General Santos Hope Christian School

Blk. 8, Salvani st., Dadiangas Heights Subdivision

General Santos City

Antibacterial and Phytochemical Screening of Synergistic

Nuts Extract of Talisay (Terminalia catappa) and Indian Tree

(Polyalthnia longifolia)

Presented by:

Ayronn Ginson B. Young

Antibacterial and Phytochemical Screening of Synergistic Nuts Extract of Talisay

(Terminalia catappa) and Indian Tree (Polyalthnia longifolia)

An Investigatory Project Presented to the Association of Science

Educators in the Philippines in the 17th National Science Quest

Teacher’s Camp, Baguio City

Presented by:

Ayronn Ginson B. Young

March 2020

ii
 ACKNOWLEDGEMENT

For accomplishing this great endeavour, the investigators would like to acknowledge

the following people:

Our compassionate, our subject facilitator who has been with us every step of the way

under the guidance and wisdom that God provides.

Our group mates who sacrificed countless hours in accomplishing the investigation and

through thick and thin, trusted each other.

Our parents who under God, pushed us to become better individuals through moral

guidance and most specially, love.

Our respected panelists who meticulously critiqued the investigation and help strengthen

the validity and transparency of the investigation

And finally, almighty God who endlessly loved and gave us the power to accomplish

such feat.

Ayronn Ginson B. Young

iii
 DEDICATION

To those who were a big part in completing this investigation,

This is for you.

iv
 Table of Contents

Cover Page………………………………………………………………………………...……i

Title Page……………………………………………………………………………….…..…..ii

Acknowledgement…………………………………………………………………………..…iii

Dedication……………………………………………………………………………..………iv

Table of Contents………………………………………………………………………...…….v

List of Figures………………………………………………………………………………..viii

Abstract………………………………………………………………………………………..ix

Chapter 1. Problem and Its setting

Introduction………………………………………………………………………………….1

Background of the Study……………………………………………………………………1

Review of Related Literature………………………………………………………………..1

Antibacterial……………………………………...………………………………..….2

Phytochemical Screening…………………………...…………………………...........2

Synergism……………………………………………………………………………..2

Polyalthnia longifolia……………………………...……………………………………..3

Terminalia catappa……..……………………………………………………………..3

Staphylococcus aureus……………………..………………………………...……….3

Escherichia coli……………………………………………………………………….4

Statement of the Problem………………………..……………...…………………………….4

v
Hypothesis………………………………………………………………………….………….5

Significance of the Study……………………………………………………………………...5

Definition of Terms……………………………………………………………………….…...6

Chapter 2. Methodology

Materials……………………………………………………………………………………….8

Solvents………………………………………………………………………………..8

Equipment.………………………………………………………………………….....8

Procedures……………………………………………………………………………….....….9

Identification of the nuts……………………………………………………...…….....9

Collection of the nuts…………….…………………...….……………………………9

Extraction of the Substrate……………..…………....…………………………..……9

Phytochemical Screening…………………………...…….…………………………..9

Phytochemical Tests Performed………………………………………………………11

Antibacterial Testing………………………………………………….........................12

Chapter 3. Results and Discussion

Phytochemical Analysis results of Terminalia catappa…….……………....................14

Phytochemical Analysis results of Polyalthnia longifolia.……………………………..18

Terminalia catappa against Staphylococcus aureus.…………....……….....................21

Terminalia catappa against Escherichia coli…………………….……...……………..23

Polyalthnia longifolia against Staphylococcus aureus...……………….…..................25

Polyalthnia longifolia against Escherichia coli.……………………….…..................28

vi
 Synergized extract against Staphylococcus aureus………...........................................31

Synergized extract against Escherichia coli………………..…………………………33

Chapter 4. Summary of the Findings, Conclusions, and Recommendtaions

Summary of the Findings…………………………………………………………….36

Conclusion……………………………………………………………………………36

Recommendations……………………………………………………………………37

Appendix………………………...……………………………………………………38

References……………...………………………………………………………….….47

vii
 List of Figures

Figure 1. Wagner’s Test of Terminalia catappa nut extract…………..……………….……….39

Figure 2. Benedict’s Test Terminalia catappa nut extract…………………………….............39

Figure 3. Froth’s Test of Terminalia catappa nut extract……………………………………..40

Figure 4. Salkowski’s Test of Terminalia catappa nut extract ……………………………….40

Figure 5. Ferric Chloride Test of Terminalia catappa nut extract ……………….……………41

Figure 6. Gelatin Test of Terminalia catappa nut extract…….. ……….…………………….41

Figure 7. Lead Acetate Test of Terminalia catappa nut extract ……………..……….……...42

Figure 8. Copper Acetate Test of Terminalia catappa nut extract …….………..…………...42

Figure 9. Wagner’s Test of Polyalthnia longifolia nut extract .......………..………………..43

Figure 10. Benedict’s Test Polyalthnia longifolia nut extract ………..…………………......43

Figure 11. Froth’s Test of Polyalthnia longifolia nut extract ………..………………………44

Figure 12. Salkowski’s Test of Polyalthnia longifolia nut extract ………..…………………44

Figure 13. Ferric Chloride Test of Polyalthnia longifolia nut extract ………..…………...…45

Figure 14. Gelatin Test of Polyalthnia longifolia nut extract………..………………………45

Figure 15. Lead Acetate Test of Polyathnia longifolia Leaf Extract……………………...…46

Figure 16. Copper Acetate Test of Polyalthnia longifolia Leaf Extract……….…….………46

viii
 ABSTRACT

Terminalia Catappa and Polyalthnia Longifolia are plants that are widespread

across several parts of Asia, especially in the Philippines. There has been various

claims about the therapeutic and antibacterial properties of these plants. This study

looks to certify those claims.

