BMC Complementary and

Alternative Medicine BioMed Central

Research article Open Access

Mosquito larvicidal activities of Solanum villosum berry extract
against the dengue vector Stegomyia aegypti
Nandita Chowdhury, Anupam Ghosh and Goutam Chandra*

Address: Mosquito and Microbiology Research Units, Parasitology Laboratory, Department of Zoology, Burdwan University, West Bengal, India
Email: Nandita Chowdhury - [email protected]; Anupam Ghosh - [email protected];
Goutam Chandra* - [email protected]
* Corresponding author

Published: 3 April 2008 Received: 17 October 2007

Accepted: 3 April 2008
BMC Complementary and Alternative Medicine 2008, 8:10 doi:10.1186/1472-6882-8-10
This article is available from: http://www.biomedcentral.com/1472-6882/8/10
© 2008 Chowdhury et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Vector control is facing a threat due to the emergence of resistance to synthetic
insecticides. Insecticides of botanical origin may serve as suitable alternative biocontrol techniques
in the future. Although several plants have been reported for mosquitocidal activity, only a few
botanicals have moved from the laboratory to field use, because they are poorly characterized, in
most cases active principals are not determined and most of the works are restricted to
preliminary screening. Solanum villosum is a common weed distributed in many parts of India with
medicinal properties, but the larvicidal activity of this plant has not been reported so far.
Methods: Aqueous and polar/non-polar solvent extract of fresh, mature, green berries of S.
villosum was tested against Stegomyia aegypti, a common vector of dengue fever. A phytochemical
analysis of chloroform:methanol extract was performed to search for the active toxic ingredient.
The lethal concentration was determined (log probit analysis) and compared with Malathion. The
chemical nature of the active substance was also evaluated following ultraviolet-visual (UV-Vis) and
infrared (IR) analysis.
Results: In a 72 hour bioassay experiment with the aqueous extract, the highest mortality was
recorded in 0.5% extract. When the mortality of different solvent extracts was compared, the
maximum (p < 0.05) mortality was recorded at a concentration of 50 ppm of chloroform:methanol
extract (1:1, v/v). The larvicidal activity was lower when compared with the chemical insecticide,
Malathion (p < 0.05). Results of regression analysis revealed that the mortality rate (Y) was
positively correlated with the period of exposure (X) and the log probit analysis (95% confidence
level) recorded lowest value (5.97 ppm) at 72 hours of exposure. Phytochemical analysis of the
chlororm:methanol extract reported the presence of many bioactive phytochemicals. Two toxic
compounds were detected having Rf = 0.82 (70% and 73.33% mortality in 24 and 48 hours,
respectively) and Rf = 0.95 (40% and 50% mortality in 24 and 48 hours, respectively). IR analysis
provided preliminary information about the steroidal nature of the active ingredient.
Conclusion: S. villosum offers promise as potential bio control agent against S. aegypti particularly
in its markedly larvicidal effect. The extract or isolated bioactive phytochemical could be used in
stagnant water bodies for the control of mosquitoes acting as vector for many communicable
diseases.

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Background rial through Whatman No 1 filter paper. Required

