Phytochemical Screening and Effectiveness of Alstonia boonei De Wild oils as an Entomocides in the Management of Cowpea Bruchid, Callosobruchus maculatus (Fab.) [Coleoptera: Chrysomelidae].  

Kayode David Ileke1 , Olusola Olasunmbo Odeyemi2 , Michael Olufemi Ashamo2
1.Department of Environmental Biology and Fisheries, Faculty of Science, Adekunle Ajasin University, PMB 001, Akungba Akoko, Ondo State, Nigeria
2.Food Storage Technology Programme, Department of Biology, School of Science, Federal University of Technology, PMB 704, Akure, Ondo State, Nigeria
Author    Correspondence author
International Journal of Horticulture, 2014, Vol. 4, No. 6   doi: 10.5376/ijh.2014.04.0006
Received: 05 Mar., 2014    Accepted: 25 Mar., 2014    Published: 28 Mar., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Ileke et al., 2014, Phytochemical Screening and Effectiveness of Alstonia boonei De Wild oils as an Entomocides in the Management of Cowpea Bruchid, Callosobruchus maculatus (Fab.) [Coleoptera: Chrysomelidae], International Journal of Horticulture, 2014, Vol.4, No.6 24-31 (doi: 10.5376/ijh.2014.04.0006)

Abstract
Oils prepared from Alstonia boonei De Wild were tested as entomocide in the management of cowpea bruchid, Callosobruchus maculatus (Fab.) [Coleoptera: Chrysomelidae].The oil of A. boonei stem barkhad the highest mortality of 100% after 4 days of application at all level of concentrations tested. The survival of the cowpea bruchid from egg to adult when treated with the plant oils showed significantly greater mortality. Oils of  the tested plant were toxic to adult insects and also prevent adult emergence of C. maculatus. The phytochemicals present in the petroleum ether extracts of A. boonei leaf, stem bark and root were identical. Flavonoids is absent in A. boonei leaf and root but present in A. boonei stem bark and this may be responsible for it high insecticidal property. The effectiveness of the plant could be arranged in this order of efficacy thus; stem bark oil>leaf oil>root oil.
Keywords
Callosobruchus maculate; Adult emergenc; Soxhlet extraction, Entomocide; Alstonia boonei; Phytochemicals

Cowpea (Vigna unguiculata (L.) walp), is an important food legume widely distributed throughout the tropics and sub tropics (Uarrota, 2010), especially in sub-Saharan Africa, Asia, and Central and South America (Singh et al., 1997). The cowpea is a principal source of protein for the rural and urban people to combat malnutrition in young children in lieu of expensive protein source such as meat, egg and fish (Ileke et al., 2013). Cowpea feeds millions of people in the developing world with annual world production estimated at 4.5 million metric tonnes on 12 to 14 million hectares (Diouf, 2011).

