A Study on the Antibacterial Activity of Kahoy Dalaga

(Mussaenda philippica) Leaves Extract Against E. coli and S.

aureus

Armona Jean B. Ongtico

IV-Faraday

Submitted in Partial Fulfilment of the Requirement in

Research II

Agusan National High School

A.D. Curato Street, Butuan City

Mrs. Marie Lou S. Paler

December, 2014

Table of Contents
Title

Page No.

Title Page ----------------------------------- 1

Table of contents----------------------------- 2
Abstract-------------------------------------- 3
Chapter I: Introduction----------------------- 4-9

Background of the Study------------Significance of the Study----------Statement of Hypotheses------------Scope and Limitations--------------Conceptual Framework---------------Definition of Key Terms-------------

4-5
5
6-7
7
8
8-9

Chapter II: Review of Related Literature------ 10-12

Chapter III: Methodology---------------------- 13-15

Material and Methods---------------Plant Extraction-------------------Preparation of Samples-------------Well Diffusion Method--------------Data Gathering---------------------Waste Disposal---------------------Data Analysis-----------------------

13
13
14
14
14
15
15

Chapter IV: Results and Discussion------------ 16- 17

Tables 1---------------------------- 16-17

Statistical Analysis----------------

Chapter V: Conclusion and Recommendation------ 18-19

Chapter VI: Bibliography---------------------- 20-22
Chapter VII: Appendix------------------------- 23-29

Documentation----------------------- 27-28
Acknowledgment-----------------------29

Abstract

The development of antibacterial agents derived from

micro-organisms and chemotherapeutic agents from plants is a
research

area

designed

to

of

the

evaluate

utmost
the

importance.

effectiveness

The

of

study

Kahoy

was

Dalaga

(Mussaenda philippica)leaves extract against Gram-positive

and

Gram-negative

Escherichia coli).

bacteria

(Staphylococcus

aureus

and

The antimicrobial testing using a Paper

Disc Diffusion shows that Kahoy Dalaga leaves extract are

partially active with E.coli with zone of inhibition of
12.99 mm and

active S. aureus with zone of inhibition of

14.89 mm. Thus, this means that the Kahoy Dalaga leaves
extract can be antibacterial agent which proves the claims
of the indigenous people making use of Kahoy Dalaga as good
emollient for stomach pains and other stomach problems.

3
Chapter I

INTRODUCTION
Background of the Study
The advocacy toward using traditional and alternative
medicine

to

deal

significantly

these

with
past

common
few

maladies

years.

has

Before,

increased

the

use

of

herbal medicine had been disregarded because of the presence

of lab-prepared and commercially available medicines. The
development
organisms

of

and

antibacterial

agents

chemotherapeutic

derived

agents

from

from
plants

microis

research area of the utmost importance. The present study

was designed to evaluate the antimicrobial activity of Kahoy
Dalaga (Mussaenda philippica) leaves extract against Grampositive and Gram-negative bacteria (Staphylococcus aureus
and Escherichia coli).
Kahoy Dalaga is a tropical shrub that grows up to 10ft
tall in tropical areas. It grows in thickets and secondary
forests at low and medium altitudes in almost all islands
and provinces in the Philippines. Its branches are slender,
lenticelled and light in color. Kahoy Dalaga is ideal for

the production of birdhouses, planters, and other twig

decors because of its softwood qualities. Aside from these
uses, Kahoy Dalaga is grown for ornamental and medicinal
purposes.

