ANTIBACTERIAL ACTIVITY OF CASSIA ALATA LINN.

(AKAPULKO) LEAF

EXTRACT ON STAPHYLOCOCCUS AUREUS- ASSOCIATED SKIN INFECTION

Research Proposal

presented to

the Faculty of Natural Sciences Department

College of Sciences and Information Technology

Ateneo de Zamboanga University

In Partial fulfillment

of the Requirements in

Research 101-B

by

Erika Anne C. Lumibao

Kristal Jane A. Torino

March 2017
 Table of Contents

CHAPTER I .....................................................................Error! Bookmark not defined.

Introduction ......................................................................Error! Bookmark not defined.
Background of the Study ................................................................................................. 1
Statement of the Problem ................................................................................................ 3
Significance of the Study ................................................................................................ 3
Scope and Limitations of the Study ............................................................................... 4
Definition of Terms ......................................................................................................... 5
Review of Related Literature .......................................................................................... 5
Synthesis of Related Literature…………….…………………………………………….9

CHAPTER II…………………………………………………………………………….11

Methodology…………………………………………………………………………......11

Research Design…………………………………………………………………….....11

Collection and Identification of Plant…………………………………………………11

Plant Extraction………………………………………………………………………..11

Preparation of Culture Media………………………………………………………….12

Kirby-Bauer Disk Diffusion Test……………………………………………………..12

Measuring of Zone of Inhibition…………………………………………………..….13

Determination of Minimum Inhibitory Concentration……………………………….13

Statistical Analysis……………………………………………………………………13
 CHAPTER 1

Introduction

Background of the Study

Over the decades, antibacterial agents are one of the most essential tools in fighting

bacterial infections. However, the continuous increase in number of drug resistant bacteria

results to less effective antibiotics. Bacteria is said to have a genetic ability to transmit and

acquire resistance to synthetic drugs (Bhalodia, N.R and Shukla, V.J, 2011). With this, it is

supposed that the investigation of new drugs with lesser bacterial resistance is essential.

Antibacterial resistance is one of the challenges that the world is facing. In fact, in the

developed and developing countries in the world, the increase antibiotic resistance is said to be

an unresolved problem and is a burden to their respective health services (Cabrera et al., 2016).

In developing countries, over one third of its population lack access to essential

medicines (WHO, 2003). In the Philippines, plants being traditional medicines have been used to

treat illnesses and serious diseases. According to the Department of Health (DOH) of the

Philippines, herbal plants may be used as alternative medicines in treating health problems. As a

matter of fact, 10 out of over 1,600 herbal plants in the Philippines have been approved by the

Department of Health through its Bureau of Food and Drugs (BFAD) following the guidelines

for drug registration.

One of the activities that can be found in traditional plants is the antibacterial activity.

Antibacterial plants are mostly herbs that contain aromatic essential oils that kill pathogenic

bacteria. The Philippines is a country with diverse flora and vast number of species, said to have

curative properties. One of the widely used medicinal plants is Cassia alata (Akapulko). It is a

shrubby legume that is commonly found in most tropical countries including the Philippines.
 This plant belongs to the family Leguminosae. Cassia alata (Akapulko) is known for its

dark green compound leaves consisting orange rachis with numbers of leaflets. Furthermore, this

type of plant is known to be an herbal plant wherein its leaves contain chrysophanic acid which

is used in treating fungal infections such as ringworm and scabies. Cassia alata (Akapulko)

leaves also contain saponin, used to treat intestinal parasites. Moreover, its flowers are associated

with its anti-fungal properties. Cassia alata is the plant used in this research in order to further

discover if its leaf extract is effective in treating bacteria, specifically those that cause skin

infections.

Bacterial skin infections are mostly caused by Staphylococcus aureus (Lyytikainen,

2002). It is frequently found in the skin; hence is part of the skin flora. Staphylococcus aureus

being a gram-positive bacterium may not be always pathogenic as those gram-negative bacteria.

