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Antifungal Activity of Star Anise (Illicium Verum) to Candida Tropicalis

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ANTIFUNGAL ACTIVITY OF STAR ANISE (Illicium verum)
TO Candida tropicalis
_______________
An Investigatory Project Presented to the Faculty
Of Talamban National High School
Talamban, Cebu City
_______________
In Partial Fulfillment of the
Requirements for
Research I
_______________
by
Queenibel S. Arriesgado
Kristine Jane A. Borces
Dominique Fatima G. Cabansay
Jobelyn B. Cogtas
Mary Rose A. Telamo

June 2014 APPROVAL SHEET
The investigatory project entitled “ANTIFUNGAL ACTIVITY OF STAR ANISE SEEDS (Illicium verum) to Candida tropicalis”, prepared and submitted by Queenibel S. Arriesgado, Kristine Jane A. Borces, Dominique Fatima G.Cabansay, Jobelyn B. Cogtas, Mary Rose A. Telamo, in partial fulfillment of the requirements for Reasearch I, has been examined for acceptance and approval for Oral Examination.

RESEARCH ADVISORY COMMITTEE
MA. JESSICA N. ABAYON, Ed. D.
Chairperson

CELIA C. GEPITULAN, M. Ed. JOCELYN C. BUTANAS, M. Ed. Adviser Member

CELIA C. GEPITULAN, M. Ed. FARAH C. CENIZA Member Member

-------------------------------------------------

PANEL OF EXAMINERS

Approved by the Committee of Oral Examination of with a grade of . MA. JESSICA N. ABAYON, Ed. D. Chairperson CELIA C. GEPITULAN, M. Ed. JOCELYN C. BUTANAS, M. Ed. Adviser Member

CELIA C. GEPITULAN, M. Ed. FARAH C. CENIZA Member Member

ACCEPTED AND APPROVED in partial fulfillment of the Requirements for Research I.

March 13, 2014ZENAIDA C. GRAPA, Chem. E.Ph.D,
Oral examination Science Coordinator

ACKNOWLEDGEMENT
The researchers would like to express their sincerest gratitude to the following:

Almighty God. Who made all things possible; for giving them wisdom, patience, hope, and for giving them the safety in all the processes they have done in the study.
Parents and Sponsors. Who consider on giving time to the researchers and supporting them to their financial problems.
Ms. Celia Gepitulan. Who offered invaluable detailed advices on grammar, organization, and for the theme of the paper. She never failed to correct mistakes. She also disciplines the researchers to do the papers . She also never failed to suggest better things to their study that contributes a lot to their success.
Mark Gilbert S. Milallos.For sharing his ideas to the researchers on what study they are to conduct and letting them borrow his cellphone to search on the web.
Mrs. Jocelyn C. Butanas and Mrs. Farah C. Ceniza. Who gave valuable comments and suggestions to the researchers.
Department of Agriculture. For verifying the star anise seeds with no payment.
Mr. Anthony S. Ilano. Who approved the researchers to conduct their experiment in Biology Department of University of San Carlos.
Mr. Rolly Viesca. Who taught and helped the researchers to do their experiments.
Borces’ Family. Who had their doors open whenever the researchers would have to do something about their research . QUEENIBEL S. ARRIESGADO
KRISTINE JANE A. BORCES
DOMINIQUE FATIMA G. CABANSAY
JOBELYN B. COGTAS MARY ROSE A. TELAMO
Researchers

TABLE OF CONTENTS Page
TITLE PAGE
APPROVAL SHEET ii
ACKNOWLEDGEMENT iii
TABLE OF CONTENTS iv
LIST OF TABLES vi
LIST OF FIGURES
ABSTRACT
Chapter
1 THE PROBLEM AND ITS SCOPE Introduction 1 Background of the study 2 Conceptual framework 3 THE PROBLEM Statement of the problem 4 Statement of the hypothesis 5 Significance of the study 5 Definition of terms 6 Scope and limitations 7 Review of related literature 8 Review of related studies 9
2 RESEARCH METHODOLOGY 10
Research design 10 Research Environment 11 Research Subject 11 Research Procedure 12 A. Assembling of Steam Distillation 13 B. Making of the product 13 C. Preparation of the Culture Media 13 D. Inoculation of Fungal Solution 13 Gathering of Data 14 Treatment of Data 14 BIBLIOGRAPHIES 15 APPENDICES 16 A. REQUEST LETTER 17 B. TRANSMITTAL LETTER 18 C. TRANSMITTAL LETTER 19 D. TRANSMITTAL LETTER 20 E. TRANSMITTAL LETTER 21