Mosquitoes transmit several public health problems, such concentrations of aqueous extracts were prepared by mix-
as malaria, filariasis, dengue and Japanese encephalitis, ing the crude extract with a suitable amount of sterilized
causing millions of deaths every year [1]. Stegomyia aegypti distilled water.
(= Aedes aegypti) is a vector for an arbovirus responsible for
dengue fever, dengue haemorrhagic fever and dengue Preparation of plant extracts in different solvent systems
shock syndrome, and with unusual manifestations such as We harvested 25 g of fresh, mature berries, which were
central nervous system involvement [2,3]. About two- rinsed with distilled water and dried in a shed. The dried
fifths of the world's populations are at risk of catching berries were put in a Soxhlet apparatus and the plant
dengue [4-6]. extracts were prepared using five solvents, namely petro-
leum ether, benzene, chloroform:methanol (1:1, v/v),
Mosquitoes in the larval stage are attractive targets for pes- acetone and absolute alcohol, applying one after another
ticides because they breed in water and, thus, are easy to (extraction period 72 hour for each solvent and the tem-
deal with them in this habitat. The use of conventional perature was < 40°C). The extracts were collected sepa-
chemical pesticides has resulted in the development of rately and the column of the Soxhlet apparatus was
resistance [7,8], undesirable effects on non-target organ- washed with 200 ml of water and 100 ml of a similar sol-
isms and fostered environmental and human health con- vent as an eluent after each type of solvent extraction pro-
cerns [9]. The use of herbal products is one of the best cedure. The eluted materials and each type of extract were
alternatives for mosquito control. The search for herbal concentrated in combination at 40°C to 100 ml of extract
preparations that do not produce any adverse effects in by evaporation in a rotary evaporator. Then each of the
the non-target organisms and are easily biodegradable extracts was filtered, solvents were evaporated and the
remains a top research issue for scientists associated with solid residues were weighed and then dissolved in a suita-
alternative vector control [10]. ble amount of sterilized distilled water for the formula-
tion of graded concentrations. The total yield of each
Solanum villosum (Solanaceae: Solanales), commonly extract from 25 g of berries was as follows: petroleum
known as red-fruit nightshade, is widely distributed in ether extract, 1.26 g; benzene extract, 2.38 g; chloro-
many parts of India. This is an Ayurvedic herb with multi- form:methanol (1:1, v/v) extract, 4.33 g; acetone extract,
ple medicinal properties [11]. The objective of the present 3.00 g; and absolute alcohol extract 2.36 g.
study was to examine the larvicidal activity of aqueous,
polar and non-polar solvent extracts of the green berries of Larvicidal bioassay
this plant against the larvae of S. aegypti mosquitoes and The larvicidal bioassay followed the World Health Organ-
to gather preliminary information about the nature of the ization (WHO) standard protocols [12] with slight modi-
active ingredient responsible for larval mortality. fications. Each of the concentrations of aqueous berry
extract (0.1–0.5%) was transferred into sterile glass Petri
Methods dishes (9 cm diameter/150 ml capacity). Ten third instar
Test mosquitoes larval form of S. aegypti were separately introduced into
The present study was conducted at Burdwan (23° 16' N, different Petri dishes containing graded concentrations
87° 54' E), West Bengal, India, during June-August 2006. and the mortality were recorded after 24, 48 and 72 hours
Larvae of S. aegypti were obtained from a laboratory col- of the exposure period. The data of mortality in 48 and 72
ony maintained in the Mosquito Research Unit, Depart- hours were expressed by the addition of the mortality at
ment of Zoology, The University of Burdwan. The colony 24 and 48 hours, respectively. Dead larvae were identified
was kept free from exposure to pathogens, insecticides or when they failed to move after probing with a needle in
repellents and maintained at 25–30°C. The larvae were the siphon or cervical region. The experiments were repli-
fed on a powdered mixture of dog biscuits and dried yeast cated three times and conducted under laboratory condi-
powder at a ratio of 3:1. The adult colony was provided tions at 25–30°C and 80–90% relative humidity. Similar
with 10% sucrose solution and 10% multivitamin syrup, types of bioassay were conducted with different polar and
and was periodically blood-fed on restrained rats. non-polar solvent extracts (concentrations of 50, 25 and
15 ppm) of the green berries and with a chemical insecti-
Preparation of aqueous extracts cide, Malathion, on third instar larval forms, and chloro-
Fresh, mature, green berries of S. villosum were randomly form:methanol (1:1, v/v) extract on first and fourth instar
harvested during the study period from plants growing on larval forms. Larval toxicity was also tested according to
the outskirts of Burdwan. All the berries were initially similar methodologies using the bioactive substances
rinsed with distilled water and dried on a paper towel. The (from chloroform:methanol extract) isolated from thin-
crude extracts were prepared by grinding the plant mate- layer chromatographic (TLC) plates.
rial in a mortar and pestle and passing the ground mate-

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Phytochemical analysis larvicidal potentiality of the plant extracts. A Student's t-

