1

Investigating the Degradation Rate of Epicatechin Compounds in Green Tea During

Long Term Storage

By

Shane Medin

A Research Paper

Submitted in Partial Fulfillment of the

Requirements for a

Master of Science Degree

In

Food and Nutritional Sciences

Approved: 6 Semester Credits

Dr. Martin G. Ondrus, PhD. Committee Chair

Dr. Cy thia Rohrer, PhD. Committee member

Dr. Thomas Lacksonen, PhD. Committee member

The Graduate School

University of Wisconsin-Stout

December, 20th 2010 2

The Graduate School

University of Wisconsin-Stout

Menomonie, WI

Author: Medin, Shane

Title: Investigating the Degradation Rate of Epictechin

Compounds il1 Green Tea During Long Term Storage.

Graduate Degree/ Major: MS Food and Nutritional Sciences

Research Adviser: Dr. Martin G. Ondrus, Ph.D.

Month/Year: December 15th 2010

Number of Pages: 79

Style Manual Used: American Psychological Association, 6th edition

Abstract

Green tea is a beverage that contributes to good health. Consumers gain nutritional benefits from the antioxidant propeliies associated with the chemical composition of a green tea infusion. Although, once prepared the quality of tea may deteriorate during storage as the infusion's chemical composition is altered. This research investigated the degradation of some polyphenol catechin compounds in Lipton® green tea with and without added fmit juice. The analytes of interest ine1uded gallic acid, caffeine and some green tea catechins: epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate. Lipton® green tea samples were prepared in triplicate according to package directions, stored for 6 days at 25°C in a water bath and shaken uncovered at 60 revolutions per minute. The analytes were measured every 24 hours 3 using reverse-phase high perfonnance liquid chromatography (RP-HPLC) with gradient elution and a photodiode array detector.

During storage epicatechin gallate and epigallocatechin gallate appeared to be enzymatically hydrolyzed releasing gallic acid, epicatechin and epigallocatechin. Aerobic and enzymatic oxidation may also have deteriorated the quality of the tea samples. Noted infusion color change, microorganism or fungal growth and infusion pH appeared to correlate with chemical degradation. Once prepared, the chemical composition of green tea infusions were altered during storage reducing predominant polyphenol antioxidant compounds that may decrease the nutritional benefits obtained from consumption. 4

The Graduate School

University of Wisconsin Stout

Menomonie, WI

Acknowledgments

Many individuals provided invaluable contributions to this paper and the research in which it reflects. First and for most, I need to thank my mother and father for constant support, enthusiasm and encouragement. You are truly tremendous individuals.

Dr. Martin G. Ondrus, thank you for dedicating your time and energy providing continuous guidance, insight and suppol1 through my graduate research. You have contributed immensely to this research and my thesis. Thanks for being an incredible teacher, eo-worker and advisor!

Dr. Cynthia Rohrer, thank you for contributing your time and energy to this research and my thesis. You have constantly provided me with suppOJ1 and detailed research advice editing my thesis. Thanks for being such a great teacher and advisor!

Dr. Thomas Lacksonen, thank you for your willingness to work with me on my research and providing essential skills needed to write a complete thesis. 1 appreciate your time invested as a committee member.

I really appreciate all of your contributions to my research. More than anything the time and tremendous amount of eff0l1 you have dedicated to my education. Thank you all very very much! 5

Table of Contents

...... Page

ABSTRACT ...... 2

List of Tables ...... 7

List of Figures ...... 8

Chapter 1: Introduction ...... 10

Problem Statement ...... 13

Research Objectives ...... 13

Assumptions of the Study ...... 14

Definition of Terms ...... 15

Chapter II: Literature Review ...... 18

History of Tea ...... 18

Tea Cultivation ...... 20

Tea Harvesting ...... 21

Tea Classifications and Processing ...... 21

Health Benefits Associated with Green Tea Consumption ...... 24

Bioavailability ...... 25

Oxidative Stress ...... 27

Obesity ...... 28

Heali Health ...... 39

Cancer ...... 30

Chemieal Composition of Green Tea Leaves ...... 30

Chemical Composition of Prepared Green Tea Infusions ...... 34 6

Factors Influencing the Chemical Composition of Green Tea Infusions ...... 41

Green Tea Leaf Enzymatic Activity ...... 42

Green Tea Leaf Microbial Activity ...... 44

Methods of Analysis ...... 44

Chapter III: Methodology ...... 46

Materials ...... 46

Tea Sample Preparation and Storage ...... 47

Sampling ...... 48

Standards ...... 48

Instrulnentation ...... 59

Data Analysis ...... 51

Chapter IV: Results and Discussion ...... 53

The Study ...... 53

Sample pH ...... 54

Lipton Green Tea Samples ...... 56

Lipton Green Tea Samples (5-mL Lemon Juice) ...... 60

Lipton Green Tea Samples (5-mL Cranbeny Juice) ...... 64

Discussion ...... 68

Chapter V: Conclusion ...... 71

Recommendations ...... 72

R.eferences ...... 73 7

List of Tables

Table I: Compounds oflnterest in this Research ...... 11

Table 2: Tea Classifications and Processing Techniques ...... 23

Table 3: Reduction Potentials and Antioxidant Activities of EC, EGC, ECG, EGCG

and Theaflavin ...... 25

Table 4: Bioavailibility Baseline and Peak Concentrations of EC, EGC, ECG and

EGCG in Human Plasma, Urine and Fecal Matter ...... 26

Table 5: Approxiamte Chemical Composition of Dry Green Tea Leaves ...... 33

Table 6: Approxiamte Chemical Composition of a Freshly Prepared Green Tea

Infusion ...... 36

Table 7: Standard Analyte Solution Concentrations ...... 49

Table 8: Solvent Programmer Settings for Mobile Phase Elution GradienL...... 51

Table 9: Average pH of Lipton® Green Tea Samples With and Without Added Fruit

Juice ...... 55

Table 10: Lipton® Green Tea Average Analyte Concentration Calculations ...... 58

Table 11: Lipton® Green Tea (5-mL Lemon Juice) Average Analyte Concentration

Calculations ...... 62

Table 12: Lipton® Green Tea (5-mL Cranberry Juice) Average Analyte Concentration

Calculations ...... 66 8

List of Figures

Figure 1: General Chemical Structure of a Flavan-3-01 ...... 13

Figure 2: General Chemical Structure of Green Tea Catechin (GTC) Compounds .... 20

Figure 3: Chemical Structure of Perdominant Catechin Compounds in Green Tea ..... 34

Figure 4: Tanase Hydrolyses ECG and EGCG to from GA, EC and EGC ...... 37

Figrue 5: Enzymatic Catechin Oxidaiton and the Formation of Catechin Quinones .. 37

Figure 6: Theaflavin Fonnation from Catechin Quinone Reacting with Gallocatechin

Quinone ...... 38

Figure 7: Theaflavic Acids Arise from Gallic Acid Quinone Reacting with Epicatechin

Quinone or Epicatechin Gallate Quinone ...... 39

Figure 8: Theaflavin Gallates Form from Catechin Quinone or Catechin Gallate

Quinone Reacting with Gallocatechin Quinone or Gallocatechin Gallate

Quinone ...... 39

Figure 9: Formation of Bisflavanols from Gallocatechin Quinones Reacting with

Gallocatechin Quinones ...... 40

Figure 10: Bulk Tea Sample Preparation and Triplicate Sample Separation ...... 47

Figure 11: Solvent Programmer Elution Curves ...... 50

Figure 12: Standard Graph Analyte Peak Area at 270 nm Plotted as a Function of

Analyte Concentration ...... 52

Figure 13: Lipton® Green Tea Triplicate Sample Average pH Readings During 144-

Hour Storage ...... 54

Figure 14: Plain Lipton® Green Tea Average Analyte Concentrations (mglL) Graph 57

Figure 15: Plain Lipton® Green Tea Sample Chromatogram (Time = 0 hours) ...... 59 9

Figure 16: Plain Lipton® Green Tea Sample Cln'omatogram (Time = 120 hours) ...... 59

Figure 17: Plain Lipton® Green Tea Sample Chromatogram (Time = 144 hours) ...... 59

Figure 18: Lipton® Green Tea (5 mL lemon Juice) Average Analyte Concentration

(mg/L) ...... 61

Figure 19: Lipton® Green Tea (5 mL Lemon Juice) Chromatogram (Time = 0 hours)

...... 63

Figure 20: Lipton® Green Tea (5 mL Lemon Juice) Chromatogram (Time 72 hours)

...... 63

Figure 21: Lipton® Green Tea (5 mL Lemon Juice) Chromatogram (Time 144

hours) ...... 63

Figure 22: Lipton® Green Tea (5 mL Cranberry Juice) Analyte Concentration (mg/L)

...... 65

Figure 23: Lipton® Green Tea (5 mL Cranberry Jnice) Chromatogram (Time 0

hours) ...... 67

Figure 24: Lipton® Green Tea (5 mL Cranberry Juice) Cln'omatogram (Time = 72

hours) ...... 67

Figure 25: Lipton® Green Tea (5 mL Cranberry Juice) Chromatogram (Time = 144

hours) ...... 67 10

Chapter I: Introduction

Tea is one of the most commonly consumed beverages on ea11h; it is cultivated in

over 30 countries and consumed worldwide. The origins of tea date back nearly 5000

years to southeastem Asia near the llorthem border of Myanmar (Caballero et aI., 2003).

Traditional Chinese medicine employed tea as a medicinal agent to treat various illnesses

(Cooper et a1. , 2005). The majority of harvested tea (Camellia sinensis) leaves are processed and classified as 78% black tea, 20% green tea and 2% oolong tea (Henning et

aI., 2003).

During leaf processing aerobic and enzymatic t1avonoid oxidation results in the unique properties associated with each tea classification. Some t1avan-3-01 green tea catechin (GTC) compounds including catechin (C), epicatechin (EC), epigallocatechin

(EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG) undergo

chemical oxidation and hydrolysis reactions in aerobic environments. Table I illustrates

the chemical structure of the GTCs of interest in this study along with gallic acid, caffeine, and ascorbic acid. During processing black tea leaves are oxidized the longest,

while oolong tealeaves are p311ially oxidized and green tea leaves are minimally oxidized. Oxidation and hydrolysis reactions initiated by enzymes during processing convert some GTCs and gallic acid to by-products including theat1avins, theaflavic acids, theaflavin gallates, bist1avanols and thearubigins. These chemical by-products created during processing account for red, orange and brown pigments in black and oolong teas, and also contribute to the aroma, color, t1avor and nutritional composition of the infusions (Francis, 2000; Caballero et a1., 2003; Graham, 1992). 11

Table 1

Compounds ofInterest in this Research

Compound Chemical Formula Structural Configuration

Gallic Acid (GA) C7H60S

o 0 Ascorbic Acid ~~ C6Hs 06 HO

HO OH

Hj:, N Caffeine (CAF) CSHlON 402 O~"Jet'" I ') 1 CH,

GTCs

OH

OOH EC C1s H140 6 ooyo:1 # OH OH

OH OH EGC ClsH1407 ~:#OOa

OH

OH &OHI"" HO~",\\# ECG Cn H1sOlO ""," OH

OH ")-Q-OH

OH

OH (x0HI"" tlO ""\ # OH EGCG C22 H1S0 11 WI OH : \ "'"OH ""'0-Q-C''\OH !J ~ OH 12

In green tea manufacturing, it is essential to minimize oxidation by thermally heat-treating leaves to inactivate enzymes. In prepared green tea infusions, chemical oxidation and hydrolysis by-products may produce undesirable characteristics. During storage in aerobic environments, the quality of infused green tea may deteriorate as complex chemical reactions alter the nutritional content, flavor, aroma and appearance by darkening the infusions (Maga, Mikhael, Skobeleva, 1980).

