Medina College

Maningcol, Ozamiz City

Philippines, 7200

A Research Paper
Presented To
The Faculty on the College Of Pharmacy
Medina College
Ozamiz City, Philippines
ANTIMICROBIAL ACTIVITY OF AN OINMENT PREPARATION OF
YELLOW DRAGON FRUIT PEEL AGAINST STAPHYLOCOCCUS
AUREUS

A Research
Presented to
the Faculty of the College of Pharmacy
Medina College
Ozamiz City, Philippines

In Partial Fulfillment
of the Requirements for the Degree
BACHELOR OF SCIENCE IN PHARMACY

by

Malinis, Rush Godwin R.

Pormilos, Marizel G.
Garcia, Oshin Mae

APRIL 2022
 CHAPTER 1
INTRODUCTION
Rationale of the Study

Dragon fruit is also known as “pitaya” or “strawberry pear”, it grows on a

climbing cactus plant that can grow from 15-20 feet high and can live for as long
as two decades. In Asian nations, where traditional practitioners utilize herbal
medicines to prevent and cure ailments, dragon fruit is also regarded a medicinal
plant (Sofowora et al. 2013).
Pitaya or Dragon fruit is also known to have some health benefits that
include preventing memory loss, anticancer activity, control of blood glucose
level in diabetes, anti-oxidant properties, aids in healing of wounds and others
(Meixner, M. 2018). As the need for pharmaceuticals grows in response to our
growing population and increasing variety of ailments, researchers continue to
look for new and more effective drug sourced from this medicinal plant.
In medicine, infectious disease is a condition caused by an agent, usually
bacteria that compromise a person's health. In many circumstances, infectious
disease can be transmitted from one person to another, either or indirectly.
Infectious diseases are a leading cause of death worldwide, particularly in low-
income countries, especially in young children (Baylor College of Medicine,
2021).
Dragon fruit pulp appears to be a promising source of pectin
(Mahattanatawee et al., 2006) even in the peel of a dragon fruit (Jamilah et al.,
2011). Dietary fiber can also be found in the peel. Fiber, which accounts for 69
grams per 100 grams of dried peel (Jamilah et al., 2011). Dragon fruit peel is the
major by-product left over from either fresh consumption or fruit processing. It
makes up about a one-fourth of the entire fruit. Because the dragon fruit skin may
contain some useful components, its qualities must be exploited and understood.
Both dragon fruit peels contained a substantial level of betacyanin (a red/purple
pigment) in the study. The level of total betacyanin in the red-flesh dragon fruit
peel is double in white-flesh peel (20 mg/100 g vs. 10 mg/100 g). The peel of two
kinds of dragon fruit has been identified as a possible natural source of

1
betacyanin. Betanin, phyllocactin, hylocerenin, and related derivatives make up
the majority of the betacyanin pigment in dragon fruit (Kim et al., 2011).
Plants contain compounds which has the potential as an antibacterial with a
new and unexplored mechanism of action resistance. One of the plants that
possess antibacterial property is the red dragon fruit (Hylocereus polyrhizus). The
peel of red dragon fruit (Hylocereus polyrhizus) is one of the plants that contains
antibacterial compounds. The phytochemical components found in red dragon
fruit peel extract included flavonoids, phenols, hydroquinone, steroids,
triterpenoids, saponin, and tannins. Effectiveness of the red dragon fruit
(Hylocereus Polyrhizus) peel extract as the colorant, antioxidant, and
antimicrobial on beef sausage. (F. M. Manihuruk et. al., 2017).

Staphylococcus aureus is a highly virulent pathogen posing an increasing

challenge both in community-acquired and nosocomial infections. (Emori, T. G.
et .al.1993). Staphylococcus aureus has generated a lot of interest over the last
half century due to its ability to rapidly adapt to antibiotic pressure and develop
antibiotic resistance. (Chambers HF, FR DL2009). One of the main causes of
pathogenic microorganism resistance is inappropriate antibiotic use, which leads
to inadequate treatment. Staphylococcus aureus is one of these resistant germs.
Thus staphylococci are easily spread between animals and under certain
conditions to humans as well as by skin to skin contact, but also by contact with
excretions which contain staphylococci, such as saliva, or aerosols released during
sneezing and coughing. Moreover, staphylococci may be spread by animal
products, such as non-pasteurized milk (Werckenthin, C. 2001). This study aimed
to analyze the effectiveness of red dragon fruit peel extracts (Hylocereus
polyrhizus) as antibacterial agent against Staphylococcus aureus.

