i

Aloe barbadensis LEAF EXTRACT IN INHIBITING THE

MUTATION OF Salmonella typhimurium

A Research Paper

of

CAVITE NATIONAL SCIENCE HIGH SCHOOL

(Regional Science High School, Region IV-A CALABARZON)

Maragondon, Cavite

Presented to

The Honorable Board of Judges

as an Official Entry for Division Science Fair

Life Science- Individual Category

ROMELLA C. LINA

IV- Nitrogen

RUBY H. BAUTISTA

Research II Adviser

September 2009

ii

ABSTRACT

Aloe vera leaf extract was said to be a potential natural way of cancer chemoprevention

and treatment. To validate the concept, a research study was conducted to know the inhibitory effect of aloe vera on the mutation of Salmonella typhimurium caused by tobacco extract.

S. typhimurium TA 100 which required histidine for growth was utilized in the

experiment. Aloe vera leaf extract and tobacco leaf extract were obtained. One-hundredth mL of the tester strain was spread on each agar plate and was incubated for 48 hours with the leaf samples of different dilution. Each set-up was observed for the presence of colonies formed as indicator of mutation.

Results revealed that aloe vera has inhibitory effect on the mutation of S. typhimurium

since the number of colonies observed on the tobacco only arm was greater than that of tobacco + aloe gel arm, 6.8 * 103 vs. 1.3 * 103.

iii

ACKNOWLEDGEMENT

This research study will not be made possible without the utmost support, help,

trust, and guidance of the following:

First and foremost, the Almighty God, our Lord and Savior who showered

countless blessings, guidance, and love. Without Him, the researcher will not have the

chance to meet the people behind the success of her research study and she will not

have the chance to experience what life offers;

The researcher’s parents, Mrs. Corazon C. Lina and Mr. Romulo A. Lina, for the

moral and financial support;

Dr. Edsel Maurice T. Salvana, Dr. Lucille C. Villegas and Professor Ernesta G.

Quintana, the researcher’s consultants who guided her throughout the conceptualization

and experimentation and for letting her know that life is not always about winning but it

is also about learning from our mistakes;

Mrs. Ruby H. Bautista, Research II adviser, for the pieces of advice, motivation

and patience that she has given;

With this, the researcher would like to extend her profound gratitude for these

people who made this research study a worthwhile endeavor.

iv

TABLE OF CONTENTS

Contents Page

Title Page i

Abstract ii

Acknowledgement iii

Table of Contents iv

Chapter I: Introduction

A. Background of the Study 1-2

B. Statement of the Problem 2

C. Hypothesis 2

D. Significance of the Study 3

E. Scope and Limitation 3- 4

F. Review of related Literature 4- 9

G. Definition of Terms 9

Chapter II: Methodology

A. Materials/ Equipment 10- 11

B. General Procedure/ Treatment 11- 12

C. Disposal of Waste Materials 12

v

D. Diagram of Assay Set-up 12

E. Experimental Design Diagram 13- 14

F. Flowchart 15

Chapter III: Results and Discussions 16- 19

Chapter IV: Conclusion 20- 21

Bibliography 22

Appendices

A. Ghantt Chart 23

B. Research Plan 24- 25

C. Letters 26- 32

D. Photographs of the Experimentation 33- 36

E. Certificate of Analysis 37- 38

F. Raw Data 39- 40

G. Expenses Incurred and Receipts 41- 42

H. Certificates of Participation and Merits 43

I. Curriculum Vitae 44- 45

1

CHAPTER I

INTRODUCTION

A. Background of the Study

Sophistication of high-tech era has been accompanied by environmental

deterioration. This poses a high risk on the health of mankind. Air pollution which includes

the exhaust fumes of vehicles and the smoke from factories, radiation, and even cigarette

or tobacco smoke can cause mutation that leads to the development of cancer.

A water extract of a crude preparation of Aloe ferox, a product similar to Aloe

barbadensis was positive in Bacillus subtilis spore rec-assay carried out in strains H17 and

M45; a corresponding test on methanol extract was negative. These assays were

conducted without S-9. Neither the water extract nor the methanol extract produced

mutations in Salmonella typhimurium strains TA98 or TA 100 with or without S-9. Cell killing

was noted in half the assays (Morimoto et al., 1982)

Dealing with the anti-tumor assays and studying rodents such as mice, in particular,

is very strenuous and includes very technical cell culture techniques and other molecular

assays. To make it more efficient, a modified Ames test was utilized in the experiment

which used bacteria to detect carcinogenic compounds. It can be modified using a known

carcinogenic compound such as tobacco extract and incubating it with a specific strain of

bacteria, Salmonella typhimurium TA 100, with and without aloe gel. Through this, the

inhibitory effect of aloe gel on the mutation of S. typhimurium could be determined.

