Justice Cecilia Muñoz Palma High SchoolScience 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. MaeOcampo, Kyle LesterPaca-anas, Melody

Panis, Christine Iesra MaePanis, Joshua MarkPattaui, Karen Joyce

Payapag, AldrinPorcare, JimwellQuiamco, Jeanky

Reyes, Charles GevieSertimo, Heide

Sevilla, Cecille Kay

Submitted to:Mrs. Estela M. Galolo

February 2013TABLE OF CONTENTS

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Title Page ………………………………………………………………………………..…….... 1

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

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

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

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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

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.

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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 1RESEARCH PROBLEM AND ITS BACKGROUND

Background of the Study:

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.

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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.

Significance of the Study:

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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 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).

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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

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.

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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.

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

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

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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:

Honey

Origanum vulgare (Oregano) leaves

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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 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

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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 solutionFirst, 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 Origanum vulgare (Oregano) extract

Measurement of honey

Solution

1st bottle 15 ml 35 ml 50 ml2nd bottle 25 ml 25 ml 50 ml3rd bottle 35 ml 15 ml 50 ml

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B. Measurement of Origanum vulgare ( Oregano) and honey in every solutionto be put at every Petri dish

Cultured bacteria Measurement of Origanum vulgare (Oregano) extract

Measurement of honey

1. Petri Dish 1 15 ml 35 ml2. Petri Dish 2 25 ml 25 ml3. Petri Dish 3 35 ml 15 ml

C. Observation on the cultured bacteria before testing the solutions

Cultured bacteria ObservationsNumber of colonies Shape Color

1. Petri Dish 12. Petri Dish 23. Petri Dish 3

D. Observation on the number of colonies with the antiseptic solutionsAfter two-weeks exposure

Cultured bacteria Number of colonies

After 1 week After 2 weeks After 3 weeks1. Petri Dish 12. Petri Dish 23. 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.

This table shows the observation on the cultured bacteria before testing the solutions.

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Title: “ ! ! “

Cultured bacteria ObservationsNumber of colonies Shape Color

1. Petri Dish 1 11 Filamentous Black and green2. Petri Dish 2 20 Filamentous Black3. 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 weeks1. Petri Dish 1 11 11 112. Petri Dish 2 20 20 203. 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.

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

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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,

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

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

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

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