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Persistence of active compounds of essential oils of clausena anisata (Rutaceae) and Plectranthus Glandulosus (Labiateae) used as insecticides on maize grains and flour.

INTRODUCTION

Regulations for food safety and quality management are world widely enforced by laws to protect consumers. If products are properly treated with insecticides, the side effect residues on consumers and mostly mammals are acceptably low or under control. Because of their persistence many chlorinated pesticides (such as DDT) in spite of their insecticidal efficiency are nowadays prohibited [1]. Aromatic plants locally used as crop protectant by local farmers could be sources of new pesticides. Their essential oils or other extracts are used for crop protection as alternative to hazardous pesticides because of their availability, their insecticidal efficiency, and their biodegradability [2]. To achieve a good control of the pest, the protectant having high biodegradability potential must be applied frequently. This has the consequence of increasing the amount of residue on the treated products. The present work aims to consider the case of essential oils of 2 local aromatic plants which will be used for the control of 2 major pests of cereals grain or flour during storage. Their damages due to insects affect the quality, the quantity, the commercial and agronomic value of the product [1]. The targeted pests Sitophilus zeamais Motschulsky and Tribolium castaneum Herbst are major pests of stored grains and grain products in the tropics [3]. Coming back to natural products as plant extracts could be an alternative to synthetic insecticides. In the northern part of Cameroon, peasants frequently introduce local aromatic plants in their granaries while filling them up with grain as a means of protecting them against insect attack during storage [4]. Studies of essential oils extracted from these active plants have already shown their potential for pest control and may contribute to alleviate losses recorded during grain storage [5, 6, 7, 8].

Previous investigations have already shown the potential activity of the essential oil of Clausena anisata and Plectranthus glandulosus that can lead to more than 90% mortality by direct application on adults of S. zeamais and T. castaneum at a lower concentration [9].

Despite the previous study on bioactivity of C. anisata and P. glandulosus to various insects, its persistence and residuals components against T. castaneum and S. zeamais had not been determined. The present research was therefore undertaken to investigate the bioactivity of the essential oil of C. anisata and P. glandulosus against adults of T. castaneum and S. zeamais, two important stored product insect pests in grain storage in Northern Cameroon.

MATERIALS AND METHODS

1. Stored product insect pests used

Two pests, S. zeamais and T. castaneum were used at adult stage and with known age. They were obtained from laboratory permanent rearing kept in the dark in incubators at 28 [+ or -] 2.2 [degrees]C and 65 [+ or -] 5.7% r.h. S. zeamais reared on white maize (CMS 8504), while T. castaneum was kept on flour of the same maize strain mixed with yeast (10:1, w:w). Adults of the two species of insects used for bio assays were 2 to 4 weeks old. Before being tested, these insects were starved for a 24 h period.

2. Extraction of essential oils of Clausena anisata and Plectranthus glandulosus

2.1 Extraction from leaves of aromatic plants

Plant materials were collected in the Guinean savannah surrounding the campus of the University of Ngaoundere in the Adamawa Region of Cameroon, near the point referenced latitude 07[degrees]25.11N and longitude 13[degrees]22.5E and the altitude 1,036 m. Leaves of two plants were collected and dried without sun light for 2 days and thereafter were cut in pieces with a knife. These leaves cut in pieces were introduced separately in a Clevenger apparatus for a four hours period distillation.

2.2 Extraction from treated flours

Flour treated with essential oils on the day 0 was collected on the day 14 and considered for this study. An amount of 200g of flour was introduced in a Clevenger apparatus for a four hour distillation period. The essential oil obtained was recovered with hexane and the chemical analysis made within a chromatograph and a GS/MS/FID process as that used for the former essential oils obtained from leaves.

The essential oil obtained for each distillation was put in separate flask and kept in a refrigerator at 4[degrees]C till use for tests or for analysis.