However, it is said that this type of bacterium may also affect the health of human in which it can

be associated with skin infections such as abscesses and eczema. The emergence of the

antiobacterial agenrt-resistant strains of Staphyloccus aureus has always been a problem of the

clinical medicine. In fact, the Philippines reported 58% cases of Methicillin-resistant

Staphylococcus aureus (MRSA) in 2013. This MRSA is also resistant to penicillin, which may

lead to more serious skin infections that may further result to complications or death (Pascual,

2014).

MRSA is said to be present within the skin of 33% of the population of the world (Gomf,

2016). This alarming result leads to the need of studying plant extract that may contr ibute to

decrease this rate. In this study, Cassia alata being known to be an antifungal medicine will be

used to treat skin infection caused by Staphylococcus aureus.

Statement of the Problem:

This study aims to determine the antibacterial activity of Cassia alata (Akapulko) leaf

extract. Specifically, this study aims to answer the following questions:

1. What will be the effect of the leaf extract of Cassia alata against a skin infection causing

bacterium, Staphylococcus aureus?

2. What will be the Minimum Inhibitory Concentration (MIC) of the Cassia alata leaf

extract inhibiting the growth of the gram-positive bacteria Staphylococcus aureus?

3. What is the effect of the different concentrations of the plant leaf extract against

S.aureus?

4. Do the different levels of leaf extract concentration affects the zone of inhibition of

S.aureus?

5. Which concentration level will give effective inhibition and less inhibition on S.aureus?

Significance of the Study

Plants have been widely used as herbal medicine in the Philippines. However, an

alarming increase of drug-resistant bacteria leads to the need to seek for new antibacterial agents

will lesser bacterial resistance. Despite these efforts to provide better or improved antibiotics,

bacteria continue to progress in response to the new antimicrobial agents that they encounter.

With this, the present study aims to determine the antibacterial activity of Cassia alata Linn leaf

extract against Staphylococcus aureus.

The analysis and investigation of the antibacterial activities of Cassia alata leaf extract

will contribute to the pharmaceutical industry and will lead them to the development of new
drugs with lesser bacterial resistance. Furthermore, this study will be useful in formulating new

herbal treatments against Staphylococcus aureus. The analysis of this study will also support the

institution such as the Department of Health (DOH) and the Department of Science of

Information Technology (DOST) in supporting the utilization and the formulation of alternative

treatment from natural sources such as the Cassia alata to treat any ailments associated with

skin-infecting bacterium. This study will be beneficial in incorporating and integrating the

scientific and technological education for conducting active and effective researches in Ateneo

de Zamboanga University. Moreover, the findings of this study will aid other researches that are

associated with investigating the antibacterial activity of other plants.

Scope and Limitations of the Study

The study will be restricted on the investigation of the antibacterial activity of Cassia alata

against a gram positive bacterium, Staphylococcus aureus. This study will be focusing on the

Minimum Inhibitory Concentration (MIC) of the Cassia alata leaf extract that will inhibit

growth of the gram-positive bacteria Staphylococcus aureus. This study furthermore will be

limited on the comparison of the zone of inhibition on Stapylococcus aureus of the different

concentrations of the plant leaf extract.

This study will not include the identification and phytochemical analysis of the different

bioactive compounds that are present in the plant. Also, this study will not omit the analysis and

investigation of the toxicity level of the plant. Additionally, this study will disregard test subject

that has skin infection caused by Stapylococcus aureus.

The experiment will be conducted at the Research Laboratory of Ateneo de Zamboanga

University which will be limited to the leaf extract of the said plant.
Definition of Terms

Antibacterial Activity. Properties of the plants that destroys bacteria suppresses their growth or

their ability to reproduce.

Disk Diffusion Method. Approach that will be used in testing the susceptibility of the organism in

this study.

Minimum Inhibitory Concentration (MIC). The lowest concentration of an antibacterial agent

that will inhibit the visible growth of microorganism in the zone of inhibition, clear region

around the paper disc saturated with the antibacterial agent on the agar surface, after 24 hours of

incubation.

Review of Related Literature

Medicinal Plants

Over the decades, plants have been a source of medicinal aid to human health concerns.

Herbs have been used to treat diseases ever since the Greek and Roman civilization. Medicinal

plants are also widely used as folk medicine in most places in India. According to the World

Health Organization, 80% of the population of the world use plant extract as community

medicine. In fact, modern clinical drugs also come from natural products (Juoda, 2016).

