ABSTRACT

Tropical plants from the genus Pandanus comprises around 700 species worldwide and 52
species were identified in the Philippines. Locally, the plant P. amarylifollius is highly regarded in
cooking because of its distinctive aroma. Studies have shown that extracted secondary metabolites from
Pandanus were mostly alkaloidal in nature. These metabolites were found to have diuretic, antibacterial,
antitubercular and antioxidant properties (Dy, et al., 2009).

A recent study conducted by Ooi and colleagues(2004) was the isolation of an antiviral protein
from P. amaryllifolius. Based from these findings, the isolation of protein from P. dubius was pursued. Its
antibacterial and anti-gelatinolytic activities were tested via the Resazurin Assay and Zymography based
assay respectively.

The protein was isolated from saline leaf extract of P. dubius and precipitated using
ammonium sulfate. The presence of protein was confirmed using the Bradford protein assay with
Bovine Serum Albumin as the protein standard. The protein extract was then lyophilyzed and
subjected to the two biological assays.

In the Resazurin Assay, six fold dilutions of the crude protein extract were tested with
Streptomycin as standard. The bacteria used were standardized using 0.5 McFarland standard.
Crude protein extract with concentrations of 100 µg/mL and lower were used in the antibacterial
against organisms of E. coli, S. aureus, S. pyogenes, K. pneumoniae and P. aeruginosa.

While in gelatin zymography, the crude protein concentration used were 1000 µg/mL,
500 µg/mL and 250 µg/mL. In this assay, gelatinase from Serratia Marsecens was used.
Results showed that the extract was not able to inhibit the activity of the bacterial supernatant.
This would indicate that the protein isolated from P dubius didn’t cause inhibition of bacterial
growth and gelatinase activity at concentrations of 100 µg/mL and lower.

Keywords: Proteins, Pandanus, nsLTP’s, Chromatography, SDS PAGE, N-terminal amino acid
sequence, hemagglutinating activity, anti-proliferative activity

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 ACKNOWLEDGEMENT

This study acknowledges the following people for helping, guiding, and supporting me

throughout the duration of this study.

First of all, I would like to thank God for giving me the knowledge and guidance to finish

this study. Without Him I would not be here right now.

My family, for their undying love and support throughout my life. And for helping me in

making decisions that helped me grew as a person.

My adviser, Prof. Cristina Ramos, for teaching me throughout this study and for never

giving up on me despite the mistakes I made. Thank you mam for being patient and considerate

to me.

My thesis partner, Jessa Grace Castillo, for being there for me especially on times when I

needed someone to talk to.

I would also like to thank the UST RCNS for providing the necessary equipments and

place to conduct my thesis. To the resident researchers, Dr. Donnie Ramos for lending me the

dialysis tubing, Dr. Gina Dedeles for generously providing the bacteria that I used, Roldan de

Guia for looking after me whenever I work alone in the lab. To kuya Bennetth, kuya Paul, ate

Sittie for their support and for looking after me in the Microbiology Lab. I would also like to

thank Dr. Maribel Nonato for guiding me throughout this study and for giving me Pandan related

journals. Last but not the least; I would like to thank Valerie Christie Miclat for the moral

support you gave me and for helping me draw the chemical structures I needed for my write up.

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 TABLE OF CONTENTS

TITLE Page
APPROVAL SHEET
ABSTRACT i
ACKNOWLEDGEMENT ii
TABLE OF CONTENTS iii
LIST OF FIGURES v
LIST OF TABLES vi

CHAPTER I: INTRODUCTION 1
I.1 Background of the Study 1
I.2 Objectives of the Study 3
I.3 Significance of the Study 4
I.4 Scope of the Study 5

CHAPTER II: REVIEW OF RELATED LITERATURE 6

II.1 Pandanus 6
II.2 Secondary Metabolites in Plants 9
II.3 Non-specific lipid transfer proteins 12
II.4 Microbial Infection 13
II.5 Resazurin Assay 16
II.6 Matrix Metalloproteinases (MMP’s) 17

CHAPTER III: METHODOLOGY 21

III.1 Materials and Equipment 21
III.1.1 Chemicals 21
III.1.2 Equipment 21
III.2 Isolation of Protein from P. dubius 22
III.3 Bradford Assay 23
III.4 Antimicrobial Assay 24
III.4.1 Preparation of the bacterial inoculums for
the Resazurin Assay 24
III.4.2 Resazurin Assay 25
III.5 Determination of Anti-gelatinolytic activity 27
III.6 Gelatin Zymography 27

CHAPTER IV: RESULTS AND DISCUSSION 30

IV.1 Protein Extraction 30
IV.2 Bradford Assay 32
IV.3 Resazurin Assay 34
IV.4 Gelatin Zymography Assay 40

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 IV.5 Evaluation of the Biological Assay Results 41

CHAPTER V: CONCLUSION AND RECOMMENDATION 42

V.1 Conclusion 42
V.2 Recommendation 42

BIBLIOGRAPHY 44
APPENDIX 47

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 LIST OF FIGURES

Figure Title Page

1 Pandanus Dubius 7

2 Known Alkaloids from Pandanus 8

3 Some Compounds Isolated from P. dubius 9

4 Structures of Nitrogen Containing

Heterocylic compounds 11

5 Structures of some known antimicrobial drugs 15

6 Reduction of Resazurin Dye to Resorufin 17

7 Cell Invasion via Proteolysis 18

8 Dialysis Set-up 23

9 SDS-PAGE Set-up 29

10 Absorbance shift of the Coomassie 33

11 Color of Resazurin(A), Resorufin(B) & Hydroresorufin(C) 35

12 Structure of Penicilin with the Beta-Lactam Ring 36

13 Results obtained from the antibiotic selection 37

14 Results of the protein extract from the Resazurin Assay 39

15 Zymogram of the supernatant and the protein extract 40

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 LIST OF TABLES

Table Title Page

1 Structural diversity of plant secondary metabolites 10

2 Some of the known MMP’s and its functions 20

3 Preparation of BSA Standards for Bradford Assay 24

4 Summary of Resazurin Assay with the necessary

reagents and volumes needed 26

5 Preparation of Stacking Gel 28

6 Preparation of Resolving Gel 28

7 Protein extract data in Bradford Assay 34

8 Absorbance of BSA Standards 48

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