Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-6, 2016 ISSN: 2454-1362, http://www.onlinejournal.in
Effect of Aqueous and Ethanolic Leaf Extract of Khaya Senegalensis against E.Coli, Shigella and Salmonella Species *1
Abdallah, M. S., 2Gambo, A., 3Ishaq, S., 4Mustapha, T. & 5Nasir, M. 1
Research Assistant Desert Research, Monitoring and Control Centre, Yobe State University, Damaturu, Nigeria. 2 Primary health care management board Damaturu, Yobe State, 3 Department of biological science, Sule Lamido university, Kafin Hausa, Jigawa State, 4 Department of biological sciences, Yobe State University, Damaturu, 5 Chemistry department, Yobe State University, Damaturu, Abstract: Khaya senegalensis leaf extracts contain some bioactive ingredients which include; saponins, alkaloids, tannins and flavonoids. The bioactive ingredients found in the extracts showed more activity on the tested organisms. The activity of the ethanolic leaf extracts was more pronounced in all tested isolates than in the aqueous leaf extracts, as being illustrated in fig. 1 and 2 of the result analysis. Key words: Bioactive ingredients, ethanolic, aqueous and isolates INTRODUCTION Khaya senegalensis (mahogany) belongs to Meliaceae family, comprises more than fifty genera with about 1400 species (Nakatani et al., 2001) is distributed in tropical and subtropical regions. The family is represented by seventeen genera and 72 species of which twelve species and two varieties endemic in India. Approximately 18% are endemic to peninsular India. From 19th century up to the present time, the mahoganies have been the most important species for the development of the forest industry in Asia, tropical Africa and Latin America. Many species of this family were used in traditional medicine for treatment of various diseases and also in pest control (Nakatani et al., 2001). Plants have great potential uses especially as traditional medicine and pharmacopial drugs. A large proportion of the world population depends on traditional medicine (Tagbodo and Townson, 2001). Medicinal plants have provided the modern medicine with numerous plants. Derived therapeutic agents (Evans, 2000 ).Moreover, diarrhoea is a symptom of infection caused by a host of bacterial, viral and parasitic organisms most of which can be spread by contaminated water. It is more common when there is a shortage of clean water for drinking, cooking and cleaning and basic
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hygiene is important in prevention.Water contaminated with human faeces for example from municipal sewage, septic tanks and latrines is of special concern. Animal faeces also contain microorganisms that can cause diarrhoea. Plants usually contain many biologically active structurally divers compounds which are useful as drugs, or raw materials which are used primarily for treating mild or chronic diseases (Baris et al., 2006; Gonzales et al., 2009, Kumar and Pandey, 2013 ). The earliest written record on Egyptian, Greek roman, Chinese and Indian traditional medicine have listed medicinal plants and their prescriptions used in treating various ailments. Herbal medicines are in great demand in the developed as well as developing countries for primary healthcare because of their wide biological and medicinal activities, higher safety margins and user cost (Craggy et al., 1997). Even in less developed societies medicinal recopies form plants have been passes orally from generation to generation. According to ward health organization (WHO), about 80% of the world population relies chiefly on the plant based traditional medicine especially for primary health care needs (Mishra et al., 2013). Moreover, medicinal plants are the richest bio-resource of traditional system of medicine, modern medicines, Polyphenols as catching act on different bacterial strains belonging to different species (Escherichia coli, Bordetella, Bronchise fica, Serratia mercescens, Klebsiella pnemoniae, Salmonella, Shigella, Pseudomonas aeroginosa, Staphylococcus aureus and Bacillus subtilis), by generating hydrogen peroxide and by altering the permeability of the microbial membrane (Kumar et al., 2013). Moreover, microbes stressed by exposure to polyphenols unregulate proteins related to defensive mechanism, which protect cells while simultaneously regulating various metabolic and
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Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-6, 2016 ISSN: 2454-1362, http://www.onlinejournal.in biosynthetic protein involved, as shown in the table 1 below. For example, in amino acids and proteins synthesis as well as phospholipids.Carbon and energy metabolism (Hubert et al., 2003). Polyphenols have been reported to interfere with bacterial quiorum sensing, i.e the production of small signal molecules by bacterial cells of Escherichia coli, Pseudomonas putida, Burkholderia cepacia that rigger the exponential growth of a bacterial population (Hubert et al., 2003).The objectives of this research are; to determined the presence of some bioactive ingredients found in the aqueous and ethanolic leaf extract of Khaya senegalensis and also to know how the extract is against the isolates. MATERIALS AND METHOD Preparation of plant material The leaves were air dried and grounded with the aid of pestle and mortar into a coarse powder , sieved with a 1mm2 and stored in a plastic container as described by (Fatope et al., 1993).Moreover, 20 grams of the powdered leaf of K. senegalensis was weighted, which has then mixed with 200 mils of the required diluents (water and ethanol) for some days. The mixtures were filtered and the filtrate, was collected separately in a clean and label beaker (Fatope et al., 1993). The phytochemical analysis has been divided into: Qualitative and Quantitative method of plants extraction (Evans, 2000).Qualitative method of phytochemical analysis for Khaya senegalensis leaves extract: Five grams of leaves extract of Khaya senegalensis powder was separately mixed with 50ml of distilled water and ethanol. Different test tubes were used for each and every phytoconstituent. The phytoconstituents tested include: Tannin, Anthraquinone, Steroids, Terpenoids, Saponins, Flavonoids, Alkaloids, and lastly Cardiac glycosides of different procedures as far below: i. Tannin test: 0.5g of both extracts were mixed in distilled water and stirred which was then filtered. 1% ferric chloride solution was added into 2ml of each filterate, blue- black and blue- green shows positive test of the extracts (Treas and Evans, 2002). ii. Anthraquinone test: 0.2g of each extract was added in a 10ml benzene, followed by a vigorous shaking and then filtered, red-pink coloration indicates a positive test (Sofowora, 1993).
