Assessment of Dose-Dependent Anti Biofilm Property of Mollugo oppositifolia (Sarsalida)

against Staphylococcus aureus and Pseudomonas aeruginosa

Juan Paolo C. Capati1,2,3,4, Russelle Divine C. Caceres1,2,3,4, Patricia Louis T. Carlos1,2,3,4,

Rizafel Joy T. Dizon1,2,3,4, Sharmella Ann A. Pacheco1,2,3,4, Joanna Marie C. Torres1,2,3,4

1
College of Pharmacy
2
Our Lady of Fatima University
3
Research Development Innovation Center
4
Undergraduate Thesis

5
Research Adviser

MARCH 2018
ASSESSMENT OF DOSE- DEPENDENT ANTI BIOLFILM PROPERTY . . .

Endorsement

This thesis entitled: “Assessment of Dose-Dependent Antibiofilm Property of Mollugo

oppositifolia (Sarsalida) against Staphylococcus aureus and Pseudomonas aeruginosa”
prepared by Capati et al. of section 4Y2-2, in partial fulfillment of the requirements for the degree
Bachelor of Science in Pharmacy has been examined and now recommended for Oral
Examination.

This is to certify that Capati et. al. are ready for the Oral Examination.

Ms. April B. Lingat

Adviser

This is to certify that the thesis: “Assessment of Dose-Dependent of Antibiofilm Property of

Mollugo oppositifolia (Sarsalida) against Staphylococcus aureus and Pseudomonas
aeruginosa” prepared and submitted by Capati et al. of section 4Y1 -2, is recommended for Oral
Examination.

Name of Panel Chair

Chair

Name of Panel Member Name of Panel Member

Member Member

Dean Olive M. de Vera

Dean, College of Pharmacy

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Certificate of Originality

We hereby declare that this thesis is our own work and that, to the best of our knowledge and
belief, it contains no material previously published or written by another person nor material to
which to a substantial extent has been accepted for award of any other degree or diploma of a
university or other institute of higher learning, except where due acknowledgement is made in the
text.

We also declare that the intellectual content of this thesis is the product of our work, even though
we may have received assistance from others on style, presentation and language expression.

Name of Principal Researcher with Signature

Juan Paolo S. Capati

Members:
Russelle Divine C. Caceres
Patricia Louis T. Carlos
Rizafel Joy T. Dizon
Sharmella Ann A. Pacheco
Joanna Marie C. Torres

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Table of Contents

Title page
Endorsement
Certificate of Originality
Table of Contents
List of Appendices
List of Tables

1.0 Introduction
1.1 Background of the Study
1.2 Statement of the problem
1.3 Hypothesis
1.4 Assumption
1.5 Significance of the study
1.6 Scope and Limitation
2.0 Literature Reviews
2.1 Conceptual Framework
2.2 Theoretical Framework
2.3 Related Literature
2.3.1 Locale Literature
2.3.2 Foreign Literature
2.4 Related Studies
2.4.1 Locale Studies
2.4.2 Foreign Studies
3.0 Research Methods
3.1 Research Design
3.2 Research Locale
3.3 Population and Sampling
3.4 Research Instrument
3.5 Data Collection
3.6 Data Analysis

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4.0 Result
4.1 Organoleptic Evaluation Result
4.2 Phytochemical Result
4.3 Microbiological Result

5.0 Reference
6.0 Curriculum Vitae

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Appendix List of Appendices Pages

A Related Review (Foreign and Locale)

B Glossary of Terminologies

C Research Instruments

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Table List of Tables Pages

1 Phytochemical Test

2 Organoleptic Test

3 Confirmatory Test Result

4 Interpretation of FTIR

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1.0 Introduction

Microorganisms attach to surfaces and develop biofilms. Bacteria have primarily been
characterized as planktonic, meaning that bacteria are dependent on a solid surface for their
proliferation and therefore found in connection in the environment. They are freely suspended
cells and described on the basis of their growth characteristics in nutritionally rich culture media.
They are first discovered by van Leeuwenhoek (Collins, 1957). These are microorganisms that
attach to and grow exponentially on exposed surfaces that led to the interest of many researchers
to develop studies of revealed surface-associated microorganisms or famously called biofilms.
Bacterial cells embedded in a biofilm can better withstand environmental stress, such as nutrient
deprivation, unphysiological temperatures and pH changes as compared to non-biofilm formers of
microorganisms.