Several plant extract are known for having great medicinal value in terms of their stem

bark decoction, such as that of Albizia gummifera, used to treat venereal diseases (Komolafe,

2014) . Traditionally used medicinal plants are now being authorized by scientific researches. In

fact, the Department of Health of the Philippines (DOH) approved 10 commonly known plants to

be used as herbal medicines for its essentials in treating some illnesses and diseases with the help

of the Philippine National Drug Formulary (PNDF) and these include: Vitex negundo (Lagundi),
Peperomia pellucida (Ulasiman-Bato), Allium sativum (Garlic), Psidium guajava (Guava),

Blumea balsamifera (Sambong), Momordica charantia (Bitter Gourd), Mentha spicata

(Spearmint), Quisqualis indica (Rangoon Creeper), Carmona retusa (Wild Tea) and Cassia alata

Linn (Akapulko) (Magno, 2012). All 10 herbs have been thoroughly tested and have been

clinically proven to have medicinal value in the relief and treatment of various ailments (dela

Cruz, 2010).

Cassia alata

The Philippines created and signed a law known as the Traditional and Alternative

Medicine Act (TAMA) of 1997 in which The Philippine Institute of Traditional and Alternative

Health Care (PITAHC) promote and advocate the use of traditional and alternative healthcare

modalities through scientific research and product development. With this, several plants were

approved to be used as an alternative natural medicine to cure infections and diseases. Of the 10

plants mentioned, Cassia alata Linn is one of the plants that have known to be medically useful

especially in treating fungal and bacterial infections.

Cassia alata is a shrubby legume that is commonly found in most tropical countries

including the Philippines. It grows for about one to two meters tall. Akapulko has several names

from various regions of the country. It is commonly known as katanda, andadasi and palochina.

This plant belongs to the family Leguminosae. Akapulko is known for its dark green compound

leaves consisting orange rachis with numbers of leaflets. The leaves also contain chrysophanic

acid which is used in treating fungal infections such as ringworm and scabies. Akapulko leaves

also contain saponin, used to treat intestinal parasites. It also serves as herbal medicine for

asthma and expectorant for bronchitis (Aruh, et. al., 2005).

 Akapulko bears golden yellow flowers with four winged ponds containing various

triangular seeds. These flowers are surrounded by bracts which are being shed in time. These

flowers are also associated with the anti-fungal properties of Akapulko. Due to these anti-fungal

properties, Akapulko is widely used as ingredients for soaps and shampoos in the Philippines. It

has also been developed as herbal medicine lotion. The methanol extracts of the leaves, flowers

and stem is being associated with the antimicrobial activity Akapulko. The activity involves the

increase on fractionation of petrol, dichloromethane and ethyl acetate. The most effective among

these fractionations is the dichloromethane fraction of flower extract.

In the Indian systems of medicine, Akapulko is well-known for its several medicinal

properties, used as vermicide, astringent, purgative, expectorant and to treat skin diseases (Aruh,

et al., 2005). According to Abubacker et al., the leaf extract of the Akapulko have been studied

and reported to possess medicinal properties that can be used to treat infections and diseases such

as ringworm, scabies, pruritis, eczema and itching. In fact, Asian countries such as Thailand

approved and recommend Akapulko to be one of their primary alternative health care treatments

against skin diseases caused by some pathogenic bacteria in their country (Ongsakul, et al.,

2004).

Staphylococcus aureus

Staphylococcus aureus is the most commonly identified agent responsible for skin and

soft tissue infection. This type of bacterium is known to be the almost-universal cause of

furuncles, carbuncles, and skin abscesses (Jernigan, 2006).

Staphylococcus aureus is gram-positive bacterium found in a grape-like cluster. It

belongs to the family Staphylococcacea which affects humans and every known mammalian

species. Since S. aures affects a wide range of species, it can freely be transmitted from a species
to another (Mandal, 2012). Forms of transmissions are through direct contact with objects

infected by this certain bacteria or via bites of contaminated animals or persons. There is

approximately 30% humans that carry Staphylococcus aureus within their nose, throat and akin.