3-Analysis of chemical composition of essential oils

The GC/FID (Chromatograph Agilent HP-6890) was carried out with HP-5MS column (5% phenyl methyl siloxane) with 30 m length and 250 um in diameter and 1 um of thickness. The carrier gas was hydrogen, the oven temperature was programmed from 40 to 230 [degrees]C with a rate of 5 [degrees]C. min-1 with a stay at 230 [degrees]C during 5 min. The pressure of the carrier gas was 49.9 KPa and the flux at 74.1 mL.min-1. Quantification was carried out by percentage of peak area calculation. The identification of single compounds was performed by comparison of the retentionindices with reference data [10, 11].

4-Insecticidal efficiency of the 2 essential oils

Quantity of each essential oil ranging from 100, 200, 300, 400 [micro]L to 500 [micro]L were dropped and diluted in 10 mL of acetone to formulate the insecticidal solution. For each preparation, 350 [micro]L was pumped and flowed regularly on a disk of filter paper (Whatman no 1) placed in a Petri dish. These concentrations of essential oils were supposed to induce insect mortality ranging from 0 to 100% after 24 h. After application, 25 insects were put on the filter paper within the dish, 4 min later and it was covered. Mortality of insect was noted 24 h after the treatment. For each trial, 4 replications were made. A control with imidacloprid was made. Imidacloprid is the active molecule present in varying proportions in certain insecticides registered in Cameroon and marketed under the name ATTAKAN, and CONFIDOR GAWA. This product is approved for the most part until 2014 [12]. It comes from the Laboratory of Entomology Evolutionary and Environmental of the Faculty of Agronomic Sciences of Gembloux (Belgium), manufactured by Bayer CropScience.

5-Persistence of insecticidal activity of the essential oils

The study of the persistence activity of 2 essential oils was made by introduction of insects on treated or flour of maize put in dishes. The mortality was noted 48 hours later. The insecticide that the persistence was to be evaluated was prepared on the day 0 and kept in Petri dishes. On the day 1 and on every 2 days till the day 20, an amount of the prepared insecticide was removed and used to treat 25 insects in new Petri dishes. Four replications were made each time. Dead insects were counted 48 hours after treatment.

Regarding imidacloprid, concentrations ranging from 10 ppm to 80 ppm were prepared in acetone and applications were made as described above in the case of essential oils.

RESULTS

1-Chemical composition of essential oils of Clausena anisata and Plectranthus glandulosus

The results of the analysis of the chemical composition of the essential oils of C. anisata and P. glandulosus are represented in the table 1. The only compounds present in the two oils were [alpha]- terpinolene and the D-germacrene, but at different proportions. The oil of C. anisata was 8 times richer in D-germacrene than the P. glandulosus one; often this last contain more than 9 times [alpha]-terpinolene than the previous.

Eighteen major compounds with concentration constituting more than 1% were identified in the essential oil of C. anisata representing 95.12% of crude oil (Table 1). Sabinene, trans-linalool oxide, estragole, (E)-caryophyllene, [beta]-copaene, [alpha]-humulene, D-germacrene and (E)-nerolidol were the major constituents and representing 71.73% of this crude essential oil.

Fifteen compounds with concentration constituting more than 1% were identified in the essential oil of P. glandulosus, were P-myrcene, limonene, fenchone, aterpinolene, and piperitenone oxide representing 74.31% of the compounds of the crude oil (Table 1).

2- Insecticidal efficiency of the tested essential oils towards Sitophilus zeamais and Tribolium castaneum

The killing activities of the two essential oils towards the targeted insect pests were evaluated by the calculation of the lethal concentration (LC). A presided LC was determined by the proportion of the experimental population killed. The [LC.sub.50] and [LC.sub.99] was not the same for both essential oils, or for the 2 pests.

In the Table 2, S. zeamais was more susceptible than T. castaneum with [LC.sub.50] of 267 ppm and, 145 ppm, respectively for C. anisata and P. glandulosus. The [LC.sub.99] was at 395 ppm for C. anisata and at 246 ppm for P. glandulosus. For T. castaneum, the [LC.sub.50] concentrations for essential oils, varied from one to the others, and were found at 294 ppm with a slope of 3.41 and at 196 ppm with a slope of 2.67, respectively for C. anisata and P. glandulosus. The [LC.sub.99] of T. castaneum were at 433 ppm for C. anisata and at 276 ppm for P. glandulosus.

