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107
Synthesis and Insecticidal Activity of Some Thiacloprid
Derivatives, against the Helicoverpa armigera (Hub)
108
CHAPTER-IV
Synthesis and Insecticidal Activity of Some Thiacloprid Derivatives, against the
Helicoverpa armigera (Hub)
4.1 Introduction
All chloronicotinyl compounds showed systemic and broad-spectrum efficacies
against sucking and chewing insects.1-2 The active component of this compound contains
6-chloropyridine present in the natural analgesic epibatidine, which was isolated from the
Ecuadorian frog, Epipedobates tricolor.3 Neonicotinoids are a sets of nicotine-based
insecticides that include the chemicals imidacloprid, clothianidin, acetamiprid,
thiacloprid, thiamethoxam, dinotefuran and nitenpyram, which possess either a
nitromethylene, nitroimine or cyanoimine groups.4-5 Thiacloprid, is targeted to control
sucking and biting insects in pome and stone fruits, small berries, cotton, vegetables,
sugar beet, potatoes, rice and ornamentals, for the control of Aphids, Whiteflies, Leaf-
hoppers, Plant-hoppers, Thrips, some Micro-lepidoptera and a number of coleopteran
pests.6-8
4.2 Summary of the work
Thiacloprid (1) was converted to the corresponding amide (2) and acid (4) by the
alkaline hydrolysis. Amide (2) was reacted with phenyl isocynate to give the
corresponding urea derivatives (3). The esterification of acid (4) yielded an ester, (5)
which was converted to the hydrazide (6) with hydrazine hydrate. The thiacloprid was
reduced by stannous chloride in dry HCl and hydrolyzed to aldehyde (7) and further
reduced by Na in alcohol to yield primary amine (8) (Scheme-1).
The efficacy of these compounds was studied on Helicoverpa armigera (Hub), the
compound thiacloprid (1.23-1.40%), 6 (1.03-2.08%), 3 (1.25-1.50%), 7 (1.06-1.83%), 8
(0.85-1.73%) and novaluron (1.51-1.61%) at 200-600 mg litre-1 concentrations, showing
higher glycogen content. The amount of protein, lipid and chitin were measured and
compared with their chitin inhibition. Thiacloprid showed the highest mortality (50%, 4th
109
day) at 600 mg litre-1 concentration. These newly synthesized thiacloprid derivatives may
serve as the potent insect chitin synthesis inhibitors.
4.3 Experimental procedure
4.3.1 Synthetic procedure
All the synthesized compounds were analyzed for their purity by TLC. Melting points
were uncorrected. Structures of the synthesized compounds have been confirmed on the
basis of LC-MS/MS (Agilent 1200 series HPLC system (Agilent technologies, USA)
hyphenated to an API-4000 Q-Trap mass spectrometer, Applied Biosystems, MDS Sciex,
Canada) and FT-IR (Perkin Elmer Spectrum one) spectral analysis.
3-[(6-Chloropyridin-3-yl)methyl]-1,3-thiazolidin-2- ylidenecyanamide (Thiacloprid)
(1): Commercially available thiacloprid has been further purified by centrifugation
method in water and again recrystallized from methanol to get a highly pure thiacloprid.
IR (KBr) νmax: 3434, 2959, 2924, 2185, 1582 cm-1. (Fig. No. 1) 1H NMR (CDCl 3) δ: 3.40(t, J=7.5 Hz, CH2), 3. 80(t, J=7.5 Hz, CH2), 4.62(s, CH2),
7.36(d, J=8 Hz, PyH), 7.67(dd, J1=8 Hz, J2=2.5 Hz, PyH), 8.32(s, PyH) ppm. (Fig. No. 2)
LCMS/MS (ESI, m/z): 252(M+) 226, 211, 188, 157, 138, 132. (Fig. No. 3)
1-{3-[(6-Chloropyridin-3-yl)methyl]-1,3-thiazolidin -2-ylidene}urea (2):
The solution of (10.08 gm., 0.04 mole) thiacloprid in 30 ml of 6N NaOH and ethanol,
refluxed for about 6-8 hrs., cooled and neutralized with H2SO4 to get a white solid which
was, filtered and dried to amorphous white powder, m.p. 1850C, yield 80%.
.
