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Critical Reviews ill Plant Sciences, 16(5):415-80 (1997)
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TABLE OF CONTENTS
I. Introduction ....................................................................................................................... 417
II. Chemical Composition, an Overview ...................................................................... ; ..... 423
A. Polyphenols .................................................................................................................. 424
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
III. 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
07352689/97/$.50
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 Tca Polyphcnols ....................................................................................... 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
1. Phenylalanine and the Shikimate Pathway ........................................................ 452
2. Chain Extension, Hydroxylation .......................................................................... 453
3. ChalconelFlavone Tautomerization ..................................................................... 454
4. FlavanolslFlavonols ............................................................................................... 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 Modcls .............................................................................. 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 a 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. Va 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.
KY WORDS: tea chemistry, polyphenol complexation, antioxidant properties.
I. INTRODUCTION
The real voyage of discovery consists
not in seeking flew landscapes but in
having new eyes.
Proust
The universe is 1I0t only queerer than
we suppose, but queerer thall 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 i ncreased awareness of the potential
health benefits associated with tea consump
tion, tea chemi stry 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
approximately 35 to 40 I of beverage. India
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., 1 994). Camellia
sinensis is a very i mportant agricultural and
commercial product with a unique horticul
ture and manufacturing process.
Chinese mythology teaches that i n 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 22 1 A.D. The first men
tion of tea is believed to occur i n the Erh Ya,
a Chinese dicti onary 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 i n 780
A.D. The history of tea is extremely relevant
because before the Tang dynasty (61 8 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 i n a soup through the Tang dy
nasty, often mixed with onions, salt, orange
peel, and/or 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 fel l
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 modem custom of brewed
tea leaves arose during the Ming dynasty
( 1 368 to 1 644), coinciding with the West' s
arrival i n China. Although there are many
variations on brewing technique, which can
i mpact 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
in the spread of tea cultivation into India
between 1 8 1 8 and 1 834, derived either
through i mport 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, 1 976).
Excellent reviews on the culture and produc
tion of tea are available (Wilson and Clifford,
1 992). Figure I 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
30C are the most favorable for growth and
agriculture of tea (Eden, 1 976).
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-
418
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 destructi ve 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 si tuated 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 i n 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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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
manufacture i s 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 i s a significant modern
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 i s 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
i n 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 1 993 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., 1 994).
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 i n 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 available is that of Sanderson
(Sanderson, 1972). Compl ementary to
Sanderson's review is that by the Russian
tea chemists Bokuchava and Skobeleva
(Bokuchava and Skobeleva, 1 980), which
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421
India 560
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FIGURE 3. Consumption of tea, 1993 (10' metric 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, f1avonols, flavonol glycosides, and
the theaflavins, have dramatically changed
the way tea chemistry i s studied, and these
methods have been reviewed in detail (Fin
ger et aI. , 1 992).
There are a variety of other reviews on
the subject of tea, including Roberts' (Rob-
422
erts, 1 942; Roberts, 1 962) and Graham's
(Graham, 1983; Graham, 1 984) reviews of
tea chemistry, a review by Stahl (Stahl, 1962),
Wickremasinghe (Wickremasinghe, 1 978),
Balentine (Balentine, 1 992), and a review by
Robertson (Robertson, 1 992). An excellent
comprehensi ve review of the work on tea
aroma to the early 1 980s i s also available
(Bokuchava and Skobeleva, 1 986), as well
as a more recent review (Robinson and
Owuor, 1 992).
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 chemi stry 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 l i terature. 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
character of the amino acids, and flowery
character of the aroma constituents, play a
much more significant role. In addition to
the nonpol yphenol i c consti tuents, the
polyphenols of black tea are a poorly under
stood but seemingly well-defined group of
compounds. The use of "thearubigen" in the
literature signifies a wide group of com
pounds whose chemical identity has not been
traced to any identifiable chemical group. Its
use was most significant as a colorimetric
indicator for plantation production, but as a
chemical identifier its use has seemed to
hamper true identifcation of the mass balance
of black tea polyphenols. Work at identifying
these compounds in a systematic fasion has
begun, and it is hoped that future research
will deemphasize the term ' thearubigen' in
favor of more chemically accurate des
criptors.
