Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-1, 2017 ISSN: 2454-1362, http://www.onlinejournal.in
Antibacterial Activity of Leaf Extract (Cassia Mimosoides Linn) On Some Bacterial Isolates from Diarrhoeal Samples of Infant Abdallah, M.S1 , Warodi, F.A2 & Gambo, R.M3 1. Research Assistant, Desert Research, Monitoring and Control Centre, Yobe State University, Damaturu, Nigeria. 2. Desert Research, Monitoring and Control Centre, Yobe State University, Damaturu, Nigeria. 3. Desert Research, Monitoring and Control Centre, Yobe State University, Damaturu, Nigeria. Abstract: The antibacterial activity of ethanolic and water extract of leave of Cassia Mimosoides Linn was analyzed using the standard procedure of (Fatope et al., 1993). Various concentrations on ethanolic and water extract viz; 10,000, 1000,100 and 10µg/ml respectively, were tested against bacterial isolates. The bacterial isolates include: Escherichia coli, Salmonella spp and Shigella spp. The leaves of Cassia Mimosoides Linn was very active against the bacterial isolates which will ensure the use of the leaves to cure diarrhoea. The result of the antibacterial activity on all the test organisms shows susceptible on ethanolic extract at 10,000, 1,000 and some up to 100µg/ml. Whereas on water extract; Shigella and Salmonella were susceptible, but with the exception of Escherichia coli which was resistance. Key words: Cassia Mimosoides Linn, isolates and susceptible. INTRODUCTION Plants have great potential uses, especially as traditional medicine and pharmacopial drugs. A large proportion of the world population depends on traditional medicine because of the scarcity and high costs of orthodox medicine( Tagbodo and Townson, 2001., Hudcub et al., 2008). Medicinal plants have provided the modern medicine with numerous plant- derived therapeutics agents (Evans, 2000 and Oledunmoye et al., 2009). Many plants contain a variety of phytopharmaceuticals which have found very important applications in the field of agriculture, human and veterinary medicine. Natural products play a dominant role in the development of novel drugs leads for the treatment and prevention of diseases (Newman et al.,2003); (Gilani and Rahman, 2005). Cassia Mimosoides linn is a common weed popular known in native Hausa as “Bagaruwar kasa”, with yellow flowers and pinnate leaves. It also occurs in
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India to southern China through Malaya of Australia. It is a low, diffuse shrub, 15 to 30 centimeters or more in height. The leaves are 7 to 10 with a solitary, sessile gland on the rachis below the leaflets. The flowers grow one or two together in the axils and are shining, small and yellow. The pods are strap- shaped, flat and about 5 centimeters long and contain rhomboid, dark brown seeds. The roots are used as cure for diarrhoea. The roots are used in tropical Africa to cure colic and spasms for stomach. It is also reported that the decoction is used in dysentery and that the entire plant is used as a remedy for eruption in the faeces (Sofowora, 1993). Diarrhoea can usefully considered as a disturbance in the balance between the mechanisms, the controlling secretion and absorption of water resulting in the loss of water in the faeces. The quality of water in food and drink is of little importance Flow of water across the gut occurs mainly to maintain isonicity with the plasma in a rapid changing chemical environment. Thus, intraluminal digestion increases the osmotic attraction of water into the bowel, while absorption of water (Fortran, 1967). Most investigators have incriminated diarrhoea syndrome as one of the major health problem worldwide, and described diarrhoeal disease as the second only to respiratory disease as a cause of adult death and also leading cause of childhood death (Lansing, 1999). However, an acute-onset of diarrhoea may be Caused by a variety of bacterial, parasitic and viral agents. Among bacterial agents include; E. coli, Bacillus cereus, Campylobacter jejuni, Shigella spp, Clostridium difficile and non typhoid Salmonella. Those among parasitic agents include; Helminthes while Cryptosporichosis has became a serious problem in patients with the acquired deficiency syndrome (AIDS),(Acholono, 1998).The objectives of this research are to;
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Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-1, 2017 ISSN: 2454-1362, http://www.onlinejournal.in Isolates and characterized the bacterial species from diarrhoeal samples collected and also to find out the activities of leaves extracts of Cassia Mimosoides linn on the organisms isolated. This is with view to understand the concentration at which the leaves extracts will be more effective. MATERIALS AND METHOD The method employed in this investigation is in line with that suggested by ( Cheesbrough, 2000). Collection and identification of plant materials Cassia Mimosoides linn leaves were collected from farm land around Wudil local government, Kano State, Nigeria. The botany identity of the plants were confirmed in the biology of department K. U.S.T. Wudil. The leaves of Cassia Mimosoides linn were grounded to powdered form using pestle and mortar as described by (Fatope et al., 1993).
