Justice Cecilia Muñoz Palma High School

Science Department
Molave St., Payatas B, Quezon City

The preparation of an antiseptic solution using Oregano (Origanum vulgare) as a

bacteriocidal agent with Honey as its bacteriostatic component

In partial fulfillment of the Requirements

in Science III (Chemistry)

Submitted by:
Group 4 (III- Hydrogen)
Nantes, Ma. Mae
Ocampo, Kyle Lester
Paca-anas, Melody
Panis, Christine Iesra Mae
Panis, Joshua Mark
Pattaui, Karen Joyce
Payapag, Aldrin
Porcare, Jimwell
Quiamco, Jeanky
Reyes, Charles Gevie
Sertimo, Heide
Sevilla, Cecille Kay

Submitted to:
Mrs. Estela M. Galolo

February 2013
TABLE OF CONTENTS
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Title Page ………………………………………………………………………………..…….... 1

Table of Contents …………………………………………………………………………....... 2

Acknowledgement…………………………………………………………………………....... 3
Abstract………………………………………………………………………………………... 4

Chapter 1. Research Problem and Its Background……………………………………….......5

 Background of the Study

 Statement of the Problem
 Hypotheses
 Significance of the Study
 Scope and Delimitations
 Definition of Terms

Chapter 2.Review of Related Literatures and Studies………………………………………. 8

Chapter 3.Research Methodology……………………………………………………….….. 11

Chapter 4. Results and Discussion ………………………………………………………….. 14

Chapter 5. Conclusion and Recommendation………………………………………...……... 15

Bibliography……………………………………………………………………...………….….16

ACKNOWLEDGEMENT

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 The researchers wish to thank the following for the completion of this project:

The researchers’ parents for the material and moral providing;

The Panis, Pattaui and Reyes Family, for providing the researchers with all available
resources;

Mrs. Estella M. Galolo, for the guidance in this project; and

Above all, to Lord God for the guidance and special talent He gave which enabled us to
successfully come up with this investigatory project.

ABSTRACT

The Philippines is rich many natural resources. One of this is the herbal plants which are
used through many generations. At the same time, Oregano (Origanum vulgare) is very

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commonly used as a medicinal plant for cough and other uses like herb spice. Likewise, honey
is used in different culinary purposes, yet studies found out that it can also kill bacteria.
The study attempted to create a natural, easy-to-prepare and affordable antiseptic solution
made from Oregano (Origanum vulgare) and honey. The antiseptic aimed to kill bacteria which
can cause some skin illnesses” ! ! “
To prepare the 3 antiseptic solutions, all ingredients and equipments were gathered and
made ready. All, the needed equipments were cleaned and the needed ingredients, especially
Origanum vulgare (oregano) leaves were washed. Next, extracts of Origanum vulgare (oregano)
leaves were gathered using a mortar and pestle.Then, the crushed leaves of Origanum vulgare
(oregano) were placed in a strainer or cheese cloth to get more extract. The extract was measured
with the measuring cups 15 milliliters (ml) of this was measured for the first bottle and was put
into a bottle using a funnel. 35 milliliters (ml) was measured and transferred into the third bottle
also using funnel.Afterwards, honey was also measured with another beaker with the same
measurement as the Origanum vulgare (oregano) extract. Then, 15ml of honey was mixed in
third bottle which measures 35ml of Origanum vulgare (oregano) extract, 25ml of honey was
mixed in the second bottle with the 25ml of Origanum vulgare (oregano) extract. 35ml of honey
was mixed in the first bottle with the 15 ml Origanum vulgare (oregano).
In testing the solution, each of the solutions was placed in 3 Petri dishes and sealed
immediately. A microscope to observe the changes in the number of bacteria after “ ! ! “ , their
color, shape and other observable characteristics were also noted.
Based on the results, the three solutions have the same effects even if they are of different
concentrations. The oregano-honey solutions prevented the growth of bacteria as the number of
colonies after three –weeks of exposure.
Findings of the study revealed that the Oregano (Origanum vulgare) extract and honey
are effective ingredients for the preparation of natural, easy-to-prepare and affordable antiseptic
solution. Result of this investigation clearly presents a positive impact on the development of
urban living. However, this study needs improvement in the measurement of the ingredients
Oregano (Origanum vulgare) extract and honey needs more manipulation, thus other
concentrations must also be considered. This product can be used as the substitute for expensive
antiseptic solution bought in the market. Also, this product can be used in different parts of the
house where bacteria are found.
CHAPTER 1
RESEARCH PROBLEM AND ITS BACKGROUND