The chemical composition of some GTCs has been associated with anticarcinogenic, antimutagenic and cardioprotective health properties (Barbosa, 2007)

Studies have shown that GTCs decreased risk of developing gastritis, obesity, diabetes, coronaIY heart disease, stroke and some f01ms of cancers such as colorectal, esophagus, lung, stomach, and prostrate (Barbosa, 2007; Cooper, 2005). Green tea has been shown to provide antioxidant health benefits to consumers.

In 2008-2009 the Lipton® tea company sold over 400 million dollars of tea products in the U. S alone (Beverage Industry, 2009). According to NHANES and the

USDA, tea is the number one contributor of flavonoid compounds to the average

American diet, providing on average 157 mg/day. GTC flavan-3-ol compounds account for 83.5% of total tea flavonoids (Song & Chun, 2008). However, it has been postulated that some GTCs in prepared green tea infusions may degrade during storage. It has also been hypothesized that treating green tea infusions with fruit juice, which contains ascorbic acid may reduce the rate of some GTC degradation. Figure 1 shows the general chemical structure offlavan-3-ol catechins, illustrating the 2-phenyl-3, 4-dihydro-2H­ chromen-3-ol chemical structure. This general flavan-3-ol structure shows the common 13 two benzene ring (A and B) configuration linked with a 3 carbon chain noted as 2, 3, and

4 on the (C ring).

3'

8 2'@r 0 2","'" ~6' 5' 70 C 3 6 OH 5 4

Figure 1. General chemical stmctme of a flavan-3-01.

Problem Statement

Gallic acid (GA), Caffeine (CAF) and epicatechins: epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC) and epigallocatechin gallate (EGCG) were investigated in Lipton@ green tea during long-tenn l44-hom (6-day) storage. Due to the large concentrations of antioxidants in infused green tea, specifically the epicatechins, it was hypothesized that these compounds may oxidize and diminish in aerobic storage environments. The green tea samples were analyzed using RP-HPLC. The HPLC instmmentation provided simultaneous analytical detection and quantification of GA,

CAF and epicatechins: EC, ECG, EGC and EGCG.

Research Objectives

The purpose of this study was to quantitatively measure the concentrations of specific analytes including: GA, CAF and epicatechins: EC, ECG, EGC and EGCG in prepared Lipton® green tea samples during long-term l44-hom (6-day) storage. The investigation also involved monitoring the preservative effects of added fruit juice on the chemical composition of green tea samples dming storage. This investigation was 14 designed to mimic typical preparation and storage teclmiques where individuals may prepare varieties of bulk tea and store them under room temperature conditions for several days. Changes in the chemical composition of green tea during storage may jeopardize nutritional benefits obtained from the consumption of the infusion. This study may provide valuable infonnation regarding variations in the chemical composition of green tea during long-tenn aerobic storage. Specifically the objectives were to:

1. Quantify concentrations of GA, CAF and several epicatechins: EC, ECG, EGC

and EGCG in prepared Lipton® green tea during long-tenn 144-hour (6-days)

storage.

2. Quantify concentrations of GA, CAF and epicatechins: EC, ECG, EGC and

EGCG in prepared Lipton® green tea with added fruit juice (5-mL lemon or 5-

mL cranbeny) during long-tcnn 144-hour (6-days) storage.

3. Compare analyte concentrations in each samplc during storage every 24 hours.

4. Monitor tea sample physical characteristics and chemical properties during

storage including: evaporative loss, infusions pH, visual color change and visual

appearance of microbial or fungal growth.

Assumptions of the Investigation

A measurable loss of catechins compounds was expected during storage at temperatures near room temperature. Catcchins of interest in this research are refened to as green tea catechin (GTC) compounds and include epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate. 15

Definition of Terms

The following definitions are referenced from Credo Reference (2010).

Polyphenol: Alcohol compounds with two or more benzene rings with at least one hydroxyl group (OH) attached. These natural antioxidant phytochemicals reduce free radical activity.

Flavonoids: A large category of natural plant products derived from y-pyrone.

Flavan-3-ols: A subclass of flavonoids.

Catechins: Flavonoid phytochemical compounds found primarily in green tea.

Catechin Gallate: Catechin compounds with a gallic acid moiety in the 3 position on the C ring of the flavan-3-o1 stmcture. This can be observed in (Figure 1) or denoted by RJ on the GTC structure in (Figure 2).

Caffeine: A methylxanthine, alkaloid that has a stmctural configuration of 1,3,7- trimethylxanthine. The compound is known as a stimulant and a diuretic.

Enzyme: Organic catalyst produced by living organisms. Enzymes are substrate specific protein complexes that promote the formation of bonds between separate substrates or break bonds within a single substrate.

Isoenzyme: An enzyme existing in multiple fonns with chemically distinct stmctures but functionally similar mechanisms of action.

Substrate: Any chemical compound in which an enzyme reacts with.

Catalyst: A compound that increases the rate of a chemical reaction while remaining chemically unchanged.

Solution: A homogeneous mixture where a solute (molecules or atoms) is completely dissolved in a medium (solvent). 16

Solute: A chemical substance that dissolves in a medium (solvent) to form a solution.

Oxidation: A chemical reaction where the loss of an electron increases the charge of the atom or molecule.

Hydrolysis: A chemical reaction that cleaves a molecule by the addition of water.

Intermediate: An essential tempormy substance formed during a chemical reaction. The substance will react to form an end product.

Infusion: A liquid extraction obtained from of any substance that has been steeped in an aqueous medium.

Analyte: Chemical characterization and measurement associated with the chemical composition of a sample being analyzed.

Hydroxyl: A covalently bonded chemical functional group consisting of an oxygen and a hydrogen atom (Oll).

Macromolecules: A large molecule characterized by stmcturally containing smaller more simple molecules.

Phytonutrients: Are natural bioactive plant chemicals, which are not essential to the human diet but are considered to be healthy.

Acid (acidic): A chemical substance capable of donating a proton (hydrogen ion).

Base (alkaline): A chemical substances capable of accepting a proton (hydrogen ion).

High Performance Liquid Chromatography (HPLC): Originally called high­ pressure liquid chromatography, HPLC is a method of chemical separation. Complex mixtures are detected and quantified by transporting a sample fraction in a liquid 17

(mobile) phase through a solid (stationary) phase which separates the individual compounds in the sample by subtle thermodynamic polar interactions. 18

Chapter II: Literature Review

History of Tea

The tenn tea commonly refers to a beverage prepared from infusing dried, processed leaves from a flowering evergreen tree Camellia sinensis with freshly boiled water (Lipinski, 1996). A variety of extracts from the Camellia sinensis plant may be considered tea (tea e, 2004). The genus Camellia, native to China and southeastem Asia belongs to the Theaceae plant family (tea, 2006). Two botanical Camellia species are used for tea production including the northern, most popular, C. sinensis and the southem

C. assamica (Maga, Mikhail, & Skobeleva, 1980).

The origins of tea have been traced to southern China near the n011hern border of

Myanmar (Caballero et al. ,2003). Chinese legends date tea back nearly 5,000 years.

However, physical evidence accredits "Confucius" a Chinese philosopher with the initial documentation (551-479 B. C.) In his writings Confucius referenced "t'u" as having a sweet flavor. T'u was eventually established as meaning tea in Chinese writings (history,

2000).

Su Jing author of Xing Xill Ben Cao, discussed the topic of tea during the Tang

Dynasty (618-907 AD). Jing stated, "tea is non-toxic, cool in nature, bitter and sweet". In traditional Chinese medicine (TCM), tea, referred to as Cha, treated urine retention, thirst, internal heat, gastrointestinal disturbances and fatigue (Harvey, 2004).

Buddhist monks introduced tea to Japan and Korea as they historically consumed tea as a stimulant during meditation (history, 2000). In 1514 the Portuguese sailed to southeastem Asia and became the first Europeans to consume tea. This group gained the right to trade with China and eventually introducing tea to Europe. During the nineteenth 19 century European countries successfully began cultivating tea (TEA, 2000). Tea is currently grown in over 30 countries worldwide (Cooper et aI., 2005). The leading tea producing countries include China, Japan, Taiwan, India, Sri Lanka and Indonesia (Tea.,

2004).

Tea is one of the most commonly consumed beverages on earth second only to water. One study showed that over a 52-week period in 2008-2009 monitoring tea sales in mass merchandise outlets across the U.S, Lipton® dominated, selling over 171 million dollars in bagged and loose leaf teas. As for ready' to' drink products, Lipton® sold nearly

250 million dollars worth of tea beverages (Beverage, 2009).

TIll'ee common types of tea exist including black, oolong and green. Processing black and oolong tealeaves requires exposing the intracellular matter to oxygen by cmshing or bmising the leaves prior to heat-treating the leaves, which stops the oxidation process. Green tea manufacturing requires initial thelmal treatment to stop the oxidation process by inactivating oxidative enzymes to ensure the leaves retain high concentrations of natural antioxidants (Maga, 1980). Green tea offers consumers nutritional benefIts, due to specific processing methods aimed to preserve polyphenol flavan-3-01 catechins in the leaves.

Polyphenols are naturally occurring chemical compounds found in the leaf tissue of plants. Various classifications of polyphenols exist based on chemical structure. One elass, flavonoids, consists of two aromatic rings bound together by a three carbon heterocyclic ring (Barbosa, 2007). GTCs are a subclass of flavonoid secondary plant metabolites refelTed to as flavan-3-0Is. Figure 2 shows the general chemical stmcture of

GTCs and some of the hydroxyl groups, which are present on the structures. Note that Rl 20 and R2 may vary depending on the GTC. R2 may represent a hydrogen (H) or a hydroxyl

(OH). Rl may represent a (H), an (OH), or a gallic acid (GA) moiety. GTCs, which are gallates, are ECG and EGCG known as catechin gallates (Henning, Choo, & Heber,

2008)

OH

OH

HO

OH

Figure 2. General Chemical Structure of Green Tea Catechin (GTC) Compounds.

By nature oxygen accepts electrons creating highly reactive oxygen species

(ROS) known as free radical compounds. GTCs have active antioxidant properties in the f01111 of hydroxyl (OH) functional groups. GTCs are capable of reducing ROS induced oxidative stress by donating one or more hydrogen atoms from a hydroxyl group to scavenge free radicals by satisfying the unbalanced electron configuration. In vivo, in vitro and epidemiological studies have revealed promising evidence regarding the biological activities of GTCs related to human health (Higdon & Frei, 2003).