cultures into a test tube of 9ml peptone water. The inoculation was incubated at 37Oc for 24hrs. After which2ml of indole reagent was added. A positive result showed dark pink or red colour formation on the top layer. Escherichia coli and Shigella are indole positive while Salmonella is indole negative (Cheesbrough, 2000) and (Mackie and MC cartney, 1997). Extraction of Cassia Mimosoides linn leaves About 50g of the powdered form of the leaves of Cassia Mimosoides linn was suspended in 500ml of ethanol (VWR) and distilled water in separate between, whereby, the suspensions were kept at room temperature and about 3 weeks with regular shaking all the days. The suspensions were then filtered and solvent removed by evaporation to dryness at room temperature (Fatope et al., 1993). Preparation of extract impregnated discs
Culturing and isolation of the test bacteria A sterile wire loop was used to inoculate stool samples on Mac conkey agar (Mark).The cultures were incubated at 37OC for 24 hrs. The pale colonies confirmed the presence of both Shigella and Salmonella. And after some times, colonies may turn to pink after 24hrs of incubation. While Escherichia coli appears pinkish in colour. Shigella, Salmonella and the Escherichia coli are gram negative bacteria (Cheesbrough, 2000). Gram staining technique Thin smears of about 20mm in diameter were made on clean grease free slide which was fixed over a burning flame. A crystal violet solution was applied to cover the smear for 30 seconds and after it was washed with distilled water. Secondly lugol’s iodine was also applied to the surface for another 30seconds, acetone was also used decolorized the stain lastly, the neutral red solution was covered on the surface for a minute, which was washed up and it was also allowed to dry at room temperature. Stains were observed under microscope with oil immersion. Red stain indicated gram-negative bacteria (Cheesbrough, 2000). Biochemical identification of bacterial isolates This is done because of to be sure of the results test which were carried out. Among the biochemical identification include; indole, betaglucoronidase, coagulase, urease test e.t.c. Indole test; was used to confirm Shigella, Salmonella and the Escherichia coli from suspected colonies. A wire loop was used to inoculate overnight growth
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One gram (1g) of each of the extracts was dissolved in 1ml of appropriate diluents (distilled water for water extract and dimethylesulphooxide (DMSO) for ethanolic extract), to yield 1000g/ml (1,000,000µg/ml) solution. And that was transferred into a tube containing 0.9ml diluents, to effect 10 times dilution, which gave a concentration of (1,000,000µg/ml).Subsequently, 0.1ml was transferred into another bottle containing 0.9ml diluents which gave a concentration of 10,000µg/ml on data basis. A 100 discs, 6.0mm in diameter of whatman no.1 filter paper were impregnated with the extracts to arrive at 10,000, 1,000, 100 and 10µg/ml discs, which were prepared and stored in refrigerator before used (Hussein and Deeni, 1991). Sensitivity testing Nutrient agar (Fluka) was prepared 28g of it was suspended into a clean conical flask containing a 1 litre of sterilized distilled water and allowed to sock and dissolved for some minutes, and then autoclaved at 121oc for 15 minutes. Two loopfuls of the standard inoculums were evenly streaked on to the plates. Discs of different concentrations as with the control discs (impregnated with only diluents) were placed firmly on the surface of the medium (Nutrient agar) by means of sterile syringe needle at about 40mm apart. The plates were incubated at 37oc for 24hrs.Diameters of the zone of inhibitions were measured with a ruler and the mean recorded in the nearest (Hussein and Deeni, 1991).
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Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-1, 2017 ISSN: 2454-1362, http://www.onlinejournal.in extract. A yield of 20.0g of ethanolic extract and that of water extract appeared greenish and oily in texture. Whereas, that of water extract was brownish and gummy in texture as given respectively.
