SCIREA Journal of Biology

http://www.scirea.org/journal/Biology

April 17, 2023

Volume 8, Issue 2, April 2023

https://doi.org/10.54647/biology180286

Investigation of the combination of neem seed oid and rotenone

extract on the inhibition of Plutella xylostella on Brassica juncea

Nguyen, Luong Hieu Hoa1, Ngo, Thi Phuong Dung1, Le, Quynh Loan2, Nguyen,
Hoang Dung1,2*
1
Nguyen Tat Thanh University, Vietnam
2
Institute of Tropical Biology, VAST, Vietnam
*
Corresponding author: Email: dung0018034@gmail.com

Abstract

The chemical pesticides being used today have adverse effects on human health and
the environment. So ,they are strictly controlled, especially for export agricultural
products. Since then, the search for pesticides derived from natural plants to replace
chemical drugs on the market have extensively done. In this study, the combination of
neem seed oil and rotenone extract on the inhibition of Plutella xylostella on Brassica
juncea was investigated. The results showed that the combination of neem seed oid
and rotenone extract has a significantly high insecticidal effect in vitro condition, the
effectiveness of which is proportional to the treatment concentration, the 70%
inhibitory concentration of Plutella xylostella was LC70 = 0.0130 %. In green house
model , it was found that after the first treatment, the efficiency of Plutella xylostella
control reached 38.31% and on the 7th day after the second treatment, the insecticide
efficiency reached 84.81%. These results showed the high potential application of the

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combination of neem seed oid and rotenone extract onvegetable cultivation to limit the
use of chemical drugs, as well as the current situation of resistant pests.

Keywords: azadirachtin, combination, rotenone, Plutella xylostella L., pesticides

Introduction

The success of the green revolution in agriculture is largely attributed to the discovery
and use of chemicals in pest control. The rapid increase in productivity would not have
been achieved without the use of synthetic chemicals. However, the use of these
chemicals also leads to serious harm to the environment and human health. Due to
increasingly strict regulations, many synthetic chemicals have been restricted from
usage. Therefore, the approach to using natural compounds with the ability to control
pests is receiving much attention. The use of biological pesticides to replace chemical
pesticides is increasingly popular. Among them, rotenone from Derris elliptica and
azadirachin from neem tree has got much attraction.

Neem tree (Azadirachta indica), a species of Indian cycad is one of the plants studied
quite early. The main active ingredient of this species is azadirachtin, which is very
safe for humans and the environment, but toxic to many plant pests. It is one of the
spearhead plants in the field of herbal pesticide research. The main effects of Neem
and its active ingredient azadirachtin on pests include: internal absorption and drainage
effects, growth inhibition, and stimulant effects. Asogwa et al. also demonstrated the
effectiveness of neem seed extract in protecting the harmful effects of the brown
planthopper Sahlbergella singularis in cocoa farms in Nigeria [1]. Neem seed extract
was found to be effective against storage and field crop pests in Sudan and is
recommended for use on small farms [2]. Another trial at the Marondera Zimbabwe
Horticultural Research Center in 2011 showed that local wild plants such as Solanum,
Lippia, Garlic, and Neem seeds could be formulated to help control aphids on rapeseed
and is used as an organic insecticide [3]. Rotenon is also considered as one of the
potential herbs to be studied as an effective insecticidal herb [4]. In 1902, Nagai first
isolated a homogenate from the plant Derris chinensis known locally as Roten, hence

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the name rotenone [5]. By 1929, Takei and Koide had determined the molecular
formula of rotenone to be C23H22O6 [6]. Rotenone is a compound that is soluble in
organic solvents except CCl4, sparingly soluble in detroleum, insoluble in water.
When exposed to light, air is easily decomposed from clear white to yellow, orange
and crimson. Rotenone can crystallize in ethanol and some other organic solvents.
Rotenone, like several other rotenoids, is highly effective against fish and many other
pests, but is most effective against leafworms. The main mechanism of action of
rotenone is to inhibit the multiplication of some insect cells that induce apoptosis [7].
Rotenone strongly inhibits electron transport in complex I of the mitochondrial
respiratory chain. Although many research on rotenone from Derris elliptica and
azadirachin from neem tree have done, the combination of these active component is
not well-studied. In this study, the combination of neem seed oid and rotenone extract
on the inhibition of Plutella xylostella on Brassica juncea was done.