With imidacloprid as essential oils, the concentrations obtained vary from one insect to another. They are: [LC.sub.80]= 60 ppm and [LC.sub.99] = 80 ppm for S. zeamais, and [LC.sub.80]= 80 ppm and [LC.sub.99]= 100 ppm for T. castaneum.

3-Persistence of insecticidal activity of the tested essential oils

The results from figure 1 show that the mortality rates increasing with time following introduction of insects, and is independent of the type of food. On the maize grains, the mortality observed with essential oils was about 100% of the experimental population of S. zeamais after 4 days of exposition (Figure 1).

On the corn flour, the mortality rates of T. castaneum were 100% at the end of day 6 of exposition (Figure 2). Beyond that time, mortality decreased continually to 80% and 62%, respectively, for C. anisata and P. glandulosus after 14 days. The analysis of this mortality rates shown a highly significant difference (p<0.0001) between the time of introduction of insects for all these essential oils. The Duncan test shows a significantly difference in the level of 5% for the two active matters.

For treatments carried out with the control, imidacloprid, mortality rates were stable at 100% until the sixth first day for S. zeamais and eighth first day for T. castaneum. Beyond these times, these mortality rates fall gradually to 38 and 32% for S. zeamais, and 42 and 30% for T. castaneum, respectively at 18 and 20 days.

4-Residues of the essential oils used on flour 14 days after application

It is evident from the table 1 that compounds of essential oils decreased after 14 days of exposition to the food. The concentration of active ingredients in the essential oil of C. anisata goes from 95.12% for the raw oil to 64.42 and 55.16%, respectively, for residual oil extracted on the corn flour and grains. The cis-p-menth-2 did not exist in the residual oil extracted from the grains. Concentration of active ingredients of P. glandulosus oil decreased from 93.82% to 61.23 and, 48.94% for residual oil on the corn flour and grains, respectively. This residual P. glandulosus oil no longer contained camphor (Table 1).

DISCUSSION

The plant species which evolve in different ecological and geographical conditions can present some variations in the concentration and the quality of their secondary metabolites [13]. The resource in water, the availability of nutriments and conditions of lighting can influence mechanisms of synthesis of essential oils. The relation exists between the chemical composition of an essential oil and the geographical variations [14]. The composition of the oil of C. anisata studied was similar to the one found by [15], but at a different concentrations.

The oil of P. glandulosus presents similar composition to that found by [16] which showed that Plectranthus genus is also a rich source of piperitone and piperitinone derivatives. The essential oil of P. incanus from India, has been found to contain 35.7% of cis-piperitone and 45% of piperitenone oxide [17], while the P. defoliatus from Burundi, contained mainly piperitenone oxide (88-53%) [18]. Essential oil of P. glandulosus differs from the two species, with a higher percentage of fenchone (29.81%), terpinolene (29.28%) and a lower percentage of piperitenone oxide (11.08%). This difference could be due to ecological conditions of the different regions and plants.

The different mortality rates between the essential oils would be due to the qualitative and quantitative specific composition of the aromatic plants from which they were extracted [19]. Other investigators have shown the anti-insect activities for these and other sources of the essential oil [6, 20, 21, 22]. One investigator suggested a synergy between all compounds [21]. The difference of efficiency observed within the different species is due to the genetic diversity within the population, which can even vary from a population to another within the same species [22]. The loss of activity of essential oils could be due to the reduction of their quantity and the quality on food [23].

The duration of insecticidal activity of the studied essential oils depends on the insect and the nature of treated product. These insecticide activities decreased quickly, because their compounds are vegetal molecules belonging to groups of monoterpenes, diterpenes, sesquiterpenes, which are volatile from their photolability (loss of molecular structure due to interaction with light)[2]. Furthermore, fast deterioration of monoterpenes hydrocarbons, such as the sabinene; 1,8 cineole, and [alpha]-pinene; as well as the alcoholic compounds, are due to a high speed of the oxidization of these essential oils [19].

