ANTIMICROBIAL PROPERTY OF GARLIC (Allium sativum) LEAVES

AGAINST Staphylococcus aureus

An Undergraduate Thesis Presented to

The Faculty of Natural Sciences

College of Arts and Sciences

San Beda College- Manila

In Partial Fulfillment of the Requirements

For BIO31

By:

Larraine Love N. Muyalde

April 2016

CHAPTER 1

Overview of Research Problem

INTRODUCTION

Statement of the Problem/Objectives of the Study

Garlic (Allium sativum) is widely known for its use as the usual condiment in various recipes.Garlic has many medicinal uses according to various studies. However Garlic leaves aren’t given attention to because there are few studies about it. A small number of people use Garlic leaves as a part of their recipe. However, garlic leaves also contain an amount of what the garlic bulb has.

Although most scientific studies have investigated the effects of garlic bulb, the leaves offer a similar profile of benefits and risks. Allicin, the primary active constituent of garlic bulb, appears in lower amounts in the leaves or chives of the plants (Russo,J. 2013).

Garlic can rightfully be called one of nature’s wonderful plants with healing power. It can inhibit and kill bacteria, fungi, lower (blood pressure, blood cholesterol and blood sugar), prevent blood clotting, and contains anti-tumor properties. It can also boost the immune system to fight off potential disease and maintain health (Abdullah et al., 1988). It has the ability to stimulate the lymphatic system which expedites the removal of waste products from the body. It is also considered an effective antioxidant to protect cells against free radical damage. It can help to prevent some forms of cancer, heart disease, strokes and viral infections. Garlic alone can provide us with over two hundred unusual chemicals that have the capability of protecting the human body from a wide variety of diseases. The sulfur containing compounds found in garlic afford the human body with protection by stimulating the production of certain beneficial enzymes (Mansell and Reckless, 1991).

Although most scientific studies have investigated the effects of garlic bulb, the leaves offer a similar profile of benefits and risks. Allicin, the primary active constituent of garlic bulb, appears in lower amounts in the leaves or chives of the plants (Russo,J. 2013).

S. aureus is one of the main causes of hospital- and community-acquired infections which can result in serious consequences (Diekema et al., 2001). Nosocomial S. aureus infections affect the bloodstream, skin, soft tissues and lower respiratory tracts. S. aureus can be a cause of central venous catheter-associated bacteremia and ventilator-assisted pneumonia. It also causes serious deep-seated infections, such as endocarditis and osteomyelitis (Schito, 2006). In addition to the infections listed above, S. aureus is often responsible for toxin-mediated diseases, such as toxic shock syndrome, scalded skin syndrome and staphylococcal foodborne diseases (SFD).

This study will determine whether there is an antimicrobial effect of Garlic leaves against Staphylococcus aureus. Since Garlic leaves are not widely known for medicinal uses, this study will focus on how the Garlic leaves give an antimicrobial effect against Staphylococcus aureus.

This study will answer the following:

1. Is there significant difference on the microbial growth of each treatment based on the amount of garlic leaves?

2. Is there an antimicrobial property in garlic leaves against Staphylococcus aureus?

Statement of Hypothesis

There is a significant antimicrobial property in the use of garlic leaves against Staphylococcus aureus.

Relevance of The study

This study, primarily benefits the individuals who has a Staphylococcus aureus infection. Since Staphylococcus aureus is an infection, this study will be focusing on how to decrease the increased cases of Staphylococcus aureus infection. Also, this study will benefit the individuals concerned in the objective of the study. This study also benefits the individuals who aren’t capable of providing the expensive medicine for Staphylococcus aureus infection.

Scope and Limitation of the Study

This study has been mainly focused on the antimicrobial property of Garlic leaves against Staphylococcus aureus infection. There are no studies about the antimicrobial property of garlic leaves. Infections such as Boils, Impetigo and cellulitis are commonly caused by S. aureus. These infections are common to humans and therefore must be treated but the treatment for such infections like these are somewhat expensive therefore this study will benefit the less fortunate when it comes to finances. This study will be conducted to know whether garlic leaves have an antimicrobial effect on Staphylococcus aureus infection.

