Effects of Added Flavor on Extraction and Antioxidant Behaviors of Catechins from Green Tea Leaves

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

Chemistry

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.

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 H2O2 Volume ...... 22 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

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

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

vii

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 H2O2 on the absorbance of the mixture of salicylic acid and 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

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, R1 and R2, can be seen on the ring C 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 the free catechins. (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG),

(+)-gallocatechin gallate (GCG) and (-)-catechin gallate (CG) are esterified catechins12 which are illustrated as Figure 3.

Figure 1 General molecular structure of catechins

(+)-Catechin (+)-gallocatechin(GC)

(-)-epicatechin(EC) (-)-epigallocatechin(EGC)

Figure 2 Molecular structures of common free catechins

2 Figure 3 Molecular structures of common esterified catechins

Among all catechins, EGCG, ECG, EGC and EC were reported as the most abundant ones present in green tea. Interestingly, the esterified catechins (gallate) contribute astringency to the tea drink with a bitter taste while free catechins have a slight sweet flavor in the taste.6c, 7b The type of solvent, brewing time, and brewing temperature have

3 strong effects on the extraction of catechins from tea leaves.13

1.2 Antioxidation Behaviors of Catechins

Catechins are well known for their antioxidation properties and capabilities to prevent free radical damaging.11a, 14 In nature, free radicals damage DNA, proteins and lipids inside the cell, causing lipid peroxidation, and ultimately leading to apoptotic cell death.11a Free radicals go through the following cycle inside the cell:

RH → R ∙ (initiation) (1)

R ∙ + O2 → RO2 ∙ (addition of O2) (2)

RO2 ∙ + RH → ROOH + R ∙ (3)

Reaction (1) initiates the formation of radicals, with the assistance of external factors, such as UV light and high temperature. The radicals formed in reaction (1) combine with

O2 and form more reactive peroxyl radical (shown as reaction (2)) and reaction (3) regenerates R· and forms peroxide once the peroxyl radical attracts an H from RH group.15 Catechin molecules are able to intervene the chain reaction mentioned above by reacting with free radicals at a faster rate than the original reactant with the radicals.

The hydroxyl groups on the aromatic rings in the structure of catechins, simplified as

ArOH, have the capability to scavenge reactive oxygen species (the radicals), such as superoxide radicals, singlet oxygen, hydroxyl radicals illustrated as reaction (4). Once the produced ArO· from reaction (4) combines with another radical, the chain reactions terminates, which is illustrated as reaction (5).14a, 16

RO2 ∙ +ArOH → ROOH + ArO ∙ (4)

ArO ∙ +X ∙→ nonradical material (5)

Despite many different types of catechins are available, there are only two antioxidation mechanisms: free radical inhibiting and scavenging. In free radical inhibiting, catechins chelate the transition metal ions such as copper (II) and iron (III) that commonly bind to the oxidase and form inactive complexes, which prevent the catalyst (in this case, the metal ions) from initiating the formation of free radicals.8a, 17The possible chelating sites are shown in Figure 4, and the complex contains a stable

5-member ring structure with ions. It has been reported that the metal chelating properties of flavonoids like catechins may play an important role in metal-overload diseases such as Wilson’s disease (copper overload) and hemochromatosis (iron overload). 11a, 18

Figure 4 Potential chelating structure between catechin and metal ion

In radical scavenging, catechins react with harmful free oxidative species, form

5 non-radical products, and prevent cell, DNA or protein damages. The general reaction is illustrated as Figure 5.12 Catechins’ free radical scavenging abilities directly relate to their chemical compound structure with ester at the 3 position of the C ring, the catechol group (3,4-dihydroxyl groups) on the B ring and/or the hydroxyl groups at the 5 and 7 positions on the A ring (see Figure 1).19 The antioxidation activity of four catechins present in green tea increases in the following order: EC < ECG < EGC < EGCG.19b

Figure 5 Free radical scavenging reaction

Additionally, catechins can increase the body’s endogenous antioxidants to reduce oxidative damage.20 Clinical evidences showed that green tea treatment increased plasma antioxidant activity which subsequently decreased oxidative damage. Rats given green tea extract orally exhibited increased levels of endogenous antioxidants such as glutathione peroxidase and reductase, superoxide dismutase and catalase.21

Furthermore, catechins can protect other antioxidants that cross cell membranes such as vitamin C, vitamin E and glutathione, which further increase antioxidation power and

6 boost the whole cell antioxidation capability.22

1.3 Applications of Tea Catechins

As discussed earlier, green tea is rich in catechins and other antioxidants. The health benefits of catechins have gained researchers’ attention into the numerous applications of tea catechins in many different areas.23

1.3.1 In Food Industry

In food industry, lipid oxidation is a major concern since it causes the development of undesirable rancidity and the formation of potentially toxic byproducts. 24 In general, meats and meat products usually have high content of lipids. Catechins have been proved to have a high potency for the prevention of lipid peroxidation inside food products.19a, 25 Application of tea catechins (300 mg/kg) in the minced muscle of fresh red meat (beef and pork) and poultry (chicken, duck, and ostrich) significantly reduced lipid oxidation for all meats during refrigerated storage.26 The addition of green tea catechins, especially EGCG, prevented oxidative deterioration of meat and effectively inhibited lipoxygenase, peroxidase, proteinase, and bacterial growth in fish and fish products.23a For the plant-based food products, the green tea catechins are applied to extend their shelf life and often adopted as antioxidant supplements.27 For example, it was demonstrated that green catechins improved the oxidative stability of the fried noodles.27 In addition, catechins significantly decreased the retro-gradation of rice starch and could be potentially utilized to enhance the quality of food products.23c