Tea Cultivation

Today, tea cultivation relies on cloning high quality parent tea plants to support mass agricultural plantations. Cultivating tea is a delicate process that requires strict growing conditions including tropical and subh'opical regions providing high rainfall, proper altitude and consistently higher temperatures to support plant growth (Tea., 2004). 21

Cultivators plUne the tea plant to induce numerous shoot fonnations while maintaining optimal heights at two to five feet tall for harvesting (Tea., 2004). Growers strategically operate plantations by coordinating ideal growing conditions/seasons with the most critical stage of plant growth. Harvesting tealeaves during specific seasonal weather patterns increases plant yield (Mmtagh, 1996).

Tea Harvesting

Traditionally, tea was harvested by hand plucking each leaf. When harvesting tea, four critical variables must be considered including target shoots, harvesting interval, leaf selectivity, and intensity of harvest. Only the youngest two leaves and the apical bud are typically harvested. These younger leaf shoots are referred to as "flushes"

(Chandramouli, Onsando, & Corley, 2007). Today, technological advances in the tea industry have developed methods of shear plucking and mechanical harvesting to increase production and reduce labor costs (Ravichandran & Parthiban, 1997).

Once harvested, tealeaves are delicately packed in specially designed plywood crates and transported to tea-collecting stations to be weighed and graded. During shipping, leaves are susceptible to foreign odors, cellular damage and premature heat induced oxidation, which can drastically reduce the quality ofthe leaves. To avoid oxidation and loss of natural antioxidants leaves must be loosely packed and the temperature in the crate must remain below 40° C (Maga et aI., 1980).

Tea Leaf Classifications and Processing

The processing oftealeaves typically results in three different classifications of tea; black, oolong and green from the same raw Camellia sinensis leaves. Distinguishing leaf characteristics separating the three classifications depend on the length which the 22

leaves are allowed to chemical oxidize during processing (tea., 2004). Hundreds of by­ products including aromatic and volatile compounds produced by chemical reactions contribute to differences in visual color/appearance, aroma, flavor, and chemical composition of each tea (Caballero et aL, 2003). During tealeaf processing leaves are withered, heated, bruised, dried, sorted and packaged (Table 2). Differences between the three tea classes are derived from variations in leaf maceration teclmiques, oxidation time and the method of heat treatment.

Green tea leaf processing requires raw leaves to be plucked, withered, and thermally heat-treated to inactivate cellular oxidative enzymes. The leaves are then fn'ed to reduce the moisture content below 6%, sorted and packaged. There are two methods of processing green tea: Chinese and Japanese (Table 2). Traditionally, the Chinese would thermally treat tealeaves by roasting or pan-flying, whereas the Japanese would steam the leaves before dlying and rolling (Maga et aL, 1980). Both the Chinese and

Japanese methods of green tea production are designed to minimize leaf oxidation, reducing by-product fonnation and allowing the leaves to retain the majority of their polyphenol flavan-3-01 catechin content.

Oolong tealeaves are allowed to partially oxidize, creating chemical by-products, which add unique color, flavor and aroma to the leaves Crable 2). Due to partial oxidation oolong tea has a light brown/reddish appearance with a slightly bitter taste. Mechanically bruising leaves releases cellular enzymes and promotes enzymatic oxidation before thermal treatment inactivates enzymes and stops oxidation (tea., 2006). The leaves are then dried to reduce moisture content, sorted and packaged. 23

Black tea production relies on extensive enzymatic oxidation. During processing,

nearly 90% of catechins compounds are oxidized providing black tea with unique characteristics associated with substantial by-product formation. When processing black tea, leaves are bruised, allowed to fully oxidize, thermally treated to inactivate cellular oxidative enzymes, dried to reduce moisture content, sorted and packaged (Table 2).

Black tea infusions have a dark brown color with a sweet smell and a full flavored body

(Maga et aI., 1980).

During processing all tealeaves are exposed to thermal treatment and maceration.

The maceration exposes the leafs cellular composition to oxygen promoting enzymatic oxidation while heat treatment inactivates enzymes stopping oxidation. The order in which these processes shown in Table 2 are carried out drastically influence the chemical and physical characteristics of the leaves. Table 2 indicates variations among leaf processing for the three main classifications of tea produced from the leaves of the

Camellia sinensis tree.

Table 2

Tea Classifications and Processing Techniques

Green Tea Roasting or Sorted and Chinese Withering ---> Pan Frying Rolled Fired Packaged Sorted and . Japanese --->Witherin~ Stearrl!1}9_ ---> Rolled ____.Ei.r.~~::~ __ E§lckag~_d_ Partially Sorted and Oxidized Fir('l~ __ ~~~F'.t:l.9~9~~ Fully Sorted and Black Tea= __ . Witb~!1rlfL_:=! Rolle(j_ ---> Oxidized ---> Fir~(j ____--->~kag~ Note. Two types of green tea production, Chinese and Japanese employ different types of heat treatment. Also, the difference between oolong and black tea is the duration leaves are oxidized. 24

Health Benefits Associated with Green Tea Consumption

Green tea is an excellent source of natural polyphenol antioxidants. Antioxidants are substances that protect cells from highly reactive oxygen species (ROSs) produced from oxygen's tendency to accept electrons. Free radical compounds cause cellular oxidative damage that may lead to disease (Antioxidant, 2009). GTCs reduce free radical activity by donating a hydrogen atom from one or more hydroxyl functional groups.

GTCs are then capable of supporting unpaired electron de1ocalization by creating a double bond with the remaining oxygen atom. Studies have shown that GTCs are capablc of scavenging several ROSs including peroxyl, alkyl peroxyl, superoxide, hydroxyl and peroxynitrite radicals reducing oxidative stress in aqueous and organic mediums

(Barbosa, 2007).

The catechin gallate compounds (ECG and EGCG) have the highest antioxidant activity due to the higher number of hydroxyl (OH) groups making them more susceptible to oxidation (see Figure 3) (Munoz-Munoz et aI., 2008). All catechin compounds undergo oxidation, but EGC and EGCG are most susceptible to free radical aerobic oxidation. EGC and EGCG have an extra hydroxyl group and catecholic hydrogen in the 5' position on the B ring (see Figure 2). By calculating bond dissociation enthalpies (BDE) one study suggests that the hydroxyl catecholic hydrogen in the 4' position on the B ring is most readily oxidized (JAOCS, 2002).

GTCs have been proven to be efficient scavengers of free radical compounds. The reactivity of an antioxidant is related to its one-electron reduction potentiaL According to trolox equivalent antioxidant activity (TEAC), a lower reduction potential indicates less energy is required for an antioxidant to stabilize free radical compounds by hydrogen or 25 electron donation (Higdon & Frei, 2003). Table 3 compares the antioxidant potential and activity of some GTCs and theaflavin. Theaflavin is a by-product of GTC oxidation. The data in Table 3 suggest that some by-products of GTC oxidation may not be as nutritionally beneficial as the natural GTCs found in green tea.

Table 3

Reduction Potentials and Antioxidant Activity ofEC, EGC, ECG, EGCG and Thea.flavin

Antioxidant Reduction••• __ PotentialM ______Antioxidant ActivitL.__ (-)-Epicatechin 0.57 2.4 (-)-Epigallocatechin 0.43 3.8 (-)-Epicatechin Gallate 0.55 4.9 (-)-Epigallocatechin Gallate 0.43 4.8 Theaflavin O. 51 2.9 Note. Both a low reduction potential and a high antioxidant activity are used to detennine the reactivity of an antioxidant.

BioavaiIability

A study investigated the bioavailability of tea polyphenols in human subjects using RP-HPLC. Plasma and urinary samples were obtained from four human subjects to analyze the presence of EC, ECG, EGC and EGCG. The detection limit for these analytes was between 0.5-1.5 ngimL. One hour after ingestion of 1.2 g of decaffeinated green tea in warm water, plasma concentrations were 48-80 nglmL EC, 82-206 ng/mL EGC and

46-268 nglmL EGCG, while ECG was not detected in plasma. The majority ofEC and

EGC were excreted within 9 hours after ingestion. In the first 24 hours L6-2.3mg of and 2.8-3.2 mg ofEGC was excreted. EGCG and EC were not detected in urine samples

(Lee et aI., 1995). Indicating that EC and ECG may not as bioavailable as EGC and

EGCG. 26

Another study, followed absorption and excretion profiles of ECG,EGC and

EGCG in human subjects consuming tea throughout the day. This investigation

administered black tea infusions four times during a 6-hour period to fifteen healthy

subjects. The peak plasma concentrations recorded EGC and EGCG at 5-hours, EC at 7-

hours and ECG at 24-hours. Peak urinary concentrations recorded ECG and EGC at

5-hours and EGCG at I-hour. Fecal excretion peak concentrations of EC, ECG, EGC and

EGCG varied from subject to subject ranging from 24-72 hours following digestion.

Table 4 shows the peak concentrations for plasma, urinary and fecal samples (Warden,

Smith, Beecher, Balantine, & Clevidence, 2001). This data indicates that ECG apparently

takes longer to digest and absorb compared to the other GTC epicatechins. Table 4 shows

peak plasma, urine and fecal concentrations of EC, EGC, ECG and EGCG in nanomoles

and micromoles.

Table 4

Bioavailability Baseline and Peak Concentrations ofEC, ECG, EGC and EGCG in

Human Plasma, Urine, and Fecal Matter (Warden et aI., 2001).

Plasma Peak Urinary Peak Analyte Baseline Plasma Baseline Urinary Baseline Peak Fecal __. __ jnmol) (nmol) (I)moIL_ _ inmol) (iJmol) (pmol) 743.0 ± EG 39.2 ± 9.5 174.0 ± 19.8 7.1 ± 4.2 87.2 0.06 ± 0.07 2.3 ± 3.2

EGG 28.7 ± 6.9 50.6 ± 13.6 18.1 ± 6.2 41.1 ± 24.4 0.07 ± 0.1 0.2 ± 0.2

EGG 72.6 ± 13.9 145.0 ± 28.8 28.7 ± 9.5 836.0 ± 4.3 0.01 ± 0.01 2.6 ± 4.3

EGGG 4.2 ± 1.2 ± 0.01 0.7 ± Note. Baseline measurements represent levels prior to the administration of the infusions.

The above studies indicate minimal amounts of some GTCs are actually

metabolized. Many in vivo, in vitro and epidemiological studies have reported health

benefits from the consumption of green tea. Some studies, acknowledge health benefits of 27 green tea and GTCs at misleading concentrations. The human digestive gastrointestinal track only absorbs and metabolizes certain amounts of theses beneficial compounds (see

Table 3). The levels ofGTCs metabolized are considerably lower than many of the levels referenced as providing nutritional benefits. Consuming tea regularly is a healthy habit, as the GTCs fi'om tea are powerful antioxidants that provide biologically active properties capable of reducing oxidative stress and decreasing risk of disease. However, some literature rep0l1s significant findings at unrealistic concentrations.

Oxidative Stress

Oxidative stress is the effect of chemical oxidation in biological systems, where

ROSs and hydroxy radicals lead to cellular damage of living tissue (Oxidative Stress,

2009). In living organism's oxygen is an imp0l1ant acceptor of electrons leading to the fOlIDation of ROSs and hydroxy radicals (Graham, 1992). Antioxidants promote human health by reducing the fOlTI1ation of free radical metabolites and scavenge radical compounds to minimize cellular damage.