Background of the Study:

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 The Filipinos, especially the elders, often use the oregano (Origanum vulgare) in food
preparations and as herbal medicine for cough because it is readily available in the Philippines.
Unfortunately, they do not know that oregano (Origanum vulgare) can also be used to help
relieve sicknesses and illnesses caused by bacteria in our environment. Studies found out that
oregano can kill different kinds of bacteria like Aeromona hydrophil, Citrobacter, Entero bacter
aerogenese and Escherichia coli.
One thing that is also commonly used for food preparation is Honey, which is also
useful food its anti-bacterial properties. Honey is used to kill bacteria by inhibiting its growth.
Because of this, the study on oregano-honey combination as an antiseptic solution is worth
looking into.

Statement of the Problem:

This study aims to create a natural, easy-to- prepare and affordable antiseptic which is
made from Oregano (Origanum vulgare) and honey. Specifically, this study sought answers to
the following problems:
1. Is the oregano-honey combination an effective component for an antiseptic solution?
2. Does changing the ratio of the amount of oregano and honey affect the antiseptic
capability of the product?
3. What is the mortality rate of identified bacteria exposed to the solution?
4. What diseases are possibly prevented by the use of the antiseptic?

Statement of the Hypotheses:

To guide the researchers for better understanding the following hypotheses were
formulated:
1. Yes, the oregano-honey combination would be an effective component for an
antiseptic solution.

2. Yes, the ratio of the oregano to honey in the prepared solutions would affect the
antiseptic capability of the product.

3. The mortality rate of the bacteria in the different oregano-honey preparations

can be determined after a continuous 2-week exposure to the solutions.

4. The prepared solutions will be specific to certain diseases-causing bacteria.

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Significance of the Study:
This study aimed to create a natural, easy-to-prepare and affordable antiseptic solution
made from Oregano (Origanum vulgare) and honey. The antiseptic aims to kill bacteria which
cause some skin illnesses” ! ! “ Having an antiseptic that is readily available is the first step to a
healthy, germ-free life in a not-so-clean urban living.

Scope and Delimitations

The solution in this study prepared in Petri dishes with the cultured bacteria, specifically
coming from the doorknob of Panis„house. The respondents of the study are those who are not
sensitive to any of the ingredients used in this research. “ ! ! “
Definition of Terms
The following words were used in this study to guide in better understanding of this
research.
Oregano is a perennial herb, growing from 20-80 cm tall with opposite leaves 1-4 cm
long. Oregano will grow will in PH range between 6.0 (mildly acid) and 9.0 (strongly alkaline)
with a preferred range between 6.0 and 8.0.

Honey is a sweet food made by bees using nectar from flowers. The variety produced
by honey bees (genus Apis) is the most commonly referenced. It is the type of honey collected
by beekeepers and consumed by humans. Honey produced by other bees and insects has
distinctly different properties.

Antiseptic are antimicrobial substances that are applied to living tissue/skin to reduce
the possibility of infection, sepsis, or putrefaction. Antiseptics are generally distinguished from
antibiotics by the latter‟s ability to be transported through the lymphatic system to destroy
bacteria within the body, and from disinfectants, which destroy microorganisms found on non-
living objects

Bacteriostatic agent or bacteriostat, abbreviated Bstatic , is a biological or chemical

agent that stops bacteria from reproducing, while not necessarily harming them otherwise.
Depending on their application, bacteriostatic antibiotics, disinfectants, antiseptics and
preservatives can be distinguished. Upon removal of the bacteriostat, the bacteria usually start to
grow again. This is in contrast to bactericides which kill bacteria.

