The phytochemical analysis and antibacterial activities of the ethanol, methanol and aqueous (cold water) extracts of the leaves of Leptadenia hastata were investigated against some selected pathogenic bacteria which include Escherichia coli, Salmonella typhi, Staphylococcus aureus and Pseudomonas aeruginosa at 62.5, 125, 250 and 500mg/ml concentrations using Agar well diffusion method. The leaves were plucked, air dried, macerated, soaked, filtered and evaporated to dryness to obtain the extracts. Chloramphenicol (125mg/ml) was used as the standard drug for positive control to determine the sensitivity of the test organisms. The results of the antibacterial activity showed that the ethanol extract is the most effective followed by the methanol extract and then the aqueous extract which is the least effective with higher zones of inhibition on E. coli and S. typhi. The MIC result showed that the MIC of the ethanol leaf extract for S. aureus, S. typhi and E. coli was 125mg/ml, while for P. aeruginosa was 250mg/ml. The MIC of the methanol leaf extract for all the test organisms was 250mg/ml. However, the MIC of the aqueous leaf extract for both S. typhi and S. aureus was 250mg/ml, the MIC for E. coli was 125mg/ml and 500mg/ml for P. aeruginosa. The MBC result showed that the MBC of the ethanol extract on both S. aureus and P. aeruginosa was 500mg/ml and 250mg/ml for both E. coli and S. typhi. The MBC of the methanol extract on S. aureus, P. aeruginosa and S. typhi was 500mg/ml while the MBC for E. coli was 250mg/ml. However, the MBC investigations of the aqueous leaf extract on S. aureus, S. typhi and E. coli was 500mg/ml while there was growth in all the methanol extract concentrations studied on P. aeruginosa thus no MBC result was obtained for P. aeruginosa. The result of the phytochemical analysis showed the presence of Tanins, Alkaloids, Flavonoids, Reducing sugar, Cardiac glycoside and Phlobatanins in all the extracts. Saponin was present in only the methanol extract, while Anthracenes were present only in the ethanol extract. Steroids and Anthraquinones were absents in all the extracts.
1.0 Introduction - - - - - - - - -1
1.1 Historical Background - - - - - - - -1
1.2 Statement of Problem - - - - - - - -4
1.3 Significance of the study - - - - - - - -5
1.4 Limitation of study - - - - - - - -6
1.5 Aims and objective of study - - - - - - -6
2.0 Literature Review - - - - - - - - -7
2.1 Taxonomy and Ecology of Leptadenia hastata - - - -7
2.2 Geographical Distribution of Leptadenia hastate - - - -8
2.3 Nutritional and Pharmaceutical uses of L. hastata extracts - -9
2.3 Brief Overview of the test organisms - - - - - -10
2.3.1 Staphylococcus aureus - - - - - - - -10
184.108.40.206 Description - - - - - - - - -10
220.127.116.11 Ecology - - - - - - - - - -10
18.104.22.168 Pathogenesis - - - - - - - - -11
2.3.2 Escherichia coli - - - - - - - - -12
22.214.171.124 Description - - - - - - - - -12
126.96.36.199 Ecology - - - - - - - - - -12
188.8.131.52 Pathogenesis - - - - - - - - -13
2.3.3 Pseudomonas aeruginosa - - - - - - -13
184.108.40.206 Description - - - - - - - - -13
220.127.116.11 Ecology - - - - - - - - - -14
18.104.22.168 Pathogenesis - - - - - - - - -15
2.3.4 Salmonella typhi - - - - - - - - -15
22.214.171.124 Description - - - - - - - - -15
126.96.36.199 Ecology - - - - - - - - - -16
188.8.131.52 Pathogenesis - - - - - - - - -16
2.4 Gas Chromatography- Mass Spectroscopy Analysis (GC-MS) - -17
3.0 Materials and Methods - - - - - - - -19
3.1 Collection and identification of experimental plant - - - -19
3.2 Sterilization of materials - - - - - - - -19
3.3 Preparation of the plant materials - - - - - -20
3.4 Extraction - - - - - - - - - -20
3.4.1 Ethanol Extraction - - - - - - - - -20
3.4.2 Methanol Extraction - - - - - - - -21
3.4.3 Aqueous Extraction - - - - - - - -21
3.5 Reconstruction of extracts and preparation of standard drug - -22
3.6 Phytochemical Analysis - - - - - - - -22