2
Objectives of the Study
This study aims to determine the Antibacterial activity of
Red Dragon fruit (Hylocereus polyrhizus) peel on Susceptibility of
Staphylococcus aureus.
Specifically, this study seeks to:
1. To determine if there is a significant differences in antibacterial activity in the
ointment formulation of Hylocereus polyrhizus fruit peel (1%, 2%, and 5%).
2. To determine the phytochemical constituent present in the plant sample.
3. To determine if Hylocereus polyrhizus fruit peel extract has effect in
Staphylococcus aureus.

Significance of the study

Medical Students .The result of this study will be greatly beneficial to
the students of Medina College, especially to those students who wants to use the
Dragon fruit (Hylocereus polyrhizus) as their future research study.
Community. This study would benefit the community to have better
understanding on how dragon fruit Hylocereus polyrhizus peel extract will
become alternative treatment on Staphylococcus aureus as ointment form.
Future Researchers. This study may serve as reference material for
future researchers as they intend to conduct a research regarding herbal medicines
that can treat bacterial infections.
Manufacturers. This study would also help the manufacturers to create a
novel and innovative product from an herbal plant like the dragon fruit plant
which would be effective, safe and affordable for the community.

Hypothesis
In this study, the researchers will use the peel extract of Hylocereus
polyrhizus to test its potential antibacterial activity on Staphylococcus aureus.
 There is no significant differences on the antibacterial activity in the
ointment formulation of Hylocereus polyrhizus fruit peel.

3
  There is no significant differences on Hylocereus polyrhizus fruit peel on
Staphylococcus aureus
Scope and limitation
The study will focuses on the effectiveness of Hylocereus polyrhizus fruit
peel on Staphylococcus aureus as an ointment. The determination of the activity
will be based on disk diffusion method (Kirby Bauer) and this will be performed
in the laboratory of Medina College, Ozamiz City.

4
Definition of Terms
Antimicrobial. This agent is used in the treatment and prevention of
infectious diseases caused by strains of bacteria through killing or inhibiting the
growth of these bacteria while the body's natural defenses work in concert to
eliminate the infection.
Dragon fruit. The fruit of a widely cultivated cactus (Hylocereus undatus)
of Mexico and Central America that usually has bright pink or red skin and white
or sometimes red, mildly sweet flesh with numerous tiny black seeds. This red
dragon fruit peel extract containing phytochemical compounds was effective as an
antibacterial agent and natural antioxidant.
Ointment. A semisolid preparation for external application to the skin or
mucous membranes. Dragon fruit’s peel extract would be made to have an
antibiotic ointment that can use to help treat or prevent infection that was caused
by of Staphylococcus aureus and Bacillus Cereus.
Phytochemical. A plant compound that is thought to have health-protecting
qualities. These are natural compounds that we can possibly observe and
determine in Hylocereus undatus, if it really consists any antibacterial compounds.
Infectious diseases- Diseases that was caused by bacteria, viruses, fungi or
parasites but this study only focuses on the infections caused by Staphylococcus
aureus and Bacillus Cereus which are all gram positive bacteria.
Minimum Inhibitory Concentration (MIC). The lowest concentration of
an antimicrobial drug that will inhibit the visible growth of a microorganism after
overnight incubation.
Glycosides. A compound formed from a simple sugar and another
compound by replacement of a hydroxyl group in the sugar molecule. Many drugs
derived from plants are glycosides.
Flavonoids. A group of natural substances with variable phenolic
structures, are found in fruits, vegetable, grains, barks, roots, stems, flowers, tea
and wine.
Alkaloid. Any of class of nitrogenous organic compounds of plant origin
which have pronounced physiological actions on humans.

