SIRJ-APBBP Volume 2 Issue 5 (October 2015)
www.scrutinyjournals.com
ISSN 2349 – 0128
Scrutiny International Research
Journal of Agriculture, Plant
Biotechnology and Bio Products
(SIRJ-APBBP)
Evaluation of antimutagenic activity of the leaves of Carica
papaya
N. Kannikaparameswari1, T. Indhumathi1, S. Suja2 and Rajathi K1
1 Post Graduate and Research Department of Biochemistry, Dr. N.G.P. Arts and Science College (Affiliated
to Bharathiar University), Coimbatore- 48
2 Department of Biochemistry, P.S.G. College of Arts and Science, Coimbatore-14
India
Article history: Submitted 6 May 2015; Accepted 4 July 2015; Published 28 October 2015
Abstract
In genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA,
of an organism and thus increases the frequency of mutations above the natural background level. As many
mutations cause cancer.Antimutagenic agents are natural or synthetic compounds which are able to lower or
abolish genotoxic effects of mutagenic and carcinogenic factors.Plants are important sources of biologically
active natural products, which differ widely in terms of structure. They are the original source of a variety of
compounds used by the pharmaceutical industry as medicines. Also, medicinal plants are candidates for
chemoprevention of cancer, because they may possess chemo preventive agents with inhibitory effects on the
initiation, promotion and progression of carcinogenesis. For this reason plants always been an important
sources of drugs.
Key words: Mutagen, Antimutagenicity, Salmonella Typhymurium , Sodium Azide
Corresponding author
Dr. N. Kannikaparameswari,
Assistant Professor,
PG and Research department of Biochemistry,
Dr. N.G.P. Arts and Science College,
Coimbatore- 48, Tamilnadu,
India.
Introduction
In genetics, a mutagen is a physical or chemical agent that changes the genetic
material, usually DNA, of an organism and thus increases the frequency of mutations above
the natural background level. As many mutations cause cancer, mutagens are therefore also
likely to be carcinogens. Mutagens cause changes to the DNA that can affect the
transcription and replication of the DNA, which in severe cases can lead to cell death. The
www.scrutinyjournals.com
Kannikaparameswariet al., / SIRJ-APBBP 2:5 (2015)
mutagen produces mutations in the DNA, and deleterious mutation can result in aberrant,
impaired or loss of function for a particular gene, and accumulation of mutations may lead to
cancer (Bennett et al., 1997).
In biology, a mutagen (Latin, literally origin of change) is a physical or chemical agent
that changes the genetic material, usually DNA, of an organism and thus increases the
frequency of mutations above the natural background level. As many mutations
cause cancer, mutagens are typically also carcinogens. Not all mutations are caused by
mutagens: so-called "spontaneous mutations" occur due to errors in DNA replication, repair
and recombination (Castro et al., 2008).Antimutagens are the agents that interferes with
the mutagenicity of a substance. The interference can be in the form of prevention of the
transformation of a mutagenic compound into mutagen, inactivation, or otherwise the
prevention of Mutagen-DNA reaction. (Bhattacharya et al., 2011).Antimutagens can be
classified into: Desmutagens, that inactivate the chemical interactions before the mutagen
attacks the genes and Bio-antimutagens, that stop the mutation process once after the genes
are damaged by mutagens.There are a number of naturally occurring antimutagens that
show their efficient action (Wallet al., 1992).
Antimutagenic agents are natural or synthetic compounds which are able to lower or
abolish genotoxic effects of mutagenic and carcinogenic factors (De Flora, 1998).Recent
research has confirmed that many common food articles contain components that possess
antimutagenic and anticarcinogenic properties. Thus it has been suggested that the use of
these compounds in everyday life will be the most effective procedure for preventing human
cancer and genetic disorders (Chang et al., 2005). Plants are important sources of
biologically active natural products, which differ widely in terms of structure. They are the
original source of a variety of compounds used by the pharmaceutical industry as medicines.
Much research has been and is being done on plants in popular use, with the objective of
identifying natural products with therapeutics potential. Also, medicinal plants are
candidates for chemoprevention of cancer, because they may possess chemo preventive
agents with inhibitory effects on the initiation, promotion and progression of
carcinogenesis. For this reason plants always been an important sources of drugs
(Freguson, 2003).