3.6.1 Test for Tannins - - - - - - - - -23
3.6.2 Test for Alkaloids - - - - - - - - -23
3.6.3 Test for Saponin - - - - - - - - -23
3.6.4 Test for Flavonoids - - - - - - - -23
3.6.5 Test for Cardiac glycosides - - - - - - -24
3.6.6 Test for Steroids - - - - - - - - -24
3.6.7 Test for Phlobatanins - - - - - - - -24
3.6.8 Test for Reducing sugar - - - - - - - -24
3.6.9 Test for Anthracenes - - - - - - - -25
3.6.10 Test for Anthraquinones - - - - - - - -25
3.7 Preparation of culture media - - - - - - -25
3.8 Collection of Test bacteria - - - - - - -25
3.9 Confirmation of Purity and Viability of test organisms - - -26
3.10 Standardization of inocula - - - - - - -27
3.11 Determination of antimicrobial activity - - - - -28
3.12 Determination of Minimum Inhibitory Concentration (MIC) - -29
3.13 Determination of Minimum Bactericidal Concentration (MBC) - -29
3.14 Statistical Analysis - - - - - - - -30
4.0 Results - - - - - - - - - -31
4.1 Physical observation of Leptadenia hastata - - - - -31
4.2 Phytochemical analysis of Leptadenia hastata - - - -33
4.3 Antimicrobial Assay - - - - - - - -35
4.4 Minimum inhibitory concentration (MIC) - - - - - 41
4.5 Minimum bactericidal concentration (MBC) - - - - - 44
5.0 Discussion - - - - - - - - - -48
5.1 Conclusion - - - - - - - - - -52
5.2 Recommendation - - - - - - - - -53
References - - - - - - - - - - -54
Appendices - - - - - - - - - - -60
LIST OF FIGURE
1. Picture of Leptadenia hastata - - - - - - -8
LIST OF TABLES
1. Comparison of the physical appearance of ethanol, aqueous and methanol extract - - - - - - - - - -32
2. The phytochemical constituents of L. hastata - - - - -34
3. Antibacterial activities of the ethanol extracts of L. hastata - -36
4. Antibacterial activities of the methanol extracts of L. hastata - -38
5. Antibacterial activities of the aqueous extracts of L. hastata - -40
6. Antibacterial activities of the standard antibiotics (chloramphenicol) -41
7. Minimum inhibitory concentration (MIC) of the ethanol, methanol and aqueous extracts of L. hastata - - - - - - -44
8. Minimum bactericidal concentration (MBC) of the ethanol, methanol and aqueous extracts of L. hastata - - - - - - -47
9. One-way ANOVA for the mean zones of inhibition obtained by the activity of the ethanol extract of L. hastata on the test organisms - - -62
10. One-way ANOVA for the mean zones of inhibition obtained by the activity of the methanol extract of L. hastata on the test organisms - -65
11. One-way ANOVA for the mean zones of inhibition obtained by the activity of the Aqueous extract of L. hastata on the test organism - - -67
1.1 Historical background
During the last century, the practice of herbal medicine became mainstreams throughout the world. In spite of great advances in modern medicine, plants still make important contributions to healthcare.
According to Cuilei (1982) and Sofowora (1996), a medicinal plant is a plant that contains chemical compounds that possess established therapeutic activity. Studies have shown that such activity is due mainly to the antimicrobial properties of the plant. Any chemical substance inhibiting the growth or causing death of a microorganism is known as antimicrobial agent and is said to possess antimicrobial activity.
Over the years, plants have been used as valuable sources of natural products for maintaining human health. Plants have been reported to contain large varieties of chemical substances that possess important preventative and curative therapies. About 80% of individuals from developing countries use traditional medicines which have compounds derived from medicinal plants (Igbinosa et al., 2009).
Due to its multidirectional promising aspects, the interest in natural product continues to this day (Kaul and Joshi, 2001; Kroll, 2001; Marriott, 2001; Bhattaram et al., 2002; Holt and Chandra, 2002). The use of herbal drugs is once more escalating in the form of complementary and alternative medicine (CAM) (Cooper, 2004).