IR (KBr) νmax: 3400, 2924, 2853, 1685 cm-1. (Fig. No. 4)
LCMS/MS (ESI, m/z): 269.80(M+) 257, 2467, 228, 181, 169, 156, 138, 117.
(Fig. No. 5)
110
N-{3-[(6-Chloropyridin-3-yl)methyl]-1,3-thiazolidin -2-ylidene}-N'phenyldicarbonim
idic diamide (3): The mixture of a thiacloprid-amide (2) (10.80 gm., 0.04 mole) and
phenyl isocyanate (4.76 gm., 0.004 moles) in 30 ml methanol was heated on a water
bath for 5 hrs., cooled to get a white amorphous product, m.p. 2100C yield 75%.
LCMS/MS (ESI, m/z): 389(M+), 380, 341, 331, 304, 277, 261, 227, 213, 209.
(Fig. No. 6)
3-[(6-Chloropyridin-3-yl)methyl]-1,3-thiazolidin-2- ylidene}carbamic acid (4):
Thiacloprid-amide (2) (10.80 gm., 0.04 moles) was refluxed with 30 ml of 6N NaOH and
ethanol for about a 13 hrs., cooled and neutralized with HCl and the separated solid was
filtered to get light yellowish amorphous powder, m.p. 2650C, yield 80%.
IR (KBr) νmax: 3396, 1672 cm-1. (Fig. No. 7)
LCMS/MS (ESI, m/z): 271(M+), 254, 228, 211,192, 186, 165, 139. (Fig. No. 8)
Methyl{3-[(6-chloropyridin-3-yl)methyl]-1,3-thiazol idin-2-ylidene}carbamate (5):
To a solution of thiacloprid acid (4) (10.84 gm, 0.04 mole) and methanol 30 ml conc.
H2SO4 (4 ml) was added drop wise and the reaction mixture refluxed on a water bath for
about a 15 hrs., cooled and neutralized with 10% sodium carbonate. After the completion
of the reaction, the mixture was neutralized to get light brown sticky compound, m.p.
2600C, yield 60%.
IR (KBr) νmax: 2927, 1750, 1660 cm-1. (Fig. No. 9)
LCMS/MS (ESI, m/z): 285(M+), 270, 240, 237, 222, 217, 169, 160. (Fig. No. 10)
N-{3-[(6-Chloropyridin-3-yl)methyl]-1,3-thiazolidin -2-ylidene}hydrazinecarboxamid
e (6) : The mixture of ester (11.40 gm., 0.04 mole) and hydrazine hydrate (2.5 gm. 0.05
mole) in methanol (30 ml.) was heated on a water bath at 700C for 5 hrs., cooled, to get
light yellowish solid, the solid which was recrystallized from DMF, m.p. 2800C, yield
70%.
111
IR (KBr) νmax: 3422, 2924, 2854, 1645 cm-1. (Fig. No. 11)
LCMS/MS (ESI, m/z): 285(M+), 284, 283, 282, 281, 279, 275, 273. (Fig. No. 12)
N-{(3-[(6-Chloropyridin-3-yl)methyl]-1,3-thiazolidi n-2-ylidene}formamide(7):
Thiacloprid (2.52 gm, 0.01 mole) and SnCl2 (1.896 gm, 0.01mole) was added in THF,
through which dry HCl, gas is passed in stirring conditions, to precipitated out the salt
within 10 hrs. The salt is further hydrolyzed with water to get desired aldehyde, light
brown amorphous solid, yield 65%.
LCMS/MS (ESI, m/z): 256(M+), 254, 228, 211, 192, 186, 165, 139. (Fig. No. 13)
N-{3-[(6-Chloropyridin-3-yl)methyl]-1,3-thiazolidin -2-ylidene}methanediamine (8):
To the solution of thiacloprid (2.52 gm., 0.01 mole) in absolute ethanol, sodium metal
(1.10 gm., 0.05 mole), was added and the reaction mixture refluxed for 5 hrs. The
solvent was removed under reduced pressure to yield an amine, reddish brown semi
solid, yield 60%.