The plants were collected and its substances were extracted. The extracts

underwent phytochemical screening and antibacterial testing over a 2 day period.

With the use of ANOVA, the experiment showed that there is a difference between

the zone of inhibition of bacteria namely E. Coli and S. Aureus, proving that the

extracts have antibacterial properties. It also showed that there was no significant

difference between most of the treatments except the synergized extracts against S.

Aureus by using Post-Hoc Analysis.

Based on the findings, the researcher concluded that the extracts of

Terminalia Catappa and Polyalthnia Longifolia have antibacterial properties and can

become a substitute in the absence of Amoxicillin.

The proponent recommends that in the absence of Amoxicillin, both

Terminalia catappa nut extract and Polyalthnia longifolia nut extract can be used as

an alternative in fighting against Staphylococcus aureus while the synergized extracts

of Terminalia catappa and Polyalthnia longifolia can be used as substitute for

Amoxicillin against Escherichia coli. Lastly, it is preferred not to synergize

Terminalia catappa and Polyalthnia longifolia when against Staphylococcus aureus.

ix
 Chapter I

INTRODUCTION

Background of the Study

Pharmaceutical drugs or medicines, have been a key component in the medical world

ever since an ancient time. Today, it is used to diagnose, treat, and prevent a disease in the

safest, cheapest, and most effective way, as long as it is used rationally. Healthcare professionals

may prescribe medicines when it is needed (Shiel Jr., 2018).

There are a multitude of people who are reliant on medication. Saying medical drugs are

important is an understatement, that’s why they are indispensable. However, it has its

disadvantages too. Pharmaceutical drugs have been so widespread and used that some have

abused it. The administration and utilization have to be done perfectly (Healthline, 2019).

Worldwide, health organizations are looking to expand access to cater to more patients

while protecting its current coverage. However, this could mean seeing a massive hike in costs

due to research. Though there are alternatives, it is yet to be found and issues are yet to be

resolved (AMA, 2018).

Review of Related Literature

This section of the study presents the related literatures, thesis, studies, and readings

from local, national, and international sources that were gathered after the extensive and in-

depth search done by the investigators.

1
Antibacterial

Antibacterial is mainly in form of chemicals, heat, or antibiotic drugs. It is the ability to

destroy or suppress the growth of bacteria and its ability to reproduce. Certain products such as

antibacterial soaps and washes are sold today to promote cleanliness and prevent bacterial

infections (Shiel, 2018).

Phytochemical Screening

Phytochemical is used to refer as a variety of plant-retrieved compounds with medicinal

and therapeutic benefits such as anti-carcinogenic, anti-mutagenic, anti-inflammatory, and

antioxidant properties (Nutraceuticals, 2011).

Photochemical screening, is the derivation, extraction, and identification of properties in plants

that give medicinal benefits. Some active substances found in plants are phenolic, antioxidants,

tannin, carotenoids, alkaloids, and flavonoid compounds (iMedPub, 2019).

Synergism

Synergism, is the interaction and combination between two or more compounds or

agents. The goal is to combine different entities, factors, and produce a greater effect than the

sum of each compound (Business Dictionary, 2019).

Also, synergistic effect can be contrasted with additive effects. The combine both

substances and create an even greater affect compared to individually used. The term synergism

is opposite to antagonism (Safeopedia, 2019)

2
Polyalthnia longifolia

Polyalthnia longifolia is a tall, magestic tree that grows or gets cultivated all over india.

Specifically, the species can also be seen in the Philippines. The plant is traditionally used for

medicinal use. It treats and heals various ailments such as fever, skin diseases, diabetes, and

hypertension. With this discovery, a comprehensive review of Polyathnia longifolia’s botanical,

photochemical, toxicological and pharmacological information was made. The tree possesses

highly significant pharmaceutical benefits that include: antioxidant properties, anti-tumor, anti-

fungal anti-ulcer, and antioxidant. The tree also has negative signs of toxicity. With this,

Polyalthnia longifolia still stands as an ancient source of remedy and cures for various ailments

(Dharmaraj, 2013).

Terminalia catappa

Terminalia catappa is abundant in the regions of Southeast Asia. This tree grows well

in the tropical and subtropical climates and are found throughout the tropics. The tree is used

for decorative purposes and its nuts are considered edible. Its fruit is nutritional and it contains

medicinal properties. Many pharmacological investigations claimed that the plant shows

antimicrobial, anti-inflammatory, antidiabetic, antioxidant, hepaprotective, and anticancer

activities which support its traditional uses.

Staphylococcus aureus

Staphylococcus aureus is a type of bacterial pathogen found in humans and has a wide

variety of clinical manifestation. Infections are commonly contracted in hospitals, communities,

or the environment. Treatments are currently challenged by antimicrobial resistant types of

3
Staphylococcus aureus, or the MRSA (Methicillin-Resistant Staphylococcus aureus). The

bacteria may also be found inside the human flora, mostly located in the epidermis, or in the

mucous. They usually do not infect when in the skin, but when it enters a persons’s internal

system such as the bloodstream, it may lead to infections. Transmission may be causesd from

person to person contact (Taylor & Unakal, 2019).

Also, Staphylococcus aureus infections can potentially turn to lethal if it invades deeper

inside the body such as the bloodstream, bones, joints, lungs and even the heart. People who

have been infected with life threatening Staphylococcus aureus infections have been seen

increasing as of today. Symptoms can be seen when infected by Staphylococcus aureus are

boils. Impetigo, which can be compared to a nasty case of rashes. Food poisoning also represents

the presence of Staphylococcus aureus septic arthritis is when Staphylococcus aureus comes in

contact and infects the joints such as the shoulder, hips, knees, etc (Mayo Clinic, 2017).