The phytochemical analysis was carried out using the test was performed to find the significance between the
chloroform:methanol extract (as it exhibited highest mor- concentration of plant extract and mortality at different
tality against S. aegypti larvae) of the green berries of S. vil- periods with different instars. Statistical analysis of the
losum using the standard methods of Harbone [13] and experimental data was performed using the computer
Stahl [14]. One or two drops of the chloroform:methanol software Statplus 2006 and MS EXCEL 2002 to find the
extract were applied (using a capillary tube) to the bottom LC50, regression equations (Y = mortality; X = concentra-
of each of the pre-coated and pre-heated (100°C for 30 tions) and regression coefficient values.
minutes) glass plates (eight glass plates), which were pre-
pared with silicagel G using Unoplan coating apparatus Results
(Shadon, London). After 5 minutes of drying, each of the The results of the present study indicate that the mortality
plates was placed in the separate glass chamber for TLC rates at a 0.5% concentration were highest amongst all
analysis, with different solvent systems as the mobile concentrations of the aqueous extracts tested for larval
phase. After the movement of solvent at the top of the mortality and it was significantly higher (p < 0.05) than
plates, each plate was removed from the glass chamber the mortality rates at 0.1%, 0.2%, 0.3% and 0.4% concen-
and separately air-dried. After 10 minutes each of plates tration of aqueous plant extract at 24, 48 and 72 hours of
was sprayed with a different spraying reagent for the iden- exposure (Table 1). The mortality of the same instar larval
tification of appropriate phytochemical. The phytochem- form with different polar and non-polar solvent extracts is
icals included in the study were sapogenins, steroid, presented in Table 2. The larvicidal potentiality of chloro-
terpenoids, flavonoids, alkaloid, essential oils, phenolics form:methanol (1:1, v/v) extract was further tested for first
and amino acids. A qualitative test was carried out to indi- and fourth instar larvae: the highest mortality was
cate the presence of saponins [15]; the remaining phyto- recorded at 15 ppm for first instar larvae and it is statisti-
chemicals were determined using TLC analysis by the cally more significant (p < 0.05) than both the 5 and 10
application of suitable solvents and spray reagents and, in ppm concentrations and at both 48 and 72 hours of expo-
each case, Rf values were recorded. sure (Table 3).

Ultraviolet-visual and infrared analysis of the active However, no significant difference was recorded for fourth
ingredient instar larvae between 15 and 10 ppm concentrations and
The chloroform:methanol extract of the green berries of S. 15 and 5 ppm concentrations. An absolute mortality
villosum was further chromatogrammed (30 plates) with- (100%) was observed within 24 hours during the expo-
out the application of spraying reagents and each of the sure to the chemical insecticide, Malathion (5 ppm con-
spots showed positive activity were separately scrapped centration). The results of regression analysis revealed that
according to their respective Rf values. Then each of the the mortality rate (Y) is positively correlated with the
spots with their distinguishing Rf value was combined period of exposure (X) having a regression coefficient
(from 30 plates) and undergoes further bioassay experi- close to one in each case (Table 4). The results of log pro-
ment to reveal the nature of active ingredient. As the spots bit analysis (95% confidence level) revealed that LC50
exhibited positive response in Liberman Buchard reagent values gradually decreased with the exposure periods hav-
recorded highest larval mortality during further bioassay
experiments, it undergoes spectral analysis by ultraviolet- Table 1: The larvicidal activity (mean mortality ± standard error)
visual (UV-Vis) and infrared (IR) spectroscopy. The UV- of different concentrations of aqueous extract of the green
Vis analysis was carried out using a UV-1601 PC, SHI- berries of S. villosum on third instar larvae of S. aegypti. Student's
t-test t = 29.42*, 5.5*, 17.0* (between 0.5% and 0.1%) 12.43*,
MADZU spectrophotometer with medium scan speed and 3.32*, 14.0* (between 0.5% and 0.2%) and 1.73*, 4.33*, 4.0*
sampling interval of 0.5 seconds. The IR spectroscopy (between mortality in 0.5% and 0.3% plant extract at 24, 48 and
analysis of the active spot was performed using KBr plates 72 hours bioassay); * denotes significant (p < 0.05); table value =
(JASCO FT-IR Model-420) with a scanning speed of 2 mm 2.92 at five degrees of freedom. M, mortality (%); SE, standard
error.
s-1.
Period of exposure (hours)
All solvents and reagents used were of analytical grade and
purchased from E. Merck, India. The TLC silica gel plates Concentration (%) 24 48 72
(0.25 mm thickness) were prepared and equilibrated with
2% (w/w) of water before use. 0.1 20 ± 5.77 26.67 ± 8.67 30 ± 8.81
0.2 30 ± 7.69 36.67 ± 5.77 40 ± 7.69
Statistical analysis 0.3 60 ± 5.57 70 ± 1.92 73.33 ± 3.84
0.4 66.66 ± 1.92 70 ± 1.92 76.66 ± 5.57
The percentage mortality observed (%M) was corrected 0.5 76.66 ± 1.92 86.66 ± 5.77 90 ± 1.92
using Abbott's formula [16] during the observation of the