LIST OF TABLES
Table Title Page
1 Research Design Composed of the conditions and treatment 20
2 Zone of inhibition in 24 hours (6 hours interval) of Candida tropicalis 23

LIST OF FIGURES
Figure Title Page
1 Conceptual framework

ABSTRACT
ANTIFUNGAL ACTIVITY OF STAR ANISE (Illicium verum)
TO Candida tropicalis The purpose of this study was to examine the antifungal activity of star anise seeds to Candida tropicalis. The researchers used three different percents of the seeds to determine its significant difference in killing Candida tropicalis. Candida tropicalis is a yeast infection that causes Candidiasis, if it will not be prevented, it may lead to death. A 10, 50, and 100 grams of Star Anise Seeds were extracted through alternative steam distillation. The extracts then were labeled as Treatment A, Treatment B and Treatment C. The treatments were put to a beaker separately as well as the distilled water (negative) and fungisol (positive). After it was soaked, it was then applied to Candida tropicalis. The making of the product was conducted at our co-researcher’s house with her parent’s permission. While the preparation of culture media and the inoculation of fungal solution was performed at University of San Carlos - Talamban Campus. The results were, Treatment A doesn’t have any zone of inhibition at all as well as the distilled water; Treatment B have zone of inhibition but it was smaller than of the treatment C. But as the time increases, the zone of inhibition decreases. The fungisol has the highest zone of inhibition as the time increases until the 24th hour.

CHAPTER 1
THE PROBLEM AND ITS SCOPE
INTRODUCTION

Candida tropicalis is one of the most common specie of the genus candida that causes human disease especially in tropical countries like the Philippines. Candidiasis is one of the fungal disease that is caused by any specie of the genus Candida. Infections (candidiasis) due to Candida tropicalis have increased dramatically on a global scale thus proclaiming this organism to be emerging pathogenic yeast. (http://jmm.sgmjournals.org/content/59/8/873.full) With the statement above, the researchers decided to make an antifungal product out of Star anise seeds (Illicium verum) which are widely available in the local markets within the city. The product is affordable, natural and has been used by several people for medicinal purposes. Illicium verum is known for its culinary uses. This is commonly called as star anise. It is dark-brown in color and has a star-like shape. The most powerful flavor component of the essential oil of anise is anethole, it is found in both anise and an unrelated spice called star anise (Illicium verum) widely used in South Asian, Southeast Asian, and East Asian dishes.( https://www.google.com.ph/?gfe_rd=cr&ei=fpOhU9b3OMOE8Qf54YGACw&gws_rd=ssl#q=composition+of+star+anise)

BACKGROUND OF THE STUDY

Some people who have such contagious fungal infections would have a trouble on buying commercial products because it is costly. Furthermore, few of these commercial antifungal products can leave severe side effects such as in Topical antifungals which may cause itching, a mild burning sensation and redness of the skin according to http://www.nhs.uk/Conditions/Antifungal-drugs/Pages/Side-effects.aspx Candida tropicalis has been reported to be one of the leading Candida species other than Candida albicans to cause fungemia in patients who have malignancy. During the 1970s and 1980s, several studies showed that C. tropicalis fungemia was common in patients with leukemia and in those who received a bone marrow transplant (Oxford Journals) Furthermore, a study which was published in Korean Journal of Medical Mycology stated that extracts from the star anise seeds (Illicium verum), showed significant promise as a natural anti-fungal. Thus, the researchers chose this study because an antifungal agent made from Star anise seeds is all natural compared to the commercial anti-fungal products.

