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Critical Reviews in Plant Sciences

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Tea Chemistry
a a b b
Matthew E. Harbowy , Douglas A. Balentine , Dr. Alan P. Davies & Dr. Ya Cai
a
Lipton, 800 Sylvan Avenue, Englewood Cliffs, NJ, 07660, USA
b
Unilever Research Colworth Lab., Colworth House, Sharnbrook, Bedford, U.K.

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To cite this article: Matthew E. Harbowy, Douglas A. Balentine, Dr. Alan P. Davies & Dr. Ya Cai (1997): Tea Chemistry, Critical
Reviews in Plant Sciences, 16:5, 415-480

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 Critical Reviews in Plant Sciences. 16(5):415480 (1997)

Tea Chemistry
Matthew E. Harbowy and Douglas A . Balentine
Lipton. 800 Sylvan Avenue. Englewood Cliffs. NJ 07660

TABLE OF CONTENTS

I. Introduction ....................................................................................................................... 417

I1. Chemical Composition. an Overview ............................................................................. 423
A. Polyphenols .................................................................................................................. 424
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B. Caffeine. the Methylxanthines. and Related Compounds ...................................... 424

1. Theobromine .......................................................................................................... 425
2. Other Derivatives of Nucleic Acids ..................................................................... 425
C. Proteins and Amino Acids ......................................................................................... 426
D. Carbohydrates. Pectins. and Fiber ........................................................................... 426
E. Organic Acids and Vitamin C ................................................................................... 426
F. Lipids. Chlorophylls. Carotenoids. and Related Compounds ............................... 427
G . Vitamins and Minerals ............................................................................................... 428
H. Aroma ........................................................................................................................... 428
I11. Polyphenols in Tea ........................................................................................................... 429
A . Chemical Classification .............................................................................................. 430
B. Green Tea Polyphenols ............................................................................................... 430
1. Catechins and Gallocatechins ............................................................................... 430
2. Flavonols ................................................................................................................. 432
3. Simple Polyphenols ................................................................................................ 432
4. Other Polyphenols ................................................................................................. 432
5. Tannins ................................................................................................................... 433
C. Black Tea Polyphenols ................................................................................................ 434
1. Residual Green Tea Polyphenols ......................................................................... 435
a . Catechins .......................................................................................................... 435
b . Flavonols .......................................................................................................... 436
2. Theaflavins and Related Products ....................................................................... 437
a. Theaflavins ....................................................................................................... 437
b . Theaflavic Acids .............................................................................................. 438

0735-2689/97/$.50
0 1997 by CRC Press LLC

415
 .
c Other Related Structures ............................................................................... 438
.
3 Further Oxidized Products The “Thearubigens” . ............................................. 439
.
a Theafulvins and Theacitrins .......................................................................... 442
.
b Gallic Acid Production ................................................................................... 444
c. Bisflavanols and Proanthocyanidins ............................................................. 444
.
d Mixed Oxidation Products of Polyphenols
and Other Compounds ................................................................................... 446
. e Aroma Formation from Polyphenol Oxidation ........................................... 447
. 4 Oolong Tea Polyphenols ....................................................................................... 447
.a Oolongtheanins and Theasinensins ............................................................... 447
IV . Biochemistry of Tea ......................................................................................................... 448
A. Caffeine Formation ...................................................................................................... 450
B. Theanine Formation ................................................................................................... 451
C . Biochemistry of Flavonoid Compound Formation ................................................. 452
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1. Phenylalanine and the Shikimate Pathway ........................................................ 452

2. Chain Extension, Hydroxylation .......................................................................... 453
3. Chalcone/Flavone Tautomerization ..................................................................... 454
4. Flavanols/Flavonols ............................................................................................... 454
5. Esterase ................................................................................................................... 455
D. Latent Enzyme Activity and the Formation of Black Tea ..................................... 456
1. Polyphenol Oxidase ............................................................................................... 456
2. Peroxidase ............................................................................................................... 458
E. Extracellular Enzymatic Activity .............................................................................. 458
V. Chemical Properties of Tea Compounds ....................................................................... 459
A. Formation of Cream and Haze in Black Tea .......................................................... 459
B. Complex Formation .................................................................................................... 460
C . Polyphenols as Antioxidants ...................................................................................... 463
1. Chemical Antioxidant Models .............................................................................. 463
2. Biological Antioxidant Models ............................................................................. 465
VI . Trends in Tea Research ................................................................................................... 466
A . Analysis by Chemical Constitution and Technical Innovation ............................. 466
B. Bulk Properties and Correlations to Tea Taster’s Profiles ................................... 467
C . Tea as an Antioxidant and as a Healthy Beverage ................................................. 467
1. Lipid Oxidation and Cardiovascular Disease .................................................... 468
2. Studies of Tobacco Nitrosamines ......................................................................... 469
3. Tea and Cancer ...................................................................................................... 470
VII . Conclusion ......................................................................................................................... 470

416
 Referee: Dr. Alan P. Davies and Dr. Ya Cai, Unilever Research Colworth Lab., Colworth House,
Sharnbrook, Bedford, U.K.

ABSTRACT: The chemistry of tea as a beverage is reviewed in depth, covering both historical
and current chemical perspectives. Special attention is given to the polyphenols in tea, although
the general composition and properties are also treated. Current trends in tea science, particularly
in the area of polyphenol complexation and antioxidant properties, are also covered. The need for
a chemically based understanding, rather than one hypothesized from generalized and indirect
observation, is stressed.
KEY WORDS: tea chemistry, polyphenol complexation, antioxidant properties.

1. INTRODUCTION approximately 35 to 40 1 of beverage. India

The real voyage of discovery consists
riot in seeking new landscapes but in
having new eyes.
- Proust
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has the largest total consumption of tea
(540,000 metric tons, 620 g per capita) and
Ireland has the largest per capita consump-
tion, at 3220 g (Anon., 1994). Camellia
sinensis is a very important agricultural and

commercial product with a unique horticul-

The universe is not only queerer than
we suppose, but queerer than we can
suppose.
-J. B. S. Haldane
It is a picture-perfect image of serenity:
relaxing with a good book, sipping a cup of
hot tea. For thousands of years, the harvest-
ing, processing, and packaging of the leaf of
Camellia sinensis, known worldwide as tea,
has developed as an integral part of society
and culture. With the advent of the post-
colonial scientific era, the perspective of
scientific investigation was added to the tra-
dition and mythology of tea cultivation.
Today, we can look at a cup of tea and
admire the complexities of this beverage and
the plant that makes it possible. Tea chemis-
try has led both consumers and researchers
to debate numerous issues and to probe for a
deeper understanding of the nature of this
beverage. With the growing popularity of
tea and increased awareness of the potential
health benefits associated with tea consump-
tion, tea chemistry promises to endure as a
growing and vibrant field.
After water, tea is the most widely con-
sumed beverage in the world today. Cur-
rently in the U.S., per capita consumption of
tea is approximately 340 g, which produces
ture and manufacturing process.
Chinese mythology teaches that in the
year 2737 B.C. Emperor Shen Nung discov-
ered tea, according to the Chinese medical
book, the Pen T’sao, written during the Han
Dynasty, circa 25 to 221 A.D. The first men-
tion of tea is believed to occur in the Erh Ya,
a Chinese dictionary circa 400 B.C., but the
modern character ch’a, signifying tea, was
not popularized until the writing of The Clas-
sic of Tea, Ch’a Ching, by Lu Yu in 780
A.D. The history of tea is extremely relevant
because before the Tang dynasty (6 I8 to 906
A.D.) tea was probably only considered as a
medicinal, but then became popular as a
beverage. As the method of brewing and
consuming tea varied as tea moved from
culture to culture, the chemical aspects driv-
ing acceptability to the consumer varied.
It is likely that tea was consumed as a
vegetable in a soup through the T’ang dy-
nasty, often mixed with onions, salt, orange
peel, andor ginger. Brick tea, still popular
among the modern Mongolians and moun-
tainous people of the Himalayas, was pre-
pared by steaming and compressing the leaf
into bricks. During the Song Dynasty (960
to 1279 A.D.), however, this practice fell
from favor and was replaced by a powdered
form of the tea, which was whipped into a

417
 froth. A bright green color and low astrin-
gency (derived from careful shading of the
plant) and a delicate aroma accompany pow-
dered green tea. This preparation method of
tea is a custom that survives today in Japan
as Mattcha. The modern custom of brewed
tea leaves arose during the Ming dynasty
(1368 to 1644), coinciding with the West’s
arrival in China. Although there are many
variations on brewing technique, which can
impact flavor and chemistry of the brew sig-
nificantly, it is the basic custom of brewing
the dried tea leaves in hot water that has
been popularized and spread throughout most
of the English-speaking world.
The modern tea industry has its origins
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in the spread of tea cultivation into India
between 1818 and 1834, derived either
through import of the tea plant from China
or through discovery of native Indian tea
var. Assamica, a varietal better adapted to
tropical production and having a larger leaf
style. Development of tea plantations, and
migration of the technology of plantation
operation from India to tropical areas in
Africa, South America, and Russia (Geor-
gia), has established a variety of localized
practices and tea products (Eden, 1976).
Excellent reviews on the culture and produc-
tion of tea are available (Wilson and Clifford,
1992). Figure 1 illustrates the major histori-
cal periods in the development of tea culti-
vation and processing worldwide.
Through cultivation, tea has become an
important agricultural product throughout the
world, particularly in regions lying close to
the equator. Geographical areas that receive
annual rainfall of at least 50 inches per year
and have a mean average temperature of
30°C are the most favorable for growth and
agriculture of tea (Eden, 1976).
Traditionally, C. sinensis has been propa-
gated, hybridized, and bred through seeds.
To maintain clonal purity and to accelerate
establishment of new productive stands of
tea, vegetative propagation is now a com-
41 8
mon practice. This involves planting leaf
cuttings in nurseries where they develop into
seedings within 6 months. The seedings are
then transplanted to the fields. The tea plant,
once established, will be economically vi-
able for decades barring disease, infestation,
or other destructive forces.
The tea tree is maintained as a shrub
during the growing season through frequent
manual harvesting, about every 8 to 12 d
during prime growing season. Tea is an ev-
ergreen tree, but it is largely dormant through
the winter season. Mechanical harvesting
methods have been developed, but these are
only popular where labor is expensive and
where tea is not grown on steep mountain
slopes. Plantations also maintain unharvested
tea trees for seed production.
Immediately after harvest, the tea leaves
(usually the flush, or first two leaves and the
bud of the growing tea shoot) are brought to
factories situated close to the tea gardens for
manufacturing. It is the manufacturing pro-
cess that determines the type of tea pro-
duced. There are three general types of manu-
factured tea: Green (unfermented), Oolong
(partially fermented), and Black (fully fer-
mented). The manufacturing processes used
to produce each type of tea differ in the
degree of enzymatic oxidation or “fermenta-
tion”. Fermentation refers not to an exog-
enous, microbial process, as with beer or
wine, but the natural browning reaction cata-
lyzed by enzymes endogenous to the plant.
The green tea manufacturing process
involves the rapid steaming or pan firing of
the freshly harvested leaves to inactivate
enzymes, preventing fermentation, produc-
ing a dry, stable product. Green teas are
typically produced in two categories: “White
tea” and “Yellow tea”, the latter is withered
(wilted), resulting in a small degree of fer-
mentation (Bokuchava and Skobeleva, 1980).
There is some variety of terminology be-
tween Chinese and Japanese green tea manu-
facture, and with the increase in popularity
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l
m
c
c
0

-
a,

419
 of green tea worldwide, pilot production of
green teas in other regions, such as Darjeeling
in India, from traditionally black tea variet-
ies have led to a wide variety of green tea
products on the market.
When oolong and black teas are to be
produced, the fresh leaves are allowed to
wither until the moisture content of the leaves
is reduced to 55 to 72% of the leaf weight.
This causes a concentration of polyphenols
in the leaves and deterioration of leaf struc-
tural integrity. Withering is important for
aroma development. The withered leaves are
rolled and crushed, initiating fermentation
of the tea polyphenols. The fermenting mass
formed from rolling and used in black tea
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manufacture is referred to as dhool. The pro-
cess used to macerate the leaf plays an
important role in the final grade of tea.
Two common methods are Orthodox and
CTC. Orthodox rolling of the leaf is per-
formed by mechanically applying weight or
compression to the leaves. Orthodox pro-
cessing is typically used for production of
large-leaf finished tea products. CTC (crush,
tear, curl) processing is a significant modem
improvement of this procedure that minces
the leaf in a continuous, high-yielding pro-
cess and produces smaller-leaf teas.
Oolong teas are prepared by firing the
leaves shortly after rolling to terminate the
oxidation process and dry the leaves. The
rolling process for oolong teas is only de-
signed to slightly damage the leaf and impart
‘twist’ to the finished product. Black teas are
prepared through a separate fermentation
process in which cooled air is circulated
through the rolled and crushed leaves to
moderate the reaction, as the onset of fer-
mentation is accompanied by a rise in tem-
perature from the exothermic fermentation
process. This fermentation process results in
the oxidation of simple polyphenols to more
complex condensed polyphenols that give
oolong and black teas their bright red colors
and brisk astringent flavor. The degree of
420
fermentation of the dhool largely determines
the flavor charateristics of the finished prod-
uct. Fermented tea leaves are then fired to
inactivate the enzymes and dry the leaves.
This procedure is accompanied by the final
chemical transformations to the product re-
sulting from the high temperatures involved
in firing. Figure 2 highlights the differences
in manufacture of the most common tea
products.
The manufactured teas are then sized,
graded, and evaluated for flavor and infu-
sion color by professional tea tasters. The
teas are packaged into sacks or wooden chests
and are sold at the world tea auctions. Total
world production of tea in 1993 was 2.58
million metric tons, of which 0.59 million
metric tons was green tea and 1.89 million
metric tons was black tea. India and China
are the major tea-producing countries, manu-
facturing 53% of tea produced (Anon., 1994).
Figure 3 illustrates available consumption
data of tea worldwide.
There are numerous polyphenolic com-
pounds produced by the growing tea plant,
and the pathways for in vivo biosynthesis of
these phytochemicals have been elucidated
generally. The chemistry of green and black
tea, therefore, typically centers on the
polyphenolic composition of these teas, with
polyphenols being the major proportion of
extracted solids in black teas. Although this
is a Western bias, both in terms of chemistry
of production and beverage as well as the
recent interest in the health-promoting as-
pects of tea, it is important that the overall
composition be given careful consideration.
The organization of this review attempts to
reemphasize these areas, in contrast to pre-
vious reviewers.
One of the most comprehensive reviews
of tea chemistry availableis that of Sanderson
(Sanderson, 1972). Complementary to
Sanderson’s review is that by the Russian
tea chemists Bokuchava and Skobeleva
(Bokuchava and Skobeleva, 1980), which
 The Tea Plant I
Jp Iu cking
pGiGl
s r e e of 'fermentation'
Withering

Withered
I"White" tea
(green tea)
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Flush
I

Rolling
t
I
"Ye IIow" tea
(green tea)

1
'Fermentation' Firing "Red" tea
(oolong tea)

