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------------- 4-5 Significance of the Study----------- 5 Statement of Hypotheses------------- 6-7 Scope and Limitations--------------- 7 Conceptual Framework---------------- 8 Definition of Key Terms------------- 8-9

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

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

Material and Methods---------------- 13 Plant Extraction-------------------- 13 Preparation of Samples-------------- 14 Well Diffusion Method--------------- 14 Data Gathering---------------------- 14 Waste Disposal---------------------- 15 Data Analysis----------------------- 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 of the utmost importance. The study was

designed to evaluate the effectiveness of Kahoy Dalaga

(Mussaenda philippica)leaves extract against Gram-positive

and Gram-negative bacteria (Staphylococcus aureus and

Escherichia coli). 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 with common maladies has increased

significantly these past few years. Before, the use of

herbal medicine had been disregarded because of the presence

of lab-prepared and commercially available medicines. The

development of antibacterial agents derived from micro-

organisms and chemotherapeutic agents from plants is a

research area of the utmost importance. The present study

was designed to evaluate the antimicrobial activity of Kahoy

Dalaga (Mussaenda philippica) leaves extract against Gram-

positive 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

4

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 the antimicrobial effect of Kahoy Dalaga

leaves extract using the paper disc diffusion with

tested bacteria of E. coli

2. Determine the antimicrobial effect of Kahoy Dalaga

leaves extract using the paper disc diffusion with

tested bacteria of S. aureus.

5

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 commercially-

available dewormer. The study will focus on the use of Kahoy

Dalaga(Mussaenda philippica), a common plant use as

perimeter barricade.

6

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 done in CSU Diagnostic

Laboratory within two weeks’ time.

7

Conceptual Framework

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 single-

celled, 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 Gram-

positive coccal bacterium that is a member of

the Firmicutes, and is frequently found in the human

respiratory tract and on the skin.

8

Kahoy Dalaga

E. coli

Kills the S. aureus bacteria

Kills the E. coli bacteria

S. aureus

Trial 1

Trial 2

Trial 3

Trial 1

Trial 2

Trial 3

5. Paper Disc Diffusion- is a 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 bacteria, including

most anaerobic organisms

9

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 lung. Its bark is used for stomach ache.

Externally, decoction of leaves can also be used as

emollient. For influenza virus, the healers of Samar,

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

10

Escherichia coli is a Gram-negative, facultatively

anaerobic, rod-shaped bacterium of the

genus Escherichia that is commonly found in the

lower intestine of warm-blooded organisms. Most E.

coli strains are harmless, but 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 a 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 a 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 a Gram-

positive coccal bacterium that is a member of

the Firmicutes, and is frequently found in the 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 40°C 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 (PEG-

400) 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 activity against pathological

cultures of E. coli and 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 37ºC 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 121°C 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 with E. coli with zone of inhibition of

12.99 mm and 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)

Degrees of Freedon (df)

Sum of Squares (SS)

Mean of Square (MS) F value

Treatment 2 66.79 33.39 2.99Error 6 305. 69 6.79

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

Anova Table for E. coli

Source of Variance (SV)

Degrees of Freedon (df)

Sum of Squares (SS)

Mean of Square (MS) F value

Treatment 2 87.87 43. 935 10.68Error 6 221.94 4. 932

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

17

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 µg /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 Sá 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 XB XC XA2 XB

2 XC2

1

15 14 15 225 196 22516 15 15 256 225 22515 14 15 225 196 225

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= Σ(Σx2 )- [ Σ(Σx) ] / rc

SSt= (706+ 617+ 675) – (46+ 43+ 45) 2 / (3)(3)

SSt= 6749- 17956/ 9 =6749- 1995.11

SSt= 4753.89

23

SSc= [Σ (Σx2)] / r- [Σ (x)]2/ rc

SSc= (Σ X2A)+ (Σ X2

B)+ (Σ 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 XB XC XA2 XB

2 XC2

1

13 12 12 169 144 14414 13 13 196 169 16913 13 14 169 169 196

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= Σ(Σx2 )- [ Σ(Σx) ] / rc

SSt= (899+ 482+ 509) – (40+ 38+ 39) 2 / (3)(3)

SSt= 1890- 13689/ 9 = 1890-210

SSt= 1680

25

SSc= [Σ (Σx2)] / r- [Σ (x)]2/ rc

SSc= (Σ X2A)+ (Σ X2

B)+ (Σ 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