CONCEPTUAL FRAMEWORK

Input
Plant Species
Star Anise seeds (Illicium verum) * 10 grams of Star Anise seeds in 100 ml of H2O * 50 grams of Star Anise seeds in 100 ml of H2O * 100grams of Star Anise seeds in 100 ml of H2O

Process
Steam Distillation of Star Anise seeds

Output Antifungal activity of Star Anise seeds (Illicium verum) to Candida tropicalis

Fig. 1
THE PROBLEM
STATEMENT OF THE PROBLEM
This study will aim to determine the antifungal activity of star anise seeds (Illicium verum) to Candida tropicalis. Specifically, this study will seek to answer the following questions.
1. Which percent (refers to weight per volume) of star anise seeds (Illicium verum) to 100 ml of water can kill more Candida tropicalis (fungus) based in the diameter of the zone of inhibition: a. 10 % b. 50 % c. 100 %
2. How effective is the percent of star anise seeds (Illicium verum) with the highest mortality rate based on the: a. number of hours the zone of inhibition appear b. diameter of the zone of inhibition
STATEMENT OF THE HYPOTHESIS
Ho1: The different proportions of star anise seeds have no significant difference to the zone of inhibition of Candida tropicalis.
Ho2: The effectiveness of the different proportions of star anise seeds solutions has no significant difference to the number of days the zone of inhibition appear and the diameter of the zone of inhibition.

SIGNIFICANCE OF THE STUDY

An organic antifungal is effective, affordable and does not cause any harm to the environment. Therefore, it is safe to use. This investigation will aim to determine the feasibility of star anise seeds (Illicium verum) against Candida tropicalis, a fungus that carries different kinds of diseases most commonly the Candidiasis.
Environment. Environment is the most important beneficiary of this study because the product is eco-friendly that can cause no harm to the environment.
Families. Families who want an environment that is safe from fungi carrying diseases.
Entrepreneurs. When proven to be effective entrepreneurs can make the products in bulk to supply the market. In doing so, they can help the public as well as gain from the commerce.
Researchers. This study helps the researchers gain more knowledge about star anise (Illicium verum) other than being an ingredient in cooking.

DEFINITION OF TERMS

For better understanding of this study and to facilitate comprehension of this work, the following terms are hereby defined.
Antifungal activity. This refers to the capacity of star anise seeds (Illicium verum) to kill more Candida tropicalis.
Candida tropicalis. Is the genus of fungus used as the subject of this study. This fungus causes an infection called Candidiasis.
Percent. These refers to the weight per volume of Illicium verum in 100 ml of water.
Star anise. Is the plant used in this study, in which it is shaped like a star.
Zone of inhibition. The clear region around the paper disc saturated with the decoction on the agar surface.

SCOPE AND LIMITATIONS

This study will investigate the effectiveness of star anise seeds as antifungal agent. It will focus on the extraction of dried star anise seeds (Illicium verum) in distilled water. The subject of this study will be a fungus called Candida tropicalis. The researchers will prepare a culture media in order for the fungi to grow. This study is proposed on March 2014 in Talamban National High School but the culturing of fungi and testing of product will be conducted at University of San Carlos-Talamban Campus where materials are available.

REVIEW OF RELATED LITERATURE Star anise is a powerful culinary and medicinal spice that has been used for thousands of years and is rich in vitamin C, calcium and iron. Star anise contains potent anti-viral, anti-bacterial, and anti-fungal properties and is particularly good for treating the flu. In fact, star anise contains a compound called shikimic acid which is used to make the common anti-viral drug Tamiflu. (http://www.medicalmedium.net/articles-members-only/star-anise) Chemical studies indicate that a major portion of this antifungal property is due to anethole present in the dried fruit. Studies with isolated anethole (compared with standard anethole) indicated that it is effective against bacteria, yeast and fungal strains according to http://onlinelibrary.wiley.com/doi/10.1002/ptr.989/abstract;jsessionid=0AC34E7A8408FA5E20A39829027FBCA4.f01t02. Anethole shows synergistic effects on the antifungal activities of phytochemicals including polygodial and (2E)-undecenal against Saccharomyces cerevisiae and Candida albicans. (http://onlinelibrary.wiley.com/doi/10.1002/ptr.2016/abstract) The antifungal activity of the oil and its main component anethole against plant pathogenic fungi were determined. Both the essential oil and anethole exhibited strong inhibitory effect against all test fungi indicating that most of the observed antifungal properties were due to the presence of anethole in the oil, which could be developed as natural fungicides for plant disease control in fruit and vegetable preservation. (http://www.ncbi.nlm.nih.gov/pubmed/21030909) Anethole has potent antimicrobial properties, against bacteria, yeast, and fungi. Reported antibacterial properties include both bacterio static and bactericidal action against Salmonella enterica but not when used against Salmonella via a fumigation method. Antifungal activity includes increasing the effectiveness of some other phytochemicals (e.g. polygodial) against Saccharomyce cerevisiae and Candida albicans; this synergistic effect has potential medical uses In vitro, anethole has antihelmintic action on eggs and larvae of the sheep gastrointestinal nematode Haemonchus contortus. Anethole also has nematicidal activity against the plant nematode Meloidogyne javanica in vitro and in pots of cucumber seedlings. (Chang KS, Ahn YJ, 2002). In the study “Antifungal Activity of star anise seeds (Illicium verum) against Dermatophyte and Saphrophite Fungi”,shows that extracts of star anise seeds inhibited growth of all Dermatophytes and Saphrophites.(http://www.researchgate.net/publication/202312370Antifungal Activity of extsacts of star anise Hook against dermatophyte and saphrophyte fungi). According to the “Korean Journal of Medical Mycology," extracts from the star anise plant, Illicium verum, showed significant promise as a natural anti-fungal. When your body’s internal balance of bacteria and fungi is disturbed, a certain type of yeast -- Candida albicans -- can grow unchecked. If this happens, it can cause candidiasis, a serious complication.Candida albicans is found naturally in your intestinal tract, mouth and throat.(http://www.livestrong.com/article/399013-health-benefits-of-star-anise/) A study published in the April 2008 issue of the Journal of Medical Virology showed that the combination of shikimic acid, flavonoids quercetin and star anise seeds ramped up immunity to fully resist fungal infection.(http://www.naturalnews.com/045643star anise superfood health benefits.html)