This type of gram-positive bacterium is one of the most common causes of skin infection

especially after surgery and injury. They are able to attack through dry or broken skin or mucous

membranes. Staphylococcus aureus then hide their antigens to avoid immune response. They kill

infection-fighting called phagocytes and survive within these cells. They also develop resistance

to antibiotics. Skin infections caused by Staphylococcus aureus is common mostly during pre-

pubertal stage of children.Most staph skin infections are treated with antibiotics; however there

are certain kinds of staphs that are resistant to particular antibiotics which make these diseases

hard to cure. Methicillin-resistant Staphylococcus aureus is one of the examples of these

bacteria. The infections caused by Staphylococcus aureus ranges from mild to life threatening. It

tends to contaminate the skin, frequently causing abscesses and eczema. This may also go

through the bloodstream and infect the endocarditis and other sites of the body (Stanway, A.,

2015).

Disk Diffusion Assay

There has been a growing interest in developing new antimicrobial agents from numerous

sources such as plants to combat the increase of microbial resistance. Many researches today

focus on investigating microbial extracts such as plant extracts for potential antimicrobial drugs.

With this, the laboratory methods are used to appraise and screen the antimicrobial activity of the

extract.

Disk Diffusion is one of the oldest approaches in microbiology. It is an approach wherein

it determines the sensitivity of microorganisms to specific antimicrobial drug. Disk diffusion is

known to be one of the most practical and commonly used antimicrobial susceptibility testing

methods in clinical laboratories in determining antibiotic resistance of microorganisms to various

antimicrobial agents (“EUCAST Disk Diffusion Test Methodology”, 2017).

The disk diffusion method is performed by the used of the Mueller-Hinton Agar (MHA)

in which it is known to be the best medium for routine susceptibility test for its good

reproducibility. This type of agar is low in sulfonamide, trimethoprim, and tetracycline inhibitors

which give adequate growth of most bacterial pathogens (Tendencia, 2004).

There are various conditions that may affect a disk diffusion test. Upon conducting this

type of test: several things: the amount of organisms used; the concentration of chemical; and the

incubation conditions such as the temperature, atmosphere and time should be held constant so

that only the size of inhibition is the variable.

Synthesis of Related Literature

According to Hansch and Shellard, plants have been used as medical agents since the

ancient times. With an increase in the antibacterial agent-resistant strains of microorganisms,

traditional plants are being explored for their antibacterial and medicinal values (Mahuli et al,

2011). Previous studies conducted by Boye, Dauda B.E.N, Oyeleke, Dauda H., Shinkafi, Priti

and Vidhi investigated for antibacterial potential of some plants against gram positive and gram

negative bacteria. In this study, the leaves of C. alata will be studied and investigated for

antibacterial activity against S.aureus.

C. alata is known to be 1 of the 10 medicinal plants that have been approved by the

Department of Health (DOH). Additionally, this plant has been investigated to have antibacterial

activity against gram positive and gram negative bacteria.

 S.aureus is a gram positive bacterium has been known to be the most common bacterium

that infects soft tissues and skin. Study shows that this type of bacterium is the common cause of

certain types of infection such as furuncles, carbuncles, and skin abscesses.

Past studies showed that C.alata exhibited antibacterial activity against gram positive

bacteria including S. aureus. However, previous studies exerted more effort on determining its

antibacterial potential using the plant’s stem and root. In this study, it will mainly focus the

antibacterial potential of the leaf extract of the said plant.

In the study of Boye, Dauda B.E.N and Oyeleke, methanol and water were used as

extractant. In this study on the contrary, ethanol with the concentration of 100%, 75% and 50%

will be used. In the study conducted by Dauda H. and Shinkafi, triple sugar iron (TSI) agar was

used as the medium which contains three sugars: glucose, sucrose and lactose. In this study,

Muller-Hinton agar will be used as the medium. In the assessment of antibacterial activity, Priti

and Vidhi used Agar Cup Double Dilution Method. In this study on the other hand, Kirby-Bauer

Disk Diffusion Method will be used to assess the antibacterial activity of the plants. In a study

conducted by Bation and Suarez, t-test and one-way ANOVA were used to validate the results.

