ABSTRACT
The antibacterial activity of plant extracts compared to a known antibiotic
(ampicillin) was evaluated against ten gram-negative and gram-positive bacteria
which can infect humans. In addition, the efficacies of different extraction methods
were studied. Soxhlet, cold aqueous, and hot aqueous extractions were used in this
study. Moreover, extracts from the following plants were utilized: Zingiber officinale
(ginger); and the leaves of Agave americana (agave), Musa acuminata (banana),
Mangifera indica (mango), Azadirachta indica (neem), and Eucalyptus sp.
(Eucalyptus). The most antibacterial activities were observed for the extracts of
Eucalyptus sp. and Mangifera indica, which inhibited 60% and 50% of the tested
bacterial species, respectively. There was significant activity against gram-positive
bacteria. Agave and banana extracts, on the other hand, did not demonstrate any
antibacterial activity. The most susceptible species of bacteria tested was
Micrococcus luteus with a total of 8 out of 17 different plant/preparation
combinations affecting it. The least susceptible species were Escherichia coli,
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Proteus vulgaris, Salmonella typhi & Klebsiella pneumoniae. Soxhlet extraction
provided the highest number of zones of inhibition by its bioactive compounds of the
three extraction methods.
Key words: soxhlet extraction, aqueous extraction, ginger root, banana leaves, neem
leaves, eucalyptus leaves, mango leaves, agave leaves, disk diffusion, Kirby-Bauer
testing, gram-positive, gram-negative, bacteria.
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Table of Contents
List of Tables………………………………………………………………………………………………………. x
List of Figures …………………………………………………………………………………………………….. x
Introduction ………………………………………………………………………………………………………… 1
Literature Review ………………………………………………………………………………………………… 2
Antimicrobial Compounds ………………………………………………………………………………… 2
Bacteria …………………………………………………………………………………………………………… 3
Klebsiella pneumoniae …………………………………………………………………………………… 3
Pseudomonas aeruginosa ………………………………………………………………………………. 3
Escherichia coli ……………………………………………………………………………………………. 3
Staphylococcus aureus …………………………………………………………………………………… 4
Staphylococcus epidermidis ……………………………………………………………………………. 5
Bacillus subtilis …………………………………………………………………………………………….. 5
Micrococcus luteus ……………………………………………………………………………………….. 5
Salmonella typhi …………………………………………………………………………………………… 6
Proteus vulgaris ……………………………………………………………………………………………. 6
Enterococcus faecalis ……………………………………………………………………………………. 6
Plants ……………………………………………………………………………………………………………… 7
Zingiber officinale…………………………………………………………………………………………. 7
Agave americana ………………………………………………………………………………………….. 7
Musa acuminata ……………………………………………………………………………………………. 9
Mangifera indica…………………………………………………………………………………………… 9
Azadirachta indica ………………………………………………………………………………………. 10
Eucalyptus sp ……………………………………………………………………………………………… 10
Modes of Extraction ……………………………………………………………………………………….. 11
Testing Methods …………………………………………………………………………………………….. 12
Luria Broth and Kirby-Bauer testing ……………………………………………………………… 12
Materials and Methods ……………………………………………………………………………………….. 13
Extractions and Diffusion Disk Preparation ……………………………………………………….. 14
Soxhlet Extraction……………………………………………………………………………………….. 14
Hot Aqueous Extraction……………………………………………………………………………….. 17
Cold Water Extraction …………………………………………………………………………………. 18
Bacterial Culture Inoculation and Agar Plate Preparation ……………………………………. 19
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LB & LBA Growth Media Preparation ………………………………………………………….. 19
Bacterial Culture …………………………………………………………………………………………. 19
Plate Preparation …………………………………………………………………………………………. 19
Results ……………………………………………………………………………………………………………… 21
Pilot Test ……………………………………………………………………………………………………….. 21
Neem ……………………………………………………………………………………………………………. 22
Mango …………………………………………………………………………………………………………… 22
Ginger …………………………………………………………………………………………………………… 23
Eucalyptus …………………………………………………………………………………………………….. 23
Agave ……………………………………………………………………………………………………………. 24
Banana ………………………………………………………………………………………………………….. 24
Control ………………………………………………………………………………………………………….. 25
Discussion ………………………………………………………………………………………………………… 26
Result Summary …………………………………………………………………………………………….. 26