Escherichia coli

Escherichia coli is a type of bacterium commonly found in the gut of humans. It is

known to cause food borne diseases and is transmitted primarily through the consumption of

raw or contaminated food (World Health Organization, 2018).

Statement of the Problem

The purpose of this experimental quantitative research is to determine the antimicrobial

properties of the synergistic nuts extract of Polyalthnia longifolia and Terminalia catappa to

find a practical and accessible antibiotic.

This study aims to investigate the following questions that serve as the study’s objective:

4
1. Do Polyalthnia longifolia and Terminalia catappa nut extracts really have antibacterial

properties?

2. What is the specific chemical of the plant that has antibacterial property?

3. How effective are the antibacterial properties of the Polyalthnia longifolia and

Terminalia catappa?

4. Can this study help to lessen the problems of expensive antibiotics?

Hypothesis

The following statements of the study were conducted with these as hypotheses:

Null: There are no antibacterial properties found in the synergistic nuts of Polyalthnia longifolia

and Terminalia catappa?

Alternative: There are antibacterial properties found in the synergistic nuts of Polyalthnia

longifolia and Terminalia catappa?

Significance of the Study

This research aims to discover the antibacterial properties of Polyalthnia longifolia and

Terminalia catappa, this is very beneficial to the following:

For budgeted families, this research would provide them an alternative antibiotic that is

affordable and readily available anywhere.

5
 For first-aid measurement, this research would show us an antibiotic that is accessible

especially for first-aid circumstances.

For patients, this research would provide an organic antibiotic that will be a substitute

for synthetic drugs.

Definition of Terms

Antibacterial. Anything that kills bacteria and suppresses their ability to reproduce. Things that

contain antibacterial properties may range from anything from heat to chemicals and antibiotic

drugs (Shiel Jr., 2018). In this study, it is the

Phytochemical Screening. Refers to the extraction and screening for the identification of

medically active substances in objects. Phytochemical screening is done in plants (Kumar,

2011).

Synergism. The interaction or activity of two or more agents in which the collective effect is

greater than the sum of its parts (Stefanović, 2017).

Terminalia catappa. A large tropical tree that makes its home throughout regions in Asia,

Africa, and Australia. It is known for its nutritional fruit and possesses medicinal benefits as

well (Anand,2015).

Polyalthnia longifolia. A tree native to India that is known for its effectiveness in alleviating

noise pollution. It is also known as “Ashoka” and is commonly cultivated in India (Katkar,

2010).

Escherichia coli. This bacterium that normally lives in your intestines. It is also found in the

gut of some animals. Most types of Escherichia coli are harmless and even help keep your

digestive tract healthy (WebMD, 2019).

6
Staphylococcus aureus. This bacterial pathogen causes a wide variety of clinical manifestation

on humans. It can be found on human flora. It can be located in the human skin (Taylor, 2017).

7
 Chapter II

METHODOLOGY

This chapter exhibits the design of the entire study. This also confers in details the

procedures done, and the instruments used in the project. It also encompasses the main subject

of the study and statistical analysis.

Materials

Solvents

Plant material ─ nuts of Polyalthnia longifolia and Terminalia catappa

Test organisms ─ The test microorganisms used for this research were

Escherichia coli and Staphylococcus aureus, obtained from General Santos

Hope Christian School. Antibiotics used ─ Amoxicillin in powder form,

obtained from General Santos Hope Christian School.

Equipment

Glass wares

Beakers, petri dish, alcohol lamp, glass tubes and rods, test tubes, vials,

measuring cylinders, round bottom flasks, pipettes, watch glasses, etc.

Metal wares

Stainless steel Spatula, mortar and pestle

Wooden wares

Wooden test tube stand and racks

8
 Other equipments

Inoculating loop, filter paper

Procedures

Identification of the plant

The plants used in this study were the Polyalthnia longifolia and Terminalia catappa

Collection of the Nuts of the Plants

These were obtained from the area around General Santos Hope Christian School and near the

City Hall. These plants are also readily available and common in General Santos. These plants

are also widespread throughout the Philippines especially in provinces.

Extraction of the Nuts

The collected nuts of the plant specimen were smashed to extract the oil substances of the

Polyalthnia longifolia and Terminalia catappa with the use of a mortar and pestle. Afterwards,

the collected extracts were stored in a flask and is prepared for screening and tests. The process

was conducted at the laboratory of General Santos Hope Christian School.

Phytochemical Screening

Phytochemical screening was carried out for hexane, chloroform and methanol soluble fractions

as per the standard methods. The following are the reagents used for phytochemical screening:

1. Mayer’s Reagent: 1.358 g of HgCl 2 was dissolved in 60 ml of water and it was mixed with

a solution of 5 g of KI in 10 ml of water.

2. Wagner’s Reagent: 16.6 g of potassium iodide was dissolved in 100 ml of distilled water and

few crystals of iodine were added to the solution and the solution was stirred properly.

9
3. Hager’s Reagent: 6 g of picric acid was dissolved in 100 ml of hot water to produce saturated

solution (URL─14).

4. Molish’s Reagent: 1 g of 1─napthol, 6 g of sodium hydroxide and 16 g of sodium carbonate

were dissolved in 100 ml of water.

5. Legal’s Reagent: 3 drops of sodium hydroxide were added to 10 ml methanol which was then

added to the solution containing 10 ml of 10% sodium nitroprusside and 10 ml of pyridine

(URL─15).