1.3.2 In Clinic Trials

The phenols in green tea like catechins have the potentials to inhibit tumor cell proliferation and induce apoptosis.28 As a result, tea catechins are often applied in the clinical cancer studies. The existing evidences have suggested that green tea catechins modulate breast cell carcinogenesis.28d, 28e Many clinical trials also suggested that green tea be protective against prostate cancer.28b In addition, it has been reported that small intestinal tumorigenesis was inhibited in a dose-dependent manner by oral administration of EGCG, which resulted in increased levels of E-cadherin and decreased levels of nuclear β-catenin, phosphorylated Akt, and phosphorylated ERK1/2 in small intestinal tumors in Apc (min/+) mice.29 Moreover, the cell growth assay indicated that tea catechins significantly inhibit cell proliferation and the flow cytometric analysis revealed green tea catechins mediate the inhibition of proliferation of lung cancer cells.30

1.3.3 In Cosmetic Industry

Free radicals are the major source for skin and tissue damage and they are often initiated by environmental factors such as long exposure to UV light. Once initiated, the number of free radicals in the skin increases, which in turn damages DNA. Catechins from green tea are popular ingredients used in cosmetics as antioxidants to fight against free radicals.31 Tea catechins have the potential to inhibit the breakdown of collagen and elastin22c and significantly affect the permeation profiles of skin,32 therefore they have been utilized in different products, including lotions, shampoos, conditioners, toners and cleansers.

1.4 Extraction of Catechins from Green Tea

Green tea is a very complicated system whose ingredients include but not limit to caffeine, phenolic compounds, amino acids, enzymes, pigments, carbohydrates, methylxanthines, minerals, and many volatile flavors and aromatic compounds.33 To make an antioxidant (catechins) enriched drink from tea leaves, a special brewing condition is desired. Catechins in green tea leaves have been extracted through cold and hot water extraction, organic solvent extraction, microwave-assisted extraction, and other extraction techniques.13, 34 Each method has its own advantages and disadvantages.

1.4.1 Water Extraction

Green tea leaves have been brewed in water using a wide range of temperatures, cold or hot.34a Cold water extraction refers to the tea brewing conducted within water at the temperatures ranging from 4 to 25 ℃, while hot water extraction refers to the brewing process above 70 ℃.13 Studies on the effect of extraction conditions on Assam green tea concluded that a great amount of polyphenols could be extracted from fresh green tea at 95 ℃ with a tea/water ratio of 1:20 (w/v) at pH 4.13 However, the research also found that the influence of temperature on catechin extraction efficiency was not significant despite five different temperatures (100, 90, 80, 70, 60℃) were used, and the concentration of EGCG, EC and ECG from tea leaves reached their maximum at 90 ℃ brewing.34b In addition, a combination of cold and hot extraction has also been applied.34a In this combination, the tea leaves were first brewed with water at a low

9 temperature from 0 to 50 ℃. Following the resulted tea solution removal, the leaves were then re-extracted with water at a higher temperature from 50 to 100 ℃. It showed that the extraction of EGCE is more time/temperature dependent than that of EGC, and the extraction separation of EGC and EGCG can thus be controlled and monitored.

Moreover, the combination method improved the stability of the extracts and reduced the denaturation of the tea catechins.34a

1.4.2 Organic Solvent Extraction

Organic solvent extraction is also a conventional method to extract catechins from green tea leaves.35 The common solvents used include ethanol, methanol, acetone and ethyl acetate, which have different polarities.35 The increase in the water content of organic solvent resulted in a decrease of catechin content in the tea extract, whereas the highest catechin contents were obtained when using a single-component alcohol solvent.

Unlike methanol and ethanol, pure acetone was proved to be an insufficient solvent for catechin extraction.35 Although organic solvents tend to be more effective than water for the extraction of catechins, they are not used in the tea leaf brewing process to make tea drinks. Instead, it is only used in the industry to produce commercial catechins.

1.4.3 Microwave-assisted Extraction

Microwave-assisted extraction (MAE) is a process that uses microwave energy to facilitate solvents to extract compounds from various matrices. MAE allows organic compounds to be extracted more rapidly and sometimes more efficiently than the conventional extraction methods.34c, 36 The main advantage of MAE methods is the

10 reduced extraction time and solvent consumption because the temperature and pressure control allows the heating to occur in a targeted and selective manner with practically no heat being lost to the environment.23c Some studies showed that MAE is very efficient for extracting major tea components, especially the catechins. It has been reported that MAE at 80°C gave the highest concentration of EGCG in the extract while still preserving antioxidant activity of the extract. However, the flavor of green tea prepared by MAE may not be favorable to drinkers due to the bitter taste given by extra amount of EGCG.34c

1.4.4 Supercritical Carbon Dioxide Extraction

Supercritical fluid extraction gives the quantitative recovery of analytes without any loss or degradation of the analytes, and has been adopted as the alternative form for

37 compound separations. Carbon dioxide (CO2) is commonly used as a supercritical fluid in that its low critical temperature 31℃, and critical pressure, 72.9 atm are relatively easy to achieve and maintain. Additionally, CO2 is non-toxic, non-flammable and

37 inexpensive to produce. Although CO2 is a good solvent for nonpolar organics, it is not a suitable solvent for extraction of polar compounds such as phenols/flavonoids. To improve the extraction efficiency of polar solutes, a polar organic modifier can be added

38 to CO2 during the extraction or prior to the extraction. Conventional extraction methods always lead to the co-extraction of caffeine and catechins from tealeaves, but supercritical carbon dioxide extraction (SC-CO2) can avoid that situation. It has been reported that SC-CO2 effectively removed caffeine from green tea in the presence of a

39 polar component in the solvent. Although the SC-CO2 is a potential method, the extraction conditions, such as temperature, pressure and the type of solvent, need to be further investigated to optimize the extraction of catechins from tea leaves. Again, this setup is mainly for industrial production of catechins from green tea and it is not utilized in tea drink production.40

1.5 Separation Analysis

Even with a very selective extraction method, the results tea drinks are still considered a complicated system. To minimize the interferences from other ingredients, a separation analysis of catechins is necessary.

1.5.1 High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is an advanced separation technique commonly used in analytical chemistry. 37 The components in a sample mixture are separated by the different retentions inside the column, resulting in different apparent migration rates. HPLC offers good separation resolution among the components and is well suitable for the separation analysis of catechins in green tea.