Both in vitro and in vivo studies have shown some GTCs significantly reduce oxidative stress. In one study (Zhang et a1.,1997), GTCs were hypothesized to protect red blood cell (RBC) membranes from free radical-induced oxidation. The study found that some GTCs, including EC, EGC, ECG and EGCG, inhibited RBC hemolysis in rats and protected against low-density lipoprotein oxidation (Zhang, Zhu, Luk, Ho, Fung, & Chen,

1997).

A study in Poland investigated the influence of green tea as a source of water­ soluble antioxidants for the prevention of oxidative stress in aged rats intoxicated with ethanol (Luczaj, Waszkiewiez, Skrzydlewska, & Roszkwaska-lakimiec, 2004). Luczaj 28

(2004) reported that liquid diets containing 7 gIL green tea extracts effectively protected

blood serum against oxidative stress induced by aging and ethanol metabolism. Green tea

protected cells against enzymatic and nonenzymatic parameters produced by ethanol and

reduced the formation of oxygen radicals (Luczaj et aI., 2004).

Obesity

A study investigated EGCG and its ability to increase 24-hour energy expenditure

(EE) and thermogenesis in humans (Oulloo et a1., 1999). It was reported that oral

administration of 90 mg EGCG stimulated thermogenesis. GTCs inhibited catechol 0-

methyltransferase (COMT) resulting in high concentrations of norepinephrine at the

synaptic junctions. The higher concentrations of norepinephrine stimulated

thermogenesis and fat metabolism by increasing 24-hour EE by 4 %. The study suggests

synergistic interactions between EGCG, caffeine and norepinephrine (Oulloo et al.,

1999).

Mechanisms of green tea action on anti-obesity were observed in rats by

monitoring the glucose uptake system in peripheral tissues. This in vivo study provided 5-

rats a bottle of commercial green and water. After 3 weeks of investigation rats were

killed and analyzed for triacylglycerol (TG), high-density lipoprotein (HOL), free fatty

acids (FFA), low-density lipoproteins (LOL) and LOL-cholesterol (Ashida et aI., 2004).

Tea acted as a modulator ofGLUT4 and maintained blood glucose levels. The cellular

uptake of glucose in adipose tissue was significantly reduced by 20% while the

concentration increased in skeletal muscle tissue by 30% (Ashida et aI., 2004). These results suggest that glucose is being used for intracellular energy in lean muscle tissue

and not being stored in adipose tissue. 29

An article investigated GTC beverage consumption on body composition and fat distribution in overweight and obese adults (Maki et a1., 2009). A 500-mL beverage containing 625 mg GTC with 39 mg of caffeine was conswned daily over a l2-week period. The study evaluated the effects of a GTC beverage with caffeine compared to a non-GTC beverage with an equal amount of caffeine. Maki et a1. (2009) reported significant reductions in total and subcutaneous abdominal fat (P = 0.019) and serum t1iglycerides (P 0.023) among the GTC induced group. These data suggest that GTC beverage consumption enhances exercise-induced fat loss in obese and overweight individuals (Maki et aI., 2009).

Heart Health

Green tea extracts containing GTCs have demonstrated inhibitory effects by reducing atherosclerotic plaque fonnation and lipid oxidation. GTCs are likely to have synergistic affects with other polyphenol compounds. The characteristics of green tea extracts may reduce the dsk of developing cardiovascular diseases (CVDs) including: coronary heart disease, heart attack, arthrosclerosis and ischemic heart disease (Cooper et a1.,2005).

Green tea (GTC extracts) reduced LDL oxidation and improved vascular function in women. Fourteen healthy women were tested for endothelial vascular function and concentrations of oxidized LDL. After 5-weeks of green tea extract consumption a significant reduction (37.4%) in the concentration of oxidized LDL was observed with an increased brachial artery vasodilatation of nearly 6% compared to the placebo (Tinahones et a1., 2008). 30

Cancer

Green Tea and cancer prevention has been extensively studied. A recent review

Boehm et a1. (2009) critically examined over 50 studies including one interventional

(RTC) study, 23 prospective cohort studies and 27 retrospective case-control studies with more than 1.6 million participants. This review investigated studies regarding green teas effect on cancers ofthe GI tract: gastric, esophagus, pancreas, colorectal and liver; uro­ genital tract: bladder, prostate, ovarian and urinary; as well as breast, lung, oral and other various types of cancer. The investigators assessed associations between green tea consumption and cancer risks and mortality. The review concluded that the studies investigated revealed conflicting evidence and lack of consistency to provide recommendations regarding green tea consumption for cancer prevention (Boehm et aI.,

2009).

Chemical Composition of Green Tea Leaves

The chemical compositions of fresh green tea leaves are influenced by factors including: plant metabolism, genetics, age and environmental conditions. The fresh leaf contains enzymes, metabolic intelmediates and structural compounds required for plant growth and photosynthesis (Francis, 2000). Biosynthetic pathways during plant growth are elaborate and too numerous to discuss, therefore the more relevant prim31Y and secondary products will be assessed. The chemical profile of a green tea leaf varies from plant to plant and leaf-to-Ieaf, therefore the general composition of the dried tealeafprior to processing is represented in Table 5.

Once the tealeaf is plucked from the plant, chemical and physical withering begins (Maga et aI., 1980). Cellular respiration reduces the moisture content in the leaf 31 while increasing the permeability of cellular membranes. This initiates many chemical and biochemical reactions influencing leaf composition (Francis, 2000). For example, during transportation from the plantation to the processing location withering continues as enzymatic activity increases, flavonols oxidize and transform, caffeine levels raise slightly, amino acid levels increase as peptidase breaks down proteins, fatty acid esters are hydrolyzed to free fatty acids (FF As) and carotenoids begin to degrade producing various aromatic compounds (Caballero et aI., 2003).

The raw tea leaves also contain epicatechin flavan-3-ol fractions, as well as other polyphenolic compounds including catechin, gallocatechin, catechin digallates, methylated catechins, chalcan-flavans, proanthocyanidins, anthocyanidins, sugar glycosides and depsides: chlorogenic acid, p-coumarylquinic acids and theogallin. Sugars such as glucose and rhamnose are present along with some organic acids including gallic acid, quinic acid and ascorbic acid (Graham, 1992). Protein contributions mainly consist of enzymes including polyphenyl oxidase, peroxidase, glucosidases, lipoxidases, glycosidases, transaminases, peptidase, and alcohol dehydrogenase (Francis, 2000).

There are many amino acids (AA) present in the raw tealeaf.

Seventeen different AA have been identified in green tea leaves. Theanine is unique to tea and contributes half the total AA composition. Theanine is an amide of L­ glutamic acid. In its pure form, theanine is a colorless needle-shaped crystalline substance

(Maga et al., 1980). Theanme plays a very impOltant biochemical role in the tea leaf. A study revealed that dOlmant seeds contained a mere 0.17% dry weight theanine, whereas seedlings after 60 days of germination contained 2. 31 % theanine dry weight (Maga et a1., 1980). This indicates that theanine is an important biologically active amino acid. 32

TIle mineral fraction consists of potassium, calcium, magnesium, fluoride, aluminum and manganese. Over 15 carotenoids have been identified such as violaxanthine, fJ-carotene, neoxanthin and lutein, which contribute to the aroma of the leaf (Graham, 1992). Caffeine is the major stimulant but there are also other methylxanthines present in low concentrations including theobromine and theophylline.

Tealeaves also contain lipids, cellulose and lignin for cellular structure and rigidity

(Lignin, 2004). Over 60 volatiles compounds exist as alcohols, carbonyls, esters, acids and cyclic compounds, which represent <0.1 % of the total chemical composition

(Graham, 1992).

Antioxidant compounds make up a large percentage of the green tea leaves dry weight. Table 5 illustrates phenolic compounds account for 30-40 % dry weight of the tealeaf, including flavanols, which comprise 25-30 % dry weight of the tealeaf.

Epicatechins represent the majority of the phenolic flavonol compounds accounting for

] 6-28 % dry weight of dry green tea leaves including EGCG 9-13%, EGC 3-6%, ECG 3-

6%and 1-3 %. Epicatechin compounds are recognized as potent antioxidants with low reduction potentials and high antioxidant capacities shown in Table 5. 33

Table 5

Approximate Chemical Composition ofDry Green Tea Leaves (Caballero et aI., 2003)

Chemical Property Chemical Substance Dry Weight (%) Water-Soluble Phenolic Compounds 30-40 Flavanols 25-30 Epigallocatechin Gallate 9-13 Epigallocatechin 3-6 Epicatechin Gallate 3-6 Epicatechin 1-3 Gallocatechin 1-2 Catechin 1-2 Flavonol Glycosides 4 Proanthocyanidins 3 Phenolic Acids 4 Caffeine 4 Amino Acids 4 Theanine 2 Others 2 Carbohydrates 19 Monosaccharides 4 Polysaccharides 14

~~_Ql'gilnic Acicl§ 0.5 Partially Water-Soluble Starch 2-5 Other Polysaccharides 12 Proteins 15 Ash 5 Water-Insoluble Cellulose 7 Lignin 6 Lipids 3.5-9 ChI orophy II/Pigments 0.5 Volatiles < 0.1 34

Chemical Composition of Prepared Green Tea Infusion

During green tea manufacturing, the GTC compounds are preserved by minimizing enzymatic oxidation during processing (Graham, 1992). In green tea, four epicatechin compounds exist in large quantities; epicatechin (EC), epigallocatechin

(EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) (Lee et al., 1995).

Figure 3 below shows the chemical structure ofEC, EGC, ECG and EGCG.

OH ~OH HO "'\\\U HO o

OH OH

Epicatechin Epigallocatechin

H OH OH ° OH ,~ I~ HO\O,0 ",\\ & # HO\OOr " ,\\cX # OH "- I., OH ~ ""10 -q-oH "-"- 'lfo -q- OH f " OH OH 'c f " OH 'c1/ _ 1/ - o ° OH OH

Epicatechin Gallate Epigallocatechin Gallate

Figure 3. Chemical Stmcture of Predominate Catechin Compounds in Green Tea (Caballero et aL,2003).

Green tea is light-yellow/greenish beverage with a mildly bitter, earthy flavor (Graham,

1992), This nutritionally dense beverage contains high coneentrations of antioxidants. As shown in Table 2, catechins significantly contribute to the ehemical composition of green tea. 35

According to NHANES and the USDA database, the estimated U.S. citizen's daily flavonoid intake is 189. 7 mg/day. Tea is the number one contributor of flavonoid compounds to the average American diet, providing I 57mg/day. GTC, flavan-3-0Is account for 83.5% of tea flavonoids (Song & Chun, 2008).

Green tea is acknowledged as a major contributor of antioxidants to the human diet.

Previous graduate research has compared the catechin content of eight commercially available green teas (Kafley, Ondrus, Rohrer, & Coker, 2008). Kafley (2008) repOlted that EGC and

EGCG constituted over half of the total polyphenols in green tea. Of the eight commercial green teas investigated by Kafley, Lipton®, Salada®, Stash® and Red Rose® were found to have the greatest total flavan-3-01 content.

