Study on Antifungal Properties of Manilkara zapota L.

(Chico) peel and leaves extract _____________________________________________ A Thesis presented to Biological Sciences Department Institute of Arts and Sciences Far Eastern University, Manila _____________________________________________ In Partial Fulfillment of the Requirements For the Degree of Bachelor of Science major in Biology

by Diaz, Kristine C. Vergara, Regenald F.

Approval Sheet This research Antifungal Properties of Manilkara zapota L. (Chico) peel and leaves extract: A Study was submitted by Diaz, Kristine C. and Vergara, Regenald F., in partial fulfillment of the requirements For the Degree of Bachelor of Science Major in Biology, has been examined for acceptance and approval.

Approved by ____________________ Prof. Dr. Glorina Orozco

Date Submitted _____________ Rating _______________

ACNOWLEDGEMENT The researchers would like to give their sincerest thanks to all the people who contributed their time, patience, and knowledge for the completion of this research. The researchers would also like to grab the opportunity to give their warmest thanks those who participated and shared a part of themselves in accomplishing this study. First and foremost, the researchers would like to give thanks to our almighty God, for giving them enough strength, patience, perseverance, and guidance in making this research successful, and giving us the opportunity to make things possible. Second are their families, for always being there and never endingly extend their support. Third is for their adviser, for Dr. Glorina Orozco, for his continuous support and for guiding them all throughout. Finally, to the friends and foes who made this study somehow an easier task. By simply contributing their time and effort for revising and editing some of the errors conducted by the researchers.

TABLE OF CONTENTS

TITLE PAGES APPROVAL SHEET ACKNOLEDGEMENT

_______________________________i _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ii _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _iii

TABLE OF CONTENTS _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _iv CHAPTER 1. THE PROBLEM AND ITS BACKGROUND_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 1 y y y y y y Introduction _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 1 Background of the Study_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 2 Statement of the Problem_ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ 4 Significance of the Study_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _5 Scope and Delimitations_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 6 Definition of Terms_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 7

2. REVIEW OF RELATED LITERATURE AND STUDIES_ _ _ _ _ _ _ _ _ _ _ _ 8 y y Related Literature_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _8 Related Studies_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _13

3. RESEARCH METHODOLOGY_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 17 y y y Research Method Used_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 17 Research Locale_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 17 Description of the Respondents_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 17

Research Instruments Used_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 19

4. PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA _ _ _ _ _ 20 y y y Presentation of the Data_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 20 Analysis_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _21 Interpretation of the Data_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 21

5. SUMMARY, CONCLUSION AND RECOMMENDATONS _ _ _ _ _ _ _ _ _ _ 25 y y y REFERENCES APPENDICES A. Questionnaires B. Survey Data Tally Sheet Curriculum Vitae Summary _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 25 Conclusions_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 26 Recommendations _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 26

CHAPTER 1 The Problem and its Background Introduction Background of the Study Medicinal plants are a source of great economic value all over the world. Nature has bestowed a very rich botanical wealth and a large number of diverse types of plants grow in different part of the country (Onyeagba, 2004). The potential of higher plants as source for new drugs is still largely unexplored. Among the estimated 250,000-500,000 plant species, only a small percentage has been investigated (Mahesh, 2008). There is a continuous and immediate need to discover new anti microbial compounds with diverse chemical structures and novel mechanisms of action per new and re-immerging infectious disease. Herbal medicine is still the mainstay of about 75-80% of the whole population, and the major part of traditional therapy involves the use of plant extract and their active constituents (Akerele, 1993). Antimicrobials of plant origin have enormous therapeutic potential. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials. Many plants are known to have medicinal value, Manilkara zapota L. or commonly known as Chico is one of the most widely used plants.

Statement of the Problem This research was undertaken to find out the Antifungal Properties of Manilkara zapota L. (Chico) peel and leaves extract Specifically, this study is conducted to seek answers to the following problems: 1. 2. 3. What will be the least and most sensitive fungi? Is there an antifungal activity of Chico peel and leaves in ethanol extract? Is Chico peel and leaves extract is significant through statistical analysis?

