur.
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102: 99-112.
Ilbas, A., Gonen, U., Yilmaz, S. and Dadandi, M. (2012) Cytotoxicity of Aloe vera gel
extracts on Allium cepa root tip cells, Turk J Bot, 36: 263-268.
Joseph, B. and Jini, D. (2013) Antidiabetic effects of Momordica charantia (bitter melon)
and its medicinal potency, Asian Pacific Journal of Tropical Disease, 3(2): 93–102.
Krawinkel, M. and Keding, G. (2006) Bitter Gourd (Momordica charantia): a Dietary
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cells, International Journal of Cytology, Cytosystematics and Cytogenetics, 66(4): 360-367.
Prajapati, R., Kalariya, M., Parmar, S. and Sheth, N. (2010) Phytochemical and
pharmacological review of Lagenaria sicereria, Journal of Ayurveda & Integrative Medicine,
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Rajkumar, V., Guha, G., Kumar, A. and Mathew, L. (2009) Evaluation of Cytotoxic Potential
of Acorus calamus Rhizome, Ethnobotanical Leaflets, 13: 832-839.
Rathnasamy, S., Mohamed, K., Sulaiman, S. and Akinboro, A. (2013) Evaluation of
cytotoxic, mutagenic and antimutagenic potential of leaf extracts of three medicinal plants
using Allium cepa chromosome assay, International Current Pharmaceutical Journal, 2(8):
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Tandon, N., Handa, S. S., Toteja, G. S., Rao, S., Satyanarayana, K., Katoch, V. M. (2012)
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