Biofilm formation is a process whereby microorganisms irreversibly attach to and grow

on a surface and produce extracellular polymers that facilitate attachment and matrix formation,
resulting in an alteration in the phenotype of the organisms with respect to growth rate and gene
transcription. The development of this study about biofilms is contributed mainly by the increase
in resistance of many biofilm-producing bacteria to several antimicrobial products that are
available in the market today and possesses a threat to public health. Biofilms have great
significance for public health, because biofilm-associated microorganisms exhibit dramatically
decreased susceptibility to antimicrobial agents.

Biofilms have been found to cause a wide variety of microbial infections in the body,
such as urinary tract infections, catheter infections, middle-ear infections, formation of dental
plaques, gingivitis, coating contact lenses. In the recent past, there has been an increased interest
in the therapeutic properties of some medicinal plants and natural compounds which have
demonstrated for their antibiofilm activities. Many plant extracts have been applied for the
treatment of inflammations, wounds, certain forms of cancer, infections due to bacteria, virus or
fungi and many more.

Clinical microbiologists have two reasons to be interested in the topic of antimicrobial

plant extracts. First, it is very likely that these phytochemicals will find their way into the arsenal
of antimicrobial drugs prescribed by physicians; several are already being tested in humans. It is

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reported that, on average, two or three antibiotics derived from microorganisms are launched each
year. After a downturn in that pace in recent decades, the pace is again quickening as scientists
realize that the effective life span of any antibiotic is limited. Worldwide spending on finding new
anti-infective agents is expected to increase 60% from the spending levels in 1993. New sources,
especially plant sources, are also being investigated. Second, the public is becoming increasingly
aware of problems with the overprescription and misuse of traditional antibiotics. In addition,
many people are interested in having more autonomy over their medical care. A multitude of
plant compounds is readily available over-the-counter from herbal suppliers and natural-food
stores, and self-medication with these substances is common (Cowan, 1999).

Application of phytochemical methods has led to the isolation of a wide range of natural
compounds from the various plant species. Researchers aiming on the isolation of tannins,
hydrolysable tannins, triterpenoid structures, organic acids, numerous flavonoids, coumarins,
polyprenols and other compounds of plant extracts.

Biofilm inhibition is considered as major drug target for the treatment of various bacterial
and fungal infections, and pharmacological development of these drugs is now extensively
studied. One promising alternative is the search for naturally occurring compounds of plant origin
capable of blocking biofilm formation. Historically, plant extracts and their biologically active
compounds have been a valuable source of natural products, which have played a central role in
the prevention and treatment of diseases, helping to maintain human health. Furthermore, they are
widely accepted due to the perception that they are safe and have a long history of use in folk
medicine to cure diseases and illnesses since ancient times.

Considering the advantages and benefits of using plant extracts as medicinal agents one
contribution is to explore the compounds present in Mollugo oppositifolia (Sarsalida) as an
antibiofilm agent. For the purpose of, that Mollugo oppositifolia (Sarsalida) may be a future
reference as an alternative medicine as to prevent biofilm formation in several clinical
manifestations

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1.1 Statement of the Problem

The research aims to answer the following questions:

1. What are the phytochemical constituents present in the leaves extract of Mollugo
oppositifolia (Sarsalida)?
2. What are the functional groups present in the leaves extract of Mollugo oppositifolia
(Sarsalida) that exhibits anti-biofilm property?
3. Which solvent yields most of the constituents of Mollugo oppositifolia?
4. At what dose will the leaves extract of Mollugo oppositifolia (Sarsalida) elicit antibiofilm
activity against biofilm-producing bacteria?
5. What method of biofilm assay can best assess biofilm inhibition?
6. Is Mollugo oppositifolia capable of inhibiting biofilm formation?
7. Is Mollugo oppositifolia capable of destroying extra polysaccharide surface (EPS)?

1.2 Hypothesis

The following hypothesis is formulated in the study:

There is an inhibition of biofilm formation of Mollugo oppositifolia (Sarsalida) extract againt

Staphylococcus aureus and Pseudomonas aeruginosa.

There is no inhibition of biofilm formation of Mollugo oppositifolia (Sarsalida) extract againt

Staphylococcus aureus and Pseudomonas aeruginosa.

1.3 Assumption

Discovery of alternative therapy against biofilm formation will be a breakthrough

in managing acute infections and will be a good alternative to conventional antibiotic
therapy as it is claimed to be safe and effective.

1.4 Significance of the Study

The significance of this study is to provide new alternatives to conventional way

of managing infections and provide innovations and advancement to science. This study
will help the following:

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Hospitals. This study will give information on how to manage biofilm formation
on certain medical devices that will lessen or make obsolete nosocomial infections caused
by biofilm-producing bacteria.

Patient. This study will help people suffering from acute infections to provide
cure to their recurrent infections.