Statement of the Problem

The

study

wants

to

investigate

the

antibacterial

activity of Kahoy Dalaga leaves extract. Specifically this

will answer the following queries:
1. Determine
leaves

the

extract

antimicrobial
using

the

paper

tested bacteria of E. coli

2. Determine the antimicrobial
leaves

extract

using

the

of

disc

effect

paper

tested bacteria of S. aureus.

effect

Dalaga

diffusion

of

disc

Kahoy

Kahoy

with

Dalaga

diffusion

with

Statement of the Hypotheses

Null Hypothesis: There is no significant difference between
the antibacterial activity of Kahoy Dalaga Leaves Extract
and two-gram bacteria (E. coli and S. aureus)
Alternative Hypothesis: There is a significant difference
between the antibacterial activity of Kahoy Dalaga Leaves
Extract and two-gram bacteria (E. coli and S. aureus)

Significance of the Study

The advocacy toward using traditional and alternative
medicine

to

deal

with

common

maladies

has

increased

significantly these past few years. Bearing this parameter

in mind, the trust of people in commercial medicine in
dealing with common diseases was shaken, opting them to seek
alternative

ways.

The

study

was

geared

toward

creating

herbal medicine in dealing with the effect of commerciallyavailable dewormer. The study will focus on the use of Kahoy
Dalaga(Mussaenda

philippica),

perimeter barricade.

common

plant

use

as

This

study

aims

to

establish

the

efficacy

of

the

antibacterial agent which could be applied through decoction

as an emollient for common sickness such as stomached pain.
This will lessen the expenses of buying synthetic medicine
by using a plant which could use also as an ornamental
plant.

Scope and Limitation

This study was limited only on the antibacterial test
of the ornamental plant known as Kahoy Dalaga which was
known to have medicinal effect among the aboriginal people
with two application of bacteria, the E.coli and S. aureus.
The

experimental

set

up

was

Laboratory within two weeks time.

done

in

CSU

Diagnostic

Conceptual Framework
Trial 1

E. coli

Trial 2

Kahoy
Dalaga

Kills the E.
coli bacteria

Trial 3
Trial 1

S.
aureus

Trial 2
Trial 3

Kills the S.
aureus bacteria

Definition of Key Terms

1. Chemotherapeutic- is a category of cancer treatment that
uses chemical substances, especially one or more anti-cancer
drugs that are given as part of a standardized chemotherapy
regimen.
2. Microorganisms- are often illustrated using singlecelled, or unicellular organisms.
3. Escherichia coli

- is a Gram-negative, rod-shaped

bacterium of the genus Escherichia that is commonly found in

the lower intestine of warm-blooded organisms.
4. Staphylococcus aureus- is a Grampositive coccal bacterium that is a member of
the Firmicutes, and is frequently found in the human
respiratory tract and on the skin.

5.

Paper

Disc

Diffusion-

is

method

used

to

see

what

antibiotics or compounds inhibit bacterial growth, or are

bacteriostatic.
6. Emollient- help keep the skin dry by reducing water loss
from the epidermis.
7. Maladies- a disease or ailment.
8. Lenticelled- are small corky spots on young bark.
9. Biological assays- is a type of scientific experiment. It
involves the use of live animal or plant or tissue or cell
to determine the biological activity of a substance
10.Ethanol- Commonly referred to simply as alcohol or
spirits. It is the principal type of alcohol found in
alcoholic beverages, produced by the fermentation of sugars
by yeasts.
11. Derwormer- used to get rid of helminths parasites, such
as roundworm, flukes and tapeworm
12. Chloramphenicol- is effective against a wide variety
of Gram-positive and Gram-negative
most anaerobic organisms

bacteria,

including

Chapter II
REVIEW OF RELATED LITERATURE

Kahoy Dalaga is a tropical shrub that grows up to 10ft

tall in tropical areas. It grows in thickets and secondary
forests at low and medium altitudes in almost all islands
and provinces in the Philippines. The cultivar is native to
the Philippines, popularly grown in most towns and cities.
Its branches are slender, lenticelled and light in color.
Leaves are opposite, broad-ovate, with short-pointed tips,
dark

green

and

glossy.

Kahoy

Dalaga

is

ideal

for

the

production of birdhouses, planters, and other twig decors

because of its softwood qualities. Aside from these uses,
Kahoy Dalaga is grown for ornamental and medicinal purposes.
Kahoy

Dalaga

is

used

for

dysentery

and

snake

bites.

