Chapter 3 Example

University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS

General Luna St. Iloilo City, Philippines 5000
ANTIBACTERIAL ACTIVITY PROFILING OF MUNG BEANS (Vigna radiata)

EXTRACT AGAINST COMMON BACTERIAL PATHOGENS – Staphylococcus
aureus and Escherichia coli
A Research Proposal Presented to the Faculty of the

College of Health & Allied Medical Professions
In partial fulfillment of the degree of Bachelor of Science in Pharmacy
By:
CIASICO, Mark Anthony L.
DEBUQUE, Ramon Paulo B.
ABUNTO, April Joanne R.
BAACO, Dorie Joy A.
BLANCAVER, Jenice Alyson D.
CAMPOSAGRADO, Emy Faye B.
CARAGAYAN, Kathleen Jade T.
CATANGCATANG, Charmane Amor M.
August 2018
ENDORSEMENT FOR PROPOSAL EVALUATION
____________________
Date
Jesusima P. Monserate
Dear Mrs. Monserate,

This research entitled,
Antibacterial Activity Profiling of Mung Beans Extract Against Common Bacterial
Pathogens – Staphyloccus aureus and Escherichia coli
Prepared and submitted by:
Abunto, April Joanne Caragayan, Kathleen Jade
Baaco, Dorie Joy Catangcatang, Charmane Amor
Blancaver, Jenice Alyson CIasico, Mark
Camposagrado, Emy Faye Debuque, Ramon Paulo
Group: 1 Course/year/section: BS Pharmacy 4C
Is hereby endorsed for evaluation.
Below is the schedule of their research proposal defense:

Date:___________ Time:____________ Venue: _____________
Sincerely,
Melissa June V. Paderog

Research Adviser
TABLE OF CONTENTS
CHAPTER 1
Background and Rationale of the study 1
Objectives 3
Hypothesis 4
Significance of the Study 4
Scope and Limitation of the Study 4
Definition of Terms 5
CHAPTER 2
Review of Related Literature 9
CHAPTER 3
Research Design 16
Sampling Collection and Drying 17
Extraction 18
Freeze drying 18
Data Gathering 19
Solvent Partitioning 20
Agar Well Diffusion Assay

MIC Determination 21
Thin layer chromatography 22
Bioauthography 24
Wastes Disposal 25
Data Statistical Analysis 26
References 27
Appendices
Appendix A – Schematic Diagram 31
Appendix B – Gantt Chart 32
Appendix C – Budgetary requirement 34
Appendix D – Sample Tables/graphs for each Assay 35
Appendix E – Curriculum Vitae of Researchers 36

Chapter I
INTRODUCTION
Background and Rationale of the Study
(You should start this by talking about the antibacterial cases which may include
some statistics)
Bacteria are living things that have only one cell. Under a microscope, they
look like balls, rods, or spirals. Some bacteria help to digest food, destroy disease-
causing cells, and give the body needed vitamins. Bacteria are also used in making
healthy foods like yogurt and cheese. Recently, much attention has been focused
to unravel the medical properties of natural products in tandem, the need to search
for effective new antibacterial and invention of novel therapeutic agents that could
benefit mankind. (Concentrate more on the negative effects of bacteria and
bacterial infection)
For a long period of time, plants have been a valuable source of natural
products for maintaining human health, especially in the last decade, with more
intensive studies for natural therapies. The use of plant compounds for
pharmaceutical purposes has gradually increased in Brazil. According to World
Health Organization, medicinal plants would be the best source to obtain a variety
of drugs. About 80% of individuals from developed countries use traditional
medicine, which has compounds derived from medicinal plants. Therefore, such
plants should be investigated to better understand their properties, safety and

efficiency (Gislene G. F. Nascimento, et al; 2000). The antimicrobial properties of
plants have been investigated by a number of researchers world wide, especially

2
in Latin America. In Argentina, a research tested 122 known plant species used
for therapeutic treatments. (Gislene G. F. Nascimento, et al; 2000).
The mung bean (Vigna radiata) has been consumed as a common food in
China for more than 2,000 years. In the book Ben Cao Qiu Zhen (本草求真), the
mung bean was recorded to be beneficial in the regulation of gastrointestinal upset
and to moisturize the skin. The seeds of mung beans are also widely used as a
fresh salad vegetable or common food in India, Bangladesh, South East Asia, and
western countries. As a food, mung beans contain balanced nutrients, including
protein and dietary fiber, and significant amounts of bioactive phytochemicals. High
levels of proteins, amino acids, oligosaccharides, and polyphenols in mung beans
are thought to be the main contributors to the antimicrobial of this food and are
involved in the regulation of lipid metabolism. (Tang et al.; licensee Chemistry
Central Ltd. 2014).
Several studies were conducted where they investigated extracts of Vigna
radiata (Camalxaman et al 2013) reported the antibacterial potentials of the
extracts of Vigna radiata against Pseudomonas aeruginosa, Escherichia coli,
Klebsiella pneumoniae and Salmonella spp. Using agar disk diffusion method and
minimum inhibitory concentration (MIC) assessment. Both extracts showed
antimicrobial activities against all the tested gram negative bacteria with the
exception of K. pneumoniae which remained resistant. (Camalxaman, et al; 2013)