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Table 2: Efficacy of different concentrations of polar and non-polar solvent extracts of the green berries of S. villosum on third instar
larvae of S. aegypti. M, mortality (%); S, survivality (%).

Type of solvents Concentrations (ppm) Period of exposure (hours)

24 48 72

M S M S M S

Petroleum ether 50 3.33 96.67 6.67 93.33 13.33 86.67

25 3.33 96.67 3.33 96.67 10 90
15 0 100 3.33 96.67 6.67 93.33
Benzene 50 6.67 93.33 13.33 86.67 23.33 76.67
25 3.33 96.67 6.67 93.33 16.67 83.33
15 3.33 96.67 6.67 93.33 13.33 86.67
Chloroform: methanol 50 70 30 73.33 26.66 76.66 23.33
25 53.33 46.66 56.66 43.33 56.66 43.33
15 40 60 43.33 56.66 43.33 56.66
Acetone 50 6.67 93.33 10 90 13.33 86.67
25 6.67 93.33 10 90 10 90
15 3.33 96.67 6.67 93.33 10 90
Absolute alcohol 50 30 70 36.67 63.63 50 50
25 13.33 86.67 23.33 76.67 33.33 66.67
15 10 90 16.67 83.33 26.67 73.33

ing the lowest value at 72 hours of exposure to third instar in two compounds. The highest mortality (at a concentra-
larvae, followed by first and fourth instar larvae. The tion of 50 ppm) was recorded in the first compound hav-
results of preliminary phytochemical analysis of the chlo- ing Rf = 0.818 (70% and 73.33% in 24 and 48 hours,
roform:methanol extract of the green berries of S. villo- respectively) followed by a second compound having Rf =
sum are presented in Table 5. A qualitative test indicated 0.946 (40% and 50% in 24 and 48 hours, respectively)
the presence of saponins and chromatographic analysis with maximum absorption at 297.50 and 361.00 nm,
revealed the presence of steroids, alkaloids, terpenoids, respectively, during UV-Vis analysis. IR analysis of two
saponins, amino acids, phenolics, flavonoids and essen- compounds and their respective functional groups are
tial oil as major phytochemicals and the absence of the shown in Figures 1 and 2.
sapogenins following the application of different solvent
systems and spraying reagents. When the isolated com- Discussion
pounds from the TLC plates were further bio-assayed Nowadays, mosquito control is mostly directed against
against the third instar larvae, the mortality was recorded larvae and only against adults when necessary. This is

Table 3: The larvicidal potentiality (mean mortality ± standard error) of different concentrations of chloroform:methanol (1:1, v/v)
extract of the green berries of S. villosum and a synthetic insecticide, Malathion, on first and fourth instars larvae of S. aegypti. For first
instar larvae: t = 2.07NS, 3.14*, 7.56* (between 15 and 10 ppm at 24, 48 and 72 hours); t = 5.2*, 26.62*, 13.99* (between 15 and 5 ppm at
24, 48 and 72 hours). For fourth instar larvae: t = 2NS, 1.99NS, 0.91NS (between 15 and 10 ppm at 24, 48 and 72 hours); t = 1.73NS, 1.89NS,
1.82NS (between 15 and 5 ppm at 24, 48 and 72 hours). * denotes significant (p < 0.05); NS, not significant (p > 0.05). Table value = 2.92
at five degrees of freedom. M, mortality (%); SE, standard error.

