Background of The Study
Background of The Study
Background of The Study
INTRODUCTION
country and elsewhere is one of the serious problems that man faced. This trend is
reflected through the progressive increase in number of medicinal drugs throughout the
market. Often the prices of manufactured drugs are high and the availability is not
always assured. The high cost of recommended drugs especially those antibiotics
besets the treatment of human diseases. This is the reason why researches have been
continuous inflation in prices of medicinal drugs motivated consumers to seek ways and
approach to this problem is the utilization of cheap but highly effective available
everywhere and it is difficult to move away from them. When people catch disease,
normally they buy antibiotic to fight it off and feel well again.
buy antibiotic to fight their sickness. That is why antibacterial and antifungal medium
with lower prices than those of commercially produced can help those people who find
themselves and their brood, to live on through the winter – as such, it’s packed with
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everything they need, including vitamins, minerals, antioxidants, and enzymes that are
beneficial for us. Honey is a sweet food made by bees using nectar from flowers. The
variety produced by honey bees is the one most commonly referred to, as it is the type
other bees (bumblebees, stingless bees) and other hymenoptera insects (e. g. honey
wasps) have different properties, and they are not discussed in this article. Honey bees
transform nectar into honey by a process of regurgitation and evaporation. They store it
This study wants to determine the efficacy of honey extract to resist and kill
Aspergillus fumigatus.
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3. Is there a significant difference on the zone of inhibition of the pathogens
different treatments?
The aim of this study is to produce and prove the capability of Honey extract in
drugs. The success of this study would provide a very wide and safe alternative
infections will give information to the public to watch, protect and preserve it.
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This can be a possible start for another innovation in enriching the knowledge
about honey achieving a greater purpose for this organism to improve the lives of
of the study can unlock new potentials of ants that can help people especially those in
Specifically, this study will be significant and helpful to the following persons:
CONSUMERS: This study will be beneficial to them for they can save a lot of money.
Instead of buying high priced commercial drugs, they can cure their skin diseases as
DRUG MANUFACTURERS: This study can serve as eye opener for them to be
RESEARCHERS: The study can serve as a basis for further researchers and
commercial, medicinal and environmental waste problem. This study will enhance their
skill and knowledge in conducting experiments which they can use for personal
STUDENTS: This study may serve as eye opener and encourage them to undertake
product development.
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Scope and Delimitations of the Study
was also conducted to produce an antimicrobial formulation for skin diseases and to use
as a drug for diarrhea out of cheap, easy to produce, and readily available materials like
honey.
The study was also limited in testing the experimental samples on one type of
The preparation of this research study was conducted from June to August 2014.
Collection honey as well as preparation of the extract was done at Brgy. Tugatog,
Bongabon, Nueva Ecija. Preparation and culturing of bacteria was done at Veterinary
University.
The measurement of the zone of Inhibition and testing of the inhibitory effect of
University.
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Chapter II
Certain books, journals, encyclopedias, and studies, which have direct and indirect
connection with the study, were carefully reviewed. These studies helped the
Medicinal Drugs
The widely held belief that modern medicine is all the product of complex
chemical syntheses is a fallacy. More than 120 current prescription drugs are still
obtained from higher plants and about 25% of all prescriptions contain one or more
to currently available antibiotics has necessitated the search for novel and effective
antimicrobial compounds. Globally, plant extracts are employed for their antibacterial,
antifungal and antiviral activities. It is known that more than 400,000 spp. of tropical
flowering plants have medicinal properties and this has made traditional medicine
cheaper than modern medicine. Some plant decoctions are of great value in the
infertility, wound and cutaneous abscesses (Meyer et al., 1996; Dimayuga and Gracia,
1991).
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Black (1996) mentioned that there is a growing recognition of the economic value
Honey
According to National Honey Board (2011), honey is a sweet food made by bees
using nectar from flowers. It is also stated to cure some allergies; particularly localized
honey to an area could help minimize seasonal allergies as bees feed on pollen from
local plants which eventually find its way to form honey. Its use for centuries is as a
treatment for sore throats and coughs, and according to recent research, may in fact be
as effective as many common cough medicines. Scientists have revealed that honey
has powerful anti-bacterial properties on at least sixty species of bacteria, and unlike
antibiotics, which are often useless against certain types of bacteria; honey is non-toxic
and has strong effects. The pH of honey is commonly between 3.2 and 4.5. This
Escherichia coli
gram negative rod-shaped bacterium that is commonly found in the lower intestine of
warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some
such as serotype O157:H7 can cause serious food poisoning in humans, and are
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E. coli are not always confined to the intestine, their ability to survive for brief
periods outside the body makes them an ideal indicator organism to test environmental
The bacteria can also grown easily and its genetics are comparatively simple
Staphylococcus aureus
Staphylococcus aureus (literally the “golden cluster seed” or “the seed gold” and
also known as golden staph and Oro staphira) is the most common cause of staph
infections. It is a spherical bacterium, frequently part of the skin flora found in the nose
and on skin. About 20% of the populations are long-term carriers of S. aureus.