Umegaki et al. employed a sensitive HPLC-electrochemical method to analyze tea catechins in human plasma.41 In this method, EC, ECG, EGC, EGCG, and ethyl gallate peaks were well resolved with high sensitivity on the chromatogram. Yasuda et al. also utilized HPLC to study the effects of metal ions towards antioxidation activities of catechins.42 In addition, HPLC could be applied to analyze the catechins and catechin

12 gallates in biological fluids.43 Different detection wavelengths were reported for HPLC-UV analysis of catechins in green tea. EI-Shahawi et al. developed a low cost reversed phase

HPLC-UV method for simultaneous determination of catechins and other flavanolic contents in green tea with the detection wavelength set at 205 nm.44 He et al. reported a wavelength of 280 nm for the determination of five catechins in Chinese green tea extract.45 In most cases, HPLC separation of catechins was made using reversed-phase

C18 column with different mobile phase. Bi et al. reported that five catechins were separated in 17 minutes using the mobile phase that consisted of acetonitrile/water

(12/88, v/v) with 1.5mmol/L beta-cyclodextrin at a flow rate of 1.0 mL/min on a conventional C18 column.46 To improve the separation resolution, Lee et.al employed two mobile phases for gradient HPLC elution: (A) 5% (v/v) acetonitrile/water containing

0.035% (v/v) trifluoroacetic acid and (B) 50% (v/v) acetonitrile/water containing 0.025%

(v/v) trifluoroacetic acid. The gradient elution profile started with A/B ratio at 90:10, and then B was gradually increased to 20% at 10 min, and then to 40% at 30 min. Beside the peaks of EC, EGC, ECG and EGCG were observed, other ingredients from tea tealeaves such caffeine, adenine, theophylline, quercetin, gallic acid and caffeic acid were well resolved in 25 minutes.

1.5.2 High Speed Counter Current Chromatography

High speed counter current chromatography (HSCCC) is a chromatographic separation technique that is based on partitioning between two immiscible liquid phases: solutes are separated precisely according to their partition coefficients between the

13 liquid mobile phase and the liquid stationary phase.47 Kumar et al. reported that HSCCC could provide a convenient method for tea catechin analysis/isolation. It is possible to obtain a high yield of pure EGC, ECG, and EGCG from a single high-speed counter-current chromatography run.48 However, separation analysis of catechins performed by HSCCC was tedious, usually over a period of hours, rather than a few minutes typical for HPLC.

Therefore, it is mainly used for the preparative purpose.49

1.5.3 Gel Filtration Chromatography

Gel filtration chromatography is a powerful separation technique that is applied to water-soluble samples that contain relatively high molecular-mass components. 37 In gel filtration chromatography, the stationary phase consists of a network with a well-defined range of pore sizes into which solute and solvent molecules can diffuse. Separations of solute molecules are based on the size of the analyte molecules with the small molecules penetrating through the porous network.37 Tan et al. reported catechins extraction from tea by gel filtration chromatography utilizing the Sephadex LH-20 or agarose gel. Eight catechins were separated and isolated, along with small amount of caffeine and theobromine, and the EGCG yield was more than 60%.50

1.5.4 Electrokinetic Separation

Electrokinetic separations are based on the different migration rates of analytes under the influence of an electric field in submillimeter diameter capillaries and/or in micro- and nanofluidic channels. Compared to HPLC, electrokinetic separation offers several advantages such as higher separation efficiency, simpler instrumentation, lower

14 consumption of reagents, and less waste generation. It is an attractive alternative means for routine analysis of catechins. There are three types of electrokinetic separations suitable for the analysis of catechins including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC) with UV detection.51 In CZE analytes are separated into different bands because of differences in their electrophoretic mobilities in the electric filed. Six different catechins were separated by CZE whose running electrolyte solution consisted of 200 mM boric acid (pH 7.7), 10 mM potassium dihydrogenphosphate, 9 mM beta-cyclodextrin and 27.5%(v/v) acetonitrile. 52 MEKC separations are based upon partitioning between micelles (pseudo-stationary phase) and surrounding aqueous buffer solution (mobile phase). Recently, Peres et al. developed and validated a sulfated-β-cyclodextrin-modified reduced flow MEKC for the determination of catechins in green tea.53 MEEKC relies on water-immiscible micromulsion droplets in nanometer-scale and forms stable pseudo-homogeneous translucent phase which has a higher solvation capacity and widens migration window. Compared to MEKC, MEEKC is applicable to a wider range of analytes and is able to provide higher separation efficiency of catechins.54 However, the surface chemistry and Joule heating associated with the electric field often affect the separation efficiency catechin analysis.55

1.6 Determination of Catechin Antioxidation Activity

As mentioned earlier, catechins are the abundant antioxidants which are present in

15 green tea. The antioxidant activity of antioxidants can be determined by many ways such as the Fenton assay, 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay and oxygen radical absorbance capacity assay. 56

1.6.1 Fenton Assay

The Fenton reaction is well known for its production of hydroxyl radicals. The reagents involved in the Fenton reaction generally consist of hydrogen peroxide and an ionic iron catalyst, which has been discovered for more than a century. However, the application of the Fenton reaction was not reported until the late 1960s.57 The reaction mechanism that involves Fenton’s reagent is still controversial. Two pathways have been proposed by different scientists.57 One pathway is that OH· is produced by

2+ one-electron reduction of H2O2 with the presence of Fe , then OH· attracts hydrogen and initiates radical chain reaction.57 The chemical reaction (6) servers as the chain initiation and the reaction (7) and (8) serve as the termination step. The cycle (8)-(9)-(10) illustrates the redox reactions between Fe (II) and Fe (III). The net effect is a formation of two different oxygen-radical species, with water (H+ + OH–) as byproducts.