Bactericide or bacteriocide sometimes abbreviated Bcidal, is a substance that kills

bacteria and ideally nothing else. Bactericides are disinfectants, antiseptics or antibiotics.

Bacterial Colony is defined as a cluster of bacteria growing on the surface of or within a

solid medium, presumably cultured from a single cell. Because all organisms within a colony

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descend from a single ancestor‟ they are genetically identical (except for mutations that occur at
a low, unavoidable frequency, as well as the more likely possibility of contamination).

Agar is used throughout the world to provide a solid surface containing medium for the
growth of bacteria and fungi. Microbial growth does not destroy the gel structure because most
microorganisms are unable to digest Agar. Agar is typically sold commercially as a powder that
can be mixed with water and prepared similarly to gelatin before use as a growth medium.

Phenol itself is an effective bactericide and anti-fungal agent, which is used as a

preservative in some injections and creams.

Antioxidant benefits include strengthening your immune system, slowing down the
aging process and most importantly fighting off free-radicals.

Flavonoids have antioxidant activity. Flavonoids are becoming very popular because
they have many health promoting effects. Some of the activities attributed to flavonoids include:
anti-allergenic, anti-cancer, antioxidant, anti-inflammatory and anti-viral.

CHAPTER 2
REVIEW OF RELATED LITERATURE AND STUDIES

REVIEW OF RELATED LITERATURE

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 According to http://www.wikipedia.com/ , oregano scientifically named Origanum
vulgare by Carolus Linnaeus, is a common species of Origanum, a genus of the mint family
(Lamiaceae). It is native to warm-temperate western and southwestern Eurasia and the
Mediterranean region.
Oregano is a perennial herb, growing from 20–80 cm tall, with opposite leaves 1–4 cm
long. Oregano will grow in a pH range between 6.0 (mildly acid) and 9.0 (strongly alkaline) with
a preferred range between 6.0 and 8.0. The flowers are purple, 3–4 mm long, produced in erect
spikes. It is sometimes called wild marjoram, and its close relative O. majorana is known as
sweet marjoram.
It prefers a hot, relatively dry climate, but will do well in other environments. To
cultivate, it should be planted in early spring, in fairly dry soil, with full sun. The plants should
be spaced 12 inches apart.
Origanum vulgare hirtum (Italian oregano, Greek oregano), is one of the notable
subspecies of oregano, a common source of cultivars with a different aroma from those of O. v.
gracile. Growth is vigorous and very hardy, with darker green, slightly hairy foliage. Generally,
it is considered the best all-purpose culinary subspecies.
Hippocrates used oregano as an antiseptic, as well as a cure for stomach and respiratory
ailments. Cretan oregano (O. dictamnus) is still used today in Greece as a palliative for sore
throat.
Oregano is high in antioxidant activity, due to a high content of phenolic acids and
flavonoids. It also has shown antimicrobial activity against strains of the food-borne pathogen
Listeria monocytogenes
Oregano is the anglicised form of the Italian word origano, or possibly of the Medieval
Latin organum; this latter is used in at least one Old English work. Both were drawn from the
Classical Latin term origanum, which probably referred specifically to sweet marjoram, and was
itself a derivation from the Greek ὀρίγανον (origanon), which simply referred to "an acrid herb".
The etymology of the Greek term is often given as oros ὄρος "mountain" + the verb ganousthai
γανοῦσθαι "delight in", but the Oxford English Dictionary notes it is quite likely a loanword
from an unknown North African language.

Also, according to http://www.wikipedia.com/, Honey is a sweet food made by bees

using nectar from flowers. The variety produced by honey bees (the genus Apis) is the one most
commonly referred to and is the type of honey collected by beekeepers and consumed by
humans. Honey produced by other bees and insects has distinctly different properties.
The average pH of honey is 3.9, but can range from 3.4 to 6.1. Honey contains many
kinds of acids, both organic and amino.
Raw honey is honey as it exists in the beehive or as obtained by extraction, settling or straining,
without adding heat (although some honey that has been "minimally processed" is often labeled
as raw honey). Raw honey contains some pollen and may contain small particles of wax. Local
raw honey is sought after by allergy sufferers as the pollen impurities are thought to lessen the
sensitivity to hay fever.
Honey has an osmotic effect. Honey is primarily a saturated mixture of two
monosaccharide, with a low water activity; most of the water molecules are associated with the
sugars and few remain available for microorganisms, so it is a poor environment for their
growth. If water is mixed with honey, it loses its low water activity, and therefore no longer
possesses this antimicrobial property.