5
 Saponins. Are water soluble phytochemicals which are characterized by
having detergent properties and produce a foam when dissolved in water.
Tannins. Are water soluble polyphenols that are present in many plants.
Disk Diffusion Method. A method to determine antimicrobial susceptibility
to anitibiotics with the use of filter paper disk containing unknown concentrations.

6
 CHAPTER 2
REVIEW OF RELATED LITERATURE AND STUDIES

Hylocereus polyrhizus
Hylocereus polyrhizus or red pitaya (Weber) is a Britton & Rose is native to
Mexico but is now cultivated worldwide. In Philippines it’s called “dragon fruit”.
A Dragon fruit belongs to the Cactaceae family. It originated in Southern Mexico
and Central America. The Bahamas, Bermuda, Indonesia, Colombia, Israel,
Philippines, Myanmar, Malaysia, Mexico, Nicaragua, northern Australia,
Okinawa (Japan), Sri Lanka, southern China, southern Florida, Taiwan, Thailand,
Vietnam, and the West Indies are among the countries where it is commercially
grown. Their appealing color and flavor, global production is rapidly expanding,
and new fruit crops in arid locations are being developed (Mizrahi, 2014, 2015).

Yellow dragon fruit’s taste can best be described as mild and sweet with floral notes, bearing
some resemblance to kiwifruit or cactus pear. The flesh also a slight crunch that’s somewhat
softer than that of watermelon.Due to its mild flavor, yellow dragonfruit a delicious snack on
its own as well as an excellent addition to smoothies.

Yellow Dragon fruit have an oblong shape and are slightly smaller in size than the more
common red varieties. The fruits measure approximately 10 centimeters long and 7
centimeters wide. Their size depends on climatic conditions and are often categorized into
three different size groups. Their thick yellow skin is covered in small knobby protrusions,
which when immature displays small spines that will naturally fall off as the fruit matures.
Beneath the skin is a dense white flesh containing numerous petite, edible black seeds. Yellow
Dragon fruit has a crisp, juicy texture and very sweet, tropical flavor with floral hints and no
acidity.

Yellow Pitaya is available year-round with a peak in the early summer and fall months.

Yellow Dragon fruit are the fruits of a climbing cactus known for its large, fragrant flowers
that bloom for only one to two nights. Botanically known as Selenicereus megalanthus or
Hylocereus megalanthus, the fruits are also known as Yellow pitahaya or Pitaya Amarillo.
This variety is considered the “real” Yellow Pitaya and is said to be the sweetest of all the
dragon fruits.

7
Yellow Dragon fruit is a good source of magnesium, phosphorus and potassium. They contain
dietary fiber and protein and have higher amounts of calcium than the red skinned varieties.
They provide small amounts of iron, vitamins A and C, and niacin. Yellow Dragon fruit are a
good source of antioxidants and beneficial fatty acids. The edible seeds contain high amounts
of omega-3 fatty acids that are beneficial for a healthy heart.

The plant is a cactus vine that

thrives in dry climates. It grows best in
soil with a high level of organic elements
due to its epiphytic nature. The plant is
also known as the "moonflower" or "Lady
of the Night" since its blossoms only bloom at night. The flowers are white and
huge, measuring 20 cm or more in length and bloom for only one night. When in
bloom, they have a bell-shaped shape and are aromatic. Pitahaya trees can have
between four to six fruiting cycles in one year. It can be reproduced by seed or by
stem cuttings (Blancke, R. 2016).
The Hylocereus polyrhizus is high in antioxidants that help to reduce
degenerative diseases such arthritis, arteriosclerosis, cancer, heart diseases,
inflammation and brain dysfunction. This fruit is also high in fiber and vitamins,
which can aid digestion, prevent colon cancer and diabetes, remove hazardous
chemicals like heavy metal, and help control cholesterol and blood pressure
(Susanti, 2012).

