SIP Sample-2

PHYTOCHEMICAL ANALYSIS AND ANTIBACTERIAL PROPERTIES
OF MADRE DE AGUA (Trichanthera gigantea) ROOT AND STEM
CRUDE EXTRACTS AGAINST METHICILLIN-RESISTANT
Staphylococcus aureus AND Escherichia coli
Submitted as an entry for the Regional Science and Technology Fair 2023
Research Proponent:
Berlie Daniel M. Yap
Research Adviser:
Brecini Faith V. Tan
REGIONAL SCIENCE HIGH SCHOOL FOR REGION – IX
Malasiga, San Roque, Zamboanga City
October 2023
ANTIBACTERIAL PROPERTIES OF MDAS AND MDAR EXTRACTS
ABSTRACT
Antibacterial resistance and prevalence of multidrug resistant bacteria is a continuous global health
danger, as well as efforts to address this complicated issue. This study aimed to determine the
antibacterial properties of Madre de agua (Trichanthera gigantea) against Methicillin-resistant
Staphylococcus aureus (MRSA) and Escherichia coli and the combined effect of the Minimum
Inhibitory Concentrations (MICs) of T. gigantea root and stem crude extracts with ciprofloxacin
against MRSA. This would be beneficial for pharmacists, biologists, and researchers finding
components for prescriptions and/or antidotes for multidrug resistant bacteria. The T. gigantea
root and stem crude extracts were collected through dehydrating, blending, pulverizing, Soxhlet
crude fat extraction, and rotary evaporation. Set-ups A1 to A4 were the root group, set-ups B1 to
B4 were the stem group, and set-up C was the control group. The experimental groups had a
concentration of 1000 μL plant crude extract and normal saline solution. Set-up A1 and B1 (25%),
set-up A2 and B2 (50%), set-up A3 and B3 (75%), set-up A4 and B4 (100%). The crude extracts
were subjected to three different tests: First, qualitative phytochemical analysis; Ferric chloride
test for tannins, Lead acetate test for flavonoids, Mayer’s test for steroids, and Foam test for
saponins. Second, agar-well diffusion antibacterial assay utilizing Escherichia coli and
Methicillin-resistant Staphylococcus aureus. Third, modified Epsilometer tests utilizing the MICs
of the T. gigantea root and stem crude extracts and ciprofloxacin. Based on the results and findings,
tannins, saponins, and steroids were found in both root and stem crude extracts. All experimental
groups exhibited antibacterial properties against MRSA and are significantly higher than
ciprofloxacin as P-values were 0.0000353529 and 0.0011611 which are lesser than 0.05. However,
T gigantea was not effective against E. coli as the experimental set-ups did not exceed the cutoff
of the control group; there were also significant differences among the experimental set-ups of
against E. coli as succeeding P-values are 9.22×10-19 and 1.08×10-26 which are lesser than 0.05.,
although did not exhibit antibacterial properties against E. coli. Combination assay results revealed
antagonism in all groups as T. gigantea MIC 25% root crude extract and ciprofloxacin revealed
ZIs of 38.333 + 40.333 > 60.667, MIC 25% stem crude extract and ciprofloxacin revealed ZIs of
39.333 + 40.167 > 59.833. Therefore, T. gigantea root, stem, and ciprofloxacin can act as an
antibacterial agent alone for MRSA.
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Table of Contents
INTRODUCTION ........................................................................................................................ 1
METHODOLOGY ..................................................................................................................... 11
RESULTS .................................................................................................................................... 19
DISCUSSIONS ............................................................................................................................ 24
CONCLUSION ........................................................................................................................... 27
REFERENCES............................................................................................................................ 29
APPENDICES ............................................................................................................................. 35
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INTRODUCTION
The significance of antibiotic resistance is a continuous global health danger, as well as
efforts to address this complicated issue (Frieri, Kumar, and Boutin, 2017). Despite breakthroughs
in antibiotic therapy, infectious complications continue to be a significant cause of death and
morbidity in hospitalized patients. Although medical practitioners can treat these individuals with
second or third-choice medications, the use of these synthetic pharmaceuticals may put the patient
at greater risk due to the chance of the drugs having adverse side effects (Zembower, 2014).
The increasing rate of antibacterial resistance and multidrug-resistant bacteria poses
significant challenges in fighting bacterial infections. The lack of effective medications,
preventative measures, and limited new antibiotics require novel alternatives (Mühlen and Dersch,
2015).
According to the first World Health Organization (WHO) regional assessment on the health
and economic impact of antimicrobial resistance (AMR), an estimated 5.2 million people in the
Western Pacific Region will die because of drug-resistant bacterial infections between today and
the end of 2030 (Nery, 2023). Methicillin-resistant Staphylococcus aureus (MRSA) is a severe
danger to global public health. In the Philippines, there have been few research addressing the
issue at hand (Valle et al., 2016). MRSA prevalence in the Philippines have been over 50% since
2010 (Masim et al. 2021).
The wide use of Ciprofloxacin, a broad-spectrum antibiotic, in treating E. coli and MRSA
related bacterial diseases has resulted in a rise in resistance among these bacteria (Nwabuife,
Andeve, and Govender, 2022).
Antibiotic availability over the counter has been demonstrated to be reflected in prevalent
pre-admission antibiotic therapy in hospitalized patients. A common predicament in the
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Philippines is that antibiotics can be bought without a physician’s prescription, yet the options and
doses are frequently inaccurate (Sombrero et al., 2008), hence, in 2016 the Philippines adopted
RA 10918, commonly referred to as the Pharmacy Law mandating a prescription for the
administration of antibiotics. Some municipal governments have implemented laws on the
Rational Use of Medicines to ban the selling of antibiotics outside of pharmacies. This policy is
being applied and monitored at the present time. The Department of Health (DOH), Research
Institute for Tropical Medicine (RITM), and experts created the Antimicrobial Stewardship
Program for hospitals, which encourages the formation of teams of service providers, pharmacists,
and microbiologists based on their specialized roles. This program has been implemented in all
the country's Level 2 and Level 3 hospitals (ICAMR, et al., 2023).
It is imperative that therapeutic antimicrobial research focuses more on the suppression of
medically relevant bacteria such as community-acquired and hospital-acquired Methicillin-
resistant Staphylococcus aureus (MRSA) (Lai et al., 2014). Antibacterial properties of medicinal
plants have been progressively documented from various regions of the world in recent years.
Antibiotic resistance requires controlling antibiotic use, understanding resistance genetics, and
developing new antibiotics and therapeutic strategies. Advances in natural antimicrobial sources
and antibiotic chemical diversity provide chemical leads for new drugs. In developing nations, it
is critical to create effective but less priced antibiotics for all patients, regardless of financial
situation, in order to reduce some of the human variables that can produce multidrug resistant
bacteria (Valle et al., 2015).
In the agricultural industry, the use of antibacterial agents as growth promoters, as well as
preventative and therapeutic treatments, has been linked to the emergence of methicillin-resistant
S. aureus (MRSA) in livestock (Badua et al., 2020).
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Trichanthera gigantea has been used medicinally for both humans and domesticated
animals (Daniel, 2015). According to Kien et al., Trichanthera gigantea is primarily used as a feed
ingredient in poultry and rabbit diets to improve farm animal products such as meat and eggs.
Stems and branches of green food plants such as Trichanthera gigantea are frequently removed
while creating leaf meal, and only fresh leaves are utilized (2020). Results of Quiceno’s study
revealed that Trichanthera gigantea contains several phytochemicals of pharmaceutical
significance. In terms of biological activity, it was discovered that the plant possesses inhibitory
action, implying that Trichanthera gigantea has pharmacologically significant qualities (2022).
The researcher hypothesized the antibacterial potential of Trichanthera gigantea roots and stems
since the leaves of the same species are proven to exhibit antibacterial properties.
There have been no published research studies online on the antibacterial properties of
Madre de agua (Trichanthera gigeantea) roots and stems. Lack of scientific information has
frequently been cited as a key impediment to considering the use of traditional herbal treatments
in combination with or as a cost-effective alternative to standard medical therapy.
This study aimed to determine the antibacterial properties of Madre de agua (Trichanthera
gigantea) root and stem crude extracts after a phytochemical analysis and determine the Minimum
Inhibitory Concentration (MIC). It also aimed to determine the combined effects of the MICs of
Madre de agua (Trichanthera gigantea) root and stem crude extracts with ciprofloxacin against
Methicillin-resistant Staphylococcus aureus.
Specifically, the researcher aimed to answer the following questions:
1. What phytochemicals are present in Madre de agua (Trichanthera gigantea) root and stem
crude extracts?
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2. Is there a significant difference in the antibacterial properties through the diameters of
zones of inhibition after treating the following bacteria: gram positive bacteria: MRSA;
gram negative bacteria: E. Coli in the following set-ups?
A. Set-up A1: 25% v/v concentration of Madre de agua (Trichanthera gigantea) root crude
extract and Normal Saline Solution
B. Set-up A2: 50% v/v concentration of Madre de agua (Trichanthera gigantea) root crude
C. Set-up A3: 75% v/v concentration of Madre de agua (Trichanthera gigantea) root crude
D. Set-up A4 100% v/v concentration of Madre de agua (Trichanthera gigantea) root
crude extract and Normal Saline Solution
E. Set-up B1: 25% v/v concentration of Madre de agua (Trichanthera gigantea) stem
F. Set-up B2 50% v/v concentration of Madre de agua (Trichanthera gigantea) stem crude
G. Set-up B3 75% v/v concentration of Madre de agua (Trichanthera gigantea) stem crude
