International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 1(7), 20-26, November (2012) I. Res. J. Biological Sci. International Science Congress Association 20 Antifungal activity of Ocimum canum Essential oil against Toxinogenic Fungi isolated from Peanut Seeds in post-harvest in Benin Adjou Euloge S., Kouton Sandrine, Dahouenon-Ahoussi Edwige, Sohounhloue Dominique C.K., Soumanou Mohamed M.* Laboratory of Research and Study in Applied Chemistry, Polytechnic School of Abomey-calavi, University of Abomey-Calavi, Cotonou, BÉNINAvailable online at: www.isca.in Received 31st August 2012, revised 10th September 2012, accepted 1st October 2012Abstract The aim of this study is to evaluate the inhibition of Aspergillus flavus and Aspergillus parasiticus isolated from peanut and their aflatoxin production exposed to the essential oils extracted from fresh leaves of Ocimum canum. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of the oil were determined. The essential oil was found to be strongly fungicidal and inhibitory to aflatoxin production. Through GC/MS analysis, an amount of 30 components were identified, representing almost 95.2% of the oil. Essential oil of O. canum was characterized by major components such as terpinene-4-ol (41.18%), linalol (14.7%) and -terpinène (6.9%). This plant offers novel approach to the management of storage fungi Key words: Bioactivity, essential oils, aflatoxin, antifungal, peanut, Benin. Introduction Investigations into the chemical and biological activities of plants during the past two centuries have yielded compounds for the development of modern synthetic organic chemistry and the emergence of medicinal chemistry as a major route for the discovery of novel and more effective therapeutic agents. Thus, plants are considered as one of the most important and interesting subjects that should be explored for the discovery and development of newer and safer drug products. In tropical areas, such as Benin, fungal deterioration of stored seeds and grains is a chronic problem. Harvested grains are colonized by various species of fungi, such as Aspergillus flavusand Aspergillus parasiticus, under such conditions leading to deterioration and mycotoxin production. Among all the mycotoxins, particularly aflatoxin B1 (AFB1) is the most toxic form for mammals and presents hepatotoxic, teratogenic and mutagenic properties, causing damage such as toxic hepatitis, hemorrhage, edema, immunosuppression and hepatic carcinoma. It has been classified as a class 1 human carcinogen by the International Agency for Research on Cancer. The presence and growth of fungi may cause spoilage of food and mycotoxin production. Therefore, the control of fungiand of aflatoxin biosynthesis is extremely important for agriculture and public health. To overcome these problems, the usual practice is to fumigate or treat the stored commodities using different synthetic preservatives. However, none of these methods has solved the problem. The increase of demand for safe and organic food, without chemical preservatives, incites many researchers to investigate the antimicrobial effects of natural compounds. Numerous investigations have confirmed the antimicrobial action of essential oils in model food systems and in real food. Essential oils are a rich source of biologically active compounds and they are potential sources of novel antimicrobial compounds. It was demonstrated that essential oils have been shown to possess antibacterial, antifungal, antiviral insecticidal and antioxidant properties. Ocimum canum is grown for its medicinal and culinary value and it is highly useful in treating various types of diseases and in lowering blood glucose, especially in type 2 diabetes levels. The traditional medicine recognized its value in the treatment of fevers, dysentary and tooth problems. It was used as an insect repellent to counter the insect damages post harvest. The herb has known antibacterial, and acts like an analgesic and rubefacient. The present study was undertaken to investigate the bioactivity potential of essential oil extracted from leaves of O. canum as antifungal agent using toxinogenic strains of Aspergillus parasiticus and Aspergillus flavus strains infecting peanut at post harvest in Benin. Material and MethodsCollection of plant leaves: Plant materials used for essential oils extraction were fresh leaves of Ocimum canum. Plants were collected at Dassa (center of Benin) and identified at the Benin national herbarium, where voucher specimens are deposited. Essential oil extraction: Essential oils tested were extracted by the hydro-distillation method using Clevenger-type apparatus. Oils