Characteristics of whole dragon fruit

The physical qualities of two types of dragon fruit were investigated: white-
flesh dragon fruit (H. undatus) and red-flesh dragon fruit (H. polyrhizus). Thirty
dragon fruits from each species were chosen to study the appearance and calculate
the average size, weight, and proportion of the fruit. The complete white-flesh
dragon fruit resembled the red-flesh dragon fruit in appearance. Both white-flesh

8
and red-flesh species have a brightly red peel on their ovary fruit. They feature
reddish scales (special leaves) with greenish tips, although red-flesh dragon fruit
scales were clearly shorter than white-flesh dragon fruit scales. The fruits have an
intriguingly exotic and oriental aspect as a result of their unusual appearance. It
shows that the physical attributes of both white-flesh and red-flesh species in
terms of dimension, weight, and proportion. The white-flesh dragon fruit was
much longer, wider, and heavier than the red-flesh dragon fruit (14 cm compared
to 13 cm, 11 cm compared to 7 cm, and 445 g compared to 358 g, respectively,
Wichienchot et al (2010).
Additionally, the weight proportion of the peel, pulp and seed of dragon
fruit was also estimated based on the weight of the whole fruit and expressed as
percentage (w/w). Despite the difference in dragon fruit size, no significant
differences in the ratio of the peel, pulp and seed between the two species of
dragon fruit were observed. Both white-flesh and red-flesh species contained
about two-thirds of pulp (67%) and around 8% of seeds. There was also a sticky
mucilage layer between the flesh and seed of dragon fruit. The dragon fruit peel
accounted for approximately 25% of the whole fruit weight. It is normally
discarded during dragon fruit processing. The results are slightly different from
the data found by (Lim et al. 2010) where dragon fruit pulp is about half and
dragon fruit peel is closely to one fourth of the whole fruit. This is probably due to
the differences in the origin, plantation and harvesting conditions.

Antibacterial Activity Test

To evaluate or screen the in vitro antimicrobial activity of an extract of a pure
compound, a variety of laboratory methods can be used. The Kirby-Bauer test,
known as the disk-diffusion method, is the most widely used antibiotic
susceptibility test in determining what choice of antibiotics should be used when
treating an infection. This method relies on the inhibition of bacterial growth
measured under standard conditions. For this test, a culture medium, specifically
the Mueller-Hinton agar, it is uniformly and aseptically inoculated with the test
organism and then filter paper discs, which are impregnated with a specific

9
concentration of a particular antibiotic, are placed on the medium. The organism
will grow on the agar plate while the antibiotic “works” to inhibit the growth. If
the organism is susceptible to a specific antibiotic, there will be no growth around
the disc containing the antibiotic. Thus, a “zone of inhibition” can be observed
and measured to determine the susceptibility to an antibiotic for that particular
organism. The measurement is compared to the criteria set by the Clinical and
Laboratory Standards Institute (CLSI). Based on the criteria, the organism can be
classified as being Resistant (R), Intermediate (I) or Susceptible (S). (Student
Health Center Manuals, 2019).
The purpose of the Kirby-Bauer disk diffusion susceptibility test is to
determine the sensitivity or resistance of pathogenic aerobic and facultative
anaerobic bacteria to various antimicrobial compounds in order to assist a
physician in selecting treatment options for his or her patients. The pathogenic
organism is grown on Mueller-Hinton agar in the presence of various
antimicrobial impregnated filter paper disks. The presence or absence of growth
around the disks is an indirect measure of the ability of that compound to inhibit
that organism (Hudzicki, J., 2009).

From previous studies, the fruit possessed antimicrobial activities against

pathogenic microbial with different assay. The use of antibiotics irrationally
causes many pathogenic microbes to adapt to their environment and become
resistant to synthetic antibiotic drugs. This has encouraged the development of
novel antimicrobial substances derived from natural products. Therefore, in this
study will conducte to evaluate the antibacterial activities of dragon fruit peel
pigments against pathogenic bacteria. (Rudi Hendra,2019)

Over the years, a wide variety of natural colour sources have been
identified. One source, the dragon fruit is known to impart colours to products,
such as food and drink. However, there have been limited studies done to
determine phenolic compounds and antibacterial activity of the dragon fruit peel
(H.polyrhizus) via Microwave Assisted Extraction (MAE) method. Both this