Previously researchers [5, 24] have shown that the essential oils of Ecalyptus calmadulensis, E. citiodora, Lippia rugosa and Ocimum gratissimum formulated with kaolin had a persistence of 10 days and 6 days for essential oils of Hyptis spicigera and L. rugosa, respectively, diluted in acetone. This weak persistence of essential oils suggests that the efficient use of these oils requires substrata that prolong in a substantial manner of their activity to improve the frequency of the successive treatments.

CONCLUSION

The present study examined persistence of active compounds of essential oils of Clausena anisata (Rutaceae) and Plectranthus glandulosus (Labiateae) for use as insecticides on maize grains and flour in northern Cameroon. This study showed that persistence of essential oils was 10 days when applied to maize and 12 days when applied to flour. At the end of this time, the concentration of active chemicals of these essential oils were still present, but reduced to one half. Local farmers can exploit this result for treatment of their products to reduce pest losses.

ACKNOWLEDGEMENTS

Authors are grateful to the Belgian Cooperation for Development for the financial support of this work through the convention Storeprotect PIC 2003.

REFERENCES

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[3.] Delobel A and M Tran The coleopterans of foodstuffs stored in the hot regions. ORSTOM / CTA. Paris. 1993; 242 p.

[4.] Anonyme. Storeprotect, Rapport semestriel Annee U.N. (periode septembre 2003-fevrier 2004).

[5.] Goudoum A Recherche de la remanence de l'activite insecticide de six huiles essentielles sur Sitophilus zeamais Motsch. (Coleoptera: Curculionidae). Memoire de maitrise, Univ. Ngaoundere. 2002; 47 pages

[6.] Kouninki H Etude de la toxicite des huiles essentielles de quelques plantes traditionnelles utilisees au Nord Cameroun contre les Bruchidae et Curculionidae. Memoire de D.E.A, Univ. Cath. Louvain. 2005; 82 pages.

[7.] Goudoum A Resistance de Tribolium castaneum Herbst (Coleoptera : Tenebrionidae) et Sitophilus zeamais Motsch. (Coleoptera : Curculionidae) au lindane et au fipronil et sensibilite de ces ravageurs a trois huiles essentielles. Memoire de D.E.A., ENSAI, Univ. de Ngaoundere. 2006 ; 91p.

[8.] Ngamo LST, Goudoum A, Ngassoum MB, Mapongmetsem PM and T Hance Chronic toxicity of essential oils of 3 local aromatic plants towards Sitophilus zeamais Motsch. (Coleoptera : Curculionidae). Afr. J. Agric. Res. 2007; Vol. 2 (4): 164-167.

[9.] Goudoum A, Ngamo LST, Ngassoum MB, Tatsadjieu NL and H Kouninki Variation in the insecticidal efficiency of pesticides towards four successive generations of Tribolium castaneum (Coleoptera: Tenebrionidae). Poster in 59th International Symposium On Crop Protection of Gent (Belgium), 29 may 2007.

[10.] Davies NW Gas Chromatographic Retention Indices of Monoterpenes and Sesquiterpenes on Methyl Silicone and Carbowax 20M Phases. J. Chromatogr. 1990;503: 1-24.

[11.] Kouroussou R, Lanara T and S Kokkini Piperitone oxide-rich essential oils from Menthalongifolia subsp. Petiolata and M. villoso-nerveta grown on the Island of Crete (Greece). J. Essent. Oil Res. 1998; 10: 375-379.

[12.] Ministere de l'Agriculture et du Developpement Rural (MINADER),). Liste des pesticides homologues au Cameroun. Fimex International. 2006 ; 16 P.

[13.] Boeke SJ, Baumgard IR, Van Huis A, Dicke M and DK Kossou Toxicity and repellence of African plants traditionally used for the protection of stored cowpea against Callosobruchus maculatus. J. Stored Prod. Res. 2004; 40: 423-438.