recovered was dried over anhydrous sodium sulphate and stored at 4°C until it was used. Gas chromatography-mass spectrometry analysis: The EOs were analysed by gas chromatography (PerkinElmer Auto XL GC, Waltham, MA, USA) equipped with a flame ionisation detector, and the GC conditions were EQUITY-5 column (60 m x 0.32 mm x 0.25 m); H was the carrier gas; column head pressure 10 psi; oven temperature program isotherm 2 min at International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(7), 20-26, November (2012) I. Res. J. Biological Sci. International Science Congress Association 21 70°C, 3°C/ min gradient 250°C, isotherm 10 min; injection temperature, 250°C; detector temperature 280°C. Gas chromatography-mass spectrometry (GC-MS) analysis was performed using PerkinElmer Turbomass GC-MS. The GC column was EQUITY-5 (60 m x 0.32 mm x 0.25 m); fused silica capillary column. The GC conditions were injection temperature, 250°C; column temperature, isothermal at 70°C for 2 min, then programmed to 250°C at 37°C /min and held at this temperature for 10 min; ion source temperature, 250°C. Helium was the carrier gas. The effluent of the GC column was introduced directly into the source of MS and spectra obtained in the EI mode with 70 eV ionisation energy. The sector mass analyzer was set to scan from 40 to 500 amu for 2 s. The identification of individual compounds is based on their retention times relative to those of authentic samples and matching spectral peaks available with the published data10. Preparation of media: Three different media were used in this study: Potato Dextrose Agar (PDA) for isolation of toxigenic fungi, Yeast Extract Sucrose Agar (YES) for testing antifungal potential of essential oil and the conventional Dessicated Coconut Agar medium (DCA) for the detection and visualization of aflatoxin production. PDA and YES was prepared as described by N'Guyen11. DCA was prepared by modification of the method of Davis et al12, as reported by Atanda13 as follows: two hundred grams of desiccated coconut were soacked in 1L of hot distillated water for 30 min and filtered through four layers of cheese clothes. Two percent of bacteriological agar was added to the filtrate and heated for boiling. The media was then sterilized at 121°C for 15 min. Fungal isolation: All target toxinogenic fungi strains were isolated originally from infected peanuts collected in different agro ecological zones of Benin14. Strains were preserved on the Potato Dextrose Agar (Oxoid Basingstoke) at 4°C. Subcultivations on Petri dishes and other manipulations with these strains were carried out in the Bio Security Level two (BSL 2) Laboratories with respect to the BSL of Aspergillus species used in our experiment. Antifungal assay (Direct method): Antifungal assay was performed by the agar medium assay15. Yeast Extract Sucrose (YES) medium with different concentrations of essential oil (1.5, 2.0 or 2.5 µL/ml) were prepared by adding appropriate quantity of essential oil and Tween 80, to melted medium, followed by manual rotation of Erlenmeyer to disperse the oil in the medium. About 20 ml of the medium were poured into glass Petri-dishes (9 cm). Each Petri-dish was inoculated at the centre with a mycelial disc (6 mm diameter) taken at the periphery of A. parasiticus and A. flavus colonies grown on PDA for 48 h. Control plates (without essential oil) were inoculated following the same procedure. Plates were incubated at 25°C for 8 days and the colony diameter was recorded each day. Minimal Inhibitory Concentration (MIC) was defined as the lowest concentration of essential oil in which no growth occurred. The inhibited fungal discs of the oil treated sets were re-inoculated into the fresh medium, and revival of their growth was observed. Minimal Fungicide Concentration (MFC) is the lowest concentration at which no growth occurred on the plates. Diameter of fungal colonies of treatment and control sets was measured, and percentage inhibition (PI) of fungal growth was calculated according to following formula16 . Dt PI = 1 - x 100 Dc Dt: the diameter of growth zone in the test plate; Dc: the diameter of growth zone in the control plate. Antifungal assay (Disk diffusion assay): Filter paper disks (6 mm diameter) containing 5.0 L of the crude essential oil of O.canum was applied on the surface of Yeast Extract Sucrose (YES) medium plates previously inoculated with A. parasiticusor Aspergillus flavus. The inoculated plates were incubated at 25 °C for 5 days. At the end of the period, antifungal activity was evaluated by measuring the zone of inhibition (mm) against tested fungi17. The fungicide Nystatine disc (Bio