antimicrobial activity of selected plants from

31 Advances in Natural and Applied Sciences, 2(1): 31-34, 2008 ISSN 1995-0772 © 2008, American Eurasian Network for Scientific Information This is a refereed journal and all articles are professionally screened and reviewed

ORIGINAL ARTICLE Antimicrobial Activity of Aqueous and Ethanolic Extracts from Tithonia diversifolia and Bryum Coronatum Collected from Ogbomoso, Oyo State. Nigeria Liasu, M.O. and Ayandele, A.A. Department of Pure and Applied Biology, Ladoke Akintola University of Teghnology, Ogbomoso. Liasu, M.O. and Ayandele, A.A.,: Antimicrobial Activity of Aqueous and Ethanolic Extracts from Tithonia diversifolia and Bryum Coronatum Collected from Ogbomoso, Oyo State. Nigeria,: Adv. in Nat. Appl. Sci., 2(1): 31-34, 2008

ABSTRACT Bioassays for antimicrobial activities were carried out using stem, leaves and flowers of Tithonia diversifolia and Byrum coronatum plants. Crude aqueous and ethanolic extracts from leaves, stem and flowers of T. diversifolia and whole plant of B. coronatum were prepared and tested against gram positive bacteria i.e. Bacillus subtilis and Staphylococcus sp, gram negative bacteria Proteus vulgaris, Pseudomonas aeruginosa and Escherichia coli and fungi i.e. Penicillium atrovenetium, Aspergillus niger, Geotrichum candidium and Fusarium flocciferum .Both the aqueous and ethanolic extracts showed considerable activity against all the test organisms while ethanolic extracts of leaves and stem of T. diversifolia and B. coronatum, aqueous extract of T. diversifolia showed no activity against Proteus vulgaris. Both the aqueous and ethanolic extracts showed considerable antifungal activity against all the test fungi except the aqueous extracts of T. diversifolia leaf and B coronatum which showed no activity against Penicillium atrovenetium. The Minimum Inhibitory Concentration MIC of the plant extracts ranged from 0.01 mg/ml to 100 mg/ml. The antibacterial and antifungal activities of both the ethanolic and aqueous plant extracts were comparable to those of selected chemical antibiotics suggesting their potential as alternatives to orthodox antibiotics in the treatment of infections caused by these microorganisms. Key words: Bioassays, Ethanolic extract, Antimicrobial activity, Agar diffusion, Minimum inhibition Concentration (MIC) INTRODUCTION Medicinal plants are gifts of nature to cure limitless number of diseases among human beings (Bushra and Ganga, 2003). The abundance of plants on the earth’s surfaces has led to an increasing interest in the investigation of different extracts obtained from traditional medicinal plants as potential sources of new antimicrobial agents. (Bonjar and Farrokhi, 2004). Researches have shown that all different parts of the plants which include; stem, root, flower, bark, leaves, e.t.c. possess antimicrobial property, work of Jalalpure et al., 2004, showed that the extracts of flowers and seeds of C. auriculata possess antidiabetic activity. Recently several workers have reported antibacterial activities of local plants i.e. Euphorbia hirta (Ogbulie et al, 2007), Kigelia africana (Omokehin et al 2007), Hibiscus sabdariffa (Olaleye 2007) and Mucuna pruriens (Salau and Odeleye, 2007). Tithonia diversifolia Hemsley. Gray is a plant belonging to the family asteraceae (compositae) found widely distributed” throughout the humid and sub-humid tropics in central and south America, Asia and Africa. Chemical Corresponding Author: Liasu, M.O., Department of Pure and Applied Biology, Ladoke Akintola University of Teghnology, Ogbomoso. E-mail: [email protected]