Table 6 shows the approximate chemical composition of a freshly prepared green tea infusion. The chemical composition of a green tea infusion differs from that a dry green tea leaf in that many of the insoluble and partially soluble fractions are absent in the infusion. The epicatechin concentrations increase slightly due to the absence of other compounds. In freshly prepared green tea epicatechins comprise a large percentage of extract solids. In solution GTCs dissolve as water soluble, colorless, astringent solutes that easily oxidize in aerobic environments. Thus, the most beneficial components are susceptible to degradation during storage (Francis, 2000). 36

Table 6

Approximate Chemical Composition of a Freshly Prepared Green Tea Infusion (Francis;

Graham, 1992)

Chemical Compo-cu~n-cd___ ~ ______We19ht % Extr§lft Solids EGCG 20 ECG 5 EGC 8 ECG 2 Other Catechins 1-7 Flavanols 3 Other Flavonoids 2-4 Methylxanthines 8 Theogallin 2 Other Depsides 1 Gallic acid 0.5 Ascorbic acid 1 Quinic acid 2 Other organic acids 4-5 Theanine 5 Other amino acids 4-6 Carbohydrates 7-15 Precipitable alcohols 12 Minerals 6-8 Volatiles < 0.1 Note: Some values have a range of weight % extract solids.

During storage, catechins are initially oxidized by the cellular enzymes polyphenol oxidase and peroxidase to form highly reactive quinone compounds. Green tea leaves typically have very little gallic acid, but once the fraction is steeped, galloyl esters may be enzymatically hydrolyzed from catechins (Caballero et al., 2003). Brenda (2009), references tannase as a hydrolase enzyme produced from microorganisms found on tealeaves. Figure 4 shows the tamlase initiated enzymatic hydrolysis of GA and EC or EGC from ECG or EGCG. Figure 5 shows the formation of catechin quinones via enzymatic oxidation. Hydrolysis and oxidation 37

reactions both contribute to the quality of green tea infusions. Reactions below in Figures 4-10

appear to occur in samples during long-tenn storage.

OH OH OH ECG '" EGCG &OH ,\ R ",0 HOtyO""UlR ~ "1/ OH O OH + HO OH OH olu~:~' OH Gallic Acid Y'OH OH

(EGCG hydrolyzes to EGC and gallic acid, R = hydroxyl); (ECG hydrolyzes to EC and gallic acid, R = hydrogen)

Figure 4. Tannase IIydrolyscs ECG and EGCG to Form GA, EC and EGC.

OH o OH Catechins Catechin Quinones Lt° HO or R' R' H01iO ,,\\\\ A Polyphenol Oxidase O tIll/ OR OR OH OH

(R = hydrogen or 3,4, 5-trihydroxybenzoyl; R' = hydrogen or hydroxyl)

Figure 5. Enzymatic Catechin Oxidation and the Fonnation of Catechin Quinones.

Polyphenol qui nones react to fonn colorful by-products: theaflavins, theaflavic acids and theaflavin gallates. Figure 6 shows the formation of thea flavin from a catechin quinone reacting with gallocatechin quinine to f01111 theaflavin. Theaflavins account for a small percentage of oxidized catechins but are recognized in solution by their bright red-orange pigment. Figure 7 shows the formation oftheaflavic acid from a gallic acid quinine reacting with gallocatechin 38

quinone. The fonnation of theaflavic acids contributes minimally as a percentage of oxidized catechins but increases the acidity of the solution while also being noticed as a bright red

pigment (Caballero et al., 2003; Francis, 2000; Graham, 1992). The presence of these colorful

by-products indieates polyphenol oxidation, whieh takes place during tealeafprocessing and

long-term storage of infused tea.

It has been postulated that catechin quinones oxidize due to their high oxidation potential from their hydroxyl group configuration. Figure 8 shows the formation of thea flavin gallates fi'om catechin quinone reacting with gallocateehin quinone. Theaflavin gallates have an astringent odor and also contribute as a dark colored pigment to infusions and processed leaves.

Figure 9 shows the fonnation ofbisflavanols from the coupling of two gallocatechin quinones.

Bisflavanols are colorless substances that contribute a small fraction to the composition of tea

(Caballero et al., 2003; Francis, 2000; Graham, 1992), During storage eellular enzymes initiate reactions that produce by-products that may alter the physical and chemical characteristics of green tea infusions.

o OH Catechin Quinone (~O H0'Q?,.,\\\l) OH "1 o IIOH OH + o OH Gallocateehin Quinone 6:0 HO HOYO\~o ,.' .# OH "IIIOH

OH OH

Figure 6. Theaflavin Formation From Catechin Quinone Reacting with Gallocatechin Quinone. 39

Gallic Acid Quinone o OH o

OH CO2 OH HO + 0 ~ Epi"",b;" Q"""", ~o """"------~ HOurO'l""U OH ~'IIIOR

OH OH

(Epitheaflavic acid, R = hydrogen); (Epitheaflavic acid-3'-gallatc, R = gallate)

Figure 7. Theaflavic Acids Arise From Gallic Acid Quinone Reacting with Epicatechin Quinone or Epicatechin Gallate Quinone. ° How·,,\\\l)Catechin Quinone (V0 OH "1/ OH o ~OR OH +

Gallocatechin Quinone (:(0° OH HO

h Ho'QOo ." \\\ /7' OH "IIIOR

OH OH

(Theaflavin gallate A; R gallate and R' hydrogen); (Theaflavin gallate B; R= hydrogen and R' = gallate); (Theaflavin digallate; R R' = gallate)

Figure 8. Theaflavin Gallates FOlID from Catechin Quinone or Catechin Gallate Quinone

Reacting with Gallocatechin Quinone or Gallocatechin Gallate Quinone. 40

OH OH OH ::l~'O~"ohlnQ:m:,&OH OH o .. VlOH "'IIOR HO OH

OH O2 Bisflavanols + OH Polyphenol Oxidase• OH :~I'Q:)locatechin:no~:\ 0 OH HO o .' 6c OH "'IIOR OH

OH OH OH

Bisflavanol A; R R' gallate; Bisflavanol B; R gallate and R' hydrogen; Bisflavanol C; R = R' hydrogen

Figure 9. Fonnation of Bisflavanols from Gallocatechin Quinones Reacting with Gallocatechin

Quinones.

Oxidation by-products including theaflavin, thea flavin acids and theaflavin gallates are responsible for the orange, red and brown pigments in black and oolong tea leaves formed during processing. These by-products are also responsible for the darkening of green tea infusions during storage. Oxidative chemical reactions diminish green tea quality during long-term storage by reducing the flavan-3-01 content and fonning undesirable aromatic and orange, red and brown pigmented polyphenols. The most significant by-product of oxidation in green tea infusions during storage are compounds known as thearubigins (Caballero et aI., 2003; Francis, 2000;

Graham,1992; Maga,1980).

Thearubigins are mixtures of compounds with a wide range of molecular weights fl:om

700 - 40,000. These compounds are large complexes molecules known to contain polysaccharides, proteins, nucleic acid anthocyanidins, cyanidin and delphinidin. Thearubigin compounds are not completely understood but are thought to be polyphenolic polymers

(Caballero et al., 2003; Francis, 2000; Graham, 1992). 41

It has been postulated that thearubigins result from catechin oxidation, catechin coupling, catechin-anthocyanidin interactions, and interactions between flavanol quinones and other macromolecules. Thearubigins are thought to further degrade and polymerize to form other thearubigins (Tanaka et aI., 2002). Thearubigins are reported to form by molecular condensation during the storage of infused tea, accounting for the majority of oxidized GTCs, and predominately represent the dark red and brown pigments, which form during degradation

(Caballero et aL, 2003).

Factors influencing the Chemical Composition of Green Tea Infusions

The chemical composition of prepared green tea and GTC mixtures may be altered by free radical activity in aerobic atmospheric conditions, microbial activity, enzymatic activity, steeping time and temperature as well as the pH of the solution (Zhu, Zhang, Tsang, Huang, &

Chen, 1997). Previous graduate research examined the effects of extraction parameters on polyphenol content in green tea. The results revealed that green tea polyphenolic content increased with steeping time ranging from 2-10 minutes and temperature which ranged from 80-

100°C (Gudala, Ondrus, Rohrer, & Barnhart, 2008). Other studies have shown that pH may also influence the chemical composition of green tea (Henning et aI., 2008).

Feruzzi has stated that "catechins are relatively unstable in non-acidic environments"

(Main, 2007). A recent mticle reported that the bioavailability of flavan-3-ols (EGCG and EGC) degraded 50% in 2-3 hours in an alkaline environment at a pH 7. In the same alkaline environment EC and ECG remained stable for 8 hours (Henning et a1., 2008). These findings indicate that the chemical stability of some GTCs is reduced in alkaline pH solutions more than others. 42

Another study investigated the degradation of GTCs in acidic or alkaline pH solutions ranging from 5.0 7.4. The results indicated that the concentration of GTCs decreased from almost 100% in a 5.0 pH to nearly 0% in the 7.0 pH solution. The color of solutions changed from a clear to dark brown as the concentration of GTCs decreased in the alkaline solutions.

EGCG and EGC were much more susceptible to degradation compared to EC and ECG which remained stable (Zhu et aI., 1997).

Green Tea Leaf Enzymatic Activity

Once tea is prepared, it has been hypothesized that microbial and enzymatic activation allows polyphenol oxidase, peroxidase, tannase and other enzymes to react targeting ester bonds and hydroxyl functional groups. When activated, enzymes compromise the quality of green tea infusions by initiating chemical and biochemical reactions that produce pungent odors and dark pigments from by-product compounds, while altering the nutritional content as well (Pruidze,

Mchedlishvili, Omiadze, & Gulua, 2003). Therefore, controlling the enzymatic activity in tea ensures optimal aroma, taste, appearance and nutritional content

Enzymes, catalysts and substrates initiate all biochemical reactions, which take place in living organisms. Green tea leaves contain a variety of active enzymes that contribute to composition, including: polyphenol oxidase (PPO), peroxidase (POD), tannase, invertase, amylase, glucosidases, pectinase, pectin methyl esterase, alcohol dehydrogenase, lipoxidases, transaminase, peptidase, phenylalanine ammonia lyase, 5-dehydroshikinmate reductase, and metalloprotein enzymes (Maga et a!., 1980; Caballero et a!., 2003; Francis, 2000; Graham,

1992).

O-diphenol oxidase or PPO is an oxygen reductase enzyme found in the cytoplasm, chloroplasts and mitochondria of the tea plant (Maga et aI., 1980). PPO oxidizes polyphenols, 43

tannins, pyrocatechnis, pyrogallol and GA accounting for up to 16.5% of total enzymatic

activity. The PPO initiated aerobic oxidation of catechins leads to the production of reactive

quinone intermediates (Caballero et a1., 2003). The Camellia sinensis cellular leaf structure

houses multiple isoenzyme forms of PPO with molecular weights ranging between 28,000

250,000, these complex protein compounds act as either oxidation enzymes or catalysts (Pruidze

et a1., 2003). The most abundantly active PPO enzyme has a molecular weight of 142,000

(Francis, 2000).