Students. This study will help aspiring medical students provided advance
knowledge about bacteria and its resistance to antibiotics.

Manufacturers. This study will help several companies to provide quality

medications that involve antibiofilm therapy.

Researchers. This study will be provided as a future reference for the

advancement of studying plant sources as anti biofilm agents to reduce the risk of AMR.

1.5 Scope and Limitation

This study aims to determine the Dose- Dependent Antibiofilm Property of Mollugo
oppositifolia (Sarsalida) Methanolic Extract limited to Staphylococcus aureus and
Pseudomonas aeruginosa. This study will use the methods Microtiter Plate Biofilm Assay,
Air Liquid Interface Coverslip Assay, and Colony Biofilm Assay in assessing the biofilm
formation and inhibition of Staphylococcus aureus and Pseudomonas aeruginosa.

2.0 Literature Reviews

In nature, microorganisms exist primarily by attaching and growing upon living and
inanimate surfaces. These surfaces may take many forms those on the spectrum of indwelling
medical devices, and those of living tissues such as tooth enamel, heart valves, or the lung, and
middle ear (Donlan, 2015). The common feature of this attached growth state is that the cells
develop a biofilm. Biofilm formation is a process whereby microorganisms irreversibly attach to
and grow on a surface and produce extracellular polymers that facilitate attachment and matrix
formation, resulting in an alteration in the phenotype of the organisms with respect to growth rate.
The formation and persistence of surface-attached microbial communities, known as biofilms, are

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responsible for 75% of human microbial infections (National Institutes of Health). Biofilm
lifestyle confers several advantages to the pathogens, notably during the colonization process of
medical devices and/or patients’ organs.

The formation and persistence of surface-attached microbial communities, known as

biofilms, are responsible for 75% of human microbial infections (National Institutes of Health).
Biofilm lifestyle confers several advantages to the pathogens, notably during the colonization
process of medical devices and/or patients’ organs (Miguel, 2016). Biofilms are considered an
important virulence factor that causes persistent chronic and recurrent infections. Biofilm
inhibition is considered as major drug target for the treatment of various bacterial and fungal
infections, and pharmacological development of these drugs is now extensively studied (Sanchez,
2016).

Antimicrobial agents have been used for the treatment of different microbial infections on
human health. Biofilm producing bacteria exist in biofilm and withstand harsh environmental
conditions and antimicrobial agents. Most microbial infections caused by biofilm producing
bacteria are resistant to conventional therapy. Researchers have been prompted to identify
alternatives for the treatment of such infections. In the recent past, there has been an increased
interest in the therapeutic properties of some medicinal plants and natural compounds which have
demonstrated for their antibiofilm activities (Kodali, 2013)

Puzon et. al (2015) confirmed factors that are responsible for the development of
multiple drug resistance in human pathogenic microorganisms include the unscientific and
impractical uses of commercial antimicrobial agents, the specific nature of the relationship of
bacteria to antibiotics, host characteristics and environmental factors.

Mollugo oppositifolia locally called Sarsalida according to Royal Botanic Gardens

indicates is an upright, spreading or prostrate very nearly glabrous diffuse subsucculent herb. It
has branches that elongates from 5–80 cm. long. Leaves are opposite or apparently verticillate or
petiolate with 10–51 mm. long and 4–12.5 mm. broad, the blade narrowly elliptic. Mollugo
oppositifolia (Sarsalida) flowers are inconspicuous, greenish or pinkish white.

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2.1 Conceptual Framework

INPUT PROCESS OUTPUT

Biofilm
Mollugo Staphylococcus
aureus & Result
oppositifolia
Pseudomonas
aeruginosa

Positive
- Microtiter
Plates Biofilm - Inhibition of
Assay Biofilm
- Method of - Destruction of
extraction Biofilm
- Air-Liquid
Interface
- Dosage Negative
Formulation - No inhibition
Coverslip Assay
of Biofilm
- No Destruction
- Colony Count of Biofilm
biofilm Assay

2.2 Theoretical Framework

It has been established that Molligo oppsotifolia or locally called Sarsalida, Papait and
popularly recognized here in the Philippines as Malagoso has been established its antibacterial
property against several strains of bacteria mostly gram negative bacteria. (Puzon, 2015)

The leaf plant extract of Mollugo oppositifolia (Sarsalida) will undergo phytochemical
screening to isolate compounds that elicits antibacterial property and be tested for blocking
biofilm formation against biofilm-producing bacteria like Staphylococcus aureus and
Pseudomonas aeruginosa as these species of bacteria are capable of producing biofilm.