H. Set-up B4 100% v/v concentration of Madre de agua (Trichanthera gigantea) stem
I. Control: 5 μg of Ciprofloxacin
3. What is the Minimum Inhibitory Concentration (MIC) of Madre de agua (Trichanthera
gigantea) root and stem crude extracts in E. Coli and MRSA?
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4. Does Madre de agua (Trichanthera gigantea) root crude extract, stem crude extract and
ciprofloxacin have a synergistic, additive, or antagonistic effect when combined?
This study focused on examining both the phytochemical analysis and antibacterial
properties of Madre de agua (Trichanthera gigantea) root and stem crude extracts as well as the
combination assay of the said components along with ciprofloxacin. This would be beneficial for
pharmacists, biologists, and researchers finding components for prescriptions and/or antidotes for
multidrug resistant bacteria. This study would provide baseline results of Trichanthera gigantea
roots and stems as there are no published studies online that have examined the phytochemicals
and antibacterial properties of Trichanthera gigantea.
The study was limited to the qualitative phytochemical analysis and antibacterial properties
of Madre de agua (Trichanthera gigantea) root and stem crude extracts. Five phytochemicals were
screened through a qualitative phytochemical analysis, namely, tannins, flavonoids, alkaloids,
saponins, and steroids; nine set-ups were made and tested on E. Coli and MRSA to determine the
antibacterial properties. The Minimum Inhibitory Concentrations (MICs) of Madre de agua
(Trichanthera gigantea) were also determined through the set-ups of the antibacterial assay. The
combination assay of the MICs of Madre de agua (Trichanthera gigantea) root and stem crude
extracts with ciprofloxacin against MRSA were screened to determine a synergistic, additive, or
antagonistic effect. The results for the qualitative phytochemical analysis were obtained by testing
the set-ups at Zamboanga State College of Marine Science and Technology Chemistry Laboratory
whereas the data for the antibacterial methods were obtained at Zamboanga City Medical Center,
Department of Pathology, Microbiology section.
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REVIEW OF RELATED LITERATURE
Madre de Agua
Nguyễn et al. discovered significant chemical compounds in Madre de agua (Trichanthera
gigantea) leaves, including flavonoids, phenols, saponins, tannins, and essential oils. Trichanthera
gigantea leaves exhibited antibacterial properties on E. coli (2022). Furthermore, secondary
metabolites such as alkaloids, steroids, and triterpenoids were also present in the leaves of
Trichanthera gigantea. The plant extract revealed antibacterial properties on E. coli and S. aureus
strains (Quiceno, 2022). Another study conducted by Delgado et al. revealed moderate presence
of tannins in Trichanthera gigantea foliage (2012). According to Gutiérrez, Posada, and Romero,
Trichanthera gigantea reproduces vegetatively by cuttings. An adult tree of Trichanthera gigantea
has a lateral root system reaching up to a depth of 1.6 m and lateral length of 5.0 m. These lateral
roots have large numbers of secondary roots which extend to distances of 4-5 m (2013).
Roots
Roots are essential for plant growth since they are the plant component that accumulates
water and nutrients from the soil (Tajima, 2021). Flavonoids, phenols, alkaloids, tannins,
phytosterols, and saponins are among the most abundant phytochemicals found in plant roots
(Ardalani et al., 2021).
Tannins
Tannins have been extensively researched in the biomedical sector of science due to its
unique antibacterial capabilities confirmed by in vitro methods. Tannins have been shown to have
antibacterial activity against both Gram-positive and Gram-negative bacteria such as S. aureus,
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and E. coli. Tannin-rich plant extracts have powerful antibacterial properties. Tannins’
antibacterial activity is explained by their capacity to permeate through the bacterial cell wall up
to the internal membrane, causing interference with the cell's metabolism and, as a result, its
demise. Tannin activity is swift in Gram positive bacteria. However, because of the presence of a
bilayered membrane, it is slower in Gram-negative bacteria. Gram negative bacteria are more
dangerous and cause specific illnesses; hence, testing for this type of bacteria is extremely
important (Kaczmarek, 2020).
Flavonoids
Flavonoids, also known as polyphenolic chemicals, have been extensively investigated for
their antibacterial properties due to their ability to inhibit the growth of a wide range of pathogenic
microorganisms, including multidrug resistant bacteria (Shamsudin et al., 2022). Flavonoid
derivatives have been proven to have antibacterial properties in various research studies (Ayele,
Akele, and Melese, 2022). Some synthetic flavonoid derivatives also demonstrated noteworthy
antibacterial properties against multidrug-resistant Gram-negative and Gram-positive bacteria,
including E. coli and S. aureus (Farhadi et al., 2019).
The most crucial element of flavonoids is that they must maintain their amphiphilic
properties to penetrate bacteria and exercise their effective antibacterial effect. As a result,
considering these significant structural properties of antibacterial flavonoids while orchestrating
novel synthetic methods may play an important role in synthesizing improved antibacterial
medications to overcome the severe hurdles associated with resistant microorganisms (Shamsudin
et al., 2022).
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Alkaloids
Previous studies on active principles have revealed that the presence of compounds such
as alkaloids is extremely beneficial on both an ecological and medicinal scale. Alkaloids have been
reported to have antibacterial properties because they interact with the cell wall and weaken its
defensive systems (Quiceno, 2022).
Saponins
Saponins are detergent-like compounds with antibacterial properties (Arabski et al., 2012).
They are secondary metabolite found in a variety of plant parts, including stems and roots
(Moghimipour and Handali, 2015). The chemical structures of distinct saponins determine their
antibacterial activity (Dong et al., 2020). A study by Ajibade et al., revealed that pure isolated
saponins from Phyllanthus niruri showed anti-MRSA activity, concluding that saponins have the
potential to be further developed into an antibacterial agent (2019).
Steroids
Steroids are one of the phytochemical compounds that are known to be biologically active
and hence help in plant antibacterial activity (Nethathe and Ndip, 2011). Plant steroids are known
to have antibacterial properties (Tambe, Pedhekar, and Harshali, 2021). Nadaraia et al. revealed
that fourteen steroid compounds exhibited excellent antibacterial behavior, with MIC ranging from
0.003-0.96 mg/mL (2019).
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Gram Positive Bacteria
Gram-positive bacteria lack an outer membrane, although their Peptidoglycan layers are
several times thicker than those of Gram-negative bacteria. Long anionic polymers termed as
teichoic acids are embedded in the inner membrane and connected to the Peptidoglycan layers,
and they serve many functions in cell envelope physiology and pathogenesis (Rajagopal and
Walker, 2015). Gram-positive bacteria develop resistance by either enzymatically degrading
antibiotics through the production of β-lactamases or by decreasing the affinity and susceptibility
of their target site, the penicillin-binding protein (PBP), through either the acquisition of
exogenous DNA or changes in the native PBP genes (Jubeh, Breijyeh, and Karaman, 2020).
Methicillin-resistant Staphylococcus aureus
Methicillin resistance developed in S. Aureus because of a chromosomal mutation in a
penicillin-binding protein. The peptidoglycan matrix is required for survival and is heavily
functionalized with other glycopolymers in gram-positive organisms. In many gram-positive
species, wall teichoic acids are the most prevalent Peptidoglycan-linked glycopolymers. Wall
teichoic acids are essential for methicillin-resistant Staphylococcus aureus (MRSA) β-lactam
resistance, and they alter sensitivity to cationic antibiotics in different organisms (Brown, Santa
Maria, and Walker, 2013). Bacteriophages spread this form of resistance across S. Aureus
organisms (Lakhundi and Zhang, 2018). According to CDC, common infections caused by the
MRSA bacteria are skin infections and lung diseases such as pneumonia (2019).
Gram-negative Bacteria
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Gram-negative bacteria have three layers in their envelope. The first layer is the outer
membrane, which serves as a protective and distinctive characteristic between Gram-negative and
Gram-positive bacteria. The peptidoglycan cell wall is the second layer, while the inner membrane
is the third. Gram-negative bacteria's outer membrane is the primary cause of resistance to a wide
range of antibiotics, including β-lactams, quinilons, colistins, and others (Breijyeh, Jubeh, and
Karaman 2020).
Escherichia Coli
It is normally found in the human intestine but can also cause intestinal and extraintestinal
sickness (Mueller and Tainter, 2023).
Colibacillosis is an illness caused by the bacteria E. coli, which is generally found in the
lower intestines of most warm-blooded mammals. In contrast, recent research has shown that
animals are substantial repositories of plasmid-mediated colistin resistance genes, which are
primarily found in E. coli isolates and may provide an additional risk to humans (Poirel et al.,
2018).
Ciprofloxacin, norfloxacin, and sulfamethoxazole-trimethoprim were 100% effective
against E. coli isolates, whereas gentamicin, streptomycin, ceftazidime, nalidxic acid,
nitrofurantoin, kanamycin, amikacin, and chloramphenicol were 89-63.4% effective (Sarba et al.,
2019). Another recently conducted study by Jafari-Sales, Hossein-Nezhad, and Shahniani, E. Coli
strain isolates were revealed to have high susceptibility levels on ceftriaxone (66.6%) and
ciprofloxacin (36.7%) (2020).
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METHODOLOGY
Research Locale
The crude extracts of Madre de Agua (Trichanthera gigantea) roots and stems were
extracted at Zamboanga State College of Marine Science and Technology Chemistry Laboratory
as well as the phytochemical analysis. The testing transpired at Zamboanga City Medical Center
Laboratory, Department of Pathology, Microbiology section.
Research Design
The researcher utilized the After only with Control design, wherein the data was collected
after the MIC assay test for each strand of bacterium, as the seven set-ups were applied to
determine which concentration exhibited the most antibacterial property.
Figure 1. Experimental Layout for the antibacterial property in After only with control design
Experimental Groups Control Group
Set-up A1- Set-up B1-

