Journal of Advanced Laboratory Research in Biology

E-ISSN: 0976-7614
Volume 5, Issue 3, July 2014
PP 79-82
https://e-journal.sospublication.co.in

Research Article

Chromatographical Analysis of Amino Acids in Sepia Mutant of Drosophila

melanogaster under Stress of Cypermethrin and Alphamethrin
Namrata Rana*, P.N. Saxena and Nishi Saxena

Department of Zoology, School of Life Sciences, Dr. B.R. Ambedkar University, Agra-282002, U.P., India.

Abstract: Insects are invertebrates that are taxonomically referred to as the class Insecta. They are the most
numerous and most widespread terrestrial taxon within the phylum Arthropoda, and indeed the most diverse group
of animals on the earth, with around 925,000 species described—more than all other animal groups combined.
Insects may be found in nearly all environments on the planet, although only a small number of species have
adapted to life in the oceans where crustaceans tend to predominate instead. HPTLC is a method commonly applied
for the identification, assay and the testing for purity, stability, dissolution or content uniformity of raw materials
(herbal and animal extracts, fermentation mixtures, drugs and excipients) and formulated products (pharmaceuticals,
cosmetics, nutrients). These flexible and cost-effective techniques present the advantage of the simultaneous
processing of standards and samples with versatile detection possibilities, including a great variety of post-
chromatographic derivatization reagents.

Keywords: HPTLC, Leucine, Glycine, Valine, Marker compounds, Pyrethroid insecticides.

1. Introduction for protection against infestation is a successful

approach to the problem of preventing damage to crop
There are approximately 5,000 dragonfly species, seeds and keeping grains free from insects (Tufail et al.,
2,000 praying mantis, 20,000 grasshoppers, 170,000 1992). Pyrethroids are synthetic derivatives of natural
butterflies and moth, 120,000 fly, 82,000 true bug, pyrethrum which is obtained from the flower heads of
350,000 beetle, and 110,000 bee and ant species Chrysanthemum cinerariaefolium and some other
described to date. Estimates of the total number of species of Chrysanthemum. Natural pyrethrum and
current species, including those not yet known to synthetic pyrethroid insecticides are considered among
science, range from two million to fifty million, with the safest class of insecticides available.
newer studies favouring a lower figure of about six to The insecticidal activity of pyrethrum is due to six
ten million. Adult modern insects range in size from esters namely pyrethrum I, II; cinerin I, II and jasmolin
0.139mm (Dicopomorpha echmepterygis) to 555mm I, II. Natural pyrethrins are light sensitive and undergo
(Phobaeticus serratipes). rapid photodegradation, while synthetic pyrethroids are
The use of chlorinated and organophosphate photostable i.e. they do not undergo photolysis in
insecticides in the household is being discouraged sunlight. Pyrethrum is a complex insecticide with the
throughout the world. These insecticides are considered following outstanding properties:
to have high persistence, adverse environmental impact
a. Rapid Action: Pyrethrum is a contact insecticide
and toxic effects on higher animals including man. On
attacking the nervous system of insects almost
the other hand, in the past decade, the synthetic
immediately and causing knockdown, soon followed
pyrethroids have emerged as a major class of
by kill.
agriculturally useful organic insecticide. Pyrethroids are
b. Low Mammalian Toxicity: No other insecticide can
a diverse group of chemicals, which have been
claim such a long record of proven safety towards
developed to kill or suppress a wide variety of insect
humans and warm blooded animals. It is one of the
pests. Application of pyrethroids and other insecticides

*Corresponding author:
E-mail: [email protected]; Mobile No.: +91-9837068940.
Chromatographical Analysis of Amino Acids in Drosophila melanogaster Rana et al

least toxic of domestic insecticides available and is University, Agra. The culture was nurtured and
rapidly metabolized if accidentally swallowed. maintained in glass culture vials of 100ml capacity, at a
c. Lack of Insect Immunity: The structure of the temperature 25 + 0.50C, 50 + 5% relative humidity in
molecule is so complex that insect resistance to B.O.D. incubator. The flies were fed on prescribed
pyrethrum is not a practical problem. A very few Drosophila food (Roberts, 1986).
isolated cases have been reported, unlike synthetic
insecticides. 2.2 Experimental protocol
d. Broad Spectrum of Activity: Because pyrethrum For the chromatographical analysis experiment was
consists of a group of related compounds, it has a conducted on three sets of Sepia mutant of Drosophila
wider spectrum of activity against insect species than melanogaster.
many single insecticides. It can be used against any
insect found in the house or garden: mosquitoes, a. Control set;
flies, cockroaches, aphids, ants. b. Cypermethrin treated set and;
e. Environment friendly: Nonresistant: Pyrethrum is c. Alphamethrin treated set.
degraded by the combination of sunlight and air and
therefore presents little of the hazards which are First set was free from treatment and considered as
usually associated with other classes of persistent control set, while the second and third sets were given
insecticide. sublethal treatments of cypermethrin and alphamethrin
f. Repellency: Pyrethrum is a powerful insect repellent respectively for 48 hours and served as treated sets.
which in combination with its low mammalian Flies of all the sets were allowed to fertilize for five
toxicity favors it in many applications, such as food days and the observation was made in the F1 generation.
and grain storage protection, personal protection
(spray-on, roll-on, mosquito coils). Beside 3. Result and Discussion
repellency, mosquito coils will inhibit target insects
from biting. An increase has been observed in area percentage
g. Flushing Action: Pyrethrum has a much greater of cypermethrin and alphamethrin treated sets as
flushing effect than any other insecticide; it disturbs compared to control set. However, the increase is more
pests in their hiding places, forcing them to get out in alphamethrin treated sets as compared to
and to get exposed to the insecticide. cypermethrin treated sets (Table 1-3; Fig. 1-4).