Adv. in Nat. Appl. Sci., 2(1): 31-34, 2008

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analysis of extracts from the leaf of Tithonia showed that they contain sesquiterpene lactones e.g. Tagitinin which possess insecticidal properties (Liasu and Achakzai 2006). It is not yet known whether these substances have antimicrobial properties. Bryum coronatum, (Schwaegr) is a lower green plant of the division bryophyta. Which is found growing profusely in moist and shaded microhabitats within rural and urban ecosystems in tropical West Africa e.g. abandoned buildings, open walls of roadside gutters and embankments. An interesting feature of this plant is that quite unlike most mosses often affected by environmental changes brought about by human activities, it persist in habitats within heavily populated human settlements irrespective of industrial emissions. Another unique feature of this moss is that its community is not easily amenable to decomposition after the completion of its annual growth cycle. Hence, the community viewed “in situ” consist of superposed layers of debris of succeeding generations overtopping one another in a descending order of age with the present year’s growth lying on the surface. The fact that this moss does not decay in nature, and its persistence in situations when other plants remains must have decayed suggest that it has innate resistance to microbial agents of degradation i.e. bacteria and fungi. No known economic use has been reported for this plant. The present investigation was carried out on stem, leaves and flowers of T. diversifolia and Byrum coronatum in other to determine the antimicrobial activity of their aqueous and ethanolic extracts against five bacterial and four fungal isolates. Materials and methods The two plants samples, Tithonia diversifolia and Byrum coronatum used for the work were collected during the rainy season from the bushes around Ladoke Akintola University of Technology, Ogbomoso, Nigeria. Fresh plants were used and all the samples were grounded in a laboratory mortal into fine particles. The extraction was done with 15 gm powdered samples in distilled water and ethanol by reflux method. The crude extracts were subjected to vapourization to remove excess solvent. The crude extracts were used for bioassay against both gram negative and gram positive bacteria and fungi by agar diffusion method. Antimicrobial assay: The antimicrobial activity of three different parts of T. diversifolia and B. coronatum extracts was tested against five pathogenic bacteria, viz, gram positive, Bacillus subtilis and Staphylococcus sp and gram negative Proteus vulgaris, Pseudomonas aeruginosa and Escherichia coli, while the fungal isolates include Penicillium atrovenetium, Aspergillus niger, Geotrichum candidium and Fusarium flocciferum. The agar diffusion method of Garg and Jain (1988) was followed for the antimicrobial assay. Inoculum was prepared from the 24 hours old culture of bacterial isolates in nutrient broth while mycelia plug was cut off from the 48 hours culture of fungal isolates. Nutrient agar plates were prepared and the inocula were seeded by spread plate method, for the fungal isolates, Potato Dextrose agar were prepared and the mycelia plugs were put at the centre of the prepared plates. The extracts were applied to sterile Whatman No. 1 filter paper discs. All the samples were done in duplicate. Both positive and negative controls were determined, for negative control the two solvents (distilled water and ethanol) were also used to determine their effect on test organisms. While six commonly antibiotics discs were also used to compare the effectiveness of the plants extracts with that of the antibiotics. Antibiotics used include; Amoxicillin, Cotrimoxazole, Ciprofloxacin, Tetracycline, Ofloxacin and Gentamycin. While Clotrimazole was used to compare the effectiveness of the extracts against fungi. After 24 hours of 370c and 48 hours of 250c for bacteria and fungi inoculation, the inhibition zone surrounding the discs by the diffusion of compounds was measured in mm diameter. The minimum inhibitory concentration (MIC) of the extracts against bacteria was also determined. This was done by soaking the paper disc in different concentration of the plant extracts using the same method of agar diffusion. Zones of inhibition in mm were also measured. Results and discussion All the plants extracts used in this study possessed antibacterial property except the ethanolic extract of T. diversifolia leaf which is only active against Staphylococcus sp and E. coli with zones of inhibition of 08.00 mm and 15.00 mm respectively (Table 1). Aqueous extract of T. diversifolia stem and the ethanolic extract of its flower had the highest zones of inhibition .The activity of 10mg/ml of both the ethanolic and aqeous extracts of all the