10
information can escalate the potential role of dragon fruit as a natural colour
source. This study aimed to identify the types of bioactive compounds (phenolic
compounds) and antibacterial activity of dragon peel. To achieve this objective,
MAE was used to extract bioactive compounds from the dragon peel as it
maintained the integrity of the compound. Based on the results, 13 types of
phenolic compounds were identified from the pitaya peel extract via qualitative
research using library database matching which include quinic acid, cinnamic
acid, quinic acid isomer, 3,4-dihydroxyvinylbenzene, isorhamnetin 3-O-
rutinoside, myricetin rhamno-hexoside, 3,30-di-O-methyl ellagic acid,
isorhamnetin aglycone monomer, apigenin, jasmonic acid, oxooctadecanoic acid,
2 (3,4-dihydroxyphenyl)-7-hydroxy-5-benzene propanoic acid and protocatechuic
hexoside conjugate.  The dragon fruit peel extract was also found to have small
antibacterial effect on the Gram-positive, Staphylococcus aureus (S.aureus) and
Gram- negative, Escherichia coli (E.coli). The SEM demonstrated that cell wall
disruption of dragon fruit peel caused by microwave radiation from MAE
appeared to be the main reason for rapid extraction of bioactive compounds. In
conclusion, the study established that dragon fruit peel extract is a natural colour
source with an abundance of phenolic compounds and minimal antibacterial
activity, which could be used in the food and cosmetic industries.
Yellow dragon fruit contains antibacterial content of betalain pigments
which belongs tobetacyanin the alkaloids, the phenols such as flavonoids and
vitamin C, while the peel of this fruit contains flavonoids, alkaloids and these
terpenoids. The content of phenols, such as flavonoids are naturally found in
plants and vegetables. Phenolic compounds may inhibit the growth of bacteria
with the ability of damaging the cytoplasmic membrane and proteins as well as
inactivated some bacterial enzymes. Polyphenols are not only beneficial to health
but they also have antimicrobial and antiviral properties.( Mat Zain.2019)
The usage of fruit is only as consumption of fruit pulp alone, about 30-35%
of the fruit peel has no further use. In fact, the results of previous studies shows
the total content of phenolic substances in the peel of red dragon fruit is larger
than in the red dragon fruit pulp with 70% ethanol extract. Red dragon fruit peel

11
extract is also proved to be able to inhibit the growth of Salmonella pullorum and
Staphylococcus aureus. Based on the explanation above, researchers are interested
in examining the activity of antibacterial extracts of dragon fruit (Hylocereus
polyrhizus) peel and pulp through it’s inhibit power against the growth of S.
aureus.

Health Benefit of Dragon Fruit

Dragon fruit mostly as fresh fruit as relieving thirst due to it contains
high water level compared with other nutrient. Dragon fruit can also take
the form of juice, jam, or preserves according to the taste needed. Regular
consumption of Dragon fruit helps in fighting against cough and asthma;
also it helps for healing wounds and cuts quickly due to it contains high
amount of vitamin C. However, the high level of vitamin C found in Dragon
fruit plays an important role to enhance immune system (Parmar, M., et a.,
2019).
Moreover, Dragon fruit is also rich in flavonoids that act against cardio
related, also dragon fruit aids to treat bleeding problems of vaginal discharge.
As Dragon fruit rich in fibers, however it aids in digestion of food. Dragon fruit is
also packed with B vitamin group (B1, B2 and B3) which possess an important
role in health benefit. Vitamin B1 helps in increasing energy production and in
carbohydrate metabolism, Vitamin B2 in Dragon Fruit acts as a multivitamin;
however, it aids to improve and recover the loss of appetite. Vitamin B3
present in dragon fruit plays an important role in lowering bad cholesterol
levels; it provides smooth and moisturizes skin appearance. As well as it
improves eyesight and prevent hypertension. Dragon fruit contains high level of
phosphorus and calcium it helps to reinforce bones and play an important role
in tissue formation and forms healthy teeth. It also contains phenols and
antibacterial property that is good for treating bacterial infections. (Cheah, L., et
al, 2016).