6. Benedict’s Reagent:

Solution I - 50 g of crystalline sodium carbonate, 50 g of crystalline sodium citrate and 31.25 g

of potassium thiocyanate were dissolved in 200 ml hot distilled water.

Solution II - 4.5 g of CuSO 4 was dissolved in 25 ml water to prepare copper sulphate solution.

Solution III - 5% solution of potassium ferrocyanate was prepared. Finally, Benedict’s Reagent

was prepared by mixing 200 ml of solution I, 25 ml of solution II and 5 g of solution III and the

final volume was adjusted to 250 ml with water.

7. Fehling’s Reagent:

Solution I - 31.66 g of CuSO 4 was dissolved in sufficient amount of water to produce 500 ml

solution.

Solution II - 176 g of sodium potassium tartarate and 77 g of sodium hydroxide was dissolved

in sufficient amount of water to produce 500 ml solution. Finally, equal volume of solution I

and II were mixed to prepare Fehling’s solution.

8. Concentrated sulfuric acid solution: 36 N concentrated sulfuric acid solution was used.

9. Dilute sulfuric acid solution: Concentrated sulfuric acid was diluted 10 times with water to

produce dilute sulfuric acid solution.

10
10. Ferric chloride solution: 15 g of ferric chloride hexahydrate was dissolved in 100 ml of

distilled water.

11. Ammonia solution: 25% of ammonia solution was used.

12. Gelatin solution (1%): 1 g of gelatin was dissolved in 100 ml of hot water.

13. Copper acetate solution: 199.65 g of copper acetate was dissolved in 1000 ml of distilled

water.

14. Sodium hydroxide solution: 20 g of NaOH was dissolved in 100 ml of distilled water.

15. Lead acetate solution: 10 g of lead acetate was dissolved in 100 ml of Carbon dioxide free

water.

16. Concentrated nitric acid solution: Nitric acid solution (69 – 72%) was used.

Phytochemical Tests Performed

(For each tests 1 ml of each of the three extracts were used)

1. Alkaloids Test: Each of the three extracts were dissolved individually in dilute hydrochloric

acid and filtered. Then the filtrates were separately treated with, Wagner’s Reagent to test if

there is the presence of alkaloids.

2. Carbohydrates Test: All three extracts were dissolved individually in 1 ml distilled water and

filtered. Then the filtrates were separately treated with Benedict’s Reagent to test for the

presence of carbohydrates.

3. Saponins Test: All three extracts were shaken vigorously with 1 ml of water in a test tube for

few minutes following a process known as Foam test.

4. Phytosterols test: It was done by Salkowski’s test method.

11
In Salkowski’s test method, extracts were treated with chloroform and filtered. The filtrates

were then treated with few drops of concentrated sulfuric acid and allowed to stand and the

result was observed.

5. Phenols test: It was done by treating the extracts with a drop of FeCl 3 solution.

6. Tannins test: It was done by gelatin test method in which extracts were treated with a drop of

1% gelatin solution containing NaCl and results were observed.

7. Diterpenes test: It was done by copper nitrate test in which a drop of copper nitrate solution

was added to the aqueous solution of extracts and observed.

8. Flavonoids test: It was done by lead acetate test in which a drop of lead acetate solution was

added to the extracts and observed. In Lead Acetate test, extracts were treated with a drop of

lead acetate solution and observed.

Antibacterial Screening

In this screening, five treatments were prepared. Each nut extract and the synergistic extract of

both were prepared with their respective bacterium to assess whether the extracts possess

antibacterial properties.

A. Preparation of Inoculum

Suspensions of organisms were prepared as per McFarland standard.

B. Preparation of the culture media

Petri plates were sterilized in Hot Air Oven at 160 0C for one hour. Cultured organism (1 ml)

was poured in it and 25 ml of cooled media was added to the plate and left for some time to

solidify. After solidification of the media, 5 bores were made in the 9 cm Petri plate separated

by at least 2.54 cm from each other. Fifty μl of each extract were poured in three bores, solution

12
of antibiotic in other bore and the solvent in the last bore. Thirty plates were prepared for each

bacterium.

C. Preparation for positive and negative control

Six plates for amoxicillin standard served as the positive control of both of the organismS and

six plates for the negative control. In positive control no antibiotics and extract solution were

kept. For negative control no microorganisms were kept to assure the sterility of the medium.

Plates were kept in freeze for one hour to let the proper diffusion of extract and antibiotic

solution in the media. Then they were incubated in BOD incubator for 24 hours at 37 °C. After

24 hours’ zone of inhibition of the extract, active drugs were measured from at least two sides

of the inhibited zone.

13
 Chapter III

RESULTS AND DISCUSSIONS

This chapter presents and analyses the data gathered after the experiment. This also

interprets the collected data in tables, which are discussed in details.

Table 1. Phytochemical Analysis results of Terminalia catappa

Phytochemical Test Reagents used Interference Result

Alkaloid Wagner’s Reagent Formation of +++

brown/reddish
precipitate
Carbohydrates Benedict’s Test Appearance of orange +++
red precipitate
Saponin Froth Test Produce foam that lasts ---
for more than 10
minutes
Phytosterol Salkowski’s Test No golden brown colour +++
obtained
Phenol Ferric Chloride Test Appearance of bluish +++
black ppt.
Tannin Gelatin Test Formation of white ppt. +++

Flavonoids Lead acetate Test No intense yellow colour +++

obtained

Diterpenes Copper Nitrate Test No formation of bright +++

green colour

Wagner’s Test (Alkaloid)

According to the Wagner’s test it was found out that Terminalia catappa has strong

detection of Alkaloids. Alkaloids have antibacterial properties which can enter the cell wall and

14
DNA of parasites that promotes the release of autocoids and prostaglandins. Alkaloids also have

antidiarrhoeal properties that processes anti-oxidating effects.

