LOS BAÑOS NATIONAL HIGH SCHOOL

Umali Subd., Brgy. Batong Malake, Los Baños, Laguna

THE EFFICACY OF KANTUTAY SHOOTEXTRACT AS ANTIBACTERIAL AGENT

AGAINST ESCHERICHIA COLI, AND STAPHYLOCOCCUS AUREUS

CLUSTER 1

Life Science

(TEAM CATEGORY)

ABSTRACT

THE EFFICACY OF KANTUTAY SHOOT EXTRACT AS ANTIBACTERIALAGENT AGAINST ESCHERICHIACOLI AND STAPHYLOCOCCUS AUREUS

The research study is all about the Efficacy of Kantutay Shoot Extract as Antibacterial Agent against Escherichia coli and Staphylococcus Aureus. It was conducted in the Institute of Biological Sciences. The two bacteria mentioned were tested to determine the efficacy of the Kantutay Extract.

Likewise, a positive control, two Kantutay shoot extracts (Pure & Boiled) and a negative control were used to determine the effects of Kantutay shoot extracts on each bacteria. The study was laid out in Kirby- Bauer method with two replications.

The experiment showed that the test result was positive on E. coli, but was proven to be negative on the Staphylococcus Aureus.

The results suggest that Kantutay shoot extract was an Effective Antibacterial Agent against E. coli only.

I

ACKNOWLEDGEMENT

This research projects has been a challenge for us. Since we have different

schedules and we experienced time conflict, we had time on doing this research project.

We were just taking our time on doing the project knowing that we still have more time

and it will all be fine. Until our research teacher told us the deadline of the project. It was

earlier than what we expected. So, we crammed.

We were on the edge of giving up having the entire obstacle that we had but still,

we were to pass our work. And we couldn’t do this without the help of a lot of people.

First and foremost, we would like to praise and express our faith to the Almighty for

giving us the patience and hope whenever we felt we lost it all. Thank you Lord God for

everything. Thank you for blessing us with knowledge and giving us with instruments of

Your love. We all love You.

And of course, we would like to thank our research teacher, Mrs. Karla Mae M.

Lopez, for giving us guidelines, tips and encouragements on doing this project.

We also would like to express our gratitude to Mr. Patrick M. Rocamora. Thank

you for helping us as our resource person.

Ii

Our gratitude to Rocamora family unselfishly letting us stay in their house in

doing our experiments and papers.

Next is to Mr. Noel Sabino our researcher consultant, mentor and friend. Thank

you of encouragement. You instilled in us the skill and the ways of a researcher.

To our loving parents, many thanks! You are our inspiration on doing this

researcher project. We could not have done this without you. Thank you very much for

allowing and understanding us when we are going home late going on weekends when

we are conducting our experiments.

Our classmates in IV- Diamond on encouraging us that we can finish this project

on time and for tour support. Thank you and good luck to us.

To our subject teachers and especially our adviser, thank you for letting us be

excused on your classes especially on the day we needed the most.

To all who have been part of this research project, words can’t express our

gratitude and happiness. Thanks a lot for being there for us.

iii

TABLE OF CONTENTS

Title

Abstract i

Acknowledgement ii-iii

Table of contents iv

I. The Problem and Its Background 1

Introduction 1

Background of the Study 2-13

Statement of the Problem 14

General Objective 14

Specific Objectives 14

Hypotheses 14

Significance of the Study 15

Scope and Delimitations 15

Definition of Terms 16

II. Methodology 17-21

III. Results and Discussion 22-23

IV. Summary of the Findings, Conclusion and Recommendations 24

V. Bibliography 25

ivTHE PROBLEM AND ITS BACKGROUND

Introduction

The pharmaceutical industry uses about 120 different compounds derived from

plants in the drugs it manufactures, and it discovered nearly three-quarters of these

compounds by studying folk remedies.

