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PHYTOCHEMICAL AND ANTIMICROBIAL ANALYSES OF Hoya spp. JEAN CLAIRE M. ESTOPACIA A Thesis Submitted to Department of Biological Sciences, College of Arts and Sciences, University of Southern Mindanao Kabacan, Cotabato in Partial Fulfilment of the Requirements for the Degree of BACHELOR OF SCIENCE IN B...

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PHYTOCHEMICAL AND ANTIMICROBIAL ANALYSES OF Hoya spp Jean Claire Estopacia

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PHYTOCHEMICAL AND ANTIMICROBIAL ANALYSES OF Hoya spp.

JEAN CLAIRE M. ESTOPACIA

A Thesis Submitted to Department of Biological Sciences, College of Arts and Sciences, University of Southern Mindanao Kabacan, Cotabato in Partial Fulfilment of the Requirements for the Degree of BACHELOR OF SCIENCE IN BIOLOGY (Microbiology)

MARCH 2015 i

Republic of the Philippines UNIVERSITY OF SOUTHERN MINDANAO Kabacan, Cotabato COLLEGE OF ARTS AND SCIENCES APPROV AL OF THESIS OUTLINE Name: JEAN CLAIRE M. ESTOPACIA Degree Sought: BS IN BIOLOGY Major: MICROBIOLOGY Specialization: Thesis Title: PHYTOCHEMICAL AND ANTIMICROBIAL ANALYSES OF Hoya spp.

APPROVED BY THE GUIDANCE COMMITTEE

TESSIE E. LERIOS, Ph.D. Adviser __________________ Date

HONEY VINCENT V. VALLE Department Statistician _________________ Date

TEODORA N. CABASAN, MSc. Department Chairperson __________________ Date

JOHN ARIES G. TABORA, M.Sc. Department Research coordinator _________________ Date

JANETTE P.SUPREMO, Ph.D. College Research Coordinator ________________ Date

EVANGELINE TANGONAN, Ph.D. Dean _________________ Date

Study No:_________ Recorded by:_________ Received: ARISTON D. CALVO, Ph.D. Director for Research and Development _______________ Date Index No.:_________ Recorded by: ___________ ii

ACCEPTANCE

The thesis outline attached hereto entitled “PHYTOCHEMICAL AND ANTIMICROBIAL ANALYSES OF Hoya spp.”, prepared and submitted by JEAN CLAIRE M. ESTOPACIA, in partial fulfillment of the requirements for the degree of Bachelor of Science in Biology (Microbiology) is hereby accepted.

TESSIE E. LERIOS, Ph.D. Adviser ___________________ Date

Accepted as partial fulfillment of the requirements for the degree of Bachelor of Science in Biology (Microbiology).

JANETTE P. SUPREMO, Ph.D. Research Coordinator, CAS ____________________ iii

Date BIOGRAPHICAL DATA

The researcher was born on January 12, 1994 in Lika Mlang Cotabato and the youngest child among three children of Mr. Mario C. Estopacia and Mrs. Lourdes M. Estopacia. She finished her elementary education at Lika Central Elementary School in 2006 and at Bacoor National High School Bacoor, Cavite for her secondary education in 2010. With her desire to have a degree, she pursued and enrolled her tertiary education in the University of Southern Mindanao, Kabacan, Cotabato taking up Bachelor of Science in Biology with Microbiology as her major. During her college days, she attended several seminars, workshops, and conventions inside and outside the campus. She is an active member of Biology Club and Philippine Society of Microbiology. She had her On- Job- Training in Bureau of Fishiries and Aquatic Resources (BFAR) at General Santos City.

JEAN CLAIRE M. ESTOPACIA Researcher iv

ACKNOWLEDGMENT

The researcher would like to express her deepest thanksgiving and appreciation to the people who gave their full support in time and in effort, for the encouragement and advices and most especially the prayers for the success of this research paper. Above all she would like to give thanks and praise to God and glory to His name for the Words of wisdom, courage, strength and constant guidance making this study successful. To Dr. Tessie E. Lerios, the researcher’s adviser for a mother’s heart that gave her the courage to make this research paper excellent. Thank you as well for all the advices and considerations. Thank you very much Ma’am Tess. To the members of the committee: Ma’am May, Ma’am Jane, Sir Kerwin, Sir Bry and Sir John Aries for the recommendations, suggestions, opinions, and valuable comments toward improving my work. To Ms. Honey Vincent V. Valle, Department Statistician for encouragement and for analysing all the necessary data needed in the study. To the faculty of the Department of Biological Sciences, Ma’am Janette, Ma’am Ellen, Ma’am Sedra, Ma’am Dory, Ma’am Jul-Annie, Sir Gelo, Sir James, Sir Wyne, Sir Krizler, thank you for the support and encouragement you have given to the researcher.