In recent years, there has been greater interest in investing compounds originating
from most of the medicinal plants and their effects on DNA. This is done with different
organism. The action of these compounds may be involved in maintaining the balance
between the consumption of mutagenic and anti-mutagenic substances, thus contributing to
increase or reduction in the incidence of cancer in the population.
Materials and Methods
Collection of Plant Material
Carica Papaya is the most widely cultivated species of the genus carica, which is the
only genus in the family caricaceae. The plants were collected from the interior parts of
Guruvayur.
Extraction of Carica Papaya Leaves
20g of Carica Papaya Leaf powder was immersed in 100ml ethanol for 72 hours at
37ºc and the extract was filtered through two layered muslin cloath and dried in vacuum.
The dried extract was solubilized by using distilled water and was used for further studies.
8
www.scrutinyjournals.com
Kannikaparameswariet al., / SIRJ-APBBP 2:5 (2015)
Experimental Organism: Salmonella Typhymurium TA98 and TA 100
Phytochemical Analysis: Phytochemical analysis was done according to the method of
Mukherjee et al., 2002.
Antimutagenic Assay: Antimutagenic assay was done by the method of Ames et al., 1973
Confirming genotypes of the salmonella strains
Histidine requirement
The Histidine character of the tester strains was confirmed by demonstrating the
Histidine requirement for growth on selective agar plates such as Histidine/biotin plate.
Biotin was also requiring by all of the tester strains because of the UVrB deletion which
extended through the bio-gene. Cotton swab was dipped in the 12 Hour broth culture and a
single sweep was made across the histidine/biotin plate. Then, the plates were incubated
overnight at 37° C and the growth was examined on the next day.
rfa mutation
Strains having the deep rough (rfa) character were tested for crystal violet
sensitivity.0.1 ml of fresh overnight culture of the tester strains (TA 98 and TA 100) was
added to a test tube containing 2 ml of moltent agar at 45 ° C. The top agar tubes were
vortexed for 3 sec at low speed and poured on nutrient agar plate without histidine and
biotin. The plates were tilted and rotated for the even distribution of the top agar on the
plates. The plates were placed on a leveled surface and allowed several times for agar to
become firm. 10 µl of 1 mg/ml solution of crystal violet was pipetted to the center of the
sterile disc (1/4 inch) and discs were transferred to each of the inoculated plates using the
sterile foceps. The discs were lightly pressed with forceps to embed it slightly in the
overlay. The plates were incubated at 37 °C and observed for crystal violet sensitivity.
UVrB Mutation
The UVrB mutation was confirmed by demonstrating UV sensitivity in strain that
contain this mutation. The R-factor strains TA 98 and non R-factor TA100were streaked in
parallel stripes with sterile swabs across the nutrient agar plate. A plate was irradiated with
a 15 W germicidal lamp approximately at a distance of 35 cm and was irradiated for 8 sec.
The irradiated plates were incubated at 37°C for 12-24 hours.
R-factor
The R-factor strains TA 98 were tested for the presence of the ampicillin resistance
factor. To test for ampicillin resistance, the cultures were streaked across of an ampicillin
plate using the procedure as described for confirming the Histidine requirement, the non Rfactor strain TA 100 was tested on the same plate as a control for ampicillin activity.
Toxicity
For the testing the toxicity minimal glucose agar medium was prepared. The plant
extract was added to the minimal glucose agar and mixed well. Minimal glucose agar
9
www.scrutinyjournals.com
Kannikaparameswariet al., / SIRJ-APBBP 2:5 (2015)
containing plant extract was poured onto the petridish. Overnight culture of strains TA 100
and TA 98 was streaked onto it. The plates were incubated for 24 hours.
Antimutagenicity assay
Antimutagenicity of the plant was tested in salmonella typhymurium strains TA 98
and TA 100 using direct acting mutagens.