Benedict’s Test (Carbohydrates)

The Benedict’s Test that was conducted on the plant extract of Terminalia catappa

determine a no distinction of carbohydrates in the plant extract of Terminalia catappa.

Carbohydrates play an important key role in the body. Carbohydrates are involved in

reproduction, the immune system, the development of diseases, and blood clotting. They are the

main energy source of the body by conducting the energy transport process. They also serve as

structural components of plants and insects.

Froth Test (Saponins)

The results from the Froth test shows a very weak distinction of saponins in Terminalia

catappa extract. Saponins are said to possess numerous properties including Antidiarrhoeal

which inhibit histamine release in vitro. They also have anthelmintic properties that leads to the

attrition of teguments. Saponins is correspondingly anticancer that means they possess

membranes permeabilizing properties.

Salkowski’s Test (Phytosterols)

According to the Salkowski’s Test conducted on the plant extract of Terminalia catappa

it was found out that the plant extract of Terminalia catappa contain a strong presence of

phytosterols. Phytosterols play an important role in a cell membrane’s function. They are also

known for their other health benefits such as reduction of cholesterol levels.

15
Ferric Chloride Test (Phenols)

The results from the Ferric Chloride Test on plant extract of Terminalia catappa shows

a strong detection of phenols. Phenols play a variety of important roles in organisms. Majority

of phenolic substance have important effects on defence against herbivores and pathogens. They

also mechanically strengthen cell walls and also affect the growth of neighbouring plants thus

have allelopathic activity.

Gelatin Test (Tannins)

The Gelatin Test conducted on the plant extract of Terminalia catappa contain strong

presences of Tannins that has properties like antimicrobial which help cells bind to adhesion,

enzyme inhibition, substrate deprivation, complex cell walls, and more. Tannins also has

Antidiarrhoeal that makes intestinal mucosa more resistant and reduces secretion, stimulates

normalization of deranged water transport across the mucosal cell and reduction of the intestinal

transit and more. Lastly the plant extract of Terminalia catappa is anthelmintic which increases

supply of digestive proteins by building proteins complexes in rumen which interferes with

energy generation by uncoupling oxidative phosphorylation that decreases the GI metabolism.

Lead acetate Test (Flavonoids)

The plant extract of Terminalia catappa contain a powerful distinction of flavonoids as

seen in the Lead acetate test conducted. Flavonoids have multiple properties like antibacterial.

It is concluded that hydroxyls at special sites on the aromatic rings of flavonoids improve the

activity. However, the methylation of the active hydroxyl groups generally decreases the

16
activity. The plant extract of Terminalia catappa also contain Antidiarrhoeal which inhibits the

release of autocoids and prostaglandins, hinder contractions caused by spasmogens, simulates

normalization of the deranged water transport across the mucosal cells, and discouraged GI

release of acetylcholine.

Copper Nitrate Test (Diterpenes)

The copper nitrate test assesses the plant extract of Terminalia catappa which concluded

that the plant extract contains a strong distinction of diterpenes. Diterpenes contain anticancer

which is used in therapy against ovarian, breast, and lung cancer. They are also ginkgolides

showing potent and selective antagonistic activity toward platelet- activating factor increasing

in conditions of shocks, burns, and more.

17
Table 2. Phytochemical Analysis results of Polyalthnia longifolia

Phytochemical Reagents used Interference Result

Test

Alkaloid Wagner’s Reagent Formation of —-

brown/reddish
precipitate

Carbohydrates Benedict’s Test Appearance of orange —-

red precipitate

Saponin Froth Test Produce foam that lasts +++

for more than 10
minutes

Phytosterol Salkowski’s Test No golden brown colour +++

obtained

Phenol Ferric Chloride Test Appearance of bluish +++

black ppt.

Tannin Gelatin Test Formation of white ppt. +++

Flavonoids Lead acetate Test No intense yellow +++
colour obtained

Diterpenes Copper Acetate Test No formation of bright +++

green colour

Wagner’s Test (Alkaloids)

Using the Wagner’s test, it shows that there are some alkaloids present. If alkaloids are

seen, then the plant extract has a potential antibacterial property. Alkaloids may act as protective

substances against animal or insect attacks. Alkaloids are also important for fighting cancer.

They are known as the second-most-used class of cancer drugs.

18
Benedict’s Test (Carbohydrates)

The Benedict’s test shows minimal to none carbohydrates present in our plant extract.

The presence of carbohydrates helps fuel up our brain, kidney, heart and muscles. It also helps

in digestion and keep the blood cholesterol in check.

Froth Test (Saponins)

The results from the froth test shows that the plant extracts contain saponins. Saponins

are possessed with numerous properties including antidiarrheal which the plant clearly contains.

Saponins are more effective against gram-positive organisms compared to gram negative

organisms.

Salkowski’s Test (Phytosterols)

The Salkowski’s test helps us in indicating that there are Phytosterols present.

Phytosterols can reduce LDL cholesterol levels by around 10%, by reducing the absorption of

cholesterol from the gut. They are particularly effective for people who have high cholesterol,

independently of whether they are taking cholesterol-lowering statin drugs or not.

Ferric Chloride (Phenols)

According to the Ferric Chloride test there are Phenols present. Phenols has

antimicrobial properties which binds adhesion, enzyme inhibition and more and it has

anthelmintic properties which increases supply of digestible proteins by animals by forming

protein complexes in rumen.

