The Antimicrobial activity of Holy Basil (Ocimum Sanctum)

Extract against Staphylococcus Aureus

Grade 12 ENHS students.

By:

Reyes, Efren Eduardo

Alegato, Chiel Anne F.

Altares, Athea Franz N.

Anog, Althea

Beseos, Triesha Kaye

Nogra, Pia Bianca

Potot, Beatrice Yssabelle

Sabete, Louella Clarisse

Researchers of Escalante National High School

June 5, 2023
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ACKNOWKNOLEGEMENT

We would like to extend our sincere appreciation to all the individuals and

organization for their invaluable support and assistance throughout the completion

of our life science capstone project. Without their support, this capstone project

would not have been possible. Their contributions have significantly impacted our

research, and we are truly grateful for their involvement.

We would first and foremost like to express our gratitude to Jesus Tabelino, the

principal of the school, for his continuous support in enabling us to carry out this

initiative. We would also like to acknowledge Mary Anne Biaquis, the Assistant

Principal, for her continuous support and enthusiasm towards our project.

We are very appreciative of our capstone adviser, Irnie Panaguition, for his advice

and knowledge during the course of our project. His insightful comments and

recommendations were really helpful in guiding and guaranteeing the success of our

research.

Special thanks are due to Negros Prawn Diagnostic for their collaboration and

provision of essential resources. Their expertise and assistance in the field of

diagnostics have been invaluable in our project's execution. We would like to express

our heartfelt appreciation to Rey Justin Lanada, the Chemist, for his generous

contributions and insights. His expertise and dedication have greatly enriched our

understanding of the subject matter.

We would also like to extend our gratitude to all the individuals who extended their

help in various capacities during this project. Their support, whether it was through

providing equipment, sharing knowledge, or lending a helping hand, has been

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instrumental in our achievements. Our parents deserve a special mention for their

unwavering support and understanding throughout this journey. Their love,

encouragement, and sacrifices have been the driving force behind our success. We

are forever grateful for their presence in our lives.

Lastly, we would like to acknowledge and express our deepest gratitude to our God.

We are thankful for the strength, inspiration, and guidance provided throughout this

project. It is through His grace that we have been able to overcome challenges and

achieve our goals.

Thank you, to God be all the Glory.

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ABSTRACT

This study aimed to evaluate the antimicrobial activity of Holy Basil extracts against

Staphylococcus aureus (S. aureus) and assess its potential as a substitute treatment

for diseases caused by this bacterium. The disc diffusion method was employed to

investigate the inhibitory effects of Holy Basil extracts on S. aureus. The experiment

included a negative control (no treatment) and a positive control (commercial

germicidal soap) for comparison. Multiple replicates were performed for each

treatment condition, and the zones of inhibition were measured in millimeters. The

results showed that the Holy Basil extracts exhibited a mean zone of inhibition of 19

mm against S. aureus, indicating antimicrobial activity. In comparison, the negative

control showed no zone of inhibition (0 mm), while the positive control (commercial

germicidal soap) demonstrated a mean zone of inhibition of 31 mm. These findings

suggest that Holy Basil extracts possess antimicrobial properties against S. aureus,

although the commercial germicidal soap showed stronger activity. Further research

is needed to explore the mechanism of action and optimize the formulation of Holy

Basil extracts to enhance their antimicrobial efficacy. This study contributes to the

understanding of alternative and natural treatment options for S. aureus infections,

highlighting the potential of Holy Basil as a natural antimicrobial agent.

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TABLE OF CONTENTS

ACKNOWLEDGEMENT……………………………………………………………………………………… ii

ABSTRACT….……………………………………………………………………………………………………iii

INTRODUCTION……………………………………………………………………………………………… 1

Objective……………………………………………………………………………………………… 3

Locale of the study……………………………………………………………………………….. 4

Significance of the study………………………………………………………………………. 5

Definition of terms……………………………………………………………………………… 6

RRL……………………………………………………………………………………………………………… 8

METHODOLOGY……………………………………………………………………………………………. 14

Materials……………………………………………………………………………………………. 14

Results and Discussion………………………………………………………………………………….. 19

Conclusion……………………………………………………………………………………………………. 22

Recommendations.……………………………………………………………………………… 23

REFERENCES………………………………………………………………………………………………... 24

APPENDICES………………………………………………………………………………………………… 26

Appendix A…………………………………………………………………………………. 26

Appendix B……………………………………………………………..………………….. 27

Appendix C…………………………………………………………………………………. 28

Appendix D………………………………………………………………………………….. 29
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INTRODUCTION

           Skin diseases are prevalent in tropical climates, developing countries, people

with poor hygiene, and polluted areas. It is a kind of disease that is visible to human

eyes which makes carriers susceptible to shame and may cause people to keep their

distance from them. Although visible, many people are unsure about the type of skin

disease they have. As a result, they seek help from dermatological clinics to obtain a

diagnosis and treatment. However, the extent of its effectiveness against S. Aureus,

Infectious diseases are typically treated by giving antibiotics to act as growth

inhibitors or killing microbes that cause infection. A supplement to support antibiotic

treatment.