Having aloe vera as the main material of the experiment would not be a problem

since it is abundant and readily available within the country.

2

B. Statement of the Problem

This research study utilized aloe gel to inhibit the tobacco extract from mutating

Salmonella typhimurium.

The investigation sought to answer the following questions:

1. What is the extent of inhibition of aloe gel on the mutation of S. typhimurium when

combined with tobacco extract?

2. What is the degree of inhibitory effect of aloe gel in relation to the mutation of S.

typhimurium?

3. What is the degree of concentration of tobacco extract that will establish the highest

mutagenic potential?

C. Hypotheses

1. Aloe gel does not inhibit the mutation of Salmonella typhimurium when combined with

tobacco extract.

2. The highest concentration of aloe gel exhibits the highest inhibitory effect.

3. The highest concentration of tobacco extract established the highest mutagenic

potential.

D. Significance of the Study

Mutations are randomly derived changes to the nucleotide sequence of the genetic

material of an organism. These are caused by copying errors in the genetic material during

cell division due to the exposure to radiation, mutagenic chemicals or viruses. Some

3

mutations do not affect the fitness of an individual yet some of them also bring harmful

effects on the fitness of human which could lead to cancer.

Cancer has been one of the major causes of mortality and morbidity all over the

world. This may be brought by the foods we eat such as charbroiled food or burnt meat and

easy to prepare and cured food. Demand for medicines and medical procedures for the

treatment of this fatal disease has increased. Yet none of these can perfectly cure cancer

instead it just brought severe side effects to the patient such as nausea and hair loss.

People suffering from cancer are spending a lot of money thinking that these apparatuses

or procedures are the solutions to their problems.

Using aloe vera, being a natural way of preventing mutation that eventually lead to

cancer once not given an immediate suppressor of carcinogenic progression. This offers no

side effects, easy to prepare and affordable.

E. Scope and Limitation

This research study involved the efficacy of aloe gel in inhibiting the mutation of

Salmonella typhimurium done by the tobacco extract. Determining which active chemical

constituent of aloe gel that could inhibit the mutation of S. typhimurium is further than the

study.

In view of the fact that tobacco is a known carcinogenic compound, it was used in

the experiment to carry mutation on S. typhimurium.

Since aloe vera is a source of two herbal preparations (aloe latex and aloe gel), it

was specified that the gel was usedl.

4

While Ames test was intended to detect the mutagenic potential of a substance, this

was modified by using a known carcinogenic compound which was the tobacco extract and

combining it with aloe vera extract which was said to have an inhibitory effect on the

mutation of S. typhimurium.

Only one trial having two replicates was conducted. Aloe gel’s efficacy was

determined through its inhibition on the mutation of Salmonella typhimurium brought by the

tobacco extract.

F. Review of Related Literature

In biology, mutation is a sudden change in the base pair sequence of the genetic

material of a living organism, whether the genetic material be deoxyribonucleic acid (DNA) or

ribonucleic acid (RNA). In multicellular organisms that reproduce sexually, mutations can be

subdivided into germ line mutations, which can be passed on to descendants, and somatic

mutations, which cannot be transmitted to descendants in animals. Plants sometimes can

transmit somatic mutations to their descendants asexually or sexually (in case when flower

buds develop in somatically mutated part of plant). A new mutation that was not inherited from

either parent is called a de novo mutation. Mutations can be caused by copying errors in the

genetic material during cell division, by exposure to ultraviolet or ionizing radiation, chemical

mutagens, or viruses, or can occur deliberately under cellular control during processes such as

hypermutation. (Taggart, R., and C. Starr. 2006)

Nearly all cancers are brought by abnormalities in the genetic material of the

transformed cells. Risk factors for the development of cancer are carcinogens, such as

5

tobacco smoke, radiation, chemicals or infectious agents. Other genetic abnormalities may be

randomly acquired through errors in DNA replication, or are inherited, and thus present in all

cells from birth. The heritability of cancers is usually affected by complex interactions between

carcinogens and host’s genome. (Vogelstein, 2002)

Genetic abnormalities found in cancer typically affect two general classes of genes:

oncogenes and tumor suppressor genes. Oncogenes are usually activated in cancer cells,

giving those cells new properties, such as hyperactive growth and division, protection against

programmed cell death, loss of respect for normal tissue boundaries, and the ability to become

established in diverse tissue environments. On the other hand, tumor suppressor genes are

then inactivated in cancer cells resulting in the loss of normal functions in those cells.

