ISSN: Mensah et al / Current Science Perspectives 2(4) (2016) iscientic.org. Current Science Perspectives 2(4) (2016)

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1 Current Science Perspectives 2(4) (2016) Assessment of the antimicrobial and antioxidant activities of the secondary metabolites produced by pure cultured Curvularia lunata, Aspergillus parasiticus and Mucor spp John Kenneth Mensah 1 * Charles Kwoseh 2, Yesman Akuoko 1, Rahinatu Ali Bawah 1, Stephen Tawiah Jr. 1, Charles Anamoah 1 and Malik Borigu 2 1 Department of Chemistry, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi 2 Department of Crop and Soil Sciences. College of Agriculture and Natural Resources, KNUST, Kumasi. *Corresponding author s jkmensah75@yahoo.com A R T I C L E I N F O A B S T R A C T Article type: Research article Article history: Received January 2016 Accepted July 2016 October 2016 Issue Keywords: Curvularia lunata Aspergillus parasiticus Mucor spp. Antimicrobial Secondary metabolites Broth dilution assay Secondary metabolites biosynthesized by pure cultures of three soil-habitat fungi: Curvularia lunata, Aspergillus parasiticus and Mucor spp. were evaluated for their antimicrobial and free radical scavenging activity in vitro. Bioactive compounds were extracted separately with ethyl acetate and hexane from the culture supernatant and from intracellular debris of each fungi. Phytochemical analysis revealed the presence of alkaloids, tannins, flavonoids, glycosides, steroids, anthraquinones, terpenoids and saponins in the ethyl acetate media and intracellular extracts. The presence of coumarins and terpenoids were recorded in the hexane extracts of media and intracellular materials. Thin layer chromatographic profiling of all the extracts confirmed the presence of multiple compounds in each extract. Antimicrobial activities of extracts were assessed via broth dilution method against an array of microorganisms that included two gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis); two gramnegative bacteria (Escherichia coli, Salmonella typhi) and one fungus (Candida albicans). For all fungi, the hexane extracts exhibited a more potent microbial inhibitory activity as demonstrated by the low recorded MIC values: 0.10 mg/ml for Curvularia lunata, mg/ml for Aspergillus parasiticus and mg/ml for Mucor spp. In the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, ethylacetate media extract and hexane media extract of Mucor spp. showed substantial antioxidant activities as demonstrated by their relatively low IC 50 values of and µg/ml respectively. Extracts of Curvularia lunata, Aspergillus parasiticus and Mucor spp. are potential new sources of novel bioactive antimicrobial and antioxidant agents International Scientific Organization: All rights reserved. Capsule Summary: Ethylacetate and hexane extracts of the secondary metabolites of pure cultured Curvularia lunata, Aspergillus parasiticus and Mucor spp. displayed variable antimicrobial and antioxidant activities. Cite This Article As: John Kenneth Mensah, Charles Kwoseh, Akuoko Yesman, Ali Rahinatu Bawah, Stephen Tawiah Jr., Charles Anamoah and Malik Borigu. Assessment of the antimicrobial and antioxidant activities of the secondary metabolites produced by pure cultured Curvularia lunata, Aspergillus parasiticus and Mucor spp. Current Science Perspectives 2(4) (2016) editorcsp@bosaljournals.com

2 INTRODUCTION Natural products biosynthesized by fungi have historically exhibited a broad range of biological activities including antimicrobial action. With the increasing prevalence of microbial infections and the growing list of multi-drug resistant microbial strains, the need for the discovery and development of new classes of new anti-infectives that work through novel mechanisms of action cannot be overemphasized (Stanner et al., 2002). The generation of reactive oxygen species (ROS) through oxidative stress is associated with multiple disease etiologies and inactivation of ROS with antioxidant are known to abate the development of myriad diseases (Farrukh and Mukhtar, 2002). Consequently, vigorous ongoing efforts to address the unmet needs for antimicrobials and antioxidants still includes the screening of fungal cultures for novel antimicrobials with unprecedented mechanism of action and for new antioxidants to complement