     The increasing prevalence of antimicrobial resistance among pathogenic

microorganisms has led to a growing interest in the development of alternative

antimicrobial agents from natural sources. Among these sources, medicinal plants

have gained significant attention due to their potential therapeutic properties.

Throughout history, plants have been valued for their natural antimicrobial

properties and have been used to treat a variety of illnesses. This is due to the

active components found within plants that can serve as a natural form of

treatment.

          

Holy basil ( Ocimum Sanctum), also known as Tulsi, is a widely used

medicinal herb in traditional medicine, it is traditionally used in Ayurvedic medicine

to treat a variety of ailments, including bacterial infections. Holy basil contains a

variety of bioactive compounds, such as eugenol, rosmarinic acid, and ursolic acid,
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which have been shown to exhibit antimicrobial activity against various bacterial

strains. And its extracts have been shown to possess various pharmacological

activities.  It is cultivated for medicinal and religious purposes. It is marked by its

strong aroma and its astringent taste, as it is regarded in Ayurveda (an ancient

medical system) as a kind of ‘elixir of life’ and believed to promote longevity. Tulsi is

mainly used in the form of extract and oil

Several studies have reported that holy basil extract exhibits antimicrobial

activity against various pathogens, including S. aureus. However, the exact

mechanisms of action and the potential clinical applications of holy basil extract

against S. aureus are still not fully understood. According to Dr. Zeel Gandhi

(Bachelor of Ayurveda, Medicine & Surgery), Chief Ayurvedic Doctor, Vedix “Tulsi

has positive effects on the skin’s immunity. Holy basil, heals skin disorders and is a

strong antimicrobial, rendering the skin free of troubles.”

Staphylococcus aureus is widely acknowledged as a significant bacterial pathogen

responsible for various diseases in humans. Staphylococcus is one of the main

bacteria that can cause skin disease. Among its common manifestations are skin

and soft tissue infections, including abscesses (boils), furuncles, and cellulitis. While

most staph infections are typically mild, S. aureus can also result in severe illnesses

such as bloodstream infections, pneumonia, or bone and joint infections. Due to the

emergence of antibiotic-resistant strains of S. aureus, there is a growing need for

alternative therapies to combat this bacterium.

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OBJECTIVE

The objective of this study is to evaluate the antimicrobial activity of Holy Basil

extracts against Staphylococcus aureus. The study aims to determine whether Holy

Basil extracts can be a potential substitute treatment for diseases caused by this

bacterium. By investigating the antimicrobial activity of Holy Basil extracts against

Staphylococcus aureus, this research seeks to provide valuable insights into the

potential use of Holy Basil as a natural antimicrobial agent against this bacterium.

The study aims to contribute to the knowledge of alternative and natural treatment

options for Staphylococcus aureus infections, ultimately leading to potential

advancements in the field of antimicrobial therapy.

Statement of the problem

               The researcher aims to evaluate the antimicrobial activity of Holy Basil

extracts against S. Aureus. The study was conducted to evaluate the possible use of

this plant as a substitute treatment for diseases brought on by this bacterium,

researchers are looking at the antimicrobial activity of holy basil extracts against

Staphylococcus Aureus and the result obtained could provide valuable insights into

the potential use of Holy Basil extracts as a natural antimicrobial agent against S.

Aureus.

Specifically, it sought to answer the following question:

         1. Does holy basil extract have a significant effect against S. Aureus bacteria?

         2. Is there any specific bioactive compound responsible for this activity?

                
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   Overall, the study findings could contribute to the development of alternative

antimicrobial strategies to combat S. Aureus infection, and the results could be of

great interest to researchers and health-care professionals working in the field of

infectious diseases.

Hypothesis

Alternative:

1. Holy basil extract is effective against Staphylococcus Aureus.

2. There is a specific bioactive compound responsible for this activity.

Null:

1. Holy basil extract is ineffective against Staphylococcus Aureus.

2. There is no specific bioactive compound responsible for this activity.  

LOCALE OF THE STUDY

The study was conducted at Escalante National High School which is located at Brgy.

Alimango, Escalante City. The school has an area of approximately one hectare with

three-thousand students and one-hundred twenty teachers in junior high and thirty-

three teachers in senior high.

SCOPE AND LIMITATIONS

The scope of this study is to assess the antimicrobial activity of Holy Basil extracts

against Staphylococcus aureus, focusing on its potential as a substitute treatment for

diseases caused by this bacterium. By conducting experiments to investigate the

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antimicrobial activity of Holy Basil extracts specifically against Staphylococcus

aureus, the research aims to provide valuable insights into the efficacy of Holy Basil

as a natural antimicrobial agent. The study seeks to contribute to the existing

knowledge of alternative and natural treatment options for Staphylococcus aureus

infections, aiming to advance the field of antimicrobial therapy.