[14.] Azevedo NR, Campos IFP, Ferreira HD, Portes TA, Santos SC, Seraphin JC, Paula JR and PH Ferri Chemical variability in the essential oil of Hyptis suaveolens. Phytochem. 2001; 57: 733-736.

[15.] Aminatou Boubakary AB Activite anti-insecte des huiles essentielles de trois souches de Clausena anisata (Hook) (Rutaceae) sur Tribolium castaneum Herbst (Coleoptera : Tenebrionidae). Memoire de Maitrise en BPA, Universite de Ngaoundere, Cameroun. 2005, 61 p.

[16.] Ngassoum MB, Jirovetz L, Buchbauer G and W Fleischhacker Investigation of essential oils of Plectranthus glandulosus Hook f. (Lamiaceae) from Cameroon. J. Essent. Oil Res. 2001; 13 (2): 73-75.

[17.] Shah GC, Bhandari R and CS Mathela 1,2-Epoxy-p-menthane, Derivatives from Some Labiatae Species. J. Essent. Oil Res. 1992; 4: 57-59.

[18.] Hari L, Bukuru J, De Pooter HL, Demyttenaere JR and H Fierens Essential oil of Plectranthus defoliatus Hoechst. ex Benth. J. Essent. Oil Res. 1996; 8: 87-89.

[19.] Kim SI, Roh JY, Kim DY, Lee HS and YJ Ahn Insecticidal activities of aromatic plant extracts and essential oils against Sitophilus oryzae and Callosobruchus chinensis. J. Stored Prod. Res. 2003; 39: 293-303.

[20.] Huang Y, Lam SL and SH Ho Bioactivities of essential oil from Elletaria cardamomum (L.) Maton. to Sitophilus zeamais (Motschulky) and Tribolium castaneum (Herbst). J. Stored Prod. Res. 2000; 36: 11-17.

[21.] Cimanga K, Kambu K, Tona L, Apers A, De Bruyne T, Hermans N, Totte J, Pieters L and AJ Vlietinck Correlation between chemical composition and antibacterial activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo. J. Ethnopharmacol. 2002; 79: 213-220.

[22.] Muck O Quand rien ne va plus. Les phenomenes de resistance aux insecticides in Protection des denrees Stockees a Madagascar. Recueil des exposes lors du Symposium tenu a Toliara du 7 au 11 Octobre 1996, GTZ Eschborn, 5 p.

[23.] Goudoum A, Ngamo LST, Ngassoum MB and CM Mbofung Antioxidant activities of essential oils of Clausena anisata (Rutaceae) and Plectranthus glandulosus (Labiateae), plants used against stored grains insects in North Cameroon. Int. J. Biochem. Sc. 2009; 3 (3): 567-577.

[24.] Ngamo LST, Goudoum A, Ngassoum MB, Mapongmetsem PM, Kouninki H and T Hance Persistence of the insecticidal activity of five essential oils on the maize weevil Sitophilus zeamais Motsch. (Coleoptera: Curculionidae). Commun. Agric. Appl. Biol. Sci. 2004; 69 (3): 145-147.

Goudoum A (1) *, Tinkeu LSN (2), Ngassoum MB (3) and CM Mbofung (3)

* Corresponding author email: goudoumaugust@gmail.com

(1) The Higher Institute of Sahel, P.O. Box 46, Maroua, Cameroon

(2) Faculty of Sciences, Department of Biological Sciences, P.O. Box 454 Ngaoundere, Cameroon

(3) National High School of Agro Industrial Sciences, P.O. Box 455 Ngaoundere, Cameroon


Table 1: Chemical composition identified by GC of the essential oil
extracted from fresh leaves Clausena anisata and Plectrantus glandulosus
from Cameroon and from treated maize flour 14 days after application