LCMS/MS (ESI, m/z): 256(M+), 247, 225, 215, 195, 181, 157, 117, 100. (Fig. No. 14)
112
N
S N ClN
N
N
S N ClN
HO
N
S N ClN
NH2O
N
S N ClN
OHO
N
SN ClN
NH
NH2
O
N
S N ClN
NH2
N
S N ClN
OO
N
S
NCl
N
NHO
NHO
1
2
3
4
56
78a
b
c
d
e
fg
a) NaOH/H2O, Reflux, b) NaOH/H2O, Reflux, c) H2SO4/MeOH,
d)NH2.NH2.H2O/MeOH, e) PhNCO, f) SnCl2/dry HCl, H2O2, g) Na/C2H5OH (dry).
Scheme-1.
4.3.2 Biological Screening
4.3.2.1 Insecticidal activity against test compound was performed on H. armigera
Second instars larvae of H. armigera (Hub) were collected from chick pea field and
maintained in the laboratory using separate containers for each larva. The standard
solutions of the above compounds were prepared by dissolving them in 0.5 ml DMF with
9.5 ml, 1% Tween-20 solution to make final concentrations 200, 400 and 600 mg litre-1.
The treatments of these compounds were given through the oral route with treated food
113
for 3 days on the daily basis, along with untreated control (0.5 ml DMF + 9.5 ml, 1%
Tween-20) and standard insecticide thiacloprid. The mortality data were collected, up to
14th days, with every two days intervals (Table-1).
4.3.2.2 Cuticle biochemical assay against H. armigera
The treatments of these compounds were done by the above method using two
untreated controls, (0.5 ml DMF + 9.5 ml, 1% tween-20 solution and water), standard
insecticide thiacloprid and standard insect chitin synthesis inhibitor novaluron. The
treated insects were collected after 72 hrs. of the treatment and frozen at -80oC. The outer
cuticular layer was separated out from the frozen insects and then oven dried at 1000C.
These were then ground to a fine powder. This procedure was repeated several times to
get sufficient sample for further analysis. Using a standard procedures, the protein
contents was estimated by Lowry method,9-11 lipids by Vanillin,12-14 carbohydrate by
Anthrone reagent15,16 and chitin by 4-dimethylaminobenzaldehyde method (Table-2).17-19
4.3.3 Statistical Analysis
Data from the experiments were square-root transformed (x + 0.5) and then subjected
to the analysis of variance.20 Means were separated by least significant difference (LSD)
(P = 0.05) and are presented in Tables-1 and 2.
114
Table-1. Efficacy of new synthesized compounds against H. armigera (Hub).
Concentrations Mortality, days after treatments Compound No.
mg/lit. 2day* 4day* 6day* 8day* 10day* 12day* 14day*
200 10.00
(18.43)
30.00
(32.30)
33.33
(34.22)
33.33
(33.22)
33.33
(34.22)
36.66
(36.15)
36.66
(36.15)
400
16.60
(23.86)
46.66
(42.29)
46.66
(42.29)
46.66
(42.29)
46.66
(42.29)
46.66
(42.29)
50.00
(45.00)
Thiacloprid
N
S N ClN
N
1. 600
16.60
(19.92)
50.00
(45.00)
50.00
(45.00)
50.00
(45.00)
50.00
(45.00)
50.00
(45.00)
50.00
(45.00)
200
03.30
(6.14)
23.33
(24.15)
30.00
(33.00)
30.00
(33.00)
33.33
(35.00)
33.33
(35.01)
36.66
(36.93)
400
10.00
(15.00)
10.00
(15.00)
26.66
(30.78)
30.00
(33.00)
36.66
(36.93)
40.00
(38.85)
40.00
(38.85)
N
S N ClN
NH2O
2.
600
13.30
(17.21)
20.00
(25.37)
26.66
(30.29)
30.00
(33.00)
33.33
(35.22)
40.00
(39.15)
46.66
(43.08)
200
26.60
(30.29)
30.00
(32.22)
36.66
(37.14)
40.00
(39.15)
43.33
(41.15)
43.33
(41.15)
43.33
(41.15)
400
03.30
(6.15)
03.33
(6.15)
16.66
(23.85)
33.33
(34.99)
33.33
(35.01)
33.33
(35.01)
33.33
(35.01)
N
S N ClN
OHO
4. 600
06.60
(12.29)
13.33
(17.71)
20.00
(26.56)
30.00
(32.99)
30.00
(33.00)
30.00
(33.00)
30.00
(33.00)
200
13.30
(13.08)
30.00
(25.37)
20.00
(25.37)
23.33
(28.07)
26.66
(30.78)
30.00
(33.00)
33.33
(35.01)
400
06.60
(12.29)
16.66
(19.22)
20.00
(25.37)
30.00
(33.22)
33.33
(34.14)
36.66
(36.15)
40.00
(38.36)
N
S N ClN
OO
5.
