Qualitative Research

SYNERGISTIC EFFECT OF GUAVA (Psidiumguajava) AND PAPAYA
(Carica papaya L.) LEAVES EXTRACTS AGAINST Streptococcus

pyogenes AND Pseudomonas aeruginosa
A THESIS
PRESENTED TO THE FACULTY OF
COLLEGE OF MEDICAL LABORATORY SCIENCE
LYCEUM-NORTHWESTERN UNIVERSITY
DAGUPAN CITY
IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR
RESEARCH PROJECT AND CAPSTONE
IN MEDICAL LABORATORY SCIENCE
BY:
ADRIAN CAYABYAB
RUTH GAÑALONGO
ANNE YVONNE REYES
CAMILLE POSADAS
PAOLO CODIÑERA
2019
CHAPTER 1
INTRODUCTION
Rationale and Background of the study
Papaya plant (Carica papaya L.) is

widely found in
Indonesia. Almost allparts of the plant
can be utilized
by humans for food or for medicinal
purposes [1-6]. Its
fruits, leaves, and flowers are edible.
Its roots can be
used as medicine for renal and urinary
bladder problem,
and its seeds have anthelmintic
activity [4-7]. Papaya is
also knownas the source of papain
enzyme, a kind of
1
enzyme that is utilized as meat
tenderizer [7]. Papaya
leaf extractshave phenolic
compounds,such as
protocatechuic acid, p-coumaric acid,
5,7-
dimethoxycoumarin, caffeic acid,
kaempferol, quercetin,
chlorogenic acid[8-11]. These
compoundshave
antimicrobial activity andhave been
proven to be able
to inhibit the growth of Rhizopus
stolonifer[3-13]. This
research was done to observe the
antibacterial activity
of papaya leaf extracts against
pathogenic bacteria.
2
widely found in
can be utilized
purposes [1-6]. Its
Its roots can be
bladder problem,
enzyme, a kind of
compounds,such as
3
5,7-
compoundshave
proven to be able
widely found in
can be utilized
4
purposes [1-6]. Its
Its roots can be
bladder problem,
enzyme, a kind of
compounds,such as
5,7-
5
compoundshave
proven to be able
widely found in
can be utilized
purposes [1-6]. Its
Its roots can be
6
bladder problem,
enzyme, a kind of
compounds,such as
5,7-
compoundshave
proven to be able
7
widely found in
can be utilized
purposes [1-6]. Its
Its roots can be
bladder problem,
8
enzyme, a kind of
compounds,such as
5,7-
compoundshave
proven to be able
9
widely found in
can be utilized
purposes [1-6]. Its
Its roots can be
bladder problem,
enzyme, a kind of
10
compounds,such as
5,7-
compoundshave
proven to be able
widely found in
11
can be utilized
purposes [1-6]. Its
Its roots can be
bladder problem,
enzyme, a kind of
compounds,such as
5,7-
12
compoundshave
proven to be able
Papaya plant (Carica Papaya) is widely found in the Philippines. Almost
all parts of the plant could be used as food or medicinal purposes. Its roots can
be used as medicine for renal and urinary bladder problem. Papaya is also
known as the source of Papain enzyme. Papain is a proteolytic enzyme present
in the leaves, latex, roots, and fruit of the papaya plant (Carica papaya) that
catalyzes the breakdown of proteins by hydrolysis (addition of a water
molecule. Papain is a popular folk remedy to reduce pain, inflammation, and
swelling. It has also been used to improve digestion and to treat infections,
13
diarrhea, and allergies. It’s also being studied for potential use in cancer and
other diseases.
Guava plant was also included because like the papaya, it is abundant in
the Philippines and it could be used as food or for medicinal puposes.
Psidium guajava L. leaf (family Myrtaceae) has a long history of folk
medicinal uses in Egypt and worldwide as a cough sedative, an anti-diarrheic,
in the management of hypertension, obesity and in the control of diabetes
mellitus. The leaf extract was found to possess anticestodal, analgesic, anti-
inflammatory propertiesantimicrobialhepatoprotective and antioxidant
activities. In addition, the leaf extract is used in many pharmaceutical
preparations as a cough sedative.
The researchers, came upon the idea of proving guava and papaya leaves
extract can be use against the emerging and reemerging infectious diseases
also known as Streptococcuspyogenesand a hospital acquired infection called
Pseudomonas aeruginosa and spread of deadly drug-resistant strains pose a
challenge to public health care services. The researchers present this study to
prove the ability of the antibacterial properties of guava and papaya leaves. The
researchers chose this plant leaves extract to go against
StreptococcuspyogenesandPseudomonas aeruginosa because it has the
antibacterial potential in crude extracts of leaves against clinical bacterial
strains.
14
Theoretical framework
To determine the antimicrobial potential of guava (Psidium guajava) and
papaya (carica papaya) leaf extracts against Streptococcus pyogenes made up of
non-motile and non-sporing cocci and Pseudomonas aeruginosa. The leaves
were extracted by pounding the leaves until it could produce its juice. Then the
leaves were placed in a clean cloth and squeezed to collect the crude extract of
the leaves. This study provides scientific understanding to further determine
the antimicrobial values and investigate other pharmacological properties.
Research Paradigm
Input Process Output
 Guava  AST  Zone of

and inhibition
papaya
leaves
extract
Statement of the Problem
This study aimed to examine the synergistic effect of guava and papaya
leaves extracts that contains antibacterial properties that can inhibit
15
Streptococcus pyogenes and Pseudomonas aeruginosa. This study aimed to
answer the following:
1.) What is the active component of guava and papaya leaves that can
inhibit Streptococcus pyogenes and Pseudomonas aeruginosa.
2.) What is the mechanism of action.
3.) What is the zone of inhibition produced by papaya and guava leaves
against Streptococcus pyogenes and Pseudomonas aeruginosa.
Significance of the Study