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(Costerton, 2000). The process of evaluating its antibiofilm property will be following the process
of Merrit et. al. on her study entitled “Growing and Analyzing Static Biofilms” following the
microtiter method of analysis.

2.3 Related Literatures

2.3.2 Foreign Literature

Bacterial Biofilms are common cause of persistent infections because of their adherence
to solid surfaces and formation of slimy, slippery coat. It was first discovered by the late
scientist, Anton van Leuwenhoek when he noticed and scraped a plaque on his teeth and observed
the “animalculi” that consists of microbial community. But the emergence of biofilm being
appreciated its existence was in 1970s when sessile bacteria, a major component of bacterial
biomass in many environments and in 1980s and 1990s that microbiologists appreciates attached
bacteria as organized and elaborate community. The efforts done by several experts have led to
the current definition of a bacterial biofilm as a structured community of bacterial cells enclosed
in a self-produced polymeric matrix and adherent to an inert or living surface.

Biofilms can survive a hostile environment. They constitute a protected mode of growth
that allows their persistence as a community. The structures that form in biofilms contain
channels on how they survive certain environments. Understanding biofilm formation and
structures can be of great help to initiate antimicrobial property.

The formation of biofilm is regulated by different genetic and environmental factors.

Genetic studies have shown that bacterial mobility, cell membrane proteins, extracellular
polysaccharides and signaling molecules play significant roles in biofilm formation. Basic
structural units of a biofilm are microcolonies, this are separate communities of bacterial cells
embedded into EPS matrix. These microcolonies are in most cases mushroom-shaped or rodlike
and they can consist of one or more types of bacteria. Depending on bacteria type, microcolonies
consist of 10–25% of cells and 79–90% of EPS matrix. EPS matrix protects biofilm cells from
various negative environmental conditions, such as UV radiation, abrupt changes in pH values
and draining.

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Biofilm is polymorphic and it can adjust its structure to changes in the amount of
nutrients, which was demonstrated by experiments with different glucose concentrations. When
glucose concentration is high, microcolonies grow fast and consequently biofilm thickness
increases significantly. When glucose concentration is decreased, biofilm biomass is reduced and
the former structure is restored.
Initial event in biofilm development is interaction between planktonic bacteria and
substrate surface. This phase is called reversible adsorption because some bacteria attach to the
substrate surface only for a brief period and then detach from it. This phase lasts a few minutes.
In the second phase, irreversible attachment, bacteria adhere firmly to substrate surface and lose
their mobility. Bacterial cells attach to each other and to the substrate surface and thus formation
of bacterial microcolonies begins. This phase lasts two hours.

Protein analysis of a first two phases in biofilm formation determined that there were
significant differences in regulation of the large number of proteins, which showed that there is
physiological difference between reversibly and irreversibly attached cells. Maturation I is the
third phase in biofilm formation. In this phase, a matrix of extracellular polysaccharide
substances (EPS) is produced. Microcolonies increase and become multi-layered, and their
thickness is up to 10 µm. This phase lasts three days. In the next phase, maturation II, bacterial
microcolonies grow to their maximum size and their thickness is about 100 µm. This phase lasts
six days. Studies of protein expression have shown a significant difference between maturation I
and maturation II phases. It is assumed that changes in protein structure are directly correlated to
phenotype adaptations of bacterial cells. Comparison of cells in maturation II phase and
planktonic cells has shown significant difference in protein structure, which proves that there is
great physiological difference between biofilm bacteria and planktonic bacteria. The last phase in
biofilm development is dispersion. In this phase, microcolony structure changes since the bacteria
cells situated in their central part regain their mobility and detach from the previously formed
structure. Microcolonies are therefore not mushroom-shaped or rodlike any longer, but adopt
shell-like structure having an inner empty cavity and the wall consisting of immobile bacteria.
The process dispersion probably takes place to allow bacterial cells better access to nutrients.
During this phase, water channels form between microcolonies. It lasts nine to twelve days.
Protein expression in the dispersion phase is similar to protein expression in planktonic cells,
which proves that some bacteria return into planktonic phenotype.