II. CHEMICAL COMPOSITION, AN
OVERVIEW
In order to divide the subject of tea chem
istry into well-ordered i ssues, it is most con
venient to do so on the basis of chemical
composition. Frequently, researchers' inter
est in specific compounds or a class of com
pounds induces a focusing of attention on a
small fraction of the total tea mass. In addi-
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 I 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,
these factors generally control the amount of
solids extracted, and to a lesser extent influ
ence the composition. A typical brew of one
tea bag in one cup of water produces a solu
tion of 0.35% wtlwt solids, and from this
value the dose expected from consumption
of one cup of tea can be calculated. This is
typically how tea phytochemicals are con
sumed. Notes on the composition of the
'flush' or the fresh tea leaves, or other parts
of the tea plant, are added for completeness
TABLE 1
The Composition of a Typical Tea
Beverage, %wtwt Solids
Green tea Black tea
Catechins 30% 9%
Theaflavins 4%
Simple polyphenols 2% 3%
Flavonols 2% 1%
Other polyphenols 6% 23%
Theanine 3% 3%
Aminoacids 3% 3%
Peptides/Protein 6% 6%
Organic acids 2% 2%
Sugars 7% 7%
Other carbohydrates 4% 4%
lipids 3% 3%
Caffeine 3% 3%
Other methylxanthines <1% <1%
Potassium 5% 5%
Other minerals/ash 5% 5%
Aroma Trace Trace
423
within the review. We have also subdivided
the issue of tea compounds by chemical class.
This is done to focus on specific aspects of
tea chemistry, such as aroma or polyphe
nols, and because the methods used for de
termination of the various chemical classes
are quite different.
The strict division of tea chemistry by
functional chemical identity has its uses, but
begins to lose value i n discussing subjects
that cross the boundaries of chemical class
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
gree of accuracy, and determination of the
synergies and 'boundary violations' present
in the chemical composition of tea bever
ages becomes a necessity.
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 1 80 to 240 mg of polyphenols
from a strong cup of tea. Recent interest in
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
a means of chemical defense against insects,
birds, and animals, which would consume
the plant as food (Beart et aI., 1 985). The
evolution of salivary proline-rich proteins,
which bi nd polyphenols effecti vely, has
ameliorated this defense mechanism, con
verting it to 'astringency' (Luck et aI., 1 994).
424
B. Cafeine, Methylxanthines, and
Related Compounds
Tea has been valued historically for
its caffeine content. Caffeine [ 1 ] i s viewed
as an important constituent of tea, bestow
ing mood and cognitive-enhancing proper
ties (Bokuchava and Skobeleva, 1 980).
Figure 4 illustrates the methylxanthines of
tea. Tea leaves contain between 2 and 5%
wt/wt caffeine depending on the variety.
1|. Caffei ne
2|.Theobromi ne

o
[3|. Theani ne
FIGURE 4. W|lrogenouslea pnylocnem|ca|s.
C irrawadensis, a member of the Camellia
family, lacks caffeine (Roberts et ai., 1 958)
but is not processed commercially because it
produces a poor finished tea product. The
quantity of caffeine that i nfuses into a tea
brew i s 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 caffeifl <on
tent of a typical tea beverage will range from
20 to 70 mg per 1 70 ml of infusion, with a
typical infusion being prepared from about 2
to 2.5 g of tea leaves. Coffee brews typically
contain from 40 to 1 55 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 ai. , 1 965).
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 ai. , 1 996). 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 ai., 1 996), discrediting the
theory that withering and fermentation have
a significant impact on caffeine content
(Sanderson, 1 972).