This study aims to determine the potential of guava leaves and papaya
leaves as an extract against Streptococcus pyogenes and Pseudomonas
aeruginosa. Findings of this study would benefit health and society in fighting
the bacteria. Its contribution to individuals is to help them fight the
Streptococcus pyogenesand Pseudomonas aeruginosathrough the use of guava
leaves and papaya leaves extract.
This study will be conducted not only to help researchers and their
research but also this study is significant to people having a strep throat as an
initial symptom to prevent damage in the heart that can lead to rheumatic
fever and to eradicate the organisms that causes pseudomonal infections.
Scope and Delimitation
16
The researcher will conduct study to determine the potential of guava
and papaya leaves extract against Streptococcus pyogenes and Pseudomonas
aeruginosa. The experiment will be performed at Lyceum-Northwestern
University Medical Technology Clinical Laboratory. The zones of inhibition
susceptibility perception will be measured in millimeters and interpreted as
resistant, intermediate or susceptible.
Definition of Terms
 Control Drug – Refers to the treatment (Erythromycin) that served as
basis of comparison on the level of effectiveness of the different
treatments.
 Crude Extraction Method -which undergoes fermentation and
generates alcohol in situ;this facilitates the extraction of the active
constituents contained in the plant material
 Guava Leaves- full of antioxidants, anti-inflammatory agents,
antibacterial, and even tannins that can have significant health
benefits, from treating stomach troubles to chronic diseases like
cancer.
 Leaf Extract Combinations of Guava and Papaya leaves – Refers to
the extracts that were used against Streptococcus pyogenes.
17
 Papaya Leaves -useful in treatment and prevention of diseases,
including cancer, allergies and as a component in some vaccines.
 Pseudomonas aeruginosa - an important cause of gram-negative
infection, especially in patients with compromised host defense
mechanisms. It is the most common pathogen isolated from patients
who have been hospitalized longer than 1 week, and it is a frequent
cause of nosocomial infections.
 Pure Leaf- Refers to the extract obtained from the leaves of each plant.
 Resistant – means that the bacterium did not show zones of
inhibitions to the different treatments used.
 Streptococcus Pyogenes – Refers to bacterium causing strep throat as
initial symptoms and when not treated effectively may damage the
heart leading to rheumatic fever.
 Susceptible – Means that the bacterium used in the study show
significant zones of inhibitions to the different treatments.
 Susceptibility Test – Refers to the procedure to find out the
susceptibility of Streptococcus Pyogenes and Pseudomonas aeruginosa
to the sensitivity disk prepared (impregnated with leaf extract
concentration)
 Synergistic - relating to the interaction or cooperation of two or more
organizations, substances, or other agents to produce a combined effect
greater than the sum of their separate effects.
18
 Treatments – Refer to the different concentration of leaf extracts from
guava and papaya.
 Zone of inhibition – Refers to the response of the bacterium to the
different treatment that were used in the study. It appears as clear
areas surrounding the disk.
Chapter II
REVIEW OF RELATED LITERATURE AND STUDIES
RELATED LITERATURE
Guava
Psidium guajava L. leaf (family Myrtaceae) has a long history of folk
medicinal uses in Egypt and worldwide as a cough sedative, an anti-diarrheic,
in the management of hypertension, obesity and in the control of diabetes
mellitus. The leaf extract was found to possess anticestodal, analgesic, anti-
inflammatory propertiesantimicrobialhepatoprotective and antioxidant
activities. In addition, the leaf extract is used in many pharmaceutical
preparations as a cough sedative.
Guava leaf extract contains flavonoids, mainly quercetin derivatives, which are
hydrolyzed in the body to give the aglycone quercetin which is responsible for
the spasmolytic activity of the leaves. Quercetin has several pharmacologic
19
actions; it inhibits the intestinal movement, reduces capillary permeability in
the abdominal cavity and possesses dose-dependent antioxidant properties,
anti-inflammatory activity, antiviral and antitumor activities. It also inhibits
the aldose reductase enzyme. It should be noticed that most of the flavonoidal
constituents of guava leaf are quercetin derivatives, namely, quercetin,
avicularin, guaijaverin, isoquercetin, hyperin, quercitrin, quercetin 3-O-
gentiobioside, quercetin 4’-glucuronoide.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2950385/
The experiments conducted in Psidium guajava for analysis of phytochemical
constituents showed the following results. Preliminary Phytochemical Analysis
Of Leaves Of Psidium Guajava: In the present study, a preliminary
phytochemical analysis was carried out to identify the active constituents such
as alkaloids, flavonoids, sterols, terpenoids, quinone, oil and fat, phenol,
starch, anthocyanin, protein, carbohydrate and cellulose present in the leaves
of guava plant. Preliminary phytochemical analysis of different extract (ethanol,
chloroform, benzene and water) of P. guajava showed the following results. The
dried and powdered leaves of P. guajava were dissolved in different solvents
viz., water, ethanol, chloroform and benzene and the extracts thus obtained
were analyzed for the presence or absence of secondary metabolites. In
P.guajava, carbohydrate, cellulose, oil & fat and phenols were observed in all
the four solvent extracts. Alkaloids, sterols and anthocyanin were completely
absent in all the four solvent extracts of leaves of P. guajava. Protein and
20
quinones were observed only in the ethanol extract. Except for water, starch
content was observed in the other three solvent extracts of P.guajava. Among
the four solvent extracts of the leaves of P.guajava, terpenoids were observed in
water, ethanol and chloroform, but the presence of flavonoids were seen only in
chloroform and benzene extracts. A study carried out on preliminary
phytochemical screening of active leaf extract of Aegle marmelos, Annona
squamosa, Ficusracemosa, Hibiscus rosasinenses and Psidium
guajavallllrevealed the presence of different type of compounds like alkaloids,
coumarins, flavonoids and steroids which could be responsible for the
antidiabetic activities. The presence of flavonoids, terpenoids, quinone, sterols,
oil and fat have been earlier studied in the plant, Vigna mungo by the use of