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The genetic transition occurs across the life cycle of the biofilm and is comprised of
seven distinct steps: Conditioning, wherein a clean surface is immediately covered with a
conditioning film of organic molecules and macromolecules. Transport of molecules and small
particles is rapid and as a result adsorption of conditioning rapid and as a result adsorption of
conditioning film occurs instantaneously. The presence of conditioning film alters the
characteristics of the substratum. Secondly is the Contact Phase where the bacteria in fluid
contact the substrate via mass transport mechanisms that is strongly influenced by mixing in the
bulk fluid and is related to flow regime and the bacteria penetrate the vicious sublayer via
diffusion. The third stage is the primary or early colonization. The biofilm process is mediated
through specific or non-specific physiochemical interactions with components of conditioning
film. The Growth step, wherein there is irreversible adsorption of cells that increases due to
replication and concentration dependent of rate-limiting nutrient important. Fifth stage is the
production of extra-cellular products wherein the affixed cells transition from planktonic form to
attached form. The processes controlled by gene encoding for the production of products.
Attachment or the secondary colonizing cells from bulk fluid attach to the existing biofilm fluid
attach to the existing biofilm that can result in species displacement of cells. The seventh and
final stage of biofilm process is the Re-entrainment where cells detach from the surface and
return to the bulk fluid and planktonic form of growth. Detachment can be an active or passive
process leading to further survival or colonization.

Biofilms have great significance for public health, because biofilm-associated

microorganisms exhibit dramatically decreased susceptibility to antimicrobial agents. This
susceptibility may be intrinsic as a natural outcome of growth in the biofilm or acquired due to
transfer of extrachromosomal elements to susceptible organisms in the biofilm. Modern-day acute
infections can often be treated effectively with antibiotics. However, biofilm develop
preferentiallyon inert surfaces or on dead tissue, and or occur more commonly on medical devices
and fragments of dead tissue. Biofilms grow slowly, in one or more locations, and biofilm
infections are often slow to produce overt symptoms that even individuals with excellent cellular
and humoral immune reactions biofilm infections rarely resolved by the host defense
mechanisms. Antibiotic therapy typically reverse the symptoms caused by planktonic cells
released from the biofilm, but fails to kill the bacteria. For this reason, biofilm infections typically
show recurring symptoms, after cycles of antibiotic property.

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Bacterial biofilms are inherently resistant to antimicrobial agents. One mechanism of

biofilm resistance to antimicrobial agents is the failure of an agent to penetrate the full depth of
the biofilm. Antibiotics have been shown to penetrate biofilms readily and in some cases and
poorly in others, depending on the particular agent and biofilm.

Second hypothesis to explain reduced biofilm susceptibility to antibiotics posits that at

least some of the cells in a biofilm experience nutrient limitation and therefore exist in a slow-
growing or starved state.

A third mechanism of reduced biofilm susceptibility is that at least some of the cells In a
biofilm adopt a distinct and protected biofilm phenotype. This phenotype is not a response to
nutrient limitation; it is biologically programmed response to growth on a surface.

Plant extracts have been applied for the treatment of inflammations, wounds, certain
forms of cancer, infections due to bacteria, virus or fungi and many more. Application of
phytochemical methods has led to the isolation of a wide range of natural compounds from the
various plant species. Researchers aiming on the isolation of tannins, hydrolysable tannins,
triterpenoid structures, organic acids, numerous flavonoids, coumarins, polyprenols and other
compounds of plant extracts. These compounds presumed to be safe and no toxic effects. In the
recent past, there has been an increased interest in the therapeutic properties of some medicinal
plants and natural compounds which have demonstrated for their antibiofilm activities. Synthetic
substances and antibiotics have been used in the control of biofilms. One promising alternative is
the search for naturally occurring compounds of plant origin capable of blocking biofilm
formation. Historically, plant extracts and their biologically active compounds have been a
valuable source of natural products, which have played a central role in the prevention and
treatment of diseases, helping to maintain human health. Furthermore, they are widely accepted
due to theperceptionthat they are safe and have a long history of use in folk medicine to cure
diseases and illnesses since ancient times.

Plants represent a huge resource of bioactive molecules. A recent study showed that some
of them contain anti-biofilm compounds that inhibit growth, interrupt quorum sensing (QS)
and/or prevent bacterial adhesion (Husain et al., 2015). Mollugo oppositifolia Linn. (Sarsalida)
with a synonym of Glinus oppositifolious belongs to the family of Mollugoginaceae. It is a
slender, spreading or ascending, branched, annual herb with branches 10 to 40centimeters.

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Common name in English is Bitter Leaf, Slender Carpet Weed and Bitter cumin. Leaves are
opposite or whorled, spatulate, oblanceolate to oblong-obovate. A common weed in and about
towns low and medium altitudes and it is also found in tropical countries. Exceptionally rich in
iron and a good source of calcium. Its properties considered a stomachic, appetizer, aperient,
uterine stimulant. Studies have shown antioxidant, immunomodulating, larvicidal, antifungal,
molluscicidal, antimicrobial, complete fixating, hepatoprotective, anti-inflammatory, antidiabetic,
anthelmintic properties. Phytochemical screening yielded alkaloids, saponins, and terpenoids in
excess; together with tannins and flavonoids. Antimicrobial study evaluated aqueous and
ethanolic extracts of leaves, flowers and stems for secondary metabolites and antimicrobial
activity. Antimicrobial activity was significant in the stem, leaf, and flower samples, the activity
attributed to saponins.

