# Insecticidal Activity and Chemical Composition of theMorinda lucida Essential Oil against Pulse Beetle Callosobruchus maculatus

Authors: Moses S. Owolabi; Eduardo Padilla-Camberos; Akintayo L. Ogundajo; Isiaka A. Ogunwande; Guido Flamini; Olaniyi K. Yusuff; Kirk Allen; Karen Isabel Flores-Fernandez; Jose Miguel Flores-Fernandez
Journal: The Scientific World Journal
 (2014)

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## Abstract

Insecticidal activity of essential oil extracted fromMorinda lucida was tested on pulse beetle Callosobruchus maculatus, which is a pest that causes serious damage to several pulses. The insecticidal activity was compared with two pesticides, Phostoxin and Primo-ban-20. 120 mixed sex adult C. maculatus were introduced, along with 30 g of cowpeas. Four concentrations (0.40, 0.20, 0.10, and 0.05 μg/mL) of the M. lucida essential oil, Phostoxin, and Primo-ban-20 were tested. Essential oil chemical composition was analyzed by GC-MS. M. lucida essential oil showed a high toxicological effect, producing 100% mortality after 72 hours at a dose of 0.20 μg/mL. M. lucida essential oil had a potent insecticidal activity (LC90 = 0.629 μg/mL) compared to both pesticides, Phostoxin (LC90 = 0.652 μg/mL) and Primo-ban-20 (LC90 = 0.726 μg/mL), at 24 h. The main compounds of the essential oil were the oxygenated monoterpenoids, 1,8-cineole (43.4%), and α-terpinyl acetate (14.5%), and the monoterpene hydrocarbons, mostly sabinene (8.2%) and β-pinene (4.0%). Results clearly indicate that M. lucida essential oil can be used as an effective alternative for pulse beetle C. maculatus control, and it could be tested against other pulse beetles affecting Asia and Africa and throughout the world, thereby reducing use of synthetic pesticides.


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## Body

## 1. Introduction

Callosobruchus maculatusis an economically important pest of several pulses, including leguminous grains such as lentils, cowpeas, green gram, chickpea, black gram, soybean, and haricot beans [1, 2]. These pulses are important sources of vegetable protein for millions of people of tropical and subtropical regions of Asia and Africa.C. maculatuslarvae bore into pulse grain or into stored crop, making it unfit for human consumption and unviable for replanting [3–5].Control of insect pestsrelies heavily on the use of synthetic insecticides such as methyl bromide or phosphine. However, their intensive use has led to the development of genetic resistance by insect species, pest resurgence, residual toxicity, environmental hazards, and serious problems arising from factors such as direct toxicity to predators, pollinators, fish, and man [6]. As such, the use of methyl bromide is being restricted because of its potential to damage the ozone layer [7]. Susceptibility of crop plants to insect pests and increasing costs of application of the presently used synthetic pesticides have directed the need for effective, biodegradable pesticides [8, 9].Because of their high volatility, plant extracts or plant compounds and the use of natural compounds with insecticidal activity provide a potential biodegradable alternative to synthetic pesticides [10–12]. The contact and fumigant insecticidal activity of several plant essential oils and their constituents have been demonstrated against stored-product pests [13–15], a number of bruchid pests [16–18], and insects and mites [19].Several botanical families for pest control are described, such as Meliaceae, Rutaceae, Asteraceae, Labiatae, and Malvaceae. Rubiaceae is a family of 630 genera and about 13,000 species [20].Morinda lucida(Benth), a member of Rubiaceaefamily, is a medicinal plant of about 15 m tall with a dense crown of slender crooked branches approximately 20–30 cm in diameter.M. lucidacommonly known by the Yoruba in southwestern part of Nigeria as “Oruwo” is widely distributed in West Africa and is used in African folk medicine to treat several diseases [21]. The leaves are bitter and are used by the natives to treat malaria, yellow fever, jaundice, hepatitis, eczema, edema, cough, hypertension, diabetes, and sexual weakness [20, 22, 23].In a previous study, another species of the same genus,M. citrifolia, showed larvicidal activity against three species of mosquito vectors [24] and the biofriendly nature ofM. lucida suggested the need to search for new properties of this plant; therefore the present study was conducted to determine the insecticidal activity of the essential oil ofM. lucida againstC. maculatus by dose response bioassays and to characterize its chemical composition for consideration as an alternative insecticide for pulse beetle.


