THE ANTIBACTERIAL ACTIVITY OF AZADIRACHTA INDICA (NEEM PLANT)
The antibacterial effect of Azadirachta indica (neem plant) against Pseudomonas aeruginosa, Klebsiella ozanae, Staphylococcus aureus and Escherichia coli was determined using the agar cup plate techniques. The phytochemical components of A. indica showed the presence of saponin and phlobatanin and the absence of alkaloids, tannis, phenolics, plycosides, flavonoids, and triterpenes, the result showed that the test organisms were susceptible to 500mg/ml, 50mg/ml and 5mg/ml of the plant extract. The minmum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined, the result showed that the MIC and MBC were 5mg and 50mg respectively for Pseudomonas aeruginosa, K. ozanae, Staphylococcus aureus and Escherichia coli. The result of the study suggest that extracts of A. indica could be suitable for the treatment of various infections caused by P. aeruginosa, K. ozanae, S. aureus and E. coli.
TABLE OF CONTENT
Title page i
Table of Content vi-vii
1. Introduction 1-2
1. Literature review 3-7
2. Materials and methods 8
1. Collection and preparation of samples 8
2. Collection of specimen 8
3. Extraction of materials 8
4. Phytochemical screening of extracts of A. indica 8
5. Anti-microbial susceptibility test 9
6. Determination of minimum inhibitory concentration (MIC) 9
1. Result 10
2. Phytochemical activities of the extracts 10
3. Antimicrobial activities of the extracts 10-12
4.0 Discussion 13-14
5.0 Conclusion 15
Plants contain many biologically active compounds which have potential for development as medicinal agents. Herbal medicines already form the basis of therapeutic use in the developing countries but on recent there has been an increase in the use of herbal medicine in the developed world too (De and Ifeoma, 2002; El-manhood et al, 2010) plants provide alternative strategy in search for new drug. There is a rich abundance of plant reported in traditional medicine to process protective and therapeutic properties (Kayode and Kayode, 2011). It is likely that plants will continue to be a valuable sources of new molecules which may, after possible chemical manipulation, provide new and improve drugs (Shah et al; 2006) Bacterial resistance to antibiotic represents a serious problem for clinicians and the pharmaceutical industry and great efforts are being made to reverse this trend and one of them is the widespread screening of medicinal plant from the traditional system to medicine hoping to get some newer, saver and more effective agents that can be used to fight infectious diseases (Natarian et al, 2003)
Azandirachta indica is one of such medical plants belonging to the meliaceae family and the indigenous to Southern Asia (Akula et al, 2003) Azadirachta indica commonly known as neem has attracted worldwide prominence in recent years, owing to its wide range of medicinal properties Neem has been extensively used in Ayurveda Umani and homoeopathic medicine and has become a cynosure of modern medicine
Neem elaborates a fast array of biologically active compound that are chemically diverse and structurally complex.
More than 140 compounds have been isolated from different parts of neem. All parts of the neem tree leaves, flowers, seeds, fruits and roots and sac bark have been used traditionally for the treatment of inflammations infections, fever, skin diseases and dental disorders. The medicinal utilities have been described especially for neem leaf. Neem leaf and its constituents have been demonstrated to exhibit immunomodulatory anti-Inflammatory, antihyperglycaemic, Antiurcer, antimalaria, antifungal, antibacterial, antiviral, antiocidant, antimutagenic and anticarcinogenic properties (Talwar et al;1997 Biswas et al 2002; Subapriya and Nagine; 2005).
The objective of this study therefore are to determine the phytochemical components of the leaf extract of A. Indica to determine the minimum inhibitory concentration (MIC) of the extract on Pseudomonas aeruginosa, Nebsiella Ozanas, Staphylococcus aureus and Escherichia coli.
1.1 LITERATURE REVIEW
Plants contain many biologically active compounds which have potential for development as medicinal agents.
Herbals medicines already form the basis of therapeutic use in the developing countries, but of recent there has been an increase in the use of herbal medicines in the developed world too (De and Ifeoma, 2002). The photochemical compound of the A. Indicial have been established in previous studies and these include tannins, saponins, alkaloids, carbohydrate, phenols, flavonoids, anthraquinones, cardiac glycosides, sterol and resins (Sundarasivara and Nazma, 1977; Bhomick and choudhary, 1982; Rao et al, 1986; Natarajan et al, 2003; De and Ifeoma 2002; Biswas et al, 2002). Several studies have linked presence of these bioactive compound is plant material to antimicrobial activity.
The presence of these secondary metabolites in plants, produce some biological activity in man and animal and it is responsible for their use as herbs. These compounds also have served to protect the plant against infection by micro organism predation by insect herbivores while some give plants their odours and or flavors and some still are responsible for their pigment (Ketkar et al, 1995; El-Mahmood et al, 2008).
In some case the activity has been associated with specific compound or classes of compounds. These active constituents can be used to search for bioactive lead compounds that could be used in the partial synthesis of more useful drugs (Ogbonnia et al, 2008).