v

To Ma’am Julie, Kuya Sam, and Kuya Joel, thank you for the assistance in the central laboratory. To Ma’am Sonia for the assistance in the USMARC laboratory. To her classmates Clovell, GB, Jastine, Eden, Nikki, Lele, Mayee, Mich, Gsel, Mari, Kathlyn, Prei, Joh, Ysai, Danica, and ate Kat for sharing your life, love and wonderful memories. Thank you for a friendship that went through hard times and good times together. To my laboratory buddy Kristelle Mari for a heart-warming kindness and a helping hands during in times of need. To Lakas Angkan family for all the prayers, encouragement and joy you brought to the researcher. The researcher also expresses her deepest thanksgiving to her family for unending love and support that motivates the researcher to finish this paper excellently. To her parents Mr. Mario C. Estopacia and Lourdes M. Estopacia for the deepest love and care, unconditional trust, timely encouragement, unfailing support and endless patience you brought to the researcher. To her tita Sylvia for the support, encouragements and love to the researcher. To her siblings: kuya Harrel and kuya Von for the love, support, courage and happiness they brought to the researcher.

vi

To her Paternal and Maternal Grandparents, Uncle, Aunty, Cousins and Friends for constant support and encouragements to the researcher. The researcher would also like to extend a sincere word of thanks to all who contributed and take part in the completion and success of this research study, thank you very much and God bless!

vii

TABLE OF CONTENTS

Pages INTRODUCTION

1

REVIEW OF RELATED LITERATURE

6

METHODOLOGY

18

Collection of the Specimen

18

Sterilization of Glasswares

18

Preparation of Extract

18

Phytochemical Analyses

19

Test Microorganisms

21

Preparation of Inocula

21

Treatment

21

Disc Diffusion Assay

22

Measurement of Zone of Inhibition

22

Data Analysis

23

RESULTS AND DISCUSSION

24

SUMMARY AND CONCLUSION

28

LITERATURE CITED

30

APPENDICES

34

viii

LIST OF TABLES

Table

Title

Page

1

Phytochemical Components of Crude Leaf Extract of Hoya spp.

24

2

Mean Zone of Inhibition (mm) of the Test Organisms as Affected by Different Treatments of Hoya spp. Crude Leaf Extract After 24 Hours of Incubation

26

3

Mean Zone of Inhibition (mm) of the Test Organisms as Affected by Different Treatment of the Hoya spp. Crude Leaf Extract After 48 Hours of Incubation

27

ix

LIST OF APPENDICES

Appendix

Title

Page

A

The map of Kabacan North Cotabato Showing the Sampling Site

35

B

Photograph and Taxonomic Description of Hoya spp.

36

C

Photograph of Hoya spp. Crude Extract

37

D

Schematic Diagram of Culture Preparation

38

E

Schematic Diagram of Disc Diffusion Assay

39

F

Measurement of Zone of Inhibition (Ontengco, 1994)

40

G

Phytochemical Components of Crude Leaf Extract of Hoya spp.

41

H

Mean Zone of Inhibition (mm) of the Test Organisms as Affected by Different Treatments of Hoya spp. Leaf Crude Extract After 24 Hours of Incubation

42

I

Mean Zone of Inhibition (mm) of the Test Organisms as Affected by Different Treatments of Hoya spp. Leaf Crude Extract After 48 Hours of Incubation

43

J

Result of Analysis of Variance Mixed Treatments

44

K

Result of Analysis of Variance of the Test Organisms

45

L

Result of Analysis Period

46

of Variance of Incubation

x

M

Photographs of Antimicrobial Activity of Hoya spp. Crude Extract After 24 Hours of Incubation Against Gram Positive Bacteria (Staphylococcus aureus)