3.0 Research Methods

This section contains enough details on the scientific methods used to provide a result. That
this study undergone specific processes and that the tools utilized in the study are based on
scientificically proven providing validity and reliability.

3.1 Research Design

The researchers utilized an Experimental Design Method. This study utilized variables which
consist of the experimental positive and negative controls. The study focused on the assessment
of dose-dependent anti-biofilm property of Mollugo oppositifolia (Sarsalida) against
Staphylococcus aureus and Pseudomonas aeruginosa.

3.2 Research Locale

Phytochemical screening was conducted in the Biological Laboratory of Our Lady of Fatima
University. The extract of Mollugo oppositifolia or locally called Sarsalida was macerated and
extracted at the Physical Laboratory of the said university.

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3.3 Population and Sampling

The study utilized a convenience sampling of non-probability method due to the limited
availability of the plant sample 44.05 g of Mollugo oppositifolia (Sarsalida) for the Methanolic
extraction.

3.4 Research Instrument

3.4.1 Fourier Transport Infrared Spectroscopy (FTIR) Analysis

This instrument will serve to be the identifier of the functional groups present in Mollugo
oppositifolia (Sarsalida) that is capable of eliciting anti-biofilm property to biofilm-forming
organisms. FTIR relies on the fact that the most molecules absorb light in the infra-red region of
the electromagnetic spectrum. This absorption corresponds specifically to the bonds present in the
molecule. The frequency range is measured as wave numbers typically over the range 4000 – 600
cm-1. The background emission spectrum of the IR source is first recorded, followed by the
emission spectrum of the IR source with the sample in place. The ratio of the sample spectrum to
the background spectrum is directly related to the sample's absorption spectrum. The resultant
absorption spectrum from the bond natural vibration frequencies indicates the presence of various
chemical bonds and functional groups present in the sample.

3.4.2 Microtiter Plate Assay Method

This method uses microtiter plate to grow and develop biofilm of specific species of
bacteria and will be assayed using spectrophotometer of certain light wavelength that the samples
will absorb. It is a simple high-throughout method used to monitor microbial attachment to an
abiotic surface. Cells are grown in a microtiter dishes for a desired period of time, and then the
wells are washed to remove planktonic bacteria. Cells remaining adhered to the wells are
subsequently stained with a dye that allows visualization of the attachment pattern. This surface-
associated dye can also be solubilized for semi-quantitative assessment of the biofilm-forming
formed.

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3.4.3 Colony Biofilm Assay

This is the alternative method of analyzing biostatic biofilms wherein a colony biofilm is
grown on a semipermeable membrane that sits on a agar plate. The bacteria in this type of biofilm
can be given a new supply of nutrients simply by relocating the membrane- grown cells to a fresh
agar plate. This assay is more analogous to growing bacteria on plates rather than in growth. This
is useful especially useful for observing the effects of antimicrobial agents, as changes in cell
number can be more easily attributed to cell death rather than detachment.

3.5 Data Collection

The researchers collected the data from a standardized phytochemical screening method
with standard result outcomes. The functional groups saponins will be identified using a froth test
and total saponin test method.

3.6 Data Analysis

The data will be analyzed using a method derived from Biofilm Formation Inhibition by
Sanchez, et. al. (2016) that measuring zone of inhibition in the Agar-Plate Method and other
method of biofilm formation using the formula SBF= (AB-CW)/G whereas SBF is the specific
biofilm formation, AB is the OD570nm of the attached and stained bacteria, CW is the OD570nm
of the stained control wells containing only bacteria- free medium, and G is the OD360nm of cell
growth in broth.

3.6.1 Organoleptic Evaluation

Appearance: Gelatinous appearance

Color: Dark green

Odor: Pungent odor

Taste: N/A

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3.6.2 Phytochemical Screening

In determining the constituents present in Mollugo oppositifolia (Sarsalida), the researchers

conducted a phytochemical screening last August 4, 2017. Test for Alkaloids, Amino acids,
Carbohydrates, Fixed oils and Fats, Glycosides, Phenolic Compounds and Tannins, Phytosterols,
Proteins, Saponins, Gum and Mucilages and Volatile oils were determined.