2+ + 3+ Fe + H2O2 + H → Fe + HO ∙ +H2O (6)

HO ∙ +H2O2 → HO2 ∙ + H2O (7)

3+ 2+ + Fe + HO2 ∙ → Fe + O2 + H (8)

2+ 3+ − Fe + HO2 ∙ → Fe + HO2 (9)

Fe2+ + HO ∙ → Fe3+ + OH− (10)

The other pathway is called non-radical pathway, which was first proposed by Bray and

Gorin. 56a They suggested that FeO2+ was the active intermediate in the Fenton reaction and iron(II) and iron(III) were converted into each other through the following chemical equilibria.

2+ 2+ Fe + H2O2 ⇌ FeO + H2O (11)

2+ 3+ − Fe + H2O2 ⇌ Fe + HO ∙ +OH (12)

The analysis of radical scavenging utilizing the Fenton reaction is referred as the Fenton assay, which has been applied toward the catechin antioxidation activity study. When the tea extract was mixed with Fenton reagent system, with the increase of catechin concentration in the tea extract, the radical scavenging effects increased as reported in the literatures.

1.6.2 DPPH Assay

2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical method is an antioxidant assay based on the electron-transfer process that produces a violet solution in ethanol.

(13)

When DPPH radicals attract a hydrogen atom from other molecules, it gives rise to the reduced form of DPPH. As a result, the purple color of the initial reactant solution (λ =

517 nm) disappears after the reaction. The DPPH assay provides an easy and rapid way

17 to evaluate antioxidant behavior by spectrophotometry.56b

Recently, to analyze the bioactivity of catechins, on-line HPLC–DPPH assay has been developed which is shown as Figure 6. 56b The analytes were first separated through the

HPLC column, followed by the reaction with DPPH radicals in the post-column. The decrease in the DPPH absorbance was monitored at 517 nm whereas phenolic compounds in tea samples were detected at 210 nm, which means the type of antioxidants and their antioxidation power could be determined simultaneously. It was found that catechins, especially EGCG, contributed greatly to the antioxidation activity of tea extract. The loss of EGCG during tea fermentation led to the reduction of antioxidation activity in DPPH assay. Unreal low antioxidation activities, however, were measured at higher water ratios (70–90% (v/v)), because part of the DPPH radicals form aggregates and will not react with catechins.56b

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

1.6.3 ORAC Assay

Oxygen radical absorbance capacity (ORAC) assay is another method of measuring antioxidant capacities on biological samples in vitro, which was first developed by Cao et al. in 1993.56c The assay measures the oxidative degradation of the fluorescent molecule

18 after being mixed with free radical generators. Following the mixing with the fluorescent molecules, peroxyl radicals produced react with the fluorescent molecule, resulting in the loss of fluorescence. With the presence of the antioxidant, the fluorescent molecules are protected from the oxidative degradation. Figure 7 indicates the principle of ORAC.

Because the peroxyl radicals mix with only fluorescent probe and it may result in the loss of fluorescence, the blank experiment should be taken into consideration in ORAC.

Therefore the calculation of ORAC capacity is followed as

ORAC Capacity = AUCsample-AUCblank

The fluorescence decay is employed to evaluate the radical-scavenging activity of many antioxidants. The ORAC assay quickly became a standard method to measure antioxidation capability of tea catechins. Zhao et al. determined the antioxidation activities of 20 tea extract samples by the ORAC assay and concluded that the galloyl catechins contributed to the main antioxidation power of tea extract.58 Carloni et al. employed ORAC assay to compare the antioxidation activity of white, green and black tea from the same tea cultivar and reported a general trend in antioxidant activity of different tea extract, i.e. green tea ≥ low-caffeine green tea > white tea ≥ black Orthodox tea > black CTC tea.59

Fluorescent probe+ Loss of flurescence AUC antioxidant antioxidant Reactive oxygen species (ROS) Fluorescent Loss of flurescence AUC blank probe+blank

Figure 7 The principle of ORAC assay

1.7 Objectives of Project

Traditionally, water is always brought to a gentle boil before pouring into a container that has tea leaves inside to brew tea drinks. Unlike brewing tea by hot water commonly seen in Eastern Asian countries, cold-brewing tea is very convenient since it does not require additional heating apparatus. Cold-brewing produces a lighter-bodied tea with less astringency and bitterness, as this method extracts fewer tannic compounds from tea leaves. In addition, different flavored tea has become popular among young generations. Therefore, it is very important to investigate the brewing conditions to make tea with a flavor and without the loss of its antioxidant function. This project is to focus on determining how the flavor (salt and sugar) added to the extracting/brewing process affect the extraction efficiency of catechins from green tea leaves and investigating how they affect antioxidation functionality of catechins.

20 Chapter 2: Materials and Methodology

2.1 Materials

All chemicals were used as received without further recrystallization or purification.

The standards of free and esterified catechins, including (-)-epicallocatechin (EGC),

(-)-epicatechin (EC) and (-)-epigallocatecatechin gallate (EGCG), were obtained from

Sigma-Aldrich (St. Louis, MO, USA). Salicylic acid, iron(II) chloride tetrahydrate (98%), hydrogen peroxide (3% w/w) , ethanol (95%) and methanol (HPLC grade) were purchased from VMR International, LLC (Randor, PA, USA). Sodium chloride (NaCl) was obtained from Fish Scientific (Fair Lawn, NJ, USA). The cane sugar from Domino Foods,

Inc. (Yonkers, NY, USA) and Bigelow, Inc. green tea (USDA organic grade) (Palmdale, CA,

USA) were purchased from the Target○R local market.

2.2 Preparation of the Fenton Reagent

A Fenton reagent consists of FeCl2, salicylic acid (SA) and H2O2. The standard stock solution of each was prepared separately. The solutions of FeCl2 (0.1 mmol/mL) and was prepared by dissolving 0.0995 g of FeCl2·4H2O in 5.00mL of DI water. The solution of SA

(1.0 mmol/mL) was prepared by dissolving 0.690 g of SA in ethanol (95% v/v). The solution of H2O2 was prepared by diluting the original 3% H2O2 solution with DI water to

0.3% (w/w). All solutions mentioned in this paragraph were prepared fresh daily before the reaction took place.