leaf extracts of medicinal plants. Earlier work have revealed the presence of
alkaloids, flavonoids, glycosides, polyphenols, reducing compounds, saponins
and tannins in the aqueous extract of Psidium guajava leaves. The
phytochemical analysis of Psidium guajava leaves showed the presence of more
than isolated compounds, including alkaloids, anthocyanins, carotenoids,
essential oils, fatty acids, lectins, phenols, saponins, tannins, triterpenes and
vitamin C. The present study is only a qualitative analysis of the medicinal
plants. Further studies on the quantitative analysis of the various bioactive
compounds present in guava could contribute significantly to the health
management of man and could be recommended in our daily need of nutrition.
http://impactfactor.org/PDF/IJPPR/6/IJPPR,Vol6,Issue2,Article34.pdf
21
Papaya
Chemical constituents of the extracts of C. papaya leaf.
Phytochemical analysis showed the presence of alkaloids and tannins in the
ethanolic extract; and steroid and quinones in the three extracts. The highest
concentrations of tannins (4.85 ± 10-3 ± 1.76 ± 10-4 M; 0.824%) and steroids
(5.98 ± 10-3 ± 2.20 ± 10-3 M; 0.231%) were found in the ethanol extract
of C. papaya leaf. Moreover, alkaloids (1.04 ± 10-4 ± 1.78 ± 10-6 M; 7.53 ± 10-
4%) and quinones (2.20 ± 10-5 ± 5.81 ± 10-7 M; 1.53 ± 10-4%) were also
identified. The C. papaya hexane extract contained both steroids (9.32 ±10-3 ±
1.45 ± 10-4 M; 0.360%) and quinones (1.27 ± 10-5 ± 4.80 ± 10-7 M; 8.87 ± 10-
5%); whereas in the chloroform extract, the steroid concentration was notable
(4.34 ±10-2 ± 5.25 ± 10-3 M; 1.678%). As can be seen from the values, the
chloroform extract showed the highest levels for both steroids and quinones.
For this reason, such extract was used for the pharmacological tests.
Effect of C. papaya leaf chloroform extract on body weight and blood
glucose of STZ-induced diabetic rats
The STZ single-dose effect to induce type 1 diabetes in rats was confirmed by
their significant weight loss and hyperglycemia as compared to non-diabetic
animals (p <0.05). The body weight was recorded every week; final data are
22
shown in Fig. 1. There was a significant decrease in the area under the curve
(AUC) of body weight of diabetic rats (3173 ± 120 g) compared with non-
diabetic animals (3988 ± 110 g). The C. papaya chloroform extract (31 and 62
mg/kg) caused a minor reduction in body weight of diabetic rats (p < 0.05)
than that displayed by control diabetic rats. In addition, this extract had a
similar effect on the body weight AUC in diabetic rats receiving insulin (3639 ±
98 g). An interesting fact is that oral administration of C. papaya chloroform
extract (31, 62 mg/kg) induced significant decreases of the glucose AUC of
diabetic rats (p < 0.05), similarly to that observed in the group receiving
insulin; both cases compared with the diabetic control group (not showed).
https://www.sciencedirect.com/science/article/pii/S0102695X14000362
All other analyses conducted on dried inaterial. were placed in a Soxhlet
extractor and continuously extracted for 25 hours, using 95 percent ethanol.
The extracts were then each diluted to 1000 ml. with 95 percent ethanol for
saponin and flavanol testing. Aliquots of these solutions representing 5 gin.
were evaporated to dryness and the residues dissolved in 25- nil. portions of
hot water and filtered through a sintered glass filter. These solutions were used
for testing alkaloids, tannins, organic acids and phenols. Tests for unsaturated
sterols were made on chloroform solutions prepared in the same manner as the
aqueous solutions. In addition to the saponin tests described by Wall,
Bamford's cholesterol inhibition test and color reaction tests with sulfuric acid
and Frohde's reagent were performed. The results of plant constituent
23
screening are compiled in Table II. Isolation and Identification of Carpaine. One
hundred gin. of dried papaya leaves were extracted by refluxing with 95
percent ethanol, containing 0.5 percent acetic acid, for 45 minutes, according
to Wall andGreshoff. The solvent was spontaneously evaporated and the
residue thoroughly extracted with 1 percent HC1. This solution1 was made
basic with ammonia and completely extracted with ether. Upon evaporation of
the ether, a negligible amount of residue was obtained. It is possible that
heating in tim presence of water caused hydrolysis of the laetone ring of
earpaine to give earpamie acid, which is only sparingly soluble in alcohol. With
this in mind, 500 gin. of dried papaya leaf (Dec. batch) were extracted by
shaking with 80 percent alcohol containing 1 percent HC1. After 48 hours, the
solvent was filtered off and the marc washed with 80 percent alcohol. The
combined extracts were then concentrated in vacuo, at a temperature not
exceeding 40 ~ C., to a wflume of 500 ml. This thick, dark green syrup was
extracted thoroughly with ether to remove chlorophyll and other extraneous
plant materiM. This partially tmrified syrup was then made basic with
potassium carbonate and extracted in a modified Palkin extractor, using fresh
ether. A yield of 0.4 gm. (0.08 percent) of brownish, slightly impure crystals
was obtained. These crystals were purified by dissolving them in a minimum
amount of hot ethanol, allowing the solution to cool somewhat, then adding
distilled water until no further precipitate was formed. This procedure was
repeated three times until practically pure white crystals were obtained. M.P.
121 ~ C.
24
https://link.springer.com/article/10.1007/BF02899003
Streptococcus pyogenes
Acute Streptococcus pyogenes infections may take the form of
pharyngitis, scarlet fever (rash), impetigo, cellulitis, or erysipelas. Invasive
infections can result in necrotizing fasciitis, myositis and streptococcal toxic
shock syndrome. Patients may also develop immune-mediated sequelae such
as acute rheumatic fever and acute glomerulonephritis. S agalactiae may cause
meningitis, neonatal sepsis, and pneumonia in neonates; adults may
experience vaginitis, puerperal fever, urinary tract infection, skin infection, and
endocarditis. Viridans streptococci can cause endocarditis, and Enterococcus is
associated with urinary tract and biliary tract infections. Anaerobic
streptococci participate in mixed infections of the abdomen, pelvis, brain, and
lungs.
https://www.ncbi.nlm.nih.gov/books/NBK7611/
Streptococcus pyogenes, also known as the flesh eating bacteria, is the
most pathogenic bacterium in the whole genus. The name pyogenes comes
from the word pyogenic, which is a classification for the streptococci that are
