1

Chapter 1

THE PROBLEM AND ITS BACKROUND

Introduction

Over the decades, antimicrobial 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 be have a genetic ability to transmit and acquired resistance to

synthetic drugs (Bhalodia, N.R and Shukla, V.L, 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 the unresolved problem and is a burden to

their respective health service (Cabrera et al., 2016).

In the Philippines, plants being traditional medicines have been used to

treat illness and serious diseases. According to 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 guideline for drug registration.

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

antimicrobial activity. Antimicrobial plants are mostly herbs that contain

aromatic essential oil that kills photogenic 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 Acapulco (Cassia alata). It is a

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shrubby legume that is commonly found in most tropical countries in including

the Philippines.

This plant belongs to the family Leguminosae. Acapulco (Cassia alata)

is known for its dark green compound leaves consisting orange rachis with

number 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. Acapulco (Cassia alata)

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

flower is associated with its antifungal properties. Acapulco 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.

Staphylococcus aureus and Bacillus subtilis are both gram-positive

bacteria that can be found in human skin. It can be the cause of some human

skin infection. In this study, Cassia alata being known to be an antimicrobial

medicine will be used to treat skin infection caused by Staphylococcus aureus

and Bacillus subtilis.

Background of the Study

It is fact that maintenance of health is very costly, thus herbal medicines

such as ointment, cream and soap provide a wise alternative to the expensive

medicines in the treatment or management of these skin diseases.

Acapulko is one of the medicinal plants endorsed by the DOH’s National

Integration Research Program on Medicinal Plants. Akapulko is widely

available in our surrounding, especially in the rural areas. Its main medicinal
 3

value is primarily for the management of fungal problems like athlete’s foot,

ringworm, scabies and other skin diseases.

A common plant, Acapulco, scientifically known as cassia alata is the

subject of this study. It is locally known as bayas-bayabasan, kapurko, katanda,

katandang aso, pakagonkon, sonting in tagalog. Acapulco has a long history of

medical usage and has a long list of benefits. Acapulco is an erect, shrubby

legume with dark green compound leaves. Acapulco leaves have orange rachis

that 16-28leaflets. Acapulco produces an axis of golden yellow flower that has

4-winged pods containing 50-60 flattened, triangular seeds. Acapulco flowers

are enclosed by yellow-orange bracts that later shed in time.

Akapulko is a shrub that grows wild on many areas of the Philippines.

The leaves contain chrysophanic acid. The leaves are reported to be sudorific,

diuretic and purgative, being used in the same manner as senna. The leaves

are commonly used for ringworm and other skin diseases. The leaves in

concoction are also used to treat bronchitis and asthma (Healing Wonder of

Philippines Medical Plant, 2015).

People phone the plant kantada, andadasi, and also palochina

throughout tagalog. The plant is one of the family of leguminosae, along with

the expand about one to two yard extra tall. Its heavy twigs along with the

foliage is accepted with 7 to twenty pamphlets which can be oblong elliptical

healthy. The medical herb that contains chrysophanic chemical, a fungicide

utilized to handle fungus infection, just like ringworm, scabies, as well as tinea

infection. Acapulco also contains saponin, any healthy laxative that attractive

expelling intestinal organisms (Benefits of Acapulco plant,2012).

 4

Theoretical Framework

The used of traditional medicine has increased in development countries

also, mainly due to the failure of modern medicine to provide effective treatment

for chronic diseases and emergence of multi-drug resistant bacteria and

parasites. The current emphasis of new drug discovery process from plants in

the development of product with new pharmacological modes of action

(Jachak, 2007).

Leilen,N. (2008), Acapulco or cassia alata is from the family of

Fabaceae. Its flower buds appear like yellow candles, each complete with a

flame thus, making it also known as Emperor’s Candle or Candle bush. In the

Philippines, it is also known as bayas-bayasan. Its leaves fold together at night.

The plant chemical component includes Anthraquinones, Chrysoeriol 7-0,

Beta-D-Allopyranoside and Rhamnetin 3-0 making it known for its antibacterial,

antifungal, antimicrobial, diuretic, laxative, analgesic, and chloretic.

Cassia alata., Linn belonging to family caesalpiniaceae is a pantropical,

ornamental shrub, distributed from tropical America to India is commonly

known as Ringworm Senna, Seven Golden Candlesticks, Candle bush,

Gelenggang (Malay) or Akapulko (Filipino). The attractive shruB is named for

its flower buds which grow in a column and look like fat yellow candles each

complete with a flame. Its leaves, which fold together at night, are bilateral and

symmetrically opposed. The leaf extracts of the plant have been reported to

possess medicinal properties and used against ringworm, scabies, ulcers and

other skin diseases such as pruritis, eczema and itching. The leaves contain

chrysophanic acid, a fungicide that is used to treat infections such as ringworm

 5

and athlete’s foot. Previous reports of antimicrobial activity against human

pathogens have been widely carried out for Cassia alata Linn. Fuzellier et al.

(1982) found that aqueous extract from the leaves of Cassia alata and some of

its components, rhein, emodol, 4,5-dihydroxy-1-hydroxy-methylanthrone and

8,9-dihydroxy-6-hydroxy-methylanthraquinone, had antifungal activity against

some dermatophytes and yeast.

Senna Alata, grows well in full sun in a wide range of soil, which remain

moisture adequately. The plant grows in waste places, often along ditches

between rice-field. The plant usually propagated by seed and distributed all

over the country up to 1,500 m above sea level; sometimes they are cultivated

for medical purposes (Farnworth and Bunyapraphatsara 1992).

Conceptual Framework

The conceptual framework of the study is presented in the form of

paradigm. The Frame 1 consist of independent variables which contains

Acapulco leaf ethanol extraction and measurement of the Acapulco leaf using

well-diffusion assay

The Frame 2 consist of dependent variable pertains to the antimicrobial effect

of Acapulco leaf against Staphylococcus aureus and Bacillus subtilis.