4.1 Preliminary Qualitative Analysis

4.1.1 Test for Alkaloids

Mayer’s Test and Wagner’s Test were used to identify the presence of
Alkaloids. A few mL of plant extract and two drops of Mayer’s reagent will
show a white creamy precipitate. On the other hand, when two drops of
Wagner’s reagent is added to a few mL of plant extract, a reddish- brown
precipitate will confirm the presence of the said constituent.

4.1.2 Test for Amino acids

Before subjecting the extract for Amino acid test, the extract (100 mg)
must be dissolved first in a 10 mL distilled water then filtered through Whatmann
No.1 filter paper.

For Ninhydrin Test, two drops of ninhydrin solution is added to a 2 mL

of aqueous filtrate. A purple color indicates a positive result.

4.1.3 Test for Carbohydrates

To detect the presence of Carbohydrates, Molisch’s test and Benedict’s

test were applied.

To 2 mL of plant extract, two drops of alpha naphthol are added. Shake

the mixture well then add few drops of concentrated sulfuric acid slowly, on the
side of the test tube. A violet ring gives a positive result for Carbohydrates.

A 0.5 mL of Benedict’s reagent is added to a 0.5 mL of filtrate. For 2

minutes, boil the solution on a boiling water bath. A characteristic colored
precipitate indicates the presence of sugar.

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4.1.4 Test for Fixed Oils and Fats

Add drop wise of plant extract on a filter paper. Permanent stain on the
paper indicates the presence of fixed oils.

4.1.5 Test for Glycosides

For 2 hours, 50 mg of plant extract will be hydrolyzed with concentrated

Hydrochloric acid on a water bath and will be filtered. The collected filtrate will
undergo Borntrager’s test and Legal’s test.

In Borntrager’s test, 2 mL of the filtered hydrolysate and 3 mL of

chloroform is combined in a test tube. Shake. Separate the chloroform layer then
add 10% of ammonia solution. A pink color confirms the presence of glycosides.

In Legal’s test, dissolve 50 mg of extract in pyridine solution, then add

sodium nitroprusside and 10% NaOH to make it alkaline. A pink color also
indicates the presence of glycosides.

4.1.6 Test for Phenol compounds and Tannins

Ferric Chloride test, Gelatin test, Lead acetate test, Alkaline reagent test
and Magnesium and Hydrochloric acid reduction will be utilize to test the
presence of phenolic compounds and tannins.

For Ferric Chloride Test, dissolve 50 mg of extract in a 5 mL of distilled

water. Add few drops of neutral 5% ferric chloride solution. A dark green color
shows a positive result.

For Gelatin test, dissolve 50 mg of extract in a 5 mL of distilled water

and then add 2 mL of 1% Gelatin solution. The solution will give off a white
precipitate which indicates the presence of phenolic compounds.

For Lead acetate test, dissolve 50 mg of extract in a 5 mL of distilled

water. 3 mL of 10% Lead acetate is added. A bulky white precipitate will be the
positive result.

For Alkaline reagent test, the extract is treated with few drops of 10%
Ammonium hydroxide solution. A yellow fluorescence indicates the presence of
flavonoids.

For Magnesium and Hydrochloric acid reduction, dissolve 50 mg of

extract in a 5 mL of alcohol. Add small fragment of Magnesium turnings, then
drop wise, add concentrated Hydrochloric acid. A pink to crimson color solution
indicates the presence of flavonol glucosides.

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4.1.7 Test for Phytosterols

In detecting phytosterols, Liebermann- Burchard’s test is used. In a test

tube, dissolve 50 mg of extract in a 2 mL of acetic anhydride. Then add slowly,
one to two drops of concentrated sulfuric acid on the sides of the test tube. The
solution will show an array of color change, blur-violet-emerald green complex,
confirms the presence of phytosterols.

4.1.8 Test for Proteins

Before subjecting the extract for test for Proteins, dissolve it first in a 10
mL of distilled water. Then filter, using Whatmann No.1 filter paper.

In a test tube, add 2 mL of filtrate and few drops of Millon’s reagent. A

white precipitate will be the positive result.

In another test tube, treat the 2 mL of filtrate with 1 drop of 2% Copper

sulfate solution. Then add 1 mL of 95% Ethanol, followed by excess Potassium
hydroxide pellets. A pink color in the Ethanolic layer indicates the presence of
Proteins.

4.1.9 Test for Saponins

In a graduated cylinder, dilute 50 mg of extract with distilled water up to

20 mL. Shake the suspension for 15 minutes. Presence of two centimeter foamy
layer indicates the presence of Saponins.