Hypothesis ??????????????????????? Manilkara zapota L. extract (chico) would not produce an antifungal substance which inhibits the growth of fungi. Significance of the Study This study hopes to enlighten the mind of the readers to understand the psychological effects of using teeth braces towards the increase and decrease of selfesteem of an FEU-IAS student. The society, this study would like to enhance possible alternatives known in fighting or preventing fungal infestation. The readers, it would like to address them to acknowledge the feelings and insights of individuals with concern on what other alternatives to use in replacement to other known product.

The biologist, this will serve as an additional knowledge and awareness so they can promote further study on the said fruit in terms of other known possible properties present to it. The Future Researchers, it would give way for them to further their knowledge about this topic and to look for other properties present to the fruit that were not tackled in this research. Scope and Delimitation of the Study The study focused only on the property of the Manilkara zapota L. (Chico) that inhibits the ability to kill fungi. Physical effects like the range of effect of the drug were used as the determinant factors in the study. The research was limited to study only the antifungal properties of Chico (Manilkara zapota L.) peel and leaves against Aspergillus flavus and Aspergillus niger and Itraconazole as control only. The study covers 2 extracts by which each extract contain 3 trails. Each trial contains 3 specific treatment by which, the researchers took the results. Professionally cultivated agar- based fungi were used by the researchers to gather information about the antifungal properties of Manilkara zapota L. (Chico) peel and leaves extract. The result cannot be generalized to all other types of fungi due to the limitation of the said research.

Definition of Terms 1. Filtration- process of separating a suspended solid from liquid in which it

is suspended by straining it. 2. Residue- also called residuum which usually in granules or solid particles

of the process either disregarded or subjected to chemical analysis. 3. Ethanolic extract- a preparation using ethanol. It contains the

pharmaceutically active components of a natural product such as plant. 4. Zone of inhibition- zone of diameter in which the fungi reacted against

the avocado extract. 5. 6. Assay- analysis to determine the quality or property. Resistant- is applied to microorganisms requiring substantially higher

concentrations. 7. Sensitive- is also used in a qualitative sense to describe organisms

susceptible to concentrations of antimicrobial drug. 8. 9. Antifungal- inhibiting the growth of fungi Disk- Diffusion method- it is also called Kirby-Bauer antibiotic testing is

a test which uses antibiotic-impregnated wafers to test whether particular fungi are susceptible to specific antibiotics.

CHAPTER 2 Review of Related Literature and Studies This chapter is about the studies related to the research topic and its division on related literature and studies. Related Literature ANTIFUNGAL STUDIES Antifungal screening were investigated through the utilization of Atis fruit

pericarp, which will be tested to different strains of fungi with the use of different methods in evaluating antifungal activity. The effects of pH and acid solvent on the antifungal activities of chitosan were studied by Zhong Qiu-Ping et al., (2005) by testing anthracnose, stem- end rot pathogens in Mango fruits and quiescent colletotrichum musae, crown rot pathogens in banana fruit. The results showed that pH4.8 and pH6.0 were both optimum to the antifungal activity of chitosan. The antifungal activity of chitosan was increased with the increase of its concentration. The activity of inhibition of chitosan in lactic acid was very strong for stem-rot pathogens, quiescent colletotrichum musae, and crown rot pathogens. The EC50 value increased as the tested pathogens increased, but they had some resistance reduced after the isolates were successively stimulated 40 times by application of chitosan. While the Botryodiplodiat heobromae Pat. exhibited the worst inhibition capability.