12
 Staphylococcus aureus
S. aureus has long been recognized as one of the most important bacteria that
cause disease in humans. It is the leading cause of skin and soft tissue infections
such as abscesses (boils), furuncles, and cellulites. Although most staph infections
are not serious, S. aureus can cause serious infections such as bloodstream
infections, pneumonia, or bone and joint infections. Most skin infections resolve
without treatment, however, some infections require incision and drainage or
antibiotic treatment to cure the infection. (Minnesota Department of Health Fact
Sheet, 2010).
S. aureus are capable of resisting to the mechanical stress, antimicrobial
therapies and the innate immune system of the host, becoming a serious chronic
condition when protected by the biofilm. .Staphylococcus aureus is the main
causative agent of biofilm-forming infections on medical devices such as
orthopedic implants, ventilators, intravenous catheters, heart valves,
pacemakers and vacuums. Periodontitis and peri-implant diseases that cause
wounds (e.g. foot and pressure ulcers, diabetes), chronic endocarditis, eye
infections (conjunctivitis, keratitis), osteomyelitis sepsis syndromes and
metastatic infections are mostly derived from biofilm-forming strains
(Stoodley, P., et al., 2015).
The fruit flesh from yellow dragon has been widely studied both regarding
its content and antioxidant activity, but dragon fruit peel which takes up to
30-35% of the fruit weight is often only disposed of as waste. Whereas based
on the results of the phytochemical component test, dragon fruit peel
contains several compounds which possess antibacterial activity, including
phenol hydroquinone, flavonoids, triterpenoids, steroids, saponins, tannins
and alkaloids. In addition, red dragon fruit peel also has proven to have a
broad antibacterial activity, both towards gram-positive and gram-negative
bacteria, one of which is against Staphylococcus aureus (Yuani Setiawati)
H. polyrhizus or known as dragon fruit is from
the Cactaceae family and has become a subject of
interest to many researchers mainly due to its unique
taste, shape and color which can be utilized as a

13
natural colorant. Initially, scarce research was done
on this plant until the last decade, in which ample
amount of articles were published on this plant.
H. polyrhizus or known as dragon fruit is from the Cactaceae family and has
become a subject of interest to many researchers mainly due to its unique taste,
shape and color which can be utilized as a natural colorant. Initially, scarce
research is done on this plant until the last decade, in which sample amount of
articles were published on this plant (Mizrahi, 2014).

CHAPTER 3
METHODOLOGY

Research Design
This study employee the experimental design with quantitative measures. In
determining the antibacterial activity of (Hylocereus polyrhizus) peel extract on

14
 Staphylococcus aureus using Disk –Diffusion Method adapted from Kirby- Bauer
technique.

Research Setting
The experiment will be conduct at the Instrumental Laboratory at Medina
College, Ozamiz City which is limit to the peel extract of the said plant. The
bacteria cultures is going to obtain from Medina General Hospital, Ozamiz City.

Collection and Identification of the Plant

Fresh Peel of Dragon fruit (Hylocereus polyrhizus) is being collect from

Brgy. Austin Heights, Kolambugan Lanao del Norte, Philippines. Sampling will
be done before sunrise.

Preparation of Plant Extract

A total of 10 Dragon fruits were purchase. The peel of Dragon fruit are
separate from peel and pulp of the fruit, cut into thin pieces, dried under the sun
cover in a black cloth, and follow by oven at a temperature of 50◦C for 3hrs or
more until dry. After that, it will blended to get dry powder of red dragon fruit’s
peel. Maceration with 95 % ethanol, a hundred grams of the powder peel soak in
500 mL of 95% ethanol in conical flask for 3 days. After 3 days, the mixture is
filter using a fine muslin cloth and follow by filter paper Whatman (No.1). The
filtrate evaporate under reduce pressure in rotary evaporator.