existing meagre list. Secondary metabolites from three species of cultured fungi were assessed for inhibition of microbial growth in anti-microbial assays in vitro and for inactivation of reactive oxygen species (ROS) in anti-oxidant assays in vitro. The studied extracts originate from pure cultures of Curvularia lunata, Aspergillus parasiticus and Mucor spp. Known studies have neither been undertaken to assess the anti-microbial activities nor to evaluate the anti-oxidant activities of the secondary metabolites produced from the sabouraud fermentation culture of all three fungi. The prevailing rationale that governed this study is that unique fungal colonies from the vast fungal population within soil habitats can be tapped for the production of bioactive secondary metabolites that may be efficacious novel antimicrobials and unprecedented antioxidants. All three examined fungi are common soil-habitat microbes implicated in diverse roles as spoilage microorganisms of many agricultural products including maize, wheat, barley and sorghum. Some strains of Aspergillus parasiticus are mycotoxins producing fungi (Bennett and Klic, 2003). Spores of Curvularia lunata are commonly found in immunocompromised individuals with an implicated etiologic role in mycetoma, keratomycosis, sinusitis and cerebral phaeohyphomycosis (Wilhelmus and Jones, 2001). Mucor spp has a more cosmopolitan distribution than the others with wide ranging infection of agricultural products (Garcia-Hermoso, 2011; Kwon and Hong, 2005). While the activities of the degradative enzymes produced by the cultured fungal strains belonging to all three species have been extensively studied in the past, much less is known on the antimicrobial and antioxidant activities of compounds biosynthesized within in vitro culture. This study provides preliminary antimicrobial susceptibility and antioxidant suitability assessments of crude extracts of secondary metabolites derived from the pure cultured fungal strains of Curvularia lunata, Aspergillus parasiticus and Mucor spp. The results show that extracts of the supernatant and of the intracellular debris display substantial antioxidant activities as demonstrated by the relatively low IC 50 in DPPH radical scavenging assay. Extracts also exhibited differential levels of anti-microbial activities, in broth dilution assays, that were substantially lower than clinically pure standard agents Ciprofloxacin (antibacterial) and Fluconazole (antifungal) against a panel of microorganisms that include Staphylococcus aureus (gram positive bacterium), Bacillus subtilis (gram positive bacterium), Escherichia coli (gram negative bacterium), Salmonella typhi (gram negative bacterium) and Candida albicans (fungus). This study provides additional alternative options to the conventional terrestrial plant-based approach to identifying new bioactive compounds for further development as antiinfections and as antioxidants. MATERIAL AND METHODS Chemical and reagents Nutrient broth, nutrient agar and sabouraud broth were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Ethanol, methanol and hexane were obtained from Merck Chemical Supplies (Damstadt, Germany). DPPH (2,2- Diphenyl-1-picrylhydrazyl) and ascorbic acid were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All reagents were of analytical grade. All flasks and media were sterilized by autoclaving at 120 o C for 30 min. All fermentation cultures were performed in 500-mL Erlenmeyer flasks containing 100 ml of sabouraud media and the flasks corked with cotton plugs. Culture and maintenance of microorganisms The microbes used for the assessment of antimicrobial activity were acquired from ATCC (USA) and were stored in liquid nitrogen in 20% glycerol. Microbes were maintained by regular sub-culturing on nutrient agar (bacterial) and on potato dextrose agar (PDA) (fungi) before storage at 4 o C prior to experimental use. Isolation of fungi from soil Soil samples from various farms found in KNUST were acquired and about 1 g of soil sample was mixed with 9 ml of sterilized distilled water and the mixture shaken vigorously. The resulting suspension was made to settle and the supernatant decanted into a test tube. A 1 ml of the supernatant was pipetted and diluted with 9 ml of sterile water. Using a petri dish, an aliquot (100 µl) of the diluted filtrate was spread uniformly on potato dextrose agar (PDA) medium supplemented with 50 µg/ml chloramphenicol and incubated at 37 C for 8 days. Developed fungal colonies were isolated and further sub-cultured till pure fungal cultures were obtained. Identification of fungi 96 editorcsp@bosaljournals.com