Independent Variables Dependent Variables

Acapulco (Cassia alata

Linn.)
Antimicrobial effect of
 Ethanol leaf Acapulco leaf against :
extraction
 Staphylococcus
 Antimicrobial
aureus
effect of Acapulco
 Bacillus subtilis
leaf using well-
diffusion assay
 6

Figure 1. Research Paradigm of the study

Statement of the Problem

This study determined whether the Acapulco (Cassia alata Linn) has

antimicrobial property against the Staphylococcus aureus and Bacillus subtilis.

Specially, the study sought to answer the following question:

1. What is the process of Acapulco leaves extraction;

2. What phytochemicals are present in the Acapulco leaf extract that shows

the antimicrobial effects on Staphylococcus aureus and Bacillus subtilis;

3. What is the mean level of antimicrobial properties of Acapulco leaf

extract against Staphylococcus aureus and Bacillus subtilis;

4. Does Acapulco leaf extract have significant effect on Staphylococcus

aureus and Bacillus subtilis according to measurement of zone of

inhibition produce around the assay?

Research Hypothesis

Acapulco leaf extract has no significant effect on Staphylococcus aureus

and Bacillus subtilis according to measurement of zone of inhibition produce

around the assay.

Significance of the Study

 7

This study aimed to find out the antimicrobial activities of Acapulco leaf

extract. The outcome of the findings in this research paper may provide insight

and information to the following target beneficiaries.

Community. This study may give knowledge to the community who are using

Acapulco leaves. Results from this research may provide insight on the benefit

they may get fro Acapulco leaves.

Students. This study may provide preliminary study from the Acapulco leaves.

It may help them to develop ideas that enhances the present study and may

give other methods to strengthen its analyses and results. The methodology

used and other insight and from the collected citations had in the paper.

Tropical Medicine Researchers. They can use this study for any further

confirmations to clarify the outcome or otherwise use this for any related

literature or related studies.

Scope and Limitation of the Study

Preliminary laboratory test and proper analysis of the study were done

at Forest Products Research and Development Institute.

The study covers Acapulco’s antimicrobial effect on Staphylococcus

aureus and Bacillus subtilis.

Definition of Terms

Terms are conceptually and operationally defined for better

understanding of the readers.

Acapulco. The plant use to make extract for determining its antimicrobial

property against Pseudomonas aeruginosa and Bacillus subtilis.

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Bacillus subtilis. A gram-positive bacterium commonly found in soil and has

an ability to form a tough protective envelope. The fast growing population of

this bacterium in the human body may pose threat to the health.

Phytochemical Screening. The plant’s extract are tested through the

chemical indicated in the standard protocol. color indications may confirm the

presence or absence of the phytochemicals.

Saponin. Are detergent-like substances showing antibacterial as well as

anticancer potential.

Staphylococcus aureus. A gram-positive bacterium sphere-shaped (coccal)

bacteria often cause skin infections but can cause pneumonia, heart valve

infections, and bone infections.

Well-Diffusion assay. Another diffusion assay in which the plant extract as

well as the control were placed in the hole in the agar; this study used agar-

well diffusion assay to collect data and statistically interpreted by the

researcher.

Zone of Inhibition. The clear region around the assay with an antimicrobial

agent on the agar surface. The clear region is an indication of the absence, or

the effective inhibition, or microbial growth by the antimicrobial agent.

Chapter 2

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter is consisting of review of related literature and review of

related studies was significant to conceptualize the finished product of the

present study.

Related literature
 9

Otto et.al (2007) Acapulco or cassia alata, especially its leaves is widely

used in tropical regions as home remedies and sometimes cultivated for

medicinal purposes. The uses includes: ayurvedic medicine, treatment of:

constipation, stomach pain, ringworm, skin disease, inguinal hernia, intestinal

parasitosis and diabetes. The antimicrobial activity reported is diverse, acting

on; bacteria, fungi and amoeba. Though it has been used blindly in treating

syphilis, the specific bacteria against which the leaves have been found to be

active are: Vibrio cholerae, Staphylococcus aureus, Streptococcus sp. and

Escherichia coli.

Sennas act as purgatives and are similar to aloe and rhubarb in having

as active ingredients anthraquinone derivatives and their glucosides. The latter

are called sennosides or senna glycosides. Senna alexandrina is used in

modern medicine as a laxative; acting on the lower bowel, it is especially useful

in alleviating constipation. It increases the peristaltic movements of the colon

by irritating the colonic mucosa (Herbal and Alternative Remedies, 2015).

It is a perennial shrub with ornate yellow flowers which blooms through

most of the year. The shrub grows up to 3–4 m tall, with leaves 50–80 cm long.

The flower looks like a yellow candle. It begins blooming in October and it has

a long blooming season, from autumn almost throughout the winter. The fruit

shaped like a straight pod is up to 25 cm long. The seed pods are dark brown

or nearly black, about 15 cm long and 15 mm wide. Pods contain 50 to 60

flattened, triangular seeds. It is grown as an ornamental plant for its pretty

yellow flowers. Cassia alata is easy to grow from seed. It is a fast grower and
 10

flowers in the first year. The plant is suspected of being poisonous to livestock

(Chukwuma Muanya, 2015).

Cassia alata is an erect tropical, annual herb with leathery compounded

leaves found in the Western Ghats of India. The plant grows quickly to a height

of 6-12 ft and can live for many years. It has waxy yellow spikes on the tip of

each branch for which reason they are also known by the name candle bush

and seven golden candle sticks. The spikes elongates as the period of blossom

progresses. Flowers are pollinated by carpenter bees and caterpillars of

migrating butterflies feed on this plant. The entire plant has both internal and

external medicinal properties. Application of the crushed leaves is used for skin

diseases and the boiled one for treatment for high blood pressure, snake bites

and venereal diseases. The flowers have anti-fungal properties. Taken

internally the plant is used as remedy for asthma symptoms, weight loss and

stomach ulcers (LaPazGroup, 2012).