Fermented
Dhool

1 BlackTea
FIGURE 2. The tea manufacturing process.

421
 India 560
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China 408
Iran 85
Pakistan 117
FIGURE 3. Consumption of tea, 1993 (lo3metric tons).
provides a variant perspective and insight
into Russian-language literature sources,
which are typically not cited in English-lan-
guage publications. HPLC techniques, now
available for rapidly measuring a number of
important tea polyphenols, including the cat-
echins, flavonols, flavonol glycosides, and
the theaflavins, have dramatically changed
the way tea chemistry is studied, and these
methods have been reviewed in detail (Fin-
ger et al., 1992).
There are a variety of other reviews on
the subject of tea, including Roberts’ (Rob-
422
13i2
tJK 151
0’
ther 22 1
:0 30
22
Turkey 124
erts, 1942; Roberts, 1962) and Graham’s
(Graham, 1983; Graham, 1984) reviews of
teachemistry,a review by Stahl (Stahl, 1962)’
Wickremasinghe (Wickremasinghe, 1978),
Balentine (Balentine, 1992), and a review by
Robertson (Robertson, 1992). An excellent
comprehensive review of the work on tea
aroma to the early 1980s is also available
(Bokuchava and Skobeleva, 1986), as well
as a more recent review (Robinson and
Owuor, 1992).
Reviews published subsequent to that of
Sanderson appear to attempt an update of the
 subject, but none have critically reassessed
the field. It is hoped that this review can take
some of the historical material, combined
with a comprehensive and critical account of
recent research, and synthesize a review of
tea chemistry as it is currently viewed and
applied in our laboratory. An overemphasis
on polyphenols, compounds that are signifi-
cant to color and astringency but less rel-
evant to teas such as shaded green tea pro-
duction, dominates the literature. Low
polyphenol content and high chlorophyll
content are important for producing an em-
erald-green and smooth finished green tea
product that lacks astringency, and the brothy
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tion, there are differences between the com-
position of the tea leaf and other phytological
components, as well as differences between
the leaf and the “brew”. It is therefore diffi-
cult to synthesize an overall picture of the
chemistry of tea without resorting to a sub-
division of the problem.
With this in mind, we have attempted to
focus on the “extract solids”, the component
of tea leaves that is extracted by boiling
water. Table 1 gives an approximate compo-
sition of black and green tea beverage solids
by chemical class. Although the length of
time for steeping and the amount of water in
which the leaves are steeped can vary widely,

character of the amino acids, and flowery these factors generally control the amount of
character of the aroma constituents, play a solids extracted, and to a lesser extent influ-
much more significant role. In addition to ence the composition. A typical brew of one
the nonpolyphenolic constituents, the tea bag in one cup of water produces a solu-
polyphenols of black tea are a poorly under- tion of 0.35% wtfwt solids, and from this
stood but seemingly well-defined group of value the dose expected from consumption
compounds. The use of “theambigen” in the of one cup of tea can be calculated. This is
literature signifies a wide group of com- typically how tea phytochemicals are con-
pounds whose chemical identity has not been sumed. Notes on the composition of the
traced to any identifiable chemical group. Its ‘flush’ or the fresh tea leaves, or other parts
use was most significant as a colorimetric of the tea plant, are added for completeness
indicator for plantation production, but as a
chemical identifier its use has seemed to
hamper true identification of the mass balance TABLE 1
The Composition of a Typical Tea
of black tea polyphenols. Work at identifying
Beverage, %wt/wt Solids
these compounds in a systematic fasion has
begun, and it is hoped that future research Green tea Black tea
will deemphasize the term ‘theambigen’ in
favor of more chemically accurate des- Catechins 30% 9%
Theaflavins 4%
criptors. Simple polyphenols 2% 3%
Flavonols 2% 1%
Other polyphenols 6% 23%
II. CHEMICAL COMPOSITION, AN Theanine 3% 3%
OVERVlEW Aminoacids 3% 3%
PeptidedProtein 6% 6%
Organic acids 2% 2%
In order to divide the subject of tea chem- Sugars 7% 7%
istry into well-ordered issues, it is most con- Other carbohydrates 4% 4%
venient to do so on the basis of chemical Lipids 3% 3%
Caffeine 3% 3%
composition. Frequently, researchers’ inter- Other methylxanthines <1Yo <1 Yo
est in specific compounds or a class of com- Potassium 5% 5%
pounds induces a focusing of attention on a Other mineraldash 5% 5%
small fraction of the total tea mass. In addi- Aroma Trace Trace

423
 within the review. We have also subdivided B. Caffeine, Methylxanthines, and
the issue of tea compounds by chemical class. Related Compounds
This is done to focus on specific aspects of
tea chemistry, such as aroma or polyphe- Tea has been valued historically for
nols, and because the methods used for de- its caffeine content. Caffeine [ 13 is viewed
termination of the various chemical classes as an important constituent of tea, bestow-
are quite different. ing mood and cognitive-enhancing proper-
The strict division of tea chemistry by ties (Bokuchava and Skobeleva, 1980).
functional chemical identity has its uses, but Figure 4 illustrates the methylxanthines of
begins to lose value in discussing subjects tea. Tea leaves contain between 2 and 5%
that cross the boundaries of chemical class wt/wt caffeine depending on the variety.
or those that have no distinctive chemical
identity, such as discussions concerning the
‘thearubigens’. Modern tea research has
reached the point where simple chemical
subdivision has been refined with a fair de-
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gree of accuracy, and determination of the

synergies and ‘boundary violations’ present
in the chemical composition of tea bever-
ages becomes a necessity.
[I]:
Caffeine
A. Polyphenols

In terms of human consumption, tea rep-

resents a major source of dietary polyphe-
nols. The polyphenolic fraction of tea repre-
sents 30 to 40% wt/wt of extract solids and
provides astringency, the ‘drying’ sensation
experienced in the mouth after consumption
of the tea beverage. A tea drinker typically
consumes 180 to 240 mg of polyphenols
from a strong cup of tea. Recent interest in [Z]: Theobromine
the health aspects associated with consump-
tion of tea beverages has grown within the
scientific community and has generated much
excitement about tea polyphenols.
The tea plant produces a diverse number
of polyphenolic constituents, presumably as \ N d COOH
a means of chemical defense against insects,
birds, and animals, which would consume 0
the plant as food (Beart et al., 1985). The
evolution of salivary proline-rich proteins,
which bind polyphenols effectively, has [3]: Theanine
ameliorated this defense mechanism, con-
verting it to ‘astringency’ (Luck et al., 1994). FIGURE 4. Nitrogenous tea phytochemicals.

424
 C. irrawadensis, a member of the Camellia
family, lacks caffeine (Roberts et al., 1958)
but is not processed commercially because it
produces a poor finished tea product. The
quantity of caffeine that infuses into a tea
brew is determined by infusion time and by
leaf style. Longer infusion times lead to
greater quantities of caffeine in a tea bever-
age. Smaller sized tea leaves give a more
rapid and stronger infusion, whereas larger
leaves and uncut leaves lead to weaker infu-
sions. This results in more or less caffeine
extraction, respectively. The caffeine con-
tent of a typical tea beverage will range from
20 to 70 mg per 170 ml of infusion, with a
typical infusion being prepared from about 2
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to 2.5 g of tea leaves. Coffee brews typically
contain from 40 to 155 mg caffeine per
170 ml beverage.
There has been little research done on
the pharmacology of tea-beverage caffeine.
One study suggests a dose of caffeine from
tea has a different physiological effect than
a pure dose of caffeine (Das et al., 1965).
This has been attributed to the amino acid
theanine, which is unique to tea. However,
there are no well-designed clinical studies to
support this position. The consensus among
scientists today is that caffeine from all bev-
erage sources has a similar physiological
effect. The actual content of caffeine de-
pends on many factors, particularly the
method of brewing. A brew prepared by the
Chinese “gong-fu” style is likely to have a
different caffeine impact compared with the
Western style of loose tea or to that from a
tea bag (Hicks et al., 1996). Some reports
have suggested that green tea contains sig-
nificantly less caffeine than black tea. This
may be influenced by the clone of leaf used
to produce the tea or by the impact of differ-
ent brewing techniques. No significant dif-
ferences have been found when brewing
green and black teas under similar condi-
tions (Hicks et al., 1996), discrediting the
theory that withering and fermentation have
a significant impact on caffeine content
(Sanderson, 1972).
Caffeine is one of the most comprehen-
sively studied ingredients in the food sup-
ply. Extensive research does not link moder-
ate caffeine intake to any health risks. Studies
are needed to better understand the physi-
ological role of tea caffeine and its associa-
tion with the popularity of tea beverages.
Those individuals who are especially sensi-
tive to caffeine can find decaffeinated teas
readily available.
1. Theobromine
Theobromine [2] is present in tea in much
lower quantities than caffeine. Theobromine
is formed as a consequence of the biosynthe-
sis of caffeine (Negishi et al., 1985a) and is
produced in abundance if the methylation
path to caffeine is absent, such as in
C. irrawadensis (Roberts et al., 1958). Theo-
phylline, a similar di-methylxanthine, has
been reported in trace quantities in tea leaves
(Michl and Haberler, 1954; Sanderson, 1972).
Recent reports contradict as to the the exist-
ence of this compound in tea, some failing to
detect these compounds (Hicks et al., 1996)
and others (Meyer et al., 1996) reporting
small quantities. The xanthine content of teas
is clearly an area that requires further, more
careful research.
2. Other Derivatives of Nucleic
Acids
The RNA and DNA in tea leaves are
metabolized naturally as well as digested
under the conditions of withering and fer-
mentation by tea nucleases, nucleosidases
(Imagawa et al., 1982), and a specific ad-
enine nucleosidase (Imagawa et al., 1979).
These catabolic reactions produce purines,
which have been detected in very small quan-
425
 tities in tea (Michl and Haberler, 1954;
Sanderson, 1972; Hicks et al., 1996).
C. Proteins and Amino Acids
While caffeine is the most well-known
nitrogenous component of tea, tea proteins/
peptides and amino acids contribute signifi-
cantly to the composition of both the leaf
and the tea extract. Figure 4 illustrates some
of the other important miscellaneous nitrog-
enous and nonnitrogenous components of
tea. Recent measurement of the amino acids
in two green teas (Liang et al., 1990) con-
firms the presence of 18 amino acids, a re-
sult that is mirrored for black tea. Amino
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acids contribute about 6% wvwt of the ex-
tract solids. Tea also contains a significant
amount of peptidic material (protein), ap-
proximately 6% wtjwt of extract solids. Ni-
trogenous materials therefore comprise about
15% wtjwt of extract solids. The free amino
acid content of tea seems to increase during
withering of the fresh tea leaves but de-
creases during fermentation to black tea
(Roberts and Sanderson, 1966), as it is
likely consumed during aroma biogenesis
and through other routes. These pathways
have a strong impact on the aroma of the
finished product and require more detailed
investigation.
In addition to the common amino acids,
there is a unique amino acid known only to
be present in tea. This amino acid, theanine
(y-N-ethyl glutamine, [3]), is believed to be
the major amino acid present in tea, com-
prising about 3% wtjwt of extract solids.
Theanine is a significant component of both
green tea (Sakato, 1950) and black tea
(Feldheim et al., 1986). Theanine has been
associated with improved flavor and a modu-
lation of the stimulative effects of caffeine
(Kimura and Murata, 1971). Recent studies
on the antihypertensive effect of theanine
found that a large quantity of this compound
was required to exhibit an effect in rats
(Yokogoshi et al., 1995).
426
Optimum production of theanine in cell
culture has been investigated (Furuya et al.,
1990; Matsuura et al., 1992). Theanine
can also be made synthetically on a com-
mercial scale in good yield (Kawagishi
and Sugiyama, 1992).
Tea leaves subjected to anaerobic condi-
tions are found to produce excess amounts
of GABA, or y-amino butyric acid (Tsushida,
1987), derived from glutamic acid due to the
action of an endogenous glutamate decar-
boxylase in tea (Tsushida and Murai, 1987).
D. Carbohydrates, Pectins, and
Fiber
Tea leaves have been shown to contain
free sugar residues in addition to pectic
subtances, polysaccharides, and fiber
(Mizuno et al., 1964; Sanderson and
Perera, 1965). Carbohydrates contribute ap-
proximately 11% wtjwt of extract solids
(Sandersonet al., 1976;Graham, 1984).High
molecular weight pectins (polygalacturonic
acid) and other polysaccharides have been
analyzed on Sephadex G-100 (Millin et al.,
1969). As the tea plant matures, increases in
the content of lignin and cellulose have been
observed (Selvendran et al., 1972), which is
consistent with their role in providing struc-
tural integrity to the growing plant.
A significant portion of the carbohydrate
fraction in tea extract has been found to
comprise the disaccharide2-O-(P-~-Arabino-
pyranosy1)-myo-inositol [4] (Sakata et al.,
1987), as shown in Figure 5. It was detected
by NMR techniques in an aqueous fraction
subjected to consecutive extraction with ethyl
acetate and butanol (Sakata et al., 1989).
E. Organic Acids and Vitamin C
Tea is a significant source of oxalic acid
(Sanderson and Selvendran, 1965) and malic
acid (Jayman and Sivasubramanian, 1975),
along with citric, isocitric, and succinic ac-
ids (Sanderson and Selvendran, 1965). Tea
 HO@"
HO 0
[4]: 2-0-( P-L-Arabinopyranosy1)-myo-inositol
OH
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[51: Spin asteroI
FIGURE 5. Miscellaneous tea phytochemicals.
also contains shikimic and quinic acids,
which are important to the biosynthesis of
the polyphenols (Zaprometov, 1961). Vita-
min C (ascorbic acid) has also been detected
in green tea (Liang et al., 1990) and black
tea (Sanderson, 1972).
6. Lipids, Chlorophylls,
Carotenoids, and Related
Compounds
The main pigments in the fresh tea leaf
are chlorophylls and carotenoids. Chloro-
phylls (e.g., [39]) are oxidized during the
course of black tea manufacture to the
pheophytins and pheophorbides (e.g., [40]),
which give the fermented leaf its character-
istic brown-black color. Some of the
pheophytins and pheophorbidesare extracted
into the black tea beverage (Sanderson,
1972). An efficient HPLC method has been
developed for analysis of these pigments
(Taylor and McDowell, 1991). The compo-
sition of these components in the green tea
leaf has been demonstrated to have a strong
impact on the quality of the beverage as
perceived by tea tasters (Taylor et al., 1992).
Tea grown in the shade has been found to
have a lower quantity of catechins (resulting
in a less astringent beverage) and increased
levels of carotenoids and chlorophyll (which
may assist in aroma production). This chemi-
cal balance is thought to contribute favor-
ably to taste (Mahanta and Baruah, 1992).
The carotenoids play a significant role in the
formation of aroma characteristic to black
tea (Bokuchava and Skobeleva, 1986). High
chlorophyll levels and low astringency are
important to some green tea manufacture,
particularly Mattcha, for which a brothy,
emerald-colored brew is very important.
Lipids, terpenoids, and saponins make
up a large portion of the fresh tea leaf, yet
because of their low water solubility, are
generally thought to be a minor portion of
427
 the water extract solids. However, plant ste-
roids such as spinasterol[5] or lipids such as
the plant cuticle wax triacontanol [6] have
been shown to comprise an important frac-
tion of tea cream, the precipitate that forms
after cooling of concentrated tea extracts
(Seshadri and Dhanaraj, 1988). It is hypoth-
esized that the hydrophobic environment
presented by the tea polyphenols and caf-
feine complex in tea provide for extra solu-
bility of the lipid components. Lipids are
approximately 3 to 4% wt/wt of leaf and has
been analyzed in detail (Anan, 1983; Bhuyan
and Mahanta, 1984). The role of hydropho-
bic plant materials in the appearance and
organoleptic properties of the brew has been
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typically disregarded in favor of the polyphe-
nols, and this is an important area for which
future research is necessary.
G. Vitimans and Minerals
The tea plant has been shown to be rich
in potassium (Sanderson et al., 1976) and
contains significant quantities of calcium and
magnesium, as well as small amounts of
manganese, iron, and phosphorus (Kalita
and Mahanta, 1993), copper and nickel
(Burke and Albright, 1970), and sodium,
boron, and molybdenum (Hasselo, 1965).
Zinc (Tolhurst, 1962) and sulfur (Pethiyagoda
and Krishnapillai, 1970) are essential ele-
ments for healthy maturation of the tea plant
as well. Cobalt, lead, and cadmium have
been detected in the plant, and concentra-
tions depend principally on soil concentra-
tions of these minerals (Ramakrishna et al.,
1987).
The tea plant is known to accumulate
aluminum (Chenery, 1955). Aluminum lev-
els can be traced by NMR techniques (Nagata
et al., 1991) and have been found in com-
plexes with fluoride and catechins in the tea
plant (Nagata et al., 1993). Tea polyphenols
are commonly thought to complex with min-
428
erals, and may be excellent chelating agents.
The large number of phenolic hydroxyl
groups provides a great number of potential
active complexation sites.
Tea beverages are also a significant
source of fluoride (Elivin-Lewiset al., 1980).
This is due, in part, to the uptake of alumi-
num fluoride (Yamada and Hattori, 1977;
Yamada and Hattori, 1980).
H. Aroma
The essential oil or aroma of tea pro-
vides much of the pleasing flavor as well as
scent of green and black tea beverages, yet
comprises only a minor fraction of the total
mass of the tea plant or the extracts. Tea
aroma contains hundreds of compounds in
trace quantities, the composition and mecha-
nism of production of which has been re-
viewed (Choudhury, 1982; Bokuchava and
Skobeleva, 1986; Robinson and Owuor,
1992).
Withering has been determined to play
an important role in aroma development, as
in oolong teas (Takeo, 1984; Kharebava,
1986) and black teas (Owuor et al., 1987).
Many of the aroma components of tea can be
found as glycoside derivatives, which are
freed during the fermentation process due to
the action of glycosidases.Fresh tea enzymes
permit the release of additional aroma con-
stituents in the leaf and extract, restoring
fresh aroma from stale tea (Guo et al., 1992).
Geranyl, linolyl, terpinyl, and neryl glyco-
sides can be found in fresh tea extracts (Guo
et al., 1993). P-Glucosidase is the enzyme
most likely to be responsible for formation
of tea aroma from these glycosides (Morita
et al., 1994). The exploitation of bound gly-
cosides of aroma components by glycosi-
dase treatement offers the possibility of fu-
ture improvements in tea quality.
Part of the aldehyde fraction may be
generated from a unique tea leaf amine oxi-
 dase (Tsushida and Takeo, 1985). Tea also
contains a fatty acid hydroperoxide lyase,
which forms volatile aldehydes from the lipid
constituents of the tea leaf (Matsui et al.,
1991). There are also many products that are
derived from the oxidizing conditionspresent
during tea fermentation (Bokuchava and
Skobeleva, 1986). Figure 6 illustrates one
such unique aroma constituent, theaspirone
[9], which is produced from oxidation of
p-carotene [7].
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[7]:p-Carotene
J
[8]: p-lonone
[91: Theaspi ro ne
FIGURE 6. Unique tea aroma constituents.
111. POLYPHENOLS IN TEA
The term ‘polyphenol’ is an inclusive
descriptor referring to the millions of natural
and synthetic aromatic molecules that are
substituted with multiple hydroxyl groups.
The polyphenols comprise one of the most
distingishing characteristics of the tea plant
and have been more thoroughly investigated
than any other class of compounds in tea.
For this reason, the polyphenols in tea are
many other aroma constituents
429
 treated in greater depth in this section and
separate from the overall review of the chemi-
cal constituents of tea.
Because of the abundance of polyphe-
nols present in tea leaves and in tea bever-
ages, it is natural that tea chemistry is often
considered to be synonymous with tea
polyphenol chemistry. The polyphenols are
principally responsible for the color and as-
tringency and partially responsible for the
flavor of the tea beverage. The compounds
are known antioxidants and are being stud-
ied as agents that might reduce risk factors
associated with cancer and heart disease.
While careful attention needs to be paid to
the overall chemistry of the tea plant and
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beverage, it is no surprise that this review
and much of the tea literature is dominated
by the discussion of the tea polyphenols.
A. Chemical Classification
The polyphenols in tea may be subdi-
vided by several chemical backbone struc-
tures. Simple tea polyphenols are those that
are synthesized during the early stages of
polyphenol biosynthesis, whereas the degree
of complexity of the polyphenols increases
as one progresses down the biosynthetic
pathway. The flavonoids, a subgrouping of
polyphenols and the dominant class of green
tea polyphenols, are synthesized in part from
the simple polyphenols and represent com-
pounds with 15 or greater carbon atoms [C,,
stage] in the basic framework. The polyphe-
nols of black tea represent further chemical
transformations of the green tea polyphenols
and therefore comprise a third level of com-
plexity. Unique black tea polyphenols are
commonly thought to be polymers of the
green tea polyphenols and therefore are
thought to be comprised of molecules of
approximately 30 carbon atoms [C,, stage]
or greater, as the simplest polymer would be
a dimer such as procyanidin.
430
All too frequently, popular writing on
tea compounds confuse the nearly hom-
onymic flavonoid classifications. Flavonoids
are the widest subgrouping, including
flavanols, which have a saturated central (C)
ring and include the catechins, the major
green tea polyphenols, and flavonols, which
have an unsaturated central (C) ring and a
ketone group. Careful attention must be paid
when reading any of these three classifica-
tions. These classifications are all inclusive
under the broader term polyphenol, which
refers to any compound that contains aro-
matic rings with multiple pendant phenolic
OH groups or derivatives thereof.
B. Green Tea Polyphenols
Green tea polyphenols consist of both
simple and complex polyphenols. The large
majority of polyphenols in green tea are fla-
vonoid monomers called catechins and fla-
vonols.
1. Catechins and Gallocatechins
The catechins [lo-131 represent the ma-
jor polyphenolic constituent of green tea and
are illustrated in Figure 7. Catechins are
members of a more general class of fla-
vonoid, the flavan-3-01s (also referred to as
flavanols). Three subgroupings of the
flavanols [afzelechin [14], catechin [ 151,
gallocatechin [16], shown in Figure 81, rep-
resenting varying degrees of B-ring hydroxy-
lation, are the dominant forms, of which the
epi-isomers of the catechins and gallo-
catechins are the principal components found
in tea. The tea catechins, a term commonly
used to refer to both catechins and gallo-
catechins, make up as much as 30%wtjwt of
dissolved solids.
A large percentage of the catechins
present in tea exist as gallic acid esters. While
 OH