However, this study has certain limitations. It will be conducted in vitro, which may

not fully reflect the outcomes in clinical settings. Additionally, the focus will be

limited to Staphylococcus aureus, and the effects of Holy Basil extracts on other

bacteria or pathogens will not be explored. The study will not investigate the

potential side effects or toxicity of Holy Basil extracts on human cells or tissues, and

the variability in Holy Basil extracts from different sources will not be considered.

Furthermore, the mechanism of action of Holy Basil extracts against Staphylococcus

aureus will not be explored as the primary aim is to assess antimicrobial activity.

  

SIGNIFICANCE OF THE STUDY:

                 This capstone project is beneficial for the community, for the reason that

the findings of this study will contribute to the existing literature of the potential use

of Holy Basil as an alternative treatment for S. Aureus infections. Furthermore, this

study may provide insights into the mechanisms of actions of holy basil extract

against S. Aureus, which could inform the development of novel antimicrobial

agents. Ultimately, the result of this study may have implications for the

management of S. Aureus infections and the development of new antimicrobial

therapies. The evaluation of Holy Basil extracts' antimicrobial activity against S.

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aureus holds great significance in our school. This study addresses the urgent need

for alternative treatments in the face of antibiotic resistance, explores the potential

of Holy Basil as a natural antimicrobial agent, supports evidence-based herbal

medicine, and offers educational and research opportunities. The findings obtained

from this study can contribute to the development of effective and sustainable

treatment options against S. aureus infections, benefiting both the local community

and the broader field of healthcare.

DEFINITION OF TERMS

Skin diseases: Medical conditions that affect the skin and are prevalent in tropical

climates, developing countries, people with poor hygiene, and polluted areas. They

are visible to the human eye, causing carriers to feel susceptible to shame and may

result in others keeping their distance. Dermatological clinics are sought for

diagnosis and treatment.

Antimicrobial resistance: The increasing prevalence of microorganisms becoming

resistant to the effects of antimicrobial drugs, which has led to the development of

alternative antimicrobial agents.

Alternative antimicrobial agents: Substances derived from natural sources, such

as medicinal plants, that possess antimicrobial properties and can be used as

alternatives to traditional antimicrobial drugs.

Medicinal plants: Plants known for their therapeutic properties, historically used to

treat various illnesses. They contain active components that serve as natural forms

of treatment.
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Holy basil (Ocimum Sanctum/Tulsi): A widely used medicinal herb in traditional

medicine, particularly in Ayurvedic medicine, known for treating bacterial infections.

Holy basil contains bioactive compounds, including eugenol, rosmarinic acid, and

ursolic acid, which exhibit antimicrobial activity against various bacterial strains. It is

cultivated for medicinal and religious purposes and is considered an "elixir of life" in

Ayurveda.

Staphylococcus aureus: A significant bacterial pathogen responsible for various

diseases in humans, including skin and soft tissue infections. It is one of the main

bacteria causing skin diseases and can also lead to severe illnesses like bloodstream

infections, pneumonia, and bone and joint infections. The emergence of antibiotic-

resistant strains of S. aureus necessitates alternative therapies.

Mechanisms of action: The specific ways in which holy basil extract interacts with

S. aureus to exert its antimicrobial effects. These mechanisms are not fully

understood and require further research.

Clinical applications: The potential use of holy basil extract in clinical settings to

treat S. aureus infections. The specific applications and effectiveness of holy basil

extract against S. aureus in clinical practice require investigation.

Skin immunity: The ability of the skin to defend against pathogens and maintain

its overall health. Holy basil has been suggested to have positive effects on the

skin's immunity, helping to heal skin disorders and providing antimicrobial benefits.

Antibiotic-resistant strains: Strains of S. aureus that have developed resistance

to commonly used antibiotics, making them less responsive to treatment and

highlighting the need for alternative therapeutic options.

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Alternative therapies: Treatment approaches other than traditional antibiotic

treatments that are being explored to combat antibiotic-resistant strains of S.

aureus. Holy basil extract is one such alternative therapy under investigation.

Clinical manifestations: The visible signs and symptoms of S. aureus infections,

including skin and soft tissue infections such as abscesses, furuncles, and cellulitis,

as well as more severe systemic infections

REVIEW OF RELATED LITERATURE

       In recent years, the appearance of antibiotic-resistant bacteria has become a

growing public health concern. One approach to combat this problem is the

exploration of alternative antimicrobial agents from natural sources. One such agent

is Ocimum Sanctum, commonly known as holy basil, a plant traditionally used for its

medicinal properties.  Studies have shown promising antimicrobial activity of

Ocimum Sanctum against a wide range of bacterial and fungal pathogens.  

According to Priyabrata Plattanayak, Pritisha Behera, Debajyoti Das, and Sangram K.