Based on the study of Caillier, two series of surfactants monomers, with a quaternary ammonium group as polar head and an acrylic function as the polymerizable moiety, were synthesized and tested to evaluate their surface active properties alongside with their antibacterial and antifungal properties. The biocidal efficacy measured by bacterial and fungal growth inhibition expressed as MIC (minimal inhibitory concentration) and MLC (minimal lethal concentration) values was discussed as a function of molecular parameters. Phytochemical investigation of the stem bark of Terminalia mollis afforded friedelin (1), catechin with epicatechin (2), gallocatechin with epigallocatechin (3) and 3O-methylellagic acid 4'-O-alpha-rhamnopyranoside (4). Arjunolic acid was conducted by Liu M et al., (2009) with 2alpha, 3beta, 23-trihydroxy-urs-12-en-28-oic acid (5), 2alphahydroxyursolic acid (6), gallic acid (7), chebulanin (8) and 2''-O-galloylvitexin (9) were isolated from the leaf. Chebulanin (8), betulinic acid (10), ursolic acid (11), catechin (12), isoorientin (13), orientin (14), isovitexin (15) and punicalagin (16) were isolated from Terminalia brachystemma leaf. The first full unambiguous NMR assignments for (4) and (8), and revised assignments for (9), are reported. Compound (16) showed good activity against three Candida species. The microbial transformation of racemic diisophorone was investigated by Ismail Kiran (2004) using the plant pathogen Aspergillus niger as a biocatalyst. Incubation of diisophorone with Aspergillus niger gave 8 -hydroxy-diisophorone, 10-

hydroxydiisophorone and 17-hydroxydiisophorone on the basis of their spectroscopic data including two-dimensional NMR analysis, nOe and an X-ray crystallographic study. The antifungal activity of diisophorone against Aspergillus niger was also examined.

The study conducted by D.N.A Tagoe et al., (2010) aim at comparing the antimicrobial (antifungal) properties of garlic, ginger and lime on Aspergillus flavus, Aspergillus niger and Cladosporium herbarum using organic and water base extraction. Pure cultures of the test fungal were isolated from rotten vegetables, air spora, and a swab of the skin surface whilst filtered plant extracts were obtained by means of organic (absolute ethanol) and aqueous (distilled water) extraction techniques. The pour plate technique was applied in the antifungal sensitivity testing using Potato Dextrose Agar (PDA) and results obtained by measuring diameter of fungal growth over a 7day incubation period. Growth results showed high but varied levels of antimicrobial effectiveness. Ginger showed the highest antimicrobial effect against all the test fungi (P<0.01) followed by garlic (P<0.05) and then lime (P<0.01) in Aspergillus species but not in effective on C. herbarum (P>0.05) using ethanol extraction. In the water (aqueous) base extraction medium, lime exhibited the highest antimicrobial (antifungal) effect (P<0.01) on all test fungi, followed by garlic (P<0.01) on Aspergillus species, (P<0.05) on C. herbarum whilst ginger was ineffective on Aspergillus species (P>0.05) but highly fungicidal on C. herbarum (P<0.01). Thus water base extraction technique was the most effective in inducing antifungal properties of lime whiles ethanol base extraction technique was the best for ginger. Garlic on the other hand exhibited a good antimicrobial (antifungal) property in both ethanol and water extraction media. The study thus confirms the antifungal properties of these medicinal plants and suggests the type of extraction to yield the best results. Investigations conducted by I.O Ezeibekwe(2009) were carried out to test the potency of Aloe vera gel extract on fungal organisms associated with yam rot. These

fungi were isolated and identified as Fusarium oxysporium Schlech ex. Fr., Rhizopus oryzae, Went, Botryodiplodia theobromae Pat and Fusarium solani. Mart sacc.

Pathogenicity test was carried out to confirm these organisms as the pathological agents of the yam rot. The Aloe vera gel at the different concentrations of 25, 50 and 100% did not inhibit the growth of these fungi when tested for its antifungal potency, p = 0.05. The result of this study showed that Aloe vera gel did not actually inhibit fungal growth as the there was progression in the growth and development of the fungi Toki Taira et al., (2005) researched on the Purification, Characterization, and Antifungal Activity of Chitinases from Pineapple (Ananas comosus) Leaf . Three chitinases, designated pineapple leaf chitinase (PL Chi)-A, -B, and -C were purified from the leaves of pineapple (Ananas comosus) using chitin affinity column chromatography followed by several column chromatographies. PL Chi-A is a class III chitinase having a molecular mass of 25 kDa and an isoelectric point of 4.4. PL Chi-B and -C are class I chitinases having molecular masses of 33 kDa and 39 kDa and isoelectric points of 7.9 and 4.6 respectively. PL Chi-C is a glycoprotein and the others are simple proteins. The optimum pHs of PL Chi-A, -B, and -C toward glycolchitin are pH 3, 4, and 9 respectively. The chitin-binding ability of PL Chi-C is higher than that of PL Chi-B, and PL Chi-A has lower chitin-binding ability than the others. At low ionic strength, PL Chi-B exhibits strong antifungal activity toward Trichoderma viride but the others do not. At high ionic strength, PL Chi-B and -C exhibit strong and weak antifungal activity respectively. PL Chi-A does not have antifungal activity.