2.3 Preparation of Standard Solutions of Catechins

The concentrations of the stock solution of catechins prepared are listed in the Table

1. The standard mixtures of three catechins at various concentrations (0.1 to 0.5 mg/mL) were prepared by diluting the stocks with DI water accordingly.

Table 1 Concentration of Standard Solution of Catechins

Standard samples of catechins Concentration (mg/mL)

EGCG 5.0

EGC 5.0

EC 0.67

2.4 Preparation of Green Tea Extract

A Bigelow, Inc. tea bag was soaked in 100 mL of DI water with constant stirring for

15 minutes under room temperature, which resulted in a green tea extraction solution

(GTE). GTE with NaCl and sugar additives were made in a similar way. A desired amount of NaCl or cane sugar was dissolved in 100 mL DI water first. The tea bag was then soaked in the solution of NaCl or cane sugar for 15 minutes with continuous stirring. All the GTE were prepared at room temperature and filtered with a VWRTM filtration system

(0.2 μm) prior to use.

2.5 Optimization of H2O2 Volume

The Fenton reaction was used to investigate the tea catechins’ antioxidation behavior. A series of the Fenton reaction were carried out, using different concentrations of H2O2, to optimize the volume of H2O2 needed. The recipe for preparing such a series of solution is listed as Table 2. All the mixture solutions were diluted to the mark of 5.00 mL volumetric flask by DI water. Then the absorbance of the mixture solutions was monitored by the Varian UV spectrophotometer from Agilent Technologies (Santa Clara,

CA, US), at the wavelength from 800 nm to 400 nm.

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

Number GTE FeCl2 SA H2O2 (mL) (mL) (mL) (mL) 1 1.000 0.100 0.100 0.070

2 1.000 0.100 0.100 0.080

3 1.000 0.100 0.100 0.090

4 1.000 0.100 0.100 0.100

5 1.000 0.100 0.100 0.110

6 1.000 0.100 0.100 0.120

7 1.000 0.100 0.100 0.130

2.6 Spectrophotometric Analysis of GTE and Fenton Reagent Mixture

Antioxidation behavior of GTE was evaluated by a method based on the Fenton assay while using catechins in the GTE as the free radical terminators. 1.000 mL of GTE prepared in Section 2.4 was mixed with the Fenton reagents (with the optimal H2O2 concentration found in Section 2.5). When NaCl and sugar were added to the GTE, their

23 amount varied as Table 3 and 4. All the mixture solutions were diluted to the mark of

5.00mL volumetric flask by DI water. The mixture solutions were then scanned by the UV spectrophotometer from 800nm to 400nm.

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

NO GTE FeCl2/(0.1mmol/mL) SA/(1.0mmol/mL) H2O2/0.3%(w/w) NaCl (mL) (mL) (mL) (mL) (g/100mL) 1 1.0 0.1 0.1 0.1 0.0

2 1.0 0.1 0.1 0.1 0.1

3 1.0 0.1 0.1 0.1 0.2

4 1.0 0.1 0.1 0.1 0.3

5 1.0 0.1 0.1 0.1 0.4

6 1.0 0.1 0.1 0.1 0.5

7 1.0 0.1 0.1 0.1 0.6

8 1.0 0.1 0.1 0.1 0.7

Table 4 Solution recipes for the mixture of GTE with sugar and the Fenton reagents

NO GTE FeCl2 SA H2O2 Sugar (mL) (mL) (mL) (mL) (g/100mL) 1 1.0 0.1 0.1 0.1 0.0

2 1.0 0.1 0.1 0.1 1.0

3 1.0 0.1 0.1 0.1 2.0

4 1.0 0.1 0.1 0.1 3.0

5 1.0 0.1 0.1 0.1 4.0

6 1.0 0.1 0.1 0.1 5.0

7 1.0 0.1 0.1 0.1 6.0

8 1.0 0.1 0.1 0.1 7.0

2.7 HPLC Analysis

Profiling of GTE was carried out by a reversed phase HPLC method. The HPLC system

(Agilent-1100, Santa Clara, CA) equipped with a StarRiseTM C-18 column with 4.6 mm ×

100 mm, a G1312A bin pump, and a G1315 A UV detector. The mobile phases were composed of (A) methanol and (B) water with the ratio of A/B at 70:30 (v/v), if not described otherwise in this project. The flow rate used was 1.0 ml/min and the absorbance of the eluate was monitored at 280 nm.

Chapter 3: Results and Discussion

3.1 HPLC Analysis of GTE

GTE samples were analyzed via the developed HPLC method to determine the amount of catechins in the samples, and a representative chromatogram of GTE is shown as Figure 8. Three catechins, EGC, EGCG and EC, were observed on the chromatogram, and the identity of catechin peaks were confirmed by comparing the retention time of each peak with the retention time of the corresponding standard catechin. The retention times and peak areas of all observed catechins are summarized in Table 5. In addition, the peak at 3.86 min was identified as caffeine, another major component in green tea. The concentrations of catechins in GTE can be determined by the single point external standard methods, and the equations for calculation are showing as followed:

푃푒푎푘 푎푟푒푎 Concentration of analyte = 푅푒푠푝표푛푠푒 푓푎푐푡표푟

푃푒푎푘 푎푟푒푎 Response factor = 퐶표푛푐푒푛푡푟푎푡푖표푛 표푓푠푡푎푛푑푎푟푑 푠푎푚푝푙푒

Table 5 Quantification of three catechins in GTE Compound Retention time Peak Area Concentration (min) (mAU*s) (mg/mL) EGC 2.78 556.71 1.41

EGCG 4.24 1904.11 0.85

EC 5.36 536.60 0.09

Figure 8 Representative HPLC chromatogram of GTE

Overall, EGC is the most abundant catechin observed in the tested samples.

3.2 The Effect of NaCl on Extracting Catechins

Salty tea is the most common beverage in Kashmir of India with a long history.60 In modern days people in western countries found that a light sprinkling of salt could elevate the taste of tea drinks. Therefore, addition of salt (NaCl) to the brewing water was adopted to investigate its effects on the catechin extraction from green tea leaves.