POD is a cationic isoenzyme that oxidizes polyphenols producing reactive radical

intermediates. POD is a cellular oxidative enzyme that also exists in the cytoplasm, chloroplasts and mitochondria of tea plants. Once activated, this enzyme may contribute up to 35% of total enzymatic activity, capable of mass oxidation reducing substrates at an incredible rate (Maga et a1., 1980).

One method, which may be particularly imp0l1ant to this study, is the production of tannase by microorganisms. Tannase (tannin acylhydrolase) is a hydrolase enzyme that hydrolyzes GA esters such as ECG and EGCG releasing free GA during the reaction while potentially increasing the concentration of EC and EGC (Osawa & Walsh, 1993). Tannase is commonly produced by some fungal strains: Aspergillus, Candida, Streptococcus, Lonepinella,

Penicillum and Bacillus species (Osawa & Walsh, 1993; Pruidze et a1. ,2003). Tannase has been acknow ledged as an imp0l1ant enzyme in the manufacturing of instant teas (Paranthaman,

VidyaJakshrni, Indhumathi, & Singaradivel, 2009). Using these active enzymes during manufacturing of tea may also increase the rate of degradation during storage. 44

Green Tea Leaf Microbial Activity

Recent studies have acknowledged a number of fungal microbes on tealeaves. Many of these fungal strains have been shown to produce enzymes and catalysts that aid in biochemical reactions that influence the quality of tea. In China various strains of algae chlorophyta and phaeophya have been incorporated into tea production (Algae, 2000). The Chinese and many other costal cultmes have consumed various forms of algae for its nutritional content and potential phannacological activity. Thus, although the ingestion of some microbes maybe nutritionally beneficial; the presence ofthem in tea can reduce the quality of the beverage.

According to Mohammad and Alireza, (2007) sixteen aerobic endospore-forming species of Bacillus were isolated from tealeaves. Some various species identified were: B. subtilis, B.

Iicheniformis, B. sphaericus and B. pumilus. The isolated fungus contributed polyphenol oxidase and peroxidase enzymes that chemically react, altering tea characteristics. Thus, monitoring fungal and enzymatic activity in the green tea infusions may contribute valuable information when investigating the chemical change and quality of the infusions dming storage.

Aspergillus ficcum is referenced in a comprehensive enzyme database (Brenda, 2009) as a green tea leaf microorganism that produces tannase. The database indicates that tannase catalyzes the hydrolysis of ester bonds in gallic acid esters liberating free gallic acid and possibly

EC or EGC.

Method of Analysis

When investigating mixtures of naturally occurring phenolic compounds, analytical detection and quantification of particular analytes of interest is commonly achieved using high perfonnance liquid chromatography (HPLC) with photo-diode array detection (Treutter, Santos­

Buelga, Gutmann, & Kolodziej, 1994). Depending on the polarity of the analytes of interest the 45 investigator may choose to use reversed-phase HPLC (RP-HPLC). The RP-HPLC system employee's a non-polar stationary phase with moderately polar aqueous mobile phases containing Milli-Q water, acetonitrile and acetic acid. The concentration of water in the mobile phases is altered to match the binding affinity of the sample analytes.

It has been stated that, "normally (RP-HPLC) is used to separate useful compounds from crude plants" (Jin & Row, 2007). The article explains that an efficient method of analyte extraction, detection and quantification was accomplished using gradient elution of binary mobile phase solutions consisting of water, acetonitrile and acetic acid. The goal of this research was to extract catechin and epicatechin compounds from green tea using the RP-HPLC (Jin &

Row, 2007). 46

Chapter III: Methodology

Commonly used, well known, Lipton® Green Tea was chosen to investigate the loss of epicatechin antioxidants during storage at room temperature (2S°C). This tea had previously been shown to have high antioxidant content as determined by (Kafley et a1. , 2008).

Materials

The analytes investigated in the tea samples were compared to standards purchased from

Sigma-Aldrich Chemical Company in St. Louis, Missouri. Standards included (-)- Epicatechin >

90% pure, Epigallocatechin > 95% pure, Epicatechin Gallate> 98% pure, (-)-Epigallocatechin

Gallate> 95% pure and caffeine. Gallic acid was purchased from MP Biomedicals (Solon,

Ohio).

Water for preparing the tea samples and standards was obtained from the UW-Stout

Chemistry Department Milli-Q water purification system, this system was purchased from the

Millipore Corporation. Reagent grade acetic acid and HPLC-grade acetonitrile were purchased from Fisher Scientific (Fairlawn, New Jersey).

The investigation also required the use of glassware including beakers, Erlenmeyer flasks, volumetric flasks, graduated cylinders, pipets and autos-sampler vials. Plastic 5-mL syringes equipped with Whatman 0.45-/.un filters were used for filtering the individual sample aliquots. A Sheldon 1227 SHEL Lab water bath shaker from VWR Scientific Products (SN:

0700301) was used to maintain constant temperature and consistent aerobic conditions for each tea sample during storage. 47

Tea Sample Preparation and Storage

All tea samples were prepared from the same box of commercially available Lipton® green tea. Each set of tea samples was prepared by steeping 3 tea bags with 800 mL of freshly boiled Milli-Q water in a large 2000-mL Erlenmeyer flask. The 800-mL sample was divided equally into three labeled 400-mL beakers. The bulk 800-mL samples were prepared in triplicate according to package directions to ensure consistency throughout the experiment. Figure 10 shows the preparation of bulk samples and the separation of the triplicate samples.

Boil MiIli-Q Water Separate Triplicate Samples

Remove from heat and steep tea bags ~

~.- c",,";:;c:.

Figure 10. Bulk Green Tea Preparation and Triplicate Sample Separation.

The tea samples investigated were plain Lipton® green tea, Lipton® green tea with 5-mL lemon juice and Lipton® green tea with 5-mL cranberry juice. Nine 400-mL beakers containing approximately 250-mL samples of Lipton® green tea were prepared and separated from three bulk 800-mL samples. Fresh fruit juice was added to six of the 250-mL samples. When preparing triplicate tea samples with fresh fruit juice, 5-mL of freshly squeezed fruit juice (lemon or cranberry) was added to each 400-mL beaker to mimic the amount a consumer may add to a cup of tea. 48

Initially, each sample was prepared in bulk and equally separated for consistency purposes. The samples were allowed to cool to room temperature and individually measured on an analytical balance to obtain the exact mass. Once the mass of each triplicate tea sample (with or without fmitjuice) was recorded, aliquots were removed and samples were measured again to determine the change in mass and monitor evaporative loss. Samples were stored in uncovered labeled beakers for 6-days in a water bath at 25° C while shaken at 60 revolutions per minute.

Sampling

Every 24 hours the sample beakers were removed from the water bath, dried and massed.

Once the mass of the sample was recorded, was measured and a sample aliquot was removed.

Table 8 shows variations in pH during storage for each sample. After the sample aliquot was removed, the mass was recorded and the beaker was rehllTled to the water bath for storage. Each

24-hour evaporative loss was calculated as the difference between the mass after sampling and the mass of the sample 24-hours later before the next aliquot was removed.

Daily, 2-mL aliquots were removed at 24-hours intervals using a syringe and membrane­ filtered into glass auto sampler vials. Before each sample aliquot was removed the syringe was rinsed with 2-3 mL of sample. The glass auto-sampler vials were filled, labeled and immediately frozcn. Once the final aliquot was removed, after 6-days or 144-hours, the frozen aliquots were thawed, categOlized and placed in the auto sampler for subsequent analysis.

Standards

Standard solutions of epicatechin and caffeine were prepared from bulk stock solutions.

From the bulk standards, fractions were removed and diluted to concentrations indicated below in Table 7. Standard solutions of epicatechin gallate, epigallocatechin, epigallocatechin gallate 49 and gallic acid were prepared by measuring a precise mass of the solid analyte anhydrous on an analytical scale and diluting with Milli-Q water to the 25-mL mark in volumetric flasks.

Table 7

Standard Analyte Solution Concentrations

II III IV V VI Analyte Epicatechin 25 50 75 100 125 150 Epigallocatechin 20 40 200 Epicatechin Gallate 20 100 200 Epigallocatechin Gallate 20 40 60 80 100 120 Gallic Acid 21 45 65 87 109 130 Caffeine 50 100 150 200

Instrumentation

A Waters high performance liquid chromatography (HPLC) system with reverse phase

(RP) column and gradient elution was used in this research. The instillment was able to quantitatively analyze GA, CAF and epicatechin compounds: EC, EGC, ECG and EGCG. The modified chromatographic separation used a PC with Windows NT operating system equipped with Empower 2 software. The components of the HPLC-RP included a Waters 1525 binary pump, 717 Waters plus auto-sampler, 2996 photodiode array detector and an RCM-100 radial compression module with a Waters radial compression ™ (10 cm x 8 mm ID) NovaPak ™ CIS colunm. The solvent programmer shown below in Table 11 represents elution gradient options.

Curves 1-5 indicate convex gradient options and curves 7-11 indicate concaved gradient options. 50

Figure 11. Solvent Programmer Elution Curves

The gradient elution required two aqueous solvents consisting of milli-Q water, acetonitrile and acetic acid. Solvent A consisted of99.75% milli-Q water and 0.25% acetic acid

(v/v). Solvent B consisted of60% solvent A and 40% acetonitrile (v/v). The acetic acid prevented polyphenol ionization and reduced peak broadening.

The solvent programmer was set for two elution curves. The concave curve 7 shown above in Figure 11 varied the mobile phase from 100% solvent A to 100% solvent B during the

30minute sample. The instrumentation required 30 minutes to run the sample through gradient elution, 1 minute to reset the initial conditions (100% solvent A) and 5 minutes to equilibrate the system prior to the next sample injection. Table 8 shows the gradient elution stages, elapsed time between each stage, flow rate in (mLimin) and the mobile phase gradient used during each 36- minute sample period. 51

Table 8

Solvent Programmer Settings for Mobile Phase Elution Gradient

Gradient Sta~ Time(~ FI(jw (mLlmin) Mobile A Mobile B Curve 1 0.00 2.00 100.00 0.00 0.00 2 30.00 2.00 0.00 100.00 7.00 3 31.00 2.00 0.00 100.00 6.00 4 32.00 2.00 100.00 0.00 6.00 5 36.00 2.00 100.00 0.00 Note. When a mobile phase changes between gradient stages it does so according to a specific solvent programmer elution curve (see Figure 11).

Data Analysis

For identification and quantitation of green tea analytes, the retention times and peak areas from sample chromatograms were compared to standard analyte chromatograms with known concentrations. Figure 12 shows analyte peak area as a function of compound concentration. A least squares linear curve fit applied to each set of standard data points in Table

6 provided linear regression line equations (y Ax) for each standard. This equation allowed the use of chromatogram peak areas for each corresponding analyte in the green tea samples (y) to determine concentration (x) using the given regression line slope (A) from the standards. All analytes produced linear best-fit correlation of 0.999 or higher. Chromatograms produced at a wavelength of 270 nm for standards and samples revealed crisp peaks, showing little tailing and shoulder formation, respectively. 52

~+------~------~------r-----~------r------~

...... Data Set IEpicatechin --- Data Set I Epigallocatechin ...... Data Set I Epicatechin Gallate -+- Data Set I Epigallocatechin Gallate -I- Data Set I Galiic Acide --- Data Set ICaffeine

g~+------~------~~~~------r------?~------~ ~ ~ I / I>.