S. aureus can cause a range of illnesses from minor skin infections, such as
shock syndrome (TSS) and bacteremia sepsis. Its incidence is from skin, soft tissue,
respiratory, bone, joint, endovascular to wound infections. It is still one of the five most
be confused with the similarly dangerous (and also medically relevant) species of the
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Aspergillus fumigatus
Aspergillus fumigates is a fungus of the genus Aspergillus, and is one of the most
soil and decaying organic matter such as compost heaps where it plays an essential
role in carbon and nitrogen recycling. Colonies of the fungus are produced from
airborne. For many years A.fumigatus was thought to only reproduce asexually, as
neither mating nor meiosis had ever been observed. However, in 2008 it was shown tha
A.fumigatus possesses a fully functional sexual reproductive cycle,145 years after its
RELATED STUDIES
Bacteria
A new research published in the July 2010 print edition of the FASEB
Journal explains for the first time how honey kills bacteria. Specifically, the research
shows that bees make a protein that they add to the honey, called defensin-1, which
could one day be used to treat burns and skin infections and to develop new drugs that
"We have completely elucidated the molecular basis of the antibacterial activity of
medicine," said Sebastian A.J. Zaat, Ph.D., a researcher involved in the work from the
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Department of Medical Microbiology at the Academic Medical Center in Amsterdam.
To make the discovery, Zaat and colleagues investigated the antibacterial activity
Ultimately, researchers isolated the defensin-1 protein, which is part of the honey bee
immune system and is added by bees to honey. After analysis, the scientists concluded
that the vast majority of honey's antibacterial properties come from that protein. This
information also sheds light on the inner workings of honey bee immune systems, which
may one day help breeders create healthier and heartier honey bees.
"We've known for millennia that honey can be good for what ails us, but we
the FASEB Journal, "Now that we've extracted a potent antibacterial ingredient from
honey, we can make it still more effective and take the sting out of bacterial infections."
Pages 554–557) on July 2012 evaluated the antifungal activity of four honeys of
different types from Algeria against pathogenic yeast i.e.Candida albicans (C. albicans)
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Four Algeria honeys of different botanical origin were analyzed to test antifungal
10%, 30%, 50% and 70% w/v) of honey were studied in vitro for their antifungal activity
honey was (7–23 mm) for Rhodotorula sp., while C. albicans showed clearly resistance
towards all concentrations used. The MICs of tested honey concentrations against C.
respectively. This study demonstrates that, in vitro, these natural products have clearly
Universidade Estadual de Feira de Santana (UEFS) in Brazil (Apr. 2013) states the
Plebeia sp. were investigated. The agar well diffusion assay demonstrated that all
honeys had antibacterial activity against Staphylococcus aureus, but only the samples
more active than that from Plebeia sp. for S. aureus and E. coli. The microorganisms
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Pseudomonas aeruginosa and Candida albicans were resistant to the all native
stingless bee honey in both assays. Honey was more effective against bacteria than a
sugar solution, suggesting that the mechanism for bacterial growth inhibition is not only
related to the osmotic effect. The results of antimicrobial activity may explain the
Candida albicans
Ethiopian food through ethno botanical survey and studies their antifungal activity
against Candida albicans. Ethno botanical survey of the selected Kebeles of Jimma,
Ethiopia was conducted using a semi structured questionnaire from October 2006 to
November 2007. Antifungal nature of the spices and combination of spices and honey
were evaluated by agar well diffusion assay from September 2008 to July 2010. Ethno
botanical survey indicated fourteen species of spices and honey play a major role in
Ethiopian food & beverages. Single plant extract of Trachyspermum copticum showed
highest activity against C. albicans. The same plant showed antagonistic effect when
combined with brown and white honey. Cinamomum zeylanicum showed highest
synergistic effect with both brown & white honey when compared to Allium ursenum,
hodiense. Thus spices used in Ethiopian food could be a preventive as well as a cure
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Relevance of Review of Related Literature and Studies
The related literature and readings provide significant information that can help
the researchers develop the study better; providing clear and reliable processes and
concept involved; like the description of the different bacterial pathogens that can be
The related studies provide the researchers the knowledge about the past
regarding the past studies about the ability and characteristics of honey.