A4 B4 E. Coli (β1) MRSA (β2)
E. Coli MRSA E. Coli MRSA

(β1) (β2) (β1) (β2)
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General Procedure
Collection of Madre de agua roots and stems
The Madre de agua (Trichanthera gigantea) roots and stems were collected at the City
Veterinarian, San Roque Road, San Jose Gusu, Zamboanga City. Both roots and stems were
thoroughly washed with water to remove all unwanted dirt. They were air-dried and stored in a
sterile air-tight container until further use.
Preparation of Madre de agua roots and stems coarse powder
The roots and stems of Trichantera gigantea were dehydrated in a food dehydrator at 40°C
until constant weight was obtained using an electronic balance. The dried materials were then
pulverized using a sterile laboratory mortar and pestle and then grinded using a blender. The
powder and debris were separated using a sterile laboratory sieve. The powder was transferred to
a sterile zip lock bag.
Soxhlet crude fat extraction of Madre de agua root and stem powder
The powder was transferred to a sterile filter paper and weighed using an electronic scale.
The Madre de agua (Trichanthera gigantea) root and stem powder were extracted separately for
16 hours by a Soxhlet extractor using 50% ethanol.
Crude extract preparation for Madre de agua roots and stems
The method of Valle et al. (2015) was used with minimal modifications. Specifically, the
resulting aqueous extracts were centrifuged in a rotary evaporator continuously for 78°C at 56
12
r/min for 5 hours, until the solvent had been completely evaporated. The crude extracts were
collected and dried at room temperature.
Phytochemical Analysis
Qualitative Phytochemical Analysis
a. Tannins
- In the determination of the presence of tannins in both Madre de agua (Trichanthera
gigantea) root and stem crude extracts, a few drops of 5%w/v FeCl3 solution was added
to 1-2mL of the crude extract. The presence of tannins was determined by the formation
of dark green or black color (Joshi, Bhobe, and Sattarkar 2013).
b. Flavonoids
- In the determination of the presence of flavonoids in both Madre de agua (Trichanthera
gigantea) root and stem crude extracts, the Lead acetate test was employed. 1 mL of
lead acetate solution was added to 2mL of the crude extract. The presence of flavonoids
was determined by the formation of yellow precipitate (Joshi, Bhobe, and Sattarkar
2013).
c. Alkaloids
- In the determination of the presence of alkaloids in both Madre de agua (Trichanthera
gigantea) root and stem crude extracts, Mayer’s test was employed. A few drops of
Mayer’s reagent were added to 2mL of the extract. The presence of alkaloids was
determined by the formation of white or pale yellow precipitate (Joshi, Bhobe, and
Sattarkar 2013).
d. Saponins
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- In the determination of the presence of saponins in both Madre de agua (Trichanthera
gigantea) root and stem crude extracts, the Foam test was employed. The crude extract
was mixed with distilled water and shaken thoroughly. The presence of saponins was
determined by the observation of persistent foam (Joshi, Bhobe, and Sattarkar 2013).
e. Steroids
- In the determination of the presence of steroids in both Madre de agua (Trichanthera
gigantea) root and stem crude extracts, 1 mL of the crude extract was dissolved in 10
mL of chloroform, and an equal volume of concentrated H2SO4 acid was added from
the test tube's side. The presence of steroids was determined by the observation of the
upper layer turning red and the H2SO4 layer turning yellow with green fluorescence or
by the formation of red rings (Joshi, Bhobe, and Sattarkar 2013).
Antibacterial experiment
Test microorganisms
This study's panel of test organisms for initial in vitro antibacterial screening comprises the
following: E. Coli and Methicillin-resistant S. Aureus. All bacterial strands were acquired at
Zamboanga City Medical Center, Department of Pathology, Microbiology section. Identification
of microorganisms were done through CRISPR-Cas9 American Type Culture Collection (ATCC).
Antibacterial assay of Madre de agua root and stem extracts
The procedure of Ayoade, Musbau, and Musa, 2007 was utilized with modifications.
Subcultured colony of MRSA and E. coli with the ATCC identification number were inoculated
with standardized innocula McFarland standard (1.5 x 108 cells/mL). At the middle of each
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Mueller-hinton agar plate, 5 mm diameter wells were bored, and 150 μL of the various
concentrations of the crude extracts were distributed into each well. At room temperature, the
extracts were allowed to diffuse into the medium for 1 hour. After incubating for 24 h at 37°C, the
zones of growth inhibition were measured using a vernier caliper and reported in millimeters. The
control was set up in the same way, but the extract was replaced with ciprofloxacin 5 μg disc.
Determination of minimum inhibitory concentration (MIC)
Each test was done in three trials. The minimum inhibitory concentration (MIC) of the
Madre de agua (Trichanthera gigantea) roots and stem were determined after the agar-well
diffusion antibacterial assay tests to which the lowest concentration resulting zones of inhibition
was the MIC.
Combination Assay
The combined interactions were evaluated by Epsilometer tests with modifications
(Lorian, 2005). Sterile Whatman filter paper strips were soaked for 1 hour in three different petri
dishes containing the MICs of root extract, stem extract, and ciprofloxacin. The soaked sterile
Whatman filter paper strips were then placed perpendicularly on the Mueller-hinton agar plates
swabbed with MRSA. The plates were incubated for 24 hours at 37°C. After incubation, the zones
of inhibition surrounding the intersections were measured using a vernier caliper.
The individual averages of zones of inhibition for Madre de agua (Trichanthera gigantea)
root and stem crude extracts and ciprofloxacin were measured then summated to compare to the
ZIs of the combined group.
Synergistic = A + B < C
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Additive = A + B = C
Antagonistic = A + B > C
Wherein;
A = Crude extract
B = Ciprofloxacin
C = Both
Statistical Analysis
Evaluation of the antibacterial activities was done by computing the average results of the
diameters of zones of inhibition of E. Coli, and MRSA. Utilization of one-way-ANOVA for
comparison of the zones of inhibitions in the experimental group – different concentrations of
Trichanthera gigantea roots and stem extracts – and the control group – ciprofloxacin – to
determine the significant difference among the set-ups. Furthermore, Tukey HSD (Honest
Significant Difference), a Post-hoc test, was done to determine which between the set-ups had the
significant difference.
Disposal treatment
The substances after the phytochemical analysis were transferred to sealed containers with
labels. Test tubes, pipettes, and beakers in the phytochemical analysis were thoroughly washed.
BSL-2 (Biosafety Level 2) precautions were taken for the disposal treatment of the antibacterial
analysis. The disposable test tubes and agar plates used for the antibacterial analysis were stored
inside a yellow bag and subjected to an autoclave prior to disposal to black bag trash can. Reusable
test tubes and glass plates were autoclaved and washed using tap water. A chemical indicator
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(autoclave tape) was used for the sterilization, once it turned black, it reached the desired PSI, and
thus, the materials were completely sterilized.
17
Figure 2. Flowchart of the methodology conducted in this study
Collection of Madre de agua (Trichanthera gigantea) roots and stems
Preparation of Madre de agua (Trichanthera gigantea) root and stem coarse powder
Soxhlet extraction of Madre de agua (Trichanthera gigantea) roots and stems
Crude extract preparation for Madre de agua (Trichanthera gigantea) roots and stems
Preliminary phytochemical analysis
Test microorganisms
Antibacterial assay of Madre de agua (Trichanthera gigantea) root and stem extracts
Determination of minimum inhibitory concentration (MIC) and Minimum bactericidal

concentration (MBC)
Combination Assay of crude extracts and ciprofloxacin against Methicillin-resistant

Staphylococcus aureus
Recording of generated data
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RESULTS
Table 1. Phytochemical Analysis Results for Root and stem Crude Extract
Phytochemicals Name of Test Stem crude extract Root crude extract

Tannins Ferric chloride test + +
Flavonoids Lead acetate test - -
Alkaloids Mayer’s test - -
Saponins Foam test + +
Steroids Salkowski’s test + +
Legend: + = presence of phytochemical - = absence of phytochemical
Table 1 shows the qualitative phytochemical analysis results for Madre de agua
(Trichanthera gigantea) root and stem crude extracts. Tannins were present in both extracts and
passed the ferric chloride test. Flavonoids exhibited a negative result during the lead acetate test.
Alkaloids showed the same the result on Mayer’s test. Saponins on the other hand, were present
in foam test. In Salkowski’s test, steroids were detected on stem crude extracts.
Figure 3. The mean data of the diameters of zones of inhibition (mm) of each set-up
containing different concentrations of Madre de agua root crude extract.
Average data for MDR Zones of Inhibition in MRSA

35
30
25
20
15
10
5
0
A1 A2 A3 A4 Control
Diameters of zones of inhibition in mm
Figure 3 shows the mean data of the zones of inhibition of each set-up containing different
concentrations of Madre de agua (Trichanthera gigantea) root crude extract against Methicillin-
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resistant Staphylococcus aureus. The average for set-up A1 is 30.667; for set-up A2, 31.667; for
set-up A3, 32.333; for set-up A4, 30.667; for the control group, 24.667.
Figure 4. The mean data of the diameters zones of inhibition of each set-up containing
different concentrations of Madre de agua stem crude extract
Average data for MDS Zones of Inhibition in MRSA

35
30
25
20
15
10
5
0
B1 B2 B3 B4 Control
concentrations of Madre de agua (Trichanthera gigantea) stem crude extract against Methicillin-
resistant Staphylococcus aureus. The average for set-up B1 is 31.667; for set-up B2, 30.333; for
set-up B3, 31.667; for set-up B4, 33.333; for the control group, 26.333.
Figure 5. The mean data of the diameters zones of inhibition of each set-up containing
different concentrations of Madre de agua root crude extract
Average data for MDR Zones of Inhibition in E. coli

35
30
25
20
15
10
5
0
A1 A2 A3 A4 Control
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concentrations of Madre de agua (Trichanthera gigantea) root crude extract against Escherichia
coli. set-up A1 showed no zones of inhibition; for set-up A2, 5.667 mm; for set-up A3, 7 mm; for
set-up A4, 8 mm; for the control group, 30.333 mm.
Figure 6. The mean data of the diameters of zones of inhibition of each set-up containing
different concentrations of Madre de agua stem crude extract
Average data for MDS Zones of Inhibition in E. coli