R1 R2
[I
01: Epicatechin EC H H
I]:
[I Epicatechin Gallate ECG Gallate H
[I
21: Epigallocatechin EGC H OH
[I
31:Epigallocatechin Gallate EGCG Gallate OH
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FIGURE 7. The principal tea catechins.

OH

R1 R3
[14]: Afzelechin H H
[I 51: Catechin C OH H
[ I 61: Gallocatechin GC OH OH
FIGURE 8. The flavan-3-01s.

gallation is found to occur principally at the
3-positionYvarious other gallated species
have been isolated, including the epigallo-
catechin digallates (Nonaka et al., 1983), and
epicatechin digallate (Coxon et al., 1972;
Hashimoto et al., 1987). 3-Methyl gallates
of EC and EGC have also been reported
(Saijo, 1982). EGCG was once thought to be
unique to the tea plant (Graham, 1983) but
now has been isolated from other sources
(Danne et al., 1994).
The four most common catechins are
epigallocatechin gallate (EGCG, [ 13]),
epigallocatechin (EGC, [12]), epicatechin
gallate (ECG, [ 1l]), and epicatechin (EC,
[ 101). Catechin (C, [ 151) and gallocatechin
(GC, [16]) are also present in smaller quan-
tities. While gallocatechin gallate (GCG) and

431
 catechin gallate (CG) have also been ob-
served, it is likely that these are products of
racemization and not ‘native’ to the tea plant
(Roberts, 1962; Robertson, 1992).
2. Flavonols
The flavonols [kaempferol,quercitin, and
myricitin] and their glycosides [ 17-19] have
only been recognized recently as significant
components in tea, although their presence
as trace constituents has always been ac-
knowledged. The flavonols are illustrated in
Figure 9 Analyses of the flavonol glycosides
in general (McDowell et al., 1990) and of
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the flavonol diglycosides (Finger et al.,
1991a) and triglycosides (Finger et al.,
1991b) in tea leaf and of flavonol glycosides
in tea seed (Sekine et al., 1991; Sekine et a1
1993) have been performed.
Use of hydrolysis to determine flavonol
and flavonol glycoside content as their agly-
cones (Hertog et al., 1993) has proven to be
useful in determining overall flavonol con-
tent (about 0.5 to 2.5% wt/wt extract, as
aglycone) of tea infusions.
[17]: Kaempferol Glycoside KaG
[I81:Quercitin Glycoside QuG
[I91: Myricitin Glycoside MyG
FIGURE 9. The flavonol glycosides.
432
3. Simple Polyphenols
Gallic acid [20] and its quinic acid ester
(or depside, as quinic acid esters are com-
monly referred), theogallin [21], have been
identified in tea (Cartwright and Roberts,
1954; Cartwright and Roberts, 1955) and
have been detected by HPLC (Bailey et al.,
1990; Hashimoto et al., 1992). The simple
polyphenols and their depsides are shown in
Figure 10.
Cinnamic acid derivatives of quinic acid,
the coumaryl and caffeoyl- quinic acids (in-
cluding chlorogenic acid or 5-caffeoylquinic
acid [23]) have also been identified in tea
(Cartwright et al., 1955). Chlorogenic acid
and 4-coumarylquinic acid [22] have been
detected by HPLC (Bailey et al., 1990).
4. Other Polyphenols
Flavones and their glycosides (Engel-
hardt etal., 1993), such as apigenin [24],
have been detected in tea but represent a
very small fraction of the polyphenols
present. Flavone glycosides can potentially
R1 R3
H H
OH H
OH OH
 [20]: Gallic Acid

.OH

\
\ C A
H O O OH
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O H V O H [21]: Theogallin
OH

o ? O H

0
[22]: Coumarylquinic acid, R=H
H oOHo c ~ o , , [23]: Chlorogenic acid, R=OH
OH

FIGURE 10. Gallic acid and the depsides.

be measured quantitatively as aglycones Flavan-3,4-diols such as leucocyanidin [27]

(Hertog et al., 1993). Careful resolution of
individual flavone glycosides (Engelhardt
et al., 1993) has confirmed their presence in
tea. Apigenin is illustrated in Figure 11.
A number of proanthocyanidin species,
such as prodelphinidin B2 [25] gallates, have
been isolated and are present in green tea
(Nonaka et al., 1983; Nonaka et al., 1984).
The assamicains, such as assamicain A [26]
isolated from C. sinensis var. assarnica, ap-
pear to be ring-opened products of the
proanthocyanidins (Hashimoto et al., 1989a).
have also been reported (Roberts et al., 1956).
These are illustrated in Figure 12.
5. Tannins
Although it is commonly stated that there
are no tannins (meaning hydrolyzable tannins
such as pentagalloylglucose [28]) in tea, this
statement is not strictly true. In addition to
the gallic acid esters of the catechins and
their oxidation products (which can be hy-
drolyzed to produce gallic acid readily and

433
 [24]: Flavone (Apigenin)
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HO

[25]: Proant hocyanidin

(EpigaIlocat echin-4-a-Epigallocatechin,
or Prodelphinidin B2)

FIGURE 11. Miscellaneous polyphenols.

precipitate proteins), there is also a small C. Black Tea Polyphenols

quantity of hydrolyzable tannin (Nonaka
et al., 1984; Yoshida et al., 1990; Hatano
et al., 1991; Han et al., 1994). The unique
hydrolyzable tannins in tea are typically
“hybrid” tannins such as camelliatannin A
[29], which is a galloylglucose derivative
with pendant catechins. The tannic acid de-
rivatives common to gall-nuts and tree bark
are not present in significant quantities in tea
infusions. The tea tannins are illustrated in
Figure 13.
Black tea polyphenols are produced
from the controlled enzymatic reactions
involved in the fermentation of green leaf
during commercial and model black tea
production. The extent and conditions un-
der which fermentation occurs determines
the degree to which the polyphenols of green
tea are transformed to those unique to black
tea. It is reasonable to expect that black tea
should contain an amount of polyphenols

434
 OH
i)H
[26]: Assarnicain A
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[27]: Flavan-3,4-diol
(Leucocyan id in)
FIGURE 12. Miscellaneous polyphenols.
similar to green tea. However, the complex
nature of these polyphenols, some of which
are “polymeric” in nature, has largely re-
sisted chemical identification. The uniden-
tified polyphenolic constituents are often
referred to as thearubigens. Despite their
complexity, some of the unique black tea
polyphenols have been identified and char-
acterized.
1. Residual Green Tea Polyphenols
During the course of fermentation, the
polyphenols of green tea are rapidly con-
verted to the polyphenols of black tea. De-
Ga=Gallate
OH
pending on the degree of fermentation, how-
ever, some green tea polyphenols remain
unconverted. This is particularly true in the
case of oolong teas and some Darjeeling
teas, which have been known to resemble
green tea both in chemical constitution (Ding
et al., 1992a) and astringency.
a. Catechins
The catechins represent the major por-
tion of green tea polyphenols and conse-
quently are thought to be the building blocks
of black tea polyphenols. Some of the green
tea catechins survive the fermentation pro-
435
 OH
I

-OH

[28]: Hydrolyzable Tannin

(Pe ntagaIIoyIgIu cose)
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OH

HO
OH
OH
HO
OH HO

[29]: Camelliatannin A

FIGURE 13. Tannins.

cess and are detected in black tea (Bailey
et al., 1990). Due to the oxidation reactions
and thermal conditions experienced by the
tea leaf during black tea production, it is
hypothesized some of the catechins are also
epimerized and/or degallated, which explains
the appearance of free gallic acid as well as
increased levels of non-epi isomers of the
catechins (Coggon et al., 1973).
b. Flavonols
It is likely that the majority of flavonols
(free as well as glycosides) present in the

436
 initial fresh green leaf remain unoxidized
and are likewise present in black tea in simi-
lar quantities. There is some evidence that
some of the flavonols are oxidized during
fermentation. It has been suggested that
myricitin and myricitin glycosides are the
most likely oxidized of the three flavonols
(kaempferol, quercitin, myricitin) (McDowell
et al., 1990).
Despite this, in a recent analysis of fla-
vonols and their glycosides (Hertog et al.,
1993), no significant difference between the
green teas and the black teas was found in
terms of total flavonol as aglycone, except
for myricitin, which was found to be slightly
reduced in the black tea samples compared
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with green teas. Differences between these
samples might have also been derived from
different origins for the two analyzed mate-
rials, however, owing to the variations in
flavonol content of teas grown from differ-
ent clones and from different regions of the
world.
2. Theaflavins and Related Products
One of the key distinctions of black teas
compared with green tea is the production of
a new type of polyphenol, the theaflavins.
The fermentation of green tea leaf also re-
sults in the development of characteristic
aroma components, a darkening of color of
the leaf and extracts, and a decreasing astrin-
gency with increased fermentation time.
a. Theaflavins
Best known of the fermentation prod-
ucts is the class of compounds known as the
theaflavins [30-331, comprising about 3 to
5% wtlwt of the extract solids. They are
illustrated in Figure 14. Theaflavin provides
a bright, red-orange appearance to the tea
beverage and has long been positively corre-
lated with market value of tea (Roberts,
1958). Market value for tea is also influ-
enced by secondary factors such as aroma,
as observed with Kenyan teas that normally
contain higher theaflavin contents (Owuor
et al., 1986) and therefore not a distinctive
characteristic.
While characterized by a unique benzo-
tropolone ring structure resulting from the
dimerization of a catechin and a gallo-
catechin, there are a series of related com-
pounds, including the isotheaflavins,
neotheaflavins, and theaflavic acids, which
also possess a similar benzotropolone unit.
The benzotropolone ring provides the red
color and makes the theaflavins easily dis-
tinguishable from other components.
Analysis of theaflavins began with ex-
traction of water extracts (Roberts, 1958;
Spiro et al., 1987; Robertson and Hall, 1989)
into isobutyl methyl ketone (Roberts and
Smith, 1963) or ethyl acetate (Ullah, 1972),
followed by spectrophotometric measure-
ments. Later spectrophotometric methods
improved on this technique (Xiao and Li,
1992). The Flavognost method (Hilton, 1973)
uses diphenyl boric acid ethanolamine to
induce a spectrophotometric shift in the
benzotropolone ring for better accuracy.
Recent improvements in analytical technol-
ogy include the use of GC (Collier and
Mallows, 1971), and HPLC (Robertson and
Bendall, 1983; Steinhaus and Engelhardt,
1989), as well as NIR measurements on the
leaf (Hall et al., 1988) and absorption onto
cartridge columns (Whitehead and Temple,
1992).
The mechanism of theaflavin formation
was fairly well defined in early papers on
purpurogallin formation and related experi-
ments (Horner et al., 1961; Critchlow et al.,
1967; Takino and Imagawa, 1964b). Papers
investigating the fermentation reaction lead-
ing to the characteristic benzotropolone ring
reported the production of a series of
theaflavin-like compounds, most notably
erycitrin (Takino and Imagawa, 1963),
categallin, and pyrogallin (Takino and
437
 PH
[30]: Theaflavin
[31]: Theaflavin 3-Gallate
[32]: Theaflavin 3'-Gallate
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[33J : T heaflavin 3,3'- DigaIIate
FIGURE 14. The theaflavins.
Imagawa, 1964a). Oxidations involving na-
tive tea enzymes (Roberts, 1958), bicarbon-
ate/ferric ammonium sulfate (Takino and
Imagawa, 1964a), potassium iodate (Takino
et al., 1964), and peroxidase (Takino et al.,
1967; Finger, 1994) have all produced
theaflavins and theaflavin-like compounds
in varying yields. Precise NMR analyses of
the theaflavins are available (Bryce et al.,
1970; Cai et al., 1995).
b. Theaflavic Acids
Theaflavic acids such as epitheaflavic
acid [34], shown in Figure 15, are formed
from oxidative condensation of a gallic acid
molecule and a catechin (Coxon et al.,
1970b). In the production of epitheaflavic
acids, gallic acid provides the tri-hydroxy
structure and thus mimics the role of a
gallocatechin in the mechanism of theaflavin
formation. Theaflavic acids are formed from
438
R1 R2
TF H H
TF3G Gallate H
TF3'G H Gallate
TF DG GaIlate GalIate
condensation of the non-epi forms of the
catechins with gallic acid through an identi-
cal mechanism. Similarly,theaflagallins such
as epitheaflagallin [35] arise from gallo-
catechins and gallic acid (Nonaka et al.,
1986), where the carboxylic acid moiety
becomes a leaving group and mimics the
catechin group in the mechanism of
theaflavin formation.
c. Other Related Structures
Isotheaflavins (Coxon et al., 1970a) and
neotheaflavins (Bryce et al., 1972;Robertson,
1992) are formed in the same manner as the
theaflavins, except that they arise in part
from the non-epi forms of the pairs of cat-
echins. The abundance of non-epi forms of
catechins in the green tea leaf is small, and
therefore these black tea components are
present in significantly lower concentration
compared with the theaflavins.
 OH
OH
[34]: Epitheaflavic Acid
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OH
[351: Epit heaf lagaIIin
FIGURE 15. The epitheaflavic acids and related
compounds
3. Further Oxidized Products: The
“Thearubigens”
From the early stages of development,
simple solvent extractions attempted to quan-
tify the amount of colored tea compounds
present in the black tea brew (Roberts et al.,
1957). The theaflavins, a group of bright
orange-red compounds, were quickly sepa-
rated from the remainder. Paper chromatog-
raphy experimentsconfirmed that subsequent
to solvent extraction, a series of brown-red
compounds remained in the aqueous phase,
which were not well resolved by 2-D paper
chromatography, but were roughly quantifi-
able by simple spectrophotometric tech-
niques. These compounds were given the
label of thearubigens. However, subsequent
research began to compromise the under-
standing of the thearubigens as a well-de-
fined group of compounds.
Refinements on the procedure for quan-
tifying thearubigens were made beyond the
early paper chromatography techniques.
Solvent extraction methods, employing ethyl
acetate and butanol, or methyl iso-butyl ke-
tone and butanol, used colorimetry differ-
ences at -450 nm (for theaflavins) and -350
nm (for thearubigens), with each successive
method building on the early assumptions of
Roberts. This method was later converted to
use C-18 cartridge columns (Whitehead and
Temple, 1992) and a series of solvents to
elute the appropriate fractions and measure
thearubigens and theaflavins by colorimetry.
The significant limitation of use of this
method is that there is no clear evidence,
other than a correlation, that these measured-
”theambigens” and the “thearubigens” iden-
tified by Roberts are one and the same. This
becomes more evident in later methods. The
thearubigens were divided into three sub-
classes on the basis of paper chromatogra-
phy: SI, SIIa, and SIIb (Roberts et al., 1957).
Later HPLC techniques reinterpreted the is-
sue with division of the thearubigens into
groups I, II, and 111(Bailey et al., 1991;Bailey
et al., 1994a). In both papers, the presence of
an unresolved mass (as illustrated in Figure
16)is taken to represent the same compounds,
the “unresolved thearubigens”. This is based
on loose similarities of HPLC with paper
chromatography as well as more recent work
with cartridge column fractions (Wellum and
Kirby, 1981).
The arrival of HPLC chromatography
led to the belief that for the first time indi-
vidual thearubigens would be separated and
isolated (Hoefler and Coggon, 1976;
Robertson and Bendall, 1983). However, it
439
 c
Cinnamic Acids