                                           C. anisata

RT      Compounds                  Crude oil       After 14days

851     1-hexanol
943     [alpha]-pinene
977     sabinene                     4.91              1.51
991     [beta]-myrcene
1008    [delta]-3-carene
1027    limonene
1076    trans-linaloooxide           4.25              3.76
1089    fenchone
1090    [alpha]-terpinolene          2.94              1.92
1091    cis linalooloxide            1.08              0.86
1100    linalool                     1.21              1.18
1127    cis-p-menth-2-en-1-ol        1.73              0.34
1142    camphor
1146    terpinene-4-ol
1179    [rho]-cymene-8-ol
1193    estragole                    23.68             20.04
1201    methyl salicylate            2.12              1.85
1234    Z-ocimenone                  2.11              2.03
1243    E-ocimenone                  2.08              1.34
1247    cis-piperitoneoxide
1292    thymol                       6.07              3.06
1315    piperitenone
1348    [DELTA]-elemene              2.07              0.85
1353    piperitenoneoxide
1389    [alpha]-copaene              1.11              0.64
1399    isopulegone-4-methyl
1438    E-caryophyllene              4.68              1.61
1445    [beta]-copaene               4.57              1.34
1473    [alpha]-humulene             9.78              8.24
1499    germacrene D                 10.61             5.42
1571    E-nerolidol                  10.12             8.43

        Total                        95.12             64.42

                                         P. glandulosus

RT      Compounds                  Crude oil       After 14days

851     1-hexanol                    1.23              0.56
943     [alpha]-pinene               1.06              0.87
977     sabinene
991     [beta]-myrcene               5.13              2.32
1008    [delta]-3-carene              1.1              0.51
1027    limonene                      2.7              0.93
1076    trans-linaloooxide
1089    fenchone                     29.81             27.55
1090    [alpha]-terpinolene          28.29             14.67
1091    cis linalooloxide
1100    linalool
1127    cis-p-menth-2-en-1-ol
1142    camphor                      1.34
1146    terpinene-4-ol               2.51              1.33
1179    [rho]-cymene-8-ol             2.8              0.87
1193    estragole
1201    methyl salicylate
1234    Z-ocimenone
1243    E-ocimenone
1247    cis-piperitoneoxide          2.82              0.88
1292    thymol
1315    piperitenone                 1.23              0.27
1348    [DELTA]-elemene
1353    piperitenoneoxide            11.08             8.86
1389    [alpha]-copaene
1399    isopulegone-4-methyl         1.11              0.98
1438    E-caryophyllene
1445    [beta]-copaene
1473    [alpha]-humulene
1499    germacrene D                 1.61              0.63
1571    E-nerolidol

        Total                        93.82             61.23

RT = retention time; Compounds considered in this analysis have
their rate above 1%.

Table 2: Insecticidal efficiency through calculation of lethal
concentration of essential oils of Clausena anisata and Plectrantus
glandulosus used against Sitophilus zeamais and Tribolium castaneum.

                                        Imidacloprid

S. zeamais        [LC.sub.50]                60
                  [LC.sub.99]                80
                  IC                   40 < IC < 100
                  slope                     1,88

T. castaneum      [LC.sub.50]                80
                  [LC.sub.99]               100
                  IC                   60 < IC < 120
                  slope                     2,10

                                         C. anisata

S. zeamais        [LC.sub.50]               267
                  [LC.sub.99]               395
                  IC                   245 < IC < 427
                  slope                     2.32

T. castaneum      [LC.sub.50]               294
                  [LC.sub.99]               433
                  IC                   269 < IC < 458
                  slope                     3.41

                                       P. glandulosus

S. zeamais        [LC.sub.50]               145
                  [LC.sub.99]               246
                  IC                   127 < IC < 298
                  slope                     2.07

T. castaneum      [LC.sub.50]               196
                  [LC.sub.99]               276
                  IC                   163 < IC < 309
                  slope                     2.67

IC : Confidence Interval.
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Author:Goudoum, A.; Tinkeu, L.S.N.; Ngassoum, M.B.; Mbofung, C.M.
Publication:African Journal of Food, Agriculture, Nutrition and Development
Article Type:Report
Geographic Code:6CAME
Date:Jan 1, 2013
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