Antimicrobial properties of the podocarpus costalis (arius) leaf extract againts escherichia coli

ANTIMICROBIAL PROPERTIES OF THE Podocarpus costalis (ARIUS) LEAF
EXTRACT AGAINTS Escherichia coli
ELSA B. BISWELAN
DON MARIANO MARCOS MEMORIAL STATE UNIVERSITY
OPEN UNIVERSITY SYSTEM
CITY OF SAN FERNANDO, LA UNION
MASTER OF ARTS IN SCIENCE EDUCATION
MAY 2018

ACKNOWLEDGEMENT
The researcher is grateful for having a chance to meet so wonderful people and
professionals who led her through this study. It is her genuine pleasure to express her
deep sense of thanks and gratitude to the following people who helped her a lot in
finalizing this study, to wit:
Dr. Priscilla S. Abellera, Adviser, whose guidance and assistance helped her
bring this study into success;
Mrs. Armida B. Reyes, external evaluator from Lorma Colleges, whose expertise
in the field of technical Research gave light to the processes involved in this study;
Dr. Ophelia RL Cardenas, Director of DMMMSU-OUS, for her consistent
encouragement;
Dr. Mario B. Mendoza and Dr. Virgilio B. Karganilla, Members of Oral
Examination Committee, for their constructive comments, suggestions and critiquing;
Mrs Marianne Menecio, Dean of College of Pharmacy, Lorma Colleges, for
giving permission to conduct laboratory experiments in the college;
Ms. Winnielyn P. Reyes, Dean of College of Medical Technology, for her
assistance during the conduct of the susceptibility test of the plant extract.
Mr. Narciso B. Colcolen, foster parent, for his pieces of advices to finish my
master’s degree.
Mr. Severo P. Bitongan, parent, for his accompaniment during the gathering of
the (Arius) leaves along the river.
EBB

My achievement is usually born of sacrifice and the result of selflessness.
All the hard works, all the sleepless nights, struggles and downfalls, it all pays off.
In dedication to the almighty God, for providing all my needs,
my parents for making me be who I am, and my husband and son for supporting me all
the way!

TABLE OF CONTENTS
Page
TITLE PAGE…………................................................................................................ i
APPROVAL SHEET.................................................................................................... ii
ACKNOWLEDGEMENT .......................................................................................... iii
DEDICATION ............................................................................................................ iv
LIST OF TABLES........................................................................................................ v
ABSTRACT………………………………………………………………………….. vi
CHAPTER
1 INTRODUCTION
Situation Analysis .......................................................................................... 1
Objectives ..................................................................................................... 14
Time and Place of Study................................................................................ 15
Definition of Terms ..................................................................................... 15
2 METHODS AND PROCEDURES ........................................................................ 19
Research Design ........................................................................................... 19
Materials and Procedures ............................................................................. 19
Data Gathered .............................................................................................. 20
Analysis of Data............................................................................................ 27
3 RESULTS AND DISCUSSIONS ........................................................................... 28

Phytochemical Test on the Constituents of Podocarpus costalis (Arius)
Leaf extract ................................................................................................ 29
Degree of susceptibility of Escherichia coli to the different concentrations
of Podocarpus costalis (Arius) Leaf extract ……………………………… 33
Effectiveness of Podocarpus costalis (Arius) leaf extract in Various
and the Control Drug based on the zone of inhibition…. ………………. 33
Difference between the Extent of Effectiveness of the Podocarpus costalis
(Arius) Leaf Extract and Control Drug………………………................ 33
4. SUMMARY, CONCLUSIONS AND RECOMMENDATION
Summary ...................................................................................................... 36
Conclusions .................................................................................................. 37
Recommendations.......................................................................................... 39
LITERATURE CITED ............................................................................................ 41
APPENDICES ......................................................................................................... 46
a Plant Identification Certificate .............................................................. 47
b Phytochemical Testing Results ............................................................. 48
c Phytochemical Test Standard Procedures .............................................. 49
d Sensitivity Test Results Certificate ........................................................ 51
e Statistical Treatment ............................................................................. 52
f Plates ....................................................................................................... 63
g Certification of Statistician ................................................................... 64

h Certification of the English Critic .......................................................... 65
CURRICULUM VITAE ........................................................................... 66
LIST OF TABLE
Table Page
No
1 Phytochemical Test on the Constituents of Podocarpus costalis (Arius)
Leaf Extract ………………………………………………………………… 29
2 Effectiveness of Podocarpus costalis (Arius) Leaf Extract in Various
Concentrations and the Control Drug Based on the Zone of
Inhibition…………………………………………………………………… 33
3 Difference Between the Extent of Effectiveness of the Podocarpus costalis
(Arius) Leaf Extract and Control Drug…………………………….................. 3

ABSTRACT
Biswelan, Elsa B. (2018), Antimicrobial Properties of Podocarpus costalis
(Arius) Leaf Extract Against Escherichia coli. Master of Arts in Science Education.
Don Mariano Memorial State University, Open University System, City of San Fernando,
La Union.
Adviser: Abellera, Priscilla S., Ph. D.
The primary aim of this study was to determine the antimicrobial properties
and the properties of Podocarpus costalis (ARIUS) Leaf Extract. It utilized the
experimental research design. Specifically, it aimed to: (a) determine the phytochemicals
present in the ethanolic extract of Podocarpus costalis leaves;(b) determine the
antimicrobial properties and the effectiveness of Podocarpus costalis leaf extract in
various concentrations and the control drug against Escherichia coli based on the zone
of inhibition; and (c) determine the significant difference in the effectiveness of the
Podocarpus costalis leaf extract in different concentrations and the control drug.
The study found out that the leaf extract of Podocarpus costalis (ARIUS) contains
the following: phenol, tannins, flavonoids, carbohydrates and reducing sugars, Most of
these phytochemical properties are antimicrobial. Moreover, the 100 per cent leaf extract
had the highest measure of anti-Escherichia coli with a mean zone of inhibition of
11.38mm as compared to the other concentrations of 25 %, 50 & and 75 %. ARIUS leaf
extract showed significant difference in the extent of effectiveness between and among the
various concentrations and the control drug, ciprofloxacin. On the basis of the present
findings, Arius leaf-extract might be a good candicate in the search for natural
antimicrobial agent. This study provides scientific understanding to further determine the
antimicrobial values and investigate other pharmacological properties.
Keywords: Antimicrobial, phytochemical properties, zone of inhibition