CHAPTER 2

LITERATURE REVIEW AND RELATED STUDIES

Garlic products are used as sources of medicine in many ways in human beings in their day today life. As a result, researchers from various disciplines are now directing their efforts towards discovering the medicinal values of garlic on human health. The main interest of researchers in the medicinal values of garlic is its broad-spectrum therapeutic effect with minimal toxicity. Garlic extract has antimicrobial activity against many genera of bacteria, fungi and viruses. Garlic contains a higher concentration of sulfur compounds which are responsible for its medicinal effects. The chemical constituents of garlic have also been investigated for treatment of cardiovascular disease, cancer, diabetes, blood pressure, atherosclerosis and hyperlipidaemia and highly praised by several authors (Gebreyohannes G. Gebreyohannes,M. 2013)

The antimicrobial properties of garlic were first described by Pasteur (1958), and since then, many researches had demonstrated its effectiveness and broad spectrum antimicrobial activity against many species of bacteria, viruses, parasites, protozoan and fungi (Jaber and AlMossawi, 2007). Garlic is more effective with least side effects as compared to commercial antibiotics; as a result, they are used as an alternative remedy for treatment of various infections (Tepe et al., 2004). Out of the many medicinal plants, garlic has an antimicrobial property which protects the host from other pathogens highlighting the importance of search for natural antimicrobial drugs (Bajpai et al., 2005; Wojdylo et al., 2007). Previously conducted researches confirmed that garlic is not only effective against Gram positive and Gram negative bacteria but also possess antiviral and antifungal activities (Tsao and Yin, 2001).

Garlic's use in medicine predates modern civilization. According to the University of Maryland Medical Center, records have traced the use of garlic to the ancient Egyptians, who used it as food and medicine during the time of the pharaohs. Although most scientific studies have investigated the effects of garlic bulb, the leaves offer a similar profile of benefits and risks. Allicin, the primary active constituent of garlic bulb, appears in lower amounts in the leaves or chives of the plants (Russo J. 2013)

It’s declared that garlic, as an anti-bacterial agent, is effective against many more gram negative and gram positive bacteria like Helicobacter pylori, E. coli, Lactobacillus casei and that this effect is sourced from allicin inside it (Cellini et al., 1996; Lemar et al., 2005). It is also declared that components including sulphur in garlic and also bioflavonoids like quercetin and cyanidin in it have great value in preventing diseases and infections. It’s revealed that active substances like allistatin I and allistatin II in garlic are powerful agents against staphylococcus and E. coli bacteria (Baytop, 1999; Ayaz and Alpsoy, 2007; Ankri and Mirelman, 1999; Hanafy et al., 1994; Yoshida et al., 1998). It’s predicted that antimicrobial effect of garlic has revealed in conclusion that thiosulphonates in garlic inhibit enzymes including thiol, which can take place in micro-organisms, as a result of the ability of quick reaction giving with thiol groups (Ayaz and Alpsoy, 2007; Imai et al., 1994).

Allicin is the major biologically active component of garlic. First reported by Cavallito and Bailey in 1944, allicin is the key ingredient responsible for the broad-spectrum of anti-bacterial activity in garlic. Research also showed that allicin is responsible for lipid-lowering, anti-blood coagulation, anti-hypertension, anti-cancer, antioxidant and anti-microbial effects.

Microbes found on the skin are usually regarded as pathogens, potential pathogens or innocuous symbiotic organisms. Advances in microbiology and immunology are revising our understanding of the molecular mechanisms of microbial virulence and the specific events involved in the host–microbe interaction. Current data contradict some historical classifications of cutaneous microbiota and suggest that these organisms may protect the host, defining them not as simple symbiotic microbes but rather as mutualistic. This review will summarize current information on bacterial skin flora including Staphylococcus, Corynebacterium, Propioni-bacterium, Streptococcus and Pseudomonas. Specifically, the review will discuss our current understanding of the cutaneous microbiota as well as shifting paradigms in the interpretation of the roles microbes play in skin health and disease (Cogen, A. L., NIzet, V., & Gallo, R. L. (2009))