Results
Table 3.1 below shows different physical characteristics on leaves extracts of Cassia Mimosoides linn with their individual weight in grams. Initially the grams were 50g for each Table 3.1 Shows the physical characteristics on ethanolic and water extract on leaves of Cassia Mimosoides linn
EXTRACTS
INITIAL
OF LEAVES
WEIGHT (g)
FINAL WEIGHT (g)
PHYSICAL
CHARACTERISTICS
APPEARANCE
TEXTURES
Ethanolic
50.0
20.0
Greenish
oily
Water
50.0
24.3
Brownish
Gummy
Table 3.2 below shows more activities on E.coli and very few activities on Salmonella and Shigella spp. The most active disc showed was 10,000 µg on E.coli. While the most active discs showed on Salmonella and Shigella spp. were on 10,000 µg/ml and 1,000 µg/ml. Table 3.2 Sensitivity of impregnated paper discs on ethanolic leaves extracts of Cassia Mimosoides linn
TEST ORGANISMS USED
DIAMETER OF ZONE OF INHIBITION (mm) 10,000 µg
1,000 µg
Escherichia coli
6.0
4.0
3.0
0.0
Salmonella spp
3.0
2.0
0.0
0.0
0.0
0.0
0.0
Shigella
spp
1.0
100 µg
10 µg
Table 3.3 below shows a unique activity on E.coli while some activities were on Salmonella and Shigella spp. The unique disc showed 10,000 µg/ml and the most active disc showed on 10,000 µg/ml and 1,000 µg/ml of water extract on both Salmonella and Shigella spp. Table 3.3 Sensitivity of impregnated paper discs of water on leaves extracts of Cassia Mimosoides linn
TEST ORGANISMS USED
DIAMETER OF ZONE OF INHIBITION (mm) 10,000 µg
1,000 µg
Escherichia coli
2.0
0.0
0.0
0.0
Salmonella spp
2.0
1.0
0.0
0.0
Shigella
4.0
3.0
0.0
0.0
spp
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100 µg
10 µg
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Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-1, 2017 ISSN: 2454-1362, http://www.onlinejournal.in Table 3.4 below shows that the tested organisms are all gram negative bacteria with the appearance of red color stains. E.coli and Shigella are indole positives with the appearance of dark pink coloration, while Salmonella spp are indole negative with the a red coloration. Table 3.4 Identification of organisms tested Name of organisms
Gram staining
Indole
Escherichia coli
negative
positive
Salmonella spp
negative
positive
Shigella
negative
negative
spp
DICUSSION The extract of both ethanolic and aqueous extracts on leaves of Cassia Mimosoides linn, were measured at 50g each. A yield of 20.0g of ethanolic extract and 24.3g of water extract were obtained. The ethanolic extract appeared dark green after evaporated to dryness and this shows that the presence of enough phytochemicals in the extracts. While, the water extract appeared dark brown when evaporated to dryness. The results obtained from the antibacterial sensitivity tests of Cassia Mimosoides linn from the tables justified that the use of the plants to cure diarrhoea was very effective. This is in lined with fact that ( Ayo et al.,2009) has discovered that to very effective in the case of diarrheoa. However, herbalism being an ancient approach of healing and curing of human ailments in Kano, in which the work of (Hussein and Karatele, 1991), was the first source of reference published.
Fordtran, J.S. (1967). Speculations on the pathogenesis of diarrhoea. Fed., Proc., 26, 14051414. Fatope, A.O., Ibrahim, H and Jakada, Y.(1993). Screening of higher plant reputed as pesticides using brine shinmp lethality broassigly international journal of Phamacognosy, 3(4) 250254p. Gilani, A.H., Rahman, A.U.(2000).Trends in ethnopharmacology. J.Ethnopharmacol., 100: 4349. Hussein, H.S.N.S and Deeni, Y.Y (1991).Antibiotic sensitivity testing. Journal of ethnopharmacognosy.PP. PP .PP. 91-96. Lansin, I., Watson, T. and Ryan, R.W (1999), Journal of medical microbiology VOL-12 on chronic diarrhoea in children nestle nutrition. Merck, KGaA, (2006), 64271 darmstadt, Germany. WWW..merck.
References Acholono and Wozuzu , A.D (1991). Agent responsible for diarrhoeal diseases. Sixth international conference on global impacts of applied Microbiology (G. I .A.M.V1) 30th August, 7th September, academic press London pp 435 – 445.
Mackie and MC cartney (1998).Practical medical microbiology.Living stone newyork PP441-443.
Cheesbrough, (2001).Medical laboratory manual for tropical countries.Vol.11 Microbiology tropic health technology. Thalford press LTD. England PP. 58-67 and District laboratory practice in tropical countries part (2); printed in india and reprinted, 2002(Thrice); 2003 (Twice). Evans, W.C. (2000).Trease and Evans Phamacognosy (1) Edition. W.B. saundners company L.T.D., London, PP.1920.
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