Flavonol Glycosides
Catechins
Theaflavins
‘Thearubigens’

I
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Diffuse spot on bo h paper

chromatography a i d on
HPLC chromatogram taker
\
to be synonymous
Both identified as
‘unresolved thearu Digens’
100 atechins
76
‘ \
50

25

FIGURE 16. Paper chromatography vs. HPLC.

became clear early on that chromatography
using reversed-phase materials was not an
ideal solution, due in part to the observation
that some tea components were not eluted
from cartridge columns.
Strategies for separation of the thearu-
bigens on normal-phase chromatography
(Wedzicha and Donovan, 1989) and using
the technique of counter-current chromatog-
raphy (Okuda et al., 1988; Wedzicha et al.,
1990) have shown some promise in separa-
tion and identification. The absence of re-
cent reports suggests that use of such “alter-
native” technologies has fallen into disfavor,

440
 due in part to the extreme simplicity of re-
versed-phase techniques. Use of reverse
phase colums with a step-gradient (Putman
and Butler, 1989) might present a potential
useful technique for these compounds as well.
Use of reverse-phase technology has been
expanded systematically by a series of re-
cent papers (Opie et al., 1990; Bailey et al.,
1990;Bailey et al., 1991;Bailey et al., 1994b)
and from thesis work (Opie, 1992). The re-
verse-phase technique seemed to point to the
thearubigens in one of two classes: a series
of red (-450 nm) compounds eluting dis-
cretely and a diffuse peak appearing as a
‘rising baseline’ across the same chromato-
gram.
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Investigations into the thearubigen frac-
tions identified by early techniques revealed
that part of the thearubigens was the fla-
vonol glycosides (McDowell et al., 1990).
Being present in green tea, these compounds
are clearly not thearubigens. They contrib-
ute significantly to the absorbance around
-350 nm in both the solvent partitioning and
cartridge column techniques and must be
excluded from these measurements if the
term thearubigen is to retain its original
meaning, that is, as a product of oxidation of
green tea polyphenols.
Early model fermentation systems suc-
cessfully identified the paired role of
gallocatechins and catechins in theaflavin
formation (Sanderson et al., 1972). The
model fermentation approach was expanded
to investigate the role of purified tea PPO
(Coggon et al., 1973) and peroxidase (Dix
et al., 1981) on formation of the theaflavins
and thearubigens. After the arrival of HPLC,
this technique was used in the attempt to
justify the individual thearubigin peaks’ ori-
gin (Robertson and Bendall, 1983;Robertson,
1983). Model fermentation of individual
catechins was performed (Opie et al., 1990),
and peaks were identified that are potential
thearubigens, but attention was drawn away
from individual analysis and focused on a
diffuse rising baseline. A study of a model
fermentation of a mixed system of theaflavins
and epicatechin in the presence of polyphe-
no1 oxidase showed that epicatechin and the
theaflavins, but not the theaflavins alone,
resulted in the degradation of the theaflavins
(Opie et al., 1993). In another study, the pres-
ence of a diffuse peak on reverse-phase chro-
matography (having become a de-facto quali-
fier for thearubigens) was most significantly
formed during model oxidation of epicatechin
alone, and such a fermentation brew has been
suggested as a strategy for thearubigen iso-
lation (Opie et al., 1995). This approach,
however, seems limited, as epicatechin is a
minor constituent in tea relative to other
catechins, and to suggest that studies of its
fermentation products will lead to identifi-
cation of thearubigens can only result in a
very minor portion of the said thearubigens
being identified.
Model fermentation systems seem to be
the most reasonable approach for determin-
ing the origin of individual thearubigens as
discrete chemical identities. The use of model
fermentation systems eliminates some of
the confusion concerning the origin of
thearubigen-like compounds that may have
been “left over” from the green tea polyphe-
nols. However, until better techniques for
analysis are discovered, little information can
be gleaned from model fermentation studies.
In addition, model fermentation systems will
always suffer the question of whether the
model is accurately representing thearubigen
production.
Use of ultrafiltration to separate a high-
molecular-weight thearubigen fraction
confirmed the presence of a high-molecular-
weight “polymer”, but these high-molecular-
weight polymers represent at most only 2%
of the total brew solids (Kuhr et al., 1994).
The crude historical definition of
thearubigens on the basis of poorly resolved
paper chromatograms would seem to
represent a stumbling block for systematic
441
 chemical identification of tea compounds,
because no one unique chemical structure
seems to be indicative of a thearubigen to
date. It is therefore the preference of the
authors to avoid the use of the broad term
“thearubigens” except as a historical artifact
and as a semiquantitative analytical number
used by tea tasters. One reason is that if the
solvent extraction/colorimetry technique
(Roberts et al., 1957) is used successfully to
measure thearubigens, the thearubigens mea-
sured are, in part, composed of flavonol
glycosides (McDowell et al., 1990). Thus,
flavonol glycosides are in some sense
thearubigens. This places the definition of
thearubigens as oxidation products of cat-
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echins in an awkward position.
Some of the color attributed to the
thearubigens may in fact not be flavonoid in
nature at all. Figure 17 illustrates some pos-
sible alternative explanations for the brown
coloration and “acidity” as described in the
historical reports. They may be highly rear-
ranged compounds such as catechinic acid
[36] (Sears et al., 1974). Another explana-
tion is that thearubigens are overoxidation
products of theaflavins or direct products of
peroxidase, a theory that is well supported
by model studies of PPO and peroxidase. In
this respect, they may be ring-opened prod-
ucts similar to muconic acid [37,38]
(Hayaishi and Hashimoto, 1950; Speier et al.,
1993) or derivatives thereof (Critchlow et al.,
1967). The brown color attributed to the
thearubigens may also be attributed in part
to pheophorbide (e.g., [40]) and the
pheophytins (Sanderson, 1972), or polysac-
charides such as [4] and polymers thereof
(Millin et al., 1969). None of these conjec-
tures as shown in Figure 17 have been inves-
tigated. However, the presence of brown-
colored products that partition into all of the
phases of solvent extraction, including the
aqueous phase, butanol phase, and ethyl ac-
etate phase, seems to require that there be a
number of possible chemical moieties in-
442
volved and not a simple, single central struc-
ture as exhibited by the theaflavins.
a. Theafulvins and Theacitrins
Early reports suggested that the thearubi-
gens, after acid hydrolysis, produced
anthocyanidins (Brown et al., 1969). This
would lead one to believe that at least some
of the thearubigens are a class of condensed
tannin, possessing linkages at the 4-position,
which may have benzotropolone units or
other chromophores to give the characteristic
dark brown appearance of the thearubigens.
Recent work on this approach has iso-
lated a fraction believed to be part of the
thearubigens, termed theafulvin (Bailey et al.,
1992). The buff-tan appearance resembled
that of the condensed tannins, and the mate-
rials gave similar behavior on C- 18 chroma-
tography when compared with cider and wine
proanthocyanidins. However, acid hydroly-
sis of the two materials gave widely differ-
ing results. Condensed tannins gave reason-
able yields of anthocyanidins derived from
the hydrolysis products, a behavior that is
typical of polymeric flavan-3-01s. The
anthocyanidins are readily identified by their
bright color and characteristic absorption
spectrum and behavior on PRP-phase chro-
matography. The products of hydrolysis of
the theafulvin fraction, on the other hand,
yielded similar colored materials, but which
were unretained by PRP chromatography.
This led the authors to believe that only the
end groups of the polymer were converted to
anthocyanidin and that linkage occurred
through an alternate location, such as the 3’-
position (Bailey et al., 1994a).
In another report, Porter’s reagents were
used to achieve hydrolysis of the pro-
anthocyanidin fraction, as well as analyze
gallate and flavonol content by HPLC
(Powell et al., 1995). Both the theafulvin
fraction, as well as the caffeine-precipitable
 -
Hov-''x "'OH
OH HO
[101: Epicatechin [36]: Catechinic Acid

GKl-
(colorless) (brown)

'COOH ~ @ OH
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x - x - 0 - 0
bx@
OH OH OH
[371 [30]:Theaflavin
(brown, acidic) (red) (Brown, acidic)

FIGURE 17. Alternative brown pigments.

fraction, were analyzed by this method. In the basis of proanthocyanidins observed in

both cases, the proanthocyanidin content of green tea polyphenols (Hashimoto et al.,
these fractions seemed to be explainable on 1987), and the gallate content of these

443
 proanthocyanidins would explain most of
the hydrolyzed gallate in the fractions. This
result weighs heavily against the possibility
that these fractions are thearubigens derived
solely from catechin or theaflavin precur-
sors, unless the chemical characteristics of
these “thearubigens” had been transformed
drastically in chemical nature.
It should be noted that the theafulvin
fraction, a buff-tan solid, is isolated in ap-
proximately 3% yield from tea extract sol-
ids, of which 10% is proanthocyanidin in
nature and 3% is derived from gallate esters.
The low color impact this fraction is likely
to have on the overall beverage should be
considered as small evidence for support of
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this fraction as a thearubigen. The thearubi-
gens have been suggested to be dark brown
and strongly influencing the color of the
beverage, in addition to being -20% of the
total extractable solids. Also, gallate esters
are the predominant form of catechins in
green tea, comprising -15% wt/wt of ex-
tract, of which -35% of the mass of these is
gallic acid. Therefore, it is reasonable, to
expect -5% of the mass of black tea extract
to be gallate, as confirmed by tannase hy-
drolysis (unpublished results). Therefore, one
would expect to find much greater than
-0.3% of the extract from which a thearu-
bigen fraction was derived to be present as
thearubigen gallate esters. Given this criti-
cism, the theafulvin fraction should not be
identified conclusivelyas a major thearubigen
until stronger, direct evidence exists.
Attempts at isolation of the thearubigens
have generated at least two other new frac-
tions: the theacitrins (Powell et al., 1994)
and a caffeine precipitable thearubigen frac-
tion (Powell et al., 1993).
b. Gallic Acid Production
One of the products of the fermentation
process is the appearance of gallic acid [12],
constituting approximately 1% wt/wt of the
444
extract solids (Graham, 1984). This simple
polyphenol is thought to be a product of
degallation of the 3-galloyl substituted cat-
echins and gallocatechins that are abundant
in the natural beverage. Although the pro-
duction of gallic acid is not well understood,
either native esterase (tannase) activity or
oxidative degallation during the fermenta-
tion is a likely pathway to its formation.
A possible method of investigation of
thearubigens is to use the presence of gallated
polyphenols to establish mass balance of the
polyphenolic fraction. Tannase treatment of
both green and black tea produced from the
same clone along with measurement of gal-
lic acid content should establish the amount
of gallic acid oxidized into polymers. Sub-
tracting the free gallic acid (released in the
original black tea extract) and the consumed
gallic acid (computed from the gallic acid
released by tannase treatment) from the gal-
lic acid released from tannase treatment, one
can establish the molar concentration of cat-
echins involved in black tea polyphenols.
By then subtracting the molar quantity of
known gallated polyphenols such as catechins
and theaflavins, one can establish a molecu-
lar basis for the thearubigens and better hy-
pothesize on the true chemical constitution
of the thearubigens. Further, the difference
in mass balance between recovered gallic
acid and original gallated species in the green
leaf would establish the amount of gallic
acid polymerized into the “thearubigen
mass”. Good establishment of cycles of mass
balance such as this are notoriously absent
from much of the tea chemistry literature,
and this failure might explain why true iden-
tification of the thearubigens has eluded sci-
entific inquiry for so long.
c. Bisflavanols and Proanthocyanidins
The bisflavanols, such as bisflavanol A
[41], arise from paired condensation of two
gallocatechins (Vuataz and Brandenberger,
 1961). It was initially expected that these
would be intermediates in the formation of
theaflavins. The extra OH substitution from
the use of two gallocatechins, as opposed to
a catechin/gallocatechin pair, provides a
mechanistic barrier by replacing a hydrogen
atom, which is lost easily in tautomerization,
with an OH group (see Figure 21). This does
not rule out the possibility of further rear-
rangement or condensation, possibly to
thearubigins.
The bisflavanols were later rediscovered
and reclassified under the wider name of
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theasinensins. They were found to be present
in green tea leaf (Nonaka et al., 1983) and
oolong tea (Hashimoto et al., 1988). As well
as the gallocatechin-gallocatechin dimer
products, the theasinensins include catechin-
gallocatechin dimer products such as
theasinensin F [42]. The theasinensin family
is depicted in Figure 18.
Basic work on the separation, character-
ization, and chemical identification of black
tea constituents has been well advanced in
recent literature. In a massive compendium
of work on tannins and related compounds,

HO
OH

OH
OH OH

[41]: Bisflavanol A, or Theasinensin A

OH OH

[42]: Theasinensin F

FIGURE 18. The theasinensisns.

445
 best summarized in brief by one member of
this series (Hashimoto et al., 1992), a great
number of oxidation products from both
oolong and black tea sources have been char-
acterized, as well as novel polyphenols from
green tea. This approach is immensely use-
ful, but it is a tedious and expensive route,
and it is anticipated that a good HPLC tech-
nique that quantitatively establishes a good
mass balance of the unknown tea polyphe-
nols will require many more years of dedi-
cated work unless a working hypothesis for
their formation can be established.
The presence of proanthocyanidins and
theasinensins in green tea as well as black
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rial present in black tea. They are the prod-
uct of the condensation of catechins (which
are the likely oxidized species) with
theogallin and myricitrin, respectively.
Erycitrin [45] (Takino and Imagawa, 1963)
is a theaflavonin, the discovery of which
predates that of the theaflavonins, lending
futher confusion to the terminology. These
oxidation products are shown in Figure 19.
eH
HGOH

tea indicates that great care must be taken to

exclude the nascent green polyphenols in the
quantitation of thearubigens. Furthermore, O a- o H
these compounds do not possess significant OH
absorption in the visible region of the spec-
trum, which rules them out as contributors to
mass of the brown thearubigens. “ ‘ OH
6 O H
bH
d. Mixed Oxidation Products of [43] : T heogallinin
Polyphenols and Other Compounds

While the most widely known coupled

oxidation products of green tea fermentation
are the theaflavins, resulting exclusively from OH
the reaction of catechins and gallocatechins,
there are other possible oxidation products
in the fermentation system not arising solely
from these materials.
The o-quinone group, once formed, is a
highly reactive species and therefore has little
selectivity for condensation. It is likely there-
fore that other nucleophilic species, includ-
ing active thiols and amine groups, as well
as other unoxidized polyphenolic species,
can condense with the o-quinone (Van
Sumere et al., 1975). [44]: Theaflavonin, R=Galloyl
Theogallinin [43] and the theaflavonins [45]: Erycitrin, R=H
such as [44](Hashimoto et al., 1992) repre-
sent two recently identified species that con-
tribute to the balance of polyphenolic mate- FIGURE 19. Mixed oxidation products.