Panda (A reservoir plant for therapeutic application) “The chemical composition of

Tulsi (Ocimum Sanctum L.) is highly complex, containing many nutrients and other

biologically active compounds” (Rev. 2010 Jan-Jun:4(7); 95-105). It is a well-known

medicinal herb with a wide range of therapeutic properties; it also possesses

insecticidal and antibacterial properties. Chemicals in Holy Basil are thought to

decrease pain and swelling. They also might lower blood sugar in people with

diabetes.
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According to Pramod K. Raghav & Mitu Saini (Antimicrobial Properties of

Tulsi (Ocimum sanctum) in Relation to Shelf Life Enhancement of Fruits &

Vegetables)  “Ocimum sanctum has many beneficial properties namely anti-

oxidative, antimicrobial, anti-stress , anti-diabetic, antiviral and many others that’s

why this plant is also given the term “Queen of Herbs” (International Journal of

Green and Herbal Chemistry, 2018). Tulsi extracts are used in Ayurvedic remedies

for common colds, headache, stomach disorder, inflammation, heart disease, various

forms of poisoning and malaria.

      

    In this review of related literature, we will explore the existing studies on the anti-

microbial properties of Ocimum Sanctum specifically against S. Aureus, with the aim

of synthesizing and evaluating the current state of knowledge on this topic. Through

this review, we hope to shed light on the potential of Ocimum Sanctum as a natural

alternative to antibiotics for the prevention and treatment of S. Aureus i infections.

The aim of this paper is to discuss studies that are relevant to the following topic:

1. Chemical Composition of Ocimum Sanctum

 2. What is S. Aureus And its effects in the body?      

2.1 Chemical Composition of Ocimum Sanctum

     According to the study of  Deshmukh , Girishkumar B, and Pratibha D Shirole

(2015) “the leaves of Ocimum Sanctum  contain 0.7% volatile oil comprising about

71% eugenol and 20% methyl eugenol.” The study discusses that the oil also

contains carvacrol and sesquiterpene hydrocarbon caryophyllene.  Fresh leaves and

stem of OS extract yielded some phenolic compounds (antioxidants) such as

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cirsilineol, cirsimaritin, isothymusin, apigenin and rosameric acid, and appreciable

quantities of eugenol. Two flavonoids, viz., orientin and vicenin from aqueous leaf

extract of OS have been isolated. Ursolic acid, apigenin, luteolin, apigenin-7-O-

glucuronide, luteolin-7-O glucuronide, orientin and molludistin have also been

isolated from the leaf extract.  OS also contains a number of sesquiterpenes and

monoterpenes viz., bornyl acetate, -elemene, neral, - and -pinenes, camphene,

campesterol, cholesterol, stigmasterol and –sitosterol

2.1 A.   Antimicrobial properties of Ocimum Sanctum.

The study of Ashok Kumar Sah, M Vijaysimha, Md. Mahamood (June 2018) stated

that the Holy Basil or Ocimum sanctum has been investigated to possess various

pharmacological properties like anti-toxic, antioxidant, anticancer, antimicrobial,

antihypertensive, anti-inflammatory, anticoagulant analgesic and anti-thyroid 18,21 .

The Phenolic constituents of O. sanctum leaf extract like isothymusin, apigenin,

rosmarinic acid, cirsineol and eugenol16 . The aqueous extracts of O. sanctum

leaves are more effective against pathogens as compared to methanolic extract22-

26. The leaf extract of O. sanctum shows potent antioxidant activity and it inhibits

the growth of E. coli, Klebsiella, Staphylococcus aureus and Proteus25 .

2.1 B.  Antibacterial properties

        The study of Pramod K. Raghav & Mitu Saini(2017) found that  the

antibacterial properties of O. sanctum fixed oil are attributed to its high content of

Linolenic acid. The study discusses that extracts from tulsi leaves have been found

to inhibit the growth of various bacteria such as E. coli, S. aureus, Klebsiella, and

Vibrio cholera.Devi,  Naik, P. Shyam, Marx and Baskari (Antimicrobial Activity and
 11

Phytochemical Analysis of Ocimum tenuiflorum Leaf Extract) investigated that the O.

sanctum leaves extract is excellent antimicrobial agent; it is highly effective against

gram positive and gram negative bacteria such as Staphylococcus aureus, Klebsiella

pneumoniae, P. putida, B. subtilis and E. coli.

2.1 C. Antifungal Properties

          The study of Pramod, et al. also stated that Ocimum sanctum shows an

important property like antifungal activity. Aqueous, hexane, chloroform, n-butanol

and other solvent extracts of Ocimum sanctum showed antifungal activity. Ocimum

sanctum acts against biodeterioration of food stuff during storage. Aqueous and

acetone extract of Ocimum sanctum L. were also found to be sensitive to many plant

fungi such as Alternaria tenuis, Helminthosporium sp. And Curvularia penniseli.

    

           According to Siva M.1, Shanmugam KR1*, Shanmugam B.2, Venkata

Subbaiah G.2, Ravi S.2, Sathyavelu Reddy K.2, Mallikarjuna K3 (International Journal

of Basic & Clinical Pharmacology) "Ocimum sanctum essential oil and eugenol can be

used as plant based safe preservatives against fungal spoilage of food stuff during

storage".(May-June 2016 | Vol 5 | Issue 3 Page 560)

2.2 What is S. Aureus and its effects in the body?

            Staphylococcus aureus, often abbreviated as S. aureus, is a bacterium that

commonly resides on the skin and mucous membranes of humans and animals. It is

a Gram-positive, round-shaped bacterium that belongs to the Staphylococcaceae

family. S. aureus is known for its ability to cause various infections in humans,
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ranging from mild skin infections to more severe and potentially life-threatening

conditions.

