Antibacterial Activity Of Annona Muricata Leaf N-Hexane Extract On Some Pathogenic Bacteria

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ABSTRACT

This research work was aimed at investigating the antibacterial efficacy of N-hexane leaf extract of Annona muricata using agar-well diffusion method for four (4) clinical isolates of bacteria consisting of one (1) gram positive (staphylococcus aureus and three gram negative (Escharichia coli, salmonella. Typhi and P.aeruginosa) bacteria which have been implicated in the most common types of bacterial infections such as diarrhea, typhoid fever, tooth decay, etc. the antibacterial activity was measured by the diameter of zone of inhibition (mm) observed and the extract was found to exhibit antibacterial activities against the test organism with each organism showing different patterns of formation of zone of inhibition at different concentration of the extracts.

The result of this study reveals that P. aeruginosa was the most susceptible gram negative bacteria. Which indicate that this plant contains compound with wide antibacterial activity which validate the use for treatment of various microbial infections in traditional medicines. The findings in this study provide the basis for further study on the plant with the aim of isolating and identifying the active substance responsible for its antimicrobial activities. The plant could also be standardized to develop cheap, safer, culturally acceptable herbal medicine to help combat the problem of antibiotic resistance. The of N-hexane extraction leaf of A. muricata which was done by the use of soxhelet apparatus yield 15% respectively the different between the extract when compare of the standard antibiotic (ciprofloxacin) was significant against E.Coli, S. typhi and S. aureus. Conversely P.aeruginosa showed no significant different in the response to extract (100mg/ml) and the standard drug. This shows that Annona muricate N-hexane extract can be potential antimicrobial agent directed against P.aeruginosa.

CHAPTER ONE

1.0.            Introduction

1.1  Background

Nature has been a source of medical agents for thousands of years and the use of plants for preventing and treatment of various health ailments has been in practice from time immemorial. It is estimated that about 25% of drugs prescribed are derived from plants. In recent years, secondary metabolites previously with unknown pharmacological activities have been extensively investigated as source of medicinal agents (Devil et al.,  2013).

Annonaceae, the custard apple family with about 2500 species and more than 130 genera is a family of flowering plants consisting of trees, shrubs or rarely lianas (Slik, 2003, Pier, 2008). Biolologically active components of the family showed interesting biological properties like anti HIV, anti-cancer effects (Prescot et al., 2002), insecticidal properties (Ashok Kumar et al., 2010) and useful cytotoxic compounds (Kasahara et al., 2005).

Annona muricata locally called “mama in hausa” is a small upright evergreen tropical fruit tree it is commonly called soursop or Apre. It is grown mainly for fruits. Annona muricata leaves contain several groups of substance called annonaceae acetogenins. The leaves of Annona muricata have astringent, anti-plasmodic and gastric properties (Khan et al., 2007), parasiticidal, anti-diarrhea, rheumatologically and anti-neurologic properties (Glye et al., 2008). It is also used to treat diabetes (Adebolu et al 2005), Jaundice (Balick et al., 2000) and kidney ailments (Duke, 2007).

N-hexane extract of Annona muricata leaves have anti- bacterial activity against some strains of E-coli (Shulgin et al., 2006). An important chemical compound annonacin is found in Annona muricata seeds (Ramanthan et al., 2009). Annona reticulate a tall deciduous tree with many branches bearing nutritious fruits is a highly medicinal plant used for the treatment of epilepsy, constipation, cardiac problems, worm infestation, antibacterial infection, dysentery, fever, dysuria, hemorrhage and ulcer (Nadkarmi, 2000).

The ripe fruits from Annona muricata is a sweet, cooling good tonic and sedatives. it enriches the blood, increase muscular strength, lesions burning sensation, tendency to biliousness and vomiting. Leaf can be used for destroying lice (Murray, 2000). The leaves are also used as anthelmintic, styptics and insecticide and the chemical compound acetogenins present in the leaves are responsible for specific cytotoxic to certain human’s tumors (Stein et al., 2011). The bark is a powerful astringent and is used as anti-dysentric and vermifuge. Root bark and stem of this plant possess isoquinoline alkaloids (Nadkarni, 2002). Phytochemical constituent from the plant are anonaine, carvone, lina lool,samoquasine A, squamocin-L, squamocin-B, squamocenin, mortilin, Kaurenoic acid, phenolic and nonphenolic alkaloids, two crystalline alkaloids-muricine, muricinine92,4-cis and trans)-squamolinone, (2,4-cis and trans)-9-oxoasimicinone, bullacin B etc. (Chopra et al., 2002).