Infections caused by S. aureus range from minor skin disorders such as wound infections, furuncles and carbuncles, and bullous impetigo, through locally invasive diseases such as cellulitis, osteomyelitis, sinusitis, and pneumonia, to major life-threatening septicemia and meningitis. It is also a frequent cause of medical device-related infections such as intravascular line sepsis and prosthetic joint infections. Although minor skin infections may resolve naturally without antibiotic intervention, once S. aureus invades deeper structures, it often spreads hematogenously to other organ systems, leading to metastatic infection. Endocarditis and septicemia have significant morbidity and mortality despite aggressive antimicrobial therapy (Turnidge, J., Rao, N., Chang, F., Fowler, V. G., Jr., Kellie, S. M., Arnold, S., Tristan, A. (2008)).

Staphylococcus aureus is facultative anaerobic gram-positive cocci which occur singly, in pairs, and irregulular clusters. S. aureus is nonmotile, non-spore forming, catalase and coagulase positive. Typical colonies are yellow to golden yellow in color, smooth, entire, slightly raised, and hemolytic on 5% sheep blood agar. However, many strains may appear dirty white and nonhemolytic. It also gives a positive mannitol fermentation and deoxyribonuclease test (Turnidge, J., Rao, N., Chang, F., Fowler, V. G., Jr., Kellie, S. M., Arnold, S., Tristan, A. (2008)).

The major criterion for identification is the organism’s ability to clot plasma. There are three coagulase-positive Staphylococcal species; S. aureus in humans and animals, andS. intermedius, and S. hyicus in animals. The presence of the enzyme coagulase separates the virulent pathogen, S. aureus, from the less virulent coagulase-negative Staphylococci species. There are two different tests that can be performed to detect the presence of coagulase: a tube test to detect free coagulase and a slide test to detect bound coagulase. The slide test is a rapid test; however, a small percentage of S. aureus strains may yield a negative result. If the organism is suspected as S. aureus, negative slide tests should be followed up with a tube test (Turnidge, J., Rao, N., Chang, F., Fowler, V. G., Jr., Kellie, S. M., Arnold, S., Tristan, A. (2008)).

CHAPTER 3

RESEARCH METHODOLOGY

Materials

Culture of Staphylococcus aureus

• Petri dishes

• Inoculating loop

Preparation of Garlic leaves Extract

• Erlenmeyer flask

• Stirring rod

• Mechanical grinder

• Jar & containers

• Filter paper and cotton

Experimentation

• Mueller Hinton Agar (MHA)

• Alcohol lamp

• Digital weighing machine

• Dropper

• Ethanol 95%

Sanitation

• Autoclave

• Ethanol

• Alcohol lamp

Methods of Research

Culture of Staphylococcus aureus

In this Experiment of the study, the culture medium will be prepared first. Sabouraud Dextrose Agar will be the culture medium to be used in this experiment. Fifty grams of Sabouraud Dextrose Agar will be the amount of the culture medium. The medium will be used for subculturing the Microbe. Subculturing the Staphylococcus aureus in the subcultured microbe will be the next step of this experiment.

Preparation of Garlic leaves Extract

5 kilos of green garlic leaves will be used in controlling the microbe. The garlic leaves will be washed in tap water and will be rinsed using distilled water. The garlic leaves will be air dried for 5-10 days. The finished dried product of the garlic leaves will be mechanically grounded using the mechanical grinder. The powder will be weighed 50g using a digital weighing machine. The resulting powder (50 g) will be placed in an Erlenmeyer flask and soaked in 450 mL 95% ethanol for three days at room temperature (30 ± 2 °C). The suspension will be shaken intensely for the powder to mix with ethanol. After three days the the mixture will be filtered using a filter paper and the filtrate was distilled under reduced pressure in a rotary evaporator. The extract was kept at -20 °C prior to use (Trusheva B, Trunkova D, Bankova V. 2007)

Population and Sample

In this study, there will be five treatments of experimentation to be used. In each treatment, Five petri dishes will be used containing Stapylococcus aureus. Treatment one (1) will be the controlled group. Treatment two (2) will be treated with 20% Garlic leaves extract. Treatment three (3) will be treated with 40% Garlic leaves extract. Treatment four (4) will be treated with 60% Garlic leaves extract and lastly Treatment five (5) will be treated with 80% Garlic leaves extract.