It is a perennial shrub with ornate yellow flowers which blooms through

most of the year. The shrub grows up to 3–4 m tall, with leaves 50–80 cm long.

The flower looks like a yellow candle. It begins blooming in October and it has

a long blooming season, from autumn almost throughout the winter. The fruit

shaped like a straight pod is up to 25 cm long. The seed pods are dark brown

or nearly black, about 15 cm long and 15 mm wide. Pods contain 50 to 60

flattened, triangular seeds. It is grown as an ornamental plant for its pretty

yellow flowers. Cassia alata is easy to grow from seed. It is a fast grower and

flowers in the first year. The plant is suspected of being poisonous to livestock.

Cassia alata or Senna alata has anti-helminthic, antibacterial, laxative, diuretic,

 11

Antifungal, Analgesic, alterative, Antiseptic, Anti-inflammatory, Antibacterial,

Hepatoprotective, abortificent, aperient, purgative, Antimicrobial, diuretic, anti-

herpetic, soporific properties (Nature Magnificent, 2012).

The above mentioned literatures are related to the present study since

they define the main material used in the study.

Tortora (2008), B. subtilis known also as the hay bacillus or grass

bacillus is a gram positive, catalese-positive bacterium, found in soil and the

gastrointestinal tract of humans. A member of the genus bacillus, B. subtilis is

a rod shaped and can form a tough, protective endospore, allowing it to tolerate

extreme environmental condition. B. subtilis has has a historically been

classified as an obligate aerobe, though evidence exists that it is a facultative

aerobe. B. subtilis is considered the best studied gram positive bacterium and

a model organism to study bacterial chromosome replication and cell

differentiation.

B. subtilis is viable for decades and is resistant to unfavorable

environmental conditions such as drought, salinity, extreme pH, radiation, and

solvents. The endospore is formed at time of nutritional stress, allowing the

organisms to persist in the environment until conditions become favorable.

Prior to the process of sporulation the cells might become motile by producing

flagella (Magno, et. al, 2016).

Bacillus subtilis is a Gram-positive, Catalase-positive bacterium

commonly found in soil. A member of the genus Bacillus, Bacillus subtilis has

the ability to form a tough, protective endospore, allowing the organism to

tolerate extreme environmental conditions. Unlike several other well-known

 12

species, Bacillus subtilis has historically been classified as an obligate aerobe,

though recent research has demonstrated that this is not strictly correct. It has

also been called Bacillus globigii, Hay bacillus or Grass bacillus.

B. subtilis is a bacterium found on skin, in the digestive tract, in epithelial

wounds, on extremities of the human body, in livestock and in soil (Earl et al.,

2008).

B. subtilis is ubiquitous, it has developed adaptive strategies to subsist

in diverse environments via the production and secretion of a large number of

genetically encoded molecules that control the growth of neighbouring

organisms ((Liu et al., 2010).

B. subtilis is sold commercially as a skincare product, a food ingredient

for human consumption, animal feed, fertilizer and an antibiotic substitute. The

molecular mechanisms by which these products work are poorly understood.

The above mentioned literatures are related to the present study since

they define the main material used in the study.

Staphylococcus aureus, a pre-eminent human pathogen causing an

array of serious hospital- or community-acquired infections worldwide (Geng et

al., 2010; Klevens et al., 2007; Otto, 2010), is found on human skin, digestive

tracts, nares, livestock and surgical instrumentation (Iwase et al., 2010; Otto,

2010). At minimum, 30 % of the world population is colonized with S. aureus, a

bona fide pathogen that has developed significant resistance against a variety

of antibiotics and is the cause of more fatalities in the USA than HIV/AIDS

(Klevens et al., 2007).

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S. aureus colonizes a large proportion of the world’s inhabitants, it only

produces clinical infection in a subset of this population. One attractive

hypothesis is that neighbouring organisms occupying the same environmental

niche (e.g. skin) respond by secreting an array of antibiotic-type molecules to

control S. aureus developmental phenotypes and thereby alter its ability to

proliferate in the host. The study described herein reveals that B. subtilis, a

bacterium that is nearly ubiquitous in nature, occupying many environmental

niches and therefore a common but transient skin microbe (Ara et al., 2006),

can inhibit the growth of an epidemic S. aureus isolate and possess the ability

to directionally release a molecule with antimicrobial and metabolism-altering

properties. Ultimately, the study indicates a utility for IMS in dynamic analysis

of interspecies metabolic exchange and perhaps a paradigm for future

discovery of novel antibiotic candidates.

As mentioned by Lindsey (2008), staphylococci are important

component pathogenic bacteria responsible for variety of diseases in human

and in other animals. They the most cause of hospital acquired infection and

antibiotic resistant strain (MRSA) have become endemic in hospital in most

country causing major health issue. In addition the incidence of new strain the

cause severe community-acquired infections healthy people is increasing and

MRSA staring are emerging in agricultural and domestic animals. In the race

to understand staphylococcal pathogenesis the focus has been on genetics, as

a bacterium can only do what its genes allow. The publication of the first

staphylococcal whole genome sequence in 2001 paved the way for a greater
 14

understanding of the molecular basis of its virulence evolution and

epidemiology and drug resistance.

As stated by Spiliopoulou (2014), staphylococcus aureus and especially

methicillin-resistant strain cause out break worldwide in the community, in the

hospital setting, in livestock and among companion animals, as well as human

in close contact, Virulence determinants contributing to infection are widely

spread among S. aureus strain, also associated with certain clonal lineages.

Epidemiological studies based on the molecular characteristic of the strain

causing outbreaks have revealed the wide dissemination of successful clones,

while other have identified certain clones in more districted area. In order to

inhibit hospital outbreak among high-risk patiencts, it is important to identify

carriers and to implement guidelines for limitation or elimination of methicillin-

resistant S. aureus. Since the discovery of S. aureus by Sir. Alexander Agston

in 1880, it remains one of the major human pathogens causing a variety of

infection, ranging from superficial to invasive, with a severe impact in patients

and healthcare system worldwide.