(Vogelstein, 2002)

The aloe plant is the source of two herbal preparations: aloe gel (AG) and aloe latex.

Aloe gel is often called aloe vera and refers to the clear gel or mucilaginous substance

produced by parenchymal cells located in the central region of the leaf. Diluted aloe gel is

commonly referred to as “aloe vera extract”. The gel is composed mainly of water (99%) and

mono- and polysaccharides (25% of the dry weight of the gel). Several recent studies have

demonstrated direct inhibitory effects of AG on both tumor initiation and promotion. (Barcroft

and Myskja, 2003)

A polysaccharide fraction of AG inhibited the binding of benzopyrene to primary rat

hepatocytes and thus prevented the formation of potentially cancer-initiating benzopyrene-

DNA adducts. This effect was also demonstrated in vivo, where adduct formation was reduced

in various organs. Benzopyrene is found in coal tar, in automobile exhaust fumes (especially

6

from diesel engines), tobacco smoke, cannabis smoke, incense smoke, wood smoke and in

charbroiled food. Thus, it can possibly result to lung cancer. (Combest, 2002)

A follow-up study published in 1999 by the same investigators showed that several

other plant-derived polysaccharides were also able to block benzopyrene-DNA adducts. They

also reported in this study an induction of glutathione S-transferase and an inhibition of the

tumor-promoting effects of phorbol myristic acetate by AG. These two studies suggest a

possible benefit of using aloe gel in cancer chemoprevention. (Combest, 2002)

Aloe vera, most commonly known as Medicinal Aloe, is a species of succulent plant that

originated in northern Africa. (Reynolds, 2004)

Aloe vera is widely used as herbal medicine and cosmetics due to its rejuvenating,

healing, and soothing properties. However, some studies contradict the positive effects

brought by aloe plant. Despite this fact, there is some preliminary evidence that A. vera extract

may have a hypoglycemic effect, thus, it can treat diabetes and elevated blood lipids, due to

the presence of compounds such as mannans, anthraquinones and lectins.

Ames test is a biological assay developed by Bruce Ames and his group at the

University of California, Berkeley in 1970s. The test is performed to assess the mutagenic

potential of chemical compounds. A positive test indicates that the chemical might be

carcinogenic. As cancer is often linked to DNA damage, the test also serves as a quick assay

to estimate the carcinogenic potential of a compound since it is difficult to ascertain whether

standard carcinogen assays on rodents were successful. (Bruce N. Ames, E. G. Gurney,

James A. Miller, and H. Bartsch, 1972)

The test uses several strains of the bacterium Salmonella typhimurium that carry

mutations in genes involved in histidine synthesis, meaning they require histidine for growth.

7

The variable being tested is the mutagen's ability to cause a reversion to growth on a histidine-

free medium. The tester strains are specially constructed to have both frameshift and point

mutations in the genes required to synthesize histidine, which allows for the detection of

mutagens acting via different mechanisms. Some compounds are quite specific, causing

reversions in just one or two strains. The tester strains also carry mutations in the genes

responsible for lipopolysaccharide synthesis, making the cell wall of the bacteria more

permeable, and in the excision repair system to make the test more sensitive. Since some

compounds, like benzopyrene, are not mutagenic themselves but their metabolic products are,

rat liver extract may be added to stimulate the effect of metabolism. (Bruce N. Ames, E. G.

Gurney, James A. Miller, and H. Bartsch, 1972)

The abundance of naturally occurring and artificially synthesized chemicals that are

potentially mutagenic and carcinogenic led to the search for a simple method to assay for

carcinogenicity. The Ames test has become an acceptable bacterial system used in such

assays. It is based on the principle that a chemical which can revert his- auxotroph to his+ is a

mutagen. A reversion rate of two fold the background (spontaneous reversion) is considered

mutagenic. (Raymundo, 2000)

Salmonella typhimurium is a pathogenic Gram-negative bacteria predominately found in

the intestinal lumen. Its toxicity is due to an outer membrane consisting largely of

lipopolysaccharides (LPS) which protect the bacteria from the environment. The LPS is made

up of an O-antigen, a polysaccharide core, and lipid A, which connects it to the outer

membrane. Lipid A is made up of two phosphorylated glucosamines which are attached to fatty

acids. These phosphate groups determine bacterial toxicity. Animals carry an enzyme that

specifically removes these phosphate groups in an attempt to protect themselves from these

8

pathogens. The O-antigen, being on the outermost part of the LPS complex is responsible for

the host immune response. S. typhimurium has the ability to undergo acetylation of this O-

antigen, which changes its conformation, and makes it difficult for antibodies to recognize.