Medicinal Plants are sources of enormous quantities of chemical substances

which are able to initiate different biological activities including those useful in the

treatment of human diseases. Scientific investigations of medicinal plants used in folklore

remedies have attracted increased attention in the “medical world,” especially in a bid to

finding solutions to the problems of multiple resistances to the existing synthetic

antimicrobials. Most of the synthetic antibiotics now available in the market have major

setbacks due to the accompanying side effects on patients and the multiple resistances

developed by pathogenic organisms to them. Hence, there is a justifiable need to explore

for new and more potent antimicrobial compounds of natural origin to combat these

pathogens. A pathogen is a biological agent that causes disease or illness to its host. The

essential oil composition of the Kantutay shoots contains the parts that are needed for the

application of this experiment. It contains cinecol, sabinene, and caryophylene.

Basically, in testing Gram-positive bacteria, there is a need for the Gram-negative

bacteria. The pathogenic capability of E. coli is usually associated with certain

components of Gram-negative cell walls, in particular the lipopolysaccharide (also know

n as LPS or endotoxin) layer. Unlike E. coli has a Gram-positive spherical cell.

Background of the study

Lantana camara, also known as Spanish Flag, is native to tropical regions in

Central and South America. Lantana camara has been introduced into other parts of the

world as an ornamental plant; it is considered an invasive species in many tropical areas.

It is sometimes known as "red (yellow, wild) sage", despite its classification in a separate

family to sage (Lamiaceae), and a different order to sagebrush (Asterales).

The essential oil of L. camara from different regions of the world has been

reported by many workers (Da Silva et al., 1999: Sefidkon, 2002: Kasali et al., 2004).

The oils differ in their chemical compositions according to geographic origin of the

plants. Da Silva et al. (1999) reported differences in essential oil composition of L.

camara collected at different places in the Amazon region of North Brazil.

The essential oil obtained from leaves of Lantana camara from Cuba was

analyzed by GC-FID and GC/Ms. The oil was characterized by the high percentage of

sesquiterpenes. The major components were (E)-nerolidol (43.4%), lambdacadinene

(7.6%), alpha-humulene (4.9%) and beta-caryphyllene (4.8%) (Journal of Essential Oil

research, Cuba, 2004).

The essential oil composition of Lantana camara leaves from Nigeria was

reported to have 1,8-cineol (15.8%), sabinene (14.7%) and P-caryophyllene (8.9%) out of

78% of oil that were identified.

E. coli is normally present in the human intestine, but the variant E. coli O157:H7

produces toxins that cause bloody diarrhea and, in some cases, far more severe problems,

including kidney failure and death .A person can become infected by eating contaminated

meat (Microsoft Encarta, 2006). This pathogenic capability is usually associated with

certain components of Gram-negative cell walls, in particular the lipopolysaccharide

(also known as LPS or endotoxin) layer. In humans, LPS triggers an innate immune

response characterized by cytokine production and immune system activation.

Inflammation is a common result of cytokine production; which can also produce host

toxicity.

Staphylococcus aureus, literally the "golden cluster seed" or "the seed gold" and

also known as golden staph) is the most common cause of staph infections. It is a

spherical bacterium, frequently part of the skin flora found in the nose and on skin. S.

aureus was discovered in Aberdeen, Scotland in 1880 by the surgeon Sir Alexander

Ogston in pus from surgical abscesses. Each year some 500,000 patients in American

hospitals contract a staphylococcal infection.

In this study, the efficacy of Lantana camara, locally known as “Kantutay”

against E. coli and S. aureus will be determined.

3

Review of Related Literature

Lantana camara is a low erect or subscandent, vigorous shrub with stout recurved

prickles and a strong odour of black currents; it grows to 1.2-2.4 meters (or even more);

its root system is very strong, and it gives out a new flush of shoots even after repeated

cuttings; Leaf ovate or ovate-oblong, acute or subacute, crenate-serrate, rugose above,

scabrid on both sides; Flower small, usually orange, sometimes varying from white to red

in various shades and having a yellow throat, in auxiliary heads, almost throughout the

year; Fruit small, greenish-blue black, blackish, drupaceous, shining, with two nutlets,

almost throughout the year, dispersed by birds. Seeds germinate very easily. (Sastri and

Kavathekar, 1990).