47

N

Photographs of Antimicrobial Activity of Hoya spp. Crude Extract After 24 Hours of Incubation Against Gram Negative Bacteria (Pseudomonas aeruginosa)

50

O

Photographs of Antimicrobial Activity of Hoya spp. Crude Extract After 48 Hours of Incubation Against Gram Positive Bacteria (Staphylococcus aureus)

53

P

Photographs of Antimicrobial Activity of Hoya spp. Crude Extract After 48 Hours of Incubation Against Gram Negative Bacteria (Pseudomonas aeruginosa)

56

xi

ABSTRACT

ESTOPACIA, JEAN CLAIRE MARZO. 2015. Phytochemical and antimicrobial analyses of Hoya spp. BSBio Thesis. Department of Biological Sciences. College of Arts and Sciences, University of Southern Mindanao, Kabacan, Cotabato. 60 p.

Adviser: TESSIE EVANGELISTA – LERIOS, Ph. D.

The study was conducted to determine the phytochemical component and antimicrobial activity of Hoya spp. Phytochemical analysis revealed the presence of alkaloids, flavonoids and tannins in the plant extract. Hoya spp. was evaluated for their antimicrobial property through Disc Diffusion Assay. Hoya spp. exhibited inhibitory activity against the growth of Staphylococcus aureus while in Pseudomonas aeruginosa showed no inhibitory effect. Overall result for the susceptibility of test organisms to the plant extract at varying concentrations showed highly significant difference as well as the overall result for the Hoya treatments including controls. In addition, there was highly significant difference (p17

++Moderate

12-16

+Weak

7-12

-Negative

0-6

41

Appendix G. Phytochemical Components of Crude Leaf Extract of Hoya spp.

42

Appendix H. Mean zone of inhibition (mm) of the test organism as affected by different treatments of Hoya spp. crude leaf extract after 24 hours of incubation.

Test

Organisms

Staphylococcus aureus Pseudomonas aeruginosa

Leaf Extract Concentration 25% 50% 75% 100%

Control + -

9.41 6

8.68 6

11.41

12

34.68

6

6

6

32

6

43

Appendix I. Mean zone of inhibition (mm) of the test organism as affected by different treatments of Hoya spp. crude leaf extract after 48 hours of incubation.

Test

Organisms

Staphylococcus aureus Pseudomonas aeruginosa

Leaf Extract Concentration 25% 50% 75% 100% 8.92 6

8.30 6

10.90 6

Control + -

11.48

32.60

6

6

31.59

6

44

Appendix J. Result of Analysis of Variance Mixed Treatments

(I) CONCENTRATIN (J) CONCENTRATION 25%

Mean Difference (I-J) .34

.436

.986

75%

-.99

.436

.416

100%

-1.29

.436

.163

-25.13*

.436

.000

1.58*

.436

.049

-.34

.436

.986

75%

-1.34

.436

.135

100%

-1.63*

.436

.040

-25.47*

.436

.000

1.24

.436

.192

25%

.99

.436

.416

50%

1.34

.436

.135

100%

-.29

.436

.993

-24.13*

.436

.000

2.58*

.436

.000

25%

1.29

.436

.163

50%

1.63*

.436

.040

75%

.29

.436

.993

-23.84*

.436

.000

2.87*

.436

.000

25%

25.13*

.436

.000

50%

25.47*

.436

.000

75%

24.13*

.436

.000

100%

23.84*

.436

.000

Distilled water

26.71*

.436

.000

25%

-1.58*

.436

.049

50%

-1.24

.436

.192

75%

-2.58*

.436

.000

100%

-2.87*

.436

.000

-26.71*

.436

.000

Distilled water 25%

Ciprofloxacin Distilled water 75%

Ciprofloxacin Distilled water 100%

Ciprofloxacin Distilled water Ciprofloxacin

Distilled water

Sig.

50%

Ciprofloxacin 50%

Std. Error

Ciprofloxacin Based on observed means. The error term is Mean Square(Error) = .569. *. The mean difference is significant at the .05 level.

45

Appendix K. Result of Analysis of Variance of the Test Organisms

95% Confidence Interval for Differencea

Mean (I)

(J)

Difference (I-

Std.