Chapter I
INTRODUCTION
Situation Analysis
The plant kingdom is a treasure house of potential drugs and in the recent years
there has been an increasing awareness about the importance of medicinal plants. Drugs
from the plants are easily available, less expensive, safe and efficient and rarely have side
effects. The plants which have selected for medicinal use over thousands of years
constitute the most obvious choice of examining the current search for therapeutically
effective new drugs such as anticancer drugs, antimicrobial drugs, antihepatotoxic
compounds. According to the World Health Organization (WHO), medicinal plants
would be the best source to obtain variety of drugs. About 80% of individuals from
developed countries use traditional medicines, which has compounds derived from
medicinal plants. However, such plants should be investigated to better understand their
properties, safety and efficiency.
The search for newer sources of antibiotics is a global challenge preoccupying
research institutions, pharmaceutical companies and academia, since many infectious
agents are becoming resistant to synthetic drugs (Latha and Kannabiran, 2006). Infectious
diseases are the world’s major threat to human health and account for almost 50 000
deaths every day (Ahmad and Beg, 2001). The situation has further been complicated
with the rapid development of multidrug resistance by the microorganisms to the

antimicrobial agents available. Plants have the major advantage of still being the most
effective and cheaper alternative sources of drugs (Pretorious and Watt, 2001). The local
use of natural plants as primary health remedies, due to their pharmacological properties,
is quite common in Asia, Latin America and Africa (Bibitha et al., 2002). Podocarpaceae
are also known to have medicinal properties that benefit humans and animals . The
receptacles and leaves contain a variety of bio–active compounds such as antioxidants,
nordi–terpenes, podocarpic acid, and tatarol. Some of these compounds have
antimicrobial, fungistatic, or bacteriostatic properties. Other compounds have cytotoxic
properties that may be useful in destroying cancer.
In Germany, about 600-700 plant based medicines are available and are
prescribed by some 70% German physicians. Attention has also been drawn to the
antimicrobial properties of plants and their metabolites due to the growing incidences of
drug-resistant pathogens of clinical importance. Medicinal plants have their intrinsic
ability to resist pathogenic microorganisms. This has enabled exploitation of medicinal
plants for the treatment of microbial infections of humans by developing new
antimicrobial agents (Das et al. 2010).
The importance of herbs in the management of human ailments cannot be over
emphasized. It is clear that the plant kingdom harbors an inexhaustible source of active
ingredients invaluable in the management of many intractable diseases. Furthermore, the
active components of herbal remedies have the advantage of being combined with other
substances that appear to be inactive. However, these complementary components give

the plant as a whole a safety and efficiency much superior to that of its isolated and pure
active components (Ahmad, 2001).
The use of medicinal plants predates the introduction of antibiotics and other
modern drugs into the African continent. Since medicinal plants do not merely save
people from feeling pain but also permit them to emerge unscathed, then they deserve
investigation. The active components in these medicinal attribute are expected to be
inimical to the growth of at least some microorganisms especially the disease causing
ones e.g. Escherichia coli, Staphylococcus aureus, Proteus mirabilis, Klebsiella
pneumonia and Pseudomonas aeruginosa, therefore, many studies and researches had
been done on the antimicrobial properties of many plants.
Recently, the International Trading Corporation reported that based on their
survey, Philippines has the most expensive cost of medicines thus marginal Filipinos or
low income families cannot afford to buy their medicine. To ease medicinal needs of the
poor Filipino populace, medicinal plants support the “Health in the Hands of the People
by 2020.”
Modern medicine, with its arsenal of manufactured drugs and advanced
technological devices, presents a big disparity from the folk traditional healing with the
use of medicinal plants through comparison of their practices and principles. Suffused in
the concept of modernity is the idea of efficiency in treatment, reproducibility
of medications and predictability of results; as such, modern practitioners have criticized
traditional medicine as inferior. However, high cost of modern medicine, especially those
manufactured abroad, and their unavailability in remote areas led to the continued

dependence of rural folks on medicinal plants as their primary therapeutic
means and has resulted in the need to re-evaluate the potential of these medicinal plants
as alternative treatment resource.
Despite advancements in technologies and coming of the modern era, poverty is
still do not have access to basic necessities, such as food shelter and education; and in
such state of economic deprivation, it has been difficult for the low income group to
prioritize one of the necessities in life, health care. This compelled the
Department of Health of the Philippines to reappraise traditional medicinal
therapy, and endorse the use of the ten herbal medicines clinically proven to have
therapeutic value in the relief and treatment of various ailments in place of expensive
pharmaceutical preparations, known as the “Sampung Halamang
Gamot program.” Moreover, medicinal plants address not only the need for access to
medicine as a component of health services but also to the need increased income for
farmers and as a significant contribution to the national economy.
(http://www.docstoc.com/docs/27321322/PHILIPPINE#).
The importance of traditional medicine in improving essential health care to the
people should be recognized. At present, its use had tremendously expanded and gained
wide global acceptance and popularity. In the Philippines, the practice of traditional
medicine which was passed from generation to generation has gained a deep significance
in health delivery considering the expensive western medical treatment which most
Filipinos could not afford.

The timely of the law ((R.A 8423) otherwise known as the Traditional and
Alternative Medicine Act (TAMA) of the 1997 gave rise to the creation of the Philippines
Institute of Traditional and Alternative Care ( PITAHC) a corporation attached to DOH
to answer the present needs of the people on health care through the provision
and delivery of traditional and alternative health care (TAHC) products, services and
technologies that have been proven safe, effective and affordable. PITAHC’s upholds the
right of every Filipino for better health through the provision of safe, effective and
affordable traditional and alternative health car e products, services and technologies.
Foremost among its objectives are: (1) to encourage scientific research on and
develop traditional and alternative health care system that has a direct impact on public
health care; (2) to promote and advocate the use of traditional or alternative health care
modalities that have been proven, safe, effective, cost-effective and consistent with the
government standards on health care practice and (3) to develop and coordinate skills
training course of various forms of traditional alternative health care modalities
( www.gov.ph/pitahc/pitahcmain.htm).
The emergence of conventional antimicrobial is a serious problem that physicians
face. This necessitates constant development of newer agents, which can kill or inhibit
the growth of resistant microorganisms.
The problem of microbial resistance is growing and the outlook for the use of
antimicrobial drugs in the future is still uncertain. Therefore, action must be taken to
reduce this problem, for example the control the use of antibiotics, develop research to
better understand the generic mechanisms of resistance, and to continue studies to

develop the new drugs, either synthetic or natural. The ultimate goal is to offer
appropriate and efficient antimicrobial drugs to the patient, (Oania, 2009).
Numerous studies have been carried out extract various natural products for
screening antimicrobial property but attention has not been focused intensively on
studying the combinations of these products for their antimicrobial activity (Nita, et al.
2002).
Podocarpus is a genus of conifers, the most numerous and widely distributed of
the podocarp family, Podocarpaceae. Podocarpus are evergreen shrubs or trees usually
from 1 to 25 meters tall, known to reach 40 meters at times. The leaves are 0.5 to 15 cm
long, lanceolate to oblong or falcate (sickle-shaped) in some species, with a distinct
midrib. They are arranged spirally, though in some species twisted to appear in two
horizontal ranks. The cones have two to five fused scales, of which only one, rarely two,
are fertile, each fertile scale has one apical seed. At maturity, the scales become berry-
like, swollen, brightly coloured red to purple and fleshy, and are eaten by birds which
then disperse the seeds in their droppings. The male (pollen) cones are 5 to 20 mm long,
often clustered several together. Many species, though not all, are dioecious. There are
approximately 104 to 107 species in the genus (International Journal of Advances in
Chemical Engg., & Biological Sciences, (IJACEBS) Vol. 3, Issue 2 (2016).
Several species of Podocarpus (Podocarpaceae) are utilized in treating ailments
across the world. In Africa, four species are used traditionally in both animal and human
health. Furthermore, Podocarpus costalis have found to have inhibition against gram-
positive bacteria was stronger with a minimum inhibitory concentration (MIC) value of