S. aureus is a versatile pathogen and can cause a wide range of illnesses depending

on the site of infection. It is a common cause of skin and soft tissue infections such

as boils, abscesses, cellulitis, and wound infections. It can also cause more serious

infections such as pneumonia, bloodstream infections (septicemia), bone and joint

infections, and infections of the heart (endocarditis)

One of the concerning characteristics of S. aureus is its ability to develop resistance

to multiple antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) is a strain

of S. aureus that has become resistant to many commonly used antibiotics, making

it more difficult to treat infections caused by this bacterium.

S. aureus can spread from person to person through direct contact with infected

individuals or by touching contaminated objects or surfaces. It can also colonize the

nasal passages of healthy individuals, serving as a reservoir for further infections.

According to the result of the study conducted by Mittal R, Kumar R and , Chahal

HS, all different types of extracts obtained from Ocimum sanctum leaves

shown to be with antibacterial activity against tested microbial pathogens.

Ocimum sanctum extract has found to be with antimicrobial properties

against Staphylococcus aureus and Kleibsiella pneumonia. The study concludes that

Ocimum sanctum possesses potential antimicrobial properties especially to

Staphylococcus aureus and Kleibsiella pneumonia, making it a rich source of

phytochemical constituents with potential applications as an antimicrobial agent. The

widespread availability of Ocimum sanctum in India suggests that it can be

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recommended as an easily accessible and renewable source of antimicrobial

compounds, offering an alternative to synthetic chemicals.

The study of Hanaa A. Yamani, Edwin C. Pang, Nitin Mantri, and Margaret A.

Deighton stated that tulsi essential oil could serve as a valuable topical antimicrobial

agent for managing skin infections caused by S. aureus (including MRSA), P.

aeruginosa, and E. coli. It could be used either directly on the affected area or as a

component of wound dressings to prevent infection. The study shows that early

treatment or preventative measures with Tulsi oil may help halt the progression of

skin infections and reduce the need for systemic antibiotic therapy. Additionally,

using Tulsi oil in such applications may help minimize the risk of developing

resistance to conventional antibiotics.

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METHODOLOGY

The extraction process for this project was conducted at the premises of Negros

Prawn Producers Cooperative, situated at Door 1 and 2 of the NEDF Building, located

at the intersection of 6th Street and Lacson Street, Bacolod, Negros Occidental, with

the postal code 6100.

I. MATERIALS

These materials were used in extracting, and performing qualitative phytochemical

analysis.

1. Leaves of Ocimum sanctum L. (tulsi): These were collected from Brgy.

Washington Purok Linao.

2. Sterile water: Used to wash the collected tulsi leaves.

3. Mechanical grinder: Used to powder the dried tulsi leaves.

4. Soxhlet apparatus: Used for extraction.

5. Distilled water: Used as a solvent for extraction.

6. 70% ethanol: Used as a solvent for extraction.

7. Rotary evaporator: Used to concentrate the extract.

8. Digital water bath: Used for drying the extract.

9. Biuret reagent: Used for testing proteins.

10. Fehling A and Fehling B reagents: Used for testing carbohydrates.

11. Alcohol: Used for testing phenols.

12. Ferric chloride solution: Used for testing phenols and tannins.

13. Magnesium: Used for testing flavonoids.

14. Concentrated HCl: Used for testing flavonoids.

15. Test tubes: Used for conducting the tests.

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16. Distilled water: Used for dilution and mixing.

17. Heating apparatus: Used for heating the samples during testing.

II. Materials and Methods

Collection of plant material: Leaves of Ocimum sanctum L. (tulsi) were collected

from Brgy. Washington Purok Linao washed with sterile water and oven dried for

several days to take away the moisture of the Holy Basil. Then the samples were

powered in mechanical grinder.

Ethanol Extract: The dried tulsi (50g) powder was placed in the thimble of Soxhlet

apparatus and 500- 700 ml of distilled water, 70% ethanol was used for extraction

procedure. A clear solvent or water was seen in the thimble after a while of

extraction. Through the use of a rotary evaporator, the extract was concentrated.

Once a dark green residue had been obtained, the extract was dried in a digital

water bath.
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Qualitative phytochemical analysis: The extract was tested following standard

biochemical methods

Test for proteins:

2ml of Biuret reagent was added to 2ml of extract. The mixture was shaken well

and warm for 5 min.Appearance of red or violet colour indicated presence of

proteins.

Test for carbohydrates:

Equal amount of Fehling A and Fehling B reagents were mixed and 2ml of it was

added to the plant extract and then gently heated the sample. Appearance of brick

red precipitate indicated the presence of reducing sugars.