REVIEW OF RELATED STUDIES In another study published in June 2007 at the Department of Medical Microbiology & Parasitology, University College Hospital, Ibadan, Nigeria by Ogbolu DO, et. al, tested coconut oil verses 17 different isolates of Candida albicans, 9 isolates of Candida glabrata, 7 isolates of Candida tropicalis, 7 isolates of Candida parapsilosis, 6 isolates of Candida stellatoidea, and 6 isolates of Candida krusei. They found that Candida albicans had the highest susceptibility to coconut oil for yeast infections (100%). (http://www.yeastinfectionadvisor.com/coconutoilforyeast.html) Candida krusei showed the highest resistance to coconut oil with a MIC of 100% (undiluted), while fluconazole had a MIC of > 128 microg/mL. Candida krusei is actually more resistant to fluconazole than it is coconut oil.It is noteworthy that coconut oil was active against all species of Candida at 100% concentration compared to fluconazole. Coconut oil should be used in the treatment of fungal infections in view of emerging drug-resistant Candida species. The other species fell somewhere in the middle as far as being susceptible to coconut oils fungal killing ability. In "The Yeast Connection Handbook", coconut oil is recommended by the author, (William Crook M.D) Many natural health practitioners will prescribe caprylic acid for yeast infection treatment but as the studies indicate, you’re much better off with all the active ingredients rather than just the one. So always take the whole form of coconut oil and not the extracts and make sure it is a virgin cold pressed natural organic oil. (http://www.yeastinfectionadvisor.com/coconutoilforyeast.html)
CHAPTER 2
RESEARCH METHODOLOGY This chapter dealt with the methods in extracting the star anise seeds and the process of testing it against Candida tropicalis. RESEARCH DESIGN The research design used in this study was experimental; where in there was a positive control (fungisol) and a negative control (distilled water) and the different percents of star anise seeds (Illicium verum). The independent variable in this study is the concentration of star anise and the dependent variable is the zone of inhibition of Candida Tropicalis.
Table 1.Research Design Composed of the conditions and treatment Conditions | Set-up A(10 g star anise seeds in 100 ml water) | Set-up B(50 g star anise seeds in 100 ml water) | Set-up C(100 g star anise seeds in 100 ml water | Distilled water(negative control) | Fungisol(positive control) | Discs rinse in each solution | 3 | 3 | 3 | 3 | 3 | Culture media-Sabouraud agar containing Candida tropicalis | 0.91g | 0.91g | 0.91g | 0.91g | 0.91 g | Measuring the zone of inhibition | (Every 6 hours interval)6th ,12th, 18th and 24th Hour | (Every 6 hours interval)6th ,12th, 18th and 24th Hour | (Every 6 hours interval)6th ,12th, 18th and 24th Hour | (Every 6 hours interval)6th ,12th, 18th and 24th Hour | (Every 6 hours interval)6th ,12th, 18th and 24th Hour | RESEARCH PROCEDURE A. Making of the Product The 10 g, 50 g and 100 g of Star anise seeds were weighed and pounded separately. The 10 g were put to a kettle that contained 100 ml of water. A 2 m hose was connected to the mouth of the kettle and was taped tightly including the opening of the kettle (this is to make sure that the steam will only pass to the hose.) The middle part of the hose was rolled and put to a basin that contained water with ice cubes (this serves as the condenser) and the end part of the hose was connected to a container where in the distillate was collected. (The process will take 5 hours and it requires medium fire. The procedure was repeated respectively to 50 g and 100 g of seeds.) B. Preparation of the Culture Media A 4.55 g of Sabouraud agar was mixed to 70 ml water. The solution was covered with foil and was put to the Pressure Steam Sterilizer as well as the 3 Petri dishes (served as the plates) and 5 beakers (soaking materials) covered with papers. When the agar was sterilized (it solidified) , it was put to a beaker with 500 ml water and heated it until it melted. Afterwards, the solution was divided into 3 sets of 20 ml. Using the pour-plate method, the 20 ml agar solution was poured to the Petri dish and shook slowly then was sterilized using an alcohol lamp and was set aside for 45 minutes under the UV rays. A 10 ml distilled water was put to a sterilized test tube. The inoculating loop was used to get the spores of the Candida Tropicalis and was mixed to the 10 ml distilled water. The solution was compared to the 0.5 McFarland standard. The burette was used to get 0.5 ml of the fungal solution and was poured to the agar surface in the plate. It was spread using a Glass spreader and was set aside. D. Inoculation of Fungal Solution There were 15 discs which will be used in the study which are punched filter paper. And the discs was soaked in 5 set-ups; Set-up A, Set-up B, Set-up C, 2 ml fungisol and 25 ml distilled water and was left overnight. One disc from each of the 5 set-ups was dispensed on the surface of the Petri dish using flame sterilized forceps. Therefore, it made 5 discs per dish. The flame-sterilized forceps was used to press each disc to the agar to ensure that the disc was attached to the agar. The plates were wrapped with a paper followed by plastic and a cartolina then was put inside a shoebox. The shoebox was placed to an underground dark room and was checked every 6 hours. E. GATHERING OF DATA After the different discs will be placed to the fungal colony, the changes of the colony will be determined after 12hours and 24 hours by: * Measuring the zone of inhibition * Describing the colonies F. TREATMENT OF DATA The data will be collected, statistically treated, analyzed and interpreted. ANOVA (Analysis of Variance) will be used for statistical treatment. The selection of star anise seeds will be analyzed for its capacity in killing Candida Tropicalis compared to the positive control which is the Fungisol.