One major problem encountered during storage of farm products is insect pest infestation (Adedire et al., 2011). This often leads to loss in both quality and quantity of the products (Ogunleye, 2000; Ojo and ogunleye, 2013a; 2013b; Ileke et al., 2013a). The Cowpea bruchid, Callosobruchus maculatus, is a major post harvest insect pest of grain legumes under storage conditions (Gbaye and Holloway, 2011). The larvae bore into the seeds which becomes unsuitable for human consumption, and loose it viability (Taylor, 1981).
The most effective insect pest control measure is the use of synthetic chemical insecticides. The continued and intensive usage of these insecticides has produced some undesirable toxic effects on man handling them and also on non-target biotic components of the ecosystem (Ojo and Ogunleye, 2013b). Other potential difficulties are the limited efficacy in warm-humid climates and the development of resistant pest populations. In a reaction to this problem, entomologists all over the world have resorted to testing available and environmentally friendly botanicals for this purpose (Isman, 2006). Several botanicals have been screened for insecticidal activities. These include among others: Zanthozylum zanthoxyloides, Nicotiana tabacum, Eugenia aromatica, Azadirachta indica and Dennetias tripetela (Ogunleye et al., 2004; Adedire et al., 2011; Ojo and Ogunleye, 2013b). As part of the quest for an alternative to synthetic chemical insecticides, research efforts are currently being focused on the use of plant products, such as ashes, plant powder and latex, extracts and oils, which are cheaper, safe, biodegradables and eco-friendly (Adedire and Ajayi, 1996, Adedire et al., 2011; Ileke and Oni, 2011; Ojo and Ogunleye, 2013a; 2013b). Farmers and researchers often claim successful use of plant materials in insect pest control including vegetable oil, spices and plant powders or extracts (Isman, 2006; Rajapakse and Van Emden, 1997). Their main advantage is that these materials are cheap and readily available to farmers and small scale industries in form of crude or partially purified extracts. It was reported that when mixed with stored-grains; leaf, bark, seed powder or oil extracts of plants reduced oviposition rate and suppressed adult emergence of bruchids and also reduced seed damage rate (Onu and Aliyu, 1995, Shaaya et al., 1997; Keita et al., 2001, Ojo and Ogunleye, 2013b). For these reasons, there is need to search for other plants which could comparably contend with synthetic chemical insecticides since tropical region of the world including Nigeria, are well endowed with numbers of plant species that could have entomocidal properties. The cheese wood, A. boonei De Wild (Apocyanaceae) is an African large evergreen deciduous crude medicinal tree that shed its leaves annually. The plant is about 45 m tall and 1.2 m in diameter. It possess roots, stems, barks, leaves, fruits, seeds, flowers, and latex, which are claimed to have medicinal values in some cultures in African countries. The plant stem bark and its latex are applied in traditional medicine for treating many diseases (Moronkola and Kunle, 2012).Therefore, this study investigated the phytochemical vetting and effectiveness of A. boonei oil in the management of cowpea bruchid, C. maculatus.
1 Results
1.1 Toxicity of A. boonei oils obtained by Soxhlet method on mortality of adult C. maculatus
Table 1 presented the effects of A. boonei oils obtained by soxhlet method on adult mortality of C. maculatus after 4 of application. The petroleum ether oils of A. boonei stem bark at rate 2%, 3% and 4%/20g of cowpea seeds completely caused 100% mortality of adult C. maculatus after 4 days of application. This is followed by oil of A. boonei leaf caused 100% adult cowpea bruchid at rate 4% while the least toxic was oil of A. boonei root evoked 95% adult C. maculatus at rate 4% / 20g of cowpea seeds after 4 days of application. The results obtained on oils treated seeds is significantly difference from solvent treated and untreated cowpea seeds. The stem bark oils completely caused 100% adult mortality of C. maculatus at all tested concentrations after 4 days of application.


Table 1 Mortality of adult C. maculatus in cowpea seeds treated with 4% A. boonei oils after 4 days of application


1.2 Effect of A. boonei stem bark oils obtained by Soxhlet method on oviposition and adult emergence of adult C. maculatus
Table 2, Table 3, Table 4 and Table 5 presented the effects of A. boonei oils obtained by soxhlet method on oviposition and adult emergence of adult C. maculatus after 4 of application. There was no adult emergence of C. maculatus in seed treated with Stem bark oil at all levels of concentration tested after 4 days of application (Tables 2, Table 3, Table 4; Table 5). Similar result was also obtained for oil of A. boonei stem root at rate 4% after 4 days of treatment. The results obtained on cowpea seeds treated with oils were significantly different from solvents treated cowpea seeds (Tables 2, Table 3, Table 4; Table 5).


Table 2 Oviposition and adult emergence of adult C. maculatus in cowpea seeds treated with 1% oils of A. boonei



Table 3 Oviposition and adult emergence of adult C. maculatus in cowpea seeds treated with 2% oil of A. boonei



Table 4 Oviposition and adult emergence of adult C. maculatus in cowpea seeds treated with 3% oils of A. boonei



Table 5 Oviposition and adult emergence of adult C. maculatus in cowpea seeds treated with 4% oils of A. boonei


1.3 Phytochemicals screening of Alstonia boonei
Table 6 presented the result of the phytochemical screening of the petroleum ether extracts of A. boonei leaf, stem bark and root. The phytochemicals present in the petroleum ether extracts of A. boonei leaf, stem bark and root were identical. Flavonoids is absent in A. boonei leaf and root but present in A. boonei stem bark and this may be responsible for it high insecticidal property.