30
25
20
15
10
5
0
B1 B2 B3 B4 Control
concentrations of Madre de agua (Trichanthera gigantea) root crude extract against Escherichia
coli. All experimental groups revealed no zones of inhibition whereas the control group revealed
30.333 mm.
21
Figure 7. The mean data of combination assay of each set-up from the Madre de Agua roots
and ciprofloxacin group
Average Data for Combination assay in Madre de Agua

roots group
100
80
60
40
20
0
Sum of the Individual ZIs Combined ZI
Zones of inhibition in mm
Figure 7 shows the mean data of the combination assay containing the minimum inhibitory
concentration (MIC) of Madre de agua (Trichanthera gigantea) root crude extract which was the
25% concentration and ciprofloxacin. The sum of the individual ZIs is 78.667 and the combined
ZI is 60.667.
Figure 8. The mean data of combination assay of each set-up from the Madre de Agua stem
and ciprofloxacin group
Average Data for Combination assay in Madre de Agua

stem group
100
80
60
40
20
0
Sum of Individual Zis Combined ZI
Zones of inhibition in mm
Figure 8 shows the mean data of the combination assay containing the minimum inhibitory
concentration (MIC) of Madre de agua (Trichanthera gigantea) stem crude extract which was the
22
25% concentration and ciprofloxacin. The sum of the individual ZIs is 79.500 and the combined
ZI is 59.833.
23
DISCUSSIONS
Phytochemicals and Antibacterial Assay
Based on table 1, tannins, saponins, and steroids are the common phytochemicals
discovered in Madre de agua (Trichanthera gigantea) root and stem crude extracts. In comparison
to a similar study by Nguyễn et al., to which leaf crude extracts were used, it revealed that tannins
and saponins were present, however alkaloids were absent (2022). The crude extracts passed the
ferric chloride test. It functions on the principle that phenolics react with iron salts to produce a
blue or green black result, hence tannins are present (Bharudin, Zakaria, and Chia 2013). The red
color in the positive result for Salkowski test in the screening for steroids is caused by the creation
of bi-sulfonic acid of bi-cholestadiene. In this reaction, concentrate sulfuric acid removes two
molecules of water from two molecules of cholesterol, resulting in the formation of bicholestadien.
If cholesterol is present, sulfuric acid sulfonates this, resulting in the synthesis of bi-sulfonic acid
of bi-cholestadiene red color (Gupta, 2019). In the foam test for saponin screening, saponin
molecules align vertically on the surface of an aqueous solution of distilled water and plant extract,
with their hydrophobic ends directed away from the water. This causes the water's surface tension
to decrease, causing it to foam (Kregiel et al., 2017). The Mayer’s test for alkaloids is explained
through nitrogen atoms with free electron pairs of alkaloids. A covalent bond coordinate is formed
when this pair of free electrons combines with the metal. The reaction of alkaloid test with this
reagent may occur when the electrons of the nitrogen atom react with the K+ ion of dipotassium
tetraIodomercurate, resulting in the formation of a potassium-alkaloid complex with white
precipitate (Warsi and Sholichah 2017). Madre de agua (Trichanthera gigantea) root and stem
crude extracts failed in the Mayer’s test for alkaloids, thus, this reaction did not occur.
24
Madre de agua (Trichanthera gigantea) root and stem crude extracts were proven to be an
efficient antibacterial agent against MRSA as the diameters of ZIs were significantly higher than
the control group as shown in tables 5 and 6 as succeeding P-values are 0.0000353529 and
0.0011611 which are lesser than 0.05. However, Trichanthera gigantea is not effective against E.
coli as the experimental set-ups did not exceed the cutoff of the control group in reference to
figures 5 and 6. In reference to tables 9 and 10, there are also significant differences among the
experimental set-ups of Trichanthera gigantea root and stem crude extracts against E. coli as
succeeding P-values are 9.22 × 10!"# and 1.08 × 10!$% which are lesser than 0.05.
Antibacterial activity towards MRSA has three stages: (1) cell membrane attachment; (2)
cell membrane fluidity modification; and (3) cell membrane structure disruption (Chew et al.
2018). According to Safitri et al., Gram-positive bacteria's cell wall is mostly composed of
peptidoglycans, teichoic acids, and plasma membranes. Inhibiting bacterial cell wall synthesis can
be undertaken by eliminating peptidoglycans or teichoic acids. The hydrogen bonds formed
between teichoic acids and saponins cause the phosphate groups in teichoic acids to become
unstable, leading the synthesis of cell wall bacteria to be stopped and the cell wall to lyse (2018).
Hence, this supports the susceptibility of MRSA to both Madre de agua (Trichanthera gigantea)
root and stem crude extracts due to the presence of saponin phytochemicals. Tannins’ antibacterial
activity is explained by their capacity to permeate through the bacterial cell wall up to the
internal membrane, causing interference with the cell's metabolism and, as a result, its demise.
Tannin activity is swift in Gram positive bacteria. However, it is slower in Gram-negative bacteria
due to the presence of a bilayered membrane (Kaczmarek, 2020). Thus, the Madre de agua
(Trichanthera gigantea) root and stem crude extracts had antibacterial properties on gram-positive
MRSA and otherwise on gram-negative E. coli.
25
Combination Assay
Synergy testing is performed using advanced susceptibility testing methodologies that
account for antimicrobial combinations and quantify their cumulative effectiveness. The purpose
of synergy testing is to establish if the impact of two antibacterial agents is larger than the sum of
their separate activities by assessing their in vitro interaction. Antimicrobial combinations can
function additively, where the cumulative antimicrobial impact is just the sum of the two
antimicrobials operating together, or synergistically, where the combined activity exceeds the sum
of their separate activities. These approaches, on the other hand, can find antagonistic pairings
(Doern 2014, 2). On table 14, Madre de agua (Trichanthera gigantea) root crude extracts and
ciprofloxacin showed antagonism as both treatments for all trials had greater zones of inhibition