The diverse and broad geographic distribution of lantana is a reflection of its wide

ecological tolerances. It occurs in diverse habitats and on a variety of soil types. It

generally grows best in open unshaded situations such as wastelands, rainforest edges,

beachfronts, and forests recovering from fire or logging. Disturbed areas such as beside

roads, railway tracks and canals are also favorable for the species (Thaman 1974; Winder

and Harley 1983; Thakur et al. 1992, Munir 1996, in Day et al. 2003). Lantana does not

invade intact rainforests, but is found on its margins (Diatloff 1975; Humphries and

Stanton 1992, in Day et al. 2003). Where wet sclerophyll forests and rainforests have

been disturbed through logging, gaps are created; this allows lantana to encroach on the

forests. Further logging aggravates the condition and allows the lantana to spread or

become thicker (Waterhouse 1970, in Day et al. 2003).

4

Kantutay has several uses, mainly as herbal medicine and in some areas as

firewood and mulch. The leaves are used to reduce itching. Other used are against flu,

colds coughs, fever, yellow fever, dysentery and jaundice. The roots are used for

gonorrhea.

Lantana oil is sometimes used for the treatment of skin itches, as an antiseptic for

wounds and externally for leprosy and scabies. Lantana repels other plants and other

groups of organisms such as insects. Lantana oil is used externally for leprosy and

scabies. Plant extracts are used as medicine for the treatment of cancers, chicken pox,

measles, asthma, ulcers, swellings, eczema, tumors, high blood pressure, bilious fevers,

catarrhal infections, tetanus, rheumatism, malaria and atoxy of abdominal viscera.

The toxic and repellent effects of two tropical plants, Lantana camara L. and

Tephrosa vogelli Hook were Evaluated against Sitophilus zeamais Motschulsky in stored

maize grain.s Five rates (1.0, 2.5, 5.0, 7.5 and 10.0% w/w) of each powdered plant

material, an untreated control and a synthetic insecticide (Actellic Super 2% dust) were

used to investigate treatment efficacy on mortality of the adult insect (five to eight days

old), F1 progeny emergence and repellence against zeamais adults.

In many regions, lantana has become dominant component of natural and

agricultural ecosystems. The rapid removal of natural forests without replacement by

structurally similar native vegetation may be partially replaced with tickets of lantana.

Consequently, the amount of available habit for native animals may decrease. In some

areas, weeds such as lantana may provide shelter and vital winter food for many native

birds. A number of endangered birds species utilize lantana thickets when their natural

habitat is unavailable. In Australia, the vulnerable black-breasted buttonquail, Turnix

melanogaster, feeds and roots in lantana thickets adjacent to its more favored habitat,

vine forest.

(Smith et al. 1998, in Day et al. 2003.). While buttonquails prefer intact vine

forests, lantana provides an important temporary refuge for them between forest

remnants. In Central Kenya, where natural riverine thickets have been almost completely

cleared, the endangered Hinde’s babbler, Turdoides hindei, has become dependent on

lantana thickets, and unless sufficient suitable natural habitat can be restored the survival

of this species depends on the retention of lantana infestations (Njoroge et al. 1998).

Apart from benefiting some bird species, lantana is a major nectar source for many

species of butterflies and moths.

Lantana camara is a highly variable species. It has been cultivated for over 300

years and now has a hundred of cultivars and hybrids. These belong mostly to the L.

camara complex. Cultivars can be distinguished morphologically (variation in: flower

size, shape and color, leaf size, hairiness and color; stem thorniness), physiologically

(variation in: growth rates, toxicity to livestock) and by their chromosome number and

DNA content (Pierre Binggeli, 1999).

The key to good management of lantana is constant vigilance (Day et al. 2003.).

Repeated control of regrowth is critical to success. Control of new infestations should be

a priority because the species is able to expand its range during good seasons.