98 μg/ml while for Gram-negative bacteria, the highest inhibition was against Klebsiella
pneumoniae with an MIC value of 0.33 mg/ml.
(www.sciencedirect.com/science/article/Retrieved May 13, 2017).
In nature, different types of plants produce certain chemicals that are naturally
toxic to microorganisms. These chemicals produced by plants play an essential role in the
natural defence and well-being of plants, and belong to a wide range of classes which
include the flavonoids and isoflavonoids.
Flavonoids can be classified into flavanones, flavones, flavonols, and biflavones.
Biflavonoids are linkages of flavone–flavone, flavanone–flavones or flavanone–
flavanone subunits. Naturally occurring flavonoids are polyphenolic compounds which
can be found in different parts of plants such as flowers, fruits, nuts, seeds, stems and
leaves. They can also be found in wine, honey and commonly consumed beverages such
as tea. Apart from the phytonutritional role of flavonoids in providing beneficial health
effects by the alteration of various metabolic processes, these classes of compounds have
been acclaimed for their neuroprotective effect, antiparasitic activity ,protective effect
against DNA damage and lipoperoxidation , antiviral activity, antimicrobial activity, anti-
inflammatory activity, antioxidant activity and many more.
The biflavonoids Podocarpus flavone A (PFA) and isoginkgetin (IGG) have
previously been isolated from Podocarpus neriifolius tetramethoxy amentoflavone
(TMA) on the other hand has been isolated from Dacrydium cupressinum and
Araucaria) in addition to its inhibition of tumour cell invasion by regulating
phosphatidylinositol 3-kinase/Akt -dependent matrix metalloproteinase-9 expression . It

also has an inhibitory effect on pre-mRNA splicing and some neuroprotective effects in
vitro, (Bagla, et al., 2006)
In Gregorio Del Pilar Ilocos Sur, there are abundant plants near the river. The
plant is described a small evergreen conifer which is native in the Philippines. In the town
the plant is locally called dumanay .The people in the village are not aware on the use
of the said leaves. At this point of view, it is very important for them to know about the
use of the leaves of these native plants as alternative antimicrobial medicine.
The presence of diverse molecules represented in the class of bioflavonoids and
their symmetrical or asymmetrical nature, offers an opportunity for manipulation by
synthetic chemists to further potentiate the biological activity of these useful classes of
compounds. Despite the promise and potential therapeutic relevance of this class of
compounds, very few bioflavonoids have been investigated either for their biological
activity, toxicity or as leads for the development of new drugs. To date, no information
on antimicrobial activity of these biflavonoids is apparently available. Hence, this study
was aimed at evaluating the antibacterial activity of the compounds, present in
Podocarpus costalis (Arius).
Bacterial infection is one common condition, affecting millions of individuals in
the world classified by their shapes as bacilli (shaped like a rod with a length of around
0.03 mm. Illnesses such as typhoid and cystitis are caused by bacilli (strains), cocci
(shaped like a sphere with a diameter of around 0.001 mm), spirochetes (shaped like tiny
spirals, bacteria are responsible for a range of diseases, including the sexually
transmissible infection syphilis) and vibrio (shaped like comma). Bacterial infections can

be caused by a wide range of bacteria, resulting in mild to life-threatening illnesses that
require immediate intervention. Common bacterial infections include pneumonia, ear
infections, diarrhea, urinary tract infections and skin disorders.
Urinary tract infections (UTIs) are a common type of infection caused by bacteria
(most often E. coli) that travel up the urethra to the bladder. A bladder infection is called
cystitis. If bacterial infection spreads to the kidneys and ureters, the condition is called
pyelonephritis. Cystitis is considered a lower urinary tract infection. Pyelonephritis is an
upper urinary tract infection and is much more serious.
Urinary tract infections (UTIs) are one of the most frequent clinical bacterial
infections in women, accounting for nearly 25% of all infections. Around 50–60% of
women will develop UTIs in their lifetimes. Escherichia coli is the organism that causes
UTIs in most patients. Recurrent UTIs (RUTI) are mainly caused by reinfection by the
same pathogen. Having frequent sexual intercourse is one of the greatest risk factors for
RUTIs. In a subgroup of individuals with coexisting morbid conditions, complicated
RUTIs can lead to upper tract infections or urosepsis. Although the initial treatment is
antimicrobial therapy, use of different prophylactic regimens and alternative strategies
are available to reduce exposure to antibiotics.
Escherichia (E.) coli is responsible for most uncomplicated cystitis cases in
women, especially in younger women. E. coli is generally a harmless microorganism
originating in the intestines. If it spreads to the vaginal opening, it may invade and
colonize the bladder, causing an infection. The spread of E. coli to the vaginal opening
most commonly occurs when women or girls wipe themselves from back to front after

urinating, or after sexual activity. Women are more susceptible to urinary tract infections
than men, and their infections tend to recur. One reason is that the urethra (the tube that
carries urine away from the bladder) is shorter in women than in men. Frequent sexual
intercourse also increases a woman’s risk of developing UTIs. Contraceptive spermicides
and diaphragm use are other risk factors. When women reach menopause, the decrease in
estrogen thins the lining of the urinary tract, which increases susceptibility to bacterial
infections.
Pregnancy does not increase the risk of getting a urinary tract infection but it can
increase the risk of developing a serious infection that could potentially harm the mother
and fetus. Pregnant women should report any symptoms of UTIs to their doctors, and
should get screened for asymptomatic bacteriuria (presence of significant numbers of
bacteria without symptoms).
Men become more susceptible to UTIs after age 50, when they begin to develop
prostate problems. Benign prostatic hyperplasia (BPH), enlargement of the prostate
gland, can produce obstruction in the urinary tract and increase the risk for infection. In
men, recurrent urinary tract infections are also associated with prostatitis, an infection of
the prostate gland. Although only about 20% of UTIs occur in men, these infections can
cause more serious problems than they do in women. Men with UTIs are far more likely
to require hospitalization than women.
Each year, about 3% of American children develop urinary tract infections.
During the first few months of life, UTIs are more common in boys than in girls. Boys
who are uncircumcised are about 10 - 12 times more likely than circumcised boys to

develop UTIs by the time they are 1 year old. After the age of 2 years, UTIs are far more
common in girls. As with adults, E. coli is the most common cause of UTIs in children.
(www.ncbi.nlm.nih.gov/pmc/articles/PMC3749018 Retrieved May 15, 2017).
Records showed the leading causes of morbidity since 2008 continued to be top
maladies until 2010. Pneumonia with 1,057,845 cases in 2010 top the list as the cause of
morbidity. Other leading causes of morbidity and their corresponding number were
diarrheal diseases (268,900), hypertension (265,052), urinary tract infection (219,270),
bronchitis (216,665), and, influenza (163,912). Tuberculosis (30,288) was also counted as
among the top cause of morbidity in 2010. (Health and Welfare, National Statistics
Office (NSO), Philippine Yearbook 2011 and Social Security System (SSS).
E. coli that usually cause UTIs crawl up the urinary tract, they can cause kidney
failure and fatal blood poisoning. But antibiotics may not be saving us from UTIs for
very much longer. Scientists tracking UTIs from 2000 to 2010 found a dramatic uptick in
cases caused by E. coli that do not respond to the drugs that are our first line of defense.
In examining more than 12 million urine analyses from that period, they found that cases
caused by E. coli resistant to ciprofloxacin grew five-fold, from 3% to 17.1% of cases.
And E. coli resistant to the drug trimethoprim-sulfame-thoxazole jumped from 17.9% to
24.2%. These are two of the most commonly prescribed antibiotics used to treat UTIs.
When they are not effective, doctors must turn to more toxic drugs, and the more those
drugs are used, the less effective they in turn become. When those drugs stop working,
doctors will be left with a drastically reduced toolkit with which to fight infection.