3
Objectives
To determine the antibacterial activity of Mung bean aqueous extract
against Staphylococcus aureus and Escherichia coli as a promising source of
antibacterial compounds.
Specifically, this study aims to:
1. Determine the antibacterial activity of Mung bean aqueous extract against
Staphylococcus aureus and Escherichia coli by measuring its zone of
inhibition through agar well diffusion assays.
2. Profile the extract’s antibacterial activity in terms of concentration by
determining its MIC90 value through micro broth dilution assay.
3. Determine the class of compounds responsible for the antibacterial activity
of the aqueous extract using bioassay-guided separation technique
through bioautography.
4. Initially profile the class of compounds responsible for the antibacterial
activity of the extract through color-reaction test and Fourier-transform
infrared spectroscopy analysis.
4
Hypothesis
There is no antibacterial property present in Munggo beans and the
phytochemical analysis conducted on extracts of Vigna radiata seeds.
Significance of the Study
The results of this study will be of great benefit to the following:
Student Researchers. This study will mold the skills and capabilities of the
researchers in their respective work places in pursuit of information and knowledge
concerning the discovery of new drugs. (Insert some Agustinian values related to
the pursuit of truth etc. because amo na ang ginapangita ni maam sima)
Community. This study will serve as a basis for the discovery and manufacture of
a new anti-bacterial drug that will be available for the community.
Farmers. This study will help increase the income in the farming industry due to
the increasing demand set by the manufacturers in the discovery of anti-bacterial
drugs.
Scope and Limitation of the Study
5
The Vigna radiata will be collected at Barotac, Viejo during the month of
November. Artificial drying via oven to dry the Vigna radiata and then pulverize
using a herbal grinder. It will be decocted and filtered using filter paper. Freeze
drying will then be performed to remove moisture from the Vigna radiata aqueous
extract. It will be followed by Preliminary Anti-bacterial screening, particularly Agar

well diffusion method. Solvent Partitioning will be used to separate the freezed
dried extract using Methanol as a polar solvent and Hexane as a non polar solvent
to classify and separate their constituents according to their polarity. The anti-
bacterial property of the subextract of each polarity using Agar well method. Thin
Layer Chromatography will be then be used to characterize the active subextract
that exhibited the antibacterial property. To further identify the components of the
subextracts, spray reagents such as polyethylene glycol reagent, iron (III) chloride
and dragendorff reagent will be used. Bioautography will then be performed to
confirm the bioactive bond that is responsible for the antibacterial activity. FTIR
analysis will be performed to identify the functional groups present in the bioactive
bond.
Definition of Terms
The following terms used in the study are defined according to conceptual
and operational definitions for the purpose of clarity.
Mungbean, Vigna radiata (L.)Wilczek. Has been grown in India since
ancient times. It is still widely grown in Southeast Asia, Africa, South America and
Australia. It was apparently grown in the United States as early as 1835 as the
Chickasaw pea. It is also referred to as green gram, golden gram and chop suey
bean. Mung beans are grown widely for use as a human food (as dry beans or
fresh sprouts), but can be used as a green manure crop and as forage for livestock.
(Oplinger et al., 1997)

6
In this study the Mung Bean (Vigna radiata) seeds will be used as the plant
material.
Escherichia coli. Is a type of bacteria that normally lives in your intestines.
It’s also found in the gut of some animals. (Webmd, 2018)
In this study the antimicrobial properties of Mung bean will be evaluated
against Escherichia coli.
Staphylococcus aureus. Is a major bacterial human pathogen that
causes a wide variety of clinical manifestations. Infections are common both in
community-acquired as well as hospital-acquired settings and treatment remains
challenging to manage due to the emergence of multi-drug resistant strains such
as MRSA (Methicillin-Resistant Staphylococcus aureus). (Taylor & Unakal, 2017)
In this study the antimicrobial properties of Mung bean will be evaluated
against Staphylococcus aureus.
Agar well diffusion method. Is widely used to evaluate the antimicrobial
activity of plants or microbial extracts. (Balouiri et al., 2015).
In this study, agar well diffusion method will be used to assess the
antimicrobial activity of Mung bean (Vigna radiata)
Zone of Inhibition. Also called a Kirby-Bauer Test, is a qualitative method
used clinically to measure antibiotic resistance and industrially to test the ability of
solids and textiles to inhibit microbial growth (Microchemlab, 2018).
With this method, zone diameters should be measured from the back of the
plate while it is resting on or held 2 to 3 inches above, a black, nonreflecting, flat
surface, illuminated by a reflected light source. (Barry, 1979)

7
Thin Layer Chromatography .Thin layer chromatography, or TLC, is a
method for analyzing mixtures by separating the compounds in the mixture. TLC
can be used to help determine the number of components in a mixture, the identity
of compounds, and the purity of a compound. By observing the appearance of a
product or the disappearance of a reactant, it can also be used to monitor the
progress of a reaction.
In this study the thin layer chromatography will be used in identification of
class of compounds that exhibit the antibacterial activity.
Bioautography .A bioassay based upon the ability of some compounds
(For example, vitamin B12) to enhance the growth of some organisms or
compounds and to repress the growth of others; used to assay certain antibiotics.
(Chemistry-dictionary, 2012)
In this study, Bioautography will be used to determine the class of
compounds that will exhibit the antibacterial activity of the Mung bean (Vigna
radiata).
Fourier-transform Infrared Spectroscopy.Is widely used in organic
synthesis, polymer science, petrochemical engineering, pharmaceutical industry
and food analysis. In addition, since FTIR spectrometers can be hyphenated to
chromatography, the mechanism of chemical reactions and the detection of
unstable substances can be investigated with such instruments. (Chemistry
LibreTexts, 2015)
In this study, FTIR spectrometer will be used to determine the antibacterial
activity of the Mung bean (Vigna radiata).