446
 e. Aroma Formation from Polyphenol
Oxidation
The aldehyde aroma components arising
in black tea aroma have been attributed to
condensation of corresponding amino acids
with an o-quinone to form an amine-substi-
tuted derivative (Bokuchava and Popov,
1954) during polyphenol oxidation. The ci-
tation of this mechanism is interesting as
some products (aldehydes, carbon dioxide,
and ammonia) have been observed (Popov,
1956; Skobeleva and Popov, 1962). Tea leaf
amine oxidase produces aldehydes and am-
monia as byproducts (Tsushida and Takeo,
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1985). Detection of these byproducts in fer-
mentation studies does not constitute proof
that polyphenol oxidation is involved in their
production, because these studies do not ex-
clude the possibility of the presence of amine
oxidase as a contaminant. Nonetheless, there
is compelling evidence for the role of cat-
echins in aroma generation in black tea. The
mechanism also suggests the formation of a
nitrogen-substituted catechin [46]. A similar
nitrogen-substituted catechin is formed by
heating epicatechin with alanine in a model
sy stem producing 7 - C (N-e t h y lami no) -
epicatechin [47] (Anan et al., 1987), which
is a brown compound suggestive of the
thearubigens. This is consistent with obser-
vations of nitrogen incorporation into cat-
echins (Kito et al., 1968; Konishi, 1969).
These studies lead one to believe that a small
fraction of the thearubigen fraction may
possess nitrogen content derived from these
compounds. The nitrogen substituted cat-
echins are depicted in Figure 20.
4. Oolong Tea Polyphenols
Typically understood as an intermediate
between green and black teas, oolong tea is
characterized by a much shorter fermenta-
tion time under gentler conditions such that
partial oxidation, rather than total fermenta-
tion, occurs. This characterization is also
representative of some Darjeeling teas, whose
oxidase activity is not permitted to come to
full expression. Gentler oxidation apparently
creates its own unique set of aroma and
polyphenolic compounds.
One residual question concerning the
mechanism of theaflavin formation is the
concerted oxidation of the catechins. Com-
pounds such as the bisflavonols, also known
as theasinensins A-E, represent “dead end”
condensation products because they cannot
proceed mechanistically to the theaflavins.
Figure 21 illustrates the most plausible
mechanism of theasinensin and theaflavin
formation. It is likely that the quinone groups
are present in low concentrations at any one
given time due to their high reactivity. It
seems more reasonable that the quinone re-
acts with an unoxidized polyphenol at low
quinone concentrations, and is then oxidized
further to theaflavin. Theaflavins may be
formed directly from the quinones provided
they are present in sufficiently large quanti-
ties but might be interrupted at an intermedi-
ate stage. Thus, Figure 21 also shows how
the theasinensins may arise during their for-
mation.
a. Oolongtheanin and Theasinensins
Oolongtheanin and theasinensins F [42]
and G (Hashimoto et al., 1988) may be unique
products of the oolong tea fermentation
system and are shown in Figure 22.
Theasinensins A/B and D/E are atropisomers
about the biphenyl link of the same conden-
sation products (the gallocatechins). Oolong-
theanin is likely to be a further oxidized
form of one of these theasinensins. Intrigu-
ing to note, however, is the detection of
theasinensins F and G, atropisomers of a
mixed condensation of epicatechin gallate
and epigallocatechin gallate. Such a mixture
447
 OH

OH

1461: Nitrogen Substituted Catechin,

R=derived from amino acid NH2CHRCOOH
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OH

OH

[47]:7-C(N-ethylamino)-epicatechin
FIGURE 20. Nitrogen-substituted catechins.

is expected to produce theaflavin digallate,
as epicatechin [ 101and epigallocatechin [ 121
will produce theaflavin [30].However, the
gentler oxidation present in oolong tea sys-
tems may have permitted the detection of
this intermediate.
In addition to oolongtheanin [55], two
oolonghomobisflavans, and a vitamin C
derivative of EGCG, 8-ascorbyl EGCG
[56],were also isolated from oolong tea and
may be unique oolong tea polyphenols
(Hashimoto et al., 1989b). It is difficult at
this time to place oolong tea polyphenols as
either unique polyphenols of the divergent
path of oolong tea production or as interme-
diates on the path between green and black
tea, with green representing the unfermented
leaf most closely and black tea representing
either complete or near-complete fermenta-
tion.
IV. BIOCHEMISTRY OF TEA
Tea is the subject of many biochemical
investigations because it produces a number
of unique natural products. In contrast to the
isolation and quantification of the various
components of tea, the mechanisms by which
the tea plant forms these compounds, and

448
 PPO

"OH '' 'OH

b H [lo]: EC- R=H /JH [48]: EC-R=H
[12]: EGC-R=OH [49]: EGC- R=OH

HO

/ - - PPO
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X OH

OH
OH [50] OH
Low quinone concentration Theasinensin intermediate
(two possible atropisomers)

1521
High quinone concentration

f--

Theaflavin

FIGURE 21. Mechanism of theaflavin formation.

449
 HO

on

on
[55]:Oolongtheanin
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bH

[561: 8-C-A scorby1 Ep igaII ocatechin GaII ate

FIGURE 22. Oolong tea polyphenols.

the enzymes that activate these mechanisms,
provide their own set of challenges to tea
chemistry.
A. Caffeine Formation
Caffeine had been thought to be princi-
pally synthesized during the withering stage
of freshly plucked tea leaves (Roberts and
Sanderson, 1966), although is probably syn-
thesized throughout the life of the plant.
Caffeine is most likely synthesized from
adenine nucleotides, the dominant free pu-
rine forms in tea (Takino et al., 1972). Ad-
enosine is a major product of RNA metabo-
lism in tea (Imagawa et al., 1976).Adenosine
[57] is converted to adenine [58] (Imagawa
et al., 1979), and through hypoxanthine [59]
(or inosine) to xanthine [60] (or xanthosine),
from which xanthosine [61] is the starting
branch of caffeine biosynthesis (Negishi
et al., 1992). Guanosine is also converted to
xanthosine, but plays an apparently minor
role in caffeine biosynthesis (Negishi et al.,
1992). Xanthosine is methylated at the 7-PO-
sition (Negishi et al., 1985) to 7-methyl xan-
thosine [62]. 7-Methyl xanthosine is then
hydrolyzed to 7-methyl xanthine [63]
(Negishi et al., 1988), which is subsequently
methylated to theobromine [2] and caffeine
[ l ] (Suzuki and Takahashi, 1975; Suzuki
and Takahashi, 1976). The final methylation
can be terminated, as found in the variant
C. irrawadensis (Roberts et al., 1958). Fig-
ure 23 illustrates the mechanism of forma-
tion of theobromine and caffeine in the tea
plant.

450
 Hypoxanthine
HO
Adenosine [591 HO
Xanthosine [61]
(571
Xanthine[GO]
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H”KNW
0
7-Methyl-xanthosine [62]

A
7-Methylxanthine [63]

C H3 CH3
Theobromine [2] Caffeine [ l ]

FIGURE 23. Biosynthesis of caffeine.

B. Theanine Formation glutamic acid. Theanine degradation prod-

Theanine [3] (methyl glutamine) is
formed from the action of theanine synthetase
[L-G1utamate:Ethylamine ligase] (Sasaoka,
1965; Sasaoka et al., 1965) on ethylamine
derived from alanine (Takeo, 1974), and
ucts may serve as precursors for synthesis of
the catechin A ring, apparently from utiliza-
tion of the N-ethyl group (Kito et al., 1968;
Feldheim et al., 1986). It is likely that the
N-ethyl group enters the general glycolyte
pathway, forming the catechin precursor ma-

451
 lonyl-CoA. Theanine formation is a light-
dependent process requiring the presence of
ATP as a cofactor (Sasaoka, 1965). Theanine
synthesis and accumulation is inhibited by
production of glutamine (Matsuura and
Kakuda, 1990).
C. Biochemistry of Flavonoid
Compound Formation
The pathways for the de novo biosynthe-
sis of flavonoids in both soft and woody
plants have been generally elucidated and
reviewed in detail elsewhere (Jain and Takeo,
1984; Heller and Forkmann, 1994). Similar
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pathways for biosynthesis are used by a wide
variety of plant species. The regulation and
control of these pathways in tea and the
nature of the enzymes involved in synthesis
in tea have not been studied exhaustively.
The following discussion extracts critical
information from the pertinent tea literature
and otherwise assumes that flavonoid pro-
duction by Camellia sinensis generally fol-
lows the pathways used in other plant spe-
cies.
1. Phenylalanine and the Shikimate
Pathway
Phenylalanine is thought to be the direct
precursor of polyphenols in tea plants
(Nikolaeva et al., 1982); however, tyrosine
may also participate in polyphenol produc-
tion through a light-dependent pathway
(Zaprometov and Bukhlaeva, 1971). Deami-
nation of phenylalanine [64] forms cinnamic
acid [65] by the action of phenylalanine
ammonia lyase (Iwasa, 1976; Shiplova and
Zaprometov, 1977; Zagoskina et al., 1990).
Hydroxylation of cinnamic acid forms
coumaric acid [66], which is generated from
an enzyme complex referred to as cinnamate
452
4-hydroxylate or cinnamate 4-monooxy-
genase. Hydroxylation is achieved typically
by a cytochrome P-450 enzyme activity as-
sociated with the enzyme complex (Heller
and Forkmann, 1988), and a light-indepen-
dent cinnamate 4-monooxygenase activity
has been reported from tea shoots (Saijo,
1980).The coumaric acid aromatic ring forms
the ‘B’ ring of the flavonoids. Figure 24
illustrates the biosynthesis of coumaric acid
and the various functions this compound
performs in the tea plant.
The gallic and quinic acids originate via
the shikimatekogenate pathway. The key
enzymes in shikimic acid biosynthesis have
been detected in tea (Saijo and Takeo, 1979).
Carbohydrates play an important role, pre-
sumably as a precursor to shikimic acid,
because radiolabels from both myo-inositol
and glucose are incorporated into catechins
(Wang and Huang, 1987). Gallic acid and
quinic acid play key roles in forming esters
with various polyphenols. Gallic acid is a
key component of tannins and gives the cat-
echins their tannin-like qualities.
Light has been shown to strongly induce
biosynthesis of the phenolics by stimulation
of the various enzymes along the biosyn-
thetic path. Photosynthesis is associated with
production of acetyl-CoA, ATP, and reduc-
ing agents such as NADPH, all of which
play important roles as various stages of the
biosynthesis (Zaprometov, 1987). Light is
very important for catechin production, and
shaded tea plants produce finished tea
products with reduced astringency due to
the lower concentrations of polyphenols
(Mahanta and Baruah, 1992).
Coumaric acid, along with other cinnamic
acid derivatives and phenylalanine, is also a
precursor to tea plant lignins (Zagoskina and
Zaprometov, 1976; Strekova et al., 1980).
Lignins are ubiquitous throughout the plant
kingdom. Tea lignins are methylated (rein-
forcing low solubility) by the action of
 [641: Pheny laIanine

1 Phenylalanine
ammonia-lyase

rcooH
[65]: Cinnamic Acid r nCOOH
HO&/J
1 Cinnamate
4-monooxygenase
AH2
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r C " H / [671: Tyrosine

Tyrosine Ammonia-Lyase?
HO
[66]: Coumaric Acid

/ \ \
to catechins and flavonols to lignins

(through flavonoid biosynlhesis) (through lignin condensation)

+ quinic acid

H O O 6 5

OH
OH
OH
[22]: Coumarylquinic acid

FIGURE 24. Cinnamate biosynthesis.

methyl transferase enzymes (Nikolaeva and
Zaprometov, 1990).
2. Chain Extension, Hydroxylation
After formation of the B ring moiety, the
biosynthesis of catechins proceeds with a
series of chain extensions to form the C and
A ring backbone. 4-Coumaroyl-CoA is the
preferred substrate for chain extension in
most plant species, typically formed by a
hydroxycinnamate:CoA-ligase (Heller and
Forkmann, 1988).
Malonyl-CoA is produced by carboxyla-
tion of the glycolysis product acetyl-CoA
via acetyl-CoA carboxylase (Lowenstein,
1981). A hydroxycinnamic acid-CoA deriva-
tive typically combines with three molecules
of malonyl-CoA to form chalcones via chal-

453
 cone synthase (Ebel and Hahlbrock, 1982).
This enzyme apparently requires no cofac-
tors and performs the condensation of the
malonyl units and the cyclization to form
the phloroglucinol ‘A’ ring (Heller and
Forkmann, 1994).
Theanine has also been demonstrated to
be a significant factor in the synthesis of the
phloroglucinol nucleus (Kito et al., 1968;
Feldheim et al., 1986). Incorporation of the
ethyl group of theanine into the catechins
has been demonstrated by radiolabeling
ethylamine, a theanine precursor (Sasaoka
et al., 1962; Kit0 et al., 1968). Ethylamine is
the best demonstrated substrate of tea amine
oxidase (Tsushida and Takeo, 1985), which
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is converted to acetaldehyde. Theanine may
be a storage mechanism for this amine, with
acetaldehyde being the first intermediate on
the path to catechin B-ring synthesis, fol-
lowed by conversion to acetyl-CoA and/or
malonyl-CoA.
Camellia sinensis produces flavonoids
in which the tri-hydroxy B ring species pre-
dominates; however, within the flavonols the
di-hydroxy B ring is more common. It is
unclear whether 3’ and/or 5’ hydroxylation
is occurring before or after chalcone syn-
thase (Hahlbrock and Grisebach, 1975).
Because 3’,4’-hydroxylated forms of all the
species can be found throughout, and 4’-
mono hydroxylated species are found more
commonly in the earlier branches of biosyn-
thesis, it is possible that tea contains nonspe-
cific or numerous 3’-hydroxylases (Heller
and Forkmann, 1988), accounting for the
presence of caffeic acids, quercitin, and cat-
echin derivatives; a much more specific 3’,5’-
hydroxylase (Heller and Forkmann, 1988)
accounts for the gallocatechins and the
smaller quantity of myricetin derivatives.
3. Chalcone/Flavone
Tautomerization
Subsequent to formation of the chalcone,
the final step in formation of the catechin
454
basic structure is tautomerization of the chal-
cone structure to a flavanone. The enzyme in
this step is chalcone isomerase, which usu-
ally bypasses the natural, racemic, anti-ad-
dition (Heller and Forkmann, 1988) to form
a characteristic 2s-stereochemistry (Heller
et al., 1979) of the resulting flavone by syn
addition (Ebel and Hahlbrock, 1982).
Ring closure of chalcones followed by
hydroxylation results in formation of
dihydroflavonols. The flavanone is typically
hydroxylated by the enzyme flavanone 3-
hydroxylase, an oxoglutarate-dependent
dioxygenase (Heller and Forkmann, 1988).
The formation of the 2R,3R dihydroflavonol
has been shown to form either the flavonols
or the non-epi flavan-3-ols, whereas it is
believed that a competing hydroxylase forms
the 2R, 3s dihydroflavonol [70], which has
been isolated (Nonaka et al., 1987).
4. FlavanoldFIavonols
The dihydroflavonols are enzymatically
transformed to the flavonols through action
of a 2-oxoglutarate-dependent dioxygenase
(Heller and Forkmann, 1994), forming a
double bond through abstraction of vicinal
hydrogens on the 2R,3R dihydroflavonols as
proposed for flavone synthase (Britsch,
1990). This activity is referred to as flavonol
synthase.
Formation of the 2R, 3 s dihydroflavonols
is likely to be preferred in the tea plant, as
formation of the epi-forms of the flavan-3-
01s is the preferred biosynthesis product.
However, while the route to formation of the
flavonols and non-epi-flavan-3-01s is well
defined, the formation of the epi-flavan-3-
01s remains conjecture (Stafford, 1988). Fig-
ure 25 illustrates the proposed biosynthetic
pathway from cinnamic acids to flavonols
and flavan-3-01s.
The formation of proanthocyanidin poly-
mers has also been considered, although it is
unclear if epicatechin [ 101 or leucocyanidin
[27] is the significant precursor (Nikolaeva
 OYSCOA CHIYO p O H

3x Acetyl CoA CH3

CoASKCH3
CoAS 0
SCoA

d
1661: Coumaric acid
(as CoA esler)

Chalcone
synthase

H O f l o H

0
-
I691: 2R-Flavanone
OH 0 [68]: chalcone OH 0
Flavanone

/
hydroxylase
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Dihydmflavonol
redudase?