It is in this context and premises that this study was proposed to investigate the
antibacterial potential of Podocarpus costalis (Arius) leaf extract against infection caused
by Escherichia coli by measuring the zone of inhibition and the level of activity
exhibited by the plant leaf extract, since this plant has not been extensively used as a
subject of any studying involving Podocarpus. Therefore, the researcher wanted to help
most women who are suffering from the most common problem of urinary system which
is urinary tract infection and to discover antimicrobial therapy which is the core treatment
for UTIs, with the main objective of eradicating bacteria growth in the urinary tract
through an efficacious, safe and cost effective antimicrobial agent from plants. Hence,
this study.
The Input consists of the antimicrobial properties of Podocarpus costalis (Arius)
leaf extract; its effectiveness based on the zone of inhibition and the difference between
the extent of effectiveness of the Podocarpus costalis (Arius) leaves extract and the
control drug.
The Process consists of the analysis of antimicrobial components of the
Podocarpus costalis (Arius) Leaves extract; effectiveness of the Podocarpus costalis
(Arius) leaves extract based on the zone of inhibition; antimicrobial susceptibility testing;
and differences between the of the extent of effectiveness between Podocarpus costalis
(Arius) leaves extract and control drug. Consequently, the Output is “Antimicrobial
Properties Podocarpus costalis (Arius) leaves Extract.

INPUT PROCESS OUTPUT
FEEDBACK
Fig. 1. Phytochemical Analysis of Podocarpus costalis (Arius) Leaves extract
INPUT PROCESS OUTPUT
FEEDBACK
Fig. 2. Sensitivity test of the Podocarpus costalis (Arius) leaves extract
THE RESEARCH PARADIGM
Macerated Fresh
Podocarpus costalis
(Arius) Leaves
Extract
Phytochemical
analysis
Tests for:
a. Phenol
b. Saponin
c. Flavonoids
d. Quinones
e. Terpenoids
f. Carbohydrates
g. Coumarin
h. Acid
i. Alkaloids
Crude medical
ingredients of
Podocarpus costalis
(Arius) leaves extract
Podocarpus costalis
(Arius) leaves
extracts in the
following
concentrations:
1. 25%
2. 50%
3. 75%
4. 100%
Test organism -
Eschirechia coli
Sensitivity test of the
Podocarpus costalis
(Arius) leaves extract
in different
concentrations and
the control drug
ciprofloxacin against
the test organism,
Escherichia coli
Efficacy of the
Podocarpus costalis
(Arius) leaves
extract against
Escherichia coli

Objectives
This study aimed to determine the antimicrobial properties of Podocarpus costalis
(Arius) against Escherichia coli. Specifically, it sought to answer the following specific
objectives:
1. To determine the phytochemical properties of Podocarpus costalis (Arius)
leaves extract;
2. To measure the degree susceptibility of Escherichia coli to the different
concentrations of the Podocarpus costalis (Arius) leaves extract in terms of
the zone of inhibition:
a. 25 %
b. 50 %
c. 75 %
d. 100 %
3. To determine the significant differences among the different concentrations of
the crude extract of Podocarpus costalis (Arius) and the control drug against
Escherichia coli.
Time and Place of Study
The researcher will conduct her study from May 2017 to April 2018. The plant
extraction will be conducted at # 20 Bagani Gabor, Candon City, Ilocos Sur while the
phytochemical analysis was conducted at Medical Laboratory Science and antimicrobial

testing was conducted at College of Pharmacy of Lorma Colleges, City of San Fernando,
La Union.
Definition of Terms
Antimicrobial is an agent that kills microorganisms or inhibits their growth.
Bactericidal activity is the property of a substance or chemical to destroy or
neutralize bacteria which is expressed as the zone of inhibition.
Concentration refers to the quantity or dosage of leaf extract found on each
treatment.
Control is the standard at which experimental observations will be compared. The
study will consists of two groups, negative and positive.
Ciprofloxacin is the control drug which will serve in inhibiting the growth of
Escherichia coli cultured test organism.
Crude ethanolic extract is a greenish colored solution which will be obtained
from ethanol extraction of matured leaves of Podocarpus costalis (Arius).
Escherichia coli is a bacteria found in the environment, foods, and intestines of
people and animals. It is often abbreviated as E. coli. In this study E. coli will be used
and will be cultured from the Medical Laboratory of Lorma Colleges, City of San
Fernando, La Union.

Extract refers to a concentrated preparation of vegetable drugs obtained by
removing the active constituents of the respective drugs with a suitable menstruum, by
evaporation of all or nearly all the solvent and adjusting their residual mass or powder to
a prescribed standard. (U.S. Pharmacopeia).
Herbal Medicine is a medication prepared from plants, including most of the
world's traditional remedies for disease.
(www.who.int/medicines/areas/traditional/definitions/en/)
In vitro is an artificial environment, as in a laboratory setting; often used in
reference to what occurs outside an organism.
Maceration refers to the process of softening a solid by soaking or by the action
of liquid. (http://medical-dictionary.thefreedictionary.com/maceration).
Microbial testing is the test to determine bactericidal activity. Sensitivity or
susceptibility test will be utilized in this study.
Minimum inhibitory concentration is the lowest concentration of an antimicrobial
that inhibit the visible growth of Escherichia coli after incubation.
Percolation is the process of extracting the soluble parts of powdered substances
by passing through them successive quantities of a solvent which yields a clear extract
free from insoluble matters.(http://medical-dictionary.thefreedictionary.com/percolation).
Phytochemical analysis is a series of tests for alkaloids, flavonoids, glycosides,
organic acids, saponins, sterols, tannins and triterpenes.

Susceptibility or sensitivity test is a bacterial activity of P. costalis leaf extract by
measuring the zone of inhibition. It is classified into three based on the zone diameter
breakpoints and the following interpretations: Susceptible (>1-12) which indicates that
the antimicrobial is likely to inhibit growth of the pathogens, E.coli: Intermediate (20-11)
indicates that the microorganism is not fully susceptible to the concentrations of P.
costalis leaf extract; Resistant (<1-10) indicates that the varied concentrations of P.
costalis leaf extract are not likely to inhibit growth of the pathogen
Zone of inhibition refers to the estimated amount of Escherichia coli which will
be destroyed or neutralized by the different concentrations of Podocarpus costalis (Arius)
Leaves extracts and the control drug, Ciprofloxacin. It is expressed in millimetres.

Chapter 2
METHOD AND PROCEDURE
ResearchDesign
Phytochemical Screening or Preliminary Test is the first thing to be done before
major discoveries of molecules or drugs entities are known. It is used to provide concrete
knowledge and research to what plant active constituents have potential to benefit
mankind.
The preliminary screening test may be useful in the detection of the bioactive
principles and subsequently may lead to the drug discovery and development (Doss 2009,
cited in the International Journal of Innovative Research in Science, Engineering and
Technology, (2013). Further, these tests facilitate their quantitative estimation and
qualitative separation of pharmacologically active chemical compounds, (P.Varadarajan
et al., 2008).
This study utilized the experimental method. As described by Blakstad (2008),
“an experimental method is a systematic and scientific approach to research in which the
researcher manipulated one or more variables, and controls and measures any change in
other variables. This method used to show the antimicrobial properties of Podocarpus
costalis (Arius) leaf extract is as an antimicrobial agent. It was further showed the
differences of the degree of effectiveness of the different concentrations of the leaf
extract of Podocarpus costalis (Arius) plant which was tested on Escherichia (E.) coli.