Table 6 Phytochemicals in different petroleum extracts of A. boonei


2 Discussion
It is evident from the results of this study that among the three oils tested for insecticidal activity, A. boonei stem bark oil was the most effective in controlling the population of C. maculatus. However, the effectiveness of A. boonei oils were dependent on dosage rate and period of application. Previous studies by Ileke and Oni (2011); Ileke et al. (2012); Ileke et al. (2013b); Ileke et al. (2014a; 2014b) have shown insecticidal activity of A. boonei powders against Sitophilus zeamais, A. boonei stem bark oil obtained by cold extraction using five different solvents on the mortality of cowpea bruchid, C. maculatus, A. boonei latex and extracts of A. boonei obtained by kneading extraction against C. maculatus respectively. A. boonei stem bark extract completely protected cowpea seeds against C. maculatus infestation and also prevented oviposition, adult emergence and reduction in F1 progeny of C. maculatus.
The phytochemicals present in the petroleum extract of A. boonei leaf, stem bark and root includes; alkaloids, saponins, tannins, flavoniods and cardiac glycosides. Fernando et al. (2005) reported that most plants are known to possess chemical substances like terpenoides, saponins, tannins, flavonoids and alkaloids among others which have found to have reasonable efficacy against insect pests.
Plant alkaloids are a major source of bio-insecticides especially since the discovery of Azadirachtin from neem tree, Azadirachta indica (Maala et al., 2000; James et al., 2003; Bruce et al., 2004). The toxicity rate of A boonei stem bark to cowpea bruchid, C. maculatus may be as a result of alkanoids which may be more concentrated in the stem bark of the plant than other parts. Fasola and Egunyomi (2005) reported that the major phytochemicals in the stem bark of A. boonei are saponins, alkaloids, tannins, flavonoids and cardiac glycosides. Alstonia boonei bark is known to contain some chemical compounds of the indole alkaloid group namely alstonine, porphine and alstonidine as well as triterpenoids (Phillipson et al., 1987; Anonymous 1992; 2001; Moronkola and Kunle, 2012). Facknath and Lalljee (2008) reported that alkaloids and tannins from Ayapa triplinervis (Vahl) exhibited feeding deterrence against Plutella xylostella and Crocidolomia binotalis. It have been reported that tannins help in growth regulation and also protect the plants from predators (Fasola et al., 2013). Flavonoids help in protecting the plants from microbes and insect attacks (Fasola et al., 2013). The stem bark of A. boonei was the most effective part for the control of C. maculatus and this was the only part that flavonoids was detected.
The effect of the oil on oviposition could be due to respiratory impairment which probably affects the process of metabolism and consequently other systems of the body of the bruchid (Osisiogu and Agbakwuru, 1978; Onolemhemhem and Oigiangbe, 1991; Adedire et al., 2011 Ileke et al., 2013a; 2013b; Ojo and Ogunleye 2013b; Ileke et al., 2014b). Oils have been reported to inhibits locomotion (Adedire et al., 2011; Ileke et al., 2014b); hence, the beetle were unable to move freely thereby affecting mating activities and sexual communication (Adedire et al., 2011, Ileke et al., 2012; Ileke et al., 2014b).
3 Conclusion
This study has further revealed the entomocidal activity of A. boonei oils extracted by soxhlet method in the management of C. maculatus and could serve as an alternative to synthetic chemical insecticides.
4 Materials and Methods
4.1 Rearing of Insects