In contrary, this study will only apply one-way ANOVA as statistical method in validating the

results. This will be used in comparing the zone of inhibitions of the plant extract against the S.

aureus.
 CHAPTER 2

Methodology

Research Design

This study will employ the experimental design with quantitative measures. This would

determine the antibacterial activity of Cassia alata leaf extract against Staphylococcus aureus

using Disk-Diffusion Method adopted from the Kirby-Bauer technique. In this study, six set-ups

will be used: 100%, 75% and 50% concentrations of the leaf extract; 100%, 75%, and 50%

concentrations of Penicillin-ethanol solution as the positive control. Furthermore, the range of

the antibacterial property of the leaf extract will be determined by measuring the diameter of the

clear zone of inhibition surrounding the paper disk. Aseptic technique will also be used

throughout the experimental phase.

Collection and Identification of the Plant

The leaves of the plant which will be used in this study will be collected from Taloptap,

Vitali, Zamboanga City. Only leaves judged to be mature and disease-free will be harvested in

morning hours. The plant specimen will be assigned with a voucher number CA/0001/17/LT and

will be kept in the Research Laboratory of Ateneo de Zammbong University.

Plant Extraction

The plant material will be cleaned to ameliorate the quality of the leaves which will then

be air-dried in a shade at room temperature. The dried leaves will be ground into powder using

sterile blender. A sample of 60 grams of powder leaves of Cassia alata will then be used for

extraction.

The powdered sample material will be soaked in ethanol in different concentrations for

48 hours. 20g of powdered sample will be soaked in 20ml of 100% ethanol; 20g in 20ml of 75%
ethanol; and 20g in 20ml 50% ethanol. The extract will then be filtered using whatman paper

number one.

Preparation of Culture Media

In preparing the Muller-Hinton media; 2.0 g beef extract, 17.5g of Acid Hydrolysate of

Casein, 1.5g of starch and 17.0g of agar will be needed. These components will then be

combined to have a 38g of solid mixture which will be suspended in 1L of purified water. The

medium will be boiled for 1 minute to completely dissolve the components and will be

autoclaved at 121oc for 15 minutes. The medium will be cooled to 45oc. This will then be poured

into sterile petri dishes on level, horizontal surface to give uniform depth of 4mm. the medium

will be left at room temperature to solidify.

Kirby-Bauer Disk Diffusion Test

Using a puncher, 4mm diameter discs of filter papers will be prepared. The discs will be

sterilized for 15 minutes at 15 PSI. The sterile discs will then be soaked in beakers containing

20ml of the prepared Cassia alata-ethanol solution of different concentrations; and prepared

commercialized drug-ethanol solution for 12 hours. Using sterile forceps, the pre-soaked discs

containing the plant-ethanol solution; penicillin-ethanol solution will be placed on the pre-

inoculated agar plates. The plates will then be inoculated at 37oc for 24 hours. Following the 24

hours incubation, microbial clearing will be observed on each of the agar plates.
Measuring of Zone of Inhibition

The clearing will be measured as the zone of inhibition. The diameter of the zone of the

growth of inhibition around the disk will be measured to the nearest whole millimeter using a

ruler. Measurement will be done from one end of the microbial clearing towards the opposite end

passing through approximately the middle of the disc. The date will then be collected using the

appropriate statistical analysis.

Determination of Minimum Inhibitory Concentration

Samples will be taken from the nutrient agar plates after 24 hours upon incubation. The

concentration that will exhibit the least clear zone of inhibition will be considered as the MIC for

that isolate.

Statistical Analysis

The data gathered will be statistically treated to support the results of variation of growth

of inhibition of the extract concentration. In this study, One-way ANOVA will be used to

validate the results. It will be used to test the means of zone of inhibition of Cassia alata that will

inhibit the growth of S. aureus. One-way ANOVA will also be used to compare the zone of

inhibition between the different concentrations of C. alata. This will also be used to compare the

zone inhibition between the different concentrations of C. alata and the zone of inhibitions of the

different concentrations of Penicillin. This will further analyze the relationship of the

concentration of extract to the zone of inhibition.