Instars of mosquito larvae Concentrations (ppm) Period of exposure (hours)

24 48 72

First 15 60 ± 1.92 66.67 ± 5.67 70 ± 2.93

10 40 ± 5.57 43.33 ± 3.84 46.66 ± 3.84
5 40 ± 5.57 40 ± 5.67 53.33 ± 1.92
Malathion (5 ppm) 100 ± 0.00 100 ± 0.00 100 ± 0.00

Fourth 15 40 ± 1.92 43.33 ± 3.84 46.66 ± 5.57

10 33.33 ± 3.84 40 ± 1.92 43.33 ± 5.56
5 30 ± 5.57 33.33 ± 2.93 36.67 ± 2.84
Malathion (5 ppm) 100 ± 0.00 100 ± 0.00 100 ± 0.00

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Table 4: Log probit analysis of the larvicidal activity of chloroform:methanol extract of the green berries of S. villosum on different
instar larvae of S. aegypti. LC, lethal concentration; R, coefficient of regression equations.

Type of instars of Period of bioassay Regression equations R2 LC50 values (ppm) Lower and upper
mosquito larvae (hours) fiducidal limits (ppm)

First 24 Y = 29.82 + 0.820x 0.97 22.06 16.05–27.66

48 Y = 33.16 + 0.820x 0.97 19.58 14.36–24.20
72 Y = 31.19 + 0.923x 0.98 19.19 13.97–23.45

Third 24 Y = 29.83 + 0.820x 0.97 11.67 8.49–14.84

48 Y = 33.16 + 0.820x 0.97 9.54 6.82–12.25
72 Y = 31.19 + 0.923x 0.98 5.97 2.15–9.79

Fourth 24 Y = 25.98 + 0.282x 0.99 49.84 44.53–54.77

48 Y = 31.19 + 0.256x 0.82 21.22 13.30–29.13
72 Y = 34.53 + 0.256x 0.82 21.02 15.97–73.94

because the fight against adult is temporary, unsatisfac- impact of phenolic compounds on the mosquito larvae
tory and polluting for the environment, while larval treat- has also been reported by many authors [21,22]. Alumin-
ment is more localized in time and space resulting in less- ium chloride obtained from alder leaf, known for its phe-
dangerous outcomes. Larval control can be an effective nolic complexing activity, is also reported to have the
control tool due to the low mobility of larval mosquitoes, larvicidal activity against S. aegypti [23]. Isoflavonoids
especially where the principal breeding habitats are man- from tubers of Neorautanenia mitis had a larvicidal effect
made and can be easily identified [17]. against the malaria and filariasis transmitting mosquitoes,
Anopheles gambiae and Cx. quinquefaciatus, respectively
The secondary compounds of plants make up a vast repos- [24]. Essential oils extracted from Brazilian plants exhib-
itory of compounds with a wide range of biological activ- ited larvicidal activity against S. aegypti, with LC50 values
ities. Most studies report active compounds as steroidal ranging from 60 to 538 ppm (see [25]). Studies with Lip-
saponins. Saponins are freely soluble in both organic sol- pia sidoides [26] and Cymbopogon citrates [27] essentials oils
vents and water, and they work by interacting with the suggested that they are a promising biocontrol agent
cuticle membrane of the larvae, ultimately disarranging against S. aegypti. Rohini et al [28] isolated D-pinitol,
the membrane, which is the most probable reason for lar- from the EtOH extract of Acacia nilotica, which showed
val death [18]. Wiesman and Chapagain [19] reported larvicidal activity. Alkaloids derived from Piper longum
that saponin extracted from the fruit of Balanites aegyptica fruit [29] and Triphyophyllum pellatum [30] showed larvi-
showed 100% mortality against larvae of S. aegypti. The cidal activity against C. pipiens and A. stephensi, respec-
larvicidal property of a saponin mixture isolated from Ces- tively. Khanna and Kannabiran [31] reported the role of
trum diurnum was also evaluated against Anopheles tannin compounds extracted from Hemidesmus indicus,
stephensi mosquito by Ghosh and Chandra [20]. The

Table 5: Phytochemical analysis of the chloroform:methanol extract of the green berries of S. villosum