600
10.00
(15.00)
13.33
(17.22)
20.00
(26.07)
26.66
(30.99)
26.66
(30.99)
26.66
(30.99)
26.66
(30.99)
200
10.00
(11.07)
10.00
(11.07)
26.66
(90.88)
30.00
(32.99)
33.33
(35.22)
36.66
(37.22)
43.33
(41.07)
400
00.00
(00.00)
13.33
(17.22)
36.66
(31.92)
43.33
(34.08)
46.66
(42.29)
46.66
(42.29)
46.66
(42.29)
N
SN ClN
NH
NH2
O
6. 600
00.00
(00.00)
10.00
(18.44)
13.33
(21.15)
16.66
(23.36)
20.00
(25.37)
23.33
(28.08)
30.00
(31.92)
115
* Mean of three replications, Figures in parenthesis are arcsine transformed values.
200
06.60
(12.29)
16.66
(19.92)
20.00
(22.14)
20.00
(22.14)
23.33
(24.15)
30.00
(27.99)
33.33
(34.14)
400
16.60
(15.00)
23.33
(23.07)
30.00
(31.22)
30.00
(31.22)
33.33
(33.93)
36.66
(36.15)
36.66
(36.15) N
S
NC l
N
N HO
NHO
3.
600
13.30
(17.21)
16.66
(23.36)
20.00
(26.07)
20.00
(26.07)
23.33
(28.78)
23.33
(28.78)
30.00
(33.00)
200
06.60
(12.29)
16.66
(23.85)
23.33
(28.78)
30.00
(33.00)
30.00
(33.00)
36.66
(36.93)
43.33
(41.07)
400
10.00
(17.19)
16.66
(19.22)
26.66
(30.78)
30.00
(33.00)
36.66
(37.22)
36.66
(12.22)
36.66
(37.22)
N
S N ClN
HO
7. 600
03.30
(6.15)
13.33
(17.71)
20.00
(22.14)
26.66
(26.07)
36.66
(32.19)
40.00
(34.92)
40.00
(34.92)
200
10.00
(18.44)
30.00
(31.92)
40.00
(38.40)
43.33
(41.07)
43.33
(41.07)
43.33
(14.44)
46.66
(47.22)
400
03.30
(18.44)
20.00
(26.07)
26.66
(30.78)
30.00
(32.71)
33.33
(34.92)
43.33
(61.17)
43.33
(40.78)
N
S N ClN
NH2
8. 600
03.30
(6.15)
10.00
(18.44)
16.66
(28.33)
23.99
(28.08)
26.66
(30.29)
36.66
(36.15)
36.66
(36.15)
0.5mlDMF+9.5ml
1% Tween-20.
-
00.00
(00.00)
10.00
(6.15)
06.66
(12.37)
10.00
(15.00)
10.00
(15.00)
10.00
(15.00)
13.33
(17.71)
Water -
03.30
(6.15)
10.00
(6.15)
10.00
(15.04)
16.66
(19.92)
16.66
(19.92)
16.66
(19.92)
20.00
(22.14)
116
Table 2. Percent variation in protein, lipid, glycogen and chitin contents on cuticle of H.
armigera after treatment with synthesized compounds.