8
Minimum Inhibitory Concentration. Is the lowest concentration of a given
antibiotic that inhibits the growth of a specific organism. (Medical Dictionary, 2012)
In this study, Minimum Inhibitory Concentration of the extract of the Mung
bean (Vigna radiata) is being determined to discover its lowest concentration in

order to inhibit the growth of the organism. Micro Broth Dilution Assay will be used
to determine the Minimum Inhibitory Concentration.
Chapter II 9
Review of Related Literature
Escherichia coli
Escherichia coli is a rod‐shaped, Gram‐negative bacterium, and classified
as a member of the family Enterobacteriaceae. (Tenaillon et al. 2016). E. coli is
frequently used as a faecal indicator bacterium (FIB) for assessing water quality.
In addition, E. coli strains and serotypes can cause human diseases,
understanding the ecology of this bacterium is important to prevent infection and
spread of this pathogen to food, soil, and water.( Ishii and Sadowsky 2008)
Escherichia coli is initially believed to mainly inhabit the lower intestinal
tract of warm‐blooded animals, including humans, and be discharged to the
environment through faeces and waste water treatment plants (Berthe et al. 2013).
Escherichia coli is one of the predominant facultative aerobic bacterium in the
intestinal tract, even though the anaerobic bacteria outnumber E. coli by many
orders of magnitude (Berg 1996).
Staphylococcus aureus
Staphylococcus aureus -- a gram-positive coccus found singly, in pairs,
tetrads, and irregular grapelike clusters. It is non-motile, non-spore forming,
catalase positive, coagulase positive, and facultative anaerobe. It is both a
colonizer (e.g., nares (primary reservoir), pharynx, axilla, groin, and/or damaged
skin surfaces) and a disease-producing pathogen. (Catherine Liu, 2013)

10
Staphylococcus aureus is a major human pathogen that causes a wide range of
clinical infections. It is a leading cause of bacteremia and infective endocarditis as
well as osteoarticular, skin and soft tissue, pleuropulmonary, and device-related
infections. The past 2 decades have witnessed two clear shifts in the epidemiology
of S. aureus infections: first, a growing number of health care-associated

infections, particularly seen in infective endocarditis and prosthetic device
infections, and second, an epidemic of community-associated skin and soft tissue
infections driven by strains with certain virulence factors and resistance to β-lactam
antibiotics. (Tong, 2015)
Plant as a source of antibacterial agent (make a separate one for antibacterial
agent)
The use of and search for drugs and dietary supplements derived from
plants have accelerated in recent years. Ethnopharmacologists, botanists,
microbiologists, and natural-products chemists are combing the Earth for
phytochemicals and “leads” which could be developed for treatment of infectious
diseases. While 25 to 50% of current pharmaceuticals are derived from plants,
none are used as antimicrobials. Traditional healers have long used plants to
prevent or cure infectious conditions; Western medicine is trying to duplicate their
successes. Plants are rich in a wide variety of secondary metabolites, such as
tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to
have antimicrobial properties. (Kubo, 2009)
Finding healing powers in plants is an ancient idea. People on all continents
have long applied poultices and imbibed infusions of hundreds, if not thousands,
of indigenous plants, dating back to prehistory. There is evidence that
Neanderthals living 60,000 years ago in present-day Iraq used plants such as
hollyhock, these plants are still widely used in ethnomedicine around the world.
Historically, therapeutic results have been mixed; quite often cures or symptom
relief resulted. (Hutchinson , 1988.)

It is estimated that there are 250,000 to 500,000 species of plants on Earth
(Amoros et al., 1992). Hippocrates (in the late fifth century B.C.) mentioned 300 to
400 medicinal plants (Schultes, 1996). In the first century A.D., Dioscorides
wrote De Materia Medica, a medicinal plant catalogue which became the prototype
for modern pharmacopoeias. The Bible offers descriptions of approximately 30
healing plants. Relatively small percentages (1 to 10%) of these are used as foods
by both humans and other animal species. It is possible that even more are used
for medicinal purpose (Moerman, 2006).
Plants have an almost limitless ability to synthesize aromatic substances,
most of which are phenols or their oxygen-substituted derivatives (Geissman,
2003). Most are secondary metabolites, of which at least 12,000 have been
isolated, a number estimated to be less than 10% of the total (Schultes, 1997). In
many cases, these substances serve as plant defense mechanisms against
predation by microorganisms, insects, and herbivores. Some, such as terpenoids,
give plants their odors; others (quinones and tannins) are responsible for plant
pigment. Many compounds are responsible for plant flavor (e.g., the terpenoid
capsaicin from chili peppers), and some of the same herbs and spices used by
humans to season food yield useful medicinal compounds.
12
Figure 2.1 Major group of Antibacterial compounds from plants (if you
can add chemical structures the better)
Common Scientific name Compound Class Activity Relative Reference(s)
name toxicity
Aloe Aloe Latex Complex Corynebacterium, 2.7 Martinez,199
barbadensis, Alo mixture Salmonella, Strept
e vera ococcus, S. aureus
6
Apple Malus sylvestris Phloretin Flavonoid General 3.0 Hunter, 1993