The cite literature explains the virulence of the staphylococcus. It is

important to know and what are the carries of these bacteria to immediately

stop it to spread more.

Staphylococcus aureus is a gram positive bacterium found in a grape-

like cluster. It is belong to the family Staphylococcacae which affect human and

every known mammalian species. Since S. aureus affects a wide range of

species, it can freely be transmitted from a species to another (Mandal, 2012).

Forms of transmission are through direct contact with objects infected by this
 15

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

approximately 30% humans that carry Staphylococcus aureus within their

nose, throat and skin. 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 membrane.

Staphylococcus aureus then hide their antigen to avoid immune response.

They kill infection-fighting called phagocytes and survive within these cells.

They also develop resistance to antibiotics. Skin infection caused by

Staphylococcus aureus is common mostly during pre-pubertal stage of

children. Most staph skin infection are treated with antibiotics; however there

are some kind of staph that are resistant to particular antibiotics which make

these diseases hard to cure. Methicillin-resistant 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).

The above mentioned literatures are related to the present study since

they define the main material used in the study.

These are active compound which form colloidal solution in water that

produces lather on shaking and precipitate cholesterol. It occurs as glycosides

whose aglycone is tripenoid or steroidal structure. The reason of detergent

characteristic or soap-like effect on membrane and skin is that the combination

of lipophilic sugar at the end gives them the ability to lower surface tension. It

has many physiochemical like foaming, emulsification, solubilization,

sweetness and bitterness. Also it has biological properties like haemolytic,

 16

antimicrobial, antioxidant, moluscacide, insecticide and ichthyocide. These are

exploited in many applications in food, cosmetics, pharmaceutical industries

and soil bioremediation. Saponins properties are greatly influenced by

variables such as temperature, salt concentration, aqueous phase, pH, solvent

concentration and type like ethanol or methanol (Kabera et al., 2014).

Moreover, these are pharmacologically active compounds that shown in

studies that it ameliorate abnormal physiological processes and be aptly

applied in folklore for the treatment of maladies caused by infectious agents

(Akaniro-Ejim et al., 2016).

On the other hand, there is no clear relationship between the plant origin

and the type of saponin, nor is there evidence that specific saponins are

associated with particular plants part (Vincken et al., 2007). But studies show

that saponins are stored in plants like vegetable, beans and herbs as a form of

chemical defense (Kathiruel 2016; Van Dyck et al., 2016). And yet saponins

are discovered also in marine organisms like sea cucumber Bohadschia

cousteaui. If found that it contains triterpene saponins. The triterpene saponins

are other soursce of saponin and these because a potential interest for the

body health and food industry (Elbandy et al., 2014). Triterpene saponins are

predominantly found in dicotyledons and steroidal. Saponins are found in

monocotyledons (Netala et al., 2014).

The above mentioned literatures are related to the present study since

they define the main material used in the study.

Related Studies
 17

Balbabosa (2013) Crude ethanol and aqueous extract of Cassia Alata-

based soap exhibited high antimicrobial potency against staphylococcus

aureus, the organism most widely encounter and undesirable of the human skin

flora. At a reduction time of 5 mins. The herbal soap made a 94.78% reduction

of the microbial load, findings of economic, industrial and medicinal

significance.

The Akapulko leaves contain chrysophanic acid, a fungicide that is used

to treat fungal infections, like ringworms, scabies and eczema.. Akapulko

leaves are also known to be sudorific, diuretic and purgative, used to treat

intestinal problems including intestinal parasites. Akapulko is also used as

herbal medicine to treat bronchitis and asthma. Because of Akapulko’s anti-

fungal properties, it is a common ingredient in soaps, shampoos, and lotions in

the Philippines. The Philippine Council for Health Research and Development

(PCHRD) has helped develop the technology for a Akapulko herbal medicine

lotion (Pinoy Health Guide, 2017).

The essential oils, tannins and other compounds in senna possess anti-

bacterial properties. These can inhibit the growth and proliferation of

microorganisms such as bacteria, fungi and parasites. Chewing senna leaf like

tobacco can cure mouth infections and gingivitis. It also possesses mild

inflammatory properties, which can soothe internal and external swelling (Saba

Naaz, 2015).

The above mentioned studies are related to the present study since they

define the main material used in the study.

 18

Bacillus subtilis is a bacterium found on skin, in digestive tract, in

epithelial wound, on extremities of the human body, in livestock and in soil (Earl

et al., 2008).

Bacillus subtilis is ubiquitous,it has developed adaptive strategist to

subsist in diverse environment via the production and secretion of a large

number of genetically encoded molecules that control the growth of neighboring

organisms (Liu et al., 2010).

Bacillus subtilis is sold commercially as a skincare product, a food

ingredient for human consumption, animal feed, fertilizer and an antibiotic

substitute. The molecular mechanism by which these products work are poorly

understood.

The above mentioned studies are related to the present study since they

define the main material used in the study.

Staphylococcus aureus responsible for acne, were found in anaerobic

cultures. Since the most frequent bacteria isolated from acne patients were

Staphylococcus aureus, it is possible that acne vulgaris is mainly caused by

Staphylococcus aureus (Dhillon KS et al., 2013).

Acne is a multifactorial disease of as yet incompletely elucidated etiology

and pathogenesis (Knor T. 2005).A microbial etiology of acne has been

suggested since the beginning of the last century( Shaheen, et al. 2011).

Elucidating the ambiguous determinants of this phenomenon is of major public

health interest. As a first step towards understanding the microbial etiology of

 19

acne, we have assessed S. aureus colonization and antibiotic resistance in

acne patients.