associated with pus formation. The effects of this microbe range from mild
illnesses such as strep throat and impetigo to more serious diseases such as
scarlet fever, glomerulonephritis, and necrotizing fasciitis. However, if strep
25
throat is untreated, it will lead to rheumatic fever, which is pretty rare now in
the United States but was a more serious problem before the 20th century.
S. pyogenes usually begins infection on the surface of the skin or in the throat.
From there, the bacterium begins to spread into deeper areas of the skin,
which can potentially lead to life-threatening diseases. Because of its versatility
in the human host, researchers have struggled developing a vaccine that would
stop bacterial infection. This is why so much research is being done trying to
understand the different cellular components of this organism as well as
sequencing the genome. The more characteristics that are discovered and
analyzed, the better the understanding we will have to fight this disease. Much
progress has been made, because the genome for a strain has already been
sequenced, and many conclusions have been drawn from the study.
https://microbewiki.kenyon.edu/index.php/Streptococcus_pyogenes
The most optimal fluoroquinolones are those with enhanced gram-
positive activity including levofloxacin and moxifloxacin. Levofloxacin or
moxifloxacin parenterally or orally at doses of 500mg and 400mg, respectively,
once a day for 5 to 10 days depending on the severity of illness. The prevalence
of GAS clinical isolates with reduced susceptibility to fluoroquinolones are less
than 1% in North America, but as high as 10% in some parts of Europe.
Reduced susceptibility to fluoroquinolones is mediated by point mutations in
the quinolone resistance determining region of the parC gene, whereas high-
26
level resistance has been associated with mutations in the quinolone resistance
determining region of both parC and gyrA genes.
Tetracyclines: Tetracycline can be administered orally or parenterally at doses
of 250–500mg four times a day, and doxycycline can be administered orally or
parenterally at a dose of 100mg twice a day, both for 5 to 10 days depending
on the severity of illness. Organisms susceptible to tetracycline can also be
considered susceptible to doxycycline. In GAS, the prevalence of resistance
varies from 1 to 10% in North America and is typically conferred by ribosome
protection genes such astet(M) and tet(O). Since tetracycline resistance genes
can reside on mobile genetic elements that carry macrolide resistance genes,
the co-occurrence of resistance to both classes of drugs often occurs.
Linezolid: Linezolid can be administered orally or parenterally at doses of
600mg twice a day for 5 to 10 days depending on the severity of illness.
Resistance rates are less than 1%.
Vancomycin: Vancomycin can be administered parenterally at a dose of
30mg/kg/day twice a day until step down to another oral agent is clinically
indicated.
https://www.infectiousdiseaseadvisor.com/home/decision-support-in-
medicine/infectious-diseases/streptocococcus-pyogenes/
Pseudomonas aeruginosa
27
Pseudomonas aeruginosa is a Gram-negative, rod-shaped, asporogenous,
and monoflagellated bacterium. It has a pearlescent appearance and grape-like
or tortilla-like odour. P. aeruginosa grows well at 25°C to 37°C, and its ability
to grow at 42°C helps distinguish it from many other Pseudomonas species. P.
aeruginosa is a ubiquitous microorganism which has the ability to survive
under a variety of environmental conditions. It not only causes disease in
plants and animals, but also in humans, causing serious infections in
immunocompromised patients with cancer and patients suffering from severe
burns and cystic fibrosis (CF).
Most strains of P. aeruginosa produce one or more pigments, including
pyocyanin (blue-green), pyoverdine (yellow-green and fluorescent), and
pyorubin (red-brown). Previous investigations have suggested that pyocyanin
not only contributes to the persistence of P. aeruginosa in the lungs of CF
patients, but also interferes with many mammalian cell functions, including
cell respiration, ciliary beating, epidermal cell growth, calcium homeostasis and
prostacyclin release from lung endothelial cells. However, the precise molecular
mechanism mediated by pyocyanin pathology is unknown.
P. aeruginosa strains produce two distinct types of O antigen (O-Ag): a common
polysaccharide antigen (A-band) composed of a homopolymer of d-rhamnose,
and an O-specific antigen (B-band) composed of a heteropolymer of three to five
distinct sugars in its repeat units. So far, P. aeruginosa isolates have been
classified into 20 serotypes by the International Antigenic Typing Scheme
(IATS). The lipopolysaccharide (LPS) of P. aeruginosa is less toxic than that of
28
other Gram-negative rods, facilitating its establishment of chronic infections by
eliciting a low inflammatory response.
The genome of P. aeruginosa consists of a single circular chromosome. P.
aeruginosa has a relatively large genome (5.5–7 Mb) and high G+C content (65–
67%). Because of the large genome, P. aeruginosa encodes a large number of
enzymes for various metabolic pathways, conferring high nutritional versatility.
In addition, about 8% of the genome encodes regulatory genes, which enables
the bacterium to adapt to complex growth environments.
Pseudomonas aeruginosa is a gram-negative bacillus found widely in nature, in
soil and water. Classified as an opportunistic pathogen, P. aeruginosa causes
disease infrequently in normal hosts but is a major cause of infection in
patients with underlying or immunocompromising conditions. The genome of P.
aeruginosa, which is especially large for a prokaryote, has provided an
understanding of the metabolic and pathogenic mechanisms that underlie the
success of this versatile pathogen, and it has become a model for
understanding microbial genomic variation and evolution in chronic disease. P.
aeruginosa has few nutritional requirements and can adapt to conditions not
tolerated by other organisms. It does not ferment lactose or other
carbohydrates but oxidizes glucose and xylose. Organisms grow aerobically or
anaerobically if nitrate is available as an inorganic electron acceptor, as is the
case in the lungs of patients with cystic fibrosis (CF). 1 P. aeruginosa gene
expression responds to environmental conditions with discrete patterns typical
of environmental isolates: motility, piliation, and expression of numerous
29
exoproducts. In subacute and chronic infections, the accumulation of