(Barich, 2005)

This bacterium is able to secrete small signaling molecules called autoinducers. The

LuxS gene is responsible for initiating a series of phosphate transfer reactions that produce

this molecule and allow for cell to cell communication. Sugar compounds, preferably glucose,

activate LuxS and the resulting autoinducer concentration increases with the bacterial

concentration till the substrate is depleted. At this point the autoinducer is degraded and can

be recycled by the bacterial cell. This quorum sensing allows cells to determine the metabolic

potential of the environment. This has been used to investigate its ability to deliver DNA to

antigen presenting cells (APCs) in order to produce cancer vaccines. Studies indicate that S.

typhimurium has been able to initiate an immune response as a direct result of the DNA

containing “eukaryotic expression vectors.” Hopefully these experiments will lead to the ability

to send specific DNA sequences that will elicit the appropriate immune response to eliminate

tumor or cancer cells. (Barich, 2005)

G. Definition of Terms

Mutation- randomly derive changes in the DNA sequence of an organism

Inhibition- prevention of growth or increasing population of an organism

Histidine auxotroph- organisms that require amino acid histidine for growth

Ames test- biological assay used to detect the mutagenic potential of a certain

substance.

9

CHAPTER II

METHODOLOGY

The efficacy of aloe gel to reverse, suppress or prevent carcinogenic progression to

invasive cancer was tested through a modified Ames test which utilized Salmonella

typhimurium TA 100. This type of bacteria carried mutations in genes involved in histidine

synthesis, thus, they required histidine for growth. The variable that was tested was the aloe

gel’s ability to inhibit the mutagen from causing a reversion on the growth of S. typhimurium TA

100 on a histidine-free medium.

A. Materials/ Equipment

The main materials used in this research study were aloe vera leaves and tobacco

leaves. Salmonella typhimurium TA 100 was utilized in this experiment as the tester strain.

In the experimentation centrifuge, eppendorf tubes, test tubes, beakers and flasks were

also used. The culture media used in the experiment were made up of Ames Top Agar which

consists of Difco Agar (0.6 %), NaCl (0.6 %), Biotin (0.50 mM) and Histidine (0.05 mM);

L-broth/ Agar which consists of Bacto Tryptone (10 g), Bacto Yeast Extract (5 g) and NaCl (5

g); and M9 Minimal Medium which consists of Na2HPO4 (6 g), KH2PO4 (3 g), NaCl (0.5 g) and

NH4Cl (1.0 g).

The solutions were autoclaved and sterile solutions of 1 M MgSO4 (1.0 mL), 10 mM

CaCl2 (10.0 mL) and 20 % Glucose (10.0 mL) were added.

Fifteen g/L minimal agar was also prepared. The agar was autoclaved in 930 mL water

and when it cooled to 50- 60 oC, the other solutions were added.

10

After the medium has cooled, antibiotics and amino acids were added.

B. General Procedure/ Treatment

Extraction of Aloe vera and Tobacco Leaves

Five grams of tobacco leaves was obtained and cut into small pieces. These were

ground using a sterile mortar and pestle and was suspended in 15 mL sterile distilled water.

The sample was spun at 6400 rpm in a centrifuge for 2 minutes to pellet plant debris. The

supernatant was transferred into a sterile eppendorf tube and was spun again at same rate

for 15 minutes to pellet contaminating bacteria. The aqueous portion was carefully separated

from the debris. These aliquots served as the plant extract which was used in the experiment.

Certain amount of aloe gel was also obtained.

Preparation of Control and Experimental Set-ups

Minimal agar was poured a day ahead. One plate was incubated at 30 oC and the rest

were kept in the refrigerator. This was done to ensure that the agar plates were free from

contaminants. Furthermore, an overnight culture of S. typhimurium TA 100 was prepared in L-

broth.

Each sample was prepared carefully by dissolving aseptically in sterile water. Ames top

agar was prepared and 2.5 mL of the solution was dispensed to sterile tubes.