than the zones of inhibition combined. As per definition, A + B > C is antagonism. Data from
figure 6 shows average of roots ZI + average of ciprofloxacin ZI > average of both combined:
38.333 + 40.333 > 60.667. The same is also the case for Madre de agua (Trichanthera gigantea)
stem crude extracts and ciprofloxacin as shown on table 15. Data from figure 7 shows average of
stem ZI + average of ciprofloxacin ZI > average of both combined: 39.333 + 40.167 > 59.833.
Therefore, Madre de agua (Trichanthera gigantea) roots, stems, and ciprofloxacin can act alone
as antibacterial agents.
26
CONCLUSION
Based on the analysis of results and findings, tannins, saponins, and steroids are present in
Madre de agua (Trichanthera gigantea) root and stem crude extracts with antibacterial properties
against Methicillin-resistant Staphylococcus aureus although ineffective against Escherichia coli.
In reference to tables 5 and 6, there are significant differences among the experimental set-
ups of Madre de agua (Trichanthera gigantea) root and stem crude extracts as succeeding P-values
are 0.0000353529 and 0.0011611 which are lesser than 0.05. In reference to tables 9 and 10, there
are also significant difference among the experimental set-ups of Trichanthera gigantea root and
stem crude extracts as succeeding P-values are 9.22 × 10!"# and 1.08 × 10!$% which are lesser
than 0.05. The minimum inhibitory concentration (MIC) of Trichanthera gigantea root and stem
crude extracts is 25% v/v concentration. Trichanthera gigantea was proven to be a remarkable
antibacterial treatment for MRSA as the experimental groups had ZIs higher than the ZI of
ciprofloxacin and showed significant differences between the control groups.
Trichanthera gigantea and ciprofloxacin revealed antagonistic effects as the individual
averages were greater than the combined average. Results for root crude extracts revealed that
38.333 + 40.333 > 60.667 and for stem crude extracts, 39.333 + 40.167 > 59.833. Trichanthera
gigantea and ciprofloxacin are antagonistic and can work alone as an antibacterial component.
Although, it remains unclear what mechanism causes the antagonism of Trichanthera gigantea
root and stem crude extracts in combination. Thus, further procedures must be employed to
identify if this antagonism can be beneficial for Trichanthera gigantea roots and stems being
components for antibiotics.
27
RECCOMENDATIONS
The researcher recommends the following for improvement and a better understanding of the
study:
• Compute the percentage yield of the plant extracts.
• Conduct a quantitative phytochemical analysis.
• Examine the interactions of Madre de agua (Trichanthera gigantea) root and stem crude
extracts to Methicillin-resistant Staphylococcus aureus and Escherichia coli to a
microscopic level.
• Isolate the phytochemicals of Madre de agua (Trichanthera gigantea) root and stem crude
extracts and examine its interactions on the mechanisms of Methicillin-resistant
Staphylococcus aureus and Escherichia coli.
• Utilize the checkerboard method for the combination assay and calculate the Fractional
Inhibitory Concentration Index (FICI).
• Investigate the interactions of Madre de agua (Trichanthera gigantea) root and stem crude
extracts with ciprofloxacin on the mechanism of their antagonistic effect to a microscopic
level.
• Conduct a physicochemical analysis for the plant extracts.
• Investigate the utilization of Madre de agua (Trichanthera gigantea) stem and/or root as a
component for an antibiotic or antidote for Methicillin-resistant Staphylococcus aureus
related diseases.
28
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https://www.whatisbiotechnology.org/index.php/science/summary/crispr.
ATCC. “About Us.” Accessed August 21, 2023. https://www.atcc.org/about-us.
Online Forum/Question
Dr. Gupta, Amit. (2019). Re: How does Salkowski's Test for Steroid content work?. Retrieved
from: https://www.researchgate.net/post/How-does-Salkowskis-Test-for-Steroid-content-
work/5c77d3dbf8ea5296bb2ce192/citation/download.
33
ACKNOWLEDGEMENTS
• To the Almighty God, for providing me sufficient and steadfast physical and emotional
strength throughout the timeframe of this study despite shortcomings and challenges. I was
not alone, for I was with Him all throughout.
• To Berlie Gamaliel Yap, my older brother, for being my primary consultant, designated
supervisor, and supporting my objectives for this study, helping me define unfamiliar terms
and bridging the qualities of this study; his patience and prowess in the field of medicine,
guiding me throughout my thesis.
• To my parents Berlie G. Yap and Delia M. Yap, for providing financial and emotional
support despite the extravagant expenses.
• To Sir Nico Palaran, for helping in the procurement of the samples and speeding up the
preparation process.
• To Sir Anthon Glenn Antonio, for heading the extraction and phytochemical analysis while
providing explanations on concepts about such.
• To Sir Richard, for approving my study for experimentation at Zamboanga City Medical
Center Laboratory, Department of Pathology, Microbiology section.
• To my adviser, Ma’am Brecini Faith Tan, for directing me throughout the study and
offering availability despite a busy schedule.
• To myself writing this right now, I am proud of you for accomplishing great feats. Continue
to strive, explore, and do your best not only in the field of science, but also throughout your
life; this marks as a significant milestone of your RSHS-IX journey.
34
APPENDICES
Appendix 1. Expenses and Schedule of Activities
A. Proposed Budget
SERVICE FEE COST (PHP)
Consultation Fee PHP 2000.00
Ethanol PHP 4500.00
Transportation fee PHP 300.00
Lab technician fee (All bacteria included) PHP 3000.00
TOTAL PHP 7000.00
B. Schedule of Activities
ACTIVITIES TIME FRAME (IN DAYS)
A. Planning Stage
Choosing of Study 1
Gathered Information 6
Subtotal 7
B. Implementation Stage
Combining Data 7
Encoding 3
Subtotal 10
C. Experimentation data
Procurement of Materials 1
35
Processing of raw materials 3
Extraction 3
Experiment 4
Observations/Data Collection 1
Subtotal 12
D. Analysis Stage
Analyze Data 2
Analyze the Research Report 4
Subtotal 6
E. Revising Stage
Schoolwide oral defense 1
Final revisions 1
Subtotal 2
Total 37
36
Appendix 2. Certificate for Plant Verification
20,
37
Appendix 3. ISEF Forms