derivative
Barberry Berberis vulgaris Berberine Alkaloid Bacteria, protozoa 2.0 Murakami,
1993
Basil Ocimum Essential oils Terpenoids Salmonella, 2.5 Wan, 1998
basilicum bacteria
Black pepper Piper nigrum Piperine Alkaloid Fungi, Lactobacill 1.0 Ghoshal et
us, Micrococcus,
E. coli, E. faecalis
al., 1993
Chamomile Matricaria Anthemic acid Phenolic M. tuberculosis, S. 2.3 Bose, 2008
chamomilla acid typhimurium, S.
aureus, helminths
Chili peppers, Capsicum Capsaicin Terpenoid Bacteria 2.0 Jones, 1996
paprika annuum
Garlic Allium sativum Allicin, ajoene Sulfoxide General Naganawa,
1996
(Japanese) Rabdosia Trichorabdal A Terpene Helicobacter Kadota, 1997
herb trichocarpa pylori
Legume (West Millettia Alpinumisoflav Flavone Schistosoma Perrett, 1995
Africa) thonningii one
Olive oil Olea europaea Hexanal Aldehyde General Kubo, 1995
Onion Allium cepa Allicin Sulfoxide Bacteria, Candida Vohora, 1973
Papaya Carica papaya Latex Mix of General 3.0 Satrijl, 1995
terpenoids,
organic
acids,
alkaloids
Purple prairie Petalostemum Petalostemumol Flavonol Bacteria, fungi Hufford,
clover
1997
Sainfoin Onobrychis Tannins Polyphenol Ruminal bacteria Jones, 1994
viciifolia s
Savory Satureja montana Carvacrol Terpenoid General 2.0 Ali-Shtayeh,
1997
Turmeric Curcuma longa Curcumin Terpenoids Bacteria, protozoa Apisariyakul
et al, 1999
13
Figure 2.2 Major classes of antimicrobial compounds from plants
Class Subclass Example(s) Mechanism Reference(s)
Phenolics Simple phenols Catechol Substrate deprivation Peres, 1997
Epicatechin Membrane disruption Toda, 2002
Phenolic acids Cinnamic acid Fernandez, 1996
Quinones Hypericin Bind to adhesins, King, 1994
complex with cell wall,
inactivate enzymes
Flavonoids Chrysin Bind to adhesins Perrett, 1995
Flavones Complex with cell wall
Abyssinone Inactivate enzymes Fujioka et al., 1997
Inhibit HIV reverse Harris, 2007
transcriptase
Tannins Ellagitannin Bind to proteins Hamburger, 1998
Bind to adhesins
Enzyme inhibition
Substrate deprivation
Complex with cell wall Hasegawa, 2000
Membrane disruption
Terpenoids, Capsaicin Membrane disruption Cichewicz, 1996
essential oils
Alkaloids Berberine Intercalate into cell wall Atta-ur-Rahman et al., 2009
and/or DNA
Piperine
Lectins and Mannose- Block viral fusion or Meyer, 1997
polypeptides specific adsorption
agglutinin
14
Nutritional and Medicinal Benefits of Vigna radiata
Vigna radiata are small green beans, that belong to the legume family. It is
one of the most popular dish here in the Philippines. They aren’t as popular in the
US but can be purchased from most health food stores as a great source of
different nutrients. One of the studies mentioned that the Mung beans are rich in
vitamins and minerals. One cup (7 ounces or 202 grams) of boiled mung beans
contains: Calories: 212, Fat: 0.8 grams, Protein: 14.2 grams, Carbs: 38.7 grams,
Fiber: 15.4 grams, Folate (B9): 80% of the Reference Daily Intake (RDI),
Manganese: 30% of the RDI, Magnesium: 24% of the RDI, Vitamin B1: 22% of the
RDI, Phosphorus: 20% of the RDI, Iron: 16% of the RDI, Copper: 16% of the RDI,
Potassium: 15% of the RDI, Zinc: 11% of the RDI,Vitamins B2, B3, B5, B6 and
selenium (Raman, 2018)
Mung bean (Vigna radiata) contains abundant nutrients with biological
activities. It is believed that Mung beans are a good source of antioxidants, which
may reduce your risk of chronic diseases, such as heart disease, diabetes and
certain cancers. However, more human-based research is needed before making
health recommendations. (Raman, 2018) It could also be a good source of
potassium, magnesium and fiber, which have been linked to lower blood pressure
levels in adults with and without high blood pressure. (Sonawane,2017)
Pharmacologic study displayed that high in folate, iron and protein, all of which
15
women need more of during pregnancy. Avoid raw mung bean sprouts to pregnant
woman, as they may contain harmful bacteria. (Hopkins&Stepp,2012)
Mung Bean Vigna radiata has also been proved to have high in fiber and
protein, which can help curb hunger by lowering levels of hunger hormones, such
as ghrelin, and raising fullness hormones, such as peptide YY, GLP-1 and
cholecystokinin. In fact, a review of nine studies found that people felt an average
31% fuller after eating legumes like beans than after eating other staple foods like
pasta and bread. (Swarup, et al, 2018) Animal studies have shown that mung
bean antioxidants may lower “bad” LDL cholesterol, while human studies have
linked higher legume consumption to lower LDL cholesterol levels. (Ruzainah, et
al, 2009) This species has been pronounced that Vigna radiata is not only a good
source of antibacterial activity but also could been an antioxidant and treatment of
cardiovascular disease.
Chapter 3 16
MATERIALS AND METHODS
3.1. Research Design
To obain the objectives of this study, the researchers will be collecting 4
kg of Munggo beans (Vigna radiata) around Barotac Viejo, Iloilo City. It will be
then be brought to the Department of Agriculture Region VI Iloilo City for the
plant authentication. The samples will undergo drying in an oven, will then be
pulverized by an herbal grinder and then will be decocted and filtered to obtain
the aqueous extract. The prepared extracts will then be freeze dried to obtain the
dried crude extract. The extract will then be screened for its antibacterial activity
using agar well diffusion assay to confirm its bioactivity. It will then undergo
solvent partitioning with hexane and methanol. After which, the antibacterial
acitivity of the subextract will be determined and the most active sub-extract will
be used for further investigation. Minimum inhibitory concentration determination
of the most active sub-extract will be performed using microbroth dilution assay
and then will be screened for the presence of antibacterial phytochemicals using
thin layer chromatography separation and identification techniques.
Bioautography will then be conducted to identify the band that exhibits
antibacterial activity. Prep TLC will then be performed to isolate the bioactive
band for FTIR analysis. For a clearer understanding on the design of this study, a
schematic diagram is presented below (Figure 1).