J)

Error

BACTERIUM BACTERIUM S.Aureus

P.Aeruginosa

P.Aeruginos S.Aureus a

Sig.a

Lower Bound

Upper Bound

3.062*

.251

.000

2.543

3.581

-3.062*

.251

.000

-3.581

-2.543

Based on estimated marginal means *. The mean difference is significant at the .05 level.

Between-Subjects Factors Value Label BACTERIUM

CONCENTRATION

N

1

S.Aureus

18

2

P.Aeruginosa

18

1

25%

6

2

50%

6

3

75%

6

4

100%

6

5

Ciprofloxacin

6

6

Distilled water

6

46

Appendix L. Result of Analysis of Variance of Incubation Period

(I)

95% Confidence Interval for

(J)

HOUR HOUR

Differencea

Mean Difference (I-J)

Std. Error

Sig.a

S

S

1

2

.368*

.028

.000

.309

.426

2

1

-.368*

.028

.000

-.426

-.309

Based on estimated marginal means *. The mean difference is significant at the .05 level.

Within-Subjects Factors

HOURS

Dependent Variable

1

HR24

2

HR48

Lower Bound

Upper Bound

47

Appendix M. Photographs of antimicrobial activity of Hoya spp. crude extract after 24 hours of incubation against gram positive bacteria. Staphylococcus aureus

48

Appendix M. Photographs of antimicrobial activity of Hoya spp. crude extract after 24 hours of incubation against gram positive bacteria. Staphylococcus aureus

49

Appendix M. Photographs of antimicrobial activity of Hoya spp. crude extract after 24 hours of incubation against gram positive bacteria. Staphylococcus aureus

50

Appendix N. Photographs of antimicrobial activity of Hoya spp. crude extract after 24 hours of incubation against gram negative bacteria. Pseudomonas aeruginosa

51

Appendix N. Photographs of antimicrobial activity of Hoya spp. crude extract after 24 hours of incubation against gram negative bacteria. Pseudomonas aeruginosa

52

Appendix N. Photographs of antimicrobial activity of Hoya spp. crude extract after 24 hours of incubation against gram negative bacteria. Pseudomonas aeruginosa

53

Appendix O. Photographs of antimicrobial activity of Hoya spp.crude extract after 48 hours of incubation against gram positive bacteria. Staphylococcus aureus

54

Appendix O. Photographs of antimicrobial activity of Hoya spp.crude extract after 48 hours of incubation against gram positive bacteria. Staphylococcus aureus

55

Appendix O. Photographs of antimicrobial activity of Hoya spp.crude extract after 48 hours of incubation against gram positive bacteria. Staphylococcus aureus

56

Appendix P. Photographs of antimicrobial activity of Hoya spp. crude extract after 48 hours of incubation against gram negative bacteria. Pseudomonas aeruginosa

57

Appendix P. Photographs of antimicrobial activity of Hoya spp. crude extract after 48 hours of incubation against gram negative bacteria. Pseudomonas aeruginosa Pseudom seudomonas aeruginosa onas

aeruginosa

58

Appendix P. Photographs of antimicrobial activity of Hoya spp. crude extract after 48 hours of incubation against gram negative bacteria. Pseudoomonas aeruginosa

59

Republic of the Philippines University of Southern Mindanao College of Arts and Sciences Kabacan, Cotabato APPLICATION FOR ORAL DEFENSE OF THESIS MANUSCRIPT NAME: JEAN CLAIRE M. ESTOPACIA DEGREE: BACHELOR OF SCIENCE IN BIOLOGY TITLE: PHYTOCHEMICAL AND ANTIMICROBIAL ANALYSES OF Hoya spp. Indicate whether: first: ___________ Second: _______________ Date of Defense: March 09, 2015 Place of Defense: Department of Biology, CAS, USM. MEMBERS OF DEFENSE COMMITTEE Signature

Name

Date

Dr. May Eva Z. Garcia Renee Jane A. Ele Kerwin Jade A. Mallorca TESSIE E. LERIOS, Ph.D. Adviser

Approved :

JANETTE P. SUPREMO, Ph.D. JOHN ARIES G. TABORA, M.Sc. College Research Coordinator Dep’t Research Coordinator REPORT THE RESULT EXAMINATION Name

Signature

Dr. May Eva Z. Garcia Dr. Tessie E. Lerios Renee Jane A. Ele Kerwin Jade A. Mallorca