CHAPTER 3
RESULTS AND DISCUSSION
Zone of inhibition in 24 hours (6 hours interval) of Candida tropicalis Time | Set-ups | TREATMENTS | | | A(10%) | B(50%) | C(100%) | D(Distilled water) | E(Fungisol) | 6th hour | 1 | 0 | 0 | 7 | 0 | 12 | | 2 | 0 | 7 | 7 | 0 | 14 | | 3 | 0 | 7 | 8 | 0 | 12 | 12th hour | 1 | 0 | 8 | 14 | 0 | 18 | | 2 | 0 | 10 | 12 | 0 | 19 | | 3 | 0 | 8 | 15 | 0 | 19 | 18th hour | 1 | 0 | 10 | 20 | 0 | 21 | | 2 | 0 | 13 | 15 | 0 | 25 | | 3 | 0 | 10 | 20 | 0 | 26 | 24th hour | 1 | 0 | 0 | 45 | 0 | 44 | | 2 | 0 | 0 | 10 | 0 | 40 | | 3 | 0 | 0 | 0 | 0 | 35 |
Table 3.1 shows the zone of inhibition of Candida tropicalis to the 5 treatments applied to it for 24 hours. The table above is all about the zone of inhibition of Candida tropicalis to the 5 treatments applied to it within the 6th, 12th, 18th and 24th hour. It shows that in treatment A (10%), there is no zone of inhibition in any hour. In treatment B (50%), the zone of inhibition increases from 6th-8th hour but decreases around the 24th hour. In treatment C (100%), the zone of inhibition increases from 6th- 24th hour in Set-up A but decreases to Set-up B and C. In treatment D (distilled water), there is no zone of inhibition at all then in treatment E (Fungisol), the zone of inhibition increases until the 24th hour.