Merieux) was used as a positive control. All treatments consisted of three replicates, and the averages of the experimental results were determined. Antiaflatoxin assay: Antiaflatoxin assay was performed using DCA medium according to the method described by Atanda et al18 as followed: DCA medium with different concentrations of essential oil (1.0, 1.5, 2.0, 2.5, 3.0 or 3.5 µl/ml) were prepared by adding appropriate quantity of essential oil and Tween 80 to melted medium, followed by manual rotation to disperse the oil in the medium. About 20 ml of the medium were poured into glass Petri-dishes. Care was taken to avoid trapping air bubbles in the media. Each Petri-dish was inoculated with single spores of Aspergillus parasiticus or Aspergillus flavus and incubated at 30°C for 48 hours. Control plates (without essential oil) were inoculated following the same procedure. Thereafter, the plates were examined with some media characteristics. The reverse side of each plate, which consists of a single large colony, was observed under the long wave (365mn) UV light for blue / blue green fluorescence11,18,19. Statistical analysis: Experiments were performed in triplicate, and data analyzed are mean ± SE subjected to one-way ANOVA. Means are separated by the Tukey’s multiple range test when ANOVA was significant (P 0.05) (SPSS 10.0; Chicago, IL, USA). Results and Discussion By hydrodistillation, leaves of Ocimum canum yielded 1.2% (v/w) of essential oils. Chemical analysis by GC/MS of the components of the oils led to identification of 30 components, representing 95.2% of the essential oils of Ocimum canum. The results are given in table-1. Ocimum. canum oil has chemical compositions characterized by terpinene-4-ol (41.18%), linalol (14.7%), -terpinène (6.9%),as the major components.Essential International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(7), 20-26, November (2012) I. Res. J. Biological Sci. International Science Congress Association 22 oils exhibited pronounced antifungal activity against the growth of Aspergillus flavus and A.parasiticus. The results are given in table-2 and 3. MIC of essential oil of O.canum, was found to be 1.5 l/ml and 2.0 l/ml respectively against toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. The MFC was recorded to be 2.0l/ml and 2.5 l/ml respectively against Aspergillus flavus and Aspergillus parasiticus. The results of mycelial percentage growth inhibition (PI) are given in table-4 and indicated thatthe radial growth of strains was totally inhibited by the essential oil. Percentage of growth inhibition (PI) was significantly (P 0.05) influenced by incubation time and essential oil concentration. Mycelia growth was considerably reduced with increasing concentration of essential oil while their growth increased with incubation time. The oil was more active on the mycelia growth of A. flavus than A. parasiticus. 21.33%, 72.33%, 83.44% and 100% were the PI of the oil respectively at 1, 1.5, 2.0, 2.5l/ml on A. parasiticus after 8 days of incubation. The influence of standard fungicide (Nystatine) and the essential oil on the inhibitory zone against A. parasiticus, given in table-5, was measured at 3.2 mm and 2.4 mm (average n=3) for the fungicide and the essential oil respectively. The results obtained by the disk diffusion method showed 75% of inhibition of A. parasiticus growth for the essential oil when compared with control (Nystatine). The results of antiaflatoxinogenic assay, given in table-6, showed that EO of O. canum has important aflatoxin inhibition potential on toxigenic strain Aspergillus parasiticus. At 1.5l/ml, aflatoxin production by A. parasiticus was inhibited. Table- 1 Major components identified as constituents of essential oil of Ocimum canum Compounds RT [%] -thujène 928 1,3 -pinène 937 2,0 camphène 952 0,3 sabinène 968 0,2 -pinène 972 0,1 acetate de (Z)-3-hexényle 979 0,2 myrcène 985 2,1 -phellandrène 1016 1,4 -terpinène 1020 1,7 limonène 1030 3,4 -terpinène 1058 6,9 hydrate de sabinène 1065 1,0 terpinolène 1087 1,3 linalol 1097 14,7 acétate d’octen-3-yle 1101 0,6 camphre 1139 1,0 bornèol 1150 0,3 terpinèn-4-ol 1189 41,1 p-cymèn-8-ol 1192 0,6 -terpinéol 1205 0,4 acétate de fenchyle 1219 0,9 acétate de phenyl éthyle 1238 0,2 acétate de bornyle 1282 0,4 acétate de myrtényle 1318 0,4 butyrate de (Z)-3-hexényle 1368 0,2 -caryophylléne 1439 4,1 trans- –bergamotène 1446 4,8 -humulène 1470 0,5 germacrène D 1486 2,4 -bisabolène 1510 0,2 nérolidol 1598 0,4 oxyde de caryophyllène 1611 0,1 Total 95.2 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(7), 20-26, November (2012) I. Res. J. Biological Sci. International Science Congress