Chemical Composition and Insecticidal and Repellent Effect of Essential Oils of Two Premna Species against Sitotroga cerealella
Abstract
This work aims to study for the first time the chemical composition and evaluate insecticidal and repellent effects of essential oils of Premna angolensis and Premna quadrifolia leaves, against Sitotroga cerealella, an insect pest of rice stocks as alternatives to synthetic pesticides. The GC-MS analysis showed that essential oil of P. angolensis contains 29 compounds representing 96.1% of the oil and 42 compounds corresponding to 91% for the essential oil of P. quadrifolia. The main constituents regardless of the species were β-caryophyllene (13.1%), (E)-β-caryophyllene (13.5%), octen-3-ol (3.2%–28%), phytol (3.7%–4.9%), β-elemene (1.4%–21%), globulol (11.2%), germacrene-D (8.9%), α-humulene (2.9%–6.4%), α-pinene (5%), sabinene (3.7%), δ-cadinene (0.4%–3.3%), and linalool (3.3%). The results of laboratory tests showed that both essential oils have insecticidal and repellent effects on S. cerealella. Presenting the results, the damage caused by the adults and larvae of S. cerealella was evaluated by calculating the percentage of grains attacked and weight loss thereof. The results suggest that volatile extracts of P. angolensis and P. quadrifolia can be used as alternatives to synthetic chemicals in paddy protection against S. cerealella.
1. 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 IC50 values between 0.5 and 5 μg·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 and repellent activities were tested on the Angoumois grain moth Sitotroga cerealella (L.) (Lepidoptera: Gelechiidae) in rice storage.
2. Material and Methods
2.1. 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.
2.2. Test Organisms
Individuals of S. cerealella used for mass rearing for this study come from the reserve WARDA (Benin). Rearing conditions were T = 29 ± 2°C, RH = 70 ± 10%.
2.3. Analysis of the Volatile Constituents
2.3.1. 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 μm), 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.
2.3.2. 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 μm), 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 [8–10].
2.4. Test
All tests were performed at a temperature of 29 ± 2°C and natural photoperiod with relative humidity 70 ± 10%.
2.5. 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 μL 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.
2.6. 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.
The average percentage of repulsion for the essential oil was calculated and assigned according to previous ranking [14] to one of several repulsive classes ranging from 0 to V: class 0 (Percentage Repellency (PR) < 0,1%), class I (PR = 0,1–20%), class II (PR = 20,1–40%), class III (PR = 40,1–60%), class IV (PR = 60,1–80%), and class V (PR = 80,1–100%).
2.7. 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%.
3. Results
Chromatographic analysis of the essential oil extracted from leaves of P. angolensis detected 29 compounds representing 96.1% of the weight of the essential oil (Table 1). However sesquiterpenes hydrogenated (26.6%) and oxygen (20%) were predominant. As for P. quadrifolia, 43 compounds were identified representing 91% of the essential oil. It was dominated by the hydrocarbon sesquiterpenes (65.5%) (Table 1). The predominant compounds of the two essential oils, regardless of species and their origin, were β-caryophyllene (13.1%), (E)-β-caryophyllene (13.5%), octen-3-ol (3.2%–28%), phytol (3.7%–4.9%), β-elemene (1.4%–21%), globulol (11.2%), germacrene-D (8.9%), α-humulene (2.9%–6.4%), α-pinene (5%), sabinene (3.7%), δ-cadinene (0.4%–3.3%), and Linalool (3.3%).