CHAPTER 4 CONCLUSION AND RECOMMENDATIONS
CONCLUSION
Therefore, the different proportions of Star anise seeds has a significant difference to the zone of inhibition of Candida tropicalis. As the percent of Star anise seeds increases the diameter of the inhibition also increases. The distilled water which is the negative control, is not effective in killing Candida tropicalis , while the Fungisol which is the positive control, is the most effective in killing Candida tropicalis because it has the highest zone of inhibition among the 5 treatments. RECOMMENDATIONS
The following recommendations are offered to help improve the study. * To extract much anethole, the study is required to do a proper steam distillation where in it will be held inside a laboratory. Steam distillation is more effective in distilling temperature sensitive materials such as aromatic compounds like Star anise. * To maintain optimal temperature for the culturing of the fungi, the study is required to use an incubator. It maintains humidity or the temperature to grow the fungi. * To get the exact diameter of the zone of inhibition, a caliper is required to be used.

BIBLIOGRAPHY A. Journals
Korean Journal of Medical Mycology
Oxford Journals B. Internet Sources
(http://jmm.sgmjournals.org/content/59/8/873.full)
(https://www.google.com.ph/?gfe_rd=cr&ei=fpOhU9b3OMOE8Qf54YGACw&gws_rd=ssl#q=composition+of+star+anise) http://www.nhs.uk/Conditions/Antifungal-drugs/Pages/Side-effects.aspx
(http://www.medicalmedium.net/articles-members-only/star-anise)
(http://onlinelibrary.wiley.com/doi/10.1002/ptr.2016/abstract)
(http://www.ncbi.nlm.nih.gov/pubmed/21030909)
.(http://www.researchgate.net/publication/202312370Antifungal Activity of extsacts of star anise Hook against dermatophyte and saphrophyte fungi).
.(http://www.livestrong.com/article/399013-health-benefits-of-star-anise/)
.(http://www.naturalnews.com/045643star anise superfood health benefits.html)
(http://www.yeastinfectionadvisor.com/coconutoilforyeast.html)

APPENDICES

APPENDIX A
REQUEST LETTER for Adviser

March 12, 2014

Ms. Celia C. Gepitulan
Research I Teacher
Talamban National High School
Talamban, Cebu City

Madam:
We the undersigned students of Research I, who are conducting a research entitled “Antifungal Activity of Star Anise Seeds (Illicium verum) to Candida tropicalis”. It is quite difficult to undergo the study without the assistance of a mentor who can guide us along this research. In this connection, we ask you to be our research adviser.
We are hoping for your positive response to this matter.
Thank you and God bless.

Sincerely yours,
QUEENIBEL S. ARRIESGADO
KRISTINE JANE A. BORCES
DOMINIQUE FATIMA G. CABANSAY
JOBELYN B. COGTAS
MARY ROSE A. TELAMO

Approved:
CELIA GEPITULAN
Research Teacher

APPENDIX B
TRANSMITTAL LETTER

March 12, 2014

Dr. Ma. Jessica N. Abayon
Principal III
Talamban National High School
Talamban Cebu, City

Madam:

As a requirement for our Research II subject, we are asking your permission that we, the researchers, would be allowed to use the equipment in Science Laboratory for our Investigatory Projects since the school has the necessary materials for this the study.

Your approval would highly be appreciated. We are hoping for a positive response.

Thank you and God bless.

Sincerely yours,

QUEENIBEL S. ARRIESGADO
KRISTINE JANE A. BORCES
DOMINIQUE FATIMA G. CABANSAY
JOBELYN B. COGTAS
MARY ROSE A. TELAMO

Noted by:
CELIA GEPITULAN
Research Adviser

APPENDIX C
TRANSMITTAL LETTER

TALAMBAN NATIONAL HIGH SCHOOL
TALAMBAN, CEBU CITY
MARCH 12, 2014

DR. ZENAIDA C. GRAPA
Master Teacher II
Talamban National High School
Talamban Cebu, City

Madam:

As a requirement for our Research II subject, se are asking for your permission that we, the researchers would be allowed to use the equipment in Science Laboratory for our Investigatory Projects since the school has the necessary materials for this the study. Moreover, we are asking from your good office financial assistance to make this project successful.

Your approval would highly be appreciated. We are hoping for a positive response.