intracellular dinucleotides (cyclic diguanidine monophosphate) favors a biofilm
mode of growth with the formation of an extracellular polysaccharide
matrix,2 thus enabling the organisms to avoid innate immune clearance
mechanisms and persist in human airways.3
The organism produces the fluorescent siderophores pyoverdin and pyochelin,
which function to scavenge iron. Redox-active phenazines such as pyocyanin,
the pigment that gives P. aeruginosa its characteristic blue color, play an
important role in electron transport especially under microaerophilic
conditions, increase the bioavailability of iron, and enhance virulence through
oxidative stress.4,5 P. aeruginosa can be identified biochemically as having
indophenol oxidase-positive, citrate-positive, and l-arginine dehydrolase-
positive activity. Differentiation of P. aeruginosa from other pseudomonads or
organisms such as Burkholderia species, Stenotrophomonas
maltophilia, and Achromobacter spp. occasionally can require testing for DNAse
activity, growth at 42°C, and differential carbohydrate metabolism or using
molecular methods.
https://www.sciencedirect.com/topics/medicine-and-dentistry/pseudomonas-
aeruginosa
30
CHAPTER III
RESEARCH METHODOLOGY
Research Design
The researchers used the experimental design to test the potential of the
Guava and Papaya Leaves extracts as a mouth wash and test the product
using anti susceptibility test (AST).
There were seven (7) treatments each replicated two (2) times. The
treatments were as follows.
a. G= Pure leaf extract of guava
b. P = Pure leaf extract of papaya
c. PG= Pure leaf extract combination of papaya and guava
d. Ery= Erythromycin (control drug for S. pyogenes)
e. Am= Amoxcicillin(control drug for S. pyogenes)
31
f. Lev= Levoflaxacin (control drug for P. aeruginosa)
g. Cl= Clindamycin (control drug for P. aeruginosa)
Materials for Papaya and Guava extraction
Ingredients
 100g Guava Leaves (Mature)
 100g Papaya Leaves (Mature)
 40ml of Water
Tools
 Glass jars
 Graduated cylinder
 Erlenmyer flask
 Scissors
 Strainer
 Weight measuring device
 Bowl
32
 Electric fan
 Gloves
 Mortar and pestle
 Clean cloth
Procedure of collecting extracts
1. Collect leaves from a papaya tree and guava tree. Makes sure to
collect only the mature ones.
2. Weigh the leaves. You must get 100 grams each.
3. Clean the leaves and put in a bowl then air dry it using an electric fan
to dry quickly.
4. After drying, use the mortar and pestle and mash the papaya leaves.
5. Get a clean dry cloth and put all the mashed papaya leaves there.
6. Twist the cloth and squeeze it until it produces juices. The juice must
drip into the strainer and into the graduated cylinder to measure how
much was collected.
7. After measuring place it in the Erlenmyer flask for storage.
8. Repeat steps 4-7 for extraction of guava leaves but add 40ml of water
while mashing the guava.
Materials for AST
Ingredients
 Mueller Hinton agar (MHA)
33
 Nutrient broth
 Cultured Streptococcus pyogenes
 CulturedPseudomonas aeruginosa
Tools
 Autoclave
 Incubator
 Erlenmyer flask
 Stirring rod
 Casserole
 Stove
 6 petridish
 6 cotton tip applicator stick
 Applicator sticks
 Cotton balls
 Paper
 Alcohol lamp
 Inoculating loop
 Test tube
 Test tube rack
 Dishwashing soap
 Lysol spray
34
 Sponge
Sterilization of Equipments
1. Place the petridish, flasks, and testube with the testube rack in the
autoclave at a temperature of 121 degree Celsius at 15lbs psi for 15-30
mins.
2. Wait until finished.
AST PROCEDURE
1. Select a sub cultured plate of one of the organisms to be tested.
2. Aseptically emulsify a colony from the plate in the sterile saline solution.
3. Mix it thoroughly to ensure that no solid material from the colony is
visible in the saline solution.
4. Repeat until the turbidity of the saline solution visually match that of the
standard turbidity.
5. Take a sterile swab and dip it into broth culture of organism.
6. Gently squeeze the swab against the inside of the tube in order to remove
excess fluid in the swab.
7. Take a sterile Mueller- Hinton agar ( MHA) plate or a nutrient agar (NA)
plate.
8. Use the swab with the test organism to streak a MHA Plate or a NA plate
for lawn of growth.
35
9. After the streaking is complete, allow the plate to dry for 5 minutes.
10.Antibiotic discs can be placed on the surface of the agar using sterilized
forceps.
11. Gently press the discs onto the surface of the agar using flame sterilized
forceps or inoculation loop.
12.Carefully invert the inoculated plates and incubate for 24 hours at 37°C
13. After incubation, use a metric ruler to measure the diameter of the zone
of inhibition for each antibiotic used.
14. Compare the measurement obtained from the individual antibiotics with
the standard table to determine the sensitivity zone.
15.Compare the measurement obtained from the individual antibiotics to
the standard table to determine whether the tested bacterial species is
sensitive or resistant to the tested antibiotics.
Testing and Evaluation
Evaluation was done by using the anti susceptibility test. This experiment
asked to use the prepared Guava and Papaya Leaves extraction as a product.
After using the product evaluate the product by using the prepared rating
scale.
Data Gathering Instrument and Procedure
36
Kirby-Bauer Disc Method, also called the agar diffusion method or
the disk diffusion method, was used to test the antibacterial effectiveness of
the individual leaf and combination leaf extracts of guava and papaya. First,
the disks impregnated with different leaf extracts were applied to the surface of
an agar plate which contained S. pyogenesand another plate that contains
Pseudomonas aeruginosa. The agar plates used were of the MH agar plate type
with 5% sheep blood. Then the plates were incubated at 37°C for 24 hours.
Agar diffusion method depends upon diffusion rate of a substance in the
agar medium. As the substance diffuses from the filter paper into the agar, the
concentration decreases as a function of the square of the distance of diffusion.
At some particular distance from each disk, the concentration lessens to the
point that it no longer inhibits microbial growth. The effectiveness of a
particular leaf extracts disk is shown by the presence of growth-inhibition
zones. These zones of inhibition (ZOI’s) appear as clear areas surrounding the
disk from which substances with antimicrobial activity diffused. The diameter
of the ZOI is measured with a ruler and the results of such an experiment
constitute an antibiogram. (Bassiri, 2012).
Descriptive Equivalent
Zone of Inhibition Descriptive Equivalent

