Effects of added flavor on extraction and antioxidant behaviors of Catechins from green tea leaves

62 Pages·2016·1.52 MB·English

by  Yue Zhou

EFFECTS OF ADDED FLAVOR ON EXTRACTION AND ANTIOXIDANT BEHAVIORS OF
CATECHINS FROM GREEN TEA LEAVES
A Thesis
Presented to the
Faculty of
California State Polytechnic University, Pomona
In Partial Fulfillment
Of the Requirements for the Degree
Master of Science
In
Chemistry
By
Yue Zhou
2016
SIGNATURE PAGE
THESIS: EFFECTS OF ADDED FLAVOR ON EXTRACTION AND
ANTIOXIDANT BEHAVIORS OF CATECHINS FROM
GREEN TEA LEAVES
AUTHOR: Yue Zhou
DATE SUBMITTED: Winter 2016
Chemistry and Biochemistry Department
Dr. Yan Liu
Thesis Committee Chair
Chemistry and Biochemistry
Dr. Gregory A. Barding, Jr.
Chemistry and Biochemistry
Dr. Olive Y. Li
College of Agriculture
ii
ACKNOWLEDGMENTS
Foremost, I would like to express my sincere gratitude to my advisor, Dr. Yan Liu,
for his continuous support throughout this study. His guidance helped me go through
the research project and the thesis writing.
I am also thankful to the rest of my thesis committee: Dr. Gregory Barding and Dr.
Olive Li, for their time, encouragement and insightful comments.
Last, I would like to thank my family: my parents, my husband and my daughters
for supporting me spiritually throughout writing this thesis and my life in general.
This project was supported by the Chemistry and Biochemistry Department at
California State Polytechnic University. Moreover, I greatly appreciate the Goldstein
Graduate Research Fellowship Program at the Chemistry and Biochemistry Department
for the additional support.

iii
ABSTRACT
Catechins, a group of phenolic compounds present in green tea leaves, can
eliminate damaging free radicals inside organisms and are the main contributors to
green tea’s antioxidation benefits. Different flavors are often added to the tea brewing
process to increase the taste of tea drinks. In this project, green tea extracts with salty
and sweet flavor were analyzed by a reversed-phase HPLC method using a C18 column
and a mixture of methanol/water (30:70) as the mobile phase with the
spectrophotometric detection wavelength at 280 nm. Three major catechins,
Epigallocatechin, Epigallocatechin gallate, and Epicatechin, were observed in the green
tea extract. The introduction of salt to the brewing process greatly increased the
extraction efficiency for all three catechins, while the introduction of sugar affected the
extraction differently on individual catechins. The results from the Fenton assay with
spectrophotometric detection demonstrated the effect of salt on antioxidation power of
catechins from green tea leaves. When employing the Fenton Assay to determine the
effect of sugar on antioxidation power of catechins in GTE, both catechins and sugar had
the potentials to contribute to the radical scavenging through different routes. More
studies are still desired to elucidate the reaction mechanisms.

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TABLE OF CONTENTS

Signature Page .............................................................................................................. ii

Acknowledgements ....................................................................................................... iii

Abstract ......................................................................................................................... iv

List of Tables ............................................................................................................... vii

List of Figures .............................................................................................................. viii

Chapter 1: Introduction .............................................................................................. 1

1.1 Structures of Catechins Present in Tea ..................................................... 1
1.2 Antioxidation Behaviors of Catechins ....................................................... 4
1.3 Applications of Tea Catechins ................................................................... 7
1.4 Extraction of Catechins from Green Tea ................................................... 8
1.5 Separation Analysis ................................................................................... 12
1.6 Determination of Catechin Antioxidation Activity .................................... 15
1.7 Objectives of Project ................................................................................. 20

Chapter 2: Materials and Methodology ..................................................................... 21

2.1 Materials ................................................................................................... 21
2.2 Preparation of the Fenton Reagents ........................................................ 21
2.3 Preparation of Standard Solutions of Catechins ....................................... 22
2.4 Preparation of Green Tea Extract ............................................................. 22
2.5 Optimization of H O Volume ................................................................... 22
2 2
2.6 Spectrophotometric Analysis of GTE and Fenton Reagent Mixture ......... 23
2.7 HPLC Analysis ............................................................................................ 25

Chapter 3: Results and Discussion .............................................................................. 26

3.1 HPLC Analysis of GTE ................................................................................. 26
3.2 The Effect of NaCl on Extracting Catechins ............................................... 27
3.3 Antioxidation Behavior ............................................................................. 32
3.4 The effect of NaCl on Antioxidant Activity of Catechins ........................... 34
v
3.5 The Effect of Sucrose on Extraction .......................................................... 38
3.6 The Effect of Sugar on Antioxidant Activity of Catechins ......................... 41