3 Table 1: Thin layer chromatography (TLC) report and phytochemical contents of the ethyl acetate extracts of the fungi culture. Fungi Number of spots from TLC and Rf values Phytochemicals present in extract Media Intracellular Media extract Intracellular extract extract extract C. lunata one Rf: 0.90 two Rf: 0.17, 0.73 Alkaloids, Flavonoids, Saponins and Glycosides Alkaloids, Flavonoids, Saponins and Glycosides A. parasiticus two Rf: 0.81, 0.83 two Rf: 0.70, 0.75 Alkaloids, Tannins, Flavonoids, Glycosides Steroids, Terpenoids and Saponins Alkaloids, Tannins Flavonoids, Glycosides, Anthraquinones, Terpenoids and Saponins Mucor spp. one Rf: 0.71 three Rf: 0.22, 0.55, 0.89 Alkaloids, Glycosides, Terpenoids and Steroids Alkaloids, Glycosides, Saponins, Tannins, Terpenoids and Steroids Table 2: Thin layer chromatography (TLC) report and phytochemical contents of the hexane extracts of the fungi culture. Fungi Number of spots from TLC and Rf values Phytochemicals present in extract Media extract Intracellular extract Media extract Intracellular extract C. lunata two Rf: 0.13, 0.85 two Rf: 0.66, 0.89 Terpenoids Terpenoids A. parasiticus one Rf: 0.22 one Rf: 0.24 Mucor spp. one Rf: 0.20 one Rf: 0.18 Strands of pure fungal culture were placed on a microscope slide and examined under a light microscope (Leica, Germany). Identification of individual fungal culture(s) was on the basis of morphological and cultural characteristics (Barnett and Hunter, 1986). Inoculation of media with fungal isolate Sterilized sabouraud broth was inoculated with a single isolated fungal colony and incubated at room temperature (27 o C) with daily shaking for 21 days. Extraction of secondary metabolites Broth cultures were filtered to separate the mycelia from media. Filtered culture supernatant was divided into two equal portions; one portion was extracted three times with equal volumes of ethyl acetate and the other portion extracted using hexane. The mycelia residue bearing intracellular metabolites was likewise divided into two portions; one half extracted three times with equal volumes of ethyl acetate and the other half extracted with hexane. Extracts were dried over anhydrous calcium sulphate, decanted and evaporated to dryness on a rotary evaporator. Dried residues were kept at -20 ºC until needed for bioactivity studies. In all cases, four different extracts were obtained for each cultured fungi. Quality control To ascertain that extracts obtained were solely from the cultured fungi of interest, a 100 µl aliquot of fermentation culture was pipetted and cultured on PDA. Developed fungal strains were identified under microscopic. All microscopically observed fungal strains contained only the fungi of interest and were not contaminated with different microbial species. Diagnostic TLC To ascertain the number of individual chemical compounds present in each extract, 20 µl of each extract was dissolved in 100 µl of chloroform. About 10 µl of the resulting solution was applied to a TLC plate coated with silica gel (Fisons Scientific silica gel S/0790/53, Loughborough England) approximately 500 µm thick. The spots were allowed to dry and developed in a chloroform mobile phase for 1 hr. Developed TLC plates were air- dried and the spots were visualized under iodine vapor. The retention factor (Rf) for each observed spot was subsequently computed editorcsp@bosaljournals.com

4 % Free radical scavenging activity % Free radical scavenging activity ISSN: Mensah et al / Current Science Perspectives 2(4) (2016) iscientic.org Fig. 1: DPPH radical scavenging activity of hexane and ethyl acetate extracts of C. lunata and control (Ascorbic acid) Hexane media extract Hexane intracellular extract Ethyl acetate media extract Ethyl acetate intracellular extract Ascorbic acid Concentration/ µgml Concentration/ µgml-1 Hexane media extract Hexane intracellular extract Ethyl acetate media extract Ethyl acetate intracellular extract Ascorbic acid Fig. 2: DPPH radical scavenging activity of hexane and ethyl acetate extracts of A. parasiticus