S. aureus was detected in 21.7% of the subjects in acne, and in 26.6%

of control groups. There was no statistical difference in colonization rates

between two groups (P=0.3). In patient group, most of S. aureus isolates were

resistant to doxicycline and tetracycline (P=0.001), and were more sensitive to

rifampicin compared to other drugs. In control samples, the isolated

demonstrated higher resistance to cotrimoxazole compared to patient samples

(P=0.0001). There was no difference between groups regarding resistance to

rifampicin, vancomycin, methicillin, and oxacillin. It is still unclear whether S.

aureus is actually a causal agent in the pathogenesis of acne. Based on

microbiological data of both healthy and acne-affected persons, we propose

that contribution of S. aureus in acne pathogenesis is controversial.(Farzin, et

al. 2012).

There is significant in vitro evidence suggesting a possible pathogenetic

role for S. aureus in acne vulgaris. This is in contrast to some studies which

implicated both S. epidermidis and P. acnes as bacteria-causing acne vulgaris

(Hassanzadeh P., et al. 2008). S. aureus is the most common nosocomial

pathogen (Mertz D., et al. 2009), with mortality rates ranging from 6% to 40 %

(Frank DN., et al 2010). It is a pathogen of more concern because of its ability

to cause a various array of life-threatening infections and its capacity to adapt

fast to the different environmental conditions (Onanuga A., et al. 2011). The

organism is normally present in the nasal vestibule of about 35% of apparently

healthy individuals.( Adesida SA., et al. 2007). One cross-sectional study of

 20

patients who were undergoing evaluation for acne, showed that 43%

participants were colonized with S. aureus (Fanelli M., et al. 2007).

The above mentioned studies are related to the present study since they

define the main material used in the study.

Chapter 3

RESEARCH METHODOLOGY

This chapter consists of the research design, subject of the study,

population and sample, data gathering procedures, list of materials, tools and

equipment, construction procedure, trials and revisions, construction time

frame, cost and production, research instrument, and statistical treatment used

in conducting the research

Research Design
 21

Burns and Grove (2010) define a research design as “a blueprint for

conducting a study with maximum control over factors that may interfere with

the validity of the findings”. Paraboo (2009) describe a research design as “a

plan that describe how, when, and where data are to be collected and

analyzed”.

The experimental method of research was used in this study to introduce

Acapulco (Cassia alata) leaf extract against Staphylococcus aureus and

Bacillus subtilis and to determine its antimicrobial activity.

Miranda (2008), the experimental method involves the manipulation of

one variable to determine if changes in one variable may cause changes in

another variable. The method relies on controlled methods random

assignment, and the manipulating of variable to test the hypothesis.

Subject of the Study

The main subject of the study is Acapulco leaves to make extract

against Staphylococcus aureus and Bacillus subtilis and find its antimicrobial

activity.

http://akosiandrewibot.blogspot.com/2014/06/akapulko.html
 22

Figure 2. Acapulco leaves

Population and Sampling techniques

The researcher used Staphylococcus aureus and Bacillus subtilis are

obtained from Forest Products Research and Development Institute, College

Laguna. The said bacteria’s were used to determine the antimicrobial effect of

Acapulco leaves.

Data Gathering Procedure

Before the proponent arrived at the stated subject of the study, the

researcher took many readings, researches, gathering of information and

thinks of what problem needed to be solved.

In preparing Acapulco leaf extract was carefully planned starting to the

preparation of needed tools, equipment and materials.

Materials, Tools, and Equipment

This experimental study was conducted with use of different supplies

and tools in making Acapulco leaf extract.

Table 1. List of Materials, Tools and Equipment

Materials Quantities Function

Acapulco Laeves 1 kg Used as raw materials in

producing Acapulco leaf
extract
Ethanol 300 grams Used in dilution of HCL
and washing the
Acapulco leaves.
Weighing scale 1 Used for measuring raw
material.
Scissor 1 Used for pounding the
dried Acapulco leaves.
 23

Bottles 1 Used as container of

finish product

Construction Procedure

Preparation of Plant Extraction

The researcher used the plant homogenization in ethanol as the method

for preparation of the plant extracts. Fresh or dried plant material can be used

as a source for the extraction. The basic principle is to cut the plant material to

finer particles, which increasing the surface area for extraction thereby

increasing the rate of extraction. The cut plant material were put in ethanol in a

volume double the weight of the cut leaves and left for five (5) days.

After the incubation time, the extract was filtered. The filtrate then dried

under rotary evaporator and redissolved in the ethanol determine and adjust

the concentration of the extract in uniform concentration. Concentrated extracts

were stored until use.

Preparation of Ethanol Extract

Planning

Gathering

Preparing

Washing
 24

Cutting

Extracting

Filtering

Bottling

Figure 3. Preparation of Ethanol Extract

Procedure in determining the presence of Saponin

The figure the procedure in determining the presence of saponin, an

amount of 0.5 ml ethanolic extracts of plant sample were diluted with 5 ml

distilled water in a test tube and vigorously shaken for 10 minutes. Formation

of foam indicates the presence of saponins.

0.5 ml ethanolic extract of plant sample

Diluted in 5ml distilled water

Shaken vigorously for 10 minutes

Formation of foam
 25

Figure 4. Procedure in determining the presence of Saponin

The figure the procedure in determining the presence of saponin, an

amount of 0.5 ml ethanolic extracts of plant sample were diluted with 5 ml

distilled water in a test tube and vigorously shaken for 10 minutes. Formation

of foam indicates the presence of saponins.

Determination of Zone of Inhibition (Well-Diffusion Assay)

The 40µl of broth subcultures of the test organisms were inoculated for

every 50ml LBA. The inoculum suspension were poured in the plates and

placed in a laminar flow hood of room temperature for extract against the two

(2) test organism.

The system was incubated for 24 hours at room temperature, under

aerobic condition.

Preparation of determining the zone of inhibition

The figure shows the preparation of determining the zone of inhibition.

Preparation of Planting/pouri
ng of the Drying of
the inoculum
inoculum plates
suspension
suspension

Impregnation Creating well

Incubation of test in the agar
samples using cork
borer
 26

Figure 5. Process of determining the zone of inhibition

Trials and Revisions

Table 2 below shows the trials and revisions done in production of

Acapulco leaves extract.