Caffeine is one of the most comprehen
sively studied ingredients in the food sup
ply. Extensive research does not l i nk 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] i s present in tea i n much
lower quantities than caffeine. Theobromine
is formed as a consequence of the biosynthe
sis of caffeine (Negishi et ai., 1 985a) and is
produced in abundance if the methylation
path to caffei ne i s absent, such as i n
C irrawadensis (Roberts et ai., 1 958). Theo
phylline, a similar di-methylxanthine, has
been reported in trace quantities in tea leaves
(Michl and Haberler, 1 954; Sanderson, 1 972).
Recent reports contradict as to the the exist
ence of this compound in tea, some fai l i ng to
detect these compounds (Hicks et ai., 1 996)
and others (Meyer et ai., 1 996) 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 ai . , 1 982), and a specific ad
enine nucleosidase (Imagawa et ai . , 1 979).
These catabolic reactions produce purines,
which have been detected in very small quan-
425
tities i n tea (Michl and Haberler, 1 954;
Sanderson, 1 972; Hicks et aI. , 1 996).
C. Proteins and Amino Acids
While caffeine is the most well -known
ni trogenous component of tea, tea proteins/
pep tides and amino acids contribute signifi
cantly to the composition of both the leaf
and the tea extract. Figure 4 i l lustrates some
of the other important miscellaneous nitrog
enous and non nitrogenous components of
tea. Recent measurement of the amino acids
in two green teas (Liang et aI., 1 990) con
firms the presence of 1 8 amino acids, a re
sult that is mirrored for black tea. Amino
acids contribute about 6% wt/wt of the ex
tract solids. Tea also contains a significant
amount of peptidic material (protein), ap
proximately 6% wt/wt of extract solids. Ni
trogenous materials therefore comprise about
1 5% wt/wt of extract solids. The free amino
acid content of tea seems to increase during
withering of the fresh tea leaves but de
creases duri ny fermentation to black tea
(Roberts and Sanderson, 1 966), as it i s
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% wt/wt of extract solids.
Theanine is a significant component of both
green tea (Sakato, 1 950) and black tea
(Feldheim et aI. , 1 986). Theanine has been
associated with improved flavor and a modu
lation of the stimulative effects of caffeine
(Kimura and Murata, 1 97 1 ). Recent studies
on the antihypertensive effect of theanine
found that a large quantity of this compound
was required to exhibit an effect i n rats
(Yokogoshi et aI., 1 995).
426
Optimum production of theanine i n cell
culture has been investigated (Furuya et aI.,
1 990; Matsuura et ai . , 1 992). Theanine
can also be made synthetically on a com
mercial scale in good yield (Kawagishi
and Sugiyama, 1 992).
Tea leaves subjected to anaerobic condi
tions are found to produce excess amounts
of GABA, ory-amino butyric acid (Tsushida,
1 987), derived from glutamic acid due to the
action of an endogenous glutamate decar
boxylase in tea (Tsushida and Murai, 1 987).
D. Carbohydrates, Pectins, and
Fiber
Tea leaves have been shown to contain
free sugar residues in addition to pectic
subtances, pol ysaccharides, and fiber
( Mi zuno et ai . , 1 964; Sanderson and
Perera, 1 965). Carbohydrates contribute ap
proximately I 1 % wtwt of extract solids
(Sanderson et aI., 1 976; Graham, 1 984). High
molecular weight pectins (polygalacturonic
acid) and other polysaccharides have been
analyzed on Sephadex G- 1 00 (Millin et aI.,
1 969). As the tea plant matures, increases in
the content of lignin and cellulose have been
observed (Selvendran et aI., 1 972), 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 disaccharide 2-0-(-L-Arabino
pyranosyl)-myo-inositol [4] (Sakata et ai.,
1 987), 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 aI., 1 989).