One-hundredth mL of S. typhimurium TA 100 was added. It was mixed well by rotating

between the palms and was poured to cover the entire surface of the minimal agar.

The following samples having a replicate for each were prepared: Minimal medium

alone, TA 100 alone, Pure aloe gel alone, Tobacco extract alone, TA 100 + Tobacco extract

11

(1:3), TA 100 + Tobacco extract (1:50), TA 100 + pure AG, TA 100 + AG (1:50), TA 100 +

Tobacco extract (1:3) + pure AG and TA 100 + Tobacco extract (1:50) + AG (1:50)

All plates were incubated at 37 0C for 48-72 hours. The background growth on each

plate was observed and the effects of the aloe vera and tobacco extracts were determined and

compared. The colonies formed were counted and the colony forming unit/ mL (cfu/ mL)

exhibited by S. typhimurium in each sample was computed.

The colony forming unit of S. typhimurium was computed using the formula:

cfu/mL= average no. of colonies * reciprocal of the dilution factor volume of tester strain

Disposal

All the glasswares used are decontaminated and microorganisms were killed through a

moist heat, 115 degrees Celsius at 15 psi for 20 minutes, using a pressure cooker. The

disposal team of Microbiology Division- UPLB took charge of the waste materials and the

equipment used were washed and dried and was then kept in designated cabinets.

12

Diagram for assay set-up

TA 100- tester strain

AG- aloe gel

Minimal

medium

alone

TA 100

alone

(tester strain)

Pure AG

alone

Tobacco

extract

alone

TA 100 +

Tobacco

extract

(1:3)

TA 100 +

Tobacco

extract

(1:3)

TA 100 +

Tobacco

extract

(1:50)

TA 100 +

Tobacco

extract

(1:50)

TA 100 +

pure AG

TA 100 +

pure AG

TA 100 +

AG (1:50)

TA 100 +

AG (1:50)

TA 100 +

Tobacco

extract (1:3) +

pure AG

TA 100 + Tobacco

extract (1:3) +

pure AG

TA 100 +

Tobacco

extract (1:50)

+ AG (1:50)

TA 100 + Tobacco

extract (1:50)

+ AG (1:50)

13

Experimental Design Diagram

Title: Aloe barbadensis Leaf Extract in Inhibiting the Mutation of Salmonella

typhimurium

Hypothesis: Aloe vera can prevent tobacco from mutating Salmonella typhimurium.

Independent

Variable

Aloe barbadensis leaf extract and Tobacco Leaf Extract

Levels of Independent

variable

1.5 mL Tobacco Extract (1:3)

1.5 mL Tobacco Extract (1:50)

1.5 mL

Pure Aloe Gel

1.5 mL Aloe Gel

(1:50)

1.5 mL

Tobacco extract (1:3)

+ 1.5 mL Pure

Aloe Gel

1.5 mL Tobacco

extract (1:50) +

1.5 mL Aloe Gel

(1:50)

Number of replicates

2

2

2

2

2

2

Dependent Variable: Mutation of Salmonella typhimurium

Controlled Variables: amount of S. typhimurium TA 100 (0.1 mL)

amount of samples (1.5 mL)

strain of S. typhimurium (TA 100)

species of aloe vera (Aloe barbadensis)

amount of Ames top agar (2.5 mL)

equipment used in the experiment

temperature and time of incubation (37 0C for 48-72 hours)

rate of centrifugation (6400 rpm)

14

Flow Chart

Incubation of set-ups for 48-72 hours at 37 0C

Preparation of control and experimental set-

ups

Gathering of the

materials and extraction of leaf samples (aloe

vera leaves and tobacco leaves)

Preparation of culture

media (Ames top agar + L-

broth + M9 minimal medium)

Incubation of set-ups for 48-72 hours at 37 0C

Observation of the set-ups

Recording and analyses of results

Disposing the materials used

15

CHAPTER III

RESULTS AND DISCUSSIONS

The following data verified the results obtained from the experimentation intended to

test the inhibition of the mutation of S. typhimurium TA 100.