Decoction of roots and leaves used for affections of the

chest

and

Externally,
emollient.

lung.

Its

decoction
For

bark

is

used

of

leaves

can

influenza

virus,

the

for
also

stomach
be

healers

ache.

used
of

as

Samar,

Philippines, use the scrapings of the bark of Kahoy Dalaga.

10

Escherichia

coli

is

a Gram-negative, facultatively

anaerobic, rod-shaped
genus Escherichia that

bacterium of
is

commonly

the

found

in

lower intestine of warm-blooded organisms. Most

coli strains are

harmless,

but

the
E.

some serotypes can

cause

serious food poisoning in their hosts, and are occasionally

responsible for product recalls due to food contamination.
Among

the

many

strains

of

E.

coli,

only

few

trigger

diarrhea. One group of E. coli which includes O157:H7

produces a powerful toxin that damages the lining of the
small

intestine,

which

can

cause

bloody

diarrhea.

You

develop an E. coli infection when you ingest this strain of

bacteria. Unlike many other disease-causing bacteria, E.
coli can cause an infection even if you ingest only small
amounts. Because of this, you can be sickened by E. coli
from

eating

slightly

undercooked

hamburger

or

from

swallowing a mouthful of contaminated pool water. Potential

sources of exposure include contaminated food or water and
person-to-person contact.

11

Staphylococcus

aureus is

positive coccal bacterium that

the Firmicutes,

and

is

is

frequently

a Grama

found

member
in

the

of
human

respiratory tract and on the skin. Although S. aureus is not

always pathogenic, it is a common cause of skin infections,
respiratory disease, and food poisoning. Almost any organ
system can be infected by S. aureus. Most frequently, S.
aureus strains first infect the skin and it structures or
invades

damaged

skin.

Sometimes

the

infections

are

relatively limited, but other times they may spread to other

skin

areas.

Unfortunately,

these

bacteria

can

reach

the

bloodstream and end up in many different body sites, causing

infections

(wound

infections,

abscesses,

osteomyelitis,

endocarditis, pneumonia) that may severely harm or kill the

infected person.

12

Chapter III
METHODOLOGY

Material and Methods

Plant collecting and processing: Five kilograms of Kahoy
Dalaga leaves were

collected using systematic sampling. In

the systematic sampling, every third leaf from the left were
picked

in

the

Kahoy

Dalaga

plant

located

in

the

CSU

botanical garden. The leaves were placed in a clean oven

plate and were dried under the heat of the sun for four to
five

days.

After

drying,

all

the

leaves

samples

were

preserved in wax-quoted paper bags and was brought to CSU

diagnostic laboratory for biological assays.

Plant extraction: The fresh dried plant of Kahoy Dalaga

(5kg) will be ground and soaked in ethanol (commercial,
doubly distilled 50 lit). The filtrate will be concentrated
under reduced pressure at 40C to a gum. This crude gum will
be used for activity purpose.

13

Preparation

of

samples:

The

concentrated

residues

were

suspended in an emulsifier, polyethylene glycol 400 (PEG400) which has no inhibitory effect on fungal and bacterial
growth. The organic extracts were dissolved in this solvent.
Aliquots of this solution (5% extract solution) were used to
test

for

antibacterial

cultures of E. coli and

activity

against

pathological

S. aureus.

Well diffusion method: 24 hrs fresh broth cultures (media;

nutrient broth, merk) will be used. Seeding of media (Muller
Hinton-Merk) with test cultures will initially done and then
well will be dug in media. Extract will be filled in well
and plates will be incubated at 37C for 48 hrs. The clear
zone will be measured against the test culture.

Data Gathering: After more or less 48 hours, the data were

collected and analyzed.

14

Waste Disposal: Sterilization using autoclave or pressure

cooker. The steam under pressure was used to produce a
temperature

of

121C

and

after

15

minutes,

all

the

microorganisms, including bacterial endospores, were killed.