and control (Ascorbic acid). Basic phytochemical screening The extracts were evaluated for their phytochemical constituents using protocols of Trease and Evans (1984). Test for alkaloids. Acidified solution of alkaloid produced a whiteyellowish precipitate upon addition of a few drops of Mayer s reagents. Extracts were initially heated on a boiling water bath with 2% hydrochloric acid. The mixture was allowed to cool and few drops of Mayer s reagent added. A yellow precipitate indicated the presence of alkaloids. Test for terpenoids and steroids: To a small amount of the extract 2 mg was added 250 µl of acetic anhydride and 250 µl of chloroform. Concentrated sulphuric acid (1 ml) was added slowly and a red-violet color indicated the presence of terpenes while a green-bluish color was indicative of the presence of steroids. Test for flavonoids: To a small amount of the extract was added 1.5 ml of 50% methanol and the solution warmed. Metal magnesium was then added followed by 5-6 drops of concentrated hydrochloric acid. The formation of a red color indicated the presence of flavonoids while an orange color indicated the presence of flavones. Test for tannins: To 0.5 ml of an aqueous solution of the extract was added 1-2 drops of ferric chloride solution. A blue color indicated the presence of gallic tannins while a greenish-black color indicated the presence of catecholic tannins. Test for coumarins: To 500 µl of an aqueous solution of the extract was added 750 µl of 10% NaOH. The formation of a yellow colour is indicative of the presence of coumarins editorcsp@bosaljournals.com

5 Table 3: Broth dilution for the ethyl acetate media extract Extract Organism Concentration (mg/ml) C. lunata E. Coli B. subtilis S. aureus S. typhi C. albicans A. parasiticus E. Coli B. subtilis S. aureus S. typhi C. albicans Mucor spp. E. Coli B. subtilis S. aureus S. typhi C. albicans indicates microbial growth; - indicates no microbial growth Table 4: Broth dilution for the hexane media extract Extract Organism Concentration (mg/ml) C. lunata E. Coli B. subtilis S. aureus S. typhi C. albicans A. parasiticus E. Coli B. subtilis S. aureus S. typhi C. albicans Mucor spp. E. Coli B. subtilis S. aureus S. typhi C. albicans indicates microbial growth; - indicates no microbial growth Test for saponins: To 500 µl of an aqueous solution of the extract was added 1 ml of 1% sodium bicarbonate and the solution shaken. A persistent froth is indicative of the presence of saponins. Test for anthraquinones: To 500 µl of an aqueous solution of the extract was added 10 ml of sulphuric acid (H 2SO 4) and filtered while hot. The filtrate was shaken with 5 ml of chloroform. The chloroform layer was pipetted into another test tube and 1 ml of dilute ammonia was added. The resulting solution was observed for colour changes. A delicate rose pink colour is indicative of the presence of anthraquinones. Test for glycosides: To a small amount of the extract in a test tube was added few drops of acetic anhydride. To the resulting solution was added 2-3 drops of concentrated sulphuric acid. A blue-green color indicates the presence of glycosides. Antimicrobial assay A modified protocol prescribed by Murray et al., 1999 was used in performing the broth dilution assay. Briefly, Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923) and Candida albicans (strain NCPF 113; National Collection of 99 editorcsp@bosaljournals.com

6 Table 5: Broth dilution for the ethyl acetate intracellular extract Extract Organism Concentration (mg/ml) C. lunata E. Coli B. subtilis S. aureus S. typhi C. albicans A. parasiticus E. Coli B. subtilis S. aureus S. typhi C. albicans Mucor spp. E. Coli B. subtilis S. aureus S. typhi C. albicans indicates microbial growth; - indicates no microbial growth Table 6: Broth dilution for the hexane intracellular extract Extract Organism Concentration (mg/ml) C. lunata E. Coli B. subtilis S. aureus S. typhi C. albicans A. parasiticus E. Coli B. subtilis S. aureus S. typhi C. albicans Mucor spp. E. Coli B. subtilis S. aureus S. typhi C. albicans indicates microbial growth; - indicates no microbial growth Type Cultures, London, England) were grown in Mueller- Hinton agar and broth (Difco Laboratories). Autoclaved nutrient broth was inoculated with the microbial culture and incubated at 37 o C for 24 hours. Then a 