HPTLC is the method commonly applied for the Table 1. TLC data of glycine amino acid in sepia mutant of
identification, assay and testing for purity, stability, Drosophila melanogaster.
dissolution or content uniformity of raw materials
(herbal and animal extracts, fermentation mixtures, Treated
Parameters Control
drugs and excipients) and formulated product Alphamethrin Cypermethrin
(pharmaceuticals, cosmetics, nutrients). This flexible Start Rf 0.10 0.09 0.09
and cost effective technique presents the advantage of Start height 41.8 17.4 25.2
m
the simultaneous processing of standards and samples Max Rf 0.16 0.15 0.14
m
with versatile detection possibilities, including a great Max height 240.6 228.7 329.0
m
variety of post chromatographic derivatization reagents. Max % 22.22 22.68 23.34
End Rf 0.16 0.15 0.15
The validation of analytical methods is largely
End height (mm) 240.6 228.7 307.8
recognized as the best safeguard against the generation 2
Area (mm ) 6549.7 5478.3 8436.8
of unreliable data and is becoming an absolute Area percentage 18.34 17.26 17.82
requirement in many fields. Validation is the process by
which it is established, by laboratory studies, that the Table 2. TLC data of valine amino acid in sepia mutant of
performance characteristics of an analytical method Drosophila melanogaster.
meet the requirements for the intended application.
HPTLC is finding increasing use in various fields Treated
today. The development of scanning, densitometry and Parameters Control
Alphamethrin Cypermethrin
derivatization have also helped in making it more Start Rf 0.28 0.27 0.27
useful. Alteration in HPTLC of treated sets is an index Start height 28.3 25.0 26.6
m
of toxicity so that the outcome of present investigation Max Rf 0.33 0.31 0.32
m
becomes fruitful and genetically evidenced. Max height 114.2 128.4 213.9
m
Max % 10.55 12.73 15.18
2. Materials and Methods End Rf 0.36 0.35 0.36
End height (mm) 60.3 42.5 75.0
2.1 Laboratory culture of experimental mutants Area (mm )
2
4331.0 4448.6 7835.3
The Sepia mutant form of Drosophila Area percentage 12.13 14.02 16.55
melanogaster was acquired from toxicology laboratory,
Department of Zoology, Dr. B.R. Ambedkar

J. Adv. Lab. Res. Biol. 80

Chromatographical Analysis of Amino Acids in Drosophila melanogaster Rana et al

Table 3. TLC data of leucine amino acid in sepia mutant of performance characteristics of an analytical method
Drosophila melanogaster. meet the requirements for the intended applications.
Depending on the objective of the analytical procedure,
Treated
Parameters Control the typical validation characteristics which can be
Alphamethrin Cypermethrin
considered through a statistical approach are accuracy,
Start Rf 0.47 0.46 0.47
precision, specificity or selectivity, detection limit,
Start height 105.3 119.3 203.3
m quantification limit, linearity and ruggedness. The
Max Rf 0.51 0.49 0.49
m
Max height 178.9 172.8 233.2
concept of validation applied to densitometric
Max %
m
16.52 17.14 16.54 determinations on high-performance thin-layer
End Rf 0.59 0.54 0.55 chromatography (HPTLC) indeed varies according to
End height (mm) 2.0 16.3 25.6 the goal of the analysis and the steps required for a
Area (mm )
2
7191.6 6605.0 9338.5 formal validation have been thoroughly investigated,
Area percentage 20.14 20.81 19.72 notably for purity testing, pharmaceutical dosage forms
assay and herbals fingerprinting (Biringanine et al.,
2005).

Fig. 1. Chromatogram showing glycine, valine and leucine amino

acids in control set of sepia mutant.
Fig. 3. Chromatogram showing glycine, valine and leucine amino
acids in control set of sepia mutant.