| N° | Name of the compound | RI | Premna angolensis | Premna quadrifolia |
|---|---|---|---|---|
| (%) | (%) | |||
| 1 | (Z)-3-Hexen-1-ol | 850 | 1.0 | — |
| 2 | α-Pinene | 933 | 5.0 | — |
| 3 | Sabinene | 965 | — | 3.7 |
| 4 | β-Pinene | 970 | — | 0.6 |
| 5 | Octen-3-ol | 973 | 28.0 | 3.2 |
| 7 | Octan-3-one | 977 | 2.7 | — |
| 8 | Myrcene | 987 | 0.3 | — |
| 9 | Octan-3-ol | 991 | 1.4 | — |
| 10 | α-Phellandrene | 996 | — | 1.0 |
| 11 | Limonene | 1022 | — | 1.9 |
| 12 | β-Phellandrene | 1023 | — | 0.6 |
| 13 | p-Cymene | 1024 | 0.5 | 0.8 |
| 14 | Eucalyptol | 1025 | — | t |
| 15 | Limonene | 1027 | 1.4 | — |
| 16 | (E)-β-Ocimene | 1039 | — | 0.1 |
| 17 | γ-Terpinene | 1051 | — | 0.1 |
| 18 | (Z)-Sabinene hydrate | 1064 | — | 0.1 |
| 19 | Linalool | 1096 | 3.3 | — |
| 20 | Triacetonamine | 1108 | — | 2.8 |
| 21 | Terpinen-4-ol | 1174 | 2.5 | 0.5 |
| 22 | Naphthalene | 1178 | — | 0.9 |
| 23 | α-Terpineol | 1188 | — | 0.1 |
| 24 | Methyl salicylate | 1194 | 2.2 | — |
| 25 | Nerol | 1221 | t | — |
| 26 | Ethyl salicylate | 1266 | t | — |
| 27 | α-Cubebene | 1339 | — | 0.2 |
| 28 | α-Ylangene | 1362 | — | 0.1 |
| 29 | α-Copaene | 1369 | — | 1.7 |
| 30 | β-Elemene | 1374 | 1.4 | 21 |
| 31 | (E)-β-Damascenone | 1384 | 1.2 | — |
| 32 | α-Gurjunene | 1400 | — | 2.0 |
| 33 | β-Caryophyllene | 1414 | — | 13.1 |
| 34 | γ-Elemene | 1421 | — | 1.1 |
| 35 | (E)-β-Caryophyllene | 1424 | 13.5 | — |
| 36 | (E)-α-Bergamotene | 1424 | — | 0.8 |
| 37 | Aromadendrene | 1443 | 0.6 | — |
| 38 | (Z)-β-Farnesene | 1443 | — | 0.2 |
| 39 | Alloaromadendrene | 1454 | — | 0.4 |
| 40 | α-Humulene | 1458 | 6.4 | 2.9 |
| 41 | Selina-4,11-diene | 1466 | — | 1.1 |
| 42 | Germacrene-D | 1475 | — | 8.9 |
| 43 | β-Selinene | 1486 | 0.6 | 1.6 |
| 44 | γ-Patchoulene | 1490 | 2.8 | — |
| 45 | α-Bulnesene | 1490 | — | 1.8 |
| 46 | α-Farnesene | 1494 | — | 0.8 |
| 47 | δ-cadinene | 1499 | 0.9 | — |
| 48 | β-Bisabolene | 1499 | — | 1.9 |
| 49 | Germacrene A | 1501 | — | 0.2 |
| 50 | γ-Cadinene | 1506 | — | 0.9 |
| 51 | (Z)-Calamenene | 1512 | — | 0.4 |
| 52 | δ-Cadinene | 1525 | 0.4 | 3.3 |
| 53 | Germacrene B | 1553 | — | 1.1 |
| 54 | (E)-Nerolidol | 1562 | 0.7 | — |
| 55 | Epiglobulol | 1565 | 2.2 | — |
| 56 | Spathulenol | 1571 | — | 0.2 |
| 57 | Caryophyllene oxide | 1577 | — | 1.3 |
| 58 | Globulol | 1590 | 11.2 | — |
| 59 | Humulene epoxide II | 1616 | 0.9 | — |
| 60 | Epi-α-muurolol | 1649 | — | 1.2 |
| 61 | Selin-11-en-4-α-ol | 1653 | — | 1.5 |
| 62 | α-Cadinol | 1658 | 1.0 | — |
| 63 | Intermedeol | 1673 | 0.3 | — |
| 64 | Phytol | 2048 | 3.7 | 4.9 |
| Total (%) | 96.1 | 91% | ||
| Yield (%) | 0.056 | 0.102 | ||
| Hydrogenated monoterpenes (%) | 8.6 | 8.9 | ||
| Oxygenated monoterpenes (%) | 9.2 | 4.3 | ||
| Hydrogenated sesquiterpenes (%) | 26.6 | 65.5 | ||
| Oxygenated sesquiterpenes (%) | 20 | 9.1 | ||
| Oxygenated aliphatic compounds (%) | 31.7 | 3.2 | ||
- t (traces) = 0.1%; RI: retention index.