Chapter 4: Summary ................................................................................................... 44

Chapter 5: Future work ............................................................................................... 46

References .................................................................................................................. 47

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LIST OF TABLES

Table 1 Concentration of Standard Solution of Catechins .................................... 22

Table 2 Solution recipes for the mixture of GTE and the Fenton Reagents .......... 23

Table 3 Solution recipes for the mixture of GTE with NaCl and the Fenton
reagents ................................................................................................... 24

Table 4 Solution recipes for the mixture of GTE with sugar and the Fenton
Reagents ................................................................................................... 24

Table 5 Quantification of the three catechins from GTE ......................................... 26

Table 6 Signals of catechins in GTE with added NaCl ........................................... 29

Table 7 Increase in catechin contents with Introduction of NaCl ......................... 29

Table 8 The antioxidant activity of GTE with NaCl ............................................... 37

Table 9 Signals of catechins in GTE with sugar ...................................................... 39

Table 10 Changes in catechin contents in GTE with sugar ...................................... 40

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LIST OF FIGURES

Figure 1 General molecular structure of catechins .................................................. 2

Figure 2 Molecular structures of common free catechins ....................................... 2

Figure 3 Molecular structures of common esterified catechins ............................... 3

Figure 4 Potential chelating structure between catechin and metal ions ............... 5

Figure 5 Free radical scavenging reaction ................................................................ 6

Figure 6 Schematics of the on-line HPLC-DPPH assay .............................................. 18

Figure 7 The principle of ORAC assay ....................................................................... 19

Figure 8 Representative HPLC chromatogram of GTE .............................................. 27

Figure 9 HPLC chromatogram of GTE (a) without and (b) with NaCl ....................... 28

Figure 10 The effect of NaCl on extraction of total three catechins from tea leaves
.................................................................................................................... 30

Figure 11 Structure of pectin and pigment of theaflavin in green tea ....................... 31

Figure 12 Torsion of B, D-rings of EGCG ..................................................................... 32

Figure 13 Effects of H O on the absorbance of the mixture of salicylic acid and
2 2
Fenton reagents ........................................................................................ 33

Figure 14 HPLC chromatogram of (a) the Fenton reagent with salicylic acid, (b) GTE
with the Fenton reagents and salicylic acid, and (c) GTE ........................... 34

Figure 15 Spectra of the mixture of … salicylic acid, GTE with salt, and the Fenton
reagents, — salicylic acid, and the Fenton reagents, and --- salicylic acid,
GTE, and the Fenton reagent .................................................................... 35

Figure 16 Spectra of the Fenton reagent … with NaCl , — without NaCl ................. 36
viii
Figure 17 Effects of added salt on the antioxidant activity of GTE ........................... 38

Figure 18 Representative HPLC chromatogram of (a) pure GTE and (b) GTE with
Sugar ......................................................................................................... 39

Figure 19 Spectra of the mixture of … salicylic acid, GTE with sugar, and the Fenton
reagents, — salicylic acid and the Fenton reagents, and --- salicylic acid,
GTE, and the Fenton reagents .................................................................. 41

Figure 20 The effects of sugar on antioxidant activity of catechins ........................... 42

Figure 21 Spectra of the Fenton reagents with — salicylic acid, and --- with
salicylic acid and sugar .............................................................................. 43
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Chapter 1: Introduction
Green tea is a brewed drink from the dried leaves of Camellia sinensis and is one of
the most commonly consumed beverages around the world.1 The first use of tea leaves
dated back to more than 3,000 years ago.2 Recent studies found that green tea drinks
have the potential to increase fat metabolism,3 improve physical performance4 and brain
function.5 In addition, green tea can help patients to lose weight and lower their risk of
various diseases, such as influenza,6 HIV,7 obesity6b, 8 and Parkinson’s and Alzheimer’s
diseases.9 Such health benefits of green tea mainly come from a group of phenolic
compounds that reduce the formation of free radicals in the body, protecting cells and
molecules from damage.10
1.1 Structures of Catechins Present in Tea
The most abundant phenolic compounds found in green tea beverage (% wt/wt
solids) are catechins,6c, 11 whose general molecular structure is demonstrated as Figure 1.
Two hydroxyl groups are attached to the aromatic ring A, while another two are
attached to the ring C. Different functional groups, R and R , can be seen on the ring C
1 2
and ring B respectively. The numerical values around the molecules stand for the
positions for its nomenclature. Based on the different functional groups on the two
aromatic rings, catechins can be categorized into free catechins (with only hydroxyl
functional groups) and esterified catechins (with ester structure). Illustrated in Figure 2,
(+)-catechin, (+)-gallocatechin (GC), (-)-epicatechin (EC), (-)-epigallocatechin (EGC) are
1