Materials and Equipment
The researcher used the Podocarpus costalis (Arius) leaf extract, 95 % ethyl
alcohol, ethyl acetate, distilled water, Blood agar plate, Mueller Hinton Agar, Mc Farland
Standard, control disk, bacterial smears of Escherichia Coli, test tubes, Erlenmeyer flask,
beaker, graduated cylinder, stirring rod, tweezers, filter paper, funnel gauze, digital
balance. oven and electric juicer.
All laboratory equipment were washed with antibacterial dishwashing soap and
rinsed thoroughly with water as described by Cheesbrough, (2000). They were sterilized.
All media that used were in accordance to medical laboratory science’s specification.
Data Gathering Procedures
A. Plant Identification
Fresh leaves Podocarpus costalis (Arius) were harvested from the home
village of the researcher and/or from the researcher’s own hometown at Gregorio
Del Pilar, Ilocos Sur one, of the historical places in the Philippines. The plant
was identified by the Department of Environment and Natural Resources in
Tagudin Ilocos Sur, Region I. This study involved the leaves that were subjected
for proper identification for the assurance of obtaining the right information of
the study. The plant trunk and fruits were presented to assure proper
identification of the plant species.

B. Collection Process
The fresh leaves were properly washed in tap water, and then rinsed in
sterile distilled water. The leaves were air dried for 72 hours. The dried leaves
pulverized, using sterile laboratory mortar and pestle, to obtain their powered
form.

Figure 2. Flowchart of the Procedure
Identification and gathering of young one month old Podocarpus costalis
(Arius) leaves
Preparation of Podocarpus costalis (Arius) leaves
A. Washing of outer parts
B. Drying by wiping and air drying
C. Maceration
D. Filtration
Preparation of Leaf Alcoholic extract
A. The pulverized leaves extracted by 95% alcohol
B. Filtration
Dilution of Leaf Extract
a) 25 %; b) 50 %; c) 75 % and d) 100 %
Identification of Escherichia (E.) coli
McFarland Standard Comparison
Antimicrobial Susceptibility Testing
Data Gathering
Treatment of Data

The dry leaves were stored in airtight glass dark containers protected from
sunlight at room temperature until required for analysis.
C. Extraction Process
To obtain the pure extract, the procedure is as follows:
Alcoholic Extraction Process
The 100 grams dried leaves which were mechanically pulverized into a
coarse powder , extracted with ethanol of 95% for 7 days. After exhaustive
extraction, the leaves extract were filtered and concentrated. The extract-
ethanol solutions were heated in a water bath until all the ethanol evaporated
and the pure extract was left. Ethanol was used as an extracting medium due
to its ability to easily penetrate the cellular membrane to extract the
intercellular ingredients from the plant material. Organic compounds are most
often obtained through initial ethanol or methanol extraction (www.
Ipharmascientia.com).
Evaporation by water bath was the method used to remove or separate the
alcohol from the pure extract. The vapor pressure of water is 0.03 atm and the
vapor pressure of ethanol was 0.08 atm. This means that ethanol has a greater
tendency than water to be in the gas phase. The alcohol molecules are less
strongly attracted to each other than water molecules. It will take more heat to
evaporate the water than alcohol because one must supply more energy to
separate water molecules from each other.

D. Extract Dilution
The extracts were diluted to a specific concentration in order to determine
which concentration of the extracts is effective against Escherichia (E.) coli.
The different extracts were diluted into 25 %, 50 %, 75 % and 100 %
concentration. Distilled water was added to dilute the extracts.
The ratios of distilled water which added to the extracts was calculated
using the Concentration-Dilution Principle as shown in the equation 1.
Equation 1: C1V1 = C2V2
Whereas, C1 is the concentration of the pure extract;
C2 is the final concentration of the diluted extract;
V1 is the volume of the pure extract; and
V2 is the final volume of the diluted extract.
For the 100 % concentration of extract, 5ml of ethanolic extract
was used. In 75% concentration, 3.75 ml of ethanolic extract was diluted using
1.25 ml of distilled water. In 50%, 2.5 ml of ethanolic extract was diluted with 2.5
ml distilled water, while 1.25 ml of ethanolic extract diluted with 3.75 ml of
distilled water to yield 25% solution.
The extract dilution process was applied in the leaf extract of Podocarpus
costalis (Arius)

E. Microorganism
The microorganisms which was used in the study is Escherichia (E.) coli;
; a gram negative ,rod-shaped with a small tail appear pinkish or red that
have cell wall composed of a thin layer substance (called Peptidoglican). The
bases for the selection of the bacteria were availability, cultural characteristics and
clinical significance of the above mentioned organism.
F. Media Preparation
1. Blood Agar Plate (BAP)
The agar consists of a base containing a protein source (e.g. Tryptones),
Soybean protein digest, Sodium chloride (NaCl), agar and 5% whole blood. In
1000 ml of distilled water, 33.2 grams of Blood Agar Base were suspended. The
researcher used 8.3 grams of Blood Agar Base in 250 ml of distilled water. The
agar was prepared aseptically with pH of 7.2 to 7.6 at room temperature. The
solution was dispensed in petri dishes.
2. Tyrpticase Soy Broth
The solution contains trypton, soytone, glucose, sodium chloride and
dipotassium hydrogen phosphate (DHP). It was aseptically prepared using 2.1
grams of Trypticase Soy Broth base and 70 ml of distilled water. The solution (10
ml) was transferred into a sterile, medium sized Durham tube, capped, autoclaved
and stored in the refrigerator.

2. McFarland Standard Comparison
The solution was prepared using 0.5 ml of 1% Barium Sulfate (BaSO4)
and 99.5 ml of 1% Sulfuric Acid (H2SO4) and stored in refrigerator temperature.
3. Mueller Hinton Agar
In making Mueller Hinton Agar, the weighed 3.3 grams of agar and
dissolved in 100 ml of distilled water were placed in an Erlenmeyer flask. The
mixture was heated to boil in a water bath to further facilitate the dissolving
process. The boiled mixture was autoclaved to eliminate the unwanted
microorganisms present. Afterwards, the mixture was poured into sterilized plates
aseptically, then allowed it to be solidified.
G. Method of Assay
McFarland Comparison
0.5 McFarland Standard provides an optical density comparable to the
density of a bacterial suspension of 1.5 x 108 colony forming units (CFU)/ ml.
Pure cultures are grown or are directly prepared for agar plates to match the
turbidity of the McFarland Standard.

Principle
The density of the bacterial suspension was determined by direct visual
comparison of the suspensions with the McFarland barium sulfate standards. The
diameter of the tube in which the suspension was measured was comparable with the
standard. The standards were thoroughly shaken to insure complete suspension of the
barium sulfate each time they were used. Visual comparison of turbidity was easily and
accurately made by viewing the test suspensions and standard side by side against a
black-lined background, (Forbes, et al. 2010).
Antimicrobial Susceptibility Testing
The standardized Kirby Bauer Disc Diffusion Test was employed. The
antimicrobial assay was performed by streaking the inoculum on Petri dishes containing
Mueller-Hinton agar. Four (6 mm) sterilized filter papers disks were impregnated with
the filtered leaf extract of different concentrations ranging from 25%, 50%, 75% and
100%.
The impregnated discs together were placed onto the surface of Mueller-Hinton
agar pre-inoculated with test organisms. The bacterial plates were incubated for 24 hours
at 37 OC. After incubation, diameter of zone of inhibition (clearing) were measured to
millimeter units using vernier caliper at the point where there is a prominent reduction of
80% growth. The diameter inhibition was observed and measured. All tests were
performed under sterile conditions, (Forbes, et al, 2010).