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plants extracts against Staphylococcus aureus was similar to that of 10µg disc of ciprofloxacin except the ethanolic extract of T. diversifolia which had diameter of zone of inhibition of 08.0mm.The effectiveness of the commercial antibiotics discs used against the test organisms were as follows, 0%, 0%, 100%, 20%, 60%, and 60% for amoxicillin (25µg), cotrimoxazole (25µg), ciprofloxacin (10µg), tetracycline (30µg), ofloxacin (30µg), and gentamycin (10µg) respectively (Table 1). Distilled water and ethanol which served as negative controls produced no zones of inhibition. Table 2 shows that all the plants extracts used possess antifungal properties. Ethanolic extract of T. diversifolia was able to compare favourably with the standard antifungal agent (Clotrimazole) used. While the MIC for the plants extracts ranged from 0.01 mg/ml to 100 mg/ml. E. coli recorded the least MIC of 0.01 to 1.0 mg/ml (Table 3). Table 1: Antibacterial Activity of Plant Exracts Against Tets Organisms DIAMETER OF ZONE OF INHIBITION IN MM --------------------------------------------------------------------------------------------------------------------------------Plant Extract Staphylococcus Proteus Bacillus Pseudomonas Escherichia sp. vulgaris subtilis aeruginosa coli Ethanolic extract of T. diver flower 20.0 10.0 19.0 12.0 11.0 Aqueous extract of T. diver flower 22.0 20.0 17.0 12.0 Ethanolic extract of T. diver stem 18.0 15.0 16.0 Aqueous extract of T. diver stem 19.0 13.0 24.0 15.0 14.0 Ethanolic extract of T. diver leaf 08.0 15.0 Aqueous extract of T. diver leaf 20.0 11.0 13.0 14.0 17.0 Ethanolic extract of Byrum 22.0 17.0 15.0 14.0 Aqueous extract of Byrum 20 15.0 12.0 18.0 Control(water) Control(ethanol) Amoxicillin 25µg Cotrimoxazole 25µ Ciprofloxacin 10µg 21.0 25.5 31.5 21.0 25.5 Tetracycline 30µg 13.5 Ofloxacin 30µg 17.0 27.0 26.0 Gentamycin 10µg 13.5 12.5 14.0 Key = no growth Table 2: Antifungal Activity of the Plant Extracts DIAMETER OF ZONE OF INHIBITION IN MM Plant Extract ------------------------------------------------------------------------------------------------------------------------------Penicillium atrovenetium Aspergillus niger Geotrichum candidium Fusarium flocciferum Ethanolic extract of T. diver flower 16.0 13.8 16.0 20.0 Aqueous extract of T. diver flower 14.5 10.5 13.5 12.5 Ethanolic extract of T. diver stem 17.3 14.0 18.6 15.0 Aqueous extract of T. diver stem 16.5 08.6 10.0 08.7 Ethanolic extract of T. diver leaf 10.5 07.6 09.5 16.5 Aqueous extract of T. diver leaf 05.6 05.0 09.0 Ethanolic extract of Byrum 09.0 10.0 12.0 10.4 Aqueous extract of Byrum 06.8 09.5 08.0 Clotrimazole 21.5 18.0 21.0 19.0 Key: = no growth Table 3: Minimum Inhibition Concentration of Plant Extracts (MIC) MIC (mg/ml) Plant Extract -----------------------------------------------------------------------------------------------------------------------------------Staphylococcus Proteus Bacillus Pseudomonas E. coli aureus vulgaris subtilis aeruginosa Ethanolic extract of T. diver flower 1.0 > 100 0.01 100.0 0.01 Aqueous extract of T. diver flower 1.0 > 100 0.1 0.01 0.01 Ethanolic extract of T. diver stem 10.0 >100 100. >100 0.1 Aqueous extract of T. diver stem 0.01 1.0 0.01 0.01 0.01 Ethanolic extract of T. diver leaf > 100 >100 >100 >100 0.01 Aqueous extract of T. diver leaf 0.01 100.0 10.0 1.0 1.0 0.01 >100 0.1 10.0 0.1 Ethanolic extract of Byrum Aqueous extract of Byrum 1.0 0.1 >100 100.0 0.1