20 ml fresh broth was seeded with 0.25 ml of the 24-hour broth cultures and a twofold serial dilution method was performed as described below. The dried fungal extract was dissolved in 85% methanol and the resulting solution sterilized by filtration through a 0.45 μm membrane filter. A 0.2 ml solution of the material was added to 1.8 ml of the seeded broth to obtain the first dilution. A 1 ml of this dilution was further diluted with 1 ml of the seeded broth to produce the second dilution, and the process was repeated until eight dilutions were obtained. Tubes containing only seeded broth and 85% methanol were kept as controls. All tubes were incubated for 24 hr at 37 o C. Ciprofloxacin and fluconazole (at concentrations specified in Table 9) were used as positive controls. MICs (expressed in mg/ml) were taken as the lowest extract concentrations that showed complete growth inhibition and were represented by the last tube with no visible violet color from the addition of 0.1 ml of MTT. Antioxidant capacity determination assays 2,2-Diphenyl-1-picrylhydrazyl: The antioxidant activity for each extract was determined by the DPPH method using protocol prescribed by (Govindarajan et al., 2003; Govindarajan et al., 2005). Briefly, 1 ml solutions of the various concentrations (0.7812, , 3.125, 6.25, 12.5, 25, 50 and 100 μg/ml in methanol) of the hexane and ethyl editorcsp@bosaljournals.com

7 Table 7: MIC values of media extracts of Test fungi. Organisms Ethyl acetate media extract Hexane media extract C. lunata Mucor spp. A. parasiticus C. lunata Mucor spp. A. parasiticus E. Coli 1.49 No inhibition B. subtilis 1.49 No inhibition S. aureus 1.49 No inhibition S. typhi 1.49 No inhibition C. albicans 2.99 No inhibition No inhibition 0.14 Table 8: MIC values of intracellular extracts of Test fungi. Organisms Ethyl acetate intracellular extract Hexane intracellular extract C. lunata Mucor spp. A. parasiticus C. lunata Mucor spp. A. parasiticus E. Coli B. subtilis S. aureus S. typhi C. albicans acetate extracts were placed in different test tubes. A 3 ml 0.1 mm methanolic solution of 2,2 diphenyl-1- picrylhydrazyl radical (DPPH) (20 mg/l) was added to the tubes, shaken vigorously and kept in the dark for 30 minutes to allow for reaction. Ascorbic acid ( mg/ ml) was used as a standard free radical scavenger, the control, and was prepared as above without any extract. After reaction, absorbance was read at 517nm using a UV-Vis spectrophotometer with methanol for the baseline correction. Radical scavenging activity was expressed as the inhibition percentage and was computed using the formula shown in Eq. 1. ( ) { } (1) Where, A s is the absorbance of sample and A c is the absorbance of the control. The antioxidant activity for each extract was expressed as IC 50; the concentration (mg/ml) of sample required to scavenge DPPH radical formation by 50% and was computed from the dose response curve plotted between % inhibition and extract concentrations. RESULTS AND DISCUSSIONS Phytochemical screening and thin layer chromatography Phytochemical screening of the extracts revealed the presence of alkaloids, tannins, flavonoids, glycosides, steroids, anthraquinones, terpenoids, coumarins and saponins in media supernatant and in intracellular extracts. Each fungal specimen displayed an identical set of phytochemicals for its media and intracellular extracts (Tables 1 and 2). Generally, the ethyl acetate extracts were observed to contain a higher number of phytochemicals than the hexane extracts. Phytochemicals that are considerably polar like saponins and glycosides flavonoids and anthraquinones were noticeably absent in the hexane extracts of all three studied fungi. Notably, none of the ethylacetate extracts showed the presence of coumarins. TLC analyses on the ethylacetate extracts generally displayed multiple compounds with the intracellular extracts of Mucor spp. showing the highest number of spots (three spots). However, the ethyl acetate media extracts of C. lunata, and Mucor spp as well as the hexane extracts of the media and intracellular debris of A. parasiticus and Mucor spp could not be resolved by the solvent mixture (methanol) used as the mobile phase and thus appear as single spots on the TLC plate (Tables 1 and 2). Antimicrobial activities - Broth Dilution Assays Ethylacetate media extract