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 Likewise, according to http://www.wikipedia.com/, Antiseptics are antimicrobial
substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or
putrefaction. Antiseptics are generally distinguished from antibiotics by the latter's ability to be
transported through the lymphatic system to destroy bacteria within the body, and from
disinfectants, which destroy microorganisms found on non-living objects.
Some antiseptics are true germicides, capable of destroying microbes (bacteriocidal),
while others are bacteriostatic and only prevent or inhibit their growth.
Antibacterials are antiseptics that have the proven ability to act against bacteria.
Microbicides which destroy virus particles are called viricides or antivirals.

Similarly, according to http://www.wikipedia.com/, bacteriostatic agent or bacteriostat,

abbreviated Bstatic, is a biological or chemical agent that stops bacteria from reproducing, while
not necessarily harming them otherwise. Depending on their application, bacteriostatic
antibiotics, disinfectants, antiseptics and preservatives can be distinguished. Upon removal of the
bacteriostat, the bacteria usually start to grow again. This is in contrast to bactericides, which kill
bacteria. Bacteriostats are often used in plastics to prevent growth of bacteria on surfaces.
Bacteriostats commonly used in laboratory work include sodium azide (which is acutely toxic)
and thiomersal (which is a mutagen in mammalian cells).

Moreover, according to http://www.wikipedia.com/, a bactericide or bacteriocide,

sometimes abbreviated Bcidal, is a substance that kills bacteria and, ideally, nothing else.
Bactericides are disinfectants, antiseptics, or antibiotics.

.
REVIEW OF RELATED STUDIES

According to http://www.ncbi.nlm.nih.gov/pubmed/10736000, the volatile oils of black

pepper [Piper nigrum L. (Piperaceae)], clove [Syzygium aromaticum (L.) Merr. & Perry
(Myrtaceae)], geranium [Pelargonium graveolens L'Herit (Geraniaceae)] , nutmeg [Myristica
fragrans Houtt. (Myristicaceae), oregano [Origanum vulgare ssp. hirtum (Link) Letsw.
(Lamiaceae)] and thyme [Thymus vulgaris L. (Lamiaceae)] was assessed for antibacterial
activity against 25 different genera of bacteria. These included animal and plant pathogens, food
poisoning and spoilage bacteria. The volatile oils exhibited considerable inhibitory effects
against all the organisms under test while their major components demonstrated various degrees
of growth inhibition.
In addition, according to
http://www.pakbs.org/pjbot/PDFs/39%282%29/PJB39%282%29609.pdf : the oil , aqueous
infusion and decoction of oregano (Origanum vulgare), of the family Limiaceae, were asessed
for antibacterial activity against 11 different genera of Gram-ve bacilli viz., Aeromonas
hydrophila, Citrobacter sp., Klebsiella ozaenae, K. pneumonia, Proteus mirabilis,Pseudomonas
aeruginosa, Salmonella typhi, S. paratyphi B, Serratia marcescens and Shigella dysenteriae, by
disc diffusion method . Oregano oil exhibited the highest activity against Citrobacter species
with mean zone of inhibition of 24.0 mm + 0.5 . The acqueous infusion also showed significant
inhibitory activity against Klebsiella pneumoniae (20.1 mm + 6.1 SD), Klebsiella ozaenae(19.5

9
mm + 0.5 SD) and Enterobacter aerogenes (18.0 mm). Besides, all isolates were found resistant
to the aqueous decoction of oregano seeds.