Flavonol
synthase

1711: flavan-3,4-diol
'"OH

1721. flavonot
'OH

171 Kaemplerol glycostdc "' (141 flavan-3-01 (afzelechin)

FIGURE 25. Biosynthesis of flavonoids.

et al., 1982). Proanthocyanidin polymer for- 5. Esferase

mation has been attributed to polyphenol
oxidase and the formation of the thearubigens
(Brown et a]., 1969). Further work is neces-
sary to determine the origin of proantho-
cyanidin polymers and their role in the tea
plant and beverage.
Shikimic acid has been shown to be a
good precursor for biosynthesis of the gal-
late group (Zaprometov, 1962). Gallic acid,
found principally in its esterified form in
green tea, is thought to be principally syn-

455
 thesized through this pathway, although phe-
nylalanine and the cinnamic acids may also
be precursors (Saijo, 1983).
A significant quantity of the small
polyphenols exists as quinic acid esters
(depsides), particularly the depside of gallic
acid, theogallin (Stagg and Swaine, 1971).
Quinic acid is derived from the shikimic
acid pathway via dehydroquinic acid, an in-
termediate on the pathway to shikimic acid
(Sprinson, 1960; Neish, 1964). Quinic acid
esters of coumaric and caffeic acid (chloro-
genic and caffeoylquinic acid) have also been
detected in tea (Sanderson, 1972).
Gallated flavan-3-01s (catechins) are the
major flavonoids produced by Camellia
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sinensis (Sanderson, 1972). It is of interest
that Cumellia sinensis forms gallated esters
rather than sugar esters, or glycosides, of
catechins. Glycosides are the typical plant
strategy for rendering the major flavonoids
water soluble (Heller and Forkmann, 1994),
although proanthocyanidin and catechin gly-
cosides, while not rare, are not known to be
common (Achmadi et al., 1994). It is likely
that the catechins are acylated with an acti-
vated form of gallic acid, presumably galloyl-
CoA, similar to the aromatic acylations oc-
curring with cinnamic acid acyltransferases
in other systems (Kamsteeg et al., 1980;
Heller and Forkmann, 1988). Gallic acid es-
terification is thought to be a slow process
compared with its biosynthesis (Zaprometov
and Bukhlaeva, 1963) and has been demon-
strated through tracer experiments in tea
shoots (Saijo, 1983).
D. Latent Enzyme Activity and the
Formation of Black Tea
Conversion of tyrosine and related
monophenols to diphenolic compounds is
typically accomplished by tyrosinase or
monophenol monooxidase. Whether tyrosi-
nase activity or a related enzyme activity is
456
responsible for polyphenol oxidase activity
(observed in leaf browning and the forma-
tion of black tea polyphenols) is unclear.
1. Polyphenol Oxidase
Polyphenol oxidase (EC 1.14.18.1;
monophenol monooxygenase [tyrosinase] or
EC 1.10.3.2; o-diphenol: o2 oxidoreductase)
is one of the more important enzymes in-
volved with the formation of black tea
polyphenols. The enzyme is a metallo pro-
tein, thought to contain a binuclear copper
active site. Polyphenol oxidase (PPO) is an
oligomeric particulate protein that is thought
to be bound to the plant membranes. The
bound form of the enzyme is latent and
activation is likely to be dependent on
solubilization of the protein (Tolbert, 1973).
PPO is distributed throughout the plant
(Durmishidzern and hridze, 1980) and has
been identified and analyzed in the floral
organs (Singh and Ravindranath, 1994). PPO
is localized within plant cells in the mito-
chondria (Bokuchava et al., 1970), the chlo-
roplasts (Roberts, 1941),and the peroxisomes
(Kato et al., 1976). Using antibody tech-
niques, polyphenol oxidase activity has also
been localized in the epidermis palisade cells
(Wickremasinghe et al., 1967). Recent re-
views on the subject of PPO are available
(Zawistowski et al., 1991; Whitaker, 1994;
Steffens et al., 1994).
The biological role of PPO in plants is
thought to be associated with a plant defense
mechanism and root development. PPO be-
comes activated and available when plant
tissue is damaged due to injury or infection,
catalyzing the formation of insoluble phe-
nolic polymers that aid in wound healing
and help prevent the spread of infection
(Zawistowski et al., 1991). In tea, key reac-
tions occurring during “fermentation” are
catalyzed by PPO. These reactions are initi-
ated by crushing andor tearing withered tea
 leaves and lead to the formation of black tea
polyphenols and aroma compounds charac-
teristic of black tea.
Polyphenol oxidase catalyzes two gen-
eral reactions: the hydroxylation of mono-
phenols to o-diphenols, and the oxidation of
diphenols via dehydrogenation to o-quino-
nes, both 2-electron transfer reactions. The
formation of o-quinones is then followed by
condensation to form a wide range of com-
plex products (Mayer and Harel, 1991). The
copper cofactor associated with the metallo
protein is the essential component involved
with electron transfer and oxidation of the
substrate. The reaction mechanisms associ-
ated with PPO of Camellia sinensis have not
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been determined; however, the reaction
mechanisms of PPO from other plants have
been characterized (Zawistowski et al., 1991;
Whitaker, 1994; Steffens et al., 1994). PPO
activity is dependent on oxygen concentra-
tion (Robertson, 1983). The o-quinone spe-
cies from tea has been looked for in a model
system (Korver et al., 1973).
Polyphenol oxidase is difficult to purify,
due in part to the presence of polyphenols
that have a strong affinity for proteins. Work
on isolation and characterization of PPO has
been reviewed (Mayer, 1987). The enzyme
appears to exist in multiple forms and is
comprised of a number of subunit proteins.
Reports on the molecular weight of PPO in
plants vary widely. The early work on isola-
tion of PPO from green tea leaves reported a
molecular weight in the range of 130 to 160
kDa (Gregory and Bendall, 1966). Reports
on isolation of PPO from other plant species
using modern biochemical methods state
molecular weights ranging from 40 to
72 kDa, whereas the primary isoform of typi-
cal PPO has a molecular weight of 45 kDa
(Steffens et al., 1994). The molecular size of
PPO isoforms based on cloned genes indi-
cates that PPO has a molecular weight of
between 57 and 65 kDa and includes puni-
tive transit peptides of a molecular weight
of 18 to 20 kDa. Mushroom tyrosinase has
been found to have a quarternary structure
comprised of two different subunits of 43
kDa and 13.5 kDa, with a total molecular
weight of 120 kDa and the formula b H 2
(Zawistowskiet al., 1991). These facts would
suggest that the PPO from tea is also com-
prised of four protein subunits and therefore
has a possible molecular weight of -144
kDa. This is consistent with a report of
isoforms of 117,56,41.5, and 35 kDa (Buzun
et al., 1974) and another report with isoforms
at 118 and 41 kDa (Durmishidzern and
Puridze, 1980). The copper content has been
measured at 0.26 wtfwt%, or about 4 to 5
copper atoms for the complex, which is con-
sistent with other literature (Vamos-Vigyazo,
1981; Interesse et al., 1983; Zawistowski
et al., 1991). This number is probably low
because copper diffuses readily away from
the enzyme, resulting in a loss of activity
(Mayer, 1987; Zawistowski et al., 1991).
Consistent with observed sigmoidal activity
(Pruidze, 1975), multiple active sites in the
enzyme complex are likely to exist.
Polyphenol oxidases act on a broad range
of phenolic substrates with both mono-,
di-, and trihydroxy substitutions. There is
evidence that PPOs from different plant spe-
cies have preferential substrates (Whitaker,
1994; Steffens et al., 1994). Studies of PPO
from tea have mainly been based on model
fermentation studies that utilize purified or
semipurified substrates (Dix et al., 1981;
Robertson and Bendall, 1983; Opie et al.,
1990; Finger, 1994). It is clear from these
reports that PPO from tea reacts effectively
with both di- and tri-hydroxylated catechins,
reacting with specificity for the o-dihydroxy
moiety (Gregory and Bendall, 1966; Coggon
et al., 1973). Studies that define the kinet-
ics of PPO from tea in relation to substrate
type are needed to better define the enzy-
mology of tea PPO. PPO can be inhibited
by a range of chemical constituents that
include cyanide, carbon monoxide, EDTA,
457
 sulfites, ascorbic acid, erythrobic acid, and
thiol-containing compounds (Zawistowski
et al., 1991). In general, compounds that
block oxygen, act as antioxidants, and/or
bind copper are good inhibitors of PPO.
The effect of natural flavonoids and end
products of fermentation as inhibitors of
tea PPO remains to be defined, although
there is evidence of theaflavins and
thearubigens as feedback inhibitors (Pruidze
and Grigorashvili, 1975). Most PPO en-
zymes have a pH optimum in the range of
5.0 to 7.0. Typical solutions of tea polyphe-
nols have a pH of approximately 4.5, and
increasing the pH of tea fermentation reac-
tions leads to stronger PPO activity. Tea
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PPO has an observed optimum pH in the
range 4.6 to 5.6 (Takeo, 1965; Takeo and
Uritani, 1966; Gregory and Bendall, 1966;
Perera and Wickremasinghe, 1972; Coggon
et al., 1975; Robertson, 1983).
2. Peroxidase
Peroxidase (EC 1.11.1.7) is thought to
play an integral role in the fermentation pro-
cess and is found in fresh green leaf
(Bokuchava and Popov, 1948; Gregory and
Bendall, 1966; Bokuchava and Skobeleva,
1969). Coupled with PPO, which is thought
to produce the peroxide, an activator of this
enzyme system in other systems (Jiang and
Miles, 1993), peroxidase is also thought to
play a role in the oxidation and formation of
the black tea compounds.
Peroxidase is a haem-based enzyme that
catalyzes the reductive decomposition of
hydrogen peroxide to water and organic per-
oxide species to the corresponding alcohol.
In contrast to superoxide dismutase, whose
activity is cycled by coupled oxidation and
reduction of peroxide to oxygen and water,
respectively, peroxidase cycles its activity
through oxidation of a wide variety of sub-
strates.
458
Model system studies using peroxidase
and PPO for production of black tea polyphe-
nols suggest that peroxidase activity is re-
sponsible not only for decomposition of
theaflavins but the formation of thearubigens
as well (Dix et al., 1981). Reactions of
epicatechin and PPO have been proposed as
a model for thearubigen formation (Opie
et al., 1993) and epicatechin oxidation prod-
ucts by this system have been suggested as a
good model of thearubigens (Opie et al.,
1995). This is likely due to the fact that the
isolation procedure used for purification of
PPO (Opie et al., 1990) was found to contain
peroxidase activity in an earlier report
(Robertson and Bendall, 1983). Model fer-
mentations using PPO and peroxidase, both
separately and in conjunction, show the
coupled nature of these enzymes (Finger,
1994) and point to the need for further inves-
tigation of the role of both of these enzymes
in the development of the unique black tea
polyphenols and other compounds present
in black tea extracts.
E. Extracellular Enzymatic Activity
Many tea products, such as Kombu-cha
or Pu-Erh tea, take advantage of enzymatic
systems external to the tea plant. In the case
of Kombu-cha, sweetened tea brew is seeded
with a yeast that produces ethanol and acetic
acid. The yeast generates a mushroom-like
mass that is typically reused as the seed for
future beverage production. Pu-Erh tea is
produced by burying green tea leaves in a
confined space, thereby allowing microbio-
logical fermentation to blacken the leaves
(Shao et al., 1995). Pu-Erh is thought to pos-
sess many health benefits and is often com-
pressed into bricks and called Tuo-cha tea.
Tea, because of its unique phytochemi-
cal composition, offers many opportunities
for study of exogenous enzyme systems. One
enzyme that has gained a reputation in the
 field of tea chemistry is tannase. Tannase
enzymes (Gallic acid esterase, Tannin acyl
hydrolase, EC 3.1.1.20) such as those iso-
lated from fermentation broths of Aspergil-
lus Flavus, A. Niger, or A. Oryzae represent
the most significant of these extracellular
enzymes. Tannase is significant in terms of
its demonstrated effect of hydrolyzing the
gallic acid group from tea polyphenols (Deijs
and Dijkman, 1936; Roberts and Wood,
1951; Roberts and Myers, 1959) and its ben-
eficial impact on manufacture of cold water
soluble instant teas (Coggon et al., 1975).
The use of tannase significantly inhibits the
ability of tea polyphenols to complex and
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precipitate, and reduces the ability of black
tea to form tea cream (Nagalakshmi et al.,
1985). Tannase activity and thermal stability
in the presence of tea extracts has been de-
termined (Thomas and Murtagh, 1985).
V. CHEMICAL PROPERTIES OF TEA
COMPOUNDS
Beyond the inherent ecological proper-
ties of tea compounds in the tea plant, tea
phytochemicals exhibit unusual properties
that make them interesting subjects for
chemical study.
A. Formation of Cream and Haze in
Black Tea
The onset of cooling in a freshly brewed
cup of black tea is accompanied by produc-
tion of a dark redbrown cloud. This cloud is
particularly notable in concentrated brews
of tea, where the beverage moves from a
dark brown to a milky-red color. This appar-
ent ‘lightening’ in the concentrated solution
resembles the milky-red color of tea to which
cream has been added and thus was dubbed
‘tea cream’ (Bradfield and Penney, 1944;
Roberts, 1963; Smith, 1968).
Tea cream can be separated from the
brew most conveniently by centrifugation of
a concentrated beverage (Smith, 1968).
Tea cream was demonstrated by early
researchers to contain caffeine, theaflavin,
and thearubigens, as well as traces of a
number of other substances (Roberts, 1963;
Smith, 1968). Studies on cream composi-
tion (Nagalakshmi and Seshadri, 1983)
and decreaming (Nagalakshmi et al., 1985)
show that protein and caffeine contribute
significantly to cream formation. Addition-
al contributions from lipids in tea such as
triacontanol and spinasterol have been
shown (Seshadri and Dhanaraj, 1988). Simple
carbohydrates have been shown to assist in
solubilization of cream (Nagalakshmi et al.,
1984) but do not appear to complex signifi-
cantly with polyphenols in model studies
(Williamson et al., 1995). High-molecular-
weight thearubigens have been associated
with cream formation (Hazarika et al., 1984).
HPLC analysis of the phenolic portion of tea
cream (Powell et al., 1993) revealed 86%
‘thearubigens’, 12%theaflavins, and 2% fla-
vonol glycosides.
While the polyphenolic constituents’
role in cream formation is well under-
stood, the presence of proteins/peptides,
carbohydrates, leaf fines (fragments of
leaves), and other materials needs to be
studied further to fully determine the
mechanism behind cream formation.
Analysis of the pH optimum for cream
formation (Smith, 1968), which closely
mirrors tannin-BSA pH curves (Hagerman
and Klucher, 1986), combined with knowl-
edge of the significant contribution of pro-
tein to the mass of cream (Nagalakshmi
and Seshadri, 1983), provides strong justi-
fication for better analysis of the contribu-
tion of protein to cream formation.
The study of the physicochemical prop-
erties of cream is reviewed and expanded
significantly in thesis work (Rutter, 1971)
and in the literature (Rutter and Stainsby,
459
 1975; Bee et al., 1987). Tea cream has been
shown to be comprised of ultrafine spherical
colloidal droplets that are clearly visualized
in a range of concentrations of tea in water,
increasing in size with increasing concentra-
tion. At very high concentrations, the drop-
lets begin to aggregate further, forming ir-
regular shapes. The colloidal, spherical nature
of the tea cream particles distinguishes this
material from typical “crystalline” precipi-
tates and is referred to as a coacervate (Bee
et al., 1987). Tea cream formation has been
interpreted in terms of phase separation and
is likened to two liquids miscible at high
temperatures but immiscible when cooled
(Harbron et al., 1989).
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B. Complex Formation
The onset of cream formation in tea is
driven by complexation of the black tea
polyphenols. Green tea contains simple
polyphenols when compared with the com-
plex polyphenols present in black tea. Green
tea exhibits formation of a haze but does not
‘cream’ to the same extent as black tea.
Polyphenols have been observed to have a
strong precipitating effect on proteins, dena-
turing and reducing enzymatic activity
(Sekiya et al., 1984; Ozawa et al., 1987).
Immobilized polyphenols bind proteins re-
versibly, with restoration of enzyme activity
after elution (Oh et al., 1985). Dissolved ash
metals such as hard water calcium may in-
fluence complexation, as in the case of tea
scum formation (Spiro and Jaganyi, 1994).
The mechanism of complex formation is
a subject of some dispute in the literature. It
has been asserted that polyphenols form a
“hydrophobic bond” between the flat aro-
matic surface of the polyphenol rings and
quasi-planar hydrophobic groups such as
proline in peptides and proteins. Evidence
for this assertion is derived from crystal struc-
tures of co-crystallized model polyphenols
460
with caffeine (McManus et al., 1981;Gaffney
et al., 1986; Martin et al., 1986), and of caf-
feine with 1-tryptophan(Nishijo et al., 1990).
Hydrogen bonding also plays a prominent
role (Van Sumere et al., 1975; Borazan and
Al-Ani, 1980). Although a full thermody-
namic treatment of the issue is beyond the
scope of this review, it is hoped that further
investigation of the following issues will lead
to a better understanding of the mechanism
of cream formation.
Polyphenols demonstrate hydrophobic
character by partitioning into the cyclodextrin
cavity (Spencer et al., 1988; Cai et al., 1990)
and by their octanol-water partition coeffi-
cient (Martin et al., 1990). In addition to
hydrophobic stacking, there is evidence for
a coplanar-typehydrogen bond (Martin et al.,
1986). There is an apparent correlation be-
tween hydrophobicity and complexation for
hydrolyzable tannins such as pentagalloyl-
glucose and vescalagin (Haslam, 1993).
Hydrophobic association has also been used
to explain copigmentation effects observed
in solutions of anthocyanins and polyphe-
nols (Mistry et al., 1991; Liao et al., 1992)
or xanthylium dyes with caffeine or
cyclodextrins (Dangles and Brouillard, 1993).
Polyphenols bind significantly to proline-
rich proteins, and this complexation is
thought to drive taste perception and differ-
ences among various methods of tea bever-
age consumption (Luck et al., 1994).
Closer inspection of data on condensed
tannins (Martin et al., 1990) reveals interest-
ing correlations. To illustrate, an increase of
one hydroxyl group decreases apparent hy-
drophobicity yet increases association with
caffeine for epicatechin/epigallocatechin,
catechidgallocatechin, and catechin gallate/
epigallocatechin gallate pairs. Despite their
hydrophilic nature and twisted structure,
procyanidins show similar complexation with
caffeine. The additional hydroxy group may
increase the hydrogen-bonding character,
thereby inducing greater association with
 caffeine. Those polyphenols should also ex-
hibit less hydrophobicity because the extra
hydroxy group should prefer a hydrophilic
environment. Hydrogen bonding has been
demonstrated to be a strong driving force for
association, particularly in systems such as
cyanuric acid with melamine (Mathias et al.,
1993) and p-cresol with caffeine (Borazan
and Al-Ani, 1980). The hydrogen-bonding
character of catechidpolyproline association
has been demonstrated in model systems (Sun
and Mattice, 1996).
Simple hydrophobic association would
seem to be insufficient for explanation of
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protein complexation, precipitation, and
cream formation. A simplified model for the
complexation of polyphenols and caffeine
(Figure 26) demonstrates that association of
the two hydrophobic surfaces results in a net
decrease of hydrophobicity because the
polyphenols have a hydrophobic core sur-
rounded at its edge by hydrophilic groups.
All (or most) of the hydrophilic surface is
available for solvation, which should result
in a soluble, not precipitated, complex. This
mirrors the formation of micelles, which are
commonly understood to form a dispersed
“pseudophase”. It also mirrors the observed