Newly emerged adult C. maculatus used for this studywere obtained from already existing culture in the Postgraduate Research Laboratory of the Department of Biology, Federal University of Technology, Akure, Nigeria. They were subsequently reared inside 1 litre Kilner jars, on un-infested cowpea seeds Vigna unguiculata variety Ife brown obtained from International Institute for Tropical Agriculture, Ibadan, Nigeria. The culture was placed in an insect rearing cage at ambient temperature of 28+2oC and 75+5% relative humidity.
4.2 Identification and sexing of adult Callosobruchus maculatus
The identification and sexing of C. maculatus were carried out in the Postgraduate Research Laboratory, Department of Biology, Federal University of Techno- logy, Akure, Ondo State using Binocular Microscope based on observations of Halstead (1963), Appert (1987), Odeyemi and Daramola (2000). Male have comparative shorter abdomen and the dorsal side of the terminal segment is sharply curved downward and inward. In contrast the females have comparatively longer abdomen and the dorsal side of the terminal segment is only slightly bent downward (Ileke et al., 2013a). The female also has two dark visible spots on their elytra (Halstead, 1963; Odeyemi and Daramola, 2000).
4.3 Preparation of Alstonia boonei
Leaf, stem bark and root of Alstonia boonei used for this study were sourced fresh from Akola farm at Igbara-Odo Ekiti, Ekiti State, Nigeria. These plant parts were rinsed in clean water to remove sand and other impurities, cut into smaller pieces before air-dried in the laboratory. The cleaned dried plant parts were pulverised into very fine powder using an electric blender (Supermaster ®, Model SMB 2977, Japan). The powders were further sieved to pass through 1mm2 perforation. The powders were packed in plastic containers with tight lids and stored in a refrigerator at 4oC prior to use.
4.4 Soxhlet Extraction of Alstonia boonei
The solvents used for the extraction was Petroleum ether. Thereafter, 200 g of the pulverised plant leaves, stem bark and root were measured separately into a beaker and packed in a thimble using muslin cloth and extracted with 500 ml of Petroleum ether in a soxhlet apparatus. In each case, the extraction was carried out between 40~60oC. Excess solvent was recovered using rotary evaporator vacuum. The resulting extract was air dried in order to remove traces of solvent. From this stock solution, different extract concentration of 1%, 2%, 3% and 4% were prepared separately as follows: 1% concentration was made by diluting 0.1ml of oil in 9.9ml of solvent; 2% concentration was made by diluting 0.2 ml of oil in 9.8ml of solvent; 3% concentration was made by diluting 0.3ml of oil in 9.7ml of solvent. Similarly, 4% concentration was made by diluting 0.4ml of oil in 9.6ml of solvent (Ashamo and Akinnowonu, 2012; Ileke et al., 2013b).
4.5 The contact effect of A. boonei latex, oils and extracts on C. maculatus
1%, 2%, 3% and 4% concentrations of each oils of A. booneiwas mixed separately with 20g of un-infested cowpea seeds in 250ml plastic containers. The oils and seeds were thoroughly mixed using a glass rod and then agitated for 5-10 min to ensure uniform coating. The containers were left open for 30 min to allow solvent traces to evaporate off. Two control experiments were also set up without solvent and oil and solvent treated. Ten pairs of teneral adult C. maculatus were introduced into each of the containers and covered. Four replicates of the treated and untreated controls were laid out in Complete Randomized Design in insect cage. Beetle mortality was observed after 4 days of application. The beetles were confirmed dead when there was no response to probing with sharp pin at the abdomen. The total number of eggs laid per replicate was recorded after 4 days of application. Percentage adult mortality were corrected using Abbott (1925) formular.
 