2000000+------~~~------~~------r_------r_------~

50 100 150 200 259 (l-l.9. 1I\6lS3) Concentration (rg/l)

Figure 12. Standard Analyte Peak Area at 270 nm Plotted as a Function of Analyte

Concentration. 53

Chapter IV: Results and Discussion

The Study

Analytes including GA, CAF and epicatechin compounds: EC, EGC, ECG and EGCG were detected and quantified in green tea samples during 144-hour (6-day) storage. Lipton® green tea was prepared in bulk according to package directions and separated into triplicate samples. The triplicate samples were categorized as plain green tea, green tea with 5-mL lemon juice and green tea with 5-mL cranberry juice. Samples were stored uncovered up to 144-hours in a 25°C water bath and shaken at 60 revolutions per minute. Aliquot samples were removed cvery 24 hours and immediately frozen for analysis. Analytical detection was accomplished using gradient RP-HPLC method with a photodiode array detector set at a wavelength of270 nm.

Analyte concentrations were calculated using a least square regression fit equation (y =

Ax). Using the least square regression slope (A) provided for each standard (Figure 12) and each sample chromatogram analyte peak area (y), the concentrations (x) ofGA, CAF, EC, EGC, ECG and EGCG were calculated. Analyte concentrations were detennined as milligram per liter

(mglL) and millimole per liter (mmol/L). The unit's mmol/L is useful in comparing the ratios of products to reactant in possible hydrolysis reactions.

Visual observations and the pH of each tea sample were noted during storage. Over time, during 144-hour storage all tea samples with and without added fruit juice experienced variations in chemical and physical characteristics including: pH, color change and noted visual microbial or fungal growth that was not identified. 54

SampJepH

Variations of infusion pH during storage appear to correlate with analyte degradation.

Figure 13 is a graphical representation of the average pH plotted over the 144-hour storage

period. Table 9 shows average pH change in triplicate tea samples during the 144-hour storage

period. Plain Lipton® green tea samples averaged slightly acidic pH levels around at 5.3. Tea

samples with added lemon juice averaged a more acidic pH level at approximately 3.3. Tea

samples with added cranberry juice also averaged more acidic pH levels near 3.4. Samples with

added fruit juice maintained more constant pH levels during storage. The plain tea samples

gradually became more acidic during storage exhibiting a pH decrease of about O.6-pH unit.

Data Set I Lipton pH • Data Set I Lipton (lemon Juice) pH • Data Set I Lipton (cranberry juice) pH

Figure 13. Lipton® Green Tea Triplicate Sample Average pH Readings During 144-Hour

Storage. 55

Table 9

Average pH ofLipton@Green Tea Samples With and Without Added Fruit Juice

. TimeJh°tlr~ _ Lipt

EC was not detected in several samples between 0-72 hours. Consequently, some initial

concentrations of EC in Tables 10, 11 and 12 reference no data or not detected (NO). Although

not detected, the EC peak is visible as a shoulder at the base of the EGCG peak as noticed in

sample chromatograms, Figures 15, 19 and 23. Over time as the concentration of EGCG

decreased, EGCG peak areas also decreased and the presence of EC were detected in sample

aliquots. In addition, some aesthetic characteristics such as color change, infusion odor, and

microbial or fungal growth, which have been related to catechin oxidation and by-product

formation, were also observed.

Visually, all green tea samples with and without added fmit juice aesthetically

deteriorated, darkening in color during storage. Once infused, green tea appears pale green with

almost a light yellow color. Green tea infusions do not have dark red/brown pigments which are

color characteristics associated with the formation of thea flavin, theaflavic acids, theaflavin

gallates, bisflavanols and theambigins during catechin oxidation in black and oolong tea leaf

processing (Caballero, 2003). During storage, each tea sample infusion became dark and cloudy 56 with noted pungent odors and visible fungal or microbial growth forming in the sample

infusions.

Lipton Green Tea Samples

Figure 13 is a graphical representation ofthe average analyte concentrations (mg/L) in

Lipton® green tea samples plotted over 144-hour storage period. Table 10 contains average analyte peak areas and average concentrations (mg/L) and (mmol/L) oftriplicate data used to generate Figure 14.

The two-epicatechin compounds that exhibited marked decreases in concentration during storage were the gallates (ECG and EGCG). Two analytes that exhibited a corresponding increase in concentration during storage were GA and Ee. Figures 15-17 clearly show the loss of

EGCG and ECG along with corresponding increases in GA and EC during storage. EGC increased for the first 72 hours and then decreased rapidly. It's notewOlihy that the total GA increase (approximately 1.0 mmol/L increase) is nearly equal to the total loss ofEGCG (0.9 nunol/L decrease) and ECG (0.2 mmol/L decrease). 57

Data Set I Gallic Acid Data Set I EGC III Data Set I Caffeine A. Data Set I • Data Set I EGCG Data Set ECG

200+------········-················

I

I o 50 100 150

Figure 14. Plain Lipton® Green Tea Average Analyte Concentrations (mg/L). 58

Table 10

Lipton® Green Tea Average Analyte Concentrations Calculations

Hours Peak Area 0 1.75E+05 6.2 0.04 24 1.88E+05 6.6 0.04 48 2.09E+05 7.2 0.04 GA" 72 3.02E+05 9.7 0.06 96 1.22E+06 35.1 0.21 120 3.44E+06 96.2 0.57 144 0 1.06E+06 535.7 1.75 24 1.09E+06 549.3 1.79 48 1.09E+06 546.6 1.78 EGCb 72 1.16E+06 583.7 1.91 96 1.08E+06 541.4 1.77 120 9.35E+05 470.6 1.54

0 8.15E+06 238.4 1.23 24 8.56E+06 250.6 1.29 48 8.70E+06 254.5 1.31 CAFc 72 9.30E+06 272.1 1.40 96 9.09E+06 265.9 1.37 120 8.97E+06 262.4 1.35

0 NO NO 24 1.I3E+05 15.4 0.05 48 1.78E+05 27.1 0.09 72 NO NO NO 96 2.97E+05 48.7 0.17 120 6.08E+05 105.1 036 144 0.53 0 8.13E+06 581.5 1.27 24 8.22E+06 587.3 1.28 48 7.97E+06 569.7 1.24 EGCGc 72 8.45£+06 603.9 1.32 96 633E+06 453.4 0.99 120 3.87£+06 279.4 0.61 144 0 2.37E+06 117.3 0.27 24 2.42E+06 119.8 0.27 48 2.45E+06 121.4 0.27 ECG f 72 2.54E+06 126.0 0.28 96 2.11£+06 103.7 0.23 120 132£+06 63.2 0.14 144 0.05 Note. EGC concentrations increased between 0-72 hours and then decreased between 72-144 hours. EC concentrations at 0 & 72 hours were not detected.

d e Note. GA"-Gallic Acid; EGCb-Epigallocatechin; CAFc-Caffeine; EC -Epicatechin, EGCG - Epigallocatechin Gallate; ECGf-Epicatechin Gallate, NDg-NoData. 59

CAF EGCG

Figure 15. Plain Lipton® Green Tea Sample Chromatogram Crime = 0 hours)

CAF

Figure 16. Plain Lipton® Green Tea Sample Chromatogram (Time = 120 hours)

CAF

1 "'1 1

EGCG

Figure 17. Plain Lipton® Green Tea Sample Chromatogram (Time 144 hours) 60

Lipton Green Tea Samples (5-mL Lemon Juice)

Figure 18 is a graphical representation ofthe average concentrations (mg/L) in triplicate

Lipton® green tea samples (5-mL lemon juice) plotted over 144-hour storage period. Table 11 contains average analyte peak areas and average concentrations (mg/L) and (mmol/L) of

triplicate data used to generate Figure 18.

The two-epicatechin compounds that exhibited marked decreases in concentrations during storage were the gallates (ECG and EGCG). Three analytes that exhibited a corresponding increase in concentration during storage were GA, EC and EGC. One notable difference is that EGC increased throughout storage but did not decrease after 72-hours as was noted in the tea samples not treated with fruit juice. Figures 19-21 clearly show the loss of ECG and EGCG along with cOlTesponding increases in GA, EC and EGC during storage. It is also noteworthy in tea samples treated with lemon juice, that the total average increase of approximately (1.75 nnl1ollL) GA is nearly equal to the loss of(1.35 mmollL) EGCG and (0.23

Imnol/L) ECG. 61

~~ ~~~~~~~~--~~~r-~~~~~~~~-~~~~

III ~ ~ ~ ~~~~ ~~~ ~~~~~ ~ ~~ Data Set I Gallic Acid ...• Data Set I EGC • Data Set I Caffeine A.. Data Set I • Data Set I EGCG 1000 I Data Set I ECG

::::J .s0, c o ~ C Q) u c o o 500----

O-IF==T==~~~=====4~~. _~==~~~~==~~i=~==~~_~ o

Figure 18. Lipton@ Green Tea (5-mL Lemon Juice) Average Analyte Concentrations (mglL). 62

Table 11

Lipton@ Green Tea (5-mL Lemon Juice) Average Analyte Concentration Calculations

Anal,}'te Hours Peak Area ~- Concentration(mgJLL.~_~~onc~.Iltratiol!(Jl1mo1!I.,)____ 0 I.72E+05 6.1 0.04 24 1.82E+05 6.4 0.04 48 7.70E+05 22.6 0.13 GN 72 8.54E+06 236.6 1.39 96 9.80E+06 271.3 1.59 120 1.03E+07 286.1 1.68 I II 1 0 Ll5E+06 577.8 1.89 24 1.16E+06 584.0 1.91 48 1.19E+06 600.4 1.96 EGCb 72 2.25E+06 1137.3 3.71 96 2.36E+06 1189.7 3.88 120 2.38E+06 1203.4 3.93 144 II 0 8.46E+06 247.6 1.27 24 8.66E+06 253.3 1.30 48 8.57E+06 250.9 1.29 CAFe 72 9.17E+06 268.4 1.38 96 8.89E+06 260.2 1.34 120 8.99E+06 263.0 1.35 144 0 ND ND 24 ND ND ND 48 ND ND ND ECd 72 9.53E+05 167.7 0.58 96 1.08E+06 191.0 0.66 120 1.01E+06 179.0 0.62 144 1.03E+06 1 0 8.63E+06 616.5 1.34 24 8.71E+06 622.3 1.36 48 8.08E+06 577.6 1.26 EGCGc 72 I.70E+06 125.0 0.27 96 2.67E+05 23.7 0.05 120 ND ND ND 144 ND ND -~- ND ...... ----~- .... ------,-,-.-.. ~-~------...... ---..... 0 2.52E+06 124.6 0.28 24 2.62E+06 129.8 0.29 48 2.3IE+06 113.8 0.26 ECG f 72 6.12E+05 26.8 0.06 96 5.29E+05 22.6 0.05 120 4.77E+05 19.9 0.04 144 Note. On average EC concentrations between 0-48 hours were not detected.

b d c Note. GA"-Gallic Acid; EGC - Epigallocatechin; CAFe-Caffeine; EC - Epicatechin, EGCG - Epigallocatechin Gallate; ECGf-Epicatechin Gallate and NDg-No Data. 63

C F

Figure 19. Lipton® Green Tea (5-mL Lemon Juice) Chromatogram at (Time 0 hours)

CAF

GA

Figure 20. Lipton® Green Tea (5-mL Lemon Juice) Chromatogram (Time 72 hours)

GA

Figure 21. Lipton® Green Tea (5-mL Lemon Juice) Chromatogram (Time =144 hours) 64

Green Tea Samples (S-mL Cranberry Juice)

Figure 22 is a graphical representation of the average concentrations (mglL) in triplicate

Lipton@ green tea samples (5-mL cranberry juice) over I 44-hour storage. Table 12 contains

average analyte peak areas and average concentrations (mglL) and (mmoI/L) oftriplicate data

used to generate Figure 22.