17
Collection of plant samples
Munggo beans (Vigna radiata)
Plant authentication
Processing of Samples (cleaning and oven drying)
Pulverization
Decoction for 30 minutes
Series of Filtration
Freeze-drying
Confirmatory test for Antibacterial Activity: Agar Well

Diffusion Method
Solvent partitioning (Hexane and methanol)
Identification of the Antibacterial

Subextract: Agar well diffusion
method
Antibacterial Profiling the most active subextract
Thin Layer Chromatography separation

Minimum inhibitory and identification of antibacterial
concentration determination phytochemicals
Bioautography
Figure 1. Schematic Diagram
Prep TLC
FTIR analysis
18
3.2 Sampling Collection and Drying
The researchers will randomly hand-pick the beans of Munggo around
Barotac Viejo, Iloilo City which will then be authenticated at the Department of
Agriculture-Region 6. The plant sample will then be cleaned and dried using an
oven at 44.5 oC for 4 hours. (Azwanida NN., 2015)
3.2.1. Plant Extraction via Decoction Method
The dried Mung beans sample will be pulverized by an herbal grinder and
5 grams of dried powder will be decocted with 100ml of water at 100 oC for about
30 minutes. Until about 50% of the water is lost. After decoction, the extract is will
be removed from heat and filtered by a series of filtration. (Arunkumar
Nagalingam, 2017).
3.3. Freeze-drying
The aqueous extract will be subjected to freezing at -20 oC for 24 hours
(Kunal et al., 2015) and will then undergo freeze-drying. The drying is done by
clearing the air with vacuum pump to lower down the pressure to about 10-4 atm
with the temperature of -45 to -20 oC (Kunal et al., 2015). The temperature of the
aqueous extract is raised to about 38 oC (Smith and Brian, 1994) by direct
conduction through the bottom trays radiation from the heat lamp. When the
chamber is already emptied by air, pressure will be below the threshold at which
water can simultaneously exist in a solid, liquid, and gaseous phase (vapour);
commonly known as triple point of water. Once the pressure reaches below this
point, the heat causes the ice crystals trapped in the frozen aqueous extract to
change directly into water vapour. Then vapour is drawn off and condensed,
19
leaving the freeze-dried aqueous extract behind. (World Journal of Pharmaceutical
Research, 2015)
3.4. Data Gathering Procedure
3.4.1. Confirmatory Antibacterial Screening (Agar Well Diffusion
Method)
In preparing the agar, streak plate method will be used and Mueller Hinton
agar will be used as the medium since it supports the growth of most non-fastidious
pathogens. The agar will be heated in a temperature of 40-50 oC and poured in a
petri dish/plate then allow to set on a level surface, to a depth of approximately
4mm. A 150mm plate requires approximately 25ml of medium. When the agar has
solidified, the plates will be incubated in an inverted position at 37 oC for 18-24
hours (Acharya, 2013).
Twenty-four hour cultures of the test organisms namely Staphylococcus
aureus and Escherichia coli will be prepared. A loop full of the culture will be
streaked uniformly over the surface of sterile Mueller Hinton Agar (MHA) for S.
aureus and E.coli. With pipette method, 2mg, 5mg, 10mg of freeze dried samples
of each various fractions will be prepared in pure Dimethyl sulfoxide (DMSO).
Tetracycline 10mg/ml will be used as the positive control for the assay. About 25ul
of the crude extracts from freeze-drying will be used to fill holes bored by 6mm
cork borer in the inoculated agar. The plates where made triplicate and incubated
at 37 oC for 18-24 hours and the evaluation for antibacterial activity of the extract
will confirmed by the zone of inhibition (Oseni Lateef, 2013).

20
3.4.2. Solvent Partitioning
The aqueous extract will undergo solvent partitioning with hexane and
methanol. The dried extract will be suspended in pure methanol and equal volume
of hexane will be added to separate the compounds. Using a separatory funnel,
the two immiscible liquids will be thoroughly shaken, then allow to stand for several
minutes until a distinct separation line between the solvents is observed. The
methanol fraction will be drained off unto a clean and properly labeled erlenmeyer
flask and the hexane fraction will be poured unto a separate flask.
The exhaustive solvent partitioning will be done until the appearance of the
hexane fraction becomes clear. The polar and non-polar fractions will undergo
rotary evaporation. The fractions will then be concentrated to about 5 mL volume
in a rotary evaporator and will be further dried using nitrogen gas to obtain dried
subextracts. The dried sub-extracts will be stored at 4°C in air tight containers. (H.
Usman, M.Sc., 2000)
3.4.3. Determination of the Most Active Antibacterial Subextract: Agar well
diffusion method
21
The same procedure with 3.4.1. will be done to identify which subextract will
exhibit the antibacterial activity and the bacteria which is most susceptible to that
said subextract. The dried subextracts will be reconstituted with DMSO to obtain a
concentration based on the confirmatory agar well which exhibited the greatest
antibacterial activity. A portion from the stock solution will be transferred onto their
corresponding wells to evaluate their antibacterial activity. The same positive
control will also be used in this assay( M. Aldawsari, 2014).