Thank you and God bless.

Sincerely yours,

QUEENIBEL S. ARRIESGADO
KRISTINE JANE A. BORCES
DOMINIQUE FATIMA G. CABANSAY
JOBELYN B. COGTAS
MARY ROSE A. TELAMO

Noted by:
CELIA GEPITULAN
Research Adviser
APPENDIX E
TRANSMITTAL LETTER
Dr. Anthony Ilano
Chair, Biology Department
University of San Carlos
Nasipit, Talamban
Cebu City 6000
Dear Dr. Ilano,

Greetings of Christian Peace!

We, the undersigned, are high school students of Talamban National High School which is located in Borbajo Street, Talamban, Cebu City. We are currently required to make a research paper since we belong to the graduating science class of the aforementioned school. Our study will deal with star anise seeds against Candida tropicalis. Yet, our school does not have the mentioned fungi or the facilities to grow them. In this connection, we are requesting your good office that we will be allowed to purchase them and to incubate them in your biology lab since it is equipped for the biological study. We do understand that we need to pay for everything. We hope that we can have them at an affordable price. We also asked for the help a lab technician who can assist us in performing the experiment since we are not very knowledgeable in performing the laboratory experiment.

We know that your help can greatly make our study successful.

Thank you so much.

Respectfully yours,

Queenibel S. Arriesgado

Kristine Jane A. Borces

Dominique Fatima G. Cabansay

Jobelyn B. Cogtas

Mary Rose A. Telamo

Noted:
CELIA C. GEPITULAN, M. Ed.
Research Teacher
DOCUMENTATION

Making of the Product

Preparation of Culture Media

Inoculation of Fungal Solution

CURRICULUM VITAE
OF
QUEENIBEL S. ARRIESGADO

PERSONAL BACKGROUND
Name: Queenibel S. Arriesgado
Age: 15
Date of Birth: January 15, 1999
Place of Birth: Sta. Cruz, Ronda Cebu
Stature: 147 cm
Weight: 41.6 kg
Address: Villa Leyson Bacayan, Cebu City
Father: Nixon L. Arriesgado
Mother: Annabella S. Arriesgado

EDUCATIONAL BACKGROUND

Kinder: Sta. Cruz Kindergarten
Elementary: Banilad Elementary School
High School: Talamban National High School

CURRICULUM VITAE
OF
KRISTINE JANE A. BORCES

PERSONAL BACKGROUND
Name: Kristine Jane A. Borces
Age: 15
Date of Birth: October 12, 1998
Place of Birth: Cebu City
Stature: 157 cm
Weight: 44.1 kg.
Address: Bacayan, Cebu City
Father: Jorge B. Borces
Mother: Flordeliza A. Borces

EDUCATIONAL BACKGROUND

Kinder: Talamban Kindergarten
Elementary: Talamban Elementary School
High School: Talamban National High school

CURRICULUM VITAE
OF
DOMINQUE FATIME G. CABANSAY

PERSONAL BACKGROUND
Name: Dominique Fatima G. Cabansay
Age: 15
Date of Birth: May 13, 1998
Place of Birth: Cebu City
Stature: 149 cm
Weight: 35.7 kg
Address: Villa Leyson Subdivision Bacayan, Cebu City
Father: Hermes F. Cabansay
Mother: Annabelle G. Cabansay

EDUCATIONAL BACKGROUND

Kinder:
Elementary: Talamban Elementary School
High School: Talamban National High School

CURRICULUM VITAE
OF
JOBELYN B. COGTAS

PERSONAL BACKGROUND
Name: Jobelyn B. Cogtas
Age: 15
Date of Birth: June 9, 1998
Place of Birth: Cebu City
Stature: 147 cm
Weight: 43.8
Address: Purok 2 San Jose, Cebu City
Father: Elias Cogtas
Mother: Rodriga Cogtas

EDUCATIONAL BACKGROUND

Kinder: (None)
Elementary: Talamban Elementary School
High School: Talamban National High School

CURRICULUM VITAE
OF
MARY ROSE A. TELAMO

PERSONAL BACKGROUND
Name: Mary Rose A. Telamo
Age: 15
Date of Birth:
Place of Birth: Stature:
Weight:
Address:
Father:
Mother:

EDUCATIONAL BACKGROUND

Kinder:
Elementary: Talamban Elementary School
High School: Talamban National High school

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