Test for phenol:

2 ml of alcohol and 2-3 drops of ferric chloride solution was added to 1 ml of crude

extract, bluegreen or black coloration indicated the presence of phenols

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Test for tannin:

1 ml of distilled water and 2-3 drops of ferric chloride solution was added to 0.5 ml

of crude extract. A black coluration indicated the presence of tannin.

Test for flavonoids:

Crude extract was mixed with small amount of magnesium and concentrated HCl

was added drop wise. Appearance of yellow coloration after few minutes indicated

the presence of flavonoids.

Test for saponins:

1ml of crude extract was mixed with 5ml of distilled water in a test tube and it was

shaken vigorously. The formation of stable foam was taken as an indication for the

presence of saponins.

Test for glycosides:

0.5 ml of crude extract was mixed with 2ml of glacial acetic acid containing 2-3

drops of 2% solution of FeCl3. Then 2ml of concentrated H2SO4 was poured into the

mixture. A color of upper layer -bluish green color, lower layer- brownish-red color

Test for terpenoids:

Crude extract was mixed in 2ml of chloroform and evaporated to dryness. To this,

2ml of concentrated H2SO4 was added and heated for about 2 minutes. Presence of

terpenoids was indicated by a reddish brown interface.

Test for alkaloids:

2ml of 1% HCl was mixed with crude extract and heated gently. After heating,

Mayer’s and Wagner’s reagents were added to the mixture. If precipitate was
 18

observed in the reaction mixture creamy white precipitate which indicated the

presence of alkaloids.

Method Description for culture and sensitivity

Preparation of McFarland’s Standard: 100mL of McFarland’s Standard was

prepared by mixing 99.5 mL of 1% sulfuric acid and 0.5 mL of 1.175% BaCl2.2H2O.

The turbidity is equivalent to 1.0 x108 CFU/ml bacteria. Bacterial Isolates from pure

culture of S. aureus was then transferred to sterile buffered phosphate or distilled

water solution and the turbidity was adjusted using a spectrophotometer at 600 nm

wavelength in comparison with the absorbance of the McFarland’s standard, 0.089.

Sensitivity Testing for S. aureus. A sterile swab was dipped it into the broth

culture of organism and was gently squeezed against the inside of the tube in order

to remove excess fluid in the swab. The swab with the test organism was streaked

evenly on each of the sterile and dried.

Mueller-Hinton agar (MHA) plates. After the streaking was completed, the

plates were made to dry for 5 minutes, then the discs from the (+) control

(Ciprofloxacin), (-) control, sterile distilled water, and the Basil Extract were placed

on the surface of each of the agar using sterilized forceps. The discs were gently

pressed into the surface of the agar using flame sterilized forceps or inoculation

loop. The plates were then carefully incubated for 24 hours at370 C. After

incubation, a caliper was used to measure the diameter of the zone of inhibition for

each of the discs used.

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RESULTS AND DISCUSSION

Test Holy Basil Extract Positive Reactions

Alkaloids - Creamy white precipitate

Tannins + Dark Green or black precipitate

Saponin + Continued Frothing

Proteins - Violet or Pink Coloration

Phenols + Bluish Green or Black Coloration

Flavonoids - Yellow Coloration

Glycosides + Upper layer-blush-green color

Lower layer-brownish-red color

Carbohydrates - Brick Red Precipitate

Terpenoids + Reddish Brown Interface

Table 1. Result of the phytochemical test

The holy basil extract contains the following:

Flavonoids possess a number of medicinal benefits, including anticancer,

antioxidant, anti- inflammatory, and antiviral properties. They also have

neuroprotective and cardio-protective effects. These biological activities depend

upon the type of flavonoid, its (possible) mode of action, and its bioavailability.

Phenols used to disinfect skin and to relieve itching. Phenol is also used as an oral

analgesic or anesthetic in products such as Chloraseptic to treat pharyngitis.

Additionally, phenol and its related compounds are used in surgical ingrown toenail

treatment, a process termed phenolization.

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Tannins accelerate blood clotting, reduce blood pressure, decrease the serum lipid

level, produce liver necrosis, and modulate immune responses. The dosage and kind

of tannins are critical to these effects.

Saponins decrease blood lipids, lower cancer risks, and lower blood glucose

response. A high saponin diet can be used in the inhibition of dental caries and

platelet aggregation, in the treatment of hypercalciuria in humans, and as an

antidote against acute lead poisoning.

Alkaloids have diverse physiological effects: antibacterial, antimitotic, anti-

inflammatory, analgesic, local anesthetic, hypnotic, psychotropic, and antitumor

activity and many others

Glycosides discussed in the present review have potential analgesic and anti-

inflammatory effect; thus, these compounds may act as pain mediators

Proteins help repair and build your body's tissues. It drives metabolic reactions,

maintains pH and fluid balance, and keeps the immune system strong. It also

transports and stores nutrients and can act as an energy source. Protein is crucial to

good health

Carbohydrate helps fuel your brain, kidneys, heart muscles, and central nervous

system. For instance, fiber is a carbohydrate that aids in digestion, helps you feel

full, and keeps blood cholesterol levels in check.