Determination of antimutagenicity against direct acting mutagens
Plate incorporation method was done for antimutagenicity assay without
microsomal activation. Fresh cultures of Salmonella typhymurium strains TA 100 and TA 98
(1-2x 10 9 cells/ml) were mixed with 2 ml of molten agar containing 0.5 m M
Histidine/biotin solution, different concentration of plant extract (0.1-1 mg/plate) and
direct acting mutagens such as sodium azide (2.5µg/plate). Further it was spread over
minimal glucose agar plates. Plates were incubated for 48 hours at 37° C and relevant
colonies were counted.
Results
Phytochemical Investigations
In the present study the ethanolic extract of Carica Papaya were subjected to
phytochemical investigations.
Table No.1: Phytochemical Investigations
Constituents
Alkaloids
Glycosides
Steroids
Tannins
Flavonoids
Saponins
Carbohydrates
Proteins
Resins
Ethanol extract
+
+
+
+
+
+
+
+
-
Note: Key: “+”= Present; “-“= Absent
Table No. 2: Antimutagenicity of the plant extract against Sodium azide on
Salmonella Typhymurium strain TA 98
Group
Average no. of
Colonies present
Sodium azide
Control 180
0.2 mg 30
0.4 mg 17
0.6 mg 5
%Inhibition
Sodium azide
84
91
97
10
www.scrutinyjournals.com
Kannikaparameswariet al., / SIRJ-APBBP 2:5 (2015)
Table No.3: Antimutagenicity of the plant extract against Sodium azide on
Salmonella Typhymurium strain TA 100
Group
Control
0.2 mg
0.4 mg
0.6 mg
Average no.of
Colonies
present
Sodium azide
182
40
20
8
%Inhibition
Sodium azide
79
89
96
A decrease in antimutagenicity from the irradiated samples may be a strong
indication that the ethanol extract of the Carica papaya leaves possesses mutagenic
properties. This was confirmed in repeat investigation but it was so far only investigated in
the Ames assay.
Discussion
A preliminary phytochemical analysis of Carica papaya leaves revealed the presence of
alkaloids, flavonoids, saponins, tannins, glycosides, proteins and steroids.
In the present investigation we have checked the potential activity of plant extract
for its antimutagenicity using Salmonella Typhymurium assay. This mainly based on the
reversion of mutant cells by mutagenic agents, which is Ames test.It was found that the
plant extract would inhibit the revertant formation produced by direct acting mutagen such
as Sodium azide. The plant extract showed 95% inhibition of mutagenicity at 0.6 mg/plate
and the activity decreased with concentration.
Conclusion
It has been suggested that the use of antimutagen in daily life will be the most
effective procedure for preventing human cancer and genetic diseases. These compounds
interfere with mutagen metabolism or they may act as mutagen scavengers. They may also
inhibit either the initiation or promotion phase of the carcinogenic phases. In this study we
have critically evaluate the antimutagenic activities of leaf extract from the plant Carica
Papaya using Salmonella Typhymurium strains (Ames test).Plant extracts show to have the
antioxidant activity and has been shown to be useful in reducing the oxidative stress
induced by UV radiation and protects the macular. Many carcinogenic agents have been
shown to induce mutation through intera interaction with genetic material. In this work we
have studied the effect of Carica Papaya leaves on reverse the mutation with Sodium azide in
Salmonella Typhymurium strains TA 98 and TA 100 (Ames test).
Reference
Bennett, R.N., Kiddle, G. and Wallsgrove, R.M., 1997. Biosynthesis of benzylglucosinolate,
cyanogenicglucosides and phenylpropanoids in Carica papaya. Phytochemistry, 45(1): 5966.
11
www.scrutinyjournals.com
Kannikaparameswariet al., / SIRJ-APBBP 2:5 (2015)
Castro, I.M, Anjos and M.R., 2008. Determinação De isotiocianato de benzilaemCarica papaya
utilizandocromatografiagasosa com detectoresseletivos. Quim. Nova, 31(8):1953-1959.
Fergnson, L.R., 1994. Antimutagens as cancer chemopreventive agents in diet. Mutation
Research, 307: 395–410.
Wall Monroe, 1992. Antimutagenic Agents from Natural Products. Journal of Natural
Products, 55(11): 1561–1568.
12