Table 2. Trials and Revisions

Trial Defect Revision
1 Insufficient amount of Cut more pieces of
extract from the leaves Acapulco leaves
of Acapulco.
2 Viscous extract of Addition of water will
Acapulco leaves and its add fluidity.
resin content were high.
3 Successful. The extract Done.
of Acapulco leaves, its
viscosity and resin
content are appropriate.
Construction Time Frame

Time frame was used to have a good result in preparing the Acapulco

leaves extract has shown in the table below.

Table 3. Construction Time Frame in Preparing Acapulco leaves extract

Procedure Time

2mins 4mins 8mins 10mins 20 30

mins mins

Preparation
 27

Washing

Drying

Cutting

Soaking

Filtering

Cost and Production

Cost analysis was done as in the table below.

Table 4. Cost and Production in Acapulco leaves extract

Quantity Unit Description Total Cost

1 Kilo Acapulco leaf 0

300 grams ethanol 700

total 700

Table 4 shows above the cost production which included the quantity,

unit, description, and total cost of Acapulco leaves extract.

Research Instrument

In order for the researcher to gather and fulfill the information in the study

the researcher would conduct experiment to test the antimicrobial effect of

Acapulco leaf extract in Staphylococcus aureus and Bacillus subtilis.

Statistical Treatment

The mean was used to know the average Measurement of the zone of

inhibition of each Staphylococcus aureus and Bacillus subtilis.

 28

T-test was used to know the significant effect of Acapulco leaf extract

against Staphylococcus aureus and Bacillus subtilis.

Chapter 4

PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA

In this chapter the result of the study from the experiment are presented,

analyzed the relations between each data collected and are interpreted to fully

understand the research.

Process of Acapulco Ethanol Extraction

 29

The flow chart below illustrates the process of producing extracted

Acapulco leaves.

Drying of Cutting plant Measure the

plant sample sample to finer dried plant
particles

Filtrate dried Filter the plant Put in Ethanol

under rotary extract and left for five
evaporator days

days

Redissolved Adjust the Refrigerate

in ethanol concentration until use

Figure 6. Process of Acapulco Leaves Extraction

Phytochemical Presence in Acapulco Leaf Extract

Table 5 shows the result of Phytochemical Analysis for the presence of

Saponin. Sample as received were tested following standard procedure of

Abbhi et. al (2011) for phytochemical analysis. Results were based on two

replicates. + sign indicates phytoconstituents is present.

Table 5. Phytochemical Present in the Acapulco Leaf Extract that Show

Antimicrobial Effect on Staphylococcus aureus and Bacillus subtilis
Phytoconstituent Acapulco Leaves Acapulco Leaves Acapulco Leaves
Ethanolic Extract 1 EthanolicExtract 2 Ethanolic Extract 3
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

Saponin + + + + + +
(Froth Test)
 30

The Mean Level of Antimicrobial Properties of Acapulco Leaf Extract

Against Staphylococcus aureus and Bacillus subtilis According to
Measurement of Zone of Inhibition

The table represents the mean level of antimicrobial properties of

Acapulco leaf extract against Staphylococcus aureus and Bacillus subtilis

according to measurement of zone of inhibition

Table 6. The Mean Level of Antimicrobial Properties of Acapulco Leaf Extract

Against Staphylococcus aureus and Bacillus subtilis according to
measurement of zone of inhibition
Trial Staphylococcus Bacillus subtilis 1679
aureus 1582
1 22.3 19.67

2 21.3 14.33

3 22.3 16.67

Mean 21.97 16.89

Legend:
Range
Remarks
(Zone of Inhibition)
ZOI ≥16mm. Susceptible
11mm. ≤ ZOI ≤15mm. Intermediately Susceptible
ZOI ≤ 10mm. Resistant

Table 6 shows the three trial of the indicator strain Staphylococcus

aureus 1582. The first trial has a mean of 22.3 and interpreted as

“Susceptible” for its level, the second trial has a mean of 21.3 and interpreted

as “Susceptible” for its level, the third trial has mean of 22.3 and interpreted

as “Susceptible” for its level, and have a final mean of 21.97 shows that the

mean level of antimicrobial property of Acapulco leaf extract against

Staphylococcus aureus is beyond the range of zone of inhibition. The second

indicator strain Bacillus subtilis 1679. The first trial has a mean of 19.67 and
 31

interpreted as “Susceptible” for its level, the second trial has a mean of 14.33

and interpreted as “Intermediately Susceptible” for its level and the third trials

has a mean of 16.67, interpreted as “Susceptible” for its level, and have a final

mean of 16.89 shows that the mean level of antimicrobial property of Acapulco

leaf extract against Bacillus subtilis is beyond the range of zone of inhibition.

Effect of Acapulco Leaf Extract to Staphylococcus aureus and Bacillus

subtilis According to Measurement of Zone of Inhibition

The table represents the effect of Acapulco leaf extract to

Staphylococcus aureus and Bacillus subtilis according to measurement of zone

of inhibition.

Table 7. Effect of Acapulco Leaf Extract to Staphylococcus aureus and Bacillus

subtilis According to Measurement of Zone of Inhibition
Indicator Strain Mean SD Verbal
Interpretation
Staphylococcus 21.97 0.47 Susceptible
aureus 1582
Bacillus subtilis 1679 16.89 2.18 Susceptible

Weighted Mean 19.43

Effective
Standard Deviation 2.99

Legend:

Range
Remarks Verbal Interpretation
(Zone of Inhibition)
ZOI ≥16mm. Susceptible Effective
11mm. ≤ ZOI ≤15mm. Intermediately Susceptible Moderately Effective
ZOI ≤ 10mm. Resistant Not Effective
Table 7 shows that the first indicator strains Staphylococcus aureus

1582 has a mean of 21.97 and standard deviation of 0.47, and interpreted as

“Susceptible” for its level. The second indicator strain Bacillus subtilis 1679

has a mean of 16.89 and standard deviation of 2.18, and interpreted as

“Susceptible” for its level.