 
Where PT=corrected mortality (%)
PO=observed mortality (%)
PC=control mortality (%)
The experimental set up was kept inside the insect rearing cage for further 30 days for the emergence of the first filial generation. The percentage number of adult beetle emergence was calculated according to the method described by Odeyemi and Daramola (2000).
 

 
4.6 Phytochemical screening of A. boonei
Chemical tests were carried out on the petroleum ether extract for the qualitative determination of phytochemical constituents using standard procedures as described by Harborne (1973), Trease and Evans (1985), Sofowora (1993).
4.6.1 Alkaloids
A 0.5g sample of the extracts were stirred with 5ml of 1% aqueous hydrochloric acid on a steam bath. 1ml of the filtrate was treated with a few drops of Dragendorffs reagent. Turbidity with this reagent will be taken as an evidence for the presence of alkaloids in the extract. The extract (1g) were treated with 40% Calcium hydroxide solution until the extract was distinctly alkaline to litmus paper and then extracted twice with 10ml portions of chloroform. The extracts were combined and concentrated in vacuum to 5ml. The chloroform extract was then spotted on thin layer plates. Four different solvent systems (of widely varying polarity) was used to develop each plant part extract. The presence of alkaloids in the developed chromatograms was detected by spraying the chromatograms with freshly prepared Dragendorffs spraying reagent. A positive reaction on the chromatogram (indicated by an orange or darker coloured spot against a pale yellow background) was used
4.6.2 Saponins
The ability of saponins to produce frothing in acquenous solution and to haemolyse red blood cells was used as screening tests for saponins. For frothning tests, a 0.5 g sample of the plant extract was shaken with water in a test tube. Forthning which persists on warming was taken as preliminary evidence for the presence of saponins. In order to remove false positive results, the blood haemolysis test was performed on those extracts that frothed in water. The extract (0.5 g) was boiled briefly in 50ml phosphate buffer, pH 7.4 and then allowed to cool and filtered; 5 ml of the filtrate was passed (three hours), through an asbestos disc (1.5 mm thick and 7 mm in diameter), which was previously soaked with two or three drops of 1% cholesterol in either and dried. After filtration, the disc was washed with 0.5 ml of distilled water, dried and boiled in 20 ml of oxylol for 2 hours to decompose the complex formed between cholesterol and any saponin in the extract. The disc was washed in either, dried and placed on a 7% blood nutrient agar, complete haemolysis of red blood cells around the disc after 6 hours was be taken as confirmatory evidence of the presence of saponins.
4.6.3 Tannins
A 5g sample of the plant part extract was stirred with 10 ml of distilled water, filtered, and a few drops of 10% w/v ferric chloride reagent added to the filtrate. A blue, black, green or blue-green precipitate was taken as evidence for the presence of tannins (Trease and Evans, 1985).
4.6.4 Antraquinones
A 5g sample of the plant extract was shaken with 10ml benzene, filtered and 5 ml of 10% ammonia solution added to the filtrate. The mixture was shaken and the presence of a pink, red or violet colour in the ammonia (lower) phase indicated the presence of free hydroxyl anthraquinoes. For combined antraquinones, 5g of each plant part extract was boiled in 10ml aqueous hydrochloric acid and filtered while hot. The filtrate were shaken with 5ml of benzene layer, separated and half of its own volume of 10% (v/v) ammonia solution added. A pink, red, or violet coloration in the ammonia phase (lower layer) indicated the presence of anthraquinone derivatives in the extract (Trease and Evans, 1985).
4.6.5 Cardiac Glycosides
Legal Test: The extract (0.5 g) were dissolved in pyridine and a few drops of sodium nitroprusside together with a few drops of 20% Na0H were added. A deep red colour, which faded to brownish, yellow, indicated the presence of cardenolides.
4.6.6 Flavonoids
Each of the extracts (0.5 g) were dissolved in 5ml ethanol. This will be shaken and filtered. To 1ml of the filtrate will be added few drops of 0.5N alcoholic KOH. The mixture will be observed for yellow coloration or precipitate indicating the presence of flavonoids.
4.6.7 Phlobatanins
Deposition of a red precipitation when an aqueous extract of the plant were boiled with 1% (v/v) aqueous hydrochloric acid were taken as evidence for the presence of phlobatannins (Trease and Evans, 1985).
4.7 Data Analysis
Data were subjected to analysis of variance (ANOVA) and treatment means were separated using the New Duncan’s Multiple Range Test. The ANOVA was performed with SPSS 16.0 software (SPSS, Inc. 2007).
Acknowledgement
We want to thank the Government of Ekiti State, Nigeria for financial support through the Ph.D scholarship awarded to the first author, Kayode David Ileke tenable at the Federal University of Technology, Akure, Nigeria in 2009. We thank Mr. Oguntokun of the Department of Animal Production and Health, Federal University of Technology, Akure for his technical assistance during the phytochemical screening of the plant.
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