4.1.10 Test for Gums and Mucilages

Dissolve the extract (100 mg) in a 10 mL of distilled water then add 2

mL of absolute alcohol with constant stirring. A white or cloudy precipitate
confirms the presence of Gums and Mucilages.

3.6.3 FTIR Analysis

For the sample preparation, dried powder with methanolic extract of the
Mollugooppositifolia (sarsalida) will be used for FTIR analysis. 10mg of dried extract powder
will be encapsulatedin 100mg of KBr pellet, in order to prepare translucent sample discs. The
powdered sample of each plant specimen was loaded in FTIR spectroscope with a scan range
from 400 to 4000 !" #$ with a resolution of 4 !" #$ . (Shimadzu, IR Affinity 1, Japan)

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Following to the sample preparation is to obtain a background of spectrum by collecting

an interferogram and its subsequent conversion to frequency data by inverse Fourier transform.

3.6.4 Microbiological Assay

3.6.4.1 Microtiter

Isolate Staphylococcus aureusand Pseudomonas aeroginosa in a 3-to-5ml culture and

grow to immovable phase. Dilute the cultures using ratio of 1:100 in the media. Pipet 100 mcl of
each diluted culture into each wells of the clean microtiter plate that has not been exposed/used to
any other culture. Cover the plate and incubate at the desired temperature and time.

The lids of the microtiter plate can be reused as long as the lid is cleaned with 70% (v/v)
of ethanol then air-dry it prior to each experiment.

The time course needed for the bacteria to adhere onto the wells of the plate depends on
the organism and must be determined based on observation, whereas many organism which are
commonly studied will be form a biofilm within 48 hours.

3.6.4.2 Colony Biofilm Assay

Prior to the colony biofilm assay, inoculate Staphylococcus aureus and Pseudomonas
aeruginosa in a 3-to-5 ml cultures and grow it all-night.

With the aid of sterilized forceps, place 25-mm black polycarbonate membrane with a
pore size of 0.22 mcm in a sterile Petri plate. Set the plate approximately 30 cm from a UV light
in a sterile environment for the 10 min per side, with the aid of sterilized forceps to turn over the
plate. The Petri plate must be kept covered at any time possible to keep the sterility of the
membranes. Two membranes are needed for the initial baseline count of cells(in duplicate), and
another four are required for each time point (for the duplicate sampling of treated and untreated
cells).

Dilute stationary-phase cultures in the desired medium to an OD600of 0.05 (approximately

1.64 x 108cfu/ml).

Place a membrane along its shiny side upon an agar medium plate without antibiotic, and
inoculate with 5 mcL diluted culture. Repeat this procedure for all the remaining membranes
from step 2, inoculating up to six membranes on the same agar plate. Just after the liquid on the
membranes has dried, incubate the plates in an upward position at the desired temperature for 24
hours.

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With the aid of sterile forceps, carefully lift every membrane off of its agar plate and
relocate to a fresh agar plate at up to 6 membranes per plate. Incubate the plates for 24 hours at
the desired temperature. Sterilized the forceps using flame and needed to be cooled before
touching the membrane. The whole surface of the membrane must be in direct contact with the
agar in the fresh plate. Air bubbles can be eliminated simply by carefully lifting the membrane
and repositioning it on plate. Membranes are relocated to new plates every 24 hours hereafter to
provide a fresh nutrient supply.

Aseptically transfer a membrane to each of two separate 15 ml tubes containing 10 ml

sterile PBS, such that each tube contains a single membrane. Cap tubes and vortex samples for
two pulses of 60 sec each (or as necessary)to dissociate all bacteria. Arrange the dilution series of
the vortexed samples, and plate each dilution on a different agar plate. Incubate plate all-night
long at the desired temperature and determine the average number of colony-forming units (CFU)
per membrane.

Relocate half of the remaining membrane (step 5) to fresh agar plates not containing
antibiotic, and relocate the other half to agar plates supplemented with antibiotic at the desired
concentration. Incubate all plates at desired temperature for 24 hours.

Just after the incubation is complete, sample biofilm growth ( in the step) in two
untreated membranes and two treated membranes. Relocate the remaining untreated membranes
to a fresh agar plate without antibiotic, and relocate the remaining treated membrane to fresh agar
plates enrich with antibiotic at the desired concentration. Incubate plates at desired temperature
for another 24 hr.

Repeat step 8 until all colony biofilms have been sampled.

4.0 Results

4.1 Organoleptic Evaluation Result

Appearance: Gelatinous appearance

Color: Dark green

Odor: Pungent odor

Taste: N/A

4.2 Phytochemical Result

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4.3 Microbiological Result

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