19
Gelatin test (Tannins)

The gelatin test shows that tannins are present in our plant extract. Their properties are

well remarkable. Most of the tannins fight cavities, diarrhea, and some even protect heart

diseases and cancer. They disable bacteria in the mouth, which inhibits plaque formation. Tooth

decay is also prevented, but they stain the teeth.

Lead Acetate Test (Flavonoids)

The figure above shows that there is a strong distinction of flavonoids in our plant

extract. Flavonoids are one of the reasons fruits and vegetables are so good for the body. Among

the many benefits attributed to flavonoids are reduced risk of cancer, heart disease, asthma, and

stroke. They also play a special role in protecting the brain. Flavonoids, like other antioxidants,

do their work in the body by corralling cell-damaging free radicals and metallic ions. But

flavonoids go beyond the yeoman work of your average antioxidant. Scientists have found that

certain flavonoids have antihistamine, antimicrobial, memory- and even mood-enhancing

properties.

Copper acetate (Diterpenes)

Using the copper acetate test states that diterpenes are present in our extract.

Naturally occurring diterpenes exert several biological activities such as anti-inflammatory

action, antimicrobial and antispasmodic activities. Diterpenes are believed to help with

hypertension and respiratory tract complaints such as catarrh bronchitis and colds. Some

diterpenes are believed to have tumour inhibitor properties as well as a stimulating effect on the

immune system.

20
Table 3.

Zone of inhibition of Terminalia catappa against Staphylococcus aureus.

Talisay (S. R1 R2 R3 Mean

Aureus)
Water 9.1 9.1 7.9 8.7

25% 11 12.8 10.8 11.53

50% 14.1 15.2 17.2 15.5

100% 20.4 19 17.6 19

Amoxicillin 22.5 22.7 19.6 21.6

Table 3 shows the antibacterial properties of Terminalia cattapa. The mean of

inhibition of Amoxicillin is closes to the mean of 100% Terminalia cattapa extract with

a mean difference of 2.6 mm. This table 3 also showed that there are minor differences

among the extracts and Amoxicillin, of not similar.

Table 4.

Analysis of variance of the zone of inhibition of Terminalia catappa against

Staphylococcus aureus

Source SS df MS F-Crit

Between 333.48 4 83.37 F=45.64 3.48

Treatments

Within- 18.26 10 1.82

Treatments

Total 351.75 14

21
 Table 4 shows the analysis of variance or ANOVA. This showed that the F

tabulated is greater than the F Crit. This means that the null hypothesis will be rejected.

Therefore, the alternative hypothesis was accepted that states that there is a difference

among the means of the treatments.

Table 5.

Post-Hoc test: Bonferroni-Holm analysis of the mean difference of Terminalaia catappa

against Staphylococcus aureus.

Group 1 Group 2 Critical P Significant?

50% Amoxicillin 0.01 0.010712 No

Talisay
25% 50% Talisay 0.016667 0.023171 No
Talisay
50% 100% Talisay 0.025 0.045003 No
Talisay
100% Amoxicillin 0.05 0.113495 No
Talisay

This table 5 showed the Post Hoc analysis of the anti-bacterial properties of the

Terminalia Catappa extract. Table 5 revealed that 50% Talisay has no significant

difference with all the other treatments. The 25% Talisay has no significant difference

with 50% extract. The 100% Talisay has no significant difference with Amoxicillin.

Therefore, it means that any percentage is effective against Staphylococcus aureus.

22
Table 6.

Zone of inhibition of Terminalia catappa against Escherichia coli.

Talisay (E. Coli) R1 R2 R3 Mean

Water 7.3 7.1 7.1 7.16

25% 18.6 22.8 22.7 21.36

50% 21.9 21.2 25.9 23

100% 32.8 29.8 23.9 28.8

Amoxicillin 25 24 25 24.67

Table 6 shows the antibacterial properties of Terminalia cattapa against

Escherichia coli. The mean of inhibition of Amoxicillin is closes to the mean of 100%

Terminalia cattapa extract with a mean difference of 4.6mm. This also showed that 100%

extract of Terminalia cattapa is more effective than Escherichia coli.

Table 7.

Analysis of the zone of inhibition of Terminalia catappa against Escherichia coli.

Source SS df MS F Crit

Between 810.89 4 202.72 F=30.6941 3.48

Treatments

Within- 66.04 10 6.6

Treatments

Total 876.94 14

23
 Table 7 shows the analysis of variance or ANOVA. This showed that the F

tabulated is greater than the F Crit. This means that the null hypothesis will be rejected.

Therefore, the alternative hypothesis was accepted that states that there is a difference

among the means of the treatments.

Table 8.

Post Hoc Test: Bonferroni-Holm analysis of the mean difference of Terminalia catappa

against Escherichia coli

Group 1 Group 2 Critical P Significant?

25% Amox 0.008333 0.081254 No
Talisay
25% 100% Talisay 0.01 0.138484 No
Talisay
50% 100% Talisay 0.0125 0.314487 No
Talisay
50% Amoxicillin 0.016667 0.329225 No
Talisay
25% 50% Talisay 0.025 0.462948 No
Talisay
100% Amoxicillin 0.05 0.562992 No
Talisay

Table 8 showed the Post Hoc analysis of the anti-bacterial properties of the

Terminalia Catappa extract. Table 8 revealed that 50% Talisay has no significant

difference with all the other treatments. The 25% Talisay has no significant difference

with 50% extract. The 100% Talisay has no significant difference with Amoxicillin.

Therefore, it means that any percentage is effective against Escherichia coli.