(mm)
≥21 Susceptible (S)
16-20 Intermediate (I)
37
≤15 Resistant (R)
CHAPTER IV
RESULTS AND DISCUSSIONS
Result of the AST
Table 1 below which is the result of the first trial shows that all treatments
did not produce inhibition zones except for the control drug used. All zones
measured the same at 6mm indicating that S.pyogenes was resistant to PG, P,
G. However, the control drug used Erythromycin, showed clear inhibition
zones with an average measurement of 40mm. This means that the bacterium
was susceptible to the control drug used.
Table 1. Trial 1-Level of Susceptibility of S.pyogenes to the Treatments
Treatment Mean Descriptive
Equivalent
PG 6mm Resistant
P 6mm Resistant
G 6mm Resistant
Erythromycin 40mm Susceptible
38
Table 2 which is the second trial, show that the treatments
exhibitedminimal zones of inhibitions. The zones of inhibition were measured
as follows: PG 17mm, P 20mm, G 14mm; these inhibition zones indicate that
PG and G treatments were resistant to the S. pyogenes, while the P treatment
showed intermediate activity on the bacterium. However the control drug used
which is Amoxicillin exhibited a clear zone inhibition measurement of 25mm
denoting its susceptibility to S, pyogenes.
Table 2.Trial 2-Level of Susceptibility of S.pyogenes to the Treatments
Equivalent
PG 17mm Intermediate
P 20mm Intermediate
G 14mm Resistant
Amoxicillin 25mm Susceptible
Table 3 below which is the result of the first trial against Pseudomonas
aeruginosa. The zones of inhibition were measured as follows: PG 30mm, P
26mm, G 20mm; these inhibition zones indicate that PG and P treatments were
susceptible to the P.aeruginosa, while the G treatment showed intermediate
activity on the bacterium. However the control drug used which is Levoflaxacin
exhibited a clear zone inhibition measurement of 46mm denoting its
susceptibility toP. aeruginosa.
39
Table 3.Trial 1-Level of Susceptibility of P.aeruginosa to the Treatments
Equivalent
PG 30mm Susceptible
P 26mm Susceptible
G 20mm Intermediate
Levoflaxacin 46mm Susceptible
Table 4 below which is the result of the second trial against Pseudomonas
aeruginosa. The zones of inhibition were measured as follows: PG 26mm, P
20mm, G 16mm; these inhibition zones indicate that P and G treatments were
intermediate to the P.aeruginosa, while the PG treatment showed susceptible
activity on the bacterium. However the control drug used which is Clindamycin
exhibited a clear zone inhibition measurement of 44mm denoting its
susceptibility to P. aeruginosa.
40
Table 4.Trial 2-Level of Susceptibility of P.aeruginosa to the Treatments
Equivalent
PG 26mm Susceptible
P 20mm Intermediate
G 16mm Intermediate
Clindamycin 44mm Susceptible
Based on our findings, the active component of guava and papaya leaves that
can inhibit Streptococcus pyogenes and Pseudomonas aeruginosa are tannins,
papain, and guaijaverin.
The mechanism of action of tannin is that previous studies reporting a
strong inhibition of SCFA formation with free procyanidins compared with
procyanidins enclosed in the apple matrix. One explanation to this
phenomenon is that free procyanidins inhibit bacterial carbohydrate-active
enzymes possibly because of their ability to form strong complexes with
proteins
The mechanism of action of guaijaverinis a polyphenolic substance which
exhibits some pharmacological activities such as antibacterial and antioxidant
activities.
41
The mechanism of action of papain is a proteolytic enzyme extracted from
the raw fruit of the papaya plant. Proteolytic enzymes help break proteins down
into smaller protein fragments called peptides and amino acids.
CHAPTER V
42
SUMMARY, COCLUSION, AND RECOMMENDATION
Summary
The study aimed to examine the susceptibility of Streptococcus pyogenes
and Pseudomonas aeruginosa to leaf extracts of guava (Psidium guajava),
papaya (Carica papaya), and their synergism. Specifically, it sought to measure
and compare the level of susceptibility of Streptococcus pyogenes and
Pseudomonas aeruginosa to the different treatments in terms of zone of
inhibitions.
Results showed that Streptococcus pyogenes wasresistant to various
treatments usedfrom the different leaf extracts under trial 1 and trial 2.
However, it was intermediate in the treatment P leaf extract. On the other
hand, Pseudomonas aeruginosa was susceptible to most of the treatments used
from different leaf extracts under trial 1 and 2. All the control drugs used
showed significantly clear zone of inhibitions. Some treatments did not show
clear inhibitions, this could mean that other factors could have prevented the
action of leaf extracts on S. pyogenes and Pseudomonas aeruginosa.
Conclusion
43
Based on observation gathered by the researchers, the following
conclusions were derived.
Based on the zone of inhibition,Treatment PG had 17mm, treatment G had
20mm, treatment P had 20mm, Control drug amoxicillin had 25mm, and
control drug erythromycin had 40mm against Streptococcus pyogenes
Based on the zone of inhibition, treatment PG had 30mm, treatment G had
20mm, treatment P had 26mm, control drug levoflaxacin had 46mm, and
control drug clindamycin had 44mm against Pseudomonas aeruginosa.
Findings
Based on our findings, treatment PG and P were intermediate to
Streptococcus pyogenes, while treatment G was resistant. The control drugs
erythromycin and amoxicillin were both susceptible to the organism.
On Pseudomonas aeruginosa, treatments PG and P, and control drugs
levoflaxacin and clindamycin were susceptible to the organism, however,
treatment G was the only treatment that was different which was intermediate
to the organism.
Recommendations
Based on the result of the study, the following recommendations were
made.
44
1. Increase the concentrations of the leaf extracts to ascertain their
antibacterial activities.
2. A follow up study should be carried out using different solvents for proper
extraction of important chemical components.
3. Additional studies should be conducted on the synergistic anti bacterial
effect of guava and papaya leaves.
BIBLIOGRAPHY:
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2950385/
45
 http://impactfactor.org/PDF/IJPPR/6/
IJPPR,Vol6,Issue2,Article34.pdf
 https://www.sciencedirect.com/science/article/pii/
S0102695X14000362
 https://link.springer.com/article/10.1007/BF02899003
 https://www.ncbi.nlm.nih.gov/books/NBK7611/
 https://microbewiki.kenyon.edu/index.php/
Streptococcus_pyogenes
 https://www.infectiousdiseaseadvisor.com/home/decision-
support-in-medicine/infectious-diseases/streptocococcus-
pyogenes/
 https://www.sciencedirect.com/topics/medicine-and-
dentistry/pseudomonas-aeruginosa
46
47
APPENDICES
APPENDIX A
Letter for the Monitoring Attendance Form
LYCEUM – NORTHWESTERN UNIVERISTY