 32

The weighted mean of 19.43 and standard deviation of 2.99 shows that

level of effectiveness of Acapulco leaf extract in inhibiting various strains is

“Effective”.

Test on the Difference between the Level of Effectiveness of Acapulco

Leaf Extract in Inhibiting Staphylococcus aureus and Bacillus subtilis

Table 8 represent the test on the difference between the level of

effectiveness of Acapulco leaf extract in inhibiting Staphylococcus aureus and

Bacillus subtilis.

Table 8. Test on the Difference between the Level of Effectiveness of Acapulco

Leaf Extract in Inhibiting Staphylococcus aureus and Bacillus subtilis
Indicator Extract Mean d.f Crit. Computed Remarks
Strain t- t-value
value
Staphylo- Ethanol
coccus aureus Extract of 21.96 Significant
1582 Acapulco 2 2.92 3.96
Bacillus subtilis Ethanol
1679 Extract of 16.89 Significant
Acapulco

Table 8 shows the computed t-value of 3.96 is beyond the critical t-value

of 2.92 with degree of freedom of 2 and interpreted as “Significant”.

Therefore, the null hypothesis stating “Acapulco leaf extract has no significant

effect on Staphylococcus aureus and Bacillus subtilis according to

measurement of zone of inhibition produce around the assay” was rejected.

 33

Chapter 5

SUMMARY, CONCLUSION AND RECOMMENDATION

This chapter presents the summary, findings, conclusion, and

recommendation.

Summary
 34

The purpose of this study was to determine the anti-microbial effect of

Acapulco against Staphylococcus aureus and Bacillus subtilis. It sought to

answer the following question; (1) What is the process of Acapulco leaves

extraction; (2)What phytochemicals are present in the Acapulco leaf extract that

shows the antimicrobial effects on Staphylococcus aureus and Bacillus subtilis;

(3)What is the mean level of antimicrobial properties of Acapulco leaf extract

against Staphylococcus aureus and Bacillus subtilis; (4)Does Acapulco leaf

extract have significant effect on Staphylococcus aureus and Bacillus subtilis

according to measurement of zone of inhibition produce around the assay.

Antimicrobial activity of the ethanol extract of Acapulco leaf extract was

conducted. Well diffusion assay method was used to assess the effect of the

extract on Staphylococcus aureus and Bacillus subtilis, the researcher used

mean as the statistical treatment to determine the average measurement of the

zone of inhibition produced around the assay.

The results reveal that the Acapulco ethanol extract can inhibit Bacillus

subtilis with approximately 16.89 zones of inhibition and Staphylococcus

aureus with approximately 21.97 zones of inhibition.

Findings

1. Phytochemical Present in the Acapulco Leaf Extract that Show

Antimicrobial Effect on Staphylococcus aureus and Bacillus subtilis

The result of the analysis to determine the presence of Saponin in

Acapulco leaf extract was + sign indicates phytoconstituents is present based

on the result of phytochemical analysis given by the Forest Product Research

and Development Institute.

 35

2. The Mean Level of Antimicrobial Properties of Acapulco Leaf Extract

Against Staphylococcus aureus and Bacillus subtilis According to

measurement of zone of inhibition

The mean level of antimicrobial properties of Acapulco leaf extract against

Staphylococcus aureus has a final mean of 21.97 and interpreted as

“Susceptible” according to the range of zone of inhibition. The second

indicator strain Bacillus subtilis has a final mean of 16.89 and interpreted as

“Susceptible” according to the range of zone of inhibition.

3. Effect of Acapulco Leaf Extract to Staphylococcus aureus and

Bacillus subtilis According to measurement of zone of inhibition

The effect of Acapulco leaf extract in inhibiting Staphylococcus aureus and

Bacillus subtilis according to its measurement of zone of inhibition has a

weighted mean of 19.43 and standard deviation of 2.99 it shows that level of

effectiveness of Acapulco leaf extract in inhibiting various strain is “Effective”.

4. Test on the Difference between the Level of Effectiveness of Acapulco

Leaf Extract in Inhibiting Staphylococcus auareus and

Bacillus subtilis

The test of difference between the level of effectiveness of Acapulco leaf

extract in inhibiting Staphylococcus aureus and Bacillus subtilis has a result of

3.96 for its computed t-value which is beyond to its critical t-value of 2.92 with

a degree of freedom of 2 and interpreted as “Significant”. Therefore, the null

hypothesis stating “Acapulco leaf extract has no significant effect on

Staphylococcus aureus and Bacillus subtilis according to measurement of zone

of inhibition produce around the assay” was rejected.

 36

Conclusion

Based on the findings of the study, the following conclusion was drawn:

1. The phytochemical present in Acapulco (Cassia alata) leaves is

Saponin.

2. The mean level of antimicrobial properties of Acapulco leaf extract in

inhibiting Staphylococcus aureus and Bacillus subtilis is remarked as

Susceptible.

3. There is a significant effect the Acapulco leaf extract on

Staphylococcus aureus and Bacillus subtilis according to

measurement of zone of inhibition produce around the assay.

Therefore, Acapulco (Cassia alata) leaf extract is effective as

antimicrobial against Staphylococcus aureus and Bacillus subtilis.

Recommendation

After completing the study which utilized experimentation and analyses,

the researcher recommends the following:

1. Conduct further studies on the different parts of Acapulco.

2. To use Acapulco extract in further experiments using different and more

virulent bacteria to be able to know the strength and capacity of

Acapulco to inhibit the growth of different types of bacteria.

3. Conduct additional screening of the presence of other phytochemical in

Acapulco.