## 2. Materials and Methods

### 2.1. Plant Material

The aerial parts of theM. lucidawere collected from Apomu (7°19′48N, 4°10′47E), which is located in Osun State, Nigeria, and identified at the Herbarium of the Botany Department, University of Lagos, Nigeria, where voucher specimen number LUH 2637 was kept. The samples were air-dried, pulverized, and stored in polythene bag to reduce evaporation of the volatile oil.To extract the essential oil, 500 g of pulverized sample was placed in a 5 L flask and distilled water was added to cover sample. Essential oil was obtained by hydrodistillation using glass modified Clevenger-type apparatus; the process ran for 4 h at normal atmospheric pressure and at 96-97°C inside the extractor. After the essential oil isolation, residual water was removed by filtration with anhydrous sodium sulphate, and then the essential oil was stored in an amber vial at 4°C for future analysis.


### 2.2. Gas Chromatography (GC) Analysis

GC analysis was performed with a HP-5890 Series II instrument equipped with a HP-Wax and HP-5 capillary columns (both 30 m × 0.25 mm ID × 0.25μm film thickness), working with the following temperature program: 60°C for 10 min, rising at 5°C/min to 220°C. The injector and detector temperatures were maintained at 250°C; carrier gas nitrogen (2 mL/min); detector dual, FID; split ratio 1 : 30. The volume injected was 0.5 μL. The relative proportions of the oil constituents were percentages obtained by FID peak-area normalization without the use of response factor.


### 2.3. Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS analysis was performed with a Varian CP-3800 gas chromatograph equipped with a HP-5 capillary column (30 m × 0.25 mm ID × 0.25μm film thickness) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions were injector and transfer line temperature 220°C and 240°C, respectively; oven temperature was programmed from 60 to 240°C at 3°C/min. Carrier gas was helium at 1.0 mL/min flow rate. Injection volume was 0.2 μL (10% hexane solution) using a 1 : 30 split ratio. The mass spectrometer was operated at 70-eV ionization voltage. The acquisition mass range was 30 to 300
m
/
z at 1.0 scan/s.


### 2.4. Identification of the Volatile Compounds

The identification of the volatile constituents of the essential oil sample was made using the following criteria: (1) comparison of mass spectra with the Wiley library spectra 275 L (Rev.C.00.00) electronic database; (2) injection of authentic compounds from Sigma-Aldrich with a 95% minimum purity under the same analytical conditions; and (3) comparison of the retention index (RI) obtained by GC-MS with the RI theoretical in similar phases. The molecular weights of all the identified substances were confirmed by GC-MS, using MeOH as CI ionizing gas.


### 2.5. Insect Culture

Callosobruchus maculatuswere reared on cowpea seeds (Vigna unguiculata) under controlled temperature and humidity at 28
±
1°C, 58
±
5% RH (12 h light cycle) and 25.5
±
1°C, 45
±
5% RH (12 h dark cycle). Adult insects of 8 days old were used for insecticidal test.All experiments were carried out under the same environmental conditions as described above.


### 2.6. Insecticidal Activity ofM. lucidaEssential Oil

The insecticidal activity was evaluated as described by Ilboudo et al. [25]. One hundred and twenty mixed sex adultC. maculatus (8 days old) were put into a 500 mL glass bottle with 30 g of cowpea seeds and kept in the laboratory at 28
±
1°C, 58
±
5% RH (12 h light cycle) and 25.5
±
1°C, 45
±
5% RH (12 h dark cycle) for 72 h. Concentrations of theM. lucidaessential oil diluted in acetone were tested onC. maculatus (0.05, 0.10, 0.20, and 0.40 μg/mL). The appropriate concentrations were applied to filter paper (Whatman number 1, cut into 7 cm diameter) and immediately introduced into a glass bottle that was then hermetically sealed. For the control group, the insects were placed in the glass bottles under the same conditions but without the essential oil. Each concentration and control was replicated three times. Insect mortality was determined by observing the recovery of immobilized insects in 12 h intervals up to 72 h. When no antennal or leg movements were observed, insects were considered dead.


### 2.7. Insecticidal Activity of Commercial Pesticides

Two synthetic pesticides were purchased from Chemical and Allied company, Lagos, Nigeria. Phostoxin (55% Aluminium phosphide) and Primo-ban-20 (pirimiphos-methyl, emulsifiable concentrate 50%). The first is converted to phosphine gas, which is taken by the insect through its respiratory system and the second is able to penetrate the insect cuticle. Pellets of Phostoxin were divided into small pieces and weighed to achieve the appropriate concentrations and were applied according to manufacturer’s directions [26]. Primo-ban-20 was diluted to achieve the desired concentrations, from a stock solution of 2 μg/mL. Both pesticides were evaluated at the same concentrations as the essential oil in triplicate.


### 2.8. Mortality Determination

Ten pairs of adultC. maculatus were released each 12 h after exposure of essential oil up to 72 h. After a 12 h exposure period the containers were opened and the dead beetles were removed and recorded. Knocked-down adults were regarded as alive if they showed continued movement of their appendages by the touch of a fine brush. After observation, the containers were tightly closed. After every 12 h, new batches of twenty insects were released and the numbers of dead insects were recorded and the percentage of mortality was calculated and corrected using Abbott’s formula [27].


### 2.9. Data Analysis

Average percentage insect mortality was calculated from three replicates. Dosage-mortality responses were used to estimate LC50 (concentration causing 50% mortality) and LC90 (concentration causing 90% mortality) values at 24 and 48 h with Probit analysis [28] using Statgraphics 5.1 software. Insecticidal activity was considered significantly different when the 95% CI (confidence interval) fail to overlap.


## 2.1. Plant Material

The aerial parts of theM. lucidawere collected from Apomu (7°19′48N, 4°10′47E), which is located in Osun State, Nigeria, and identified at the Herbarium of the Botany Department, University of Lagos, Nigeria, where voucher specimen number LUH 2637 was kept. The samples were air-dried, pulverized, and stored in polythene bag to reduce evaporation of the volatile oil.To extract the essential oil, 500 g of pulverized sample was placed in a 5 L flask and distilled water was added to cover sample. Essential oil was obtained by hydrodistillation using glass modified Clevenger-type apparatus; the process ran for 4 h at normal atmospheric pressure and at 96-97°C inside the extractor. After the essential oil isolation, residual water was removed by filtration with anhydrous sodium sulphate, and then the essential oil was stored in an amber vial at 4°C for future analysis.


## 2.2. Gas Chromatography (GC) Analysis

GC analysis was performed with a HP-5890 Series II instrument equipped with a HP-Wax and HP-5 capillary columns (both 30 m × 0.25 mm ID × 0.25μm film thickness), working with the following temperature program: 60°C for 10 min, rising at 5°C/min to 220°C. The injector and detector temperatures were maintained at 250°C; carrier gas nitrogen (2 mL/min); detector dual, FID; split ratio 1 : 30. The volume injected was 0.5 μL. The relative proportions of the oil constituents were percentages obtained by FID peak-area normalization without the use of response factor.


## 2.3. Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS analysis was performed with a Varian CP-3800 gas chromatograph equipped with a HP-5 capillary column (30 m × 0.25 mm ID × 0.25μm film thickness) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions were injector and transfer line temperature 220°C and 240°C, respectively; oven temperature was programmed from 60 to 240°C at 3°C/min. Carrier gas was helium at 1.0 mL/min flow rate. Injection volume was 0.2 μL (10% hexane solution) using a 1 : 30 split ratio. The mass spectrometer was operated at 70-eV ionization voltage. The acquisition mass range was 30 to 300
m
/
z at 1.0 scan/s.


## 2.4. Identification of the Volatile Compounds

The identification of the volatile constituents of the essential oil sample was made using the following criteria: (1) comparison of mass spectra with the Wiley library spectra 275 L (Rev.C.00.00) electronic database; (2) injection of authentic compounds from Sigma-Aldrich with a 95% minimum purity under the same analytical conditions; and (3) comparison of the retention index (RI) obtained by GC-MS with the RI theoretical in similar phases. The molecular weights of all the identified substances were confirmed by GC-MS, using MeOH as CI ionizing gas.


## 2.5. Insect Culture

Callosobruchus maculatuswere reared on cowpea seeds (Vigna unguiculata) under controlled temperature and humidity at 28
±
1°C, 58
±
5% RH (12 h light cycle) and 25.5
±
1°C, 45
±
5% RH (12 h dark cycle). Adult insects of 8 days old were used for insecticidal test.All experiments were carried out under the same environmental conditions as described above.


## 2.6. Insecticidal Activity ofM. lucidaEssential Oil

The insecticidal activity was evaluated as described by Ilboudo et al. [25]. One hundred and twenty mixed sex adultC. maculatus (8 days old) were put into a 500 mL glass bottle with 30 g of cowpea seeds and kept in the laboratory at 28
±
1°C, 58
±
5% RH (12 h light cycle) and 25.5
±
1°C, 45
±
5% RH (12 h dark cycle) for 72 h. Concentrations of theM. lucidaessential oil diluted in acetone were tested onC. maculatus (0.05, 0.10, 0.20, and 0.40 μg/mL). The appropriate concentrations were applied to filter paper (Whatman number 1, cut into 7 cm diameter) and immediately introduced into a glass bottle that was then hermetically sealed. For the control group, the insects were placed in the glass bottles under the same conditions but without the essential oil. Each concentration and control was replicated three times. Insect mortality was determined by observing the recovery of immobilized insects in 12 h intervals up to 72 h. When no antennal or leg movements were observed, insects were considered dead.


## 2.7. Insecticidal Activity of Commercial Pesticides

Two synthetic pesticides were purchased from Chemical and Allied company, Lagos, Nigeria. Phostoxin (55% Aluminium phosphide) and Primo-ban-20 (pirimiphos-methyl, emulsifiable concentrate 50%). The first is converted to phosphine gas, which is taken by the insect through its respiratory system and the second is able to penetrate the insect cuticle. Pellets of Phostoxin were divided into small pieces and weighed to achieve the appropriate concentrations and were applied according to manufacturer’s directions [26]. Primo-ban-20 was diluted to achieve the desired concentrations, from a stock solution of 2 μg/mL. Both pesticides were evaluated at the same concentrations as the essential oil in triplicate.


## 2.8. Mortality Determination

Ten pairs of adultC. maculatus were released each 12 h after exposure of essential oil up to 72 h. After a 12 h exposure period the containers were opened and the dead beetles were removed and recorded. Knocked-down adults were regarded as alive if they showed continued movement of their appendages by the touch of a fine brush. After observation, the containers were tightly closed. After every 12 h, new batches of twenty insects were released and the numbers of dead insects were recorded and the percentage of mortality was calculated and corrected using Abbott’s formula [27].


## 2.9. Data Analysis

Average percentage insect mortality was calculated from three replicates. Dosage-mortality responses were used to estimate LC50 (concentration causing 50% mortality) and LC90 (concentration causing 90% mortality) values at 24 and 48 h with Probit analysis [28] using Statgraphics 5.1 software. Insecticidal activity was considered significantly different when the 95% CI (confidence interval) fail to overlap.


## 3. Results

### 3.1. Chemical Composition ofMorinda lucida

The chemical composition analysis by GC-MS identified 63 components ofM. lucida essential oil, containing mainly 1,8-cineole (43.4%) followed by α-terpinyl acetate (14.5%), sabinene (8.2%), and β-pinene (4.0%; Table 1).Table 1
Retention index and relative composition of major chemical components ofM. lucida essential oil.
Component
KIa
%
α-Thujene
902
0.6
Tricyclene
908
Tr
Camphene
925
0.4
Sabinene*
950
8.2
β-Pinene*
954
4
Myrcene
966
0.9
α-phellandrene
1008
0.9
δ-3-Carene
1014
0.1
α-Terpinene
1021
0.8
o-Cymene
1027
Tr
p-Cymene*
1029
1.2
Limonene*
1034
2.4
1,8-Cineole*
1037
43.4
(E) β-Ocimene
1054
Tr
g-Terpinene
1064
1.1
Cis-Sabinene hydrate
1073
0.5
Terpinolene
1091
0.4
Linalool*
1103
2.2
Cis-p-Menth-2-en-1-ol
1127
0.3
α-Campholenal
1131
Tr
Trans-p-Menth-2-en-1-ol
1146
0.3
Camphor
1149
Tr
β-Pinene oxide
1162
Tr
Pinocarvone
1167
Tr
Borneol
1170
0.5
δ-Terpineol
1172
0.6
Terpinene-4-ol
1181
3.4
p-Cymen-8-ol
1188
Tr
Trans-p-Mentha-1(7),8-dien-2-ol
1191
Tr
α-Terpineol
1193
3.4
Myrtenal
1196
Tr
Trans-Piperitol
1211
Tr
Trans-Carveol
1223
Tr
Nerol
1228
0.2
Cuminaldehyde
1246
Tr
Carvone
1249
Tr
Cis-Verbenyl acetate
1283
0.1
Isobornyl acetate
1290
0.6
Trans-Sabinyl acetate
1292
0.1
Trans-Pinocarvyl acetate
1302
0.1
Neo-verbanol acetate
1321
0.8
α-Terpinyl acetate*
1355
14.5
Eugenol
1364
0.6
Cis-Carvyl acetate
1367
Tr
Neryl acetate
1370
0.3
Geranyl acetate
1388
Tr
β-Elemene
1394
Tr
Methyl eugenol
1409
1.4
β-Caryophyllene
1421
Tr
p-Cymen-7-ol-acetate
1423
Tr
(E)-Cinnamyl acetate
1450
Tr
(E)-Methyl isoeugenol
1495
Tr
Bicyclogermacrene
1497
Tr
α-Bulnesene
1506
Tr
β-Sesquiphellandrene
1526
Tr
Elemol
1554
Tr
Elemicin
1557
Tr
Spathulenol
1578
0.4
Caryophyllene oxide
1583
0.5
g-Eudesmol
1635
Tr
β-Eudesmol
1651
0.2
α-Eudesmol
1655
Tr
Intermedeol
1667
Tr
Total identified
95.40
Major components are shown in bold.
*These chemical components were identified by authentic compound injection.
aKI Kovats retention indices.
Tr = trace amount <0.1%.


### 3.2. Mortality ofCallosobruchus maculatus

The aerial parts of theM. lucidadistillate yielded a 0.48% (v/w) essential oil. The insecticidal bioassay of the essential oil ofM. lucidaagainstC. maculatus(Figure 1) showed that the activity of the oil was both dose dependent and exposure dependent. At a dose of 0.05 μg/mL, the essential oil produced 59.4% mortality after 72 h (Figure 1(c)). The essential oil produced 33.3%, 56.7%, and 89.9% mortality after 24, 48, and 72 h at a dose of 0.10 μg/mL, respectively, while a dose of 0.20 μg/mL yielded mortality of 46.7%, 83.3%, and 100%, respectively, over the same time duration (Figure 1). The highest concentration of 0.40 μg/mL produced a mortality of 90% and 100% after 48 and 72 h, respectively (Figures 1(b) and 1(c)).Effect of theMorinda lucida essential oil and pesticides againstCallosobruchus maculatus at different concentrations and exposure times.
(a)
(b)
(c)The synthetic pesticide Primo-ban-20 at 0.20μg/mL had a lower mortality (36.7%) at 24 h, while Phostoxin had a 46.7% and 86.7% mortality after 24 h and 48 h at 0.20 μg/mL, respectively (Figures 1(a) and 1(b)). BothM. lucida essential oil and pesticides reached 100% mortality at 72 h at 0.20 μg/mL (Figure 1(c)). The negative control showed no activity except at 72 h (1.7%).


### 3.3. Determination of Insecticidal Activity

Probit analysis showed thatM. lucida essential oil after 24 h presented an insecticidal activity (LC90 = 0.629 μg/mL) similar to Phostoxin (LC90 = 0.652 μg/mL) and Primo-ban-20 (LC90 = 0.726 μg/mL). At 24 h essential oil ofM. lucida(LC50 = 0.298 μg/mL) and Phostoxin (LC50 = 0.295 μg/mL) were more efficient than Primo-ban-20 (LC50 = 0.393 μg/mL). At 48 h after exposure, there was no significant difference in LC50 betweenM. lucida essential oil compared to Primo-ban-20 and Phostoxin (Table 2, where all confidence intervals overlap).Table 2
Insecticidal activity ofM. lucida essential oil against C. maculatus.
Test sample
24 h exposure
48 h exposure
LC
50
LC
90
χ
2a
LC
50
LC
90
χ
2a
M. lucida essential oil
0.298
0.629
18.3
0.122
0.308
36.4
(0.251–0.368)
(0.521–0.819)
(0.098–0.147)
(0.265–0.372)
Phostoxin
0.295
0.652
17.9
0.081
0.286
58.6
(0.245–0.370)
(0.532–0.870)
(0.052–0.106)
(0.241–0.357)
Primo-ban-20
0.393
0.726
12.1
0.126
0.387
41.5
(0.331–0.495)
(0.596–0.965)
(0.094–0.159)
(0.326–0.484)
UnitsLC
50 (concentration causing 50% mortality) and LC
90 (concentration causing 90% mortality) in units of μg/mL. 95% confidence interval (CI) is shown in parenthesis. Insecticidal activity is considered significantly different when the 95% CI fail to overlap. aChi-square value of Probit model fit, all significant at P
<
0.05 level.


## 3.1. Chemical Composition ofMorinda lucida

The chemical composition analysis by GC-MS identified 63 components ofM. lucida essential oil, containing mainly 1,8-cineole (43.4%) followed by α-terpinyl acetate (14.5%), sabinene (8.2%), and β-pinene (4.0%; Table 1).Table 1
Retention index and relative composition of major chemical components ofM. lucida essential oil.
Component
KIa
%
α-Thujene
902
0.6
Tricyclene
908
Tr
Camphene
925
0.4
Sabinene*
950
8.2
β-Pinene*
954
4
Myrcene
966
0.9
α-phellandrene
1008
0.9
δ-3-Carene
1014
0.1
α-Terpinene
1021
0.8
o-Cymene
1027
Tr
p-Cymene*
1029
1.2
Limonene*
1034
2.4
1,8-Cineole*
1037
43.4
(E) β-Ocimene
1054
Tr
g-Terpinene
1064
1.1
Cis-Sabinene hydrate
1073
0.5
Terpinolene
1091
0.4
Linalool*
1103
2.2
Cis-p-Menth-2-en-1-ol
1127
0.3
α-Campholenal
1131
Tr
Trans-p-Menth-2-en-1-ol
1146
0.3
Camphor
1149
Tr
β-Pinene oxide
1162
Tr
Pinocarvone
1167
Tr
Borneol
1170
0.5
δ-Terpineol
1172
0.6
Terpinene-4-ol
1181
3.4
p-Cymen-8-ol
1188
Tr
Trans-p-Mentha-1(7),8-dien-2-ol
1191
Tr
α-Terpineol
1193
3.4
Myrtenal
1196
Tr
Trans-Piperitol
1211
Tr
Trans-Carveol
1223
Tr
Nerol
1228
0.2
Cuminaldehyde
1246
Tr
Carvone
1249
Tr
Cis-Verbenyl acetate
1283
0.1
Isobornyl acetate
1290
0.6
Trans-Sabinyl acetate
1292
0.1
Trans-Pinocarvyl acetate
1302
0.1
Neo-verbanol acetate
1321
0.8
α-Terpinyl acetate*
1355
14.5
Eugenol
1364
0.6
Cis-Carvyl acetate
1367
Tr
Neryl acetate
1370
0.3
Geranyl acetate
1388
Tr
β-Elemene
1394
Tr
Methyl eugenol
1409
1.4
β-Caryophyllene
1421
Tr
p-Cymen-7-ol-acetate
1423
Tr
(E)-Cinnamyl acetate
1450
Tr
(E)-Methyl isoeugenol
1495
Tr
Bicyclogermacrene
1497
Tr
α-Bulnesene
1506
Tr
β-Sesquiphellandrene
1526
Tr
Elemol
1554
Tr
Elemicin
1557
Tr
Spathulenol
1578
0.4
Caryophyllene oxide
1583
0.5
g-Eudesmol
1635
Tr
β-Eudesmol
1651
0.2
α-Eudesmol
1655
Tr
Intermedeol
1667
Tr
Total identified
95.40
Major components are shown in bold.
*These chemical components were identified by authentic compound injection.
aKI Kovats retention indices.
Tr = trace amount <0.1%.


## 3.2. Mortality ofCallosobruchus maculatus

The aerial parts of theM. lucidadistillate yielded a 0.48% (v/w) essential oil. The insecticidal bioassay of the essential oil ofM. lucidaagainstC. maculatus(Figure 1) showed that the activity of the oil was both dose dependent and exposure dependent. At a dose of 0.05 μg/mL, the essential oil produced 59.4% mortality after 72 h (Figure 1(c)). The essential oil produced 33.3%, 56.7%, and 89.9% mortality after 24, 48, and 72 h at a dose of 0.10 μg/mL, respectively, while a dose of 0.20 μg/mL yielded mortality of 46.7%, 83.3%, and 100%, respectively, over the same time duration (Figure 1). The highest concentration of 0.40 μg/mL produced a mortality of 90% and 100% after 48 and 72 h, respectively (Figures 1(b) and 1(c)).Effect of theMorinda lucida essential oil and pesticides againstCallosobruchus maculatus at different concentrations and exposure times.
(a)
(b)
(c)The synthetic pesticide Primo-ban-20 at 0.20μg/mL had a lower mortality (36.7%) at 24 h, while Phostoxin had a 46.7% and 86.7% mortality after 24 h and 48 h at 0.20 μg/mL, respectively (Figures 1(a) and 1(b)). BothM. lucida essential oil and pesticides reached 100% mortality at 72 h at 0.20 μg/mL (Figure 1(c)). The negative control showed no activity except at 72 h (1.7%).


## 3.3. Determination of Insecticidal Activity

Probit analysis showed thatM. lucida essential oil after 24 h presented an insecticidal activity (LC90 = 0.629 μg/mL) similar to Phostoxin (LC90 = 0.652 μg/mL) and Primo-ban-20 (LC90 = 0.726 μg/mL). At 24 h essential oil ofM. lucida(LC50 = 0.298 μg/mL) and Phostoxin (LC50 = 0.295 μg/mL) were more efficient than Primo-ban-20 (LC50 = 0.393 μg/mL). At 48 h after exposure, there was no significant difference in LC50 betweenM. lucida essential oil compared to Primo-ban-20 and Phostoxin (Table 2, where all confidence intervals overlap).Table 2
Insecticidal activity ofM. lucida essential oil against C. maculatus.
Test sample
24 h exposure
48 h exposure
LC
50
LC
90
χ
2a
LC
50
LC
90
χ
2a
M. lucida essential oil
0.298
0.629
18.3
0.122
0.308
36.4
(0.251–0.368)
(0.521–0.819)
(0.098–0.147)
(0.265–0.372)
Phostoxin
0.295
0.652
17.9
0.081
0.286
58.6
(0.245–0.370)
(0.532–0.870)
(0.052–0.106)
(0.241–0.357)
Primo-ban-20
0.393
0.726
12.1
0.126
0.387
41.5
(0.331–0.495)
(0.596–0.965)
(0.094–0.159)
(0.326–0.484)
UnitsLC
50 (concentration causing 50% mortality) and LC
90 (concentration causing 90% mortality) in units of μg/mL. 95% confidence interval (CI) is shown in parenthesis. Insecticidal activity is considered significantly different when the 95% CI fail to overlap. aChi-square value of Probit model fit, all significant at P
<
0.05 level.


## 4. Discussion

Recently, biological activity has been demonstrated for theM. lucida essential oil, such as the complete suppression of aflatoxin synthesis in maize [29], cytotoxic activity on cancer cell lines [30], and antioxidant and anti-inflammatory activity [20]. This study demonstrated a potent insecticidal activity forM. lucida, showing 100% mortality onC. maculatus at concentrations of 0.20 and 0.40 μg/mL after 72 h (Figure 1).Our results forM. lucida essential oil are consistent with other reports of essential oils that exhibited insecticidal and repellency activity againstC. maculatus, such as Eucalyptus intertexta, Callistemon viminalis, Ocimum americanum, Hyptis suaveolens, Hyptis spicigera, Lippia multiflora, Cymbopogon citratus, and Lippia rugosa[25, 31–35]. Chaubey [36] evaluated the insecticidal, oviposition, egg hatching and developmental inhibitory activities of seven different essential oils againstCallosobruchus chinensisand found thatNigella sativa was the most effective at all stages. Moreover, Kéita et al. [16] found that essential oils ofOcimum basilicum andO. gratissimum at a dose of 25 μg/mL after 12 h of fumigation produced 80% and 70% mortality, respectively. Similarly, Moharramipour et al. [37] have shown a repellency of 82.4% of theThymus persicus essential oil againstC. maculatus.The chemical components of this essential oil have been previously reported [38, 39], although there were differences in the ratio of their chemicals constituents. Okoh et al. [38] identified 50 compounds where α-terpinene was the major constituent with 17.8%, while in this study it represented only 0.8%. This difference could be attributed to the number of compounds identified in each study, since in this study, 63 components were identified, representing 95.4% of the total oil content. The essential oil was found to be dominated by oxygenated monoterpenes (Table 1).Several essential oils components have shown insecticidal activity againstC. maculatus. The leaf oil ofCymbopogon schoenanthus,rich in piperitone, gave 90% mortality after 24 h at a concentration of 6.7 μL/L [40].Plectranthus grandifolius essential oil, predominantly (E)-myroxide, was shown to be toxic to both adults and eggs ofC. maculatus.Likewise,Cinnamomum aromaticumbark oil, rich incis-cinnamaldehyde, was insecticidal with an LC50 of 27.6 μg/cm2 after 24 h [41]. The essential oil fromCymbopogon giganteus, rich in limonene andp-mentha-1(7),8-dien-2-ol, demonstrated insecticidal activity againstC. maculatus andC. subinnotatus [42]. The concentrations of the major terpenoids constituents presented here forM. lucidaessential oil are consistent with use of this plant as an insecticide and insect repellent.The insecticidal activity of theM. lucida essential oil could be attributed to those known major components of oxygenated monoterpenes: 1,8-cineole (43.4%), α-terpinyl acetate (14.5%), 4-terpinen-4-ol (3.4%), α-terpinol (3.4%), and monoterpene hydrocarbons: sabinene (8.2%) and β-pinene (4.0%). These are compounds whose insecticide and repellent activities have been reported previously [32, 43, 44]. Nevertheless, it also has been shown that minor components may contribute to the biological activity [45] such as Myrcene, α-phellandrene, and Camphene.The intensive use of synthetic insecticides not only produces genetic resistance by insect species but also causes serious problems of toxicity to humans and other animal species [6, 7]. Therefore, the essential oil extracted fromM. lucida represents a botanical insecticide source, since it showed a potent insecticidal activity comparable to both pesticides, Phostoxin, and Primo-ban-20 (Table 2, where confidence intervals overlap). The lack of a statistical difference betweenM. lucida essential oil and two commercial pesticides is a positive result and demonstrates their equivalence, though we cannot conclude thatM. lucida is more effective.The results obtained indicate thatM. lucida essential oil was biocidal even at lower concentration and may, therefore, be considered to be a useful alternative to synthetic insecticides.


## 5. Conclusions

This study demonstrated that essential oil extracted fromM. lucida was toxic to pulse beetleC. maculatus. Therefore, it could be used as an alternative strategy forC. maculatuscontrol and as a substitute for synthetic pesticides. However, further studies are necessary to elucidate the mode of action and their environmental impact and develop formulations to improve the insecticidal efficacy. This would benefit agricultural sectors of developing countries as these essential oils are readily available and biodegradable. The problem of volatility can be resolved through a controlled release formulation of their active chemical compositions.



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*Source: 784613-2014-07-20.xml*