The research study conducted by Grace Onawunmi (1989) on antifungal activity of Lemon grass Oil (LGO) has been evaluated using fungistatic (MIC and agar diffusion

tests) and fungicidal (spore germination) studies. Appreciable activity was observed against various isolates of Candida and clinical isolates of Aspergillus fumigatus, Microsporum gypseum and Trichophyton mentagrophytes. The most resistant organism was A. fumigatus while M. gypseum and the Candida spp. were the most susceptible of the isolates. Comparative studies with pure samples of citral and citronellal, constituents of LGO, showed good activity against the test fungi while dipentene and myrcene showed no activity.Exposure of the spores of A. fumigatus to 0.1% LGO for 5 mins resulted in 93% of spores not germinating while lower concentrations (0.08% and 0.05%) caused 80% and 60% reduction in spore germination respectively. Challenge tests on a formulated aqueous cream containing LGO indicated that 0.25% LGO would effectively preserve the cream against fungal contamination. Jabeen made a study on the seed extracts of Moringa oleifera were assayed for the evaluation of antimicrobial activity against bacterial and fungal strains. The zones of growth of inhibition showed greater sensitivity against bacterial strains as compared to the fungal strains. MIC extracts revealed that P. multocida and B. subtilis were most sensitive strains. Maximum activity was found between temperatures 34-370 C and pH7. Mahesh also conducted a study on the methanol leaf extracts of Acacia nilotica, Sida cordifolia, Tinospora cordifolia, Withania somnifer and Ziziphus mauritiana showed significant antibacterial activity against Bacillus subtilis, E, coli, P. flourescens, S.aureus and Xanthomonas axonopodis pv. Malvacearum and antifungal activity against A.

flavus, Dreschlera turcica and Fusarium verticillioides when compare to root/bark extracts. A. nilotica and S.cordifolia leaf extract showed highest antibacterial activity

against B.subtilis and Z.mauritiana leaf extract showed significant activity against X.a.pv malvacearum. Root and leaf extract of S. cordifolia recorded significant activity against all the test bacteria. A.nilotica bark and leaf extract showed significant antifungal activity against A. flavus, Ziziphus mauritiana and Tinospora cordifolia recorded significant antifungal activity against D.turcica. The methanol extract of Sida cordifolia exhibited significant antifungal activity against F.verticillioides. However, according to Shariff et al., (2006), the systematic screening of antimicrobial plant extracts represents a continuous effort to find new compounds with potential to act against multi resistant pathogenic bacteria and fungi. Also, it have been reported that the difference in fungi toxicity towards avocado pathogens of chemical substances extracted from avocado cultivars Edranol, Duke 6 and Duke 7 (Wehner and Apostolides, 1981). On the other hand, plant extracts especially those performed with cold distilled water (CDW) had a powerful antifungal property with an important inhibition on the growth of the tested fungi and their hydrolytic enzymes (Fawzi et al., 2009).