Association 23 Table-2 Aspergillus flavus colony diameters recorded (mm) with essential oil of Ocimum canumDays Essential oil of Ocimum canum 1.l/ml 1.5l/ml 2.0l/ml 2.5l/ml 3.0l/ml 3.5l/ml 1 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 2 8.9±0.04 b 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 3 15.6±0.04 c 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 4 27.8±0.02 d 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 5 30.7±0.06 e 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6 38.6±0.08 f 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 7 44.2±0.05 g 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 8 49.8±0.04 h 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a Values are mean (n = 3) ± SE. The means followed by same letter in the same column are not significantly different according to ANOVA and Tukey’s multiple comparison tests. Table- 3 Aspergillus parasiticus colony diameters recorded (mm) with essential oil of Ocimum canumDays Essential oil of Ocimum canum 1.0l/ml 1.5l/ml 2.0l/ml 2.5l/ml 3.0l/ml 3.5l/ml 1 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 2 8.5±0.07 b 6.2±0.07 b 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 3 27.4±0.08 c 8.2±0.04 c 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 4 34.8±0.02 d 17.5±0.06 d 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 5 39.7±0.08 e 17.9±0.02 d 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6 50.4±0.06 f 24.4±0.05 f 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 7 62.4±0.08 g 24.5±0.02 f 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 8 70.8±0.06 h 24.9±0.05 f 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a 6.0±0.00 a Values are mean (n = 3) ± SE. The means followed by same letter in the same column are not significantly different according to ANOVA and Tukey’s multiple comparison tests. Table-4 Percentage of mycelial growth inhibition (PI) Concentrations of EO A. flavusA. parasiticus 1.0l/ml 44.66± 0.2 21.33± 0.5 1.5l/ml 100 ± 0.00 72.33± 0.1 2.0 l/ml 100 ± 0.00 83.44 ± 0.3 2.5 l/ml 100 ± 0.00 100 ± 0.00 3.0 l/ml 100 ± 0.00 100 ± 0.00 3.5l/ml 100 ± 0.00 100 ± 0.00 Table-5 Antifungal assay (disk diffusion method) Nystatine fungicide EO of Ocimum canum A. flavusA. parasiticusA. flavusA. parasiticus Inhibition zone (mm) 4.0 3.2 2.8 2.4 Table-6 Antiaflatoxinogenic assay with essential oil of Ocimum canum DaysFluorescence intensity (Essential oil of Ocimum canum) A. flavusAspergillus parasiticusControl 1.0l/ml 1.0l/ml 1.5l/ml 1 - - - - 2 - - - - 3 - + - - 4 - ++ - - 5 - +++ - + 6 - +++ - +++ 7 - +++ - +++ 8 - +++ - +++ Bright fluorescence (+++); moderate fluorescence (++); weak fluorescence (+); No fluorescence (-) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(7), 20-26, November (2012) I. Res. J. Biological Sci. International Science Congress Association 24 Essential oils are natural mixtures of hydrocarbons and oxygen (alcohols, aldehydes, ketones, carboxylic acids, esters, and lactones) containing organic substances of plants. Their constituents and derivatives have a long history of application as antimicrobial agents in the areas of food preservation and medicinal antimicrobial production20. Biological activities of essential oils depends on the qualitative and quantitative characteristics of their components, which is affected by the plant genotype, plant chemotype, organ of plant, geographical origin, season, environmental, agronomic conditions, extraction method and storage condition of plant and essential oils 21,22 . The present study explores the bioefficacy of essential oils of O.canum as the promising plant-based antimicrobials against toxinogenic fungi and their aflatoxin production. The essential oil was found to be effective against A. flavus and A. parasiticus. The antifungal activity was very pronounced on A. flavus than A. parasiticus. The bioactivity of the essential oil may be due to the presence of some highly fungitoxic components in the oil. Indeed O.canum essential oil has monoterpenes alcohol as the major components. Terpenes are hydrocarbons produced from combination of several isoprene units (C) and have a hydrocarbon back bone which can be rearranged into cyclic structures by cyclases, thus forming monocyclic or bicyclic structures23. The main terpenes are monoterpenes (C1016) and sesquiterpenes (C1524), but longer chains such as diterpenes (C2032), triterpenes (C3040), etc., also exist. Terpenes do not represent a group of constituents with high inherent antimicrobial activity. For example, cymene, one of the major constituents in thyme, had no antimicrobial activity against several Gram-negative pathogens even at 