Medicinal plants are of great importance to the health of individuals and communities because they contain physiologically active components which over the years have been exploited in the traditional medical practices for the treatment of various ailments. It was the advent of antibiotics in the 1950s that led to the decline of the use of plant derivatives as antimicrobials (Agedah et al., 2010). Despite the presence of various approaches to drug discovery, plants still remain the main reservoir of natural medicines (Mahomed and Ojewole, 2006).
According to Cowan (1999), clinical microbiologists have two reasons to be interested in the topic of antimicrobial plant extracts. First, it is very likely that these phytochemicals will find their way into the arsenal of antimicrobial drugs prescribed by physicians; several are already being tested in humans. It is reported that, on average, two or three antibiotics derived from microorganisms are launched each year. After a downturn in that pace in recent decades, the pace is again quickening as scientists realize that the effective life span of any antibiotic is limited. Worldwide spending on finding new anti-infective agents (including vaccines) is expected to increase 60% from the spending levels in 1993. New sources, especially plant sources, are also being investigated. Second, the public is becoming increasingly aware of problems with the over prescription and misuse of traditional antibiotics. In addition, many people are interested in having more autonomy over their medical care.
Also interest in plants with antimicrobial properties has been revived as a result of antimicrobial resistance. This resistance could be attributed to indiscriminate use of commercial drugs or not taking an antibiotic prescription according to the instruction, for example not taking all the prescription in the treatment of infectious diseases (Aliero and Afolayan, 2006). In addition, certain antibiotics present undesirable side effects such as nausea, depression of bone marrow, thrombocytopenic purpura and agranulocytosis leading to the emergence of previously uncommon diseases. This has given scientists the impetus to search for newer and alternative microbial compounds from medicinal plants. Besides, the high cost of conventional drugs, particularly in resource limited communities has led to the increased use of plants as an alternative for treatment of infectious diseases (Marchese and Shito, 2001; Poole, 2001).
Plant extracts and phytochemicals with antimicrobial properties are of great significance in therapeutic treatments. Their antimicrobial properties are due to compounds synthesized in the secondary metabolism of the plant. The screening of plant extracts and plant products for antimicrobial activity has shown that plants represent a potential source of novel antibiotic prototypes (Afolayan, 2003).
The practice of herbal medicine in modernized form is now gaining momentum in Nigeria, with various health officials and other persons coming to realize the potencies and efficacies of some of the indigenous plants (Nwaogu, 1997). Among the diseases that have been successfully managed traditionally include malaria, epilepsy, indigestion, scurvy, diarrhea, dysentery, gonorrhea, flatulence, worm infections and mental illness. Medicinal uses of these plants ranges from the administration of the plants root, barks, stem, leaves and seeds, to the use of extracts from a whole plant (Soforowa, 1996).
1.2 Statement of Problem
The discovery and development of antibiotics are among the most powerful and successful achievements of modern science and technology for the control of infectious diseases. However, the rate of resistance of pathogenic microorganisms to conventionally used antimicrobial agents is increasing at an alarming rate (Ge et al., 2002; Nair and Chanda, 2005; Neogi et al., 2008). Surveys have revealed that almost no group of antibiotics has been introduced to which resistance had not been observed (Eloff, 2000).
1.3 Significance of the study
1. The findings of this study would provide informative and educative materials to clinicians on the importance of reliable AST results in maintaining public health.
2. The research finding may kindle the interest of fellow researchers to study the impact of other serious flaws other than use of unapproved antibiotics on the results of AST in clinical laboratories. Other documented areas of flaws include:
- Improper inoculum preparation
- Incorrect media
- Too many antibiotic disks or holes per plate
- Incorrect interpretative criteria for disk zone size
- Improper or absence of confirmatory testing procedure.
1.4 Limitation of study
However the study is limited by various factors such as time, epileptic power supply and materials available within the department.
1.5 Aims and objective of study
The major aim of this project work is to determine the antimicrobial activity of the leaf extract of Leptadenia hastata on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi.
The specific objectives include:
- To obtain the leaf extract of Leptadenia hastata
- To determine the zone diameter of inhibition of the leaf extract of Leptadenia hastata on some selected microorganisms
- To determine the minimum inhibitory concentration (MIC) of the leaf extract
- To determine the minimum bactericidal concentration (MBC) of the leaf extract.