Senior High School
Tapuac Dist., Dagupan City
Sir/Ma’am:
We, the researchers, are presently conducting a study
entitled “The Potential of Guava(Psidiumguajava) leaves and
Papaya(Carica papaya L.) leaves as an Extract against
Streptococcus pyogenes” in partial fulfillments of the
requirements for the said subject.
In this regard, We the researchers are hereby request to
your good office to conduct the experimental procedures in your
laboratory through the assistance of your Microbiologist in order
to carry on the above research study.
Thank you for your kind consideration. Your favorable response is
highly appreciated.
Respectfully yours,
Adrian Cayabyab Anne Yvonne Reyes
Ruth Gañalongo
Camille Posadas
Paolo Codiñera
STEM 3-MEDTECH GRADE 12

Researchers
Endorsed
Mrs. Madelle Aquino

Mr. David Contad Noted:
Research Instructor _______________________
_______________________
48
APPENDIX B
Experimental plates
Extraction of leaves
Collection of papaya leaf extract
Collection of guava leaf extract
49
AST procedure
Extracts and control drugs
Swabbing
50
Results of AST
Trial 1 (Streptococcus pyogenes) for containing the
pure leaf extract of guava, papaya, and their combination
showed no clear inhibition zones;however Erythromycin which
is the control drug showed clear inhibition zone of about
40mm.
Trial 2 (Streptococcus pyogenes) for containing the pure
leaf extract of guava, papaya, and their combination showed
clear inhibition zones as well asAmoxicillin which is the
control drug and showed clear inhibition zone of about 25mm.
51
Trial 1 (Pseudomonas aeruginosa)forcontaining the pure leaf
extract of guava, papaya, and their combination showed clear
inhibition zones as well asLevoflaxacin which is the control
drug and showed clear inhibition zone of about 46mm.
Trial 2 (Pseudomonas aeruginosa)forcontaining the pure leaf
extract of guava, papaya, and their combination showed clear
inhibition zones as well asClindamycin which is the control
drug and showed clear inhibition zone of about 44mm.
52
CURRICULUM VITAE
NAME:Adrian R. Cayabyab
ADDRESS:Zone 9 Tuliao, Sta. Barbara, Pangasinan
PHONE #:09478758813
EMAIL ADDRESS:adrian.cybcyb@gmail.com
PERSONAL INFORMATION
AGE: 17
GENDER: Male
BIRTHDAY: May 11, 2001
CIVIL STATUS: Single
NATIONALITY: Filipino
RELIGION: Christian (Born Again)
PARENTS:Mr.and Mrs. Alfredo Q. Cayabyab Jr.
EDUCATIONAL ATTAINMENT
PRIMARY: Blessed Generation Dream Academy