Terpenoids have been found to be useful in the prevention and therapy of several

diseases, including cancer, and also to have antimicrobial, antifungal, antiparasitic,

antiviral, anti-allergenic, antispasmodic, antihyperglycemic, antiinflammatory, and

immunomodulatory properties (world j hepatology,2011)

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(+) Control
Basil Extract (-) Control Commercial
Germicidal Soap
Zone of Zone of Zone of
Inhibition Inhibition Inhibition
Disc (mm) Disc (mm) Disc (mm)
R1 19 R1 0 R1 30
Test R2 19 R2 0 R2 34
Organism: S. R3 19 R3 0 R3 32
aureus R4 20 R4 0 R4 32
UPLB-Biotech R5 19 R5 0 R5 30
R6 19 R6 0 R6 32
R7 19 R7 0 R7 28
R8 20 R8 0 R8 36
R9 21 R9 0 R9 30
R10 19 R10 0 R10 28
Mean 19 0 31

Table 2. Result of culture and sensitivity

The measurements obtained from the individual discs were recorded. This will

be compared with the standard table to determine the sensitivity zone and

determine whether the tested bacteria species are Basil Extract and the controls

used.

The zone of inhibition indicates the area of bacterial growth inhibition around

the disc. Based on these results, the holy basil extract shows some antimicrobial

activity against Staphylococcus aureus, with an average zone of inhibition of 19 mm.

However, the positive control, the commercial germicidal soap, exhibited a larger

zone of inhibition (31 mm), indicating stronger antimicrobial activity.

The results of the sensitivity test demonstrate that the holy basil extract exhibits

antimicrobial activity against Staphylococcus aureus, as indicated by the observed

zone of inhibition. The zone of inhibition represents the area around the disc where

bacterial growth is inhibited, indicating the effectiveness of the tested substance in

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preventing the growth and spread of the bacteria. In this case, the holy basil extract

shows an average zone of inhibition of 19 mm, suggesting that it has the ability to

inhibit the growth of Staphylococcus aureus to some extent.

However, it is notable that the positive control, represented by the commercial

germicidal soap, displayed a significantly larger zone of inhibition measuring 31 mm.

This result implies that the commercial germicidal soap possesses stronger

antimicrobial activity against Staphylococcus aureus compared to the holy basil

extract. The larger zone of inhibition indicates a greater ability to inhibit bacterial

growth and suggests that the commercial germicidal soap may be more effective in

eradicating Staphylococcus aureus.

CONCLUSION:

Based on the data presented, we are inclined to accept our alternative hypothesis.

The findings strongly suggest that Holy Basil extract exhibits effectiveness against

Staphylococcus Aureus, providing evidence of its potential as a natural antimicrobial

agent. Furthermore, the results indicate the presence of a specific bioactive

compound within the Holy Basil extract that is responsible for this antimicrobial

activity. These findings reject the null hypothesis, which proposed the

ineffectiveness of Holy Basil extract against Staphylococcus Aureus and the absence

of a specific bioactive compound. The data supports the notion that Holy Basil holds

promising antimicrobial properties and highlights the potential for further

investigation into the identification and characterization of the specific bioactive

compound responsible for its activity.

 23

The findings suggest that while the holy basil extract demonstrates antimicrobial

potential, it may have a comparatively milder effect against Staphylococcus aureus

compared to the commercial germicidal soap.

Overall, the results indicate that holy basil extract possesses some antimicrobial

activity against Staphylococcus aureus, although it falls short of the antimicrobial

efficacy exhibited by the commercial germicidal soap. These findings contribute to

the understanding of alternative natural treatments for Staphylococcus aureus

infections and highlight the potential for further exploration and development of holy

basil as a potential antimicrobial agent.

RECOMMENDATIONS:

To further advance the understanding and potential application of Holy Basil extracts

as a natural treatment for S. aureus infections, several recommendations for future

research are proposed. Firstly, it is essential to identify the specific compounds

within Holy Basil extracts responsible for their antimicrobial activity against S.

aureus. Additionally, exploring the effectiveness of Holy Basil extracts against other

common bacteria causing skin infections would provide valuable insights. Optimizing

the extraction methods to obtain more potent extracts with enhanced antimicrobial

properties should be pursued. Investigating the combination of Holy Basil extracts

with other natural antimicrobial agents or antibiotics could uncover synergistic

effects. Furthermore, conducting animal or human studies would provide a better

understanding of the efficacy of Holy Basil extracts in real-life situations. Unraveling

the mechanisms by which Holy Basil extracts inhibit the growth of S. aureus would

enhance our understanding of their antimicrobial action. Developing different

 24

formulations of Holy Basil extracts, such as creams or gels, would offer more

convenient and effective application on the skin. It is also crucial to assess the

safety, potential side effects, and long-term use of Holy Basil extracts. By pursuing

these research directions, we can expand our knowledge of Holy Basil extracts, their

mechanisms of action, and their potential as a natural alternative for managing S.

aureus infections.