In this study, a variety of pathogenic bacteria implicated as causative agents of eye and ear infections were selected for the screening for antibacterial activity of the crude neem seed extracts to perceive the efficacy, anti bacterial spectrum as well as authenticate. Some of the ethnomedicinal claims the susceptibility of the test bacteria [(A1 (Escherichia coli) B1 (P. aeruginosa) C1(S. pyogones) and D1 (S. aureus)] of known sensitivity extracts from both hexane and aqueous solvents. Inhibited the growth of both the test and control bacteria though to varying degree. In a similar study involving some dermatophites, Natatajam et al (2003) did not record any activity for their aqueous extracts, containing to the data present in this study, De and Ifeoma (2002) also did not record any antibacterial activity with the aqueous extracts of both the bark and the leaves of the neem bark extracts against their test bacteria. The zones of growth inhibition recorded for the methanol and acetone extract by De and Ifeoma (2002) were also smaller in size than those to influence field and biological activities of plant base products including the age of the plant time of harvest, drying and process of the materials method of extraction and the solvent used.
Some antibacterial effects of the neem seed have also been reported against S. mutans, S. faecalis, M. tuberculosis, V. cholera, S. pyogenes and K. pneumonia (Biswas et al, 2002). In another study, neem seed extract have also been observe to inhibit the growth and development of asexual and sexual stage of drug sensitive and malaria resistance human malaria parasite (P. falciparum), some fungi and some viruses (Natarajan et al; (2003). This broad spectrum activity of crude neem extracts have been linked to the presence of bioactive compounds notably azadiractin, gedunin, nimbidin, mahmoodin and nimbolide (Biswas et al; 2002) which makes the neem plant useful for the treatment of various infectious conditions including those of the eye and ear, amongst the test bacteria, S. aureus (D1) was the most susceptible, closely followed by S. pyogenes (C1), while E. coli (A1) and P. aeruginosa (A1) were less susceptible as shown by their relatively smaller sizes in several authors have also reported that plant extracts are more effective against grain-positive than gram negative bacteria and attributed this to the differences in their cell wall structures (Rabe and Van Staden 1997, Parekh and Chanda, 2006), the control bacteria were more susceptible to the toxic effects of the crude extracts than the test bacteria through the sensitivity also varied according to strains.
The effects of PH on the efficacy of the crude extracts are seen. The activity of the crude seed extracts are seen. The activity of the crude seed extracts of A. indica was optimal under acidic PH (PH2).
The effectiveness of the crude extracts was observed to decrease as the PH was raised to alkalinity (PH 10). This pattern is similar for all the other bacteria and also when hexane was used as a solvent. Activity at acidic PH is indication of acid stability while diminished activity at alkaline PH indicates less efficacy under alkaline conditions.
Practitioners of traditional medicine usually add some when treating their crude extracts. Acid and alkaline treatment was carried out in order to stimulate the situations in the stomach and the gastrointestinal tract, because the crude seed extracts are also taken orally, in addition to their being crashed and the juicy contents squeezed at the infections sites.
The effect of temperature on the efficacy of the crude seed extracts on the test bacteria as seen indicated that activity were more under elevated temperatures and this trend is similar for all bacteria, regardless whether water or hexane was used as a solvent. This supports practices of traditional healers who most of times boils the plant extracts to high temperature could inactivate volatile compounds and free radicals (Marjumdar et al; 1998). The traditional medicine practitioners were reported to sometimes crash the seeds before squeezing the fresh juices in to the affected eye and ear and after several applications, may achieve the antibacterial dosage at the infections sites; this repeated application at an infectious site is due to the lower activity of the extracts at lower temperature as depicted.
The standard antibiotic chloranphenicol, demonstrated highest activity then the crude extracts as shown.
This is because the antibiotic is in pure state and has undergone some refining processes that have established it as standard antibiotic (Prescott et al; 2005), the observed differences in efficacy may also be due the fact the extracts were in a crude form and would contain some inert substances which do not have any antibacterial activity.
The organisms used for the purposes of controls were consistently more susceptible than the test organisms. The quantitative measure of the in-vitro activity of antibiotics and non-antibiotic antibacterial agents including those agents of plant origin with anti bacterial potentials are the MIC and MBC as shown.
The growths of the organisms were inhibited at concentrations that ranged between 3.17-25 mg/ml for aqueous extract and 1.59-12.5 mg/ml for hexane extract. The study showed that E. coli (A1) and P. aeruginosa (B1) had higher MIC values, meaning that higher concentration of the extracts are required to inhibit the growth of these bacteria, while S. pyrogenes (C1) and S. aureus (D1) had lower MIC values and would require low extracts concentration to inhibit their growth and these corroborated with data presented in plant based antimicrobial substances generally have higher MIC and MBC values when compared to antimicrobial substances obtained from micro organism or those that are synthetically produced because of the presence of impurities, the MBC of the plant seed extract ranged between 6.25-50 mg/ml for hexane extracts. In this study P. aeruginosa (B1) and E. coli (A1) had higher MBC values, thus suggesting lower susceptibility to the crude extracts and lower MBC values for S. aureus (D1) S. pyrogenes (C1) thus suggesting higher activity of the extracts..