Data Analysis: The statistical tool used to analyze the data

is One-way ANOVA.

15

Chapter IV
RESULTS AND DISCUSSION
Antimicrobial Effect of Kahoy Dalaga
The Antimicrobial Activity of Kahoy Dalaga Applied to
E.coli and S. aureus

16

Table above shows the antimicrobial activity of Kahoy

Dalaga leaves extract when applied to paper disc diffusion
method. It has been found that Kahoy Dalaga leaves extract
is partially active
12.99 mm and

with E. coli with zone of inhibition of

active S. aureus with zone of inhibition of

14.89 mm.

Statistical Analysis
The Statistical tool to be used in this study is One
Way- Anova.
Anova Table for S.aureus
Source of
Variance (SV)
Treatment
Error

Degrees of
Freedon (df)
2
6

Sum of
Squares (SS)
66.79
305. 69

Mean of Square
(MS)
33.39
6.79

F
value
2.99

Since the F> 0.01 (2.99> 0.01), Ho is rejected.

Anova Table for E. coli
Source of
Variance (SV)
Treatment
Error

Degrees of
Freedon (df)
2
6

Sum of
Squares (SS)
87.87
221.94

Mean of Square
(MS)
43. 935
4. 932

Since the F> 0.01 (10.68> 0.01), Ho is rejected.

17

F
value
10.68

Chapter V
CONCLUSION AND RECOMMENDATION
CONCLUSION
This study about the efficacy of Kahoy Dalaga is an
evaluation of the indigenous knowledge of the effectivity of
a traditional medicine being used by the indigenous people
from the Mamanwa tribe. Thus, from this study wherein the
antibacterial analysis was being analyzed and it was found
out to have an antimicrobial activity of partially active
and active which means it can be used as antibacterial agent
especially

for

stomach

pains

and

problems.

Another

investigation was the nematicidal effect of kahoy dalaga and

it

was

proven

with

the

application

in

nematode-

Red

Wrigglers which has a high mortality rate with 4 minutes all

the worms were dead with concentrations of 50%,75% and 100%
dilution. Finally, the effect of the kahoy dalaga extract
with mosquitoes with the process of tenfold dilution wherein
its toxicity level was calculated as 156.77 g /ml which is
classified as moderately toxic.

18

RECOMMENDATION
Due to the effectivity of Kahoy Dalaga as antibacterial
agent, the researcher recommends to use the plant as a
dewormer and as a cure for stomach pains.

19

Chapter VI
Bibliography

Dethier VG, Barton Browne L, Smith CN. J Econ Ent, 1960; 53:
134-136.

Schoonhoven IM. EntomolExp and Appl, 1982; 31: 57-69.

Mordue AJ. Azadirachtin - A review of its mode of action in

insects. In: Kleeberg H (Ed.). Practice Oriented Results on
Use and Production of Neem-Ingredients and Pheromones
Germany, 1998; pp. 1-4.

Arnason JT, Philogene BJR, Morand P. Insecticides of plants

origin.American Chemical Society Symposium Series Vol.
387.Washington, 1989.

Jilani G, Su HCF. J Econ Entomol, 1983; 76:154-157.

Isman MB. Crop Prot, 2000; 19: 603-608.

Carlini CR, Grossi-de S MF.Toxicon, 2002; 40: 1515-1539.

20

Yang RZ, Tangs CS. Econ Bot, 1988; 42: 376-406.

Lindgren BS, Nordlander G, Birgersson G. J ApplEntomol,

1996; 120: 397-403.
. Klepzig KD, Schlyter F. J Econ Ent, 1999; 92(3): 644-650.

Govindachari TR, Suresh G, Gopalakrishnan G, Wesley SD. J

ApplEnt, 2000; 124: 287-291.

Khalequzzaman M, Khanam LAM, Talukdar D. Int Pest Control,

1994; 36: 128-130.
Park T, Frank MB. J Ecol, 1948; 29: 386-375.