The results of the estimation of the influence of different concentrations (0 to 15 μL 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 μL mL−1. At a concentration of 3 μL·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 (P < 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.
| Dose (μL mL−1) | Mortality | Emerged | A (%) | B (%) |
|---|---|---|---|---|
| 0 | 18.78 ± 0.07b | 85.71 ± 0.03a | 25.19 ± 2.41b | 22.726 ± 1.955b |
| 3 | 90.12 ± 0.30a | 1.07 ± 0.06b | 0.37 ± 0.01c | 0.003 ± 0.048c |
| 5 | 94.26 ± 0.17a | 0.40 ± 0.11c | 0.32 ± 0.020c | 0.046 ± 0.028c |
| 10 | 100 ± 0.00a | 0.33 ± 0.05cd | 0.26 ± 0.02c | 0.003 ± 0.024c |
| 15 | 100 ± 0.00a | 0.13 ± 0.10d | 0.15 ± 0.02c | 0.000 ± 0.020c |
| Probability | <0.001 ∗∗∗ | <0.001 ∗∗∗ | <0.001 ∗∗∗ | <0.001 ∗∗∗ |
| CV (%) | 13.75 | 10.58 | 18.76 | 16.85 |
- 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%.
| Dose (μL mL−1) | Mortality | Emerged | A (%) | B (%) |
|---|---|---|---|---|
| 0 | 10.82 ± 0.17d | 89.62 ± 0.01a | 25.44 ± 0.93b | 24.31 ± 0.28b |
| 3 | 57.96 ± 0.05c | 2.10 ± 0.04b | 0.61 ± 0.05c | 0.01 ± 0.00c |
| 5 | 70.38 ± 0.00bc | 1.62 ± 0.08b | 0.48 ± 0.05c | 0.05 ± 0.02c |
| 10 | 76.75 ± 0.12ab | 1.28 ± 0.05b | 0.35 ± 0.04c | 0.04 ± 0.03c |
| 15 | 88.85 ± 0.17a | 0.13 ± 0.10c | 0.07 ± 0.01c | 0.04 ± 0.01c |
| Probability | <0.001 ∗∗∗ | <0.001 ∗∗∗ | <0.001 ∗∗∗ | <0.001 ∗∗∗ |
| CV (%) | 14.21 | 7.51 | 7.90 | 6.02 |
- 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%.
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.
| Dose (%) | Repellency rate (%) (±SD) | |
|---|---|---|
| P. angolensis | P. quadrifolia | |
| 0.1 | 67.11 ± 10.95 | 65.55 ± 12.86 |
| 0.5 | 88 ± 10.95 | 83.55 ± 9.24 |
| 1 | 96 ± 8.94 | 91.55 ± 11.58 |
| Mean (±SD) | 83.70 ± 1.16 | 80.22 ± 13.31 |
| Repellence class | V | IV |
4. 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, β-cadinene, α-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 α-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 (P < 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.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
Acknowledgments
The authors are grateful to Polytechnic School of Abomey-Calavi (Benin) for financial support. They are also thankful to Professor Aristide ADOMOU from National Herbarium of Benin for plants identification. The authors thank Dr. Noudogbessi that made the chemical analysis of essential oils in the laboratory of Professor Chantal Menut in France (may he rest in peace).