weight of extract
% yield= x 100
weight of groundplant material
Phytochemical Screening
The Hylocereus polyrhizus peel extract will subject to phytochemical
screening to determine the presence of tannins, steroids, saponins, flavonoids,
terpenoids, cardiac glycosides, and alkaloids.
Test for cardiac glycosides

15
 1ml of concentrated Sulfuric Acid (H2SO4) is prepare in a test tube. 5ml of
extract from the plant sample will be mix with 2 mL of glacial acetic acid
(HCH3CO2) containing one drop of Ferric Chloride (FeCl3). The mixture will be
add carefully to 1 mL of concentrated H2SO4 so that the concentrated H2SO4
will underneath the mixture. The appearance of brown ring indicate the presence
of cardiac glycosides.
Test for tannins
5mL of extract plants will be added to 10 mL of distilled water on a test
tube and will be filter using a filter paper. 2 mL of 5% Ferric Chloride (FeCl) will
be add to the filter sample. Brownish green or blue black coloration will be
observe for the presence of tannins.
Test for steroids
2ml of acetic anhydride will be added to a 5 mL extract of plant sample
with 2 mL of Sulfuric Acid (H2SO4). Violet to blue or green precipitate will be
observe for the presence of steroids.
Test for saponins
5mL of extract plant will be added to 10mL distill water and shaken
vigorously to obtain a stable persistent froth. Persistent frothing indicate the
presence of saponin.
Test for flavonoids
Few drops of 1% Ammonia (NH3) solution will be add to 5mL extract of
plant sample in a test tube. Yellow coloration indicated the presence of
flavonoids.
Test for terpenoids
5ml extract of the plant sample will be added with 2 mL Chloroform
(CHCl3) in a test tube. Then, 3 mL of Sulfuric Acid (H2SO4) will be add
carefully to the mixture to form a layer. The formation of the reddish brown
interface indicates the presence of terpenoids.
Test for alkaloids
5ml of the extract will be prepare in a beaker and 200 mL of 10%
HCH3CO2 in ethanol (C2H5OH) will be add. The mixture will be filter and the

16
extract will allow to become concentrate in a water bath until it reach one fourth
of the original volume. Concentrated NH4OH will be add. Formation of the white
precipitate or turbidity indicate the presence of alkaloids (Trease and Evans,
1983).

Formulation of ointment
In formulation of ointment, extract of Hylocereus polyrhizus was add in
formulation. The ingredient and procedures was follow by the researchers from
the book “Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems 10th
Edition”.
Ingredient Pharmaceutical Original Percentage Working
use formula formula
Hylocereus Basis 300g 3% 9g
polyrhizus fruit peel
extract
White wax Excipients 50g 3% 1.5g
White Excipients 650g 3% 19.5g
petrolatum
q.s 1000g to make:
30g

final quantity desired

conversion fcator=
total quantity of original formula
Working formula = (quantity of original formula) * (conversion factor)

Hylocereus polyrhizus peel after the extraction process were formulate as

ointment by different concentration 1%, 2% and 5%. The ointment bases at
concentrations of 1%, 2%, and 5% were chosen as the final base for preparation.
Color, homogeneity, consistency, and phase separation were all visually checked
in the formulations. A pH meter will be used to determine the pH.
In a top loading balance, the quantities of each formulation will be precisely
weigh. A specific amount of white petrolatum will be divided among three
containers. Red dragon fruit peel extract at concentrations of 1%, 2%, and 5% will

17
be added to separate containers containing white petrolatum and mixed
thoroughly for even distribution.

Preparation of Positive and Negative Control

The positive control is amoxicillin ointment form and for the negative
control is petrolatum.

Preparation of Test Solution

The solubility test solvent serves as 100% test solution. 100% solution
was then diluted to a concentration of 1%, 2%and 5% are subjective to
analysis.