85700g/mL concentration24. In a large scale experiment, limonene, -pinene, -pinene, -3-carene, (+)-sabinene, and - terpinene showed no or low antimicrobial activity against 25 different genera of bacteria that pose problems in animals, plants, and food products25. These in vitro tests indicate that terpenes are inefficient as antimicrobials when applied as single compounds. Terpenoids are terpenes that undergo biochemical modifications via enzymes that add oxygen molecules and move or remove methyl groups23. Terpenoids can be subdivided into alcohols, esters, aldehydes, ketones, phenols, and epoxides. The antimicrobial activity of most terpenoids is linked to their functional groups, and it has been shown that the hydroxyl group of phenolic terpenoids and the presence of delocalized electrons are important for antimicrobial activity. For example, the antimicrobial activity of the carvacrol derivatives carvacrol methyl ether and -cymene were much lower than carvacrol25,26,27. Exchanging the hydroxyl group of carvacrol with methyl ether affects its hydrophobicity, antimicrobial activity, and changes how the molecule interacts with the membrane28. Carvacrol’s antimicrobial activity is comparable to that of 2-amino--cymene, which indicates that the hydroxyl group is important, but not essential for carvacrol’s activity28. The antimicrobial activity of essential oils can often be correlated to its content of phenolic constituents29. Dorman and Deans25 investigated the effect of many terpenoids against 25 different bacterial strains, and showed that all terpenoid compounds, except borneol and carvacrol methyl ester, exhibited abroad antimicrobial activity. The antimicrobial activity of carvacrol, thymol, linalool, and menthol were evaluated against Listeria monocytogenes, Enterobacter aerogenes, E. coli, and Pseudomonas aeruginosa. Themostactive compound was carvacrol followed by thymol with their highest MIC being 300 and 800g/mL, respectively30. These results confirm the high antimicrobial activity of a broad collection of terpenoids, and because their chemical structures are closely related to that of terpenes. The increased activity compared to terpenes can be attributed to the functional moieties. In our study, GC–MS data, depicted remarkable variation with the earlier reports on the oils31. The chemical profile of EOs is reported to be influenced by the harvest period. Climatic, seasonal and geographical conditions and the amount and composition of active constituent can be significantly affected32-35. Thus, the biologically active EO should be qualitatively standardized before their recommendation for practical exploitation as has been done in the present investigation. The findings of the present investigation clearly showed that aflatoxin production was significantly inhibited at concentrations lower than MIC of oil (O. canum). Hence, essential oil would be acting by two different modes of action as inhibitor of fungal growth and aflatoxin production. Based on such observation, it may be also concluded that the EO is more active as aflatoxin inhibitors than as fungal growth suppressors as emphasized by the earlier workers32. The use of natural plant extract provides an opportunity to avoid synthetic chemical preservatives and offers novel approach to the management of storage fungi. It was a promising method for preserving stored products in rural areas, which do not have access to modern storage system. Conclusion This survey underlined the bioactivity of essential oil of fresh leaves of O. canum from Beninas aflatoxin inhibitor and fungal growth suppressor. Monoterpene hydrocarbons were the main components present in the volatile extract. Based on their antifungal and antiaflatoxin potentials, essential oil of O. canum from Benin may be recommended as preservative of stored food commodities from fungal and aflatoxin contamination in storage system. This research gives also justification to the use of the leaves of O. canum intraditional medicine practices for the cure of different ailments. The leaves of this plant therefore can be used as a potential source of useful drugs. AcknowledgementsThe authors are grateful to the Department of Food Engineering and Technology of Polytechnic School of Abomey-Calavi University for their financial support. Authors wish to express their gratitude to Mr. Arnaud Sagbo for the technical assistance. References 1.Siddiqui H.H., Safety of herbal drugs-an overview, Drugs News and Views, 1(2), 7–10 (1993) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(7), 20-26, November (2012) I. Res. J. Biological Sci. 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