Data Tables, Graphs and Figures

Table 1. Aloe barbadensis Leaf Extract tested on S. typhimurium

Samples

No. of colonies observed

Remarks

MM alone

0 colony

sterile medium

TA 100 alone

(tester strain)

5 colonies

(spontaneous revertants)

a reversion rate of two fold the background (spontaneous

revertants) is considered mutagenic

AG alone (pure)

7 colonies

pure AG has contaminants

Tobacco extract alone

0 colony

tobacco extract is sterile

TA 100 + Tobacco extract

(1:3)

1

0 colony

has inhibitory effect

2

0 colony

16

TA 100 + Tobacco extract

(1:50)

1

16 colonies

established mutagenic potential

*as concentration ↓,

mutagenic potential ↑

2

11 colonies

TA 100 + pure AG

1

13 colonies

--------

2

20 colonies

TA 100 + AG (1:50)

1

4 colonies

---------

2

0 colony

TA 100 + Tobacco extract (1:3) + pure AG

1

3 colonies

---------

2

2 colonies

TA 100 + Tobacco extract

(1:50) + AG (1:50)

1

3 colonies

--------

2

2 colonies

17

*A reversion rate of two fold the background was considered to establish a mutagenic

potential.

Table 2. Colony Forming Unit/ mL of S. typhimurium in Different Treatments

Samples

Colony Forming Unit/ mL (cfu/ mL)

MM alone

0 cfu/mL

TA 100 alone (tester strain)

5.0 * 10 cfu/ mL

AG alone (pure)

7.0 * 10 cfu/ mL

Tobacco extract

alone

0 cfu/mL

TA 100 +

Tobacco extract (1:3)

0 cfu/mL

TA 100 +

Tobacco extract (1:50)

6.8 * 103 cfu/ mL

TA 100 + pure

AG

1.7 * 102 cfu/ mL

TA 100 + AG

(1:50)

1.0 * 103 cfu/ mL

18

TA 100 +

Tobacco extract (1:3) + pure AG

1.0 * 102 cfu/ mL

TA 100 +

Tobacco extract (1:50) +

AG (1:50)

1.3 * 103 cfu/ mL

Table 1 shows that A. vera extract has inhibitory effect on the mutation of S.

typhimurium TA 100 since there were more bacteria growing on the TA 100 + Tobacco extract

only arm vs. TA 100 + Tobacco extract+ AG arm (6.8*103 cfu/mL vs. 1.3*103). (See Appendix

A for the computation)

The results of the experiment were parallel to the results of a previous study wherein

Aloe ferox, a product similar to Aloe barbadensis, exhibits an inhibitory effect on the mutation

of Salmonella typhimurium TA 98 or TA 100. Though they are two different species which

belong to same genus, both showed an inhibitory effect on the mutation of S. typhimurium TA

100.

19

CHAPTER IV

CONCLUSION

To find another way of cancer chemoprevention and treatment, this research study was

done by detecting the inhibitory effect of aloe vera, Aloe barbadensis, on the mutation of

Salmonella typhimurium, particularly, on the mutation done by tobacco extract.

It was hypothesized that aloe gel did not prevent tobacco extract from mutating

Salmonella typhimurium. Since there were more bacteria growing on the tobacco only arm vs.

tobacco + aloe gel arm (6.8 * 103 cfu/ mL vs. 1.3 * 103 cfu/ mL ), there was no enough evidence

to support the hypothesis.

It was also hypothesized that greater concentration of tobacco extract would establish

higher mutagenic potential. Since there were more bacteria growing on TA 100 + tobacco

extract (1:50) arm vs. TA 100 + tobacco extract (1:3) arm (6.8*103 cfu/ mL vs. 0 cfu/ mL), there

was no enough evidence to support the hypothesis.

Principally, after the experimentation and data analyses, it was found that aloe vera

could prevent tobacco from mutating the bacteria. On the other hand, pure tobacco extract

showed an inhibitory effect on S. typhimurium since no colonies of the bacteria were observed.

Therefore, aloe vera is not just for external use such as healing wounds and burns. It is

also a potential natural way of cancer chemoprevention and treatment. Moreover, it offers no

side effects and it is an affordable method of maintaining a good health.

20

For those who are motivated to do the same study, it is recommended to use different

plant extracts, conduct more trials and determine which active chemical constituent inhibits the

growth of S. typhimurium. Other strains of the bacteria can be utilized such as TA 1535, TA

1537, TA 1538, TA 98 or TA 102. Other known carcinogenic compounds can be used such as

benzopyrene but this requires rat liver extract since this compound is not mutagenic itself. This

should be done to stimulate its metabolic byproducts which are responsible for mutation.

To achieve a better outcome, it is advised to maintain perseverance and patience

throughout the experiment. Honesty and accuracy is also essential to make the study a

successful one.