Form 1. Checklist for Adult Sponsor
38
Form 1A. Student Checklist
39
Form 1B. Approval Form
40
Form 2. Qualified Scientist
41
Form 2. Qualified Scientist
42
Form 3. Risk Assessment Form
43
Form 3. Risk Assessment Form
44
Form 6A. Potentially Hazardous Biological Agents Risk Assessment Form
45
Appendix 4. Documentations
Appendix 4A. Procurement of experimental sample
Zamboanga City Veterinarian Madre de agua stems, roots, and leaves
Appendix 4B. Preparation for crude extraction
Washing Dehydration Weighing
Pounding Grinding Separation of Debris
Weighing of powder
46
Appendix 4C: Crude extraction
Soxhlet extraction Rotary evaporation
Appendix 4D: Preliminary Phytochemical Analysis
T1 T2 T3 Control T1 T2 T3 Control
Ferric Chloride test for Tannins (MDR) Ferric Chloride test for tannins (MDS)
T1 T2 T3 Control
T1 T2 T3 Control
Lead acetate test for Flavonoids (MDR) Lead acetate test for Flavonoids (MDS)
47
T1 T2 T3 Control
T1 T2 T3 Control
Mayer’s test for Alkaloids (MDR) Mayer’s test for Alkaloids (MDS)
T1 T2 T3 Control
T1 T2 T3 Control
Salkowski’s test for Steroids (MDR) Salkowski’s test for Steroids (MDS)
T1 T2 T3 Control T1 T2 T3 Control
Foam test for Saponins (MDR) Foam test for Saponins (MDS)
48
Appendix 4E: Preparation for Antibacterial Assay
Preparation of Adjustment to Swabbing of bacterial

bacterial inoculum McFarland standard inoculum
Preparation of crude Applying the treatments Incubation

extracts
Appendix 4F: Antibacterial Assay
MRSA stem MRSA roots E. Coli
49
Appendix 4G: Preparation for Combination Assay
Soaking in stem crude Soaking in root crude Soaking in Ciprofloxacin
Appendix 4H: Combination Assay
Ciprofloxacin and stem Ciprofloxacin and root
Appendix 5. Supplementary data
Table 2. Results for Soxhlet crude fat extraction
Filter Paper Sample Weight after

Product TN
(grams) (grams) extraction (grams)
T1 2.4 30.8 26.1
T2 2.2 24.6 19
Madre de agua (Trichanthera
T3 2.6 21.8 18.6
gigantea) root
T4 2.4 25 20.4
T5 2.2 25.3 21.5
50
T6 2.8 48.8 43.7

T7 2.4 49.7 44.4
T8 2.4 24 17.9
T9 2.8 25.5 20
T10 2.2 29.3 20
T11 2.2 27.2 16.6
Madre de agua (Trichanthera T12 2.5 36.6 25.9
gigantea) stem T13 2.7 45.9 34.2
T14 2.5 37 26
T15 2.2 25.2 20.4
T16 2.2 23.3 16
Table 3. Panel of test organisms for primary antibacterial in vitro screening
Characteristic Species Source