Fig. 2. Chromatogram showing glycine, valine and leucine amino

acids in alphamethrin treated set of sepia mutant.
Fig. 4. Chromatogram showing glycine, valine and leucine amino
The validation of analytical methods is largely acids in cypermethrin treated set of sepia mutant.
recognized as the best safeguard against the generation
of unreliable data and is becoming an absolute For standardization of natural product drugs, single
requirement in many fields. Validation is the process by chemical entities, “marker compounds,” may be used as
which it is established, by laboratory studies, that the potency standards in high-performance thin-layer

J. Adv. Lab. Res. Biol. 81

Chromatographical Analysis of Amino Acids in Drosophila melanogaster Rana et al

chromatography (HPTLC) analysis. Using well- DNA in susceptible and resistant strains of Musca
characterized marker compounds, conventional domestica L. after treatment with DDT and
pharmaceutical manufacturing criteria for assay and Malathion. Natl. Acad. Sci. Lett., 25(5-6): 170-
content uniformity may be applied. These marker 175.
compounds may be used to help identify herbal [2]. Ashwell, G. (1957). In: Methods Enzymol. (eds.
materials, set specifications for raw materials, Colowick, S.P. and Kaplan, N.O.), Academic
standardize botanical preparations during all aspects of Press, New York, 87-99.
manufacturing processes, and obtain stability profiles. [3]. Finney, D.J. (1971). Probit Analysis, Cambridge
HPTLC analysis of marker compounds may provide University Press, 303 pp.
additional information in the form of “chromatographic [4]. Kaur, R. and Rup, P.J. (2003). Influence of some
fingerprints” (Natalie et al., 1998). plant growth regulators (PGR) on biochemical
Densitometric scanning of individual or all profile in the larvae of melon fruit fly Bactrocera
fractions can be repeated with the same or different cucurbitae (Coquillett) (Diptera: Tryptidae).
parameters, since all fractions remain stored on the Entomon., 28 (2): 89-95.
plate, and have many options for evaluating or [5]. Roberts, D.B. (1986). Drosophila, A practical
documenting the fractions both before and after approach, IRL Press, Oxford, OX8 IJJ, England,
derivatization. Many samples are analysed side by side 295.
and due to the offline character of the method many [6]. Saleem, M.A. and Shakoori, A.R. (1996).
samples can be chromatographed simultaneously Biochemical studies on Talcord 10EC. II. Effect
(Janchen, 1987). on some enzyme activities and macromolecules of
HPTLC is finding increasing use in various fields adult beetles of Tribolium castaneum. Pak. J.
today. The development of scanning, densitometry and Zool., 28 (2): 151-162.
derivatization have also helped in making it more [7]. Saleem, M.A. and Shakoori, A.R. (1989).
useful. Toxicity of malathion, permethrin and
HPTLC was carried out for qualitative estimation cypermethrin against resistant and susceptible
of experimental amino acids. The sequence of area strains of Tribolium castaneum (Herbst.). Pak. J.
percentage of experimental amino acids in control sets Zool., 21: 347-359.
(control sepia and control white) was found to be- [8]. Saleem, M.A., Malik, M.Z., Fayyaz, M. and
Leucine > Glycine > Valine Shakoori, A.R. (1986). The toxicity of four new
synthetic pyrethroids to sixth instar larvae of a
The sequence of area percentage remained same stored grain pest, Tribolium castaneum (Herbst.).
for the treated mutants except for alphamethrin treated Pak. J. Zool., 18: 417-431.
white mutants in which it changed to- [9]. Saleem, M.A., Shakoori, A.R. and Mantle, D.
Glycine > Leucine > Valine (1998). In vivo ripcord induced macromolecular
abnormalities in Tribolium castaneum larvae. Pak.
Area percentage of glycine decreased, whereas J. Zool., 30 (3): 233-243.
increased of valine in sepia mutants following [10]. Shakoori, A.R., Saleem, M.A. and Mantle, D.
alphamethrin and cypermethrin intoxication, but area (1998). Some macromolecular abnormalities
percentage of leucine increased in alphamethrin treated induced by a sublethal dose of cymbush 10EC in
sepia mutant which decreased in cypermethrin treated adult beetles of Tribolium castaneum. Pak. J.
sepia mutants. Zool., 30 (2): 83-90.
The area percentage decreased of both valine and [11]. Singh, R. (1982). Effect of malathion on the free
leucine amino acid in alphamethrin as well as amino acids in the haemolymph of Dysdercus
cypermethrin intoxicated white mutants whereas area koenigii (Hemiptera: Pyrrhocoridae). Entomon., 7
percentage of glycine increased in alphamethrin treated (3): 253-256.
white mutants and decreased in cypermrthrin treated [12]. Tufail, N. and Shakoori, A.R. (1992).
white mutants. Alphamethrin toxicity in Tribolium castaneum
Fluctuation in area percentage is more for valine larvae. Pak. J. Zool., 24 (1): 59-70.
amino acid in the two experimental mutants, whether [13]. Tufail, N., Saleem, M.A. and Shakoori, A.R.
treated with cypermethrin or with alphamethrin. (1992). Effectiveness of synthetic pyrethroids
Therefore, valine, an amino acid, was affected the most against susceptible and resistance strains of
among the three amino acids. Tribolium castaneum. Pak. J. Zool., 24: 199-209.
[14]. Vijayraghavan, C. and Chitra, K.C. (2002). Total
References protein and free amino acid content of Spodoptera
litura (Fabr.) due to botanicals and conventional
[1]. Akhtar, K., Naqvi, S.N.H., Ahmad, I. and Azmi, insecticides. Ind. J. Ent., 64 (1): 92-95.
M.A. (2002). Biochemical estimation of RNA and

J. Adv. Lab. Res. Biol. 82