2012 – 2013
SECONDARY: Lyceum Northwestern University Special

Science High School
2016 - 2017
53
CURRICULUM VITAE
NAME: Paolo V. Codiñera
ADDRESS: 212 CalitBinmaley, Pangasinan
PHONE #: 09770838911
EMAIL ADDRESS: codinerapaolo@gmail.com
AGE: 18
GENDER: Male
BIRTHDAY: January06, 2001
RELIGION: Roman Catholic
PARENTS: Mr. and Mrs. Luis B. Codinera Jr.
PRIMARY: Harvent School

2012 - 2013
SECONDARY: La Marea Academy

2016 – 2017
54
CURRICULUM VITAE
NAME: Anne Yvonne Q Reyes
ADDRESS: 135 A.B. Fernandez East DagupanCity
PHONE #: 09452012697
EMAIL ADDRESS: anneyvonne.reyes@gmail.com
AGE: 18
GENDER: Female
BIRTHDAY: June 23, 2000
PARENTS: Mr. and Mrs. Romulo SM. Reyes
PRIMARY: Creative Montessori Center

2012 - 2013
SECONDARY: Francisco Q. Duque Medical Foundation Special Science High

School
2016 – 2017
55
CURRICULUM VITAE
NAME: Ruth S. Ganalongo
ADDRESS: Lot 10 blk 3 Sta. Dolores S.T. Villa Sta. Barbara, Pangasinan
PHONE #: 09454786568
EMAIL ADDRESS: ruthganalongo@yahoo.com
AGE: 18
GENDER: Female
BIRTHDAY: September 15, 2000
RELIGION: Christian (Born Again)
PARENTS: Mr. and Mrs. Jun L. Ganalongo
PRIMARY: Blessed Generation Dream Academy

2012 - 2013

School
2016 – 2017
56
CURRICULUM VITAE
NAME: Camille C. Posadas
ADDRESS: #549 Malta village Dagupancity, Pangasinan
PHONE #: 09369984740
EMAIL ADDRESS: camillecposadas2000@gmail.com
AGE: 18
GENDER: Female
BIRTHDAY: June 23, 2000
PARENTS: Mr. and Mrs. Edwin E. Posadas
PRIMARY: Creative Montessori Center

2012 - 2013

School
2016 – 2017
57

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SYNERGISTIC EFFECT OF GUAVA (Psidiumguajava) AND PAPAYA

(Carica papaya L.) LEAVES EXTRACTS AGAINST Streptococcus

Rationale and Background of the study

Papaya plant (Carica papaya L.) is

known as the source of Papain enzyme. Papain is a proteolytic enzyme present

catalyzes the breakdown of proteins by hydrolysis (addition of a water

molecule. Papain is a popular folk remedy to reduce pain, inflammation, and

the Philippines and it could be used as food or for medicinal puposes.

Psidium guajava L. leaf (family Myrtaceae) has a long history of folk

medicinal uses in Egypt and worldwide as a cough sedative, an anti-diarrheic,

in the management of hypertension, obesity and in the control of diabetes

inflammatory propertiesantimicrobialhepatoprotective and antioxidant

activities. In addition, the leaf extract is used in many pharmaceutical

preparations as a cough sedative.

also known as Streptococcuspyogenesand a hospital acquired infection called

Pseudomonas aeruginosa and spread of deadly drug-resistant strains pose a

researchers chose this plant leaves extract to go against

StreptococcuspyogenesandPseudomonas aeruginosa because it has the

antibacterial potential in crude extracts of leaves against clinical bacterial

To determine the antimicrobial potential of guava (Psidium guajava) and

papaya (carica papaya) leaf extracts against Streptococcus pyogenes made up of

non-motile and non-sporing cocci and Pseudomonas aeruginosa. The leaves

the leaves. This study provides scientific understanding to further determine

the antimicrobial values and investigate other pharmacological properties.

Input Process Output

 Guava  AST  Zone of

Statement of the Problem

leaves extracts that contains antibacterial properties that can inhibit

answer the following:

inhibit Streptococcus pyogenes and Pseudomonas aeruginosa.

2.) What is the mechanism of action.

against Streptococcus pyogenes and Pseudomonas aeruginosa.

Significance of the Study

leaves as an extract against Streptococcus pyogenes and Pseudomonas

the bacteria. Its contribution to individuals is to help them fight the

Streptococcus pyogenesand Pseudomonas aeruginosathrough the use of guava

leaves and papaya leaves extract.

fever and to eradicate the organisms that causes pseudomonal infections.

Scope and Delimitation

and papaya leaves extract against Streptococcus pyogenes and Pseudomonas

aeruginosa. The experiment will be performed at Lyceum-Northwestern

University Medical Technology Clinical Laboratory. The zones of inhibition

susceptibility perception will be measured in millimeters and interpreted as

resistant, intermediate or susceptible.

 Control Drug – Refers to the treatment (Erythromycin) that served as

basis of comparison on the level of effectiveness of the different

 Crude Extraction Method -which undergoes fermentation and

generates alcohol in situ;this facilitates the extraction of the active

constituents contained in the plant material

 Guava Leaves- full of antioxidants, anti-inflammatory agents,

antibacterial, and even tannins that can have significant health

benefits, from treating stomach troubles to chronic diseases like

 Leaf Extract Combinations of Guava and Papaya leaves – Refers to

the extracts that were used against Streptococcus pyogenes.

including cancer, allergies and as a component in some vaccines.

 Pseudomonas aeruginosa - an important cause of gram-negative

infection, especially in patients with compromised host defense

mechanisms. It is the most common pathogen isolated from patients

who have been hospitalized longer than 1 week, and it is a frequent

cause of nosocomial infections.

 Resistant – means that the bacterium did not show zones of

inhibitions to the different treatments used.

 Streptococcus Pyogenes – Refers to bacterium causing strep throat as