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iii. Steroids test: 2ml acetic acid was added into 0.2g of each extract, followed by a cooling in an ice. The concentrated H2SO4 was added carefully, colour change from violet to blue or bluish- green shows a positive result (Sofowora, 1993). iv. Terpenoids test: few drops of ethanol and 1ml of acetic anhydride were added into 5ml of each extract and followed by the addition of concentrated H2SO4, pink to violet shows a positive test (Sofowora, 1993). v. Saponins test: Five mils (5ml) of distilled water was added into 2g of each extract and then boiled in a water bath and lastly filtered. Three mill (3ml) of distilled water was added into each filtrate followed by vigorous shaking for 5minutes, frothing persisting on warming indicates a positive result (Sofowora, 1993). vi. Flavonoids test: Five mils (5ml) of distilled water were added into a 0.5g of each extract, followed by boiling and then filtered. Few drops of 10% ferric chloride solution was added into a 2ml filtrate, green- blue or violet coloration indicates a positive result (Trease and Evans, 2000). vii. Alkaloids test: Two mils (2ml) filtrates of each extract was added into 1% Hcl and steamed in a water bath, brownish- red precipitate shows a positive result (Sofowora, 1993). viii. Cardiac glycosides: One mils (1ml) of glacial acetic acid, 1ml ferric chloride and 1ml of concentrated sulphuric acid have been added into 2ml of the filtrates of each sample, green blue coloration of the solution shows a positive result (Sofowora, 1993). Quantitative method of phytochemical for Khaya senegalensis leaves extract:
analysis
Determination of Flavonoids : Flavonoids level was determined by gravimetric method (Harbone, 1980). Exactly 10g of the sample was extracted with 100ml of 80% aqueous methanol at room temperature were used. It was transferred into a crucible for drying in a water bath, which was dried to a constant weight in an oven. The percentage was calculated as follows : % Flavonoids = weight of dried residue / weight of original sample Ă— 100/1 Determination of alkaloid: Alkaloids level was determined by gravimetric method (Harbone, 1980). Exactly 5g of powdered sample was dispersed in 50ml of 10% acetic acid
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Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-6, 2016 ISSN: 2454-1362, http://www.onlinejournal.in in ethanol. It was then filtered and filtrate was evaporated to 15ml of its original volume in a water bath. The alkaloid was precipitated with a drop of ammonium hydroxide and precipitated also for 10minutes, dried at 70oc for 30 minutes in a vacuum oven. The percentage was calculated as far below: % Alkaloids = weight of dried residue / weight of original sample × 100/1
negative bacteria (Cheesbrough, 2000).However, during inoculation, the plates of the media have been dried because of easier growth and identification of the colonies. The wireloop has also flamed and sterilized. The plates were placed invertedly overnight, to prevent falling of condensed water vapour on plate surface (Cheesbrough, 2000).
Determination of tannin: Tannin level was determined according to the method described by (Harbone, 1980). Exactly 0.5g of the sample A and B, were boiled in distilled water for 1hour. It was then filtered into 50ml conical flask, cooled and made to volume. The absorbance of the samples were measured in a spectrophotometer at 760nm.The level of tannins was extrapolated from a standard curve prepared from tannic acid.
Gram staining technique Thin smears of about 200mm in diameter was made on grease free slides which was also fixed over a burning flame. A crystal violet solution was used to cover the smear for 60 seconds and after it was washed with distilled water. Secondly lugol’s iodine was also used to the surface for good 60 seconds. Acetone was used to decolorize the stain and lastly, the safranin solution was applied for counter staining on the surface for a minute, which has been washed and allowed to dry at room temperature. Then the stains have been observed under microscope with oil immersion. Consequently, red stains indicate gram- negative bacteria (Cheesbrough, 2000).