association of two
hydrophobic surfaces...

1 key: results in forming- a

complex whose
hydrophobic area is
hydrophobic reduced, but
surface hydrogen-bonding area is
the same
(similar to a micelle and
caffeine self-aggregation)

hydrogen
bonding
surface
I However, hydrogen bonding reduces
hydrophilic surface area, which should
drive separation of ‘cream’ phase
.

FIGURE 26. The hydrophobic association model.

461
 self-stacking of caffeine aggregates (Thakkar
et al., 1970; Shestopa et al., 1985), which
attempt to compensate for unfavorable hy-
drophobic association of the plane by aggre-
gating in planar stacks of higher solubility.
Cream formation, however, requires the for-
mation of a discrete precipitate analogous to
phase separation.
Existence of hydrophobic bonds (Xianqi
and Haslam, 1994) is supported only by cir-
cumstantial evidence. Hydrophobicity is
driven by the loss of mutual electrostatic
stabilization of water molecules (or similar
hydrophilic groups). Hydrophilic groups are
forced to form ordered cages around a hy-
drophobic surface, placing the system at an
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entropic disadvantage. To overcome this dis-
advantage, the hydrophobic groups aggre-
gate and separate from the hydrophilic sur-
face in an attempt to minimize the surface
area of this ordered cage. The hydrophobic
surfaces are held together very loosely by
van der Waals’ forces. Aromatic surfaces
have also been described as hydrophobic,
but the mode of hydrophobicity of aromatic
molecules is apparently very different than
that of aliphatic hydrophobic surfaces
(Makhatadze and Privalov, 1994). In addi-
tion, aromatic and other unsaturated mol-
ecules can form hydrogen bonds with the n-
electrons, stabilizing the aromatic group in
the presence of water (Rzepa et al., 1994;
Ernst et al., 1995). There are also indications
that aromatic stacking interactions in aque-
ous systems have little to do with dispersion
forces or hydrophobic interaction (Newcomb
and Gellman, 1994).
A more satisfying model is one in which
hydrogen bonding of polyphenols to caf-
feine (or hydrophobically associated yet
soluble complexes to one another) causes a
net reduction of hydrophilic surface area with
little concomitant reduction of hydrophobic
surface area. This places the complex at an
electrostatic and entropic disadvantage, caus-
ing aggregation and precipitation in the
462
manner of phase separation. Proteins are most
easily precipitable at their isoelectric point
(Oh and Hoff, 1987), when the ionic charge
on a protein is lowest and the least number
of ‘water-solubilizing’ ionic factors are
present. The loose backbone of a protein can
hydrogen-bond with the polyphenol, particu-
larly at secondary amides such as those found
in proline residues, because secondary amides
have been shown to form stronger hydrogen
bonds than primary amides (Cannon, 1955;
Hagerman and Butler, 1981). After binding,
there is a reduction in hydrophilic surface
area, and the hydrophobic sidechains are
reoriented outward into aqueous solution
resulting in unfavorable interactions and pre-
cipitation. For larger proteins, binding of a
polyphenol might occur as the hydrophobic
core of the protein associates with the hy-
drophobic surface of the polyphenol. The
strong hydrogen bonding interactions will
then cause hydrophilic groups to turn in-
ward, denaturing the protein and causing
precipitation. Hydrophobic effects must also
play an important role because detergents
such as P-octyl-D-glucopyranoside can be
used to solubilize the precipitated polyphe-
no1 complex (Martin et al., 1990). Surfac-
tants are known to form ordered layers at
liquid-solid interfaces (Bigelow et al., 1946),
and the increase in entropy associated with
forming ordered monolayers may represent
a driving force for reversal of the hydrogen
bond complex and aggregate association,
resulting in increased water solubility of the
polyphenols and their complexes.
Within this model, hydrophobic associa-
tion and hydrogen bonding interactions are
given similar importance, as both are neces-
sary for driving the complex interactions
involved in the model. That the proposed
model does not currently explain apparent
differences between the condensed and hy-
drolyzable tannins is one limitation.
Vescalagin, castagalin, and pentagalloyl-
glucose have similar hydrogen bonding sites,
 although pentagalloylglucose precipitates
proteins much more readily (Haslam, 1993).
This may be related to steric restraints, but
different tannins can have different mecha-
nisms of precipitation and thus no single
model should be force-fitted to explain all
polyphenol interactions, unless the general-
ity can be rigorously proven and the excep-
tions well defined.
C. Polyphenols as Antioxidants
Research has progressed extensively on
the chemical and biological properties and
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functionality of tea polyphenols. Charcter-
ization of the antioxidant properties of tea
flavonoids is a prime example of the newer
research inititative.
There are numerous synthetic and natu-
ral compounds that block oxidative reac-
tions in vivo and in v i m by quenching free
radicals or by preventing free radical forma-
tion. These compounds have been termed
“antioxidants” (Namiki, 1990; Pokorny,
1992). Vitamins A, E, and C and the mineral
selenium are common antioxidants occur-
ring in our foods (Pokorny, 1992), and BHA
(butylated hydroxy anisole), B HT (butylated
hydroxytoluene), and propyl gallate are syn-
thetic antioxidants approved for food use
(Gates, 1987). A broad range of flavonoid or
phenolic compounds have been found to have
antioxidant activity (Ho, 1992). Polyphenols,
both synthetic and from numerous plant
sources, have also been found to be func-
tional antioxidants in numerous test systems
(Ho, 1992; Lunder, 1992; Osawa, 1992).
Antioxidants are the control agents for regu-
lation of oxidative reactions.
The antioxidant activity of tea extracts
and tea polyphenols has been studied in a
variety of model systems. It is clear that the
polyphenols from green and black tea are
effective antioxidants when evaluated in both
chemically and biochemically based test
systems. Green tea polyphenols have been
shown to be more effective than traditional
antioxidants such as BHA, BHT, ascorbic
acid, and vitamin E (Tanizawa et al., 1983;
Tanizawa et al., 1984; Namiki and Ozake,
1986; Zhao et al., 1989).
1. Chemical Antioxidant Systems
The effectivenessof purified tea polyphe-
nols and crude tea extracts as antioxidants
against the autooxidation of fats has been
studied using the Rancimat system. In one
study, purified green tea catechins were
evaluated against d2-a-tocopherol and BHA
(Ho et al., 1992). On a molar basis, the anti-
oxidant activity of the catechins was ranked
in the following ascending order: EC <ECG
<EGC <EGCG. Each of the catechins was
more effective than either d2-a-tocopherol
or BHA. A positive, synergistic effect be-
tween the catechins and ascorbic acid or dl-
a-tocopherol has also been found (Matsuzake
and Hara, 1985). Purified catechins and crude
methanol extracts of green, oolong, and black
teas have been shown to have significant
antioxidant activity. Gallic acid was found
to be a more effective antioxidant than any
of the tea extracts or purified compounds,
suggesting that the gallate moeties of the tea
polyphenols are an important part of their
expressed antioxidant acitivity (Ho et al.,
1992). Antioxidant activity of extracts of a
variety of green teas and blends of green and
black tea has been evaluated. A direct corre-
lation between the antioxidant index of a tea
extract and the concentration of EGCG in
the extract was shown (Lunder, 1992).
An alternate method to the rancimat,
based on measuring the oxidation of linoleic
acid catalyzed by bubbling air (2 h incuba-
tion at 60°C with 500 mumin air flow rate)
and determining the peroxide value (POV)
or the thiobarbituric acid value (TBAV) has
been used to study tea flavonoids (Tanizawa
463
 et al., 1983; Tanizawa et al., 1984). Epi-
catechin, green tea extract I11 (n-butanol
phase) and green tea extract IV (acetone
soluble portion of extract 111) were found to
have strong antioxidant activity equal to the
activity of BHA and were significantly more
potent than vitamin E. A similar model sys-
tem that involves the air oxidation of linoleic
acid incorporated into cetyl trimethyl-
ammonium bromide (CTAB) micelles has
also been used to study the antioxidant activ-
ity of flavonoids (Wang and Zheng, 1992).
The micellar system is a better model for
food emulsions and biological membrane
systems, and micellar studies showed that
flavonoids were active as chain-termination
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antioxidants by reacting with peroxy radi-
cals. The flavonoids had the following de-
scending order of reactivity: a-tocopherol
>morin >rutin >quercetin. There is a clear
difference between prevention of oxidation
of free linoleic acid (the crude extracts of
flavonoids were much more active than vita-
min E) and linoleic acid in micelles (fla-
vonoids were less active than vitamin E). It
is likely that vitamin E is readily incorpo-
rated into the micelles, which assures close
proximity of the antioxidant to the lipids.
Spin-trapping methods using ESR have
been employed to evaluate the ability of green
teas and other natural compounds to inter-
cept and measure free radicals. Free radicals
were generated by numerous sytems, includ-
ing stimulatedpolymorphonuclearleukocytes
(Zhao et al., 1989); hydroxy radicals pro-
duced in reaction mixture containing hydro-
gen peroxide and Fe++(from ferrous ammo-
nium sulfate) (Zhao et al., 1989; Shi and
Dalal, 1991; Uchida et al., 1987); a reaction
mixture of NADPH, potassium dichromate,
and hydrogen peroxide (Shi and Dalal, 1991);
an irradiated riboflavin system (Zhao et al.,
1989); and superoxide generated by the hy-
poxanthine-xanthine oxidase system (Uchida
et al., 1987). DMPO (5,6-dimethyl- 1-
pyrroline-N-oxide) was used to trap free radi-
464
cals and monitor their formation. These stud-
ies demonstrate tea constitutents as good
radical scavengers, suggesting that tea
polyphenols can act as antioxidants in termi-
nation of both the inititation and chain termi-
nation stages of lipid peroxidation.
A classic method of generating hydroxy
radicals is the Fenton reaction, a reaction of
iron salts and hydrogen peroxide. Studies to
determine the effect of polyphenols in scav-
enging hydroxy radicals generated by the
Fenton reaction resulted in the following
ranking of antioxidant potency: curcumin
(69% inhibition), vitamin C (56% inhibi-
tion), vitamin E (35% inhibition), rosemary
extract (16% inhibition), green tea polyphe-
no1 (12% inhibition), and so-called “green
tea fraction 6” (13% inhibition) (Zhao et al.,
1989). Caffeine was also found to be an
effective scavenger of hydroxy radicals gen-
erated by the Fenton reaction, but its effec-
tive concentration was three orders of
magnitide higher than of green tea catechins
or vitamins (Shi and Dalal, 1991).
The key active sites of flavonoids for
scavenging of free radicals and for antioxi-
dant activity are the o-dihydroxy structure in
the B ring, the 2,3-double bond in conjuga-
tion with the 4-0x0 function in the C ring in
flavonols, and the 3- and 5-hydroxy groups
with the 4-0x0 function in the A and C rings
(Nakayama et al., 1993; Pratt, 1992;
Jovanovic et al., 1994). Catechins can react
with more than one free radical to form
quinones (Zhao et al., 1992) and have a
lower reduction potentials than vitamin E
(Jovanovic et al., 1995).This should account
for the stronger radical-scavenging activity
of catechins. Tannins with a greater degree
of gallation were more effective in trapping
radicals on a molar basis than EGCG, sug-
gesting that theaflavin digallate and gallated
thearubigens from black tea could also be
effective radical scavengers. Future research
is needed to understand the potential health-
promoting properties of antioxidants, and
 better characterization and understanding of
the polyphenols in both green and black tea
extracts is needed.
2. Biological Antioxidant Models
Biologically based model systems for
the evaluation of antioxidants have been em-
ployed with tea extracts and polyphenols.
These model systems mimic the reactions
believed to be linked to the pathogenesis of
some chronic diseases.
Catechin was found to act as an effective
antioxidant in an iron-loaded rat hepatocyte
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system and was proposed to function through
an ion chelation mechanism (Morel et al.,
1993). However, other studies using catechin
as an antioxidant have suggested that its
antioxidant activity is due to a free radical-
scavenging mechanism (Fraga et al., 1987;
Ratty and Das, 1988). It is likely that both
properties of tea polyphenols contribute to
their antioxidant activity.
An erythrocyte membrane ghost system
and a rat liver microsome system were used
to study dietary antioxidants and oxidation
processes (Namiki and Ozake, 1986). The
catechins from tea were found to be very
active in both systems, with EGCG and ECG
having the greatest activity in blocking lipid
oxidation. These catechins were 10 times
more effective than vitamin E, but were less
active than BHA. EGCG and ECG were ef-
fective against peroxidation initiated by ei-
ther the chemotherapeutic agent adriamycin
or the perferyl ion, while EGC was effective
only against perferyl-ion induced oxidation
(Osawa et al., 1992). This suggests that
EGCG and ECG were effective as both radi-
cal scavengers and chelators, while EGC
appears to be a weak radical scavenger but a
good chelator. In the stimulated polymor-
phonuclear leukocyte system, a green tea
polyphenol fraction and a green tea extract
fraction called 6 (undefined) had much stron-
ger radical scavenging activity than vitamin
C, vitamin E, rosemary extract, or curcumin
(Zhao et al., 1989).
Gallic acid and gallic acid esters have
been shown to have both antioxidant and
prooxidant activity in vitro (Aruoma et al.,
1993; Cao et al., 1996). Noticeable oxida-
tion of deoxyribose was observed with ex-
cess concentrations of gallic acid, suggest-
ing that gallic acid may act as a prooxidant
and play a role in prooxidant activity in green
tea.
The cytotoxic effects of reactive oxygen
species (Oy and H202) on cells grown in
culture was used as a model system for the
evaluation of the antioxidant activity of fla-
vonoids (Nakayama et al., 1993; Ruch et al.,
1989). Chinese hamster lung fibroblast V79
cells in culture were incubated in the pres-
ence of flavonoids for 4 h to allow sufficient
time for binding of the polyphenols to the
cell surface or cellular absorption after which
the cells were washed to remove free fla-
vonoids. The washed cells were then ex-
posed to reactive oxygen species (either H20,
or superoxide produced enzymatically using
a combination of hypoxanthine and xanthine
oxidase). In a cytotoxicity test of the fla-
vonoids, catechin and taxifolin were not toxic
at concentrations up to 200 and 1000 pM,
respectively, while quercetin and kaempferol
were toxic at concentrations above 100 pM.
Quercetin and kaempferol were effective in
protecting cells from both superoxide and
peroxide at concentration of 5 pM and 20
pM,while catechin was effective at concen-
trations of 500 pM and 1000 pM (Nakayama
et al., 1993). A similar model system used
cultured mouse hepatocytes rather than ham-
ster lung fibroblasts (Ruch et al., 1989). These
studies showed that a green tea polyphenol
fraction (GTP) was effective in scavenging
H202and superoxide radicals by demonstrat-
ing the ability of GTP to inhibit cell death
induced by oxygen radicals and peroxides
produced by glucose oxidase or paraquat in
465
 a dose-dependent manner. An in vivo ex-
periment was conducted to determine the
effect of green tea on radiation-induced lipid
peroxidation and lethality (Uchida et al.,
1992). Epigallocatechin gallate (EGCG) was
provided to mice in drinking water for 1
month at 0.002% or 0.01% concentration
providing an EGCG intake of 3 or 15 mg/kg/
d, respectively. The mice were then irradi-
ated with 954 cGy X-ray and killed 3 d later.
Oral consumption of EGCG inhibited irra-
diation-induced formation of hepatic perox-
ides in a dose-dependant manner, reducing
the formation of peroxides 75% and 90% for
the 0.002% and 0.01% doses, respectively.
Oral consumption of EGCG was also found
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to significantly extend the lifespan of mice
given a lethal dose of radiation. These re-
sults support the conclusion that tea catechins
are effective scavengers of free radicals in
vivo .
