synthesis of ferulic acid derivatives using peroxidase

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SYNTHESIS OF FERULIC ACID DERIVATIVES USING PEROXIDASE ENZYME

By Raissa Melina

14212010

BACHELOR’S DEGREE in

FOOD TECHNOLOGY

FACULTY OF LIFE SCIENCES AND TECHNOLOGY

SWISS GERMAN UNIVERSITY EduTown BSD City

Tangerang 15339 Indonesia

August 2016

Revision after the Thesis Defense on 28^th July 2016

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Raissa Melina STATEMENT BY THE AUTHOR

I hereby declare that this submission is my own work and to the best of my knowledge, it contains no material previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any other degree or diploma at any educational institution, except where due acknowledgement is made in the thesis.

Raissa Melina

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Student Date

Approved by:

Hery Sutanto, M.Si.

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Thesis Advisor Date

Yulia Anita, M.Sc.

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Thesis Co-Advisor Date

Dr. Dipl.-Ing. Samuel P. Kusumocahyo

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Dean Date

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SYNTHESIS OF FERULIC ACID DERIVATIVES Page 3 of 58 USING PEROXIDASE ENZYME

Raissa Melina ABSTRACT

SYNTHESIS OF FERULIC ACID DERIVATIVES USING PEROXIDASE ENZYME

By

Raissa Melina, Students Hery Sutanto, M.Si, Advisor Yulia Anita, M.Sc, Co-Advisor

SWISS GERMAN UNIVERSITY

Ferulic acid is widely known as antioxidant due to the hydroxyl group in its phenolic structure. The synthesis reaction was done in order to increase the antioxidant activity.

It was expected to synthesize a new compound that contains more hydroxyl group.

Initially, ferulic acid was esterified to obtain maximum result of synthesis product.

Esterification product was tested using GCMS. The synthesis reaction was then assisted by peroxidase enzyme as the catalyst. Synthesis of ferulic acid derivatives using peroxidase enzyme is still limited. Peroxidase enzyme catalyzed the oxidation process by breaking down hydrogen peroxide and covert the substrate into a radical molecule. The synthesis product was suspected as tetraethyl ferulate. The LCMS spectrum supports the facts related to tetraethyl ferulate formation. Tetraethyl ferulate was found to be strong antioxidant due to its structure. The antioxidant of ferulic acid, ethyl ferulate and tetraethyl ferulate was tested against DPPH. The antioxidant activity of tetraethyl ferulate in this experiment was the highest compared to the others. In the next research, the synthesis product is recommended to be purified and analyzed further about the structure using FTIR and NMR

Keywords: synthesis, Ferulic acid, ferulic acid derivatives, tetraethyl ferulate, antioxidant activity.

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Raissa Melina

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Raissa Melina DEDICATION

I dedicate this thesis to the God Almighty, my family, friends and everyone who always support me no matter what happens and also for my advisor and co-advisor for

their endless kindness and help.

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Raissa Melina ACKNOWLEDGEMENTS

First of all, I would like to thank God for His blessings and assistance so that this thesis can be finished on time. I also feel incredibly grateful to have Hery Sutanto, M.Si to be my advisor. He always gives advice, guidance, help, assistance, inspiration, knowledge and support to make this thesis happens. I also want to thank Yulia Anita, M.Sc as my co-advisor for her knowledge, support and encouragement despite her busy schedule. I show my gratitude also for Sylvia Yusri and Tabligh Permana for their help, patience and practical assistance while working in laboratory and Agung Margiyanto, S. Pd for his non academic assistance.

My special thanks go to Levina Chandra, Andhika Nur Ramadhan, and Saineris Surya for their cooperation and support through thick and thin in our “Dimerization Team”. I am also grateful to Rachel Amanda, Michelle, Eveline, Mega Chandra, Leah Sibuyo, Felycia, Gabriela Mawi, Suwito, Gabriella Masaki, Boby Hartanto, Blandina, Riswandi Wansyah, Lucas Cendikiawan, Hamdarian Wijaya for giving me advice and endless support through bad and good times. I also want to thank to all my friends from the faculty of Life Sciences and Technology, especially Food Technology 2012.

Last but not least, I owe so much to my parents and my brother for their understanding, moral support and financial supports so that this thesis can be successfully done.

To those who read my thesis, I hope this thesis can improve any knowledge related to the subject.

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Raissa Melina TABLE OF CONTENT

STATEMENT BY THE AUTHOR ... 2

ABSTRACT ... 3

DEDICATION ... 5

ACKNOWLEDGEMENTS ... 6

TABLE OF CONTENT ... 7

LIST OF FIGURES ... 9

LIST OF TABLES ... 11

CHAPTER 1 - INTRODUCTION ... 12

1.1. Background ... 12

1.2. Research Problems ... 13

1.3. Research Objectives ... 14

1.4. Significance of Study ... 14

1.5. Research Questions ... 14

1.6. Hypothesis ... 14

CHAPTER 2 - LITERATURE REVIEW ... 15

2.1. Ferulic acid ... 15

2.2. Peroxidase Enzyme ... 16

2.3. Esterification of Ferulic Acid ... 18

2.4 Synthesis Process by Oxidative Coupling ... 18

2.5 Ultraviolet Visible Spectrophotometer ... 19

2.6 Thin Layer Chromatography ... 20

2.7 Gas Chromatography Mass Spectrometer ... 21

2.8 Liquid Chromatography Mass Spectrometer ... 22

2.9 DPPH Antioxidant Assay ... 22

CHAPTER 3 – RESEARCH METHODS ... 24

3.1. Venue and time ... 24

3.2. Materials and Equipments ... 24

3.3. Experimental Procedure ... 25

3.4. Observation ... 26

3.5. Analytical Procedure ... 26

3.5.1 Esterification of ferulic acid ... 26

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Raissa Melina

3.5.3 GCMS Analysis ... 26

3.5.4 Synthesis Reaction ... 26

3.5.5 Thin Layer Chromatography ... 27

3.5.6 Gas Chromatography Mass Spectrophotometer ... 27

3.5.7 Liquid Chromatography Mass Spectrophotometer ... 27

3.5.8 DPPH Antioxidant Assay ... 27

CHAPTER 4 – RESULTS AND DISCUSSIONS ... 29

4.1 Esterification of Ferulic Acid ... 29

4.2 Result Analysis ... 30

4.2.2 Gas Chromatography Mass Spectrometer (GCMS) ... 32

4.3 Synthesis of Ferulic acid derivatives ... 35

4.4 Result Analysis ... 39

4.4.2 Liquid Chromatography Mass Spectrometer ... 40

4.5 DPPH Antioxidant Assay ... 44

CHAPTER 5 – CONCLUSIONS AND RECOMMENDATIONS ... 48

5.1 Conclusions ... 48

5.2 Recommendations ... 48

APPENDICES ... 49

REFERENCES ... 53

CURRICULUM VITAE ... 58