Enterobacteriaceae Escherichia coli ATCC 25922
Gram-positive Methicillin-resistant ATCC 43300
coccus, MDR Staphylococcus aureus
Table 4. Diameters of zones of inhibition of set-ups against clinical isolates of bacteria
Madre de agua roots Madre de agua stem

TN
A1 A2 A3 A4 Control B1 B2 B3 B4 Control
T1 32 32 33 30 24 34 32 32 33 25
MRSA
T2 30 31 31 30 24 30 30 31 34 28
T3 30 32 33 32 26 31 29 32 33 26
T1 - 7 8 8 29 - - - - 28
E. Coli
T2 - 5 6 8 32 - - - - 29
T3 - 5 7 8 30 - - - - 28
Table 5. One-way ANOVA results for Diameters of zones of inhibition for Madre de agua root
crude extracts in MRSA
Source of SS df MS F P-value F crit

Variation
Between Groups 112.666667 4 28.1666667 24.8529412 0.0000353529 3.47804969
Within Groups 11.3333333 10 1.13333333
51
Total 124 14
Table 6. One-way ANOVA results for Diameters of zones of inhibition for Madre de agua stem
crude extracts in MRSA

Variation
Between Groups 84 4 21 10.862069 0.0011611 3.47804969
Within Groups 19.3333333 10 1.93333333
Total 103.333333 14
Table 7. Post-hoc (Tukey HSD) test results for Diameters of zones of inhibition for Madre de
agua root crude extracts in MRSA
Pairs |𝑥̄ ! − 𝑥̄ " | Critical Value Significant at

%5?
A1 + A2 1 2.21384281 Not Significant
A1 + A3 1.66666667 2.21384281 Not Significant
A1 + Control 6 2.21384281 Significant
A3 + Control 7.66666667 2.21384281 Significant
agua stem crude extracts in MRSA

%5?
B1 + B2 1.33333333 2.8914875 Not Significant
B1 + B3 0 2.8914875 Not Significant
B1 + Control 5.33333333 2.8914875 Significant
52

B2 + B4 3 2.8914875 Significant
B2 + Control 4 2.8914875 Significant
B4 + Control 7 2.8914875 Significant
Table 9. One-way ANOVA results for Diameters of zones of inhibition for Madre de agua root
crude extracts in E. coli

Variation
Between Groups 3366.57778 4 841.644444 1262.46667 9.22 × 10!"# 3.05556828
Within Groups 10 15 0.66666667
Total 3376.57778 19
Table 10. One-way ANOVA results for Diameters of zones of inhibition for Madre de agua stem
crude extracts in E. coli

Variation
Between Groups 2568.88889 4 642.222222 14450 1.08 × 10!$% 3.05556828
Within Groups 0.66666667 15 0.04444444
Total 2569.55556 19
agua root crude extracts in E. coli

%5?
A1 + A2 5.66666667 1.69793992 Significant
A1 + A3 7 1.69793992 Significant
A1 + A4 8 1.69793992 Significant
53
A2 + A4 2.33333333 1.69793992 Significant

agua stem crude extracts in E. coli

%5?
Table 13. Combination assay test raw data
Groups 1 2 3
Roots 38 + 38 37 + 38 39 + 40
Ciprofloxacin 39 + 42 40 + 38 41 + 42
Roots + Ciprofloxacin 63 + 58 62 + 60 59 + 62
Stem 40 + 40 38 + 37 41 + 40
Ciprofloxacin 40 + 39 41 + 39 40 + 42
Stem + Ciprofloxacin 61 + 59 60 + 58 59 + 62
Table 14. Combination assay test results for Madre de agua roots group
Trials Roots Ciprofloxacin Both Results Conditions Conclusion

T1 38 40.5 60.5 78.5 > 60.5 A + B > C Antagonistic
T2 37.5 39 61 76.5 > 61 A + B > C Antagonistic
T3 39.5 41.5 60.5 81 > 60.5 A + B > C Antagonistic
54
Table 15. Combination assay test results for Madre de agua stem group
Trials Stem Ciprofloxacin Both Results Conditions Conclusion

T1 40 39.5 60 79.5 > 60 A+B>C Antagonistic
T2 37.5 40 59 77.5 > 59 A+B>C Antagonistic
T3 40.5 41 60.5 81.5 > 60.5 A+B>C Antagonistic
Table 16. One-way ANOVA results for combination assay roots group

Variation
Between Groups 916.222222 2 458.111111 499.757576 2.12E-07 5.14325285
Within Groups 5.5 6 0.91666667
Total 921.722222 8
Table 17. Post-hoc (Tukey HSD) test results for combination assay for Madre de agua root
groups
Pair |𝑥̄ ! − 𝑥̄ " | Critical Significant at %5?

Value
Roots & Cipro 2.5 2.39902526 Significant
Roots & Both 22.5 2.39902526 Significant
Cipro & Both 20 2.39902526 Significant
Table 18. One-way ANOVA results for combination assay stem group

Variation
Between Groups 807.722222 2 403.861111 323.088889 7.79E-07 5.14325285
Within Groups 7.5 6 1.25
Total 815.222222 8
55
Table 19. Post-hoc (Tukey HSD) test results for combination assay for Madre de agua stem
groups
Pair |𝑥̄ ! − 𝑥̄ " | Critical Significant at %5?