21

BIBLIOGRAPHY

Research Journals

Raymundo A.K. (2000). Laboratory Manual for Microbial Genetics. Microbiology Division,

Institute of Biological Sciences. University of the Philippines Los Baños College Laguna.

40 pp.

Pecere, Teresa., et.al. (1998), Aloe-emodin Is a New Type of Anticancer Agent with Selective

Activity against Neuroectodermal Tumors

(http://cancerres.aacrjournals.org/cgi/content/abstract/60/11/2800)

Combest, Wendell L., Ph.D.(2002), Aloe Vera

(http://www.uspharmacist.com/oldformat.asp?url=newlook/files/Alte/apr00aloe.cfm&pub

_id=8&artice_id=503)

Books

Starr, Cecie (1990). Biology: The Unity and Diversity of Life., Mary Forkner, Publication

Alternatives.

Lea and Febiger (1993). Cell and Molecular Biology. 8th ed. International Copyright Union.

22

APPENDIX A

GANTT CHART

School: Cavite National Science High School

Research title: Aloe barbadensis Leaf Extract in Inhibiting the Mutation of

Salmonella typhimurium

Researcher: Romella C. Lina

Research Adviser: Ruby H. Bautista

Research Consultant: Dr. Edsel Maurice T. Salvana

Dr. Lucille C. Villegas

Activities Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Remarks

Conceptualization DONE

Formulation DONE

Gathering of Data DONE

Writing and Consolidation of

Chapter I

DONE

Research Design (Methodology)

DONE

Experimentation DONE

Consolidation of Data

DONE

Writing the Manuscript

DONE

Finalization of the Manuscript

DONE

APPROVED:

ESTRELLITA M. DE VERA

Principal III

23

APPENDIX B

RESEARCH PLAN

Date Activity Place Remarks

March 2009

Formulation of the research problem

Cavite National Science High

School

Done

May 5, 2009

Searching for a consultant

(Professor Ernesta G. Quintana)

University of the Philippines- Los Baños, Laguna

Done

May 12, 2009

Searching for a consultant through e-mail

(Dr. Ismael Noe Bacon, General and Cancer Surgeon)

Done

May 15, 2009

Searching for a consultant through letter addressed to:

Dr. Lulu C. Bravo

Executive Director

National Institutes of Health

(Dr. Edsel Maurice T. Salvana)

University of the Philippines- Manila

Done

June 2009

Completion of Chapter I

Cavite National Science High

School

Done

July 2009-

August 2009

Formulation of the methodology and conducting the

experimentation (one trial with three replicates)

University of the Philippines- Manila

Done

September 2009

Writing the first draft of the research paper, revision and

writing the final draft

Cavite National Science High

School

Done

24

Title: Aloe barbadensis Leaf Extract in Inhibiting the Mutation of Salmonella

typhimurium

ROMELLA C. LINA

Researcher

NOTED:

CORAZON C. LINA

Parents/ Guardian

DR. EDSEL MAURICE T. SALVANA

DR. LUCILLE C. VILLEGAS

Consultants

RUBY H. BAUTISTA

Research Adviser

APPROVED:

ESTRELLITA M. DE VERA

Principal III

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APPENDIX C

LETTERS

From: "Edsel Salvana" <exs128@case.edu> To: "Romella Lina" <romella_lina@yahoo.com> Romella, You need to get at least one of these S. typhimurium strains:

• TA 1535 has a base substitution that produces a missense mutation in the gene coding for the first enzyme of histidine synthesis. The mutant enzyme has a proline where a leucine is in the wild-type enzyme. • TA 100 is very similar to 1535, but is supposed to detect a different range of mutagens. • TA 1537 has a frameshift mutation (deletion of one nucleotide) in a different gene than is mutated in 1535. • TA 1538 has a different frameshift mutation (insertion of one nucleotide) in the same gene that is mutated in TA 1537. • TA 98 is similar to 1538 but is supposed to detect more mutagens than 1538 does. • TA 102 is significantly different from the others. It has an ochre mutation which means that it has a nonsense mutation. This mutation occurs in the same gene that is mutated in the strain TA 1535.