Comparison with Other Studies ……………………………………………………………………….. 26
Agave ………………………………………………………………………………………………………… 26
Ginger ……………………………………………………………………………………………………….. 26
Banana ………………………………………………………………………………………………………. 26
Eucalyptus ………………………………………………………………………………………………….. 26
Neem …………………………………………………………………………………………………………. 26
Mango ……………………………………………………………………………………………………….. 27
Problems & Limitations ………………………………………………………………………………….. 27
Way Forward ……………………………………………………………………………………………… 27
Conclusion ……………………………………………………………………………………………………….. 28
Appendices ……………………………………………………………………………………………………….. 29
A. Cold Aqueous Extraction ………………………………………………………………………….. 29
B. Soxhlet Extraction ……………………………………………………………………………………. 30
C. Hot Aqueous Extraction ……………………………………………………………………………. 30
Works Cited ……………………………………………………………………………………………………… 31
x
List of Tables
Table 1 Pilot Test ………………………………………………………………………………………………. 21
List of Figures
Figure 1 Ginger root …………………………………………………………………………………………….. 7
Figure 2 Agave plant ……………………………………………………………………………………………. 8
Figure 3 Banana tree ……………………………………………………………………………………………. 9
Figure 4 Mango tree …………………………………………………………………………………………… 10
Figure 5 Diagram of Soxhlet extraction equipment.. ………………………………………………. 15
Figure 6 Plant extracts being boiled on a heating mantle ………………………………………… 17
Figure 7 Nitrocellulose filtration apparautus …………………………………………………………. 18
Figure 8 Agar plates poured under a BSC …………………………………………………………….. 19
Figure 9 Bacteria left to grow overnight in a test tube rotator ………………………………….. 19
Figure 10 A Mueller-Hinton agar plate inoculated with S. aureus and various
antibiotics. ………………………………………………………………………………………………………… 20
Figure 11 “Brown zone” ……………………………………………………………………………………… 27
Figure 12 Bacterial contamination of the control ……………………………………………………………… 28
CHAPTER ONE
Introduction
There has been a rise in antibiotic resistant bacteria over the last decade. This is
mainly due to the mainstream misuse of them. What’s more is that these strains of
bacteria are found where the most vulnerable people in communities are, hospitals
(Wilson, et al. 2011). The combination of weak host defenses and drug-resistant
bacteria has led scientists to explore new options in antimicrobial therapy. One of
these options is the plant kingdom. For millennia, plants have been used and
marketed in one way or the other for a perceived ability to cure diseases. In fact,
60,000 years ago, a plant called hollyhock was used for medicine by Neanderthals
living in present-day Iraq (Cowan 1999). However, substantial scientific research has
not been conducted in the investigation of antibacterial activity expressed by plants.
Plants have many advantages. They are abundant, sustainable, and it is relatively
easy and inexpensive to extract potentially active compounds from them.
By broad definition an antimicrobial can be said to be any substance that antagonizes
the proliferation of microbes. “Microbe” is a general term which includes various
viruses, bacteria, fungi, and protozoan parasites – which may not necessarily be
harmful to humans. A more accurate term to describe harmful microbes is
“pathogen”. However, it is common knowledge that antiviral, antifungal,
antibacterial and antiparasitic drugs are targeted at the harmful microbes. With that
said, the antibacterial properties of different plants studied in this paper focuses on
the harmful pathogenic bacteria.
The aim of this study was to discover plant species with novel antibacterial activity.
To achieve this, locally found plants were acquired, a variety of methods were used
2
to extract their chemical contents, and possible antibacterial activity was determined
by testing the plant extracts against ten bacterial species.
Plants have coevolved with other organisms in their environments. Consequently, it
is proper to say that they must have developed mechanisms to protect themselves
from bacterial infection after an insect or animal has fed on their leaves. If this
bioactive mechanism is chemical in nature, these same chemicals can be extracted
and used directly on pathogenic or opportunistic bacteria and have some effect.
Therefore, the hypothesis for this study was that: Some plant extracts will
demonstrate significant antimicrobial activity.
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