Related Studies XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

CHAPTER 3 Research Methods and Procedures This chapter discusses the research methodology and how the researchers conducted the study. It gives information about the research method, instruments, and data gathering techniques. Research Method Used The research utilized the experimental method of research. It is a study of cause and effect. It differs from non-experimental methods in that it involves the deliberate manipulation of one variable, while trying to keep all other variables constant. (http://www.holah.karoo.net/experimental_method.htm) According to Moore, D., & McCabe, D. (1993), the best method of establishing causation is to conduct a carefully designed experiment in which the effects of possible lurking variables are controlled. According to Gay, L. R. (1992), the experimental method is the only method of research that can truly test hypotheses concerning cause-and-effect relationships. It represents the most valid approach to the solution of educational problems, both practical and theoretical, and to the advancement of education as a science Research Locale The experiment was done from January 20, 2012 to February 11, 2012 and was conducted in the Biology Research Laboratory, Science Building room 312-A, Far Eastern University, Manila.

Data Gathering Procedures All measurement information was gathered thru the use of (what machine??) to know the antifungal properties of Manilkara zapota L. (Chico) peel and leaves extract: I. Procurement of Materials A. Sample collection and preparation Manilkara zapota L. (Chico) peel and leaves samples were collected from Province of Pampanga. The fruits were thoroughly washed to remove dirt and foreign particles and were identified in the National Museum of the Philippines. B. Preparation of extracts The collected Chico peel and leaves was separately pulverized using blender. 400 grams of powdered Chico peel and leaves were soaked separately in ethanol for 24 hours. The extracts were filtered in a Watman No.-1 filter paper and evaporate to dryness using rotary evaporator. The extracts was kept in a properly sealed container and stored in the refrigerator for future use.

II. Test Specimen Pure cultures of fungi were obtained from UP-NSRI, Diliman, Quezon City. The fungi were used to test the presence of antifungal property such as Aspergillus flavus and Aspergillus niger.

III. Preparation of Media A. Culture Media

Using the Digital balance, 39g of Potato Dextrose Agar (PDA) was weighed and was transferred in a sterile 1000 ml beaker. Distilled water was added up to 1000 ml level and was mixed with the PDA. The PDA powder was dissolved by using the water bath and autoclave at 121oC for 30 minutes and was cooled and stored in the refrigerator. B. Subculture Media Using the Digital balance, 7.5 g of Sabouraud Dextrose Broth was weighed and transferred in a sterile 250 ml Erlenmeyer flask. Distilled water was added up to 250 ml level and mixed with the broth. The solution was dissolved by using water bath and autoclave at 121oC for 15 minutes. After cooling it, the broth was transferred in a sterilized test tubes having 10 ml concentration per tube. The pure culture of fungi were streaked from the test tubes using inoculating needle and immersed in the broth, observe aseptic technique while doing the procedure. The cultivation of fungi can be observed within 5-7 days period of incubation. C. Agar Well Diffusion method The melted Potato Dextrose agar was poured in the Petri dish and allowed the agar to solidify. The cotton swab was immersed in the subculture of fungi and was swabbed in the Petri plates. Wells were then bored into the agar media using a sterile 6 mm cork borer and the wells filled with the solution of the extract taking care not to allow spillage of the solution to the surface of the agar medium. Plates were incubated for 28-48 hours

and the zone of inhibition of the fungal growth was measured. The effect of the extract on fungal isolates was compared with the control plates with Itraconazole at a concentration of 1 mg /ml.

ZONE OF INHIBITION (ZI) The zone of inhibition formula was based by Li et al., (2009): ZI= (Left Horizontal + right horizontal + (Up Vertical + Down vertical) 2

CHAPTER 4 PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA This chapter shows the research presentation, interpretation and analyzing of the data that were accumulated. The chapter entirely presents the data gathered in the drugs given against the cultivated fungi. Presentation of Data The summary of the antifungal property Trial 1 A 100% B (-) C (+)

Aspergillus niger Aspergillus flavus

16mm 11.5mm

Trial 2 A 100% B (-) C (+)