Minimum Inhibitory Concentration (MIC)
It is defined as the lowest concentration of an antimicrobial agent that will inhibit
the visible growth of a microorganism after overnight incubation. It is measured using the
zone of inhibition in an agar plate.
Statistical Treatment of Data
All data gathered collected from the results of the said tests. The researcher
determined the antimicrobial property of Podocarpus costalis (ARIUS) leaf extract on
the selected organism in terms of degree of efficacy and then the degree of antimicrobial
property of Podocarpus costalis (ARIUS) leaf extract with different concentrations were
compared from each other. In order to prove that the microbial property of Podocarpus
costalis (ARIUS) is active, the medical laboratory scientist and the researcher observed
and recorded the diameter of the zone of inhibition which will form around the disks.
The researcher utilized the one-way ANOVA at .05 level of significance and
Tukey’s Honestly Significant Different (HSD). All statistical analyses, 0.05 level of
significance were used.

Chapter 3
RESULTS AND DISCUSSIONS
Phytochemical Properties of Podocarpus costalis (Arius) Leaf Extract
Table 1 shows the complete results of the phytochemical analysis of Podocarpus
costalis (Arius) leaf extract. Results revealed that the plant leaf extract has constituents
of phenols, tannins, flavonoids, carbohydrates and reducing sugars. The same
phytochemical analysis showed that there are no fixed and volatile oils, alkaloids,
saponins, nor steroids in the Podocarpus costalis (Arius) leaf extract.
Table 1. Phytochemical Constituents of Podocarpus costalis (Arius) Leaf Extract
Phytochemical Initial Color Change in Color Remarks
1. Phenol Green Blue-green +++
2. Tannins Green Bluish precipitate +++
3. Flavonoids Green Red colored precipitate ++
4. Carbohydrates Green Red precipitate +
5. Reducing Sugars Green Red precipitate +
6. Saponins Green No change -
7. Steroid Green No change -
8. Volatile Oils Green No change -
Legend: Highly present (+++) ; Moderately present (++); Low (+) ; (-) absence of
phytochemical

Specifically, it could be seen from the table 1, that the initial green color of the
Podocarpus costalis (Arius) leaf extract changed to blue-green, which indicates the
presence of phenols. The production of bluish precipitate indicates the presence of
tannins in the Ferric Chloride test.
The production of a yellow colored precipitate showed the presence of flavonoids
in the lead acetate and alkaline reagent tests. The presence of carbohydrates in the
Podocarpus costalis (Arius) leaf extract can be seen from the Benedict’s and Fehling
tests, where there was a formation of red precipitate which indicates also the presence of
reducing sugars.
Phytochemical constituents such as tannins, flavonoids, phenols and several other
aromatic compounds or secondary metabolites of plants serve as defense mechanism
against predation by many microorganisms. The curative properties of medicinal plants
are perhaps due to the presence of various secondary metabolites such as tannins,
flavonoids, phenols, etc (A.Panday and M.N. Vaisakh, 2011).
The table presents the antimicrobial components present in Podocarpus costalis
(Arius) leaf extract. The first antimicrobial component detected in the phytochemical
analysis was Phenol. Phenol is a major oxidized metabolite of benzene. Phenol is a
potent proteolytic agent. Phenol is bacteriostatic at concentrations of 0.1 – 1% and is
bactericidal and fungicidal at 1 – 2 %. It is also incorporated into cutaneous applications
for pruritus, stings, bites, burns, etc. because of its local anesthetic and antibacterial
properties to relieve itching and control infections (Edward, 2012).

Tannins are naturally occurring complex organic compounds possessing nitrogen
free polyphenols of high molecular weight. They form colloidal solution with water
giving acid reactions. They also precipitate proteins and alkaloids. The astringent in
nature of tannins is due to the fact that they can precipitate proteins and render them
resistant to enzymatic attack. When applied on a burn, sour mouth, wound, or injury,
tannins form a protective coating so as to prevent external irritation, stop local small
hemorrhages and thus promote healing. Tannins have astringent, hemostatic, antiseptic
and toning properties. Tannins are abundant in leafy vegetable and have been shown to
possess antimicrobial and antiviral activity by inhibiting cell wall synthesis resulting to
death, (Akinmoladun and Akinloye, 2007).
Flavonoids were also found present in Podocarpus costalis (Arius) leaf extract.
Flavonoids as one of the plant ingredients is known to be synthesized by plants in
response to microbial infection. It should not be surprising that they have been found in
vitro to be effective antimicrobial substances against a wide array of microorganisms.
According to Tsuchiya, et al (1999), the flavonoids compound has the ability to respond
to a wide array of microbial microorganism. Their activity is probably due to their ability
to complex with extracellular and soluble proteins and to complex with bacterial cell
walls. More lipophilic flavonoids may also disrupt microbial membranes. The finding
supports the finding of Dixon, et. al (1983); Tsuchiya, et al (1996); Toda, et al (1998),
and Afoloyan, et al (1997) as cited by Cowann (1999) and by Villar, (2005) that plants
such as oolong green teas, West African legume, apples and perennial herb Helichrysum
aureonitens were found to contain flavonoid with antimicrobial potential.

According to Ghosh, et al (2010), secondary metabolite studies have shown that the
Carbohydrates, Flavonoids, Alkaloids, Tannins, Steroids, are of great importance in the
field of drug research. These classes, Alkaloids, Saponin, Tannins, Flavonoids are known
to have activity against pathogens and therefore aid the antimicrobial activities of
medicinal plants.
Furthermore, phytochemicals such as alkaloids, triterpenoids, flavonoids, saponins,
tannins, steroids, cardiac glycosides and phenolic flavonoids, are the major classes of
antimicrobials and antioxidant in plants and are an indicative for such activities (Jedhave
et al., 2013). Thus, the detection of tannins, alkaloids and saponins in these extracts may
be responsible for their antibacterial activity, (Akinmoladun & Akinloye, 2007).
In the preliminary phytochemical screening, it was found that the ethanolic leaf
extract of Podocarpus costalis (Arius) possess flavonoids, tannins, phenol, and
carbohydrates. This suggests that leaves of Podocarpus costalis (Arius) may possess anti-
inflammatory, analgesic, antidiarrheal, antimicrobial, antioxidant, immunomodulatory,
anthelmintic, antitumor and insecticidal activities.
Based on the data gathered during the experimentation, Podocarpus costalis
(Arius) leaf extract contains physiologically active constituents into which the desired
pharmacological action can be attributed. The presence of the significant antibacterial
components in the Podocarpus costalis (Arius) leaf extract contributes to its
antimicrobial activity against Escherichia coli. However further studies are required in
this direction for its comprehensive analysis including quantitative or semi quantitative
analysis, which characterize its chemical structure and assess its biological activities.