Locale of the Study/Sampling Site

The Garlic leaves will be gathered from Marikina public market. The experimentation will be conducted on one of the Laboratories of San Beda College Manila. Safety awareness will be one of the procedures in this study.

Sampling Technique

In this study, the sampling technique will be the isolation of Staphylococcus aureus in Sabouraud Dextrose Agar medium. The prepared sample frim the sampling technique will then be treated by Garlic leaves extracts. Five (5) treatments with twenty-five (25) petri dishes, Five (5) petri dish each treatment. Each treatment will be based on the amount of concentration of Garlic leaves extracts used in each treatment except for treatment one (1) which is the controlled group.

Experimental Layout

|Treatment 1 |Treatment 2 |Treatment 3 |Treatment 4 |Treatment 5 |
|(Control group) |(20% of Garlic leaves |(40% of Garlic leaves |(60% of garlic leaves |(80% of Garlic leaves |
| |extract) |extracts) |extracts) |extracts) |
|T1S1 |T2S1 |T3S1 |T4S1 |T5S1 |
|T1S2 |T2S2 |T3S2 |T4S2 |T5S2 |
|T1S3 |T2S3 |T3S3 |T4S3 |T5S3 |
|T1S4 |T2S4 |T3S4 |T4S4 |T5S4 |
|T1S5 |T2S5 |T3S5 |T4S5 |T5S5 |

Data Gathering Procedures

In this study, gathering the of data will be for one (1) week. The Zone of Inhibition will be measured using the X and Y axis of the microbe Staphylococcus aureus will be observed and measured every day. The Formula to be used in this experiment will be X axis plus Y axis divided by two (2).

Statistical treatment

The Data of this study will be analyzed using the ANOVA style factorial. It will be computed in the lastest version of Microsoft Word Excell.

References

Gebreyohannes, G., & Gebreyohannes, M. (2013). Medicinal values of garlic: A review. International Journal of Medicine and Medical Sciences, 5(9), 401-408. Retrieved April 22, 2016.

Russo, J. (2013, August 16). Benefits & Side Effects of Garlic Leaves. Retrieved April 22, 2016, from http://www.livestrong.com/article/171913-benefits-side-effects-of-garlic-leaves/

Plata, K., Rosato, A. E., & Węgrzyn, G. (2009). Staphylococcus aureus as an infectious agent: Overview of biochemistry and molecular genetics of its pathogenicity. Acta Biochimica Polonica, 56(4), 597-612. Retrieved April 22, 2016.

What is Allicin? (2010). Retrieved April 23, 2016, from http://www.allicinfacts.com/about-allicin/what-is-allicin/

Cogen, A. L., NIzet, V., & Gallo, R. L. (2009). Skin microbiota: A source of disease or defence? PMC US National Library of Medicine National Institute of Health. Retrieved April 23, 2016.

Turnidge, J., Rao, N., Chang, F., Fowler, V. G., Jr., Kellie, S. M., Arnold, S., Tristan, A. (2008). Staphylococcus aureus. Retrieved April 23, 2016.

Goncagul, G., & Ayaz, E. (2010). Antimicrobial Effect of Garlic (Allium sativum) and Traditional Medicine. Journal of Animal and Veterinary Advances, 9(1), 1-4. Retrieved April 23, 2016.

Mandal, A. (2012, December 9). Staphylococcus Aureus Microbiology. Retrieved April 23, 2016, from http://www.news-medical.net/health/Staphylococcus-Aureus-Microbiology.aspx

Edris, A. E., & Fadel, H. M. (2002). Investigation of the volatile aroma components of garlic leaves essential oil. Possibility of utilization to enrich garlic bulb oil. European Food Research and Technology, 214(2), 105-107. Retrieved April 23, 2016.