Material and methods

Neem oil extraction

Neem seeds from Azadirachta indica were collected in Binh Thuan Province, Vietnam.
They were pre-cleaned, dried and ground to size < 1.0 mm and analyzed for physical
parameters (moisture, ash) according to the guidance and reference of Vietnamese
Pharmacopoeia standards. The seeds were pressed to obtain essential oil (200 Mpa, 15
minutes, 40oC), the neem residues after oil pressing continue to be hot-extracted with
90% ethanol to obatain the ethanol extract. The extract was then filtered and
concentrated under low pressure to remove the solvent and obtain a rich of
azadirachtin extract. The oil phase and the extract were mixed and homogenized to
form neem oil.

Rotenone extraction

Derris elliptica were collected in Binh Duong and Bac Lieu provinces 7-8 months
after planting. Raw materials are pre-treated, dried and ground to size < 1.0 mm and
analyzed for physical parameters (powder, moisture, ash) according to the instructions

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and reference of the standards of the company. Derris elliptica was then extraction
with 90% ethanol at 50oC for 60 min. The extract was then filtered and concentrated
under low pressure to remove the solvent and obtain a rich of rotenon extract.

Qualifying the presence of azadirachtin and rotenone

For azadiractin qualification: Thin plate chromatography (TLC) was performed to

determine the presence of azadirachtin in neem seed oil. The Merck SG-60
chromatographic plate was used with a mobile phase of a mixture of
dichloromethane:methanol (1:1). Azadirachin (95%) (A7430 – Merck) was used as the
standard. Detect the presence of azadirachtin by spraying with a mixture of vanillin
reagents. The azadirachin content was quantified by HPLC chromatography.

For rotenone qualification: The rotetone content was quantified by HPLC

chromatography based on Vietnamese standard (TCCS-6:2008/BVTV)

Preparing the source of Plutella xylostella L.

Plutella xylostella L.samples in leaf mustard (Brassica juncea) fields was colleted in
Chau Thanh district, Tien Giang province. Plutella xylostella were caught, put in
plastic bags containing leaf mustard. They werew then brought to the laboratory and
bred on leaf mustard plants grown in potting soil, placed in specialized cages,
providing food. The food were changes every 2 days to ensure adequate food supply
for the worms to develop well. When the pupation was carried out, they were allowed
to pair up and mate in a mesh cage supplemented with 40% honey to help the larvae
develop well and lay eggs. When the eggs hatch, the larvae of the same age were
separate into a plastic box and continued to breed on leaf mustard leaves until the
number is sufficient, then conduct the experiment. Experiments were carried out on
worms at the age of 2.

Investigation of combination of rotenone and azadirachin on Plutella xylostella L.

in in vitro

The neem oil, azadirachin and the combination of neem oil and azadirachin (1:1 ratio,
0.03%) was investigated on the kill of Plutella xylostella L. The experiment was
repeated 4 times, with 2 leaf mustard plants each time. After dropping deep into the

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trees, spray the solution on the plants in the cup, each concentration sprayed 8 plants.
The number of silk worms at 72 hours after spraying were count and calculated.

The effect of killing worms of the solution is calculated by the formula: M(%)= Ca –
Ta/ Ca

In which: M: rate of dead worms

Ca: number of live worms in the control treatment after the experiment

Ta: number of worms living in the empirical formula

Based on the obtained results, the effective insecticidal concentration (>70%) was
calculated.

Investigation of combination of rotenone and azadirachin on Plutella xylostella L.

in green house

The leaf mustard seeds was planted on separate cassettes in the net house. Each
cassette was separated by a tarpaulin so that when the silk worm is released, it could
not crawl over and when spraying, it will not diffuse to the other cassette. Watering,
fertilizing and taking care of the plants to grow well were done carefully and when the
leaf mustard was 15 days old, the experiment was conducted.

Methods: The experiment was arranged in a completely randomized design with 6

treatments, 4 replicates, 5 m2 each. When the leaf mustard was 15 days old, 2 year old
silkworms (120 heads) were released into each experimental plot the day before to
stabilize. Then proceed to spray the inoculant onto the experimental plot, spray wet
evenly - Monitoring criteria was observe and count the number of worms on 5 points
diagonally, 5 trees each at 1, 3, 5 and 7 days after each treatment; Efficacy of herbal
preparations against silkworms at 1, 3, 5 and 7 days after each treatment (%).