Aspergillus niger

10.5mm

Aspergillus flavus

9.5mm

Trial 3 A 100% Aspergillus niger B (-) C (+) 13mm

Aspergillus flavus

10.5mm

Analysis of the Data The pure extract of Chico peel and leaves, negative control and Itraconazole as positive control were used in each of the two cultures of fungi, Aspergillus flavus and Aspergillus niger. After the incubation of 1-3 days, Chico peel gave a positive result against Aspergillus flavus and gave a negative result ___________________???? The table showed that there was no antifungal property in Chico peel and leaves extract except that in positive control Itraconazole which is antibiotic. The negative control which was distilled water showed that there was no zone of inhibition. Aspergillus flavus and Aspergillus niger all gave negative result or no zone of inhibition in all treatment. The negative control which is distilled water always results to negative or no zone of inhibition. The positive control Itraconazole use to treat fungal infection of the lungs. Interpretation of the Data?????????????????????????????????????? 1. 2. 3. What will be the least and most sensitive fungi? Is there an antifungal activity of Chico peel and leaves in ethanol extract? Is Chico peel and leaves extract is significant through statistical analysis?

Advantages (need ito wla pa!) You need to have braces installed to correct dental flaws like buckteeth or a protruding jaw. In addition to this, you can use braces to align your crooked teeth. y Braces can also help strengthen the gums so that it can hold the teeth properly into position. y Braces can prevent gaps from separating your teeth as it aligns them properly. You will never have to deal with unsightly gaps if you use braces at an early age. Disadvantages (need ito wla pa!) One major disadvantage of wearing on braces is the discomfort and pain that dental patients usually experience when having braces installed. This is because of the metal frame is usually used when making the brace. It will run through the teeth and produce a sensitivity that will render the teeth prone to dental pain. y Another problem that braces can pose for the wearer is the embarrassment of having to be seen wearing braces. A person wearing braces will be regarded as a geek. Therefore, it will be highly likely for him or her to be teased or bullied in school. y Self-esteem is a big issue for young people nowadays and with braces on your teeth, your self-esteem will be greatly damaged if you do not have the courage and determination to face all your detractors.

CHAPTER 5 SUMMARY, CONCLUSIONS AND RECOMMENDATIONS This chapter presented the summary, conclusion and recommendations given by the researchers. This chapter also discusses some previous studies done to support the statements that the researchers formulated. Summary This research was conducted to know the Antifungal Properties of Manilkara zapota L. (Chico) peel and leaves extract. Likewise, this study was conceptualized to compensate on the uprising demand for alternative . This research was undertaken at Far Eastern University, Biology Research Laboratory, Science Building room 312-A from January 20, 2012 to February 11, 2012. The researchers conducted his research to give information about the Antifungal Properties of Manilkara zapota L. (Chico) peel and leaves extract and to make sure that the society would have better understanding of the topic. This research made different trials to further know the consistency on the antifungal property of the Manilkara zapota L. (Chico) peel and leaves extract. Conclusions ????????????????????????? The respondents made some alteration to their body (or in this case, the teeth alignment) that make them feel like boosting their self esteem. y Some of the respondents were found to have a past experience about being discriminated by their imperfection. Some had trouble communicating due to unease

feeling every time in a presence of another individual. Some of them merely wanted to have perfect smile. These are the reasons why these respondents used teeth brace in correcting their imperfection. y Some respondents make use of their teeth braces to increase their self esteem, making them belonged with his or her environment. y Most of the respondents perceived their lives as satisfying since they are now somewhat have felt acceptance by their peers. Contentment and patients was their central response on some of the questions given to them by the researchers. The respondents thought contented by their present situation and are now focusing on establishing good foundation of self esteem. y The respondents made several trials on how to belong to others even if it means enduring each hardship to them. Recommendations After the study was conducted and the findings had been gained, the researchers formulated some recommendations in order to serve as guide to others towards treating self esteem: The bloggers, the researchers want to suggest them to validate the specific information they throw in to the minds of individuals specifically if it wasnt proven nor has no concrete evidence just yet. y The society, the researchers want to suggest that not everything that has properties of the given drug in a said fruit can be used as alternatives for the specific use.

The local government, the researchers want to advise to the local officials to start funding on alternative herbal medicines available in our country to lessen stock price of each drug.

The biologist, the researchers want to suggest them to change the extracting methods used in extracting the kara zapota L. (Chico) peel and leaves in order to know other possible outcomes of the study.