Meanwhile, according to http://online.liebertpub.com/doi/abs/10.1089/acm.2007.6366,

honey has had a valued place in traditional medicine for centuries. Renewed interest in honey for
various therapeutic purposes, including treatment of infected wounds, has led to the search for
different types of honey with antibacterial activity. In this study, we have assessed the
antibacterial activity of different types of honey (manuka honey from Australia, heather honey
from the United Kingdom, and locally marketed Indian honey).

Also, according to http://www.hindawi.com/journals/btri/2011/917505/, the in vitro

antibacterial activity of methanol, ethanol, and ethyl acetate extracts of raw and processed honey
was tested against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Bacillus
cereus, Enterococcus faecalis, and Micrococcus luteus) and Gram-negative bacteria (Escherichia
coli, Pseudomonas aeruginosa, and Salmonella typhi). Both types of honey showed antibacterial
activity against tested organisms with the zone of inhibition (ZOI) ranging from 6.94 to
37.94 mm, while E. coli, S. typhi, and P. aeruginosa showed that sensibility towards all the
extracts with ZOI ranges between 13.09 to 37.94 mm. The methanol extract showed more potent
activity than other organic extracts. Gram-negative bacteria were found to be more susceptible as
compared to Gram-positive bacteria except E. faecalis. The broth microdilution assay gave
minimum inhibitory concentrations (MIC) value of 625 μg/mL, while the minimum bactericidal
concentration (MBC) ranges between 625 μg/mL 2500 μg/mL. The study showed that honey has
antibacterial activity (bacteriostatic and bactericidal effect), similar to antibiotics, against test
organisms and provides alternative therapy against certain bacteria.

CHAPTER 3
RESEARCH METHODOLOGY

Preparation of the solution

The ingredients and equipments needed in the research are the following:

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 Honey
Origanum vulgare (Oregano) leaves
Agar (Gelatin Powder)
Measuring cups
3 Sterilized bottles
Strainer or Cheese Cloth
Mortar and Pestle
Knife
Spoons
3 Sterilized Petri dishes
Cotton buds
Cupboard
Plastic Gloves
Funnel

Procedures in preparing the antiseptic solution

First, all ingredients and equipments were gathered and prepared. All, the needed
equipments were cleaned and the needed ingredients, especially Oreganum vulgare (oregano)
leaves were washed. Next, the Oreganum vulgare (oregano) leaves were put in a mortar and
pestle until the extract of it were gathered. Then, the crushed leaves of Oreganum vulgare
(oregano) were put in a strainer or cheese cloth to get more extract. The extract was measured
with the measuring cups 15 milliliters (ml) was measured for the first and was put into a bottle
using funnel. 35 milliliters (ml) was measured and put into the third bottle also using funnel.
Afterwards, honey was also measured with another measuring cup as the same as the
measurement in the Oreganum vulgare (oregano) extract. Then, 15ml of honey was mixed in
third bottle which measures 35ml of Oreganum vulgare (oregano) extract, 25ml of honey was
mixed in the second bottle with the 25ml of Oreganum vulgare (oregano) extract. 35ml of honey
was mixed in the first bottle with the 15 ml Oreganum vulgare (oregano).

Procedures in culturing the bacteria

First get the agar or gelatin powder and boil it according to the manufacturer‟s
instruction. Use a clean cooking thermometer to make the heat certain. Next, open a Petri dish
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and pour just enough agars into the dish to cover the bottom completely. Put the lid back on
immediately and set the dish aside until the agar firms. Fill each dish the same way, never
leaving the top of the dish off any longer than absolutely necessary. Once the agar hardens, you
may store the dishes in the refrigerator upside down to avoid the possibility of any airborne
bacteria getting into the dishes. Then, remove the Petri dishes from the refrigerator only when
you are ready to use them. Open your sterile cotton swab package and rub the cotton swab on a
surface you wish to test for bacteria. Open a Petri dish and rub your swab gently across the
surface of the agar in an “S” pattern. Close the lid on the Petri dish. Lastly, set the dish in a warm
dark cupboard for three to four days.