Disc diffusion assay

Kirby-Bauer disc diffusion assay are used for antibacterial activity to
observe the inhibitory spectrum of the extracts against Staphylococcus aureus. All
discs were ensure to fully dry before applying on the bacterial, and then the tools
and media were sterilized by autoclave at the temperature of 121oC for 15
minutes. Preparing a petri dish containing nutrient agar for S. aureus bacteria,.
Using a sterile cotton swab, the bacteria will spread on the surface of the nutrient
medium until the bacteria cover the medium. Amoxicillin are used as positive
control and petrolatum will used as negative controls. The paper disc containing
the test material will place on the surface of the media that will be treat with
bacteria and incubated for 24 hours at 37oC. Antibacterial activity will evaluate
by measuring the diameter of the inhibition zone (IZ) around the discs will
calculated using a caliper.

18
 Determination of Minimum Inhibitory Concentration (MIC)
Plates showing zones of inhibition from the disc diffusion assay will test to
determine MIC values by broth macro dilution method
A sterile 96-wells flat-bottomed microplate was filled with 100 μL of inoculum
and 100 μL sample of different concentrations. It will transfer to each microplate
disc. The final volume in the disc will adjust at 100 μL with the concentration
100mg/mL. Serial dilutions were made to obtain concentration ranging from 1
mg/mL to 100 mg/mL. The control used include blank containing bacteria,
Amoxicillin as the positive control while 0.1% distilled water as the negative
controls. Then 0.5 mL of standardized bacterial suspension will add to the tubes
containing the previously prepare 0.5 mL of diluted extract, the resulting in a
recommend final cell count of about 5 x 10 5 CFU/mL. The tubes will incubate for
24 hours at 37°C and turbidity will measure. The next day, the turbidity of each
tube's fluid will check for signs of bacterial development. The MIC value will
calculate using the lowest concentration of extract dilution that show no
observable growth (clear). The tubes were then incubate for another 24 hours
before being check for the presence or growth of bacteria after 48 hours (cloudy).
The MIC value after 48 hours will be recording. MIC is define as the lowest
concentration where no visible turbidity is observe in the test tube.
Statistical analysis
All analyses were performed in triplicate and
data reported as mean±standard deviation (SD). Data
were subjected to analysis of variance (ANOVA).
Results were processed by Excel (Microsoft Office
2007) and SPSS Version 17.0 (SPSS Inc., Chicago

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Flow Chart of Sample Collection

Collection of Plant Extract

10 pieces of Fresh red dragon fruit Hylocereus polyrhizus will collect before
sunrise and wash thoroughly with tap water

The Peel will be removed by hand and cut into thin pieces, dried under the sun
covered in a black cloth.

The dried peel will ground using milling machine.

Maceration with 95 % ethanol, a hundred grams of the powder peel soak in 500 mL
in conical flask for 3 days

After 3 days, the mixture is filter using a fine muslin cloth and follow by filter
paper Whatman (No.1).

The filtrate evaporate under reduce pressure in rotary evaporator.

All extracts were stored at 4°C until required for use. 

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 Flow Chart of Phytochemical Screening

Preparation of Positive
Phytochemical and Negative Control
Screening

Organism Positive control (+) Negative control(-)

Staphylococcus aureus Amoxicillin White Petrolatum

Test for Glycoside

Test for Terpenoids
Keller Kellani test
Salkowski test
Borntrager test

Test for Tannins Test for Flavonoids

Ferric Chloride Test Ammonia Solution Test

Test for Steroids Test for Alkaloids

Sulfuric Acid Test Mayer’s Test

Test for Saponins

21
 Kirby-Bauer disc diffusion test and Measuring of Zone of Inhibition
o All discs were ensure to fully dry before being applied on the bacterial.
o The turbidity was then adjusted to 0.5 McFarland standards (108CFU/mL) and
streaked onto Mueller-Hinton agar plates using sterile cotton swabs.
o Sterile plates containing 4mm depth of Mueller-Hinton agar were prepared and
inoculated with 0.5mL and let dry for 5 minutes.
o The plates were inverted and incubated at approximately 37°C for 12
hours.
o The diameter of zone of inhibition will measure.

Flow of Research

Preparation of Plant Extract

Phytochemical Analysis

Antibacterial Susceptibility Testing

Determination of Minimum Inhibitory Concentration

(MIC)

Measuring Zone of Inhibition

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