Because humans can only tolerate a concentration of salt at the level of less than 0.3%

(w/v), the range of concentration of NaCl to be investigated was set from 0 to 0.7% (w/v) in this project.

27 One representative chromatogram of GTE with an addition of NaCl is illustrated as

Figure 9. Comparing with the chromatogram shown in Figure 9, the addition of NaCl didn’t effect on the retention times of catechins at all. However, the signal of each catechin increased. (The signal for caffeine did not change when a small amount of NaCl was added). The numerical values of all peak areas of catechins at each NaCl condition are summarized as Table 6. Overall, the amount of every catechin was increased to certain degrees when a small amount of NaCl was added. Among the three catechins investigated, EGCG reached its highest amount (peak area) in the extract sample when the concentration of added NaCl was 0.7 g/100mL. EGC reached its highest amount in the extract sample when the concentration of NaCl was 0.5 g/100mL while EC reached its highest amount in the extract when the concentration of NaCl was 0.4 g/100mL.

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

28 Table 6 Signals of catechins in GTE with added NaCl

EGC EGCG EC

NaCl Retention Area Retention Area Retention Area (g/100mL) time (min) (mAU*s) time (min) (mAU*s) time (min) (mAU*s) 0.0 2.78 556.72 4.57 1904.11 5.73 536.60

0.1 2.73 611.94 4.48 1910.71 5.63 512.70

0.2 2.71 665.18 4.45 2390.77 5.59 617.28

0.3 2.72 663.34 4.47 2594.34 5.62 645.50

0.4 2.72 700.20 4.47 2738.89 5.63 698.81

0.5 2.73 712.73 4.49 2736.19 5.66 628.63

0.6 2.74 637.94 4.51 2610.40 5.68 634.13

0.7 2.72 667.87 4.49 2816.54 5.65 600.11

It is more straightforward when the increase of peak area is described in terms of percentile (the data are summarized as Table 8). EGC sees a maximum increase of 28%,

EC sees an increase of about 30%, and EGCG sees an approximate 48% increase.

Table 7 Increase in catechin contents with introduction of NaCl

NaCl (g/100mL) EGC EGCG EC

0.1 9.92% 0.35% -4.45%

0.2 19.48% 25.56% 15.04%

0.3 19.15% 36.25% 20.29%

0.4 25.77% 43.84% 30.23%

0.5 28.02% 43.70% 17.15%

0.6 14.59% 37.09% 18.18%

0.7 19.97% 47.92% 11.84%

The total amount of catechins in GTE samples was determined by summing the peak areas from individual catechin in GTE samples. When the total peak area from each catechin is plotted as the function of the amount of NaCl being added, shown as Figure

10, the total amount of catechins increased gradually with the increased amount of NaCl and reached a plateau when the amount of NaCl added was 0.4 g/100mL. Although the optimal amount of added NaCl is beyond the tolerance level of human being, the introduction of small amount of NaCl to water does improve the extraction efficiency of catechins from green tea leaves. 0.3 g/100mL NaCl would be a good option to improve the extraction efficiency of catechins from tealeaves.

4500

4000

3500

3000

2500

2000 Peak Area Peak 1500

1000 Total

500

0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 NaCl (g)

Figure 10 The effect of NaCl on the extraction of total three catechins from tealeaves

Zhou et al. reported that the salting-out method could help extract high purity

30 catechins, especially for catechin gallates, from green tea.61 In other words, the introduction of salt could improve the separation resolution between catechin, caffeine and pigment. Because of the similarity in their structures (ring structure and multiple hydroxyl groups), some catechins “bind” with the pigments like theaflavin, pectin (Figure

11) and other polysaccharides due to the electrostatic interaction between their hydroxyl groups and the formation of inter-molecular H-bond. After adding NaCl, dehydration occurs for polysaccharides; therefore, these polysaccharides become sediment and break the “linkage” with catechins. Therefore, more catechins, especially catechin gallates such as EGCG and ECG, are extracted through water.

Figure 11 Structure of pectin and theaflavin in green tea

On the other hand, the solvent effect may impact the solubility of catechins in water.

The introduction of NaCl increases the ionic strength of the solvent, which could bring the torsion of B and D rings to the structure of catechins33 (Figure 12). As a result it may cause the B and D rings rotate through the C-C single bond and C-O single bond with

31 specific angles. The rotation of B-ring and D-ring may increase the solubility of catechins as well. Compared with catechin gallates, the free catechins such as EC and EGC have less hydroxyl groups. As a result, the effect from the only torsion of B-rings on the solubility is expected to be weaker that that from both B- and D-rings. Therefore, when

NaCl is added, the degree of the increased extraction of EC and EGC are less than that of

EGCG.

Figure 12 Torsion of B, D-rings of EGCG

3.3 Antioxidation Behavior

The antioxidation behavior of tea catechins was investigated employing the Fenton reaction. For the Fenton reactions (Reaction (14) and (15)), radicals are formed.

2+ + 3+ Fe + H2O2 + H → Fe + HO ∙ +H2O (14)

3+ 2+ + Fe + H2O2 → Fe + HOO ∙ +H (15)

When colorless salicylic acid reacts with the free radicals produced by the Fenton reagent, a dark purple color of 2, 3-dihydroxybenzonic acid that has a strong absorbance

32 at 520 nm is formed, which is demonstrated as Reaction (16).56a

(16)

Prior to the investigation of tea catechin antioxidation behavior, the amount of hydrogen peroxide in the Fenton reagent was studied to ensure a complete reaction between hydrogen peroxide and salicylic acid. After the volume of H2O2 reached 100

L/5 mL solution, the value of absorbance of the mixed solution reached a plateau as shown in Figure 13. It is simply the indication that the H2O2 in the solution consumes all other reactants and becomes in an excess amount. Therefore, 0.1 mmol/mLFeCl2, 0.3%

(w/w) H2O2 and 1.0 mmol/mL salicylic acid were chosen to study the effects of GTE on the reaction mentioned above.