24
Table 9.

Zone of inhibition of Polyalthnia longifolia against Staphylococcus aureus

Indian Mast R1 R2 R3 Mean

Tree (S.
Aureus)
Water 8.2 8.4 8.2 8.26

25% 20.7 15.8 29.5 22

50% 31.5 28.8 28.6 29.63

100% 23.1 21.8 16.8 20.56

Amoxicillin 22.4 22.2 24.9 23.16

Table 9 shows the antibacterial properties of Polyalthnia longifolia against

Staphylococcus aureus. The mean of inhibition of Amoxicillin is closes to the mean of

50% extract with a mean difference of 6.46mm. This also showed that 50% extract of

Polyalthnia longifolia is more effective than Amoxicillin.

25
Table 10.

Analysis of variance of the zone of inhibition of Polyalthnia longifolia against

Staphylococcus aureus.

Source SS df MS F Crit

Between 726.53 4 181.63 F=14.1562 3.48

Treatments

Within- 128.30 10 12.83

Treatments

Total 854.846 14

Table 10 shows the analysis of variance or ANOVA. This showed that the F

tabulated is greater than the F Crit. This means that the null hypothesis will be rejected.

Therefore, the alternative hypothesis was accepted that states that there is a difference

among the means of the treatments.

26
Table 11.

Post Hoc Test: Bonferroni- Holm analysis of the mean difference of Polyalthnia

longifolia against Staphylococcus aureus.

Group 1 Group 2 Critical P Significant?

50% Amox 0.007143 0.007146 No

Indian
50% 100% Indian 0.008333 0.013213 No
Indian
25% 50% Indian 0.0125 0.137232 No
Indian
100% Amox 0.016667 0.284884 No
Indian
25% 100% Indian 0.025 0.763215 No
Indian
25% Amox 0.05 0.790157 No
Indian

This table 11 showed the Post Hoc analysis of the anti-bacterial properties of the

Polyalthnia longifolia extract. Table 11 revealed that 50% extract has no significant

difference with all the other treatments. The 25% extract has no significant difference

with 50% extract. The 100% extract has no significant difference with Amoxicillin.

Therefore, it means that any percentage of Polyalthnia longifolia extract is effective

against Staphylococcus aureus.

27
Table 12.

Zone of inhibition of Polyalthnia longifolia against Escherichia coli.

Indian Mast Tree (E. R1 R2 R3 Mean

Coli)

Water 8.1 8.1 8 8.07

25% 19.8 21.8 26.4 22.66

50% 25.7 25.1 19.4 23.4

100% 18.5 25.6 37.2 25.43

Amoxicillin 25 24 25 24.67

Table 12 shows the antibacterial properties of Polyalthnia longifolia against

Escherichia coli. The mean of inhibition of Amoxicillin is closes to the mean of

100% extract with a mean difference of 2.43mm. This also showed that 100% extract of

Polyalthnia longifolia showed similar effects of Amoxicillin.

28
Table 13.

Analysis of variance of the zone of inhibition of Polyalthnia catappa against Escherichia

coli.

Source SS df MS F Crit

Between 678.9 4 169.72 3.48

Treatments

Within- 225.98 10 22.59 F=7.5106

Treatments

Total 904.88 14

Table 13 shows the analysis of variance or ANOVA. This showed that the F

tabulated is greater than the F Crit. This means that the null hypothesis will be rejected.

Therefore, the alternative hypothesis was accepted that states that there is a difference

among the means of the treatments.

29
Table 14.

Post Hoc Test: Bonferroni- Holm analysis of the mean difference of Polyalthnia

longifolia against Escherichia coli.

Group 1 Group 2 Critical P Significant?

25% Amox 0.008333 0.447764 No

Indian
50% Amox 0.01 0.670302 No
Indian
25% 100% Indian 0.0125 0.756429 No
Indian
25% 50% Indian 0.016667 0.806422 No
Indian
100% Amox 0.025 0.843693 No
Indian
50% 100% Indian 0.05 0.917952 No
Indian

This table 14 showed the Post Hoc analysis of the anti-bacterial properties of the

Polyalthnia longifolia extract. Table 14 revealed that 50% extract has no significant

difference with all the other treatments. The 25% extract has no significant difference

with 50% extract. The 100% Talisay has no significant difference with Amoxicillin.

Therefore, it means that any percentage is effective against Escherichia coli.

30
Table 15.

Zone of inhibition of synergized extracts against Staphylococcus aureus

Synergized (S. R1 R2 R3 Mean

Aureus)

Water 9.8 9.3 9.2 9.43

25% 14.7 12.3 13.9 13.63

50% 17.7 12.8 16.4 15.63

100% 17.6 19.4 14.7 17.23

Amoxicillin 30.8 26.5 30.5 29.26

Table 15 shows the antibacterial properties of synergized extracts of Terminalia

cattapa and Polyalthnia longifolia against Staphylococcus aureus. The mean of inhibition

of Amoxicillin is closest to the mean of 100% extract with a mean difference of 12.03mm.

This also showed that 100% synergized extract is less effective than Amoxicillin.

Table 16.

Analysis of variance of the zone of inhibition of synergized extracts against

Staphylococcus aureus.

Source SS df MS F Crit

Between 662.92 4 165.73 F=42.655 3.48

Treatments

Within- 38.85 10 3.88

Treatments

Total 701.78 14

31
 Table 16 shows the analysis of variance or ANOVA. This showed that the F

tabulated is greater than the F Crit. This means that the null hypothesis will be rejected.

Therefore, the alternative hypothesis was accepted that states that there is a difference

among the means of the treatments.

Table 17.

Post Hoc Test: Bonferroni- Holm analysis of the mean difference of synergized extracts

against Staphylococcus aureus.

Group 1 Group 2 Critical P Significant?