Procedures in testing the solution

First, remove the dish from cupboard. There is no need to open the dish. Observe the
bacteria colonies through the top of the dish. Record their color, shape and anything else
distinguishing. Then, test the cultured bacteria with the three solutions. Put one tablespoon of
each solutions to each Petri dishes and seal it again immediately. Put the first, second and third
solution on the first, second and third Petri dish respectively. Use a microscope to see the
changes of bacteria better. Record their color, shape and anything else distinguishing.
Also note mortality rate within 3-weeks exposure.

Tables:

A. Measurement of Origanum vulgare ( Oregano) and honey in milliliters to form

antiseptic solutions

Antiseptic Solution Measurement of Measurement of Solution

Origanum vulgare honey
(Oregano) extract

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 1st bottle 15 ml 35 ml 50 ml
2nd bottle 25 ml 25 ml 50 ml
3rd bottle 35 ml 15 ml 50 ml

B. Measurement of Origanum vulgare ( Oregano) and honey in every solution

to be put at every Petri dish

Cultured bacteria Measurement of Measurement of honey

Origanum vulgare
(Oregano) extract
1. Petri Dish 1 15 ml 35 ml
2. Petri Dish 2 25 ml 25 ml
3. Petri Dish 3 35 ml 15 ml

C. Observation on the cultured bacteria before testing the solutions

Cultured bacteria Observations

Number of colonies Shape Color
1. Petri Dish 1
2. Petri Dish 2
3. Petri Dish 3

D. Observation on the number of colonies with the antiseptic solutions

After two-weeks exposure

Cultured bacteria Number of colonies

After 1 week After 2 weeks After 3 weeks
1. Petri Dish 1
2. Petri Dish 2
3. Petri Dish 3

CHAPTER 4
RESULTS AND DISCUSSSION
To test the reliability of the solution made and to know which solution was the most
effective, there is one trial conducted for about three weeks. In order to analyze the data, the
number of colonies in every Petri dish was recorded. It is recorded before and after the three
solutions was tested.
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 This table shows the observation on the cultured bacteria before testing the solutions.
Title: “ ! ! “
Cultured bacteria Observations
Number of colonies Shape Color
1. Petri Dish 1 11 Filamentous Black and green
2. Petri Dish 2 20 Filamentous Black
3. Petri Dish 3 17 Filamentous Black

For the first Petri dish, there were 11 colonies of bacteria with black and green color in
filamentous shape. For the second Petri dish, there were 20 colonies of bacteria with black color,
also in filamentous shape. For the third Petri dish, there were 17 colonies of bacteria with black
color and in filamentous shape.

The table shows the observation in the number of colonies in every Petri dish with the
antiseptic solutions.

Cultured bacteria Number of colonies

After 1 week After 2 weeks After 3 weeks
1. Petri Dish 1 11 11 11
2. Petri Dish 2 20 20 20
3. Petri Dish 3 17 17 17

The number of colonies did not increase as the three solutions were tested.
Based on the results, the three solutions have the same effects even each solution has
different amount of Origanum vulgare (Oregano) extract and honey. The oregano-honey
solutions prevent the growth of the bacteria as the number of colonies did not increase in three
weeks exposure.
CHAPTER 5

CONCLUSION AND RECOMMENDATION

Conclusion “!!“

Findings of the study revealed that the Oregano (Origanum vulgare) extract and honey
are effective ingredients to form a natural, easy-to-prepare and affordable antiseptic solution.

14
 Result of this investigation clearly presents a positive impact on the development of
urban living. Hence, the following recommendations are made by the researchers:

1. The measurement of the ingredients Oregano (Origanum vulgare) extract and honey can
be manipulated as both ingredients are effective in killing bacteria.
2. This product can be used as the substitute for expensive antiseptic solution bought in the
market.
3. This product can be used in different parts of the house where bacteria are found.

BIBLIOGRAPHY

www. google.com

www.wikipedia.com/

http://www.ncbi.nlm.nih.gov/pubmed/10736000,

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http://www.pakbs.org/pjbot/PDFs/39%282%29/PJB39%282%29609.pdf:

http://online.liebertpub.com/doi/abs/10.1089/acm.2007.6366,

: //www.hindawi.com/journals/btri/2011/917505

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