Table 2 presents the degree of effectiveness of Podocarpus costalis (Arius) leaf
extract in various concentrations and the control drug based on the zone of inhibition and
interpreted using the Clinical and Laboratory Standard Institute (CLSI) – Recommended
Standard for Escherichia coli.
Table 2. Degree of susceptibility of Escherichia coli to the different concentrations of
Podocarpus costalis (Arius) leaf extract
Concentration
Zone of Inhibition (mm)
Interpretation
Water Extraction
Test
I
Test
II
Test
III
Test
IV
Mean
Control Drug 21 21 21 21 21 Susceptible
100 % 12 11.5 11.1 10.9 11.38 Intermediate
75% 8 7.8 7 7.7 7.48 Intermediate
50 % 7 6.9 6.5 6 6.6 Intermediate
25 % 6 6.5 6.5 6 6.25 Resistant

The zone of inhibition for the control drug is 21 mm in all the four tests and is
interpreted as susceptible. The 100% concentration has a mean zone of inhibition equal to
11.38 mm which is intermediate. The 75% and 50% concentration has a mean zone of
inhibition which is interpreted also as intermediate having a 7.48 mm and 6.6mm
measurement. The 25% has 6.25 mm mean zone of inhibition which has an interpretation
of bacterial resistance to the said concentration of extract. The 100% extract
concentration has the highest effect against Escherichia coli. The 25% extract shows
bacterial resistant to the concentration of leaf extract. In comparison to this, the
antimicrobial potential of guava (Psidium guajava) leaf extracts against gram-positive
Staphylococcus aureus . The water extract had an antibacterial activity with had a mean
zone of inhibition of 6.11 and 11.0 mm respectively. On the basis of the present finding,
guava leaf-extract might be a good candidate in the search for a natural antimicrobial
agent, (Bipul Biswas ,2013).
Difference between the Extent of Effectiveness of the Podocarpus costalis (Arius)
Leaf Extract and Control Drug
Table 3 shows the significant difference of the control drug to the different
concentrations of the Podocarpus costalis (Arius) leaf extract. The means of the zone of
inhibition of the 25, 50, per cent concentrations are not significantly different at 5% level
which means that these concentrations have lesser bactericidal activity to Escherichia
coli. The 75% and 100% concentrations are significantly different to the control drug
cipfrofloxacin. Nevertheless, as shown in Table 3, the zone of inhibition of the 100%
plant extract concentration of 11.38 mm which means that its inhibitory effect against

Escherichia coli is higher than the other concentrations. Based on the comparison studies
of Antibiotics, when streptomycin and amoxicillin were tested against E.coli many strains
fell in ” intermediate”. ”. Furthermore it can be concluded that different classes of
antibiotics have unique properties in the way they destroy bacteria. That said; E. Coli
may be more tolerant to one method of destruction (antibiotic) than others, (California
State State Science Fair 2011). The category “intermediate” means a higher dose of the
antimicrobial is needed to prevent growth. If there are no known drugs available in the
susceptible group then doctors will try to compensate a drug from intermediate group
prescribing a higher dosage and longer course of treatment.
In the study conducted by Ogunjobi & Elizabeth (2011), they concluded that
acetone leaf and seed extract might effectively inhibit the growth of certain wound-
infection pathogens without any side effects when they found out in their study that at
100% concentration of both the leaf and seed extracts have a zone of inhibition of 14 mm
and 12 mm respectively.
The observed that bactericidal activity is attributed to the presence of the
antimicrobial ingredients of the one month old Podocarpus costalis (Arius) leaf extract
as shown and discussed in Table 1.

Table 3. Difference between the extent of effectiveness of the Podocarpus costalis
(Arius) Leaf Extract and Control Drug
Treatment Mean*
Control (Commercial Drug) 21.00a
100% Concentration 11.13b
75% Concentration 7.63c
50% Concentration 6.60cd
25% Concentration 6.25d
*Meanfollowed by the same letter are not significantly different at 1% level (HSD)

Chapter 4
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary
In an effort to expand the spectrum of antibacterial agents from natural
resources, this study was undertaken to determine the different phytochemicals yield and
antimicrobial properties of the leaf extract of the plant Podocarpus costalis (Arius) with
a view to contributing to the search for beneficial use .
This study was undertaken to determine the antimicrobial property of the
Podocarpus costalis (Arius) leaf extract. It also attempted to find out the best treatment
that will eliminate Escherichia coli which will serve as a basis for possible production of
products from Podocarpus costalis (Arius) leaf extract.
Specifically, it sought to answer the following queries: (a) what are the
antimicrobial properties of Podocarpus costalis (Arius) leaf extract ; (b) what is the
efficacy of the different concentrations of Podocarpus costalis (Arius) leaf extract
against Escherichia coli: (c) what is the measurement of the susceptibility of Escherichia
coli on the different concentrations of the leaf extract of Podocarpus costalis (Arius) in
terms of zone of inhibition: and (d) ) Is there a significant difference in the effectiveness
of the crude extract of Podocarpus costalis (Arius) in different concentrations and the
control drug against Escherichia coli

This study employed the experimental research method. The researcher came up
with the following salient findings:
1. The leaf extract of Podocarpus costalis (Arius) contains of phenols, tannins,
flavonoids, carbohydrates and reducing sugars. Significantly, the
antimicrobial properties of the crude extract are phenols and tannins which
are highly present and flavonoids which is moderately present.
2. The 100 percent stalk extract had the highest measure of anti-Escherichia coli
activity with a mean zone of inhibition of 11.38 mm as compared to the
different concentrations (25%, 50% and 75%) of Podocarpus costalis (Arius)
leaf extract and the control drug based from the susceptibility test.
3. Podocarpus costalis (Arius) leaf extract showed significant difference in the
extent of effectiveness between the various concentrations and the control
drug.
Conclusions
Based from the findings and observations gathered by the researcher, the
following conclusions were drawn:
1. The phytochemical components of Podocarpus costalis (Arius) leaf extract are
known to contribute to the antimicrobial capacity of the plant.

2. The 100% concentrations of Podocarpus costalis (Arius) leaf extract can be
used against Escherichia coli.
3. The antimicrobial components of P. costalis (Arius) leaf extract especially
tannins prove that it can be used as source of antimicrobial drug.
4. The 100% concentration of Podocarpus costalis (Arius) leaf extract was
significantly different on the measurement of susceptibility against Escherichia coli as
compared to the different plant extract concentrations of the Podocarpus costalis (Arius)
and the control drug Ciprofloxacin in terms of zone of inhibition. It was tested and
proven that the extract has the capability of damaging the organism.
5. The ability of Podocarpus costalis (Arius) exhibiting antimicrobial activities in
the current research work indicates a potential for alternative use of the plant as raw
materials for the production of medicine that can be used in diseases caused by
Escherichia coli.
Recommendations
In the light of the aforementioned conclusions, the following recommendations
are offered:
1. The Podocarpus costalis (Arius) leaf extract may be popularized as a herbal
medicine due to its antimicrobial properties.