25% .005556 0.000551 Yes

Mixed Amox

50 % 0.00625 0.002499 Yes

Mixed Amox

100% 0.007143 0.00349 Yes

Mixed Amox

Table 17 showed the Post Hoc analysis of the anti-bacterial properties of the

Synergized extract. Table 17 revealed that 50% Synergized extract has a significant

difference with Amoxicillin. The 25% Synergized extract has a significant difference

with Amoxicillin. The 100% extract has a significant difference with Amoxicillin.

Therefore, it means that any Synergized nut extracts is ineffective against

Staphylococcus aureus.

32
Table 18.

Zone of inhibition of synergized extracts against Escherichia coli.

Synergized R1 R2 R3 Mean
(E. Coli)
Water 8.2 8.1 8 8.1

25% 16 18.9 24.3 19.73

50% 41 30 25.8 32.26

100% 23.9 22.7 27 24.53

Amoxicillin 38 37 40 38.33

Table 18 shows the antibacterial properties of synergized extracts of Terminalia

cattapa and Polyalthnia longifolia against Escherichia coli. The mean of inhibition of

Amoxicillin is closest to the mean of 50% extract with a mean difference of 6.06mm.

This also showed that 50% synergized extract is more effective than Amoxicillin.

33
 Table 19.

Analysis of variance of the zone of inhibition of synergized extracts against Escherichia

coli.

Source SS df MS F Crit

Between 1629.96 4 407.49 F=23.5208 3.48

Treatments

Within- 173.24 10 17.32

Treatments

Total 1803.2 14

Table 19 shows the analysis of variance or ANOVA. This showed that the F

tabulated is greater than the F Crit. This means that the null hypothesis will be rejected.

Therefore, the alternative hypothesis was accepted that states that there is a difference

among the means of the treatments.

34
Table 20.

Post Hoc Test: Bonferroni- Holm analysis of the mean difference of synergized extracts

against Escherichia coli.

Group 1 Group 2 Critical P Significant?

25% 50% Mixed 0.0125 0.071448 No

Mixed
25% 100% Mixed 0.016667 0.155696 No
Mixed
50% Amox 0.025 0.175916 No
Mixed
50% Amox 0.05 0.25913 No
Mixed

Table 20 shows the Post Hoc analysis of anti-bacterial properties of the

Synergized extract of Terminalia cattapa and Polyalthnia longifolia. Table 20 revealed

that 50% Synergized Extract has no significant difference with all the other treatments.

The 25% Synergized extract has no significant difference with 50% Synergized extract

The 100% Synergized extract has no significant difference with Amoxicillin. Therefore,

it means that any percentage is effective against Escherichia coli.

35
 Chapter IV

SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS

This chapter presents the summary of findings and conclusions made from the

study and recommendations given by the researchers.

Summary of Findings

Based on the findings of the research investigation, the following conclusions

were established:

a. 50% Indian tree nut extract can substitute Amoxicillin for fighting against

Staphylococcus aureus.

b. 50% synergized extract can be used to fight against Escherichia coli.

c. Synergized extracts of Terminalia catappa and Polyalthnia longifolia is ineffective

against Staphylococcus aureus.

Conclusion

It can be concluded that the extracts of Terminalia cattapa and Polyalthnia

longifolia have antibacterial properties due to its phytochemical constituents.

Polyalthnia longifolia possesses all phytochemical constituents excepts alkaloids and

carbohydrates. Meanwhile, Terminalia catappa possesses all phytochemical

constituents except saponin. Specifically, 50% Indian Mast Tree nut extract can be used

to fight against Staphylococcus aureus. On the other hand, a 50% synergized extract is

needed to fight against Escherichia coli. According to the data gathered, this can lessen

the cost of expensive antibiotics since Terminalia catappa and Polyalthnia longifolia

are readily available.

36
Recommendations

It is recommended that in the absence of Amoxicillin, both Talisay nut extract

and Indian nut extract can be used as an alternative in fighting against Staphylococcus

aureus while the synergized extracts of Terminalia cattapa and Polyalthnia longifolia

can be used as substitute for Amoxicillin against Escherichia coli. It is also preferred not

to synergize Terminalia catappa and Polyalthnia longifolia when used against

Staphylococcus aureus. Lastly, the investigators recommend using Polyalthnia longifolia

and Terminalia catappa extracts when in rural or remote areas where antibiotics such as

Amoxicillin are scarce or unavailable.

37
APPENDIX

38
Figure 1. Wagner’s Test of Terminalia catappa Nut Extract

Figure 2. Benedict’s Test Terminalia catappa Nut Extract

39
Figure 3. Froth’s Test of Terminalia catappa Nut Extract

Figure 4. Salkowski’s Test of Terminalia catappa Nut Extract

40
Figure 5. Ferric Chloride Test of Terminalia catappa Nut Extract

Figure 6. Gelatin Test of Terminalia catappa Nut Extract

41
Figure 7. Lead Acetate Test of Terminalia catappa Nut Extract

Figure 8. Copper Acetate Test of Terminalia catappa Nut Extract

42
Figure 9. Wagner’s Test of Polyalthnia longifolia Nut Extract

Figure 10. Benedict’s Test Polyalthnia longifolia Nut Extract

43
Figure 11. Froth’s Test of Polyalthnia longifolia Nut Extract

Figure 12. Salkowski’s Test of Polyalthnia longifolia Nut Extract

44
Figure 13. Ferric Chloride Test of Polyalthnia longifolia Nut Extract

Figure 14. Gelatin Test of Polyalthnia longifolia Nut Extract

45
Figure 15. Lead Acetate Test of Polyalthnia longifolia Nut Extract

Figure 16. Copper Acetate Test of Polyalthnia longifolia Nut Extract

46
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