REFERENCES:

-Antimicrobial Properties of Tulsi (Ocimum sanctum) in Relation to Shelf Life

Enhancement of Fruits & Vegetables. (2018). International Journal of Green

and Herbal Chemistry, 7(1). https://doi.org/10.24214/ijghc/hc/7/1/02032

-Bhooshitha, A. N., Ghosh, A. R., Chandan, H. M., Nandhini, H. S., Pramod, B. R., &

Krishna, K. L. (2020). Review On Nutritional, Medicinal and CNS Activities of Tulsi

(Ocimum. Sanctum). Journal of Pharmaceutical Sciences and Research , 12(3), 420-

426.

-Deshmukh, A. S., Deshmukh, G. B., & Shirole, P. D. (2015). Ocimum sanctum: A

medicinal gift from nature. International Journal of Pharmacognosy, 2(12), 550-559.

-Hanumanthaiah, P., Panari, H., Chebte, A., Haile, A., & Belachew, G. (2020). Tulsi

(Ocimum sanctum)–a myriad medicinal plant, secrets behind the innumerable

benefits. Arabian Journal of Medicinal and Aromatic Plants, 6(1), 105-127.

-How To Use Tulsi, The Ayurvedic Elixir, For Your Skin Disorders? (2022, September

6). Vedix. https://vedix.com/blogs/articles/benefits-of-tulsi-for-skin?

 25

fbclid=IwAR0r0ZWSFnB9KrBLtPKfQ8Xgx3a03KvCaVzR4M0ksaPOGX31GIXBpL_

HcW4

-Mittal, R., Kumar, R., & Chahal, H. S. (2018). Antimicrobial activity of Ocimum

sanctum leaves extracts and oil. Journal of Drug Delivery and Therapeutics , 8(6),

201-204

-Naik, L. S., Shyam, P., Marx, K. P., Baskari, S., & Devi, V. R. (2015). Antimicrobial

activity and phytochemical analysis of Ocimum tenuiflorum leaf extract. Int. J.

PharmTech Res, 8(1), 88-95.

-Siva, M., Shanmugam, K. R., Shanmugam, B., Venkata, S. G., Ravi, S., Sathyavelu,

R. K., & Mallikarjuna, K. (2016). Ocimum sanctum: a review on the pharmacological

properties. Int. J. Basic Clin. Pharmacol, 5, 558-565.

-Tyagi, M., Tyagi, N., & Tyagi, J. (2021). A REVIEW ON: THE MAJOR

CONSTITUENTS OF TULSI (OCIMUM SANCTUM) AND THEIR ANTIMICROBIAL AND

ANTIOXIDANT ACTIVITIES AGAINST DIFFERENT MICROBES.

-Yamani, H. A., Pang, E. C., Mantri, N., & Deighton, M. A. (2016). Antimicrobial

activity of Tulsi (Ocimum tenuiflorum) essential oil and their major constituents

against three species of bacteria. Frontiers in microbiology, 7, 681.

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LIST OF APPENDICES

Appendix A: Lists of figures of Extraction method

Figure 1, 2 and 3: Powderization of Holy Basil leaves. The leaves of Holy

Basil (Ocimum sanctum) are carefully transformed into a fine powder using a

mechanical grinder. This process increases the surface area of the plant material,

facilitating efficient extraction of bioactive compounds. The resulting powdered form

of Holy Basil serves as a crucial component for further analysis and evaluation of its

potential antimicrobial activity against S. aureus. (Pages 14-15)

Figure 4: Holy Basil powder soaked in ethanol. The powdered leaves of

Holy Basil (Ocimum sanctum) are immersed in ethanol, a solvent used for extraction.

This process facilitates the release of bioactive compounds from the plant material,

which may possess potential therapeutic properties. The extraction of Holy Basil in

ethanol is a crucial step in harnessing its medicinal benefits for further analysis and

evaluation. (Pages 14-15)

Figure 5: Filtration of Holy Basil extract. The Holy Basil extract obtained

from the extraction process is subjected to filtration. This step involves passing the

extract through a filter to separate the liquid phase from solid residues or impurities.

Filtration helps ensure a purified and clarified extract, ready for subsequent analysis

and evaluation of its potential antimicrobial activity against S. aureus. (Page 14-15)

Figure 6: Rotary evaporation of Holy Basil extract. The Holy Basil

extract obtained from the filtration process is subjected to rotary evaporation. This

technique involves the application of gentle heat and vacuum to remove the solvent

(e.g., ethanol) from the extract, resulting in the concentration of bioactive

compounds. Rotary evaporation is a critical step in obtaining a concentrated and

 27

potent extract for further analysis and assessment of its antimicrobial properties

against S. aureus.(Page 14-15)

Appendix B: Documentation of phytochemical compositions

Figure 7: Flavonoids Figure 8: Saponins Figure 9: Glycosides

Figure 10: Alkaloids Figure 11: Phenols Figure 12: Proteins

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Figure 13: Terpenoids Figure 14: Carbohydrates Figure 15: Tannins

Appendix C: Documentions for Culture and sensitivity

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Appendix D: Documentations