Concentrations in Compound No. mg/lit. Protein*%
Lipid*%
Glycogen*%
Chitin*%
200
15.00
(22.78)
04.51
(12.28)
1.40
(6.79)
1.28
(6.53)
400
22.44
(28.26)
04.43
(12.15)
1.23
(6.29)
1.88
(7.92)
Thiacloprid
N
S N ClN
N 1. 600
32.44
(34.70)
03.03
(10.02)
1.28
(6.48)
5.35
(13.38)
200
15.66
(23.29)
03.35
(10.54)
0.80
(5.06)
4.85
(12.79)
400
19.00
(25.83)
02.70
(9.50)
1.08
(4.62)
2.98
(10.02)
N
S N ClN
NH2O
2. 600
25.22
(30.13)
02.70
(9.46)
1.08
(6.01)
5.31
(13.35)
200
19.66
(26.29)
02.75
(9.57)
1.18
(6.28)
4.00
(11.58)
400
09.66
(17.98)
02.56
(9.27)
1.08
(6.01)
2.88
(9.79)
N
S N ClN
OHO
4. 600
15.22
(22.89)
04.55
(12.33)
1.08
(5.92)
3.11
(10.16)
200
13.22
(21.30)
02.23
(8.65)
1.10
(5.98)
2.21
(8.57)
400
16.00
(23.55)
03.05
(10.08)
1.13
(6.11)
1.28
(6.55)
N
S N C lN
OO
5. 600
19.44
(26.13)
02.66
(9.45)
1.30
(6.44)
2.30
(8.78)
200
16.44
(23.91)
02.16
(8.52)
1.03
(5.93)
1.21
(6.54)
400
11.88
(20.12)
03.15
(10.24)
2.08
(8.31)
4.45
(12.20)
N
S N C lN
N H
NH 2
O
6.
600
12.22
(20.41)
02.16
(8.46)
1.21
(6.33)
5.88
(5.88)
117
200
19.66
(26.27)
03.38
(10.59)
1.40
(6.83)
2.11
(8.46)
400
18.44
(25.41)
03.33
(10.55)
1.50
(7.10)
2.96
(9.87) N
S
NCl
N
NHO
NHO
3. 600
15.22
(22.94)
02.21
(8.59)
1.25
(6.39)
5.22
(13.22)
200
11.22
(19.55)
03.31
(10.47)
1.06
(5.86)
3.25
(10.46)
400
17.22
(24.48)
04.66
(12.47)
1.68
(7.46)
2.05
(8.33)
N
S N ClN
HO
7. 600
10.44
(18.84)
02.86
(9.79)
1.83
(7.83)
2.30
(8.78)
200
09.66
(18.05)
02.80
(9.63)
1.73
(7.60)
2.95
(9.98)
400
14.00
(21.94)
03.70
(11.09)
1.53
(6.96)
3.66
(11.04)
N
S N ClN
NH2 8.
600
16.22
(23.64)
02.83
(9.67)
0.85
(5.26)
2.58
(9.34)
0.5mlDMF+9.5ml 1%
Tween-20.
-
14.66
(22.52)
01.88
(7.85)
1.08
(6.02)
2.55
(9.21)
Water
-
12.44
(20.61)
02.15
(8.41)
1.10
(5.95)
6.06
(14.30)
200
20.44
(26.87)
02.76
(9.62)
1.51
(7.06)
3.06
(10.07)
400
12.44
(20.61)
03.88
(11.27)
1.56
(7.25)
4.26
(11.92)
Novaluron
600
11.66
(19.96)
02.66
(8.67)
1.61
(7.28)
6.25
(14.50)
SE±
CD at 5% level
0.73
1.46
0.38
0.76
0.60
1.20
0.22
0.44
* Mean of three replications, Figures in parenthesis are arcsine transformed values,
underline values showed maximum or minimum.
118
4.4 Results and Discussion
4.4.1 Effect on Mortality
Synthesized compounds were tested against H. armigera and the results indicated that
the mortality of H. armigera, postulated no significant differences among the treatments
of the compounds. Thiacloprid has recorded highest mortality (50%, 4th day) at 600 mg
litre-1 concentration and significantly superior over the rest of the compounds, which was
linearly increased from 2 to 14th days. The compound sl. no. 2 (20.00-46.66%) at 600 mg
litre-1 and 8 (40.00%, 6th day) at 300 mg litre-1 concentrations, also observed the highest
mortality and linearly increased from 4th to 14th days next to the thiacloprid. The
compound sl. no. 7 recorded highest mortality (20.00-40.00%) at 600 mg litre-1
concentration, after 6th days and linearly increased up to 14th days. The lowest mortality
(13.33-20.00%, 14th day) was observed in both untreated controls (Table-1).