The future researchers, Try to remove each limitations of this research in your future studies. It would be better if the future researchers would tend to make some alterations on the specific methods in gathering data in this study.

REFERENCES BOOKS Ash, M.M., and Nelson, S.J. (2003) Wheelers dental anatomy, physiology and occlusion, Philadelphia, Saunders Dryden, W., (1998) Developing self- acceptance: a brief, educational, small group approach, Chichester, Willey Feist, J., and Feist, G., (2006) Theories of Personality, Asia, McGraw- Hill Lawrence, D., (2009) Enhancing self-esteem in the classroom, London, Paul Chapman PUB Martel, G., and Stewart, T. (2009) Fooled by Nature: Terrific Teeth and Awesome, Discover Communication Sarver, D.M., Contemporary Treatment of Dentofacial Deformity St. Louis, Mosby Schmidt, J.J., (2006) Social and Cultural Foundation of Counseling and human service: multiple influences on self development, Boston, Pearson Allyn and Bacon Walker, J., Payne, S., Smith, P., and Jarett, N., (2008) Psychology for nurses and caring profssion, Phillipines, McGraw-Hill Yoder, J., Practor, W., (1988) The self- confident child New York, Facts on File Zimbardo, J., Psychology: Core Concept, USA, Allyn and Bacon

INTERNET http://www.holah.karoo.net/experimental_method.htm JOURNALS Estupigan, B.C., (2002) Healthy Teeth: A National Concern, Philippine Panorama Tajfel, H., Billig, M. G., Bundy, R. P., & Flament, C. (1971). Social categorization and intergroup behavior, European Journal of Social Psychology, 1(2), 149-178. APPENDIX B Survey Data Tally Sheet NUMBER QUESTION 1. 2. 3. 4. 5. 6. 7. 8. OF STRONGLY AGREE 4 8 7 7 19 10 14 20 22 25 14 14 25 14 28 19 19 14 25 21 5 19 6 8 AGREE DISAGREE STRONGLY DISAGREE 5 3 4 8 1 7 2 2

9. 10.

13 11

28 24

6 12

3 3

CURRICULUM VITAE

BASIC INFORMATION Name: Address: Country: Telephone: Cell Phone: Email: John Benedict B. Merza 509C.Baltazar St. Tondo, Manila Philippines 254-4707 09274874138 benedictmerza@yahoo.com

PERSONAL INFORMATION Date of Birth: Place of Birth: Citizenship: Gender: October 19, 1993 Manila Filipino Male

EDUCATIONAL BACKGROUND Elementary:

Yangco Elementary School 2000- 2006 High School: Manila Science High School 2006- 2010 College: University Of Santo Tomas Bachelor of Science Major in Nursing 2010- 2011 Far Eastern University Bachelor of Science Major in Psychology 2011- Present

PERSONAL QUALIFICATIONS Good English grammar user. Computer Literate Background on First Aid Training

y y y

Graduated in a Science High School

AWARDS Graduated Salutatorian (Elementary, 2006) Consistent Honor (Elementary, 2000- 2011) Leadership Award recipient for the year 2006 (Yangco Elementary School)

y y y

LANGUAGES USED Filipino and English INTERESTS Playing Basketball, Yugioh Cards and Computer Games

CURRICULUM VITAE

BASIC INFORMATION Name: Address: Country: Telephone: Cell Phone: Email: Maria Daphne Anne Resurreccion 34c Santiago st. Pob.2, Marilao, Bulacan Philippines 711-3800 09052618445 daphne032890@yahoo.com

PERSONAL INFORMATION Date of Birth: Place of Birth: Citizenship: Gender: March 28, 1990 Bulacan Filipino female

EDUCATIONAL BACKGROUND Elementary: St. Michael School of Marilao High School: St. Paul College Of Bocaue College: Far Eastern University Bachelor of Science Major in Psychology

PERSONAL QUALIFICATIONS Good English grammar user. Computer Literate Background on First Aid Training

y y y

Graduated in a Science High School

LANGUAGES USED Filipino and English INTERESTS Cooking and reading books and magazines.