0.800

0.700

0.600

0.500

0.400

0.300

Absorbance 0.200

0.100

0.000 70 80 90 100 110 120 130

H2O2 μL/5mL

Figure 13 Effects of H2O2 on the absorbance of the mixture of salicylic acid and the Fenton reagents 3.4 The Effect of NaCl on Antioxidant Activity of Catechins

When the HPLC analysis of the solution consisted of GTE, salicylic acid, and the

Fenton reagents was performed, the overlap between the produced

2,3-dihydroxybenzonic acid and EGC was observed (shown as Figure 15), which makes it difficult to quantify the changes in the catechin content in GTE samples. (Please note that the data in Figure 14 were collected from XDB C-18 column, and the mobile phase was Methanol/Water = 20%/80%, with the flow rate as 0.8mL/min and the UV detection at 280 nm.) Therefore, a UV-vis spectrophotometric analysis of the reaction between the

Fenton reagent, salicylic acid, and GTE was employed to study the antioxidation behavior of tea catechins.

Figure 14 HPLC chromatogram of (a) the Fenton reagent with salicylic acid, (b) GTE with

34 the Fenton reagent and salicylic acid, (c) GTE Figure 15 indicates the spectrophotometric analysis results for the reaction of salicylic acid with the Fenton reagent, GTE, and GTE with NaCl. All solutions have the strongest absorbance at 520 nm, due to the presence of 2, 3-dihydroxybenzonic acid.

0.7

0.6

0.5

0.4

Absorbance 0.3

0.2

GTE with NaCl(0.4g/100mL) and Fenton reagent 0.1 Pure Fenton reagent GTE and Fenton reagent 400 500 600 700 800 Wavelength/nm

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 reagents

Smaller magnitude of the absorbance at 520 nm means less 2, 3-dihydroxybenzonic acid being formed in the reaction. In other word, more free radicals produced by H2O2 have been scavenged in the mixture. As Figure 15 shown, the magnitude of the absorbance from the mixture of salicylic acid, GTE with salt and the Fenton reagents was the smallest and the mixture of salicylic acid and the pure Fenton reagent was the largest one, which means that due to the introduction of NaCl, GTE gains stronger antioxidation power. This result further confirms that the addition of NaCl improves the extraction

35 efficiency of catechins from tea leaves.

Additionally, to demonstrate the decreased absorbance of mixture is due to the antioxidation behavior of catechins in GTE, the pure Fenton reagent and mixture of

Fenton reagent and NaCl were also analyzed by UV spectrophotometry, respectively. As shown in Figure 16, the absorption spectra of these two solutions are almost identical, indicating that NaCl itself has no effects on the Fenton reaction and less absorbance of mixture of GTE with NaCl and the Fenton reagent is indeed the result of radical scavenging from catechins in GTE.

Figure 16 Spectra of the Fenton reagent ---with NaCl and ___ without NaCl

The antioxidant activity of GTE was calculated by the equation as followed:

36 A0 − AS AA = × 100% A0 where A0is the absorbance of the blank sample and As is the absorbance of the sample after scavenging the free radical.

As shown in Table 8, the increase in antioxidant activity correlated with increased salt concentration. Figure 17 indicated that the antioxidation power reached to a plateau when the amount of NaCl reached 0.4 g/100mL. This result agrees with the result on

Table 6 that the amount of extract of catechins from GTE increased slowly when the addition of NaCl greater than 0.4 g/100 mL. The paired Student’s test verified the difference between 0 and 0.400 g NaCl/100 mL was significant and the difference between 0.300 and 0.400 g NaCl/100 mL was not significant.

Table 8 The antioxidant activity of GTE with NaCl

Concentration of NaCl (g/100mL)

Absorbance 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700

Trial 1 0.239 0.327 0.336 0.370 0.375 0.387 0.410 0.377

Trial 2 0.247 0.274 0.292 0.316 0.323 0.313 0.334 0.314

Trial 3 0.227 0.287 0.253 0.283 0.297 0.319 0.311 0.320

Average 0.238 0.296 0.294 0.323 0.332 0.340 0.352 0.337

Standard 0.010 0.028 0.042 0.044 0.040 0.041 0.052 0.035 deviation

0.450

0.400

0.350

0.300 activity 0.250

0.200

0.150 Antioxdiant

0.100 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

NaCl (g/100mL)

Figure 17 Effects of added NaCl on the antioxidant activity of GTE

3.5 The Effect of Sucrose on Extraction

Catechins have been found to be a major contributor to the astringency and bitterness of green tea.62 Increased catechin concentration enhances the antioxidation power, but it also results in a decrease in taste palatability.63 The taste intensity of gallated catechins is much stronger when compared with that of non-gallated catechins.64 The introduction of table sugar (sucrose) can decrease the astringency of teach drinks and is commonly adopted to sweeten tea drinks.

Compared with the results from pure GTE HPLC analysis, the signal intensity of each catechin in the sample of GTE with sugar changed to different degrees with some

38 increasing while others decreasing, demonstrated as Figure 18.

(a) (b) Figure 18 Representative HPLC chromatogram of (a) pure GTE and (b) GTE with sugar

Table 9 indicated that with 1.0 g table sugar being added in GTE, the peak area of EGC has a biggest jump (from 575 to 861).

Table 9 Signals of Catechins in GTE with sugar

EGC EGCG EC

Sugar(g) Retention Area Retention Area Retention Area time(min) time(min) time(min) 0 2.65 575.80 4.24 2412.41 5.36 617.82

1.0 2.66 861.98 4.29 2230.12 5.42 590.39

2.0 2.68 598.76 4.32 2373.72 5.45 625.08

3.0 2.68 592.67 4.33 2493.11 5.47 623.27

4.0 2.68 581.49 4.35 2498.94 5.49 608.11

5.0 2.70 672.92 4.39 2778.25 5.53 669.43

6.0 2.70 609.92 4.38 2512.60 5.52 602.39

7.0 2.68 558.27 4.36 2330.71 5.49 560.09

39 When the amount of sugar increased continuously, a minimal increase in terms of EGC signal was observed except when 5.0 g of table sugar was added; and for other two catechins, the peak areas decreased first with the increased amount of sugar until the amount of sugar added was 5.0 g.

