Available online at www.scholarsresearchlibrary.com Scholars Research Library J. Nat. Prod. Plant Resour., 2013, 3 (5):31-35 (http://scholarsresearchlibrary.com/archive.html) ISSN : 2231 – 3184 CODEN (USA): JNPPB7 Pesticidal potential of the leaves of Ocimum basilicum linn. and Hyptis spicigera lam. on Callosobruchus maculatus F. (cowpea weevils) family lamiaceae 1* Paul N. Olotu, 2Istifanus Y. Chindo, 3John S. Gushit, 4Ukpe Ajima, 1Temitayo L. Ohemu, 5 Lydia D. Ior, 4Noel N. Wannang, 1Marian I. Mmuo and 6Emma U. Onche. 1 Department of Pharmacognosy, University of Jos, Jos, Nigeria Department of Chemistry, Abubakar Tafawa Balewa University, Bauchi, Nigeria 3 Department of Science & Laboratory Technology, University of Jos, Jos, Nigeria 4 Department of Pharmaceutical Chemistry, University of Jos, Jos, Nigeria 5 Department of Pharmacology, University of Jos, Jos, Nigeria 6 Department of Chemistry, College of Education, Akwanga, Nigeria 2 _____________________________________________________________________________________________ ABSTRACT A laboratory study was conducted on the pesticidal potentials of the aromatic plants Ocimum basilicum Linn and Hyptis spicigera Lamarck both of the family Lamiaceae against Callosobruchus maculatus (cowpea weevils), a pest of stored cowpeas. The essential oils produced by these plants which are a complex mixture of terpenes, sesquiterpenes, their oxygenated derivatives and other aromatic compounds were obtained by steam or hydro/hot-distillation method using a soxhlet extractor. 70g portions of Ocimum basilicum and Hyptis spicigera powdered leaves were separately extracted sequentially using the solvents; Hexane, ethylacetate, acetone, methanol and water in order of increasing polarities. Phytochemical screening of the both plant leaves showed the presence of alkaloids, cardiac glycosides, in all the extracts; flavournoids, tannins, steroids and carbohydrates in most of the extracts; while saponins and anthraquinones tested virtually absent. The extracts of both plants were tested for pesticidal activity against untreated cowpea seeds using cowpea weevils. Within the experimental time of 24-72hrs using 100mg and 200mg of 100mg/ml concentration, the solvent extracts generally demonstrated a knock down and mortality activity. Hexane extracts showed 80% mortality and 66.67% knockdown while acetone extracts showed 75% mortality and 60%knockdown. The effects were significant for both plants at P>0.05. Both plants exhibited an increase in effect with increasing concentration of the extracts. Key words: Ocimum basilicum, Hyptis spicigera, pesticidal potential and Lamiaceae. _____________________________________________________________________________________________ INTRODUCTION Ocimum basilicum Linn and Hyptis spicigera Lamarck are both of the family Lamiaceae. Among all families of the plant kingdom, members of the Lamiaceae have been used for centuries in folk medicine. Lamiaceae or Labiatae, also known as the mint family, is a family of flowering plants and are aromatic herbs with environmental socioeconomic value in flavouring, cosmetics, perfumery, infectionary and medicinal preparations. It had traditionally 31 Scholars Research Library Paul N. Olotu et al J. Nat. Prod. Plant Resour., 2013, 3 (5):31-35 ______________________________________________________________________________ been considered closely related to Verbenaceae [1] but in the 1990s, phylogenetic studies showed that many genera classified in Verbenaceae do not belong to Lamiaceae. Essential oils produced by these plants are a complex mixture of terpenes, sesquiterpenes, their oxygenated derivatives and other aromatic compounds. The actual chemical composition is a function of species, chemotype, climate, soil conditions and geographical location [2]. Ocimum basilicum, or Sweet Basil, is a culinary herb of major importance. Most culinary and ornamental basils are cultivars of the species Ocimum basilicum, but other species