3.4.4. MIC Determination of the Most Active Subextract
The Minimum Inhibitory Concentration (MIC) of the most active subextract
against the most susceptible bacteria will be determined using broth microdilution
method using the 96-well microtiter plate. Exponential phase grown cultures in
agar plates of the test bacterial strains will be prepared by inoculating 2-3 colonies
onto a MHA plate and incubate it for 18-24 hrs at 37 0C. After which, a loop full of
colonies will be inoculated onto a 5 mL MHB medium and the bacterial density will
be determined using a microplate reader at 620 nm. The bacterial density of the
suspension will then be adjusted to 0.0005 OD620nm (1 x 106 cfu). The dried extract
will be prepared by reconstituting it in DMSO to obtain a concentration based on
the most active subextract.

22
10 µL (per well) of the stock solution will be delivered on the wells (first column)
of a 96-well plate. After which, 5 µL of DMSO will be delivered from column 2-9. A
volume of 5 µL of the extract will then be transferred from column 1 to 2 and will
be mixed thoroughly by sucking in and out carefully the mixture by means of a
micropipettor. Then 5 µL of the mixture in column 2 will then be transferred and
mixed onto the third column and the 2-fold serial dilution procedure will continue
until column 9. Then 5 µL of the mixture in column 9 will be discarded. In the wells
of column 10 and 11, 5 µL of DMSO (negative control) (Gbedema et al., 2010) and
5µL of 2µg/mL Tetracycline (positive control) (Cornelia Hasselman., 2003) will be
pipette respectively. After which, 195 µL of the bacterial suspension (OD620nm
0.0005) will be added and the mixture will be incubated at 37 0C for 18-24 hr. After
incubation, absorbance of the reaction mixture in each well will be determine using
a microplate reader and percent (%) inhibition will be calculated using the formula
(Baradaran et.al, 2013):
Absnegative - Absextract
% growth inhibition = -------------------------------------------------- x 100
Absnegative
High absorbance values suggest growth of the test organisms and low
absorbance values suggests antibacterial activity of the treatments (R. Wever
1980).
3.5. Identification of Antibacterial Phytochemicals using Thin Layer
Chromatographic Separation and Identification Techniques

23
Thin Layer Chromatography (TLC) using silica gel backed TLC plates will be
used to identify what antibacterial phytochemicals are present in the Mung bean
subextract. TLC plates will be prepared by cutting the plates into 1 inch (width) by
3 inch (height) dimensions. Then lines will be marked by a pencil 1 cm from the
bottom and top of the plate. Bottom line will serve as the marker for the origin spot
and top line will serve as the solvent front height. Then a stock solution of the most
active subextract will be prepared and 10uL will be spotted per plate using a
capillary tube. The chromatogram of the sample will be developed using
Butanol:Acetic acid:Water (60:15:25) as mobile phase. The TLC plate will be
placed inside the TLC jar and covered. Wait until the solvent reaches the solvent
front. The separated compounds will then be viewed under UV light at 254 nm and
365 nm. The distance traveled by each band will then be measured and retention
factor (Rf) values of each band will then be calculated using the following formula:
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑝𝑜𝑡
𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
To determine presence of antibacterial phytochemicals namely
flavonoids, phenolic compounds and amine-containing compounds, the following
spray reagents (Table 3.5.1) will be used onto the prepared TLC chromatogram of
the most active subextract.

Table 3.5.1
24
3.6. Bioautography
Reagents/s Components Compounds Visualization Results Reference
Technique
1.Dragendo -Bismuth For detection Under UV- Orange Elgerwi, 2013

rff reagent nitrate of unreactive 254 & 365 nm red/orange brown
amines lamp colour Ogbuanu et al.,
-Tartaric acid (2014)
-Potassium
iodide
2.Iron (III) -Ferric (III) For detection Under visible Blue or greenish http://www/cchem.be
chloride chloride 1g of phenols light, color rkeley.edu/rsgrp/TLC
StainGeneralReferec
-Methanol e.pdf
50mL
-Deionized
water 50mL
3. Natural Presence of Under UV- Yellow-green zone Bladt&Zgainski, 2014

products flavonoids 254 & 365 nm
and lamp
polyethylen
e glycol
reagent
(NP/PEG)
Two TLC chromatogram of the most active sub-extract will be prepared
using the same solvent system. One chromatogram will serve as reference plate
and the other chromatogram will be overlayed with a soft agar seeded with the test
bacterial strain to about 1mm thickness and then will be incubated at 37 oC for 18-
24 hours. After incubation period, the TLC plate which was overlayed with a soft
agar will be flooded with rezazurin dye (0.15mg/ml) and will be allowed to stand
for 1-2 hours inside a biosafety cabinet. A visible blue color indicates antibacterial
activity while pink color indicates no antibacterial activity (Choma et. Al,.2010).
3.6.1. Prep TLC procedure (If you are using tlc for bioautography,
shouldn’t the tlc preparation be done before explaining bioautograhy?)
An additional TLC chromatogram with a longer width will be prepared
based from the chromatogram used in bioautography. We will be applying the
same method that we used in the Table 3.5. To detect the active band which
possessed the antibacterial activity, the researchers will compare the retention
factor of the chromatogram from the reference plate in bioautography. The
researchers will then scrape off the desired band using a surgical blade and the
scraped material will be placed in an Eppendorf’s tube and will be suspended with
500 microliter of pure methanol. After which, the researchers will centrifuge the
sample at 8000rpm for 5 minutes. The supernatant liquid will be separated from
the pellets and will be purged with nitrogen gas to obtain the dried active band
which will be used for FTIR analysis.
3.7. Fourier-transform Infrared Spectroscopy