Determination of Saponins: The saponin level was determined gravimetric method (Harbone, 1980). Exactly 20g of the dried sample was dispersed in 200ml of ethanol and then heated in a water bath for 4 hours at 550C. It was then filtered and the residue was re-extracted in 100ml ethanol. The combined extract was reduced to 40ml by evaporation. Twenty milliliter of diethyl ether was added to the concentrate. The solution was evaporated in a water bath and the concentrate was dried to a constant weight in an oven. %Saponins = weight of dried residue/ weight of original sample × 100/1 Test organisms The isolates were obtained from fecal samples. The organisms include; E. coli, Shigella spp, Salmonella spp, e. t. c. The isolates were indentified using the schemes of Cheesbrough (2006) and then sub-cultured into Mac conkey agar, Eosine methylene blue and Salmonella – Shigella agar for further confirmation Cheesbrough (2006). Culturing and isolation of the test organisms A sterile wireloop has been used to inoculate stool samples on Mac conkey agar (MARK). The culture has then incubated at 370C for 24 hours. The pale colonies show the presence of both Shigella and Salmonella, while E. coli appears pinkish in colour. Shigella, Salmonella and E. coli are all gram
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Sensitivity testing Mueller Hinton agar (Fluka) was prepared, based on the manufacturer’s guide and suspended into a clean conical flask containing 1litre of sterilized distilled water and allow to sock and dissolved for some minutes, boiled for some minutes and then to be autoclaved at 121oC for 15 minutes. Furthermore, each organism (culture) was inoculated on plates using swab stick. A 6mm cork borer was used to bore holes on the medium. Six holes were made on each petri plate, adequetely spaced out. About 0.2 ml of the different concentrations (25, 50,100,150 and 200mg/ml) were introduced into the well. The petri plates were incubated at 370C for 24hrs, after which the zones of inhibition were measured using a meter rule (Idu and Igeleke, 2012). RESULTS Table:It shows that the qualitative and quantitative analysis of phytochemical screening of Khaya senegalensis leaf extract. The phytoconstituents found in the leaf extract of K. senegalensis include; tannin with a 0.044g, saponin having 0.22g, flavonoids (15.49g) and alkaloid with a gram of 1.5g.While Anthraquinone, steroid, Terpenoids and cardiac glycoside were not detected.
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Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-6, 2016 ISSN: 2454-1362, http://www.onlinejournal.in
Fig.1: Shows the bar chart of LAQ extract From the bar chart above, Salmonella Fig.2: Shows the bar chart of (LETH) extract emerges the highest activity at the extract From the bar chart above, E .coli has the highest concentration of 200mg/ml with about 10.45% . activity on the extract concentration of 200mg/ml with about 8.73%.
Key: LAQ = Leaf aqueous extract DISCUSSION The study, established the effect of the aqueous and ethanolic extracts of K. senegalensis. The results of phytochemical analysis revealed the presence of Saponins, Flavonoids, Alkaloids and Tannins. Similar studies of Kubmarawa et al ., (2008), consistently reported phytochemical bioactive ingredients of K. senegalensis to be alkaloids, tannins, saponins and Flavonoids. Therefore, the results of the phytochemical analysis of the extract of K. senegalensis obtained in this study conforms to the previous reports. The antibacterial effects of the leaf extracts of K. senegalensis were determined in comparison with the standard antibiotic (Tetracycline) against the test organisms. There was a significant difference between the zone of inhibitions by the extract and the antibiotic (control).The effect of the extracts on the isolates was due to the presence of the phytochemical components of the extracts as
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Key: LETH = Leaf ethanolic extract reported in previous study by Kubmarawa et al ., (2008).The aqueous leaf extracts had a higher activity in Shigella, followed by E. coli and shows a lesser activity in Salmonella. Moreover, this finding is in conformity with the work of Kubmarawa et al., (2008), that reported the inhibitory effect of the aqueous stem and leaf extract of K. senegalensis on the isolates. While, the ethanolic leaf extract also shows more activity in all isolates as in aqueous extract with some minute differences among them. Meanwhile, the ethanolic extract had better activity than the aqueous extracts. This shows that ethanol has extracted the bioactive ingredients than that of aqueous in this study. The result of the present study agree with that of Ahmed et al., (1998). Generally, the effect of the extracts of K. senegalensis was found to be more as the concentration of the extracts increases, as shown in fig.1 and fig. 2 which implies that the higher the
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Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-6, 2016 ISSN: 2454-1362, http://www.onlinejournal.in concentration, the more the activity by the extracts on the organisms . This is also in line with of Kubmarawa et al ., (2008). CONCLUSION The phytochemical analysis revealed the presence of some bioactive ingredients such as; Alkaloids, Flavonoids, Tannins, and Saponins in the leaf extracts. The bioactive ingredients were active enough on the tested isolates. Reference Ahmad I, Mahamood, Z., and Mohammed, F. (1998). Screening of some Indian Medicinal plants for their Antimicrobial properties. Journal of Ethnopharmacol.62:183-193. Baris, O., Gulluce, M., Sahim, F., Ozer, H. and Kilic, H. ( 2006). Biological activities of the essential oil and methods extract of Achilles biebersteinii Afan. (Asteraceae). Turk. J. Biol., 30: 65-73 Cheesbrough, M. (2000). District laboratory practice in tropical Countries low price edition. Conbridgeshire, Britain Combridge University Press. Pp. 62 -70 Cheesbrough, M. (2006) Medical laboratory Manual for tropical Countries. VOL 11 Microbiology tropical health technology press edition England. Pp. 58 – 67 and 11 30 District of laboratory practice in tropical countries part (2) Printed in India and reprinted 2002 (thrice ) 2003 (twice).
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