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Chapter III
METHODOLOGY
The chapter presents the method of research utilized in this study, the
experimentation.
Materials
The main material used in this research is the honey. Honey was bought in the
public market. The material was selected because of their relatively low cost, nutrient
content, and availability in the locality. Materials such as graduated cylinder, petri dish,
spatula, vials, alcohol lamp, weighing scale, test tube, transferpette, vernier caliper,
beaker, incubator, electric hot plate, cheese cloth and auto clave were also utilized in
the study. Lysol, Penicillin and water is also used as the positive and negative control.
Experimental Design
fumigatus were used in the study. Each microorganism was subjected to varying
percent of honey extract at 100%, 50%, and 10%. There was also positive and negative
control.
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Table 1.Treatment Assignment using Honey Extract Against
Staphylococcus aureus, E.coli (bacteria) and Aspergillus fumigatus(fungus)
Honey
PERCENTAGE Distilled
TREATMENTS Extract
CONCENTRATION Water(ml)
(ml)
Treatment 1 100% 0 5
Treatment 2 50% 2.5 2.5
Treatment 3 10% 4.5 0.5
Penicillin
Positive Control 0 0
(bacteria)
The positive control (Penicillin) for the Echerichia coli and Staphylococcus aureus
was different with the one used for the Aspergillus fumigatus, since Aspergillus
Medicine, Central Luzon State University. The organism was inoculated into separate
test tubes with sterile distilled water and incubated at 37°C for 24 hours. Suitable culture
The microbial species used were cultured. Pure bacteria (Escherichia coli and
Staphylococcus aureus) were placed in a petri dish with nutrient agar and incubated to
grow 24 hours while the fungus (Aspergillus fumigatus) used was cultured for three
days.
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Preparation of Growth Agar for the Test Organism
Nutrient agar is the type of medium that was used in this study. It was
prepared by weighing 7.2 grams of Soyabean Digest Agar for the E. coli and 5.2 grams
for the Staphylococcus aureus and dissolves it in a sterile 180 ml of water. For the
Aspergillus fumigatus 11.7 grams of Sabouraud Dextrose Agar was dissolved in 180 ml
of sterile water. After preparing the solution, it will be covered with clean cotton with
gauze and covered with foil. The prepared medium will be cooked using electric hot
plate at 25°C. After cooking, approximately 20 ml were poured sterile onto sterile petri
dish and allowed to harden. These were incubated at room temperature in a level
In-vitro Bioassay
In the eradicate test against the pathogen, the crude extracts were tested against
existing infections and measured its zones of inhibitions; presence of inhibition only
meant that the extract can kill the pathogen from an infected host. While protectant
against the pathogen meant that the extracts can stop the pathogen from attacking the
organism.
Inoculation of Pathogen
The prepared nutrient agar was sterilized for 30 minutes at 15 psi. Upon cooling,
20 ml of nutrient agar will aseptically transfer evenly in the whole plate. It is allowed to
solidify at room temperature. When the medium had already solidified, the bacterial
organisms were incorporated and incubated for a maximum 24 hours to show their
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The zone of inhibitions were determined and measured at the nearest
(mm) using a vernier caliper after 24 hours of inoculation. If the zone of inhibition
appeared in the plate, it signified the effectivity of the extracts against the pathogen.
Extracts that showed no zone of inhibition means that the extract had no inhibiting
property against the test organism. Three replicates were prepared for each treatment.
Data to be Gathered
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Chapter IV
This part presents, analyses, and interprets the data gathered in an organized
manner regarding the use of the treatments from honey extract inhibiting the growth of
M 40
E
20
A
N 0
Experimental Treatment
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The table shows the sensitivity of Escherichia coli to the experimental honey
extract against the positive control (Penicillin) and the negative control (distilled water)
after 24 hours of incubation. Disc diffusion method was the procedure done on the
experiment. Table 2.0 shows the mean measurements of the zone of inhibition of E. coli
the most effective treatment with a mean of 21.95 mm. The treatment 1(100%
concentration of Honey extract) was not effective as well as treatment 2,3 and 5. This
implied that only that positive control (penicillin) has antibacterial property against E.
coli.
among the mean zone of inhibitions of the treatments 100%, 50% and 10%
The result of the investigation showed that honey extract does not inhibit the
T5=Negative Control - - - - -
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Sensitivity of Staphylococcus aureus to Honey Extract
Measurement of the Zone of Inhibition (mm)
M 60
E 40
A 20
N
0
Experimental Treatment
Table 3 presents the result of the antibacterial test of the experimental treatment
against the positive control (Penicillin), and the negative control (distilled water) after 24
hours of incubation against Staphylococcus aureus. The table shows the mean
treatments. It shows that Treatment 4, Penicillin was significantly the most effective
treatment with a mean of 45.53mm. The 100% concentration of Honey extract was next
most effective treatment with a mean measurement of 16.23 mm. This implied that
100% honey extract has antibacterial property against E. coli. The effectivity of the
lessens.