Busvine JR. A critical review of the techniques for testing

insecticides.Commonwealth Agricultural Bureau, London.1971;
345.

Abbott WS.J econ Ent, 1925; 18: 265-267.

Edeogo HO, Okwy DE, Mbaebie BO. African Journal of

Biotechnology, 2005; 4: 685-688.

21

Mamun MSA, Shahjahan M, Ahmad M. J Bangladesh AgrilUniv,

2009; 7(1): 1-5.
Khalequzzaman M, Sultana S. J Bio-sci, 2006; 14: 107-112.
Saxena BP and Mathur A. Experientia, 1976; 32: 315-316

22

Chapter VII
Appendix

Method Used: One Way Anova

Statistical Test for S. aureus
Trials/ Respondents

XA
15
16
15

XB
14
15
14

XC
15
15
15

XA2
225
256
225

XB2
196
225
196

dft= rc-1

dfw= c(r-1)

dfc= c-1

dft= 3(3)-1

dfw= 3(3-1)

dfc= 3-1

dft= 8

dfw= 3(2)

dfc= 2

dfw= 6

SSt= (x2 )- [ (x) ] / rc

SSt= (706+ 617+ 675) (46+ 43+ 45)
SSt= 6749- 17956/ 9 =6749- 1995.11
SSt= 4753.89

23

/ (3)(3)

XC2
225
225
225

SSc= [ (x2)] / r- [ (x)]2/ rc

SSc= ( X2A)+ ( X2B)+ ( X2C)/ 3-1995.11
SSc= (46)2+ (43)2+ (45)2/ 3 -1995.11
SSc= 2249.67- 1995.11
SSc= 254.56
SSw= SSt- SSc
SSw= 4753.89-254.56 = 4499.33
MSSc= SSc/ dfc

MSSw= SSw/ dfw

MSSc= 254.56/ 2 = 127.28

MSSw= 254.6/6 = 42.43

Fc= MSSc/Mssw
Fc= 127.28/42.43 = 2.99

24

Method Used: One Way Anova

Statistical Test for E. coli
Trials/ Respondents

XA
13
14
13

XB
12
13
13

XC
12
13
14

XA2
169
196
169

XB2
144
169
169

dft= rc-1

dfw= c(r-1)

dfc= c-1

dft= 3(3)-1

dfw= 3(3-1)

dfc= 3-1

dft= 8

dfw= 3(2)

dfc= 2

dfw= 6

SSt= (x2 )- [ (x) ] / rc

SSt= (899+ 482+ 509) (40+ 38+ 39)
SSt= 1890- 13689/ 9 = 1890-210
SSt= 1680

25

/ (3)(3)

XC2
144
169
196

SSc= [ (x2)] / r- [ (x)]2/ rc

SSc= ( X2A)+ ( X2B)+ ( X2C)/ 3- 210
SSc= (40)2+ (38)2+ (39)2/ 3 210
SSc= 1521.667-210
SSc= 1311.667
SSw= SSt- SSc
SSw= 1680-1311.667 = 368.333
MSSc= SSc/ dfw

MSSw= SSw/ dfw

MSSc= 1311.667/ 2 = 655.834

MSSw= 368.33/ 6 = 61.388

Fc= MSSc/Mssw
Fc= 655.834/61.388= 10.68

26

Documentation

Figure 1. Cutting and preparing the Kahoy Dalaga leaves for

extraction

Figure 2. Filtration and Extraction of Kahoy Dalaga Leaves

27

Figure 3. Antimicrobial Testing (Well diffusion method) with

the Application for Kahoy Dalaga Leaves Extract

28

Acknowledgement

The researchers would like to acknowledge Father

God for giving us the strength to continue with our project.
Parents, for their endless support and understanding. Coach,
for helping us to make this project possible and to lastly
to our teacher for guiding us in doing our project.

29