are also grown and there are many hybrids between species. Traditionally, it is a green plant, some varieties such as 'Purple Delight' are purple [3]. The common names include Basil, sweet basil, bush basil, wild basil, European basil, French basil, scent leave, garden basil and ‘Dandoya kare’ in Northern part of Nigeria. Hyptis is a genus of flowering plant in the Lamiaceae family. These plants, known commonly as bushmints, are widespread in the tropics and warmer temperate regions of the Americas. There are 300 to 400 species, which may be annual or perennial [4]. It is called ‘Bunsuru fadama’ or ‘Dai fadama’ in Northern Nigeria. MATERIALS AND METHODS COLLECTION: The leaves of the Ocimum basilicum Linn. and Hyptis spicigera Lam. were collected from near bushes in Jos North Local Government Area, Plateau State, Nigeria on the 21st of December 2010. The plants were identified in the field using the description and keys described by the ‘Flora of West Tropical Africa [5] and the ‘Woody plants of Ghana” [6]. The identities of the plants were authenticated at the Department of Horticulture and Landscape Technology, Federal College of Forestry, Jos, Plateau State, Nigeria. PLANT PREPARATION: The leaves of Ocimum basilicum and Hyptis spicigera were separately dried under shade for about 2 weeks after which they were powdered separately using mortar and pestle. The powdered drugs were sieved with a mesh of size 20 and stored in separate airtight containers until required for extraction. EXTRACTION: Pulverized sample (70g) of Ocimum basilicum was exhaustively extracted in a soxhlet extractor using hexane and labeled as hexane extract. The marc was again extracted using ethyl acetate and labeled as ethyl acetate extract. The same procedure was followed with methanol and water and labeled as methanol and water extracts respectively. This was also done for Hyptis spicigera pulverized sample [7]. METHODS Phytochemical screening: The various extracts were subjected to phytochemical screening for the presence of chemical constituents such alkaloids, saponins, tannins, steroids, flavournoids, etc according to standard procedures [8]. Bioassay: Bioassay method was used for the determination of the pesticidal effects and potency of the extracts. Adult cowpea weevils (Callosobruchus maculatus) were used for this experiment. Forty (40) cowpea seeds were placed in sterilized and dried beakers and100mg/ml of each extract was prepared, from which 2mls and 1ml were measured separately and mixed with the cowpea in the different beakers and labeled properly. This was allowed to dry for 24hrs. Ten (10) live and active weevils (Callosobruchus maculatus) were then selected and placed in the beakers. This was covered tightly with a muslin cloth and left for observation at a total time interval of 72hrs for number of death of weevils. In the 2nd phase of the experiment, 1.0, 1.5 and 2.0g portions of crude dry powder samples were introduced into different 250ml beakers each containing 40 imperforated cowpea and 10 adult weevils (Callosobruchus maculatus). White muslin cloths were used to cover the mouth of the beakers to prevent the weevils from escaping out and to prevent suffocation and also to aid easy observation. The experiment set ups were allowed to stand for 72hrs also along side with a control set up containing no plant samples [7]. 32 Scholars Research Library Paul N. Olotu et al J. Nat. Prod. Plant Resour., 2013, 3 (5):31-35 ______________________________________________________________________________ At 48 and 72 hrs, the number of live weevils were observed and recorded. The number of survivors was calculated by subtracting the mortals from the original number of life weevils introduced. A control experiment without the powder sample, but with other experimental conditions constant was also set-up in each case. This was conducted for both plants. RESULTS Table 1: Phytochemical screening (a), Ocimum basilicum Chemicals /extract Alkaloid + Hexane + Ethyl acetate + Acetone + Methanol + Water Saponins + + Tannin + + + + (a) Hyptis spicigera Chemicals /extract Hexane Ethyl acetate Acetone Methanol Water Saponins + Tannin + + + + Alkaloid + + + + + Flavonoid + + + + + Carbo-hydrate + + + + + Steroids + + + - Anthra-quinone - Cardiac glycoside + + + + + Flavonoid Carbo-hydrate + + + + + + + Key; (-) absent (+) = present Steroids + + + - Anthra-quinone - Cardiac glycoside + + + + + Table 2: Percent recovery of the cowpea weevils for crude dry powder Crude plant Ocimum basilicum Hyptis spicigera Solvent Percentage recovery (%) Hexane Ethyl acetate Acetone 2.41 3.51 7.81 3.71 4.01 4.59 Methanol 0.89 0.33 Water 0.64 0.10 Table 3: Percent knockdown of the cowpea weevils for crude dry powder at different time interval Extract Ocimum basilicum %knockdown Hyptis spicigera %knockdown No. of survivors at 48hrs for 1.0g 1.5g 2.0g 10 7 6 0 17.65 25.0 10 6 9 0 25 5.26 No. of survivors at 72hrs for 1.0g 1.5g 2.0g 9 6 5 5.26 25.0 33.3 10 7 9 0 17.6 5.26 No. of survivors for Control at 24hrs 48hrs 6 6 25 25.0 9 8 5.26 11.1 Table 4(a-b): Bioassay of Ocimum basilicum solvent extracts at different concentrations a) 200mg Extract Average Total Mortality %mortality %knockdown Control %knockdown Hexane 8 80 66.67 17.6 Ethyl acetate 5.5 55 37.9 17.6 Acetone 3.5 35 21.2 11.1 Methanol 4 40 25 0 Water 4.5 45 37.9 14.3 Extract Average Total Mortality %mortality %knockdown Control %knockdown Hexane 4 40 25.0 17.6 Ethylacetate 2.5 25 14.3 17.6 Acetone 1 10 5.26 11.1 Methanol 1.5 15 8.1 0.0 Water 1.5 15 8.1 14.3 b) 100mg Table 5(a-b) Bioassay result of Hyptis spicigera solvent extracts at different concentrations a) 200mg Extract Average Total Mortality %mortality %knockdown Control %knockdown Hexane 4 40 25.0 17.65 Ethylacetate 6.5 65 48.1 14.3 Acetone 7.5 75 60.0 8.1 Methanol 6.5 65 48.1s 11.1 Water 1.5 15 8.1 2.6 33 Scholars Research Library Paul N. Olotu et al J. Nat. Prod. Plant Resour., 2013, 3 (5):31-35 ______________________________________________________________________________ (b) 100mg Extract Average Total Mortality %mortality %knockdown Control %knockdown Hexane 2 20.0 16.67 17.65 Ethyl acetate 3 30.0 17.65 14.3 Acetone 3.5 35 21.2 8.1 Methanol 3 30 17.6 11.1 Water 0.5 5.0 2.56 2.6 Table 6: Percentage mortality of extracts at different time intervals Extract Ocimum basilicum Hexane Ethylacetate Acetone Methanol Water Hyptis spicigera Hexane Ethylacetate Acetone Methanol Percent mortality at 24hr interval (%) 24hr 48hr 72hr Percent mortality of Solvent controls 24hr 48hr 72hr 70 30 10 40 30 90 60 30 40 40 10 0 0 0 20 20 10 20 0 30 20 20 30 20 30 10 40 50 10 40 40 0 60 80 0 70 80 20 10 20 0 Average No. of weevils = 10 20 20 20 20 20 40 40 20 90 80 50 60 80 DISCUSSION The use of natural products from plant sources as drugs has persisted despite recent advances in synthetic organic chemistry due to the observation that they are generally safer and possess fewer side effects compared to their synthetic counterparts [9]. Similarly, natural insecticides may be less harmful to the environment compared to synthetic ones hence their use is encouraged. From the results of the phytochemical screening carried out, both plants were found to contain alkaloids, tannins, saponins and flavonoids among other constituents. The presence of alkaloids, saponins and flavonoids which have insecticidal properties could explain the use of these plants as insecticides in the northern part of Nigeria [10]. The pesticidal activity test results on the whole crude dried leave powdered samples of the two plants showed a minimal progressive increase in the percentage mortality and knockdown with increased storage time and concentration; (<50%) knockdown rates. This means that the two leaves have minimal pesticidal activity on cowpea weevils as against the potent effect claimed by the herbalists in the Upper Volta and in Northern Nigeria to protect cowpea against damage by weevils for a whole season [4]. The