Escherichia coli is the most common bacterium isolated in microbiology laboratories, the most relevant facultative gram negative rod found in faeces, the most common causes of both intestinal infections (Banwart, 2000). E. coli is part of the healthy bowel faecal floral of both humans and lower animals. In 1885, a German pediatrician, Theodore Escherich, described the isolation from the faeces of healthy individuals and called it bacterium “coli” which was subsequently used for many years. However, in 1920 Rastellane and Chamlimers describe the genus Esherichia and the biochemical reaction they gave. the genus was very similar to modern description. They grow well on ordinary media forming large (2-3mm diameter), circular low convex, smooth strains are more heat resistance than other members of the enterobactriacea and will survive 60⁰_­­­ for 5 minute or 55⁰ for 60 minutes (Pelzar et al., 2000).

Staphylococcus aureus are gram positive cocci, in the family microcacea, form grape like clusters on grams’ stain. This organism is catalase positive (unlike streptococci species), non-motile, aerobic and facultative anaerobic. They are capable of prolonged survival on environmental surfaces in varying conditions and bacteriological cultures of the nose and skin of normal humans in variable yield Staphylococcus aureus.  More than 30 staphylococci species are the biochemical test. Automated diagnostic system, kits for biochemical characterization and DNA based assays are available for distinguishing among species. With few exceptions, Staphylococcus aureus is distinguished from other Staphylococcal species by its production of coagulates a surface enzyme that converts.

Salmonella are gram negative facultative rod shaped bacteria. The genus is a member of the family Enterobacteriaceae. It is composed of bacteria that relate to each other both phenotypically and genotypically. Salmonella organisms are found in virtually all animals, and humans.

Some serotypes of Salmonella are virtual species specific (Gill, 2000). Salmonella spp, cause many types of infections from mild self-Limiting to life threatening typhoid fever. The most common of Salmonella disease is self-limiting gastroenteritis with fever lasting less than 2 days and diarrhoea lasting for seven days (7 days), typhoid fever. They best studied enteric fever, is characterized by fever, headache, diarrhoea and abdominal pain and can produce fatal respiratory heptic, spleen, and neurologic damage. Bacteremia meningitis, respiratory disease, cardiac disease, osteomyelitis, and other local ingestion caused by Salmonella species. Salmonella typhi and Salmonella paratyphi A and B have been reported to cause gastroenteritis. (Bopp, 2003) normally antagonizing the activity of one another (Ekwenye and Elegalam, 2005).

In addition, despite the fact that the various extracts of Annona muricata have been tested invitro and were shown to be active against some bacteria, (Falae et al., 2000.) isolating specific strains of bacteria across other regions should be made to ascertain their invitro activity which is one of the motivation for this study display antimicrobial activity. It has been reported that the volatile oil of the plant contains mostly phenols, particularly thymol and that these are probably responsible for its reported antimicrobial action (Sofowora, 2000).

1.2 Justification

Many clinical important bacteria produce enzymes that are capable of destroying antibiotics and enzymatically degrade them to an infective form, the beta lactam bond can be hydrolyzed by a large family of enzymes known as the beta lactamases (John, 2011). However, many species of bacteria have become resistant to currently available antibiotic. There is therefore the need to search for more potent anti-bacterial agent from plant sources. In the continuation of global effort to search for drugs from plants crude seed extracts of Annona muricata against clinical isolates of Escherichia coli, Staphylococcus aureus, Salmonella typhi Pseudomonas aeruginosa being some of the endemic bacterial pathogens associated with many diseases will serve as an alternative source of antibacterial agent.

1.3 Aim

To determine the antibacterial activities of A. muricata N-hexane extract of the leaves of soursop (Annona muricata) of the Annonaceae  against  E-coli, S. aurues, S. typhi and P. aeruginoa.

1.4 objectives

1    To screen for phytochemical components of A muricata leaves using N-hexane as solvents of extraction.

2.   To evaluate the antibacterial efficacy of the extract on S.typhi, E-coli, S.aerues and P.aeruginosa.
3.   To determined the Minimum Inhibitory Concentration (MIC) and   Minimum Bactericidal Concentration (MBC).

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