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Several workers have reported that many plants possess antimicrobial properties including the parts which include; flower, bark, stem, leaf, e.t.c. It has been shown that when solvents like ethanol, hexane and methanol are used to extract plants, most of them are able to exhibit inhibitory effect on both gram positive and gram negative bacteria (Bushra and Ganga, 2003). Similar work by Omonkhelin et al., showed that ethanolic extract of Kigelia africana has minimum inhibitory concentration of 6.25 +- 1.07 mg/ml and 7.92 +- 1.52 mg/ml for S. aureus and C. albicans. This work also showed that all the plants used possessed antimicrobial activity and they can be used as broad spectrum antibiotics since they were active against both Gram positive and Gram negative bacteria. Antibacterial effects of these plants on Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa showed that the plants can be used in the treatment of gastrointestinal infection and darrhoea in man and skin diseases (Rogger et al., 1990) and they can also be used in the treatment of urinary tract infection associated with Proteus sp. (Madigan et al., 2000). Both ethanolic and aqueous extracts of these plants can be used in the treatment of boils, sores and wounds, since Staphylococcus aureus and P. aeruginosa have been implicated as causative agents of these diseases (Braude, 1982). The low MIC exhibited by these plants extracts against some test organisms showed that they can be used as an alternative to orthodox antibiotics in the treatment of infections caused by these microorganisms and since most microorganisms are developing resistance to the known antibiotics (Singleton, 1991), they will be the best therapeutics option. References Ayo, R.G., J.O. Amupitan and Yimin Zhao, 2007. Cytotoxicity and antimicrobial studies of 1, 6, 8-trihydroxy-3methyl-anthraquinone (Emodin) isolated from the leaves of Cassia nigricans Vahl. African Journal of Biotechnology, 6(11): 1276-1279. Bushra Beegum, N.R. and T. Ganga Devi, 2003. Antibacterial activity of selected Seaweeds from Kovalam south West coast of India. Asian Jr. of Microbiol. Biotech. Env. Sc., 5(3): 319-322. Fransworth, N.R, O. Akerele and A.S. Bingel, 1985. Medicinal plants in therapy. Bull. WHO., 63: 965-981. Bonjar, Glls and Farrokhi, P.R., 2004. Antibacillus activity of some plants used in traditional medicine of Iran. Niger. J. Nat. Prod. Med., 8: 34-39. Braude, A.I., 1982. Microbiology. W. B. Sauders Company, London. Garg, S.C. and R.K. Jain, 1988. Antimicrobial efficacy of Essential oil from Curcuma caesia Indian Journal of Microbiolgy., 38: 168-17. Jalalpure, S. S., M.B. Patil, A. Aruna, B.N. Shah and M.D. Salahuddin, 2004. Antidiabetic activity of Cassia auriculata seeds in alloxan induced Diabetic rats. Niger. J. Nat. Prod. Med., 8: 22-23. Liasu, M.O. and A.K.K Achakzai, 2007. Influence of Tithonia diversifolia leaf mulch and fertilizer application on growth and yield of potted tomato plants. American-Eurasian Journal of Agric. and Environmental Science, 2(4): 335-340. Madigan, M.T., J.M. Martinko and J. Parker, 2000. Brock Biology of Microorganisms.9th ed. Prentice-Hall, Inc. New Jersey., pp: 783-784. Ogbulie, J.N., I.C. Okoli and B.N. Anyanwu, 2007. Antibacterial activities and toxicological potentials of crude ethanolic`extracts of Euphorbia hirta. African Journal of Biotechnology., 6(13): 1544-1548. Olaleye, M.T., 2007. Cytotoxicity and antibacterial activity of methanolic extract of Hibiscus sabdariffa. Journal of Medicinal Plants Research, 1(1): 009-013. Omonkhelin, J.O., K.I.O. Eric and O. Osohon, 2007. Antifungal and antibacterial Activities of the ethanolic and aqueous extract of Kigelia africana (Bignoniaceae) stem bark, African Journal of Biotechnology, 6(14): 16771680. Roggers, Y.S., L.I. John and L.W. Mark, 1990. General Microbiology, 5th ed. Macmillan education Ltd London., pp: 626-642. Salau, A.O. and O.M. Odeleye, 2007. Antimicrobial activity of Mucuna pruriens on selected bacteria. African Journal of Biotechnology, 6(18): 2091-2092. Singleton, P., 1999. Bacteria in Biology, Biotechnology and Medicine. 4th Edn. John Wiley and Sons Ltd, New York.