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Treatment 2 (50% honey extract), treatment 3 (10% honey extract) and
Treatment 5 (negative control) showed no zone of inhibition against the test organism.
among the mean zone of inhibitions of the treatments 100%, 50% and 10%
honey extract especially 100% is the best concentration to show the Antibacterial
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Sensitivity of Aspergillus fumigatus to Honey Extract
Measurement of the Zone of Inhibition (mm)
50
M
40
E
30
A
20
N
10
0
T1=100 % T2=50% T3=10% T4=Positive T5=Negative
Control Control
Experimental Treatment
Table 4.0 showed the mean of the measurement of the zone of inhibition of
extract) was significantly the most effective with the mean in the zone of inhibition of
43.92 mm. This was followed by treatment 4(Positive control) with an average zone of
inhibition of 24.98 mm. This implied that the 100% honey extract has antifungal
(10% honey extract) and treatment 5 (negative control) were significantly no zone of
inhibition.
among the mean zone of inhibitions of the treatments 100%, 50% and 10%
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The result of the investigation showed that variable concentration of honey
extract has no effect on the microorganisms. The pure extract greatly inhibits the
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Chapter V
Summary
The different treatments were placed in the wells of different culture media and
Honey extract concentrations 100%, 50% and 10% were found that there is no
antibacterial property for E. coli. In the case of Staphylococcus aureus, honey extract
concentration 100% was found to have antibacterial properties. Of all the treatments,
the positive control Penicillin for E. coli and Staphylococcus aureus showed the highest
Aspergillus fumigatus. Of all the treatments, the 100% concentration of honey extract for
Distilled water, negative control for the three organisms showed no effects by
Conclusion
The results of the study showed that honey extract have anti-fungal properties
better than the positive control Lysol. Antibacterial properties of the honey extract, on
the other hand, are only showed in Staphylococcus aureus and not on the E.coli
bacteria.
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Recommendations
experimentally made honey drug. Further research must be conducted to test the anti-
fungal property and antibacterial property of the extract to other test organisms. The
period and method of application should also be modified to attain better results.
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BIBLIOGRAPHY
Black, J.K. (1996) Microbiology: Principles and Application. 3rd ed. Prentice Hall,
JESSE L. Central Luzon State University Science High School, March 2010.Common
pathogen Aspergillusfumigatus.Nature
.
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Sebastian A.J. ZaatPh.D Department of Medical Microbiology at the Academic Medical
Banaynal (1999)
Black (1996)
(Offenberg&Wiwatwitaya 2010)
Asian Pacific Journal of Tropical Biomedicine (July 2012) Antifungal activity of four
honeys of different types from Algeria against pathogenic yeast: Candida albicans and
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Appendices
28
Materials
Auto-clave Transferpette 30
Materials
31
APPENDIX FIGURES
32
Appendix Figure 3. Measurement of the Zone of Inhibtion of
Aspergillus fumigatus
Staphylococcus aureus
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Appendix Figure 5. Measurement of the Zone of Inhibtion of
Escherichia coli
34
Appendix Figure 7.0 Weighing of Nutrient Agar used in the study
wells.
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Appendix Figure 9. Microscopic view of Escherichia coli
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APPENDIX TABLES
ANOVA
Source of
Variation SS df MS F P-value F crit
Between Groups 0.039 2 0.0195 0.000202 0.999798 3.885294
Within Groups 1156.482 12 96.3735
Total 1156.521 14
ANOVA
Source of
Variation SS df MS F P-value F crit
Between Groups 1.009333 2 0.504667 0.001285 0.998716 3.885294
Within Groups 4711.444 12 392.6203
Total 4712.453 14
ANOVA
Source of
Variation SS df MS F P-value F crit
Between Groups 39.792 2 19.896 0.048288 0.953044 3.885294
Within Groups 4944.353 12 412.0294
Total 4984.145 14
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