of Mucor spp. was ineffective in inhibiting all microbial growth at all tested concentrations (Tables 3 and 7). With the exception of the high inhibitory concentration against Candida albicans (MIC 2.99 mg/ml), all ethylacetate media extract derived from C. lunata and A. parasiticus exhibited an MIC of 1.49 mg/ml against all other microbial specimens (Tables 3 and 7). Inhibitory concentrations of the hexane media extracts from all three fungi were lower than that of the ethylacetate extracts (Tables 4 and 7). The notable exception to this trend was the observed lack of inhibitory activity of the hexane media extracts of Mucor spp. against C. albicans (Tables 4 and 7). MICs of the hexane media extracts differed by fungal editorcsp@bosaljournals.com

8 % Free radical scavenging activity ISSN: Mensah et al / Current Science Perspectives 2(4) (2016) iscientic.org Hexane media extract Hexane intracellular extract Ethyl acetate media extract Ethyl acetate intracellular extract Ascorbic acid Concentration/ µgml-1 Fig. 3: DPPH radical scavenging activity of hexane and ethyl acetate extracts of Mucor spp. and control (Ascorbic acid). specimen; with Mucor spp extract demonstrating the lowest observable value of mg/ml against the bacterial test panel while that of C. lunata ranged from 0.27 to 0.54 mg/ml and that of A. parasiticus ranged from mg/ml to 0.14 mg/ml against all microbial species (Table 7). Except C. albicans that was inhibited at higher concentrations (4.28 mg/ml), inhibitory concentrations on all other microbial specimens for all ethylacetate intracellular extracts were fractionally lower than that of the ethylacetate media extract (Tables 5 and 8). MIC for the ethylacetate extract of intracellular fungal materials ranged from 1.07 for C. lunata through for Mucor spp to 0.53 for A. parasiticus (Tables 5 and 8). Inhibitory concentrations for hexane intracellular extracts for C. lunata were at least two-fold difference lower than its corresponding hexane media extract for all the panel of microbial specimen (Tables 6 and 8). Hexane intracellular extracts for C. lunata displayed a minimum MIC of 0.10 mg/ml for bacterial species and a maximum of 0.20 mg/ml for C. albicans (Tables 6 and 8). Hexane intracellular extract for Mucor spp showed MICs that were comparable to its corresponding hexane media extract for bacterial growth inhibition (0.018 mg/ml) and also displayed a three-fold increase in MIC (0.059 mg/ml) for C. albicans inhibition (Tables 6 and 8). A. parasiticus has a hexane intracellular extract that was less effective in inhibiting microbial growth compared to its corresponding hexane media extract. A three-fold increase in the hexane intracellular extract MIC compared to that of its corresponding hexane media extract was observed for all bacterial specimen treated with the hexane intracellular extract (Tables 6 and 8). The bioactivity of the standard drugs (antibacterial Ciprofloxacin and antifungal Fluconazole) used as positive controls were higher with MICs at lower concentrations (at least 2-fold to at most a 100- fold) than that of the extracts (Table 9). 2,2-Diphenyl-1-picrylhydrazyl Fungal extracts were screened for their antioxidant activity via the DPPH method and their relative scavenging activities expressed as IC 50. For all three fungal extracts, scavenging of DPPH radical was found to increase with increasing extract concentrations (Figures 1, 2 and 3). Additionally, DPPH Radical Scavenging Activities for both media and intracellular extracts for all three fungal extracts at all examined concentrations were substantially lower than that of the Ascorbic acid control as demonstrated in the lowest IC 50 value for Ascorbic acid (Table 10). The order of potency of DPPH scavenging for the extracts differed by fungal strain and no consistent discernable ordered pattern of scavenging was established from fungi to fungi. The most potent DPPH antioxidant activity among the series of extracts was displayed by the ethylacetate media extract of Mucor spp that has the lowest IC 50 value of µg/ml. Pairwise comparison of the IC 50 values extracts of Mucor spp showed that ethylacetate media and intracellular extracts exhibited much more potent antioxidant activities than their hexane counterparts. Antioxidants activities for all four extracts of Aspergillus parasiticus are