You then have three arms: One uses aloe gel alone (no tobacco), one positive control arm (tobacco only), and the tobacco + aloe gel that is the experimental arm. You also need very specific media (limited histidine in the agar) for this test. You basically incubate the aloe and the tobacco together because you want the to see if the aloe vera prevents the tobacco from mutating the bacteria. If there are more bacteria growing in the tobacco only arm versus the tobacco + aloe vera, then your experiment is a success because the aloe gel prevented the tobacco from mutating the bacteria - the bacteria are mutated if they survive. Instead of tobacco, you can use sodium azide, like they did in the protocol I sent you since this is a known mutagen, but it might be harder to get. Also, be careful handling Salmonella typhimiurium since it can cause a typhoid-like illness. Edsel On Thu, Jun 4, 2009 at 7:14 PM, Romella Lina <romella_lina@yahoo.com> wrote: I have attached the methodology I formulated which is based on your suggestion. Please give a comment for its improvement. Thank you! -- Edsel Maurice T. Salvana, MD, DTM&H Clinical Associate Professor of Medicine Section of Infectious Diseases University of the Philippines Manila, Philippines

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APPENDIX D

PHOTOGRAPHS

PHOTORAPH A

PHOTOGRAPH B

Preparation of Tobacco extract

*Photographs taken by Dr. Lucille C. Villegas

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PHOTOGRAPH C

Preparation of the Tobacco Extract

Centrifugation of Tobacco Extract

PHOTOGRAPH C

*Photographs taken by Dr. Lucille C. Villegas

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PHOTOGRAPH D

Preparation of the Culture Media

PHOTOGRAPH E

*Photographs taken by Dr. Lucille C. Villegas and the researcher

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PHOTOGRAPH F

PHOTOGRAPH G

Preparation of Control and Experimental Set-ups

*Photographs taken by the researcher

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APPENDIX E

CERTIFICATE OF ANALYSIS

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APPENDIX F

RAW DATA

Number of Colony Forming Unit/ mL

cfu/mL= average no. of colonies * reciprocal of the dilution factor volume of tester strain

TA 100 alone = 5 colonies * 1 = 5.0 * 10 cfu/ mL (50 cfu/ mL)

0.1 mL AG alone = 7 colonies * 1 = 7.0 * 10 cfu/ mL (70 cfu/ mL)

0.1 mL TA 100 + Tobacco extract (1:3) = 0 cfu/ mL TA 100 + Tobacco extract (1:50) = 16 + 11 colonies * 50 = 6.8 * 103 cfu/ mL (6750 cfu/ mL) 2 0.1 mL TA 100 + pure AG = 13 + 20 colonies * 1 = 1.7 * 102 cfu/ mL (165 cfu/ mL) 2 0.1 mL TA 100 + AG (1:50) = 4 + 0 colonies * 50 = 1.0 * 10

3 cfu/ mL

(1000 cfu/ mL)

2 0.1 mL

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TA 10 + pure AG + Tobacco extract (1:3) = 3 + 2 colonies * 4 = 1.0 * 102 cfu/ mL (100 cfu/ mL) 2 0.1 Ml TA 100 + AG (1:50) + Tobacco extract (1:50) = 3 + 2 colonies * 50 = 1.3 * 103 cfu/ mL (1300 cfu/ mL) 2 0.1 mL

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APPENDIX G

EXPENSES INCURRED AND RECEIPTS

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APPENDIX H

CERTIFICATE OF PARTICIPATION AND MERIT

Romella C. Lina

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APPENDIX I

CURRICULUM VITAE

I. Personal Information

1.1 Name: Romella C. Lina

1.2 Birthday: June 18, 1994

1.3 Age: 15 years old

1.4 Birthplace: Rosario, Cavite

1.5 Gender: Female

1.6 Address: #375 5th Street Malainen Bago Naic, Cavite

1.7 Civil Status: Single

1.8 Parents: Corazon C. Lina

Romulo A. Lina

1.9 Nationality: Filipino

1.10 Religion: Roman Catholic

1.11 Landline Phone Number: (046) 412- 1420

1.12 E-mail address: romella_lina@yahoo.com

II. Educational Background

2.1 Elementary: Naic Elementary School

2.2 Secondary: Cavite National Science High School

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III. Honors/ Awards

1.1 Honors

Grade 2: 9th honors

Grade 3: 2nd honors

Grade 4: 3rd honors

Grade 5: 2nd honors

Grade 6: 10th honors

IV. Research Studies Conducted

4.1 Second Year: Biological Staining Property of Basella rubra (Alugbati) on Onion and

Cheek Cells

4.2 Fourth Year: Aloe barbadensis Leaf Extract in Inhibiting the Mutation of Salmonella

typhimurium

V. Organizations

5.1 Science Club (2006-2008)

5.2 English Club (2008-2010)

5.3 The Molecules (2007-2010)