The potency of the drug concentrations was calculated according to the Abbott
formula (1925):

Potency (H) % = [(Ca – Ta)/Ca] x 100

In there:

H: percent potency of the drug at time points.

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Ca: Number of individuals living in the control treatment after treatment.

Ta: Number of individuals living in the experimental treatment after treatment.

Based on the obtained results, the effective insecticidal concentration (>70%) was
calculated.

Results and Discussion

Neem seed oil extraction and qualification of azadirachin.

The neem seed material was collected, dried, and finely ground to a uniform size (<1.0
mm) (Figure 1). The physical parameters analyzed include determination of total ash
reaching 4.66%; ash insoluble in HCl reached 2.91%; humidity reaches 9%. Thus, the
input materials meet the requirements in accordance with the standards of granular
medicinal herbs: (Total ash <6%, ash insoluble in HCl <4%, moisture from 8-10%).
The azadirachin content from the neem seed oil was then qualified by HPLC. The
results showed that the azadirachin content in the neem seed oil is 13.124 mg/kg. The
azadirachin content was similar to previous results.

Figure 1: Neem seed sample an neem seed oil

Rotenone extraction and qualification

Derris elliptica plants were collected, dried, and finely ground to a uniform size (<1.0
mm) (Figure 2). The physical parameters were analyzed after 3 iterations, including
the determination of total ash reaching 3.07%; ash insoluble in HCl reached 1.04%;
humidity reaches 12%. Thus, the input materials meet the requirements in accordance
with the standards of granular medicinal herbs (total ash < 6%, insoluble ash in HCl <

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4%, moisture from 12-15%). The rotenone content from Derris elliptica was then
qualified by HPLC. The results showed that the rotenone content was 3567 mg/kg.

Figure 2: Derris elliptica samples

Effect of combination of rotenone and azadirachin on Plutella xylostella L. in in

vitro

The ability to kill silkworms of different concentrations of azadirachtin, rotenone and

their combination was investigated on leaf mustard plants. The results were showed in
the Figure 3. The results showed that neem seed oil and rotenone at the concentration
0.03% could kill 80.03 and 75.13% of Plutella xylostella, respectively. The
combination of rotenone and azadirachin at the same concentration could kill 84.13%
of Plutella xylostella. It means that the combinaton of these compound have better
effect than using the single treatment.

100

80
ytilatrom mrow kliS

60

40

20

0
trol chi
n one (1:1
)
con dira ten ne
iv e Aza Ro no
gat ote
Ne :R
c hin
dira
Aza

Samples

Figure 3: Effect of azadirachin, rotenone and azadirachin: rotenone (1:1) on the mortality of silk
worm Plutella xylostella in vitro.

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Effect of combination of rotenone and azadirachin on Plutella xylostella L. in
green house

The effect of combination of rotenone and azadirachin on Plutella xylostella L. in

green house was showed in the figure 4. In green house model , it was found that after
the first treatment, the efficiency of Plutella xylostella control reached 38.31% and on
the 7th day after the second treatment, the insecticide efficiency reached 84.81%. As
showed in the figure 4, the effect of the combination azadirachin: rotenone (1:1) was
84.13% of silk worm killing. It was significant higher effect than rotenone and
azadirachin alone.

100

80
ytilatrom mrow kliS

60

40

20

0
rol hin one (1:1
)
c ont irac ten ne
iv e ad Ro no
gat Az ote
Ne :R
c hin
dira
Aza

Samples

Figure 4: Effect of azadirachin, rotenone and azadirachin: rotenone (1:1) on the mortality of silk
worm Plutella xylostella in green house

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Conclusion

These results showed the high potential application of the combination of neem seed
oil and rotenone extract on vegetable cultivation to limit the use of chemical drugs, as
well as the current situation of resistant pests. Therefore, these combination of these
plants should be used in combination with other IPM solutions in vegetable cultivation
in order to limit the use of chemical drugs and limit the current situation of resistant
pests.

Acknowledgement

The study received funding from the Department of Science and Technology of Tien
Giang province, project code: DTCN 04/2019

References

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[7] Li, N., Ragheb, K., Lawler, G., Sturgis, J., Rajwa, B., Melendez, J. A., & Robinson, J. P.
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