Tea extracts, tea polyphenol fractions,
and purified catechins have been shown to
be effective antioxidants in both chemically
and biologically based model systems. In
both types of systems, the balance between
oxidants and antioxidants is critical. Imbal-
ances between free radicals and antioxidants
can occur, caused by an increased produc-
tion of free radicals andor decreased effec-
tiveness of the antioxidants within the reac-
tion system. These imbalances can be caused
by the radicals overwhelming the antioxi-
dants within the system, or by an excess of
antioxidants leading to a prooxidant func-
tionality (Aruoma et al., 1993). It is critical
that a proper antioxidant to oxidant balance
be maintained in well-controlled biological
studies. The balance of different types of
antioxidants is also important, as demon-
strated with carotenoids, vitamin E, and vi-
tamin C (Bohm et al., 1997).
VI. TRENDS IN TEA RESEARCH
Tea chemistry as a research discipline
has been characterized and influenced by the
466
techniques and approaches of each group of
researchers. In order to understand much of
the language and notation used in the current
literature, it becomes necessary to context-
ualize the development of these terms and
theories, and, in effect, deconstruct the sci-
ence.
The application of HPLC in tea research
represents a good example of this: as a new
technology, it offered a radical new way of
looking analytically and quantitatively at tea
components. Yet, the diffuse understanding
of the compounds in tea, combined with
qualitative descriptors historically available,
has provided a challenging barrier to sur-
mount. A thearubigen is very clearly assumed
to be a complex mixture of polymerized
polyphenols that appears as a diffuse spot on
paper chromatography. The identity and
location of that spot becomes unclear
when using a new and different technology.
Without a definitive pure standard of a
thearubigen, only a qualitative assessment
can be made of an already qualitative de-
scriptor. As a new technology breaks a pre-
viously homogenous mass of compoundsinto
subdivided fractions, the relative differences
in concentration of these subdivisions among
different samples can negate the idea of ho-
mogeneity and cause dramatic reevaluations
of theories that depended on the homogene-
ity of the original mass. Tea research has
brought the thearubigens very close to this
point, and the homogeneity assumed by this
classification is beginning to show weak-
ness.
The advancement of tea chemistry is
dependent on comprehension of the litera-
ture in the context of the directions of indi-
vidual research groups.
A. Analysis by Chemical
Constitution and by Technical
Innovation
Early determinations of the chemical
constitution of tea and tea beverages
 (Kursanov, 1956; Vuataz et al., 1959; Millin
and Rustige, 1967) set the stage for a gener-
alized interpretation of the chemistry of tea
(Sanderson, 1972). The subdivisions of each
category and the analytical accuracy of new
methods have improved the understanding
of the mass balance of tea chemistry. The
subdivision technique has been propagated
in the literature (Graham, 1984; Balentine,
1992).
No single technological innovation has
propelled the efforts of tea chemistry more
in recent years than the introduction of the
HPLC (Hoefler and Coggon, 1976), and the
subsequent coupling of the technique with
diode-array detection (Bailey et al., 1990;
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Opie et al., 1990;Opie, 1992) and mass spec-
trometry (Lin et al., 1993; Bailey et al.,
1994b). Despite its power, HPLC is in a
stage of relative infancy compared with the
field of tea chemistry, and it would seem that
a large fraction of the task of repeating and
reinterpreting the historical results of tea
chemistry within the language and context
of this instrumental method still needs to be
performed. Progress and innovation in sci-
entific research is frequently the result of
dramatic breakthroughs or “revolutions”
(Kuhn, 1970). As each “revolution” propa-
gates itself through a scientific discipline,
the paradigms and assumptions that are as-
sociated with the discipline must be reevalu-
ated, and the effect of HPLC on tea chemis-
try needs further investigation in this vein.
B. Bulk Properties and Correlations
to Tea Taster’s Profiles
Efforts to understand the chemistry of
tea can be seen in part as motivated by the
desire of tea plantation owners and manu-
facturers to understand what factors drive
acceptability and market price. Tea imports
into the U.S. in 1993 totaled $124 m. (Anon.,
1994), a figure that has fluctuated but essen-
tially remained flat for 5 years. Improve-
ments in the quality, marketability, or yield
become strong driving influences for contin-
ued business growth and profitability. Im-
provements in production yield and quality
of Indian tea has been attributed to better
technological understanding of the underly-
ing factors controlling tea production
(Mahanta et al., 1993). It is therefore a busi-
ness necessity as much as an intellectual
pursuit to gain a technological advantage, an
effort that can only be underpinned by a firm
understanding of the chemistry of tea.
Many of the efforts of tea research focus
on improving the value of tea by understand-
ing the chemical parameters that deter-
mine price. Brisk teas are more astringent
and are associated with earlier fermentation
times and higher theaflavin content (Owuor
and McDowell, 1994). It follows that
thearubigens, which are a product of later
fermentation times, should be expected to be
a negative quality for production of brisk
teas. The effect of thearubigens on cream
formation (Hazarika et al., 1984) can also
determine acceptability, especially for iced
tea beverages. Principal component analysis
has determined that theaflavins are positively
correlated (and some flavonol glycosides are
negatively correlated) with tea value
(McDowell et al., 1991). Theaflavins are very
important for quality and price, owing to the
bright red color they impart (Hilton and
Palmer-Jones, 1975; Ellis and Cloughley,
198l), but among similar theaflavin con-
tents, theaflavins are not a good determining
factor (Owuor et al., 1986). Aroma can also
be expected to play an important role, espe-
cially in flowery teas (Robinson and Owuor,
1992).
C. Tea as an Antioxidant and as a
Healthy Beverage
The perception of tea as a healthy bever-
age is traced to the legendary Shen Nung,
who claimed in the Pen Tsao, a Chinese
book of herbal medicine, that tea is good for
a variety of ailments, including tumors. Tea
467
 consumption in ancient times was regarded
as healthy, likely due to the fact that the
boiling water used to make tea kills many of
the water-borne pathogens that caused ill-
nesses common in those times. Vitamin P
activity, a reduction of capillary fragility,
has been found in tea and attributed to tea
polyphenols (Kursanov et al., 1950). Today,
research into tea chemistry is a fundamental
part of studies on the role of tea as a biologi-
cal antioxidant and in prevention of chronic
diseases.
There is mounting evidence that sub-
stances called free radicals play a role in the
development of major human diseases such
as heart disease and cancer. Research sug-
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gests that antioxidants may help protect
against these diseases by minimizing the
detrimental impact of free radical damage to
cells and tissues. Much more research is
needed to better identify the processes in-
volved in free radical-mediated diseases and
to help develop nutritional strategies to op-
timize antioxidant status.
The most commonly recognized dietary
sources of antioxidants are fruits and veg-
etables, which contain vitamins C, E, and
carotenoids. Preliminary research indicates
that the flavonoids, which include the cat-
echins and flavonols found in both black and
green tea, also act as antioxidants. However,
additional research is required to determine
whether tea antioxidants substitute for or
complement some of the protective func-
tions identified with the more established
antioxidant compounds such as vitamins C,
E, and beta-carotene. The role of antioxi-
dants in heart disease is unclear, and their
potential role in cancer prevention and the
aging process remains speculative.
1. Lipid Oxidation and
Cardiovascular Disease
Cardiovascular disease is one of the lead-
ing causes of death in the Western World.
468
There is growing evidence that oxidative
injury is a major risk factor in development
of cardiovascular disease (Marx, 1987;
Grundy, 1993). Recent reports that dietary
supplementation with vitamin E (at 100 IU
and less than 400 IU) was associated with a
significant reduction in the risk for cardio-
vascular disease (36 to 54% reduction in risk
for women and a 20 to 26% reduction in risk
for men) support this hypothesis (Brody,
1993).
One of the key mechanisms of the hy-
pothesis that oxidative damage is linked to
cardiovascular disease is the theory that oxi-
dation of low-density lipoprotein (LDL) leads
to damage of the arterial wall, producing
sites for fibrous plaque formation. Antioxi-
dant vitamins are believed to inhibit this
process, and studies have shown that the
antioxidant vitamins ascorbic acid, a-toco-
pherol, and p-carotene inhibit LDL oxida-
tion in vitro and reduce the progression of
atherosclerosis in animal models.
Studiesusing a Chinese green tea polyphe-
no1 fraction (CGTP) were conducted to deter-
mine the effect of tea constituents on oxida-
tion of low-density lipoprotein (LDL) in vitro
in a model system thought to be representa-
tive of the effects of antioxidants in modulat-
ing risk factors associated with cardiovascu-
lar disease (Ding et al., 1991; Ding et al.,
1992). The antioxidant activity was deter-
mined based on lipid peroxide production,
thiobarbituric acid reactive substances
(TBARS), and the mobility of LDL in an
electrophoretic gel. The results of these stud-
ies showed that green tea polyphenols block
LDL oxidation induced by copper, UV irra-
diation, or macrophages. The mechanisms for
the antioxidant activity of the CGTP have not
been determined but are theorized to be metal
chelation, free radical scavenging, and an
antioxidant “vitamin-sparing” (whereby anti-
oxidant is preferentially consumed, ‘sparing’
the vitamin) effect. A dose-dependentinhibi-
tion of copper-induced LDL oxidation and
LDL oxidation induced by transformed
 macrophages were also found for catechin
(Mangiapane et al., 1992).
2. Studies of Tobacco Nitrosamines
Smoking of tobacco products is the main
risk factor for development of cardiovascu-
lar disease and cancer. The nitrosamines
derived from tobacco smoke are good bio-
logical oxidants that can damage cellular
lipids, LDL lipids, and DNA (Yang, 1992;
Loft et al., 1992; Marx, 1987). One outcome
of oxidative damage to DNA is the 8-hy-
droxylation of guanine bases producing
8-hydroxydeoxyguanosine (80HdG) DNA
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adducts. Repair of these DNA adducts in
vivo leads to the excretion of 80HdG in
urine that is a biomarker or index of the
current state of oxidative DNA damage and
repair (Saul and Ames, 1986; Loft et al.,
1992). Tobacco use by men and women has
been shown to cause a 50% increase in the
excretion of 80HdG, indicating that smok-
ing causes a significant increase in the level
of oxidative damage to DNA (Loft et al.,
1992). Cigarette smoking (>lo per day) was
found to significantly increase the mutation
sensitivity of chromosomes in peripheral
lymphocytes (Kim et al., 1993). This was
determined by treating the cells with mito-
gens and measuring the frequency of sister-
chromatid exchange (SCE). The degree of
SCE is a good index of the instability of
DNA and of oxidative stress.
A clinical trial was conducted to deter-
mine if consumption of green tea or coffee
would alter the sensitivity of DNA to SCE in
smokers. Patients were provided three cups
of green tea or coffee per day for 6 months.
Consumption of three cups of green tea per
day inhibited the cigarette smoking-induced
increase in frequency of SCE. Coffee con-
sumption did not change the frequency of
SCE (Kim et al., 1993). This demonstrates
that normal tea consumption should be suf-
ficient to block smoke-induced DNA dam-
age due to oxidative events.
A tobacco-specific nitrosamine, 4-(me-
thy1nitrosamino)-1(3-pyridy1)-1-butanone, or
NNK, is an oxidative carcinogen that in-
duces lung cancer in numerous laboratory
animal models (Xu et al., 1992; Wang et al.,
1992a). After metabolic activation, NNK
reacts with DNA to form oxidized adducts
(80HdG). When solutions of green tea, black
tea, or the green tea catechin epigallocatechin
gallate (EGCG) were provided to mice in
place of drinking water the numbers of NNK-
induced lung tumors were significantly re-
duced (Xu et al., 1992; Wang et al., 1992a).
Black and green tea were found to be equally
effective in reducing tumor numbers when
provided during or after NNK treatment
(Wang et al., 1992a). EGCG was found to be
almost as active as green tea infusions (Xu
et al., 1992). In vivo (Xu et al., 1992) studies
using mice and in vitro (Shi et al., 1993)
experiments showed that black and green tea
polyphenols inhibit NNK metabolism and
DNA adduct formation. EGCG was found to
be the most potent inhibitor of the tea
polyphenols.
Polyphenols containing 1,2- or 1,4-
diphenol functional groups have been found
to be potent inducers of a group of enzymes
involved with the metabolism of carcino-
gens and other xenobiotic compounds,
termed phase I1 enzymes (Sohn et al., 1994).
Phase I1 enzymes include NAD(P)H:quinone
reductase, glutathione S-transferases, and
UDP-glucuronosyltransferases (Talalay,
1992; Prochaska and Talalay, 1992). Be-
cause tea polyphenols fit the criteria for Phase
I1 enzyme inducers, it is likely that tea
polyphenols act as potent inducers of these
enzymes, as has been reported (Khan et al.,
1992). It is likely that one mechanism for the
inhibition of NNK-induced lung tumors is
blocking of NNK activation. A radical scav-
enging mechanism might also be involved in
the inhibition of NNK-induced DNA adduct
469
 formation. The effect of tea on reduction of
tumors after NNK treatment indicates that
tea can inhibit tumor formation through a
second mechanism that does not involve
NNK metabolism. Oxidative events have
been linked to the promotion and progres-
sion of initiated cells to tumors and cancers.
Tea may also act to block these events
through an antioxidant mechanism. The pro-
motion of tumor formation in mouse skin by
"PA is partially induced by formation of
peroxides or free radicals (Wei and Frenkel,
1993). Tea, GTP (Huang et al., 1992; Wang
et al., 1992b), EGCG (Fujiki et al., 1992),
and antioxidants (Kozombo et al., 1983;
Smart et al., 1987) were effective in block-
Downloaded by [Stanford University Libraries] at 03:37 09 July 2012
ing TPA-induced tumor promotion in mouse
skin. Mechanistic studies are needed to es-
tablish the role of tea in blocking these cel-
lular events.
3. Tea and Cancer
The relationship between consumption
of tea and a decreased incidence of cancer
has received a significant amount of scien-
tific and media attention during the last few
years. The role of dietary constituents
(Stavric, 1994; Wattenberg, 1992) and tea
(Yang and Wang, 1993) in prevention of
cancers has been the subject of numerous
scientific papers in recent years. While the
emerging research has stimulated a great deal
of scientific interest, evidence is still insuf-
ficient to allow any clear statements to be
made about the relationship between tea
consumption and cancer in humans.
VII. CONCLUSION
Understanding the chemistry of tea rep-
resents a key technologicalunderpinning after
which the systematic study of tea beverages
as a healthy addition to the diet can be per-
formed. Achieving mass balance of the
470
polyphenolic constituents of green and black
tea is undoubtedly a central focus of tea
research. Indirect correlations such as those
used to measure thearubigens need to be
systematicallyjustified with tangible chemi-
cal evidence, whether by isolation of indi-
vidual compounds as performed by HPLC,
or by selective hydrolysis by which the frag-
ments can be measured with quantitative
accuracy.
Tea research is moving away from its
rudimentary origins as a qualitative out-
growth of tea tasting and the tea industry and
moving into a fully quantitative and chemi-
cally interesting science. The perception of
tea not only as a plant and as a beverage but
as a healthy part of the human diet and as a
rich source of new chemical compounds will
propel tea research in new directions.
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