4.4.2 Chitin inhibitory activity
Some of the compounds showed higher glycogen content in their cuticle, it may be
due to the result of chitinase activity. The binding site interaction in chemical or atomic
resolution has been defined by comparative chemical and structural neurobiological
approach. This may be due to the change of functional cyno or nitro imino groups which
enhances the hydrophobic nature, there by nicotinic receptor further continuing study to
discover novel nicotinic insecticides with unique biological properties.21 In present
investigation, we studied the biochemical properties of treated insect cuticle due to the
change in the functionality of thiacloprid i.e. cyno group. The insect body utilizes the
glycogen for various metabolic activities as well as physiological functions such as
energy source and substrate for the chitin formation. It is suggested that the possibility of
the synthesis of carbohydrates at the expense of glycogenic amino acids. However, the
simultaneous depletion of total and neutral lipids rather intriguing in view of the
controversy regarding the conversion of lipids to carbohydrates.22 The insect cuticle
protein is a glycoprotein, one or more separate polysaccharides composed of these neutral
sugars. The chitin is a copolymer or a homopolymer of N-acetylglucosamine with
heterosaccharide branch points.23 Some insecticides affecting the energy of oxidative
phosphorylation in mitochondria, for a long time are already used the insect-acaricides
119
influencing the hormonal process of moulting and metamorphosis or inhibiting the chitin
synthesis.24 The above observations clearly showed that the chitinase activity because
treatment of these compounds, i.e. effect on cuticle glycogen, lipid, protein and chitin
concentrations. This data get characterize the properties of neonicotinoid compounds
which competes with its action on nAchR binding site.
The deformities were observed on the outer cuticle layer of H. armigera, after
treatment with insect chitin synthesis inhibitor (novaluron) and newly synthesized
compounds. These compounds may be actively involved on the biochemical process of
insect cuticle, which is an important protecting layer of insect body, its damage caused
deformities in an insect body. Due to the effect caused on outer cuticle layer, insect
become susceptible to the environmental hazards.
4.4.3 Effect of synthesized compounds on protein, lipid, carbohydrate and chitin
contents
The proteins, lipids, carbohydrates and chitins, are the major components of the insect
cuticle. It correlates with these relative variations of such components, due to the
treatments of the synthesized compounds sl. no. 2 to 8 (Table-2). For the treatment of
compound sl. no. 2 lipid content (2.70-3.35%) and protein (15.66-25.22%) content were
found to be high, as compared to rest of the compounds. The treatment of compound sl.
no. 4 the lipid content is observed (2.75-4.55%), while protein content is (9.66-19.66) as
compared to other compounds. The treatment of compound sl. no. 3 showed the highest
total glycogen (1.25-1.50%) and chitin (2.11-5.22%) contents as compared to the rest of
the compounds. This compound contain urea group similar to novaluron, responsible for
insect chitin synthesis inhibition activity. The treatment of compound sl. no. 5 showed
low chitin (1.28-2.30%), while protein (13.22-19.44%), lipid (2.23-3.05%) and glycogen
(1.10-1.30%) variation as compared to remaining test compounds. For the treatment of
compound sl. no. 6 lipids (2.16-3.15%) and chitin (1.21-5.88%) content is very low,
while glycogen contents observed to be very high (1.03-2.08%) as compared to the other
compounds. It may be due to the presence of hydrazino group responsible for the insect
chitin synthesis inhibition activity.
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The treatment of compound sl. no. 7 protein (10.44-17.22%) and chitin (2.05-3.25%)
content was found very low, while lipid (2.86-4.66%) and glycogen content (1.06-1.83%)
observed very high. The treatment of compound 8 showed the glycogen content (0.85-
1.73%) very high as compared to the treatments of other compounds. It may be due to the
presence of amino group responsible for insect chitin synthesis inhibition. The treatment
of untreated controls, showed very low lipid content (1.88%), while the treatment of
water, observed to be low lipid (2.15%) and chitin (2.58-3.66%) content i.e. high as
compared to the treatment of rest of the compounds. Thiacloprid treated insect cuticle
showed relatively higher amount of protein (15.00-32.44%), lipid (03.03-04.51%),
glycogen (1.23-1.40%) and chitin (1.28-5.35%) than other compounds. Novaluron treated
insect cuticle showed very high amount of glycogen (1.51-1.61%) as compared to the
other compounds. Here, we estimated chitin in terms of total glycogen content as a
hydrolyzed product of chitin, but we have taken total cuticle mass for estimation of
chitin, having both glycogen and chitin as a result of which % chitin was estimated
without subtracting cuticle glycogen content which may enterfere the accuracy of a
result.