The two-epicatechin compounds that exhibited marked decreases in concentrations

during storage were the gallates (ECG and EGCG). Three analytes that exhibited a

corresponding increase in concentration during storage were GA, EC and EGC. One notable

difference is that EGC concentrations increased throughout storage but did not decrease after 72- hours, similar to the lemon juice treated samples. Figures 23-25 clearly show the loss of ECG and EGCG along with corresponding increases in GA, EC and EGC during storage. It is also notewOlihy in tea samples treated with cranberry juice that the total average increase of approximately (1.90 mmol/L) GA is nearly equal to the loss of (1.40 mmol/L) EGCG and (0.25

ImnollL) ECG. 65

l1li Data Set I Gallic Acid • Data Set I EGC III Data Set I Caffeine A Data Set I EC • Data Set I EGCG I Data Set ECG

Figure 22. Lipton@ Green Tea (5-mL Cranberry Juiee) Average Analyte Concentrations (mg/L)

Graph. 66

Table 12

Lipton@ Green Tea (5-mL Cranbeny Juice) Average Analyte Concentration Calculations

Hours Area 0 1.82E+05 6.4 0.04 24 1.87E+05 6.5 0.04 48 7.70E+05 22.6 0.13 GA' 72 6.5IE+06 180.6 1.06 96 1.05E+07 289.9 1.70 120 1.08E+07 298.8 1.76

...... _------_..144 1 330.5 1.94 0 1.13£+06 568.9 1.86 24 1.55E+06 779.2 2.54 48 1.40E+06 704.7 2.30 EGCb 72 2.17E+06 1095.0 3.58 96 2.65E+06 1340.7 4.38 120 2.55E+06 1290.1 4.21

8.63E+06 252.3 1.30 8.44E+06 247.0 1.27 48 9.30E+06 272.1 lAO CAFe 72 1.01E+07 29404 1.52 96 9.92E+06 290.3 1.50 120 9.37E+06 274.3 1.41 1 0 ND ND 24 ND ND ND 48 ND ND ND ECd 72 2.81E+05 45.7 0.16 96 8.05E+05 140.9 0.49 120 1.24E+06 219.6 0.76 144 1.16E+06 204.9 0.71 ...... _--_._---_.__ ._-_._._-_._-- ._._----_._------_.. 0 8.97E+06 640.5 lAO 24 8.36E+06 597.7 1.30 48 8.97E+06 640.9 lAO EGCGe 72 4.71E+06 339.1 0.74 96 9.69E+05 73.5 0.16 120 ND ND ND 144 ND ND ND 0 2.64E+06 130.9 0.30 24 2.45E+06 121.2 0.27 48 2.58E+06 127.9 0.29 ECG f 72 1.I4E+06 53.7 0.12 96 7.02E+05 31.4 0.07 120 5.42E+05 23.2 0.05 144 5.56E+05 23.9 0.05 -.--.------Note. On average EC concentrations between 0-48 hours were not detected.

b d e Note. GA"-Gallic Acid; EGC -Epigallocatechin; CAFe -Caffeine; EC -Epicatechin, EGCG - Epigallocatechin Gallate; ECGf-Epicatechin Gallate and NDg-NoData. 67

EGCG I

Figure 23. Lipton® Green Tea (5-mL Cranberry Juice) Chromatogram (Time = 0 hours)

Figure 24. Lipton® Green Tea (5-mL Cranbeny Juice) Chromatogram (Time = 72 hours)

CAF

Figure 25. Lipton® Green Tea (5-mL Cranbeny Juice) Chromatogram (Time =144 hours) 68

Discussion

The chemical composition of Lipton@ green tea with and without added fruit juice was altered during long-tenn 144-hour (6-day) storage. In all sample infusions, chemical oxidation and hydrolysis reactions appear to deteriorate green tea quality. Sensory observations indicate chemical reactions occur during storage. In all infusions visual color change was noted as samples deteriorate from a pale greenish-yellow to a dark reddish-brown. All sample infusions also developed probable microbial or fungal growth during storage. Tea samples treated with fruit juice had a more acidic average pH compared to samples not treated with fruit juice that had a less acidic average pH readings.

In all tea samples ECG and EGCG appear to be primaIY source of GA production during storage. The fonnation of GA, EC and EGC in saInples treated with lemon and cranbeny juice show a direct relationship to the deterioration ofEGCG and ECG. However, the observations that GA increases to a greater degree than expected from the loss of ECG and EGCG suggests that the presence of other gallates may also contribute to the rising GA concentrations.

Other studies have shown that GTC oxidation produces similar unique color characteristics comparable to the physical transformations of sample infusions in this research.

Francis (2002) indicated that during oxidation catechins form quinones, which further react to fonn theat1avins, theaflavic acids, bisflavanols and thearubigins. These colorful by-products darken green tea infusions deteriorating the quality by altering the nutritional content (Maga,

1980). Infusion darkening was observed in this study but not quantitatively measured.

Lipton@ green tea samples not treated with fruit juice showed EGC concentrations increased (1.75 mmoVL to 1.91 mmol/L) from 0-72 hours shown in Table 9. This increase in

EGC concentration appears to be a result of EGCG hydrolysis. A comprehensive enzyme 69 infonnation system shows a fungi organism (Aspergillusficuum) found in green tea leaves produce tannase, a hydrolase enzyme that catalyzes hydrolysis reactions of ECG and EGCG in green tea leaves (Brenda, 2009). Tannase hydrolyzes GA and from ECG and also hydrolyzes

GA and EGC from EGCG as shown in Figure 4. The decrease of EGC from 1.91 mmol/L to 1.52 mrnol/L from 72-144 hours shown in Table 9 may be a result of oxidation due to the less acidic pH of the infusions not treated with fruit juice.

In all tea samples, the increase in GA and EC concentration during storage appears to cOlTespond with the hydrolysis ofECG. In tea samples treated with fruit juice, all three analytes

GA, EC and EGC appear to increase with the subsequent hydrolysis ofECG and EGCG. This may be due to the more acidic pH of the infusions treated with lemon and cranbeny juice that contains ascorbic acid.

The visually noted darkening of all samples during storage suggest that polyphenol catechins are possibly oxidizing and therefore producing colorful by-product pigments such as theaflavin, theaflavin acids, theaflavin gallates and thearubigins (Caballero et aI., 2003; Francis,

2000; Graham, 1992). Although, these pigmented compounds are not measured in the current study. The sensory observations suggest that both oxidation and hydrolysis reactions degrade tea quality over time by altering the ehemical composition which also alters the physical characteristics of the infusions.

During this investigation it was also visually noted that all samples became cloudy as the infusions darkened. Microbial or fungal growth was observed in each infusion during storage.

After 144-hours of storage all sample infusions including Lipton® green tea, Lipton® green tea with 5-mL added lemon juice and Lipton® green tea with 5-mL added cranbeny juice appeared to contain some type of visual fungal or mierobial growth. 70

Oxidation appeared to affect chemical composition in less acidic non-fruit juice samples but did not seem to have the same effect in the more acidic samples treated with fruit juice.

However, hydrolysis seemed to more significantly alter the chemical composition in the more acidic samples treated with fruit juice compared to the samples not treated with juice. Although, the addition of fruit juice increased sample acidity, the chemical composition of the fruit juice may have increased fungal or microorganism activity, such as Aspergillus ficuum, in tea samples, causing an increase in enzymatic (tannase) activity, which could have catalyzed the hydrolysis of

ECG and EGCG at a faster rate (Brenda, 2009). 71

Chapter V: Conclusion

GA, CAF and epicatechins: EC, EGC, ECG and EGCG concentrations were calculated using a least square regression linear fit equation (y = Ax + b). Concentrations were measured in

milligram per liter (mg/L) and millimoles per liter (mmoI/L). The concentrations changes shown

in Tables 9, 10 and 11 support the hypothesis that the epicatechin compounds will degrade during long-term 144-hour (6-day) storage. Over time, prepared green tea infusions appeared to undergo chemical oxidation and hydrolysis reactions, which reduce tea quality as, noted by the darkening of each sample infusion and variations in analyte concentrations.

The addition of fruit juice to Lipton® green tea infusions reduced the pH creating a more acidic environment. According to other findings, adding lemon juice to green tea infusions should increase the bioavailability of the chemical composition in a green tea (Main, 2007).

Cunent results show that the addition of 5-mL fmit juice (lemon or cranbeny) to tea samples did not have a preservative effect on the Epicatechin compounds during storage. The fmit juice appeared to increase hydrolysis creating larger concentrations ofGA, EC and EGC while rapidly reducing concentrations ofECG and EGCG during storage. Although oxidation did appear to contribute to the transformations of GTCs, samples not treated with fruit juice seemed to degrade at a faster rate due to oxidation compared to samples treated with fruit juice.

Therefore, prepared green tea infusions with and without added fruit juice will diminish in quality during 144-hour storage at 25°C. Green tea samples without added fmitjuice seemed to be more susceptible to oxidative transformations. Green tea samples with added lemon or cranberry juices seemed to resist oxidative transformations initially but were susceptible to hydrolysis alterations possibly due to more rapid activation of green tea fungi (Aspergillus 72 jicuum) which has previously been shown to act as a substrate initiating the hydrolysis of GA,

EC and EGC from ECG and EGCG.

Ultimately, green tea will deteriorate during long-term storage @ 25°C. Adding 5-mL of fruit juice that contains ascorbic acid did not prevent chemical transformations but did initially delay oxidative degradation.

Recommendations

There were aspects ofthis investigation that induced variability. The microorganisms present in the infusions after 144-hour storage at room temperature were not identified. Thus, the presents of tannase cannot be confinned as the enzyme responsible for the possible hydrolysis of

ECG and EGCG. To verify the presence of Aspergillus jiCUU111 in the infusions as the source of tannase, fuliher studies will need to focus on identifying the organismal growth in the infusions.

Also, to ensure the hydrolysis is due to an organism present on the tea leaf and not introduced by the milli-Q water or added fmit juice, another study may consider using an autoclave to treat the teabags, water and or fmit juice prior to sample preparation.

Also, studies have shown that infusions and tea leaf color change is due to catechin oxidation which form highly reactive quinine intennediates and eventually react to form theaflavin, theaflavic acids, theaflavin gallates, bisflavanols and thearubigins. Visible spectra may be considered in future studies to calculate the actual visual spectral changes that occurred in samples during storage. 73

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