results of the bioassay for the two plants showed that they both have greater affinity for non- polar solvents (as seen with acetone) than the polar solvents (as seen with water). This could disprove the claim by the Hausas in the Northern Nigeria that the water macerate is the only effective extracts used to protect cowpea seeds against weevils [7]. Pesticidal potentials on cowpea weevils of both plants using percent mortality at different time intervals showed an increase in activity with increasing time with the highest effects seen with Hexane extract and least effect seen with acetone for Ocimum basilicum but for Hyptis spicigera, acetone gave the highest effects while water gave the least effects. The reason could be that the chemical constituents of both plants showed greater affinities for non-polar solvents than the polar solvents. This can also be seen with the percent recovery of the plants extracts as it increases in the order- Water-methanolHexane -ethyl acetate- Acetone for Ocimum basilicum and Water-methanol-Hexane-ethyl acetate-Acetone respectively. From the foregoing, it can be said that the two plants contain more polar chemical constituents as compared to non-polar constituents. Hexane extract showed the highest pesticidal activity in terms of percent mortality and percent knockdown values (80% and 66.67% respectively for 200mg extracts and 40% and 25% respectively for 100mg) extracts while acetone showed the least pesticidal activity (35% and 21.2% respectively for 200mg extracts and 10% and 5.26%respectively for 100mg extracts). The percent knockdown at the lower concentration (100%) was generally 34 Scholars Research Library Paul N. Olotu et al J. Nat. Prod. Plant Resour., 2013, 3 (5):31-35 ______________________________________________________________________________ low (<50%) while at higher concentration of 200mg, the percent knockdown was only >50% (high) for Hexane (66.67%). CONCLUSION From the analytical data obtained, it showed that pesticidal potential of Ocimum basilicum Linn. and Hyptis spicigera Lam. have a low knock down and mortality rate potency (within the experimental time limit used) against cowpea weevils (Callosobruchus maculatus). The potencies increase with time and concentrations of the plant extracts. Also, the chemical constituents that have greater affinities for non-polar solvent exhibited greater pesticidal activity for both plants. REFERENCES [1] HM Raymond, S Atkins, AL Budantsev, PD Cantino, BJ Conn, RJ Grayer, MM Harley, RPJ de Kok, TV Krestovskaja, R Morales, AJ Paton, and PO Ryding. 2004. "Labiatae" 167-275. In: Klaus Kubitzki (editor) and Joachim W. Kadereit (volume editor). The Families and Genera of Vascular Plants volume VII. Springer-Verlag: Berlin; Heidelberg, Germany. ISBN 978-3-540-40593-1. [2] SE Kostyukovsky. 2006. Essential oils: Potency against stored Product insects and mode of action. Stewart Postharvest Review (Online) August 2006, 2(4) (Paper no. 5). http://www.stewartpostharvest.com/vol2_2006/August_2006/Shaaya. [3] HG Tilebeni. International Journal of Agronomy and Plant Production. 2011 2(1): 5-9. [4] JDH Lambert, J Grace, JT Arnason and BJR Philogene. Insect sciences its Applications, 1985 6(2): 167-170. [5] J Hutchinson and JM Dalziel. 1963. Flora of West Tropical Africa. Vol. II Crown Agents for Oversea Governments and Administrations. Milbank, London S. W. 51 – 54. [6] FR Irvine. 1961. Woody Plants of Ghana, Oxford University Press London. 81 – 83. [7] IC Okafor. “The pesticidal potentials and phytocemical studies of the leaves of Ocimum basilicum and Hyptis spicigera” 2004. 14-24. WB Saunders. [8] WC Evans. 2002. Text Book of Pharmacognogy 15th Edition. Billiere Tindall and [9] M Banu, GM Gururaja, M Deepak, TS Roopashree, S Shashidhara. J. Nat. Prod. Plant Resour. 2013, 3 (4):79-88 [10] S Abayomi. 1993. Historical review of Traditional Medicine in Africa. Spectrum Book Ltd, Ibadan. 9-25. 35 Scholars Research Library View publication stats