After comparative studies among these compounds, the percent variations of protein,
lipid, glycogen and chitin contents on the cuticle layer of H. armigera was studied.
4.4.3.1 Protein Analysis
The protein analysis, after the treatment of thiacloprid (15.00-32.44%) and compound
sl. no. 2 were observed very high amount of protein (15.66-25.22%). The treatment of
compound sl. no. 4 (9.66-19.66%) and 3 (15.22-19.66%) showed high amount of protein.
While the treatment of compound sl. no. 8 (9.66-16.22%) and untreated controls (DMF
and Tween-20), exhibited medium value (14.66%). The treatment of untreated control
(water) (12.44%), novaluron (11.66-20.44%), compound sl. no. 6 (11.88-16.44%) and
compound sl. no. 7 (10.44-17.22%) showed moderate protein content.
4.4.3.2 Lipid Analysis
After lipid analysis, it is observed that because of the treatment of novaluron (2.66-
3.88%), compound sl. no. 4 (2.75-4.55%), 7 (2.86-3.31%) and 8 (2.80-3.70%) observed
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very high amount of lipid, while the treatment of compound sl. no. 3 showed medium
amount of lipid contents (2.21-3.38%). The treatment of compound sl. no. 5 (2.23-3.05%)
showed low amount of lipid as compared with the treatment of untreated controls (12.44-
14.66%).
4.4.3.3 Analysis of Glycogen
The analysis of glycogen contents it has been observed that for the treatment of
thiacloprid (1.23-1.40%), novaluron (1.51-1.61%), compound sl. no. 6 (1.03-2.08%), 3
(1.25-1.50%), 7 (1.06-1.83%) and 8 (0.85-1.73%) showed the highest amount of
glycogen contents which may be due to the non utilization of available cuticle glycogen
for chitin synthesis, while the treatment of compound sl. no. 5 (1.10-1.30%) showed
medium amount, where as the treatment of compound sl. no. 2 (0.80-1.08%) exhibited
very low amount. The treatment of untreated controls showed the low amount of
glycogen (1.08-1.10%). The treatment of compound sl. no. 4 treated species is taken as
an average amount of glycogen content (1.08-1.18%).
4.4.3.4 Analysis of chitin
The highest amount of chitin in cuticle was observed after treatment with novaluron
(3.06-6.25%), water (6.06%) and compound sl. no. 6 (1.21-5.88%). While the treatment
of the compound sl. no. 2 (2.98-5.31%) and 3 (2.11-5.22%) higher amount of chitin was
observed. The treatment of compound sl. no. 4 (2.88-4.00%) and 8 (2.58-3.66%) showed
medium amount of chitin content. The treatment of thiacloprid (1.28-1.88%) at 200 to
400 mg litre-1 concentration and compound sl. no. 5 (1.28-2.30%) showed very less
amount of chitin contents, however thiacloprid exceptionally showed very high amount
of chitin content (5.35%) at 600 mg litre-1 concentration. The treatment of compound sl.
no. 7 (2.05-3.25%) showed medium amount of chitin over the rest of the treated
compounds.
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4.5 Conclusions
The above results it is clearly showed that H. armigera contain higher amount of
glycogen in cuticle after treatment with the compounds sl. no. 6, 3, 7, 8, novaluron and
thiacloprid. From these observations and comparison with novaluron treatment, we can
conclude that the available glycogen is not utilized for the synthesis of chitin. Therefore,
the synthesized compound sl. no. 6, 3, 7 and 8 are of potent insect chitin synthesis
inhibitors as effective as novaluron available in the market. In addition to chitinolytic
activities the treatment of compound sl. no. 8 showed good mortality and can be used as a
leading compound for the development of novel insecticide. Secondly, it can also be
concluded that the concentrations of cuticle protein, lipid, glycogen and chitin are inter
linked with each other, observed by varying the concentrations. Their profound effects
are observed which caused the cuticle deformities and insects become deformed to
perform their normal functions leading to death.
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Fig. No.15. Healthy Helicoverpa armigera is used for treatment of synthesized chemicals
as a test insect.
Fig. No.16. After treatment of synthesized chemicals Helicoverpa armigera larva
( weakened)