2. Quantitative analysis of the Podocarpus costalis (Arius) leaf extract may be
conducted since the test plant has the five (5) phytochemicals such as phenols, tannins,
flavonoids, carbohydrates and reducing sugars.
3. Other control drugs effective against Escherichia Coli may be used to compare
100 percent leaf extract concentration of Podocarpus costalis (Arius).
4. Combination of the leaf extract with other parts of Podocarpus costalis (Arius)
like its mature leaves, flowers, stem and roots is encouraged for further examination.
5. Other bacteria can also be tested for the antibacterial susceptibility testing to
find out its potential also on fungi and parasites, thereby developing an anti-fungal and
anti-parasitic drug from Podocarpus costalis (Arius) plant. And a study on its potential
natural source for drug development to treat other bacterial skin infections is encouraged.
6. Additional studies on the mechanism and adverse effect of the Podocarpus
costalis (Arius) plant to access the effectiveness in other areas of microbiology. Because
some antibiotics have been obsolete because of the problem of drug resistance. Thus
improvement of health using herbs as raw materials should be reconsidered.
7. Nutritive value and toxicity of Podocarpus costalis (Arius) leaf extract may
also be tested since the phytochemical carbohydrate and other reducing sugars were
already detected in the leaf extract.
8. Products of the Podocarpus costalis (Arius) leaf extract such as oil, ointment
and soap may be formulated for the easier consumption and economical advantage of the
community.

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APPENDIX A
Plant Identification Certificate

APPENDIX B
Phytochemical Testing Result

APPENDIX C
Phytochemical Test Standard Procedures
Test for the Presence of Flavonoids (Alkaline Reagent Test)
Reagent: Sodium hydroxide solution and lead acetate solution
Procedure:
1. One (1) ml of extract was treated with few drops of sodium hydroxide solution.
Formation of intense yellow color, which becomes colorless on addition of dilute acid,
indicates the presence of flavonoids.
Lead acetate Test:
2. One (1) ml Extracts were treated with few drops of lead acetate solution.
Formation of yellow color precipitate indicates the presence of flavonoids.
Test for the Presence of Carbohydrates and Reducing Sugars
Reagent: distilled water, alcoholic α-naphthol solution, Benedict’s reagent and
Fehling’s A & B
Procedure:
1. Extracts were dissolved individually in 5 ml distilled water and filtered. The filtrates
were used to test for the presence of carbohydrates.
2. Benedict’s Test: Filtrates were treated with Benedict’s reagent and heated gently.
Orange red precipitate indicates the presence of reducing sugars.
3. Fehling’s Test: Filtrates were hydrolysed with dillution. HCl, neutralized with alkali
and heated with Fehling’s A & B solutions. Formation of red precipitate
indicates the presence of reducing sugars.

Test for Tannins (Ferric Chloride Test)
Reagent: 10 % Sodium Chloride, Distilled water
Procedure:
1. Take the ethanolic extract equivalent to ten grams of plant material. Evaporated to
incipient dryness over a water bath and cool. Add twenty ml of hot distilled water
to the residue. Mix well with stirring rod and allow to cool at room temperature
spontaneously. Add five drops of 10% Sodium Chloride solution to salt out
undesirable constituents. Filter.
2. Add five drops of Ferric Chloride Solution. Observe any color change or
formation of precipitate. A blue-black color may indicate the presence of
hydrolysable tannins, while brownish-green color or greenish blue or greenish
black may indicate condensed tannins.
Test for Phenols (Ferric Chloride Test)
1. About 20 drops of 5% FeCl3 solution (a yellow solution) are added to a tube containing
3 drops of the extract and stir.
2. An intense color ranging from purple to reddish brown to green indicates a positive
result.

APPENDIX D
Sensitivity Test Results Certificate

APPENDIX E
Statistical Treatment
Analysis of Variance on the zone of Inhibition
Treatment Mean*
A-25% 6.25a
B-50% 6.60ab
C-75% 7.48b
D-100% 11.38c
*Means followedbythe same letterare notsignificantlydifferentat5% level of
significance,HSD.
Source of
Variation SS df MS F P-value F crit
Between Groups 66.665 3 22.22167 114.6925
4.23E-
09 3.490295
WithinGroups 2.325 12 0.19375
Total 68.99 15

APPENDIX F
Plate 1. Arius ( Podocarpus costalis) Plant
Plate 2. Gathering of Podocarpus costalis (Arius)

Plate 3. Gathered Podocarpus costalis (Arius) leaves
Plate 4. Water extraction of Podocarpus costalis (Arius) leaves using percolator

Plate 6. Phytochemical test on the constituents of Podocarpus costalis (Arius)
leaves
Plate 5. Alcoholic extraction of Podocarpus costalis (Arius) leaves

Plate 7. Boiling of ethanolic sxtract
Plate 8. Preparation of materials needed for sensitivity test

Plate 9. Autoclave all glasswares to be used
Plate 10. Measuring the estimated amount of distilled water

Plate 11. Measuring the amount of agar proportional to the number of plates
to be prepared
Plate 12. Cooking the agar until the mixture is clear

Plate 13. Autoclave the mixture for 15minutes at 15 psi and 121 OC
Plate 14. Preparing the plates while cooling down the autoclaved media

Plate 15. Dispensing the media into the plate about ¾
Plate 16. Preparation of samples, plates, bacteria, and control Plate

Plate 17. Antiseptic technique
Plate 18. Streaking plates and implanting control drug, 25%, 50%, 75%, 100%
of testing samples

Plate 18. Incubate for 24 hours at 37 OC
Plate 19. Checking and measuring for zone of inhibition after 24 hours

APPENDIX G
Certification of the Statistician

APPENDIX H
Certification of the English Critic

CURRICULUM VITAE
ELSA BITONGAN BISWELAN
# 20 BAGANI GABOR, CANDON CITY
Mobile Number- 09162328761
elsa.biswelan@deped.gov.ph
___________________________________________________________________
I. PERSONAL INFORMATION
Birthdate: November 10, 1971
Sex: Female
Civil Status: Married
Nationality: Filipino
Religion: Assemblies of God
II. EDUCATIONAL BACKGROUND
MASTER OF ARTS IN SCIENCE EDUCATION
DMMMSU-Open University System
City of San Fernando, La Union
2014-2018
BACHELOR OF ARTS MAJOR IN GENERAL SCIENCE
Union Christian College
City of San Fernando, La Union
1993-1996
CONCEPCION ADVENTIST ACADEMY
Brgy. Concepcion Gregorio Del Pilar, Ilocos Sur
1985-1990
CONCEPCION ELEMENTARY SCHOOL
Brgy. Concepcion Gregorio. Del Pilar, Ilocos Sur
1976-1984

III. WORK EXPERIENCES
Teacher 111 Candon National High School
Regular - from June 1, 2016 to present
Teacher 1 Candon National High School
Regular – from Sep. 10, 2014 to June 30, 2016
Teacher Nicosat Colleges Inc.
Regular – from June 1, 2012 – April 6, 2014
Teacher Saint Luis Colleges High School Department
Probational – from June 5,1998 –April 29,1999
IV. CIVIL SERVICE ELIGIBILITY
Title of Examination Remarks
Police Officer 1 Passed
Licensure Examination for Teachers Passed
V. SEMINARS, WORSHOP AND TRAININGS ATTENDED
Year Title
2014 Designing, Assessing and Fascilitating Grade 9 Learning
Units Based on K to 12 Standards
2014 School Based Training Workshop
2015 Training-Workshop on The Use and Care of
Advance Science Equipments and Sensors
2016 Mass Training of the Senior High School Grade 11
Teachers STEM Strand ( Science)
2017 School-Based Training/Workshop on Fundamentals
of Public Speaking and Writing

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