E. Organic Acids and Vitami n C
Tea is a significant source of oxalic acid
(Sanderson and Selvendran, 1 965) and malic
acid (Jayman and Sivasubramanian, 1 975),
along with citric, isocitric, and succinic ac
ids (Sanderson and Sel vendran, 1 965). Tea

O
H
OH
HO 0
HO 0
0H
OH
[4]: 2-0-(p-L-Arabinopyranosyl)-myo-inositol
HO
OH
HO
b]: Spinasterol
FIGURE 5. Miscellaneous tea phytochemicals.
also contains shikimic and qUIntC acids,
which are important to the biosynthesis of
the polyphenols (Zaprometov, 1 961 ). Vita
min C (ascorbic acid) has also been detected
in green tea (Liang et aI., 1 990) and black
tea (Sanderson, 1 972).
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
i stic brown-black color. Some of the
pheophytins and pheophorbides are extracted
i nto the black tea beverage (Sanderson,
1 972). An efficient HPLC method has been
developed for analysis of these pigments
(Taylor and McDowell, 199 1 ). 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 aI., 1 992).
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, 1 992).
The carotenoids play a significant role in the
formation of aroma characteristic to black
tea (Bokuchava and Skobeleva, 1 986). High
chlorophyll levels and low astringency are
important to some green tea manufacture,
particularly Mattcha, for which a brothy,
emerald-colored brew i s 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, 1 988). It is hypoth
esized that the hydrophobic environment
presented by the tea polyphenols and caf
feine complex i n tea provide for extra solu
bility of the lipid components. Lipids are
approximately 3 to 4% wtlwt of leaf and has
been analyzed in detail (Anan, 1 983; Bhuyan
and Mahanta, 1 984). The role of hydropho
bic plant materials in the appearance and
organoleptic properties of the brew has been
typically disregarded in favor of the polyphe
nols, and this is an important area for which
future research is necessary.
G. Viti mans and Minerals
The tea plant has been shown to be rich
in potassium (Sanderson ct aI., 1 976) and
contains significant quantities of calcium and
magnesium, as well as small amounts of
manganese, iron, and phosphorus (Kalita
and Mahanta, 1 993), copper and nickel
(Burke and Albright, 1 970), and sodium,
boron, and molybdenum (Hasselo, 1 965).
Zinc (Tolhurst, 1 962) and sulfur (Pethiyagoda
and Krishnapillai, 1 970) are essential ele
ments for healthy maturation of the tea plant
as wel l . Cobalt, lead, and cadmium have
been detected i n the plant, and concentra
tions depend principally on soil concentra
tions of these minerals (Ramakrishna et aI . ,
1 987).
The tea plant is known to accumulate
aluminum (Chenery, 1 955). Aluminum lev
els can be traced by NMR techniques (Nagata
et aI., 1 991 ) and have been found i n com
plexes with fluoride and catechins in the tea
plant (Nagata et aI., 1 993). 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-Lewis et aI., 1980).
This is due, in part, to the uptake of alumi
num fluoride (Yamada and Hattori, 1977;
Yamada and Hattori, 1 980).
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 i n
trace quantities, the composition and mecha
nism of production of which has been re
viewed (Choudhury, 1982; Bokuchava and
Skobeleva, 1 986; Robinson and Owuor,
1992).
Withcring has been determined to play
an important role in aroma development, as
i n oolong teas (Takeo, 1 984; Kharebava,
1 986) and black teas (Owuor et aI., 1 987).
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 aI., 1992).
Geranyl, linolyl, terpinyl, and neryl glyco
sides can be found in fresh tea extracts (Guo
et aI., 1 993). -Glucosidase is the enzyme
most likely to be responsible for formation
of tea aroma from these glycosides (Morita
et aI . , 1994). The exploitation of bound gly
cosides of aroma components by glycosi
dase treatement offers the possibility of fu
ture improvements i n 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 a!.,
1991 ) . There are also many products that are
derived from the oxidizