Chemical Composition and Insecticidal and Repellent Effect of Essential Oils of Two Premna Species against Sitotroga cerealella

1Laboratoire d’Etude et de Recherche en Chimie Appliquée (LERCA), Ecole Polytechnique d’Abomey-Calavi/Université d’Abomey-Calavi, 01 BP 2009 Cotonou, Benin 2Laboratoire de Production Végétale, Faculté des Sciences Agronomiques/Université d’Abomey-Calavi, 01 BP 526 Cotonou, Benin 3Laboratoire d’Analyse des Extraits Végétaux et des Arômes (LEXVA Analytique), 460 rue du Montant, 63110 Beaumont, Benin 4Institut des Biomolécules Max Mousseron, Equipe “Glyco et Nanovecteurs pour le Ciblage Thérapeutique”, Faculté de Pharmacie, 15 avenue Charles Flahault, BP 14491, 34093 Montpellier, France


Introduction
The genus Premna L. (Verbenaceae) contains about 200 species worldwide, which are distributed mainly in tropical and subtropical parts of Asia, Africa, Australia, and the Pacific Islands [1,2].Previous studies on chemical constituents of the genus Premna led to the isolation of triterpenes, diterpenes, and sesquiterpenes [3], some of them exhibiting significant antibacterial activities [4].When antiparasitic activity of new Caledonian medicinal plants including Premna serratifolia L. was evaluated, it was observed that P. serratifolia was active against Leishmania donovani with IC 50 values between 0.5 and 5 g⋅mL −1 [5].In addition, some preparations of plants of the genus Premna have been used in the treatment of liver disorders and antioxidant as well as for their immune modulatory effects [6] in the traditional Indian system of medicine.The Premna genus can be used in treating various ailments like rheumatism, asthma, dropsy, cough, fever, boils, and scrofulous disease.Traditionally in tropical Africa and Benin in particular, people with low purchasing power very often use odors produced by aromatic plants collected locally for the control of insects.This is the case of Premna angolensis Gürke and Premna quadrifolia Schum.& Thonn., leaves of which are burned and used by producers, as a fumigant in the attics of cereals against pests.In this study, extracts from leaves of both species P. angolensis and P. quadrifolia harvested in Benin were analyzed by gas chromatography-mass spectrometry (GC/MS) for the first time.The insecticidal

Plant Material and Distillation of the Volatile Constituents.
Leaves of P. angolensis and P. quadrifolia were collected from the municipality of Comé by the authors and certified at the National Herbarium of Abomey-Calavi University.The collected plant materials were stored in the laboratory between 18 and 20 ∘ C in the shade of the sunlight throughout the period of extraction.The essential oils were obtained by hydrodistillation of the leaves (450 g) for five hours using a Clevenger-type extractor according to British Pharmacopoeia method [7].They were then dried over anhydrous sodium sulfate and analyzed by GC/MS.

Test
Organisms.Individuals of S. cerealella used for mass rearing for this study come from the reserve WARDA (Benin).Rearing conditions were  = 29±2 ∘ C, RH = 70±10%.

GC/MS.
The essential oils were analysed on a Hewlett-Packard gas chromatograph Model 7890, coupled to a Hewlett-Packard MS Model 5875, equipped with a DB5 MS column (30 m × 0.25 mm; 0.25 m), programming from 50 ∘ C (5 min) to 300 ∘ C at 5 ∘ C/min, 5 min hold.Helium was used as carrier gas (1.0 mL min −1 ); injection was made in split mode (1 : 30); injector and detector of temperature were at 250 and 280 ∘ C, respectively.The MS working in electron impact mode was at 70 eV; electron multiplier was 2500 eV; ion source temperature was 180 ∘ C; mass spectra data were acquired in the scan mode in m/z range 33-450.

GC/FID.
The essential oils were analyzed on a Hewlett-Packard gas chromatograph Model 6890, equipped with a DB5 MS column (30 m × 0.25 mm; 0.25 m), programming from 50 ∘ C (5 min) to 300 ∘ C at 5 ∘ C/min, 5 min hold.Hydrogen was used as carrier gas (1.0 mL min −1 ); injection was in split mode (1 : 60); injector and detector temperature were 280 and 300 ∘ C, respectively.The essential oil was diluted in hexane: 1/30.The compounds assayed by GC in the different essential oils were identified by comparing their retention indices with those of reference compounds in the literature and confirmed by GC-MS by comparison of their mass spectra with those of reference substances [8][9][10].

Test.
All tests were performed at a temperature of 29±2 ∘ C and natural photoperiod with relative humidity 70 ± 10%.

Contact Toxicity
Tests with Essential Oils.Bioassays were performed using the method described by Noudogbessi et al. [11].The temperature of the test medium ranged from 25 ∘ C to 31 ∘ C and relative humidity was 80%.Five concentrations (0, 3, 5, 10, and 15 L mL −1 ) depending on the selected chemical profile of each essential oil in absolute ethanol solution were tested.The plant material, paddy rice grains of the variety "IR841," was treated with each essential oil.After 24 h, 5 males and 10 females of S. cerealella, aged 0 to 24 h, were deposited on the treated plant material.Overall, 5 doses × 3 repeats × 2 types of oil = 30 experimental units (containers) were implemented.The control sample treated only with ethanol was also infested by S. cerealella.Adult mortality was monitored 96 h after exposure to the essential oils.Then the insects were separated from the grains.The emergence of new insects was then observed at intervals of 24 h in the experimental units to the 50th day after infestation.Assessment concerned the rate of infested grains and weight loss of grains of rice.
To assess the S. cerealella damage on the rice seeds after treatment, the substrate grains were collected, counted, and weighed.The rate of weight loss of dry material of the grains was determined according to MCP (Method of Counting and Weighing) and MSVW (Method of Standard Volumetric Weight) [12].Two criteria of assessment of the damage are commonly used: the percentage of attack of grains (%) and percentage of weight loss (%) [13], respectively, calculated by the equations: where Na is number of attacked grains, Ns is number of healthy grains, Pa is weight of damaged grains, and Ps is weight of healthy grains.

Repellency Tests.
The repellent action of the plants was tested in an olfactometer (Figure 1), consisting of a 75 cm glass tube of 4 cm in diameter, with a 29 mm hole in the middle.At either end of the tube, a small jar was placed containing either 10.0 g containing paddy rice treated with only ethanol or 10.0 g of paddy rice seeds mixed with a solution of essential oil at concentrations 0.1%, 0.5%, and 1%.The hole in the middle was covered with gauze, whereas the ends of the tube were closed by putting a plastic petri dish against them.Air was gently (ca I mls) sucked away from the centre of the tube to prevent the accumulation of plant odor in the tube.Ten female S. cerealella aged 24 h to 48 h were released one after the other in the middle of the tube through the hole.Insect behavior was observed and the position was recorded for 15 min.Both volatile extracts were tested once before the second round.Insects were considered to have made a choice when entered in one of the jars containing rice grains or those who have reached the last twenty-five centimeters of the tube.For those who chose the middle of the tube, they have finally made a choice.The percentage of insects rejected was calculated using the following formula: where  is average number of insects present in the untreated portion (insects repelled) and  is average number of insects in the treated (not repelled insects) part.

Statistical Analysis.
The results from the observations were statistically processed by Analysis of Variance (ANOVA) using SAS (Statistical Analysis System) Version 9.1.Finally, an average of the percentages of the three results of the test was made.The results of statistical tests were considered significantly different if the probability of the hypothesis was less than or equal to 5%.
The results of the estimation of the influence of different concentrations (0 to 15 L mL −1 ) of the two essential oils on adult mortality of S. cerealella, the emergence of young insects, the rate of grains attacked, and loss corresponding weights are shown in Tables 2 and 3. Mortality rates of insects in Table 2 were obtained 4 days after treatment of the grains of paddy rice which ranged between 0 and concentrations 15 L mL −1 .At a concentration of 3 L⋅mL −1 P. angolensis essential oil showed over 90% mortality through its contact activity on the adults of S. cerealella.The number of grains damaged and the loss of the weight of rice decrease when the dose of essential oil increases.The insecticidal effect of P. quadrifolia essential oil was less noticed compared to the essential oil of P. angolensis, with significant difference ( < 0.001).In the controls sample, we noticed that the number of adults of S. cerealella increased rapidly and reached the rate of 89.62 ± 0.01 with P. quadrifolia and 85.71 ± 0.03 with P. angolensis.
Repellent Effect of Essential Oils Tested.The percentages of repulsion for the different doses of the essential oil of the leaves of P. quadrifolia and P. angolensis are summarized in Table 4.The different doses of essential oils (0.1%, 0.5%, and 1%) caused 67.11% to 96.84% repellency for P. angolensis and 65.55% to 91.55% for P. quadrifolia.This clearly shows that the percentage of repulsion increases with the dose.

Discussion
The output value of the essential oil from the leaves of P. quadrifolia was relatively better (0.102% ± 0.005) than that of P. angolensis (0.056% ± 0.004).No data have been reported in the literature on the essential oils of those two species.Essential oils P. quadrifolia and P. angolensis showed different chemical compositions.They were potentially rich in sesquiterpenoid compounds.During this study, insecticidal and repellent activity of both oils against S. cerealella was valued according to the treatment and the dose of applied oil.Previous studies on other species of Premna showed that their chemical compositions were rich in caryophyllene, -cadinene, -selinene, and phytol [15,16].The insecticide nature of essential oils of P. angolensis and P. quadrifolia manifested by contact adult insects S. cerealella may be linked to the main volatile compounds extracted reportedly acting alone or in synergy with other minor constituents.In fact, the essential oil of the leaves of P. angolensis contains monoterpenes and oxygenated hydrocarbon such as -pinene and Oct-1-en-3-ol and Linalool which insecticidal properties have been demonstrated against Tribolium confusum, Tribolium castaneum, Sitophilus zeamais, Callosobruchus maculatus, and Rhyzopertha dominica [11,17].
The repulsion rates evaluated of P. angolensis essential oil showed a greater repellent activity against adults of Angoumois grain moth than P. quadrifolia essential oil belonging, respectively, to the repulsive V and IV class, according to MC-Donald and Guyr [14] ranking.This can be explained by the strong offensive odor of both oil and confirm the use of the leaves by farmers in protecting their grain in some parts of Benin.Insect repellent effect of the extract of P. angolensis volatile proved more interesting than that of P. quadrifolia despite having a very low yield of essential oil.
The use of essential oils in the preservation of cereals is increasingly recognized [18].Essential oils are nowadays known as neurotoxins acutely interfering with octopaminergic transmitters in Arthropods [19].Different doses of oils caused highly significant mortality ( < 0.001) in comparison with the mortality rates in the controls.Paddy rice is the most common form of conservation; only S. cerealella species is likely to multiply in the paddy rice which confirms the results of previous work [20].Increasing doses of oil also reduced significantly the emergence of adults 50 days after infestation rates.Such a reduction in emergence rates results from the manifestation of ovicidal or larvicidal volatile extracts, which have destroyed the development of some eggs    or larvae probably due to sesquiterpenoid compounds or oxygenated aliphatic and synergy with minor compounds.

Figure 1 :
Figure 1: Olfactometer set-up.An individual female Angoumois grain moth was introduced in the center of the tube.Her position was recorded after 15 min since introduction.Treated and control paddy rice were positioned at either end of the tube.

Table 1 :
Yield and chemical composition of essential oil of P. angolensis and P. quadrifolia leaves.

Table 2 :
Rate of S. cerealella death, of emergence, of number of rice attacked (), and of weight loss () provoked by P. angolensis essential oil in contact method.* * * very highly significant difference (0.1%).The averages followed by the same letter were not significantly different at the beginning of 5%.

Table 3 :
Rate of S. cerealella death, of emergence, of number of rice attacked (), and of weight loss () provoked by P. quadrifolia essential oil in contact method.
0: ethanol treatment (control); * * * very highly significant difference (0.1%).The averages followed by the same letter were not significantly different at the beginning of 5%.

Table 4 :
Percentage (%) of the repellence with olfactometer of the essential oil of the leaves of P. angolensis and P. quadrifolia on adult Sitotroga cerealella.