25
The dried sub-extract that was purged by nitrogen gas from the preparation
will be used then TLC procedure will then be subjected to FTIR analysis to identify
the functional groups present. Radiation emitted from the source will split into two
with a big splitter in the interferometer. The fix and moving mirrors will then be
reflect of each of the beam splitter, where the two beams recombine into one and
fall to the detector. Then, the two beams will combine constructively or
destructively varying as the optical path difference, when the moving mirror is
moved combined beam will be transmitted through the sample and will be detected
as an interferogram and contains all infrared information of the sample. The
infrared spectrum will be obtained from the interferogram of fourier. Alcohol,
aldehydes, alkyne, amines, and ester are the functional groups present. Alcohols
have the highest number of waves and Esters have the lowest number of waves
(Visvesshwari M.,2017).
3.8. Waste Disposal
We will dispose the waste materials that generated during the research
by separating Mung bean waste from the materials that will be used in the initial
screening of antibacterial activity. The Mung beans will be placed in the trash bin
where it is indicated biodegradable waste and seal it with a tape. While, in
disposing materials like dishes in the trash, the bacteria should be destroyed. We
will put or soak a small amount of household bleach over the colonies while
holding the dish over the sink to sterilize. We will use autoclaving to ensure the
sterilization of materials that have been used. Then, seal with a tape and label
biohazard waste. In disposing unused plain agar in plastic plates directly into the
trash can.
26
3.9. Data Statistical Analysis
Bar graphs with distinctive color and corresponding label will be used to
analyze the results. It will help us to detect and obtain the antibacterial property
of Mung beans (Vigna radiata) using water only as solvent. (Explain the whole
process on how you will do your data analysis. Do not just simply state the
method of analysis but be detailed. Also add the positive results. Example: We
will use bar graphs……which will have a positive result of ______ in the
presence of antibacterial activity)

27
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31
Appendix A
SCHEMATIC DIAGRAM
Collection of plant samples

Munggo beans (Vigna radiata)
Plant authentication
Processing of Samples (cleaning and oven drying)
Pulverization
Decoction for 30 minutes
Series of Filtration
Freeze-drying
Confirmatory test for Antibacterial Activity: Agar Well

Diffusion Method
Solvent partitioning (Hexane and methanol)
Identification of the Antibacterial

Subextract: Agar well diffusion
method
Antibacterial Profiling the most active subextract
Minimum inhibitory concentration Thin Layer Chromatography separation and

determination identification of antibacterial phytochemicals
Bioautography 32
Appendix B
Prep TLC
GANTT CHART
Figure 1. Schematic Diagram
FTIR analysis
Jun July Aug Sept Oct Nov Dec Jan Feb March
ACTIVITIES Week Week Week Week Week Week Week Week Week Week
3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 34 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Submission of Proposal Titles
Approval of Proposed Title
Preparation of Research
Proposal
Submission of Research
Proposal to Instructor &Panel
Members for Proposal
Defense
Proposal Defense Proper
Revision of Proposal
Manuscript
Submission of Proposal
Manuscript to Instructor
for Issuance of
Certificate of Completion
Conduct of Research
Preparation of Final 33
Manuscript
Submission of Final
Manuscript to
Instructor/Adviser &
Panel Members for Final
Defense
Final Defense Proper
Revision of Final Manuscript
Submission of
Bound Manuscripts &
Tarpaulin
34
Appendix C
BUDGETARY REQUIREMENTS
DETAILS/SERVICES AMOUNT
I. PERSONNEL SERVICES
A. Honoraria
1. Panel Members (Proposal and Final PhP 3,000.00
Defense)
SUBTOTAL PhP 3,000.00
II. MAINTENANCE AND OPERATING EXPENSES
A. Materials and Laboratory Supplies PhP 10,000.00
B. Office Supplies and Printing PhP 3,000.00

C. Transportation PhP 2,500.00
SUBTOTAL PhP 15, 500.00
III. CONTINGENCY Ph 5,000.00
GRAND TOTAL PhP 20,500.00
35
Appendix D
Sample Tables/graphs for each Assay
Table 1. Phytochemical analysis
Constituents Presence/ Absence
Flavonoids
Phenols
Amines
Key: + = Present; - = Absent

36
Appendix E
Curriculum Vitae of Researchers
Name: ABUNTO, April Joanne R.
Address: Poblacion Badiangan, Iloilo
Contact number: +639389414695
E-mail address: apriljoanneabunto@yahoo.com
Mother’s Name: Lerma R. Abunto
Father’s Name: Niel M. Abunto