comparable with marginal differences between their IC 50 values. By far, extracts of Curvularia lunata displayed the weakest antioxidant potential among the series of extracts recording IC 50 values that were at least three-fold difference higher than that of Mucor spp. The top three potent DPPH radical scavengers, as provided by IC 50 values, in decreasing order is Ascorbic acid < ethylacetate media extract of Mucor spp. < hexane media extract of Mucor spp. Observed antioxidant activities of the extracts are attributable to the presence of phenolic compounds including flavonoids, tannins and saponins (Table 1) in the extracts (Grassmann, 2005), (Gulcin, 2004). Since the IC 50 of the ethylacetate media extract of Mucor spp is just about twice that of Ascorbic acid, potential bioactive antioxidant compound can be isolated and characterized from this extract. CONCLUSIONS Ethyl acetate and hexane extracts of media secreted and intracellularly biosynthesized secondary metabolites of pure cultured C. lunata, A. parasiticus and Mucor spp. displayed editorcsp@bosaljournals.com

9 Table 9: Broth dilution for Standard Drugs (Ciprofloxacin and Fluconazole) Concentration E. Coli B. subtilis S. aureus S. typhi C. albicans (mg/ml) Ciprofloxacin Fluconazole indicates microbial growth; - indicates no microbial growth Table 10: DPPH radical scavenging activities of hexane and ethyl acetate extract of the fungi C. lunata, A. parasiticus, Mucor spp. and ascorbic acid expressed as IC50. Fungal extract IC50 (µg/ml) C. lunata Hexane media extract Hexane intracellular extract Ethyl acetate media extract Ethyl acetate intracellular extract A. parasiticus Hexane media extract Hexane intracellular extract 70.9 Ethyl acetate media extract Ethyl acetate intracellular extract Mucor spp. Hexane media extract Hexane intracellular extract Ethyl acetate media extract Ethyl acetate intracellular extract Ascorbic acid remarkable phytochemical diversity with the notable presence of tannins and flavonoids that accounted primarily for their observed antioxidant activities. Levels of antimicrobial activities displayed by extracts against test microorganisms Staphylococcus aureus (gram positive bacterium), Escherichia coli (gram negative bacterium), Pseudomanas aeruginosa (gram negative bacterium) and Candida albican (fungus) were lower than that of Ciprofloxacin (antibacterial) and Ketoconazole (antifungal) clinically used agents. Similarly observed radical scavenging activities demonstrated in the DPPH assay were quantitatively lower than that recorded for ascorbic acid control. This study provides additional alternative options to the conventional terrestrial plant-based approach to identifying new bioactive compounds in Ghana for further development as anti-infections and as antioxidants. REFERENCES Ali, S.S., Kasoju, N., Luthra, A., Singh, A., Sharanabasava, H., Sahuand, A., Indian medicinal herbs as source of antioxidants. Food Research International 41(1), Bennett, J.W., Klic, M., Mycotoxins; Clinical Microbiology Reviews 16(3), Clinical and Laboratory Standards Institute (CLSI), Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically: Approved Standard, M07-A8, CLSI, Wayne, Pa, USA. Dehpour, A.A., Ebrahimzadeh, M.A., Nabavi, S.F., Nabavi, S.M., Antioxidant activity of methanol extract of Ferula assafoetida and its essential oil composition. Grasas Y Aceites 60(4), Farrukh, A., Mukhtar, H., Photochemical prevention by botanical antioxidants. Skin Pharmacology and Applied Skin Physiology 15(1), Ganesh, C. K., Mongolla, P., Joseph, Y.V.D., Nageswar, A.K., Antimicrobial activity from the extracts of fungal isolates of soil and dung samples from Kaziranga National Park, Assam. Journal on Medical Mycology 66(1), Garcia-Hermoso D., Dannaoui E., Lortholary O., Dromer F., Agents of systemic and subcutaneous mucormycosis and entomophthoromycosis. Manual of Clinical Microbiology 10(1), Govindarajan, R., Rastogi, S., Vijayakumar, M., Shirwaikar, A., Rowat, A.K., Studies on antioxidant activities of Desmodium gangeticum. Biological and Pharmaceutical Bulletin 26(1), Govindarajan, R., Vijayakumar, M., Pushpangadan, P., Antioxidant approach to disease management and the role of Rasayana herbs of Ayurveda. Journal of Ethnopharmacology 99(1), editorcsp@bosaljournals.com

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