Value
Stem & Cipro 0.83333333 2.80145795 Not Significant
Stem & Both 20.5 2.80145795 Significant
Cipro & Both 19.5 2.80145795 Significant
56
OPERATIONAL DEFINITION OF TERMS
Madre de agua. Trichanthera gigantea, common name Madre de agua, is the primary species
studied in this thesis for phytochemicals and antibacterial properties, specifically, the roots and
stems. Madre de agua (Trichanthera gigantea) is a tree or shrub found in Colombia's Andean
foothills and neighboring Central and South American nations, it adapts well to a broad range of
tropical environments and has been successfully transplanted to Vietnam, Cambodia, and the
Philippines (Mauricio, 1997).
Soxhlet crude fat extraction. Soxhlet crude fat extraction is an extraction method used for Madre
de agua (Trichanthera gigantea) roots and stems with the use of 50% ethanol to acquire the
resulting aqueous extract. According to Oliveros and Horn, the Soxhlet extractor provides efficient
results through lengthy extraction of 16 to 20 hours (2009).
Rotary evaporation. Rotary evaporation is an extraction method used for Madre de agua
(Trichanthera gigantea) roots and stems after the Soxhlet crude fat extraction to evaporate the
remaining ethanolic content and attain the pure extract. According to Pubchem, the boiling point
of ethanol (C2H6O) is 78.24°C (2014).
Crude extract. Crude extracts are pure extracts of Madre de agua (Trichanthera gigantea) root
and stems that had undergone the process of Soxhlet fat crude extraction and rotary evaporation
remove unnecessary components. Crude extracts are used in phytochemical analyses and
antibacterial studies to determine the properties of Madre de agua (Trichanthera gigantea) on such.
Phytochemicals. Tannins, flavonoids, alkaloids, steroids, and saponins are the phytochemicals
used in this study. Phytochemicals play a crucial role in antibacterial studies as they have impacts
on the mechanisms of bacteria. Thus, this study evaluated the contribution of phytochemicals
57
found in Madre de agua (Trichanthera gigantea) root and stem crude extracts to the antibacterial
properties.
Food dehydrator. Food dehydrator is used to constantly dry Madre de agua (Trichanthera
gigantea) roots and stems in three successive trials until constant weight is obtained.
Ciprofloxacin. Ciprofloxacin is a commercialized antibiotic used as a control group in this study.
Ciprofloxacin is proven to have an antibacterial effect on Methicillin-resistant Staphylococcus
aureus and Escherichia coli. Ciprofloxacin is also used as a drug in combination of Madre de agua
(Trichanthera gigantea) root and stem crude extracts.
Gram-positive Bacteria. Gram-positive bacteria have thick cell walls. Experts classify them as
Gram-positive cocci or bacilli based on their form (Sherrell, 2023). Gram-positive bacteria can be
recognized using crystal violet dye, which interacts with the bacteria to produce blue color under
a microscope; this relates to the thick peptidoglycan (PG) layer's capacity to hold the dye (Sizar et
al., 2023). Gram-positive bacteria are classified into three shapes: cocci (spherical), bacilli (rod),
and branching filaments (threadlike) (Sherrell, 2023).
Staphylococcus aureus. Staphylococcus is a Gram-positive cocci genus in the Staphylococcaceae
family, Firmicutes. It is 0.8μm in diameter, organized like a “string of grapes” under a microscope,
aerobic or anaerobic, and grows best at 37°C and pH7.4. The colonies on blood agar plate are
thick, shiny, and spherical, measuring 12mm in diameter. The majority of them are hemolytic,
generating a clear hemolytic ring around colonies on blood agar plates (Guo et al. 2020).
Gram-negative Bacteria. Gram-negative bacteria may be identified by their color because they
do not retain the complex stain and must be counter-stained with safranin to produce the pink
color. This is due to the morphology or composition of the cell wall (Moyes, Reynolds, and
58
Breakwell 2009). An example of a gram-negative bacterium is Escherichia coli which was utilized
in this study.
Escherichia coli. Escherichia Coli is a Gram-negative bacillus that comes from the family
Enterobacteriaceae (Breijyeh, Jubeh, and Karaman 2020). E. Coli grows in the optimal
temperature of 37°C and can survive at pH 3.6 (Albrecht, 2015). E. coli is a gram-negative,
straight, rod-shaped, non-sporing, acid-fast bacillus that exists in singles and pairs. Cells are
generally rod-shaped, measuring 1-3 μm 0.4-0.7 μm in length, 0.35 m in width, and 0.6-0.7 μm in
volume (Basavaraju and Gunashree, 2023).
Gram-positive Bacteria. Gram-positive bacteria can be recognized using crystal violet dye, which
interacts with the bacteria to produce blue color under a microscope; this relates to the thick
peptidoglycan (PG) layer's capacity to hold the dye (Sizar et al., 2023). An example of a gram-
positive bacterium is Methicillin-resistant Staphylococcus aureus which was utilized in this study.
Methicillin-resistant Staphylococcus aureus. Methicillin-resistant Staphylococcus aureus is a
multidrug resistant gram-positive bacterium. Its resistance is due to the β-lactam production
mechanism (Jubeh, Breijyeh, and Karaman, 2020). Wall teichoic acids are essential for MRSA β-
lactam resistance, and they alter sensitivity to cationic antibiotics in different organisms (Brown,
Santa Maria, and Walker, 2013).
CRISPR-Cas9. CRISPR/Cas9 is a technology that allows for the extremely precise and quick
editing of DNA in a genome, which is an organism's full set of genetic instructions; it is an essential
tool used for the identification of American Type Culture Collection (ATCC) bacteria (What is
Biotechnology?, n.d.)
American Type Culture Collection. American Type Culture Collection or ATCC is a private,
non-profit worldwide biological resource center and standards organization that offers scientists
59
with biomaterials and resources to undertake vital life science research (ATCC, n.d). ATCC
procured bacteria such as Methicillin-resistant Staphylococcus aureus and Escherichia coli were
used in this study.
60

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PHYTOCHEMICAL ANALYSIS AND ANTIBACTERIAL PROPERTIES

OF MADRE DE AGUA (Trichanthera gigantea) ROOT AND STEM

CRUDE EXTRACTS AGAINST METHICILLIN-RESISTANT

Staphylococcus aureus AND Escherichia coli

Berlie Daniel M. Yap

Brecini Faith V. Tan

REGIONAL SCIENCE HIGH SCHOOL FOR REGION – IX

Malasiga, San Roque, Zamboanga City

The significance of antibiotic resistance is a continuous global health danger, as well as

in antibiotic therapy, infectious complications continue to be a significant cause of death and

The increasing rate of antibacterial resistance and multidrug-resistant bacteria poses

significant challenges in fighting bacterial infections. The lack of effective medications,

2010 (Masim et al. 2021).

Andeve, and Govender, 2022).

pre-admission antibiotic therapy in hospitalized patients. A common predicament in the

administration of antibiotics. Some municipal governments have implemented laws on the

the country's Level 2 and Level 3 hospitals (ICAMR, et al., 2023).

It is imperative that therapeutic antimicrobial research focuses more on the suppression of

medically relevant bacteria such as community-acquired and hospital-acquired Methicillin-

bacteria (Valle et al., 2015).

S. aureus (MRSA) in livestock (Badua et al., 2020).

revealed that Trichanthera gigantea contains several phytochemicals of pharmaceutical

in combination with or as a cost-effective alternative to standard medical therapy.

Methicillin-resistant Staphylococcus aureus.

Specifically, the researcher aimed to answer the following questions:

2. Is there a significant difference in the antibacterial properties through the diameters of

gram negative bacteria: E. Coli in the following set-ups?

extract and Normal Saline Solution

extract and Normal Saline Solution

extract and Normal Saline Solution

D. Set-up A4 100% v/v concentration of Madre de agua (Trichanthera gigantea) root

crude extract and Normal Saline Solution

crude extract and Normal Saline Solution

extract and Normal Saline Solution

extract and Normal Saline Solution

H. Set-up B4 100% v/v concentration of Madre de agua (Trichanthera gigantea) stem

crude extract and Normal Saline Solution

3. What is the Minimum Inhibitory Concentration (MIC) of Madre de agua (Trichanthera

gigantea) root and stem crude extracts in E. Coli and MRSA?

ciprofloxacin have a synergistic, additive, or antagonistic effect when combined?

and antibacterial properties of Trichanthera gigantea.

screened through a qualitative phytochemical analysis, namely, tannins, flavonoids, alkaloids,

antibacterial properties. The Minimum Inhibitory Concentrations (MICs) of Madre de agua

Department of Pathology, Microbiology section.

REVIEW OF RELATED LITERATURE

Nguyễn et al. discovered significant chemical compounds in Madre de agua (Trichanthera

gigantea leaves exhibited antibacterial properties on E. coli (2022). Furthermore, secondary

Trichanthera gigantea reproduces vegetatively by cuttings. An adult tree of Trichanthera gigantea

(Ardalani et al., 2021).

important (Kaczmarek, 2020).

microorganisms, including multidrug resistant bacteria (Shamsudin et al., 2022). Flavonoid

antibacterial properties against multidrug-resistant Gram-negative and Gram-positive bacteria,

including E. coli and S. aureus (Farhadi et al., 2019).

considering these significant structural properties of antibacterial flavonoids while orchestrating

defensive systems (Quiceno, 2022).

potential to be further developed into an antibacterial agent (2019).

0.003-0.96 mg/mL (2019).