The data are also presented in terms of percentile changes in Table 10. Negative values in the signal increase indicated a decrease in the extraction efficiency for all three catechins when 7.0 g of sugar was added. When the concentration of sugar is low, the effects on the extraction efficiency of EGC, EGCG, and EC were different.

Table 10 Changes in catechin contents in GTE with Sugar

Sugar (g) EGC EGCG EC

1.0 49.70% -7.56% -4.44%

2.0 3.99% -1.60% 1.18%

3.0 2.93% 3.35% 0.88%

4.0 0.99% 3.59% -1.57%

5.0 16.87% 15.16% 8.35%

6.0 5.93% 4.15% -2.50%

7.0 -3.04% -3.39% -9.34%

Some increases were observed, while some decreases were observed even for the same amount of sugar being added. For example, when the amount of sugar added was 4.0 g, the extraction efficiency for EGC almost stayed same, the extraction efficiency for EGCG increased 3.59%, while the extraction efficiency for EC decreased by 1.57%. Overall,

40 different effects of sugar on catechin extraction from GTE were observed for different catechins.

3.6 The Effect of Sugar on Antioxidant Activity of Catechins

Although the spectrophotometric analysis shows that the maximum absorbance wavelength of the mixture solution of GTE, salicylic acid, and the Fenton reagents didn’t shift significantly with the presence of sugar (Figure 19), the results in terms of antioxidation activity of GTE with sugar are controversial. Figure 19 (a) and (b) were obtained under the same experimental conditions, but adding sugar to the brewing process had totally opposite effects. Figure 19(a) indicates that the absorbance of GTE with sugar was higher than GTE only while Figure 19(b) indicates a lower absorbance of

GTE with sugar.

0.7

0.6

0.5

0.4

0.3 Absrobance

0.2

Pure Fenton reagent 0.1 GTE with sugar （ 5.0g/100mL) + Fenton reagent GTE + Fenton reagent

400 500 600 700 800 Wavelength

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

Despite of the contradiction of the data one trend has been confirmed that the

41 absorbance of the solution mixture with salicylic acid and the Fenton reagents decreased when mixed with GTE and sugar. When the antioxidation activity was calcualted, there is no significant difference between the pure GTE and the GTE with sugar, demonstrated as Figure 20.

0.350

0.300

0.250 activity 0.200

0.150

Antioxidant 0.100 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Sugar (g/100mL)

Figure 20 The effects of sugar on antioxidant activity of catechins

When sugar was introduced directly into the Fenton reaction, a stronger absorbance was observed (see Figure 21), indicating the possible formation of radicals from the reaction that involved sugar. Therefore, the mechnism of sugar effects on the antioxidation behavior of catechins from gree tea leaves become very complicated. When employing the Fenton Assay to determine the effect of sugar on antioxidation power of catechins in GTE, there are both possilibities for catechins and sugar to affect the absorbance of mixture of GTE with sugar and Fenton reagent even through different routes. Further studies are required to elucidate the reaction mechanism.

0.7

0.6

0.5

0.4

0.3 Absorbance

0.2 Pure reagent 0.1 Fenton reagent+Sugar (5.0g/100mL)

400 500 600 700 800 Wavelength

Figure 21 Spectra of the Fenton reagents with ___ salicylic acid, and --- with salicylic acid and sugar

43 Chapter 4: Summary

Three catechins, EGC, EGCG and EC, along with other species were observed in cold water-brewing green tea via HPLC method. The developed HPLC employed a mixed solution of methanol and water as the mobile phase and a C-18 column as the stationary phase. The presence of NaCl and table sugar in water when preparing green tea drinks influenced the extraction efficiency and antioxidant activity of catechins differently. The introduction of NaCl increases the extraction efficiency of catechins, especially catechin gallates like ECG and EGCG. The NaCl promotes the precipitation of pigment and polysaccharides which bind with catechins, thus improving the solubility of catechins in water. When the concentration of NaCl was higher than 0.4 g/100 mL, the growth of extraction efficiency of total catehins was minimal. The sugar had different effects on catechin extraction from green tea leaves. The addition of 1.0 g/100mL sugar made the amounts of EGC increase much more than other two catechins. When the amount of sugar being added was 5 g/100 mL, the amount of total catechins reached their maximu values. However, higher concentration of sugar decreased the extraction of all three catechins. The introduction of 7 g/100 mL sugar even made the extraction of catechins less than that in GTE without sweetening.

Fenton assay was applied to determine the antioxidation power of GTE. Because of higher amounts of catechins were extracted with the assistance of NaCl, the antioxidation power of GTE with NaCl increased. However, the Fenton assay is not

44 convincible enough to prove that there was some effect of sugar on antioxidation power of catechins due to the fact that sugar can affect the Fenton reaction as well. More research is still desired to understand the reaction mechanisms involved.

Chapter 5: Future Work

Although the effect of salt and table sugar to the extraction and antioxidant activity of catechins from green tea has been established, the respective mechanism is not fully understood. More studies could be carried out.

(1) The structures of each product from individual reactions are not elucidated.

Therefore, a technique with structure identification function is desired to further study catechin extraction and antioxidation. A Liquid chromatography-mass spectrometry

(LC-MS) unit would be helpful to study the reaction mechanism and structures of product.37

(2) Milk tea is commonly consumed across the globe, and how to maximize the effective extraction of catechins in milk tea would be an interesting topic to continue on this study. Milk based products represent a very complex matrix whereas strong catechin–protein interactions may directly interfere with accurate catechin determination. Therefore, employing an inexpensive solid phase extraction method prior to HPLC analysis would be significant to increase the effective of extraction of catechins from milk tea.

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