4. Use Acapulco leaf extract in making a product that can cure skin

diseases.
 37

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C., et al. (2010). Molecular characteristics of community-acquired,
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Y. (2010). Staphylococcus epidermidis Esp inhibits Staphylococcus
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Harrison L. H., Lynfield R., Dumyati G., et al. (2007). Invasive methicillin-
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Knutsen-Larson S, Dawson AL, Dunnick CA, Dellavalle RP. Acne vulgaris:
Pathogenesis, treatment, and needs assessment. Dermatol Clin.
2012;30:99–106. [PubMed]
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Liu W. T., Yang Y. L., Xu Y., Lamsa A., Haste N. M., Yang J. Y., Ng J., Gonzalez
D., Ellermeier C. D., et al. (2010). Imaging mass spectrometry of
intraspecies metabolic exchange revealed the cannibalistic factors of
Bacillus subtilis. Proc Natl Acad Sci U S A 107, 16286–16290
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Staphylococcus aureus throat carriage: At-risk populations. Arch Intern
Med. 2009;169:172–8. [PubMed]
Onanuga A, Temedie TC. Multidrug-resistant intestinal Staphylococcus aureus
among self-medicated healthy adults in Amassoma, South-South,
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[PubMed]
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Staphylococcus aureus. Annu Rev Microbiol 64, 143–162
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with acne. Dermatol Res Pract. 2012;2012:571516. [PMC free article]
[PubMed]
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Br J Dermatol. 2011;165:474–85. [PubMed]
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Edition Benjamin-Cammngs Publishing Company, Sus OF Addison

Wesley Longman, Inc. 2008.
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Elbandy, Mohamed, Jung Rae Rho, and Raffatafifi. “Analysis of saponin as

bioactive zoochemicals from marine functional food sea cucumber
Bohadchia cousteaui.” European Food Research and Technology 238.6
(2014): 937-955

Kabera, Justin., et al. “Plants Secondary Metabolites: Biosynthesis,

Classification, Function and Pharmacological properties.” Journal of
Pharmacy and Pharmacology 2 (2014): 377-392.

Lindsay, Jodi A. (2018). The Gene for Toxic Shock: Toxin is carried by a family
of mobility, pathogenicity is lands in Staphylococcus aureus. Molecular
Microbiology 29 (2), 527-543
 40

Netala, V.R.; Ghosh, S.B.; Bobbu, P.; Anitha, D.; Tartte, V. Triterpenoid
Saponin: A review on biosynthesis, application and mechanism of their
action. Int. J. Pharm. Pharm. Sci: 204,7,24-28

Published Studies
Magno, Jovie ann, Urizza Ariane Mae, Veridiano Mark Austril, “Comparative
analysis on the antibacterial property and phytochemical constituent of
selected herbs, 2016 ”, Laguna State Polytechnic University Sta Cruz
Main Campus.

APPENDICES A
Request Letter

Republic of the Philippines

Laguna State Polytechnic University
Province of Laguna

April 13, 2018

Ms. Mariluz SP. Dionglay

Senior Science Research Specialist
CBS-MSD, DOST-FPRDI
Dear Ms. Dionglay,
Greetings of Peace and Prosperity!
I, Raquel P. Idian, a Bachelor of Secondary Education Major in
Biological Science of Laguna State Polytechnic University (LSPU) Sta. Cruz
 41

Main Campus, is currently conducting a research study entitled

“ANTIMICROBIAL EFFECTS OF ACAPULCO (Cassia alata Linn.) LEAF
EXTRACTS AGAINST STAPHYLOCOCCUS AUREUS AND BACILLUS
SUBTILIS” as a requirement for the completion of the course study of
Methods of Research.

I would like to request for the laboratory analysis on the following: a)

phytochemical constituent (Saponin), and b) the zone of inhibition.

I am hoping and patiently waiting for your participation. Thank you very
much!
Respectfully yours,
RAQUEL IDIAN
Researcher
Noted:

MARIA EVA E. DIONGCO, Ed. D.

CTE Research Unit Head

Approved/Disapproved:

Ms. Mariluz SP. Dionglay

Chief
Chemistry and Biotechnology Section
APPENDICES B
 42

Laboratory Report for Phytochemical Constituent of Sample Extract

APPENDICES C
 43

Laboratory Report for Inhibitory Property of Sample Extract against

Staphylococcus aureus

APPENDICES D
 44

Laboratory Report for Inhibitory Property of Sample Extract against

Bacillus subtilis

APPENDICES E
 45

Antimicrobial Assay for Staphylococcus aureus

Figure 7. Staphylococcus aureus trial 1

Figure 8. Staphylococcus aureus trial 2

Figure 9. Staphylococcus aureus trial 3

APPENDICES F
 46

Antimicrobial Assay for Bcillus subtilis

Figure 10. Bacillus subtilis trial 1

Figure 11. Bacillus subtilis trial 2

Figure 12. Bacillus subtilis trial 3

 47

APPENDICES G

Receipt for Ethanol

 48

Raquel P. Idian
Lot9 Blk9 Lynville East gate Phase 3
Camia St. Brgy. Calios, Sta. Cruz Laguna
Contact number: 09269222494
Email address: [email protected]

PERSONAL INFORMATION

Date of Birth : October 24, 1998

Place of Birth : Sta. Cruz Laguna
Age : 20 year’s old
Gender : Female
Height : 5’0
Weight : 40 kl’s
Religion : Aglipayan
Civil Status : Single
Father’s Name : Nicanor B. Idian
Father’s Occupation : Checker
Mother’s Name : Grace P. Idian
Mother’s Occupation : Housewife
Dialect can speak/write : Tagalog/English

EDUCATIONAL BACKGROUND

Tertiary : Laguna State Polytechnic University

Sta. Cruz Laguna
2015-2019
Bachelor of Secondary Education
Secondary : Pedro Guevara Memorial National H.S.
P. Guevara St. Sta. Cruz Laguna
2011-2015
Primary : Silangan Elementary School
Brgy. San Pablo Sur Sta. Cruz Laguna
2005-2011