Educational Background:
Primary: CenterPhil Montessori Learning Center
Secondary: CenterPhil Montessori Learning Center
Tertiary: University of San Agustin (present)
37
Name: BAACO, Dorie Joy A.
Address: Bgy. I Roxas Palawan
Contact number: 09509136444
E-mail address: spicydorie@gmail.com
Mother’s Name: Teodorica A. Baaco
Father’s Name: Rodrigo E. Baaco Jr.
Primary: Andres Soriano Memorial Elementary School
Secondary: Roxas National Comprehensive Highschool
38
Name: BLANCAVER, Jenice Alyson D.
Address: San Jose, Dingle Iloilo City
E-mail address: jeniceblancaver@gmail.com
Mother’s Name: Alice D. Blancaver
Father’s Name: Julius B. Blancaver Sr.
Primary: University of San Agustin
Secondary: University of San Agustin
39
Name: CAMPOSAGRADO, Emy Faye B.
Address: Brgy. Cagbang Oton, Iloilo
E-mail address: emycamposagrado6@gmail.com
Mother’s Name: Mary Jean Camposagrado
Father’s Name: Jose Emy Mart Camposagrado
Primary: SPED-ISEC
Secondary: Ateneo de Iloilo-SMCS

40
Name: CARAGAYAN, Kathleen Jade T.
Address: Calinog, Iloilo City
E-mail address: caragayan.k@yahoo.com
Mother’s Name: Imelda T. Caragayan
Father’s Name: Nerio L. Caragayan
Primary: Calinog Elementary School

Secondary: Calinog National Comprehensive High School
41
Name: CATANGCATANG,Charmane Amor M.
Address: Villa Cecilia Subd. Pototan, Iloilo
E-mail address: ccatangcatang@gmail.com
Mother’s Name: Elma M. Catangcatang
Father’s Name: Noe A. Catangcatang
Primary: Al Hekma International School

Secondary: CenterPhil Montessori Learning Center
42
Name: CIASICO, Mark Anthony L.
Address: Balleza Subdivision, Barotac Viejo, Iloilo
E-mail address: mark.ciasico@yahoo.com
Mother’s Name: Gerlie L. Ciasico
Father’s Name: Arnel A. Ciaisco
Primary: St. Paul School, Barotac Viejo, Iloilo

Secondary: St. Paul School, Barotac Viejo, Iloilo
Tertiary: University of San
Agustin (present)
43
Name: DEBUQUE, Ramon Paulo
Address: Gen. Luna St., Iloilo city
E-mail address: rpb_debuque@yahoo.com
Mother’s Name: Eileen Mae Debuque
Father’s Name: Roger Debuque
Primary: Wadeford School

Secondary: Sto. Nino Seminary
Tertiary: University of San Agustin

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University of San Agustin

COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS

ANTIBACTERIAL ACTIVITY PROFILING OF MUNG BEANS (Vigna radiata)

A Research Proposal Presented to the Faculty of the

Dear Mrs. Monserate,

Is hereby endorsed for evaluation.

Below is the schedule of their research proposal defense:

Melissa June V. Paderog

Background and Rationale of the study 1

Significance of the Study 4

Scope and Limitation of the Study 4

Review of Related Literature 9

Sampling Collection and Drying 17

Agar Well Diffusion Assay

Thin layer chromatography 22

Data Statistical Analysis 26

Appendix A – Schematic Diagram 31

Appendix B – Gantt Chart 32

Appendix C – Budgetary requirement 34

Appendix D – Sample Tables/graphs for each Assay 35

Appendix E – Curriculum Vitae of Researchers 36

Background and Rationale of the Study

benefit mankind. (Concentrate more on the negative effects of bacteria and

pharmaceutical purposes has gradually increased in Brazil. According to World

of drugs. About 80% of individuals from developed countries use traditional

plants should be investigated to better understand their properties, safety and

efficiency (Gislene G. F. Nascimento, et al; 2000). The antimicrobial properties of

plants have been investigated by a number of researchers world wide, especially

for therapeutic treatments. (Gislene G. F. Nascimento, et al; 2000).

mung bean was recorded to be beneficial in the regulation of gastrointestinal upset

western countries. As a food, mung beans contain balanced nutrients, including

levels of proteins, amino acids, oligosaccharides, and polyphenols in mung beans

involved in the regulation of lipid metabolism. (Tang et al.; licensee Chemistry

Central Ltd. 2014).

Several studies were conducted where they investigated extracts of Vigna

radiata (Camalxaman et al 2013) reported the antibacterial potentials of the

extracts of Vigna radiata against Pseudomonas aeruginosa, Escherichia coli,

minimum inhibitory concentration (MIC) assessment. Both extracts showed

exception of K. pneumoniae which remained resistant. (Camalxaman, et al; 2013)

To determine the antibacterial activity of Mung bean aqueous extract

against Staphylococcus aureus and Escherichia coli as a promising source of

Specifically, this study aims to:

1. Determine the antibacterial activity of Mung bean aqueous extract against

Staphylococcus aureus and Escherichia coli by measuring its zone of

inhibition through agar well diffusion assays.

2. Profile the extract’s antibacterial activity in terms of concentration by

determining its MIC90 value through micro broth dilution assay.

3. Determine the class of compounds responsible for the antibacterial activity

of the aqueous extract using bioassay-guided separation technique

4. Initially profile the class of compounds responsible for the antibacterial

activity of the extract through color-reaction test and Fourier-transform

infrared spectroscopy analysis.

There is no antibacterial property present in Munggo beans and the

phytochemical analysis conducted on extracts of Vigna radiata seeds.

Significance of the Study

The results of this study will be of great benefit to the following:

researchers in their respective work places in pursuit of information and knowledge

a new anti-bacterial drug that will be available for the community.

the increasing demand set by the manufacturers in the discovery of anti-bacterial

Scope and Limitation of the Study

extract. It will be followed by Preliminary Anti-bacterial screening, particularly Agar

Layer Chromatography will be then be used to characterize the active subextract

and dragendorff reagent will be used. Bioautography will then be performed to