Solvents used Spraying reagents Rf values (and appearance) of Presence/absence of

the positive spot phytochemicals

Acetone-hexane (4:1) Antimony chloride in concentrated - Absence of sapogenins

hydrochloric acid
Methanol-concentrated Dragendorff 0.95, 0.96 (green) Presence of alkaloids
ammonium hydroxide (200:3)
Chloroform Libermann-Buchard 0.95, 0.82, 0.68 (reddish pink) Presence of steroids
Chloroform:benzene (1:1) Vanillin-sulphuric acid 0.99 (violet blue) Presence of essential oil
Chloroform:acetic acid:water Saturated alcoholic sodium acetate 0.98 (green) Presence of flavonoids
(90:45:6)
Ethyl acetate:benzene (1:1) Folin reagent 0.98, 0.94 (blue) Presence of phenolics

Ninhydrin 0.78 Presence of protein

Chloroform on silica gel plate Antimony chloride in chloroform 0.97, 0.48 (green) Presence of terpenoids
treated with silver nitrate

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7.75
7.5

7.0

6.5

6.0

5.5

5.0

4.5

790.17
813.83
4.0

3.5
%T

3.0

2.5

2.0

1.5

1.0 1384.73

1461.64 1044.56
0.5
1656.87
1745.61
0.0
2853.33
-0.5
2924.44

-1.00
4400.0 4000 3000 2000 1500 1000 450.0
cm-1

Figure 1
Interpretation of IR spectra of the compound having Rf = 0.95
Interpretation of IR spectra of the compound having Rf = 0.95. Frequency range and probable functional groups of the
compound (Rf = 0.946): 2,924.44 and 2,853.33 cm-1, C-H (S) group; 1,745.61 cm-1, C = O (S) stretch; 1,656.87 cm-1, asymmet-
rical stretch of NO2 compounds (S); 1,461.64 cm-1, scissoring and bending of C-H compounds (V); 1,384.73 cm-1, symmetrical
stretches of NO2B Bcompounds (S); 1,044.56 cm-1, C-O stretch (S); 813.83 cm-1, phenyl ring substitution bands (S); 790.17 cm-
1, C-H bend (B). V, variable; M, medium; S, strong; Br, broad; W, weak.

Gymnema sylvestre and Eclipta prostrate that causes mortal- Conclusion

ity in Cx. quinquefasciatus larvae. In conclusion, S. villosum offers promised as a potential
bio control agent against S. aegypti particularly in its mark-
The present study indicates that green berries of S. villosum edly larvicidal effect. The biocontrol potentiality was
had biocontrol activity against S. aegypti. The highest mos- lower than chemical insecticides such as Malathion. The
quitocidal activity was noted in chloroform:methanol extract or isolated bioactive phytochemical from the plant
extract. The qualitative and chromatographic study of could be used in stagnant water bodies which are known
green berries of S. villosum revealed the presence of several to be the breeding grounds for mosquitoes. However, fur-
bioactive compounds. However, the IR spectra of the bio- ther studies on the identification of the active principals
active compounds during the present study also indicated involved and their mode of action and field trials are
that any steroid compound(s) is responsible for larval tox- needed to recommend S. villosum as an anti-mosquito
icity. product used to combat and protect from mosquitoes in a
control program.

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3.21

3.0

2.8

2.6

2.4
920.32

2.2

2.0 816.79

1.8
781.29

1.6 636.35

1.4
%T

1.2

1.0

0.8

0.6
1130.34
1461.64
0.4
1381.77
0.2 1053.43
1636.16 1074.14
1653.91
0.0

2924.44
-0.2
3422.22
-0.4
-0.50
4400.0 4000 3000 2000 1500 1000 450.0
cm-1

Figure
Interpretation
2 of IR spectra of the compound having Rf = 0.82
Interpretation of IR spectra of the compound having Rf = 0.82. Frequency range and probable functional groups of the
compound (Rf = 0.818): 3,422.22 cm-1, H bonded OH stress (B); 2,924.44 cm-1, CH (S) stretch; 1,653.91 cm-1, asymmetrical
stretch of NO2 compounds (S); 1,636.16 cm-1, NH (M) bond; 1,461.64 cm-1, scissoring and bending of C-H compounds (V);
1,381.77 cm-1, doublet isopropyl (M-W); 1,130.34, 1,074.14, 1,053.43 cm-1, CO group (S) stretch; 920.32 cm-1, alkenes (S)
bend; 816.79, 781.29 cm-1, CH phenyl ring (S) substitution bend; 636.35 cm-1, alkynes bend (B). V, variable; M, medium; S,
strong; Br, broad; W, weak.

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