Mechanical: Mechanical clearing and hand pulling are suitable for small areas and fire

can be used over large areas. Biological: Biocontrol agents have decreased the volume of

individual plants making other control methods considerably easier. None of the over 40

agents trialed have resulted in control but some have been partially successful including

Teleonemia scrupulosa Stal (Hemiptera), Octotoma scabripennis (Coleoptera), Uroplata

girardi Pic (Coleoptera) and Ophiomyia lantanae (Froggatt) (Diptera) (Dap et al. 2003)

L. camara was the first weed ever targeted for classical biological control at the

turn of the century, and since then 36 insect species have been released in 33 countries

throughout the exotic range. Despite these efforts, control of the weed has generally been

disappointing (Thomas and Ellison 2000). Many reasons have been suggested for this

failure: the great genetic diversity of the plant, its ability to hybridize, and that fact that its

origin as a hybrid ornamental plant complicates the search for its center of origin and thus

for potential agents (Thomas and Ellison 1999, Day et al. 2003). Twenty nine biotypes

exist in Australia alone (Smith and Smith 1982, in Thomas and Ellison 1999). No insect

agent released to date has caused significant damage to the very important Common Pink

biotype (Thomas and Ellison 1999). In general, the insect agents released have a

restricted host range within this complex, and, in addition, the weed is able to tolerate

wider climatic and geographical areas (Thomas and Ellison 1999). Searchers have been

made in Mexico, Central America, the West Indies, and Brazil, and insects have been

collected from several different lantana species. These insects have been host-tested and

released in Hawaii, South Africa, Australia, several countries in East Africa, South and

East Asia and the Pacific (Day. 300).

A strain of the rust Prospodium tuberculatum from Brazil was screened as a

biocontrol agent against 40 Australian L. camara forms and 52 closely related, non-

target plant species. Result under glasshouse conditions showed that the Brazilian rust

strain is pathogenic to only two flower color forms: pink ad pink-edged red. Macro- and

microsymptoms were recorded using 11 assessment categories and 4 susceptibility

ratings. No macrosymptoms were observed on any of the non-target plans (Thomas et al.

2006).

Lantana flowers in most places all year round if adequate moisture and light are

available (Gujral and Vasudevan 983, Graaff 1986, in Day et al. 2003), with flowering

peaking duringthe wet summer months. In cooler or drier regions, flowering occurs inly

in the warmer or wetter months, due to frost or drought damage (Winder 1980, Swarbrick

et al. 1998 in Day et al. 2003). Plants can flower as early as the second growing season.

Initially, lepidopteron species were thought to be primary pollinators of lantana

(Dronamraju 1958, Schemske 1976, Kugler 1980, Hilje 1985, in Day et al. 2003)

Statement of the Problem

The project aims to determine the effectiveness of the extract from Kantutay

shoots as an antibacterial agent against E. coli and Staphylococcus aureus.

General Objective

To determine the effectiveness of the extract from kantutay shoots as an

antibacterial agent against E. coli and Staphylococcus aureus.

Specific objectives

1. Is the extract from kantutay shoots an effective antibacterial agent against

E. coli and Staphylococcus aureus?

2. Is the pure extract from kantutay more effective than the boiled extract?

3. Is the antibacterial affordable?

Hypotheses

1. Kantutay shoots extract is an effective antibacterial agent against E. coli

and Staphylococcus aureus.

2. The pure extract is more effective than the boiled extract.

3. The antibacterial if affordable.

14

Significance of the study

This study was conducted due to the large spread of diseases, namely diarrhea,

cramps, meningitis, arthritis, sore throat, fever, etc. which is usually caused by the

bacteria E. coli and Staphylococcus aureus. This experiment/research study was also

conducted to prevent the spread of communicable diseases. Kantutay shoots are

commercially produced and easy to grow. They are accessible and have a high

antibacterial content. This study also provides information to the public on the causes of

diseases, its spread and prevention.

Scope and delimitation

This study focused on the use of kantutay shoot extract as antibacterial against E.

coli and Staphylococcus aureus. Other bacteria were not considered, hence the study is

focused upon those two strains of bacteria. The kantutay shoots were fresh and newly

picked. The researchers were not able to consider other plant sources due to time and

resource constraints.

15

METHODOLOGY

Antibacterial test of Kantutay shoot extract

Research design

The researchers used Kirby-Bauer Technique for the bioassay of the study. Two

bacteria: E. coli and S. aureus were used for the study. Streptomycin was used for the

positive control and distilled water for the negative control. The assay uses 3 agar plates

with 2 replications on each culture.

Research materials

The materials used in the media preparation of the activity were: 6 Petri dishes, 4

indicator strains, blender, chopping board, knife, agar plate and other necessary

glassware.

Conduction of the experiment

1. culturing the test organism

• E. Coli

• Staphylococcus aureus

The bacterial strains were placed in vials (10ml) and were inoculated for 16 to 18

hours.

2. Solvent extraction of Kantutay shoots

Solvent: water

Formulation of solution:

The weight of the solvent is 100ml (each) and the weight of the kantutay shoots

200g.

The Kantutay shoots and each kind of solvent were mixed using a blender. The

extracts undergo decantation to produce a debris-free solution.

3. Bioassay (Kirby-Bauer Method)

The sensitivity-disc method of determining which of a wide variety of antibiotics is

effective against an organism is a rapid, accurate, and inexpensive diagnostic tool. Broad

spectrum antibiotics affect a wide variety of both gram-negative and gram-positive

organisms.

The use of the carefully standardized techniques reduces the influence of

variables in the preparation of the test. This illustrates the differences in the

sensitivity of gram-positive and gram-negative bacteria to several antibiotics. This

will measure the antibiotic sensitivity of only one bacterium.

Procedure

1. The sterile swab was dipped into the one bacteria broth culture of standard

density: then rotated several times with firm pressure on the inside wall of the tube above

the fluid level to remove excess inoculums.

2. The swab was streaked over the entire Muller-Hinton agar plate surface and

repeat two more times rotating the plate about 60 degrees to insure even distribution of

inoculums. Replace plate top and allow 3-15 minutes for excess surface moisture to

absorb.

3. The discs were applied to the surface using sterile forceps and dispenser.

Distribute the discs evenly. Gently press down each disc to insure complete contact with

the agar surface.

4. Within 15 minutes, after the discs were applied, the plates were incubated at 37

degrees Celsius.

5. After 16-18 hours of incubation, each plate was examined and the diameters were

measured of the zones of complete inhibition using a ruler or template.

6. The zones of inhibition were interpreted referring to the tables provided and

reported the organisms tested to be susceptible, intermediate, or resistant to the various

agents used.

7. The plate of the second bacterium was obtained and the zones of inhibition were

measured to determine the susceptibility of these organisms to the various antibiotics.

RESULTS, FINDINGS, AND DISCUSSIONS

Antibacterial test of Kantutay extract

To test which culture was greatly affected by the crude extracts, the sensitivity

discs were put in agar plate. Data were gathered after 18 to 24 hours ofinhibitionand was

carefully analyzed by zone of interpretation as the statistical tool.

Cultures Positive

Control

1st crude extract 2nd crude extract Negative control

E. coli + + + + + + - -

Staphylococcus

aureus

+ + - + + - - -

Mean + + + -

Table 1: The results of Antibacterial test of Kantutay shoots extract

Cultures R

(positive

control)

I

(positive

control)

S

(positive

control)

N(none)

(positive

control)

E. coli + + - -

Staphylococcus

aureus

- - - -

Table 2: Zone Size Interpretation

SUMMARY OF FINDINGS, CONCLUSION AND RECOMMENDATION

Summary of findings

The results of the antibacterial test are as follows: for the positive control, both E.

coli and S. aureus were detected in both trials: for pure extract, for both trials, E. coli was

detected, but S. aureus, it was detected in only one trial. Results are the same for the

boiled extract and both bacteria were not detected in the negative control.

For the zone of inhibition, E. coli was detected up to the intermediate zone, while

there was no inhibition for S. aureus.

Conclusion

The study conducted was proven to be effective as an antibacterial agent against

E.coli only and not against Staphylococcus Aureus. The results testified this conclusion

made.

Recommendation

The researchers recommend having further experimentation using more bacteria.

The researchers suggest that the antibacterial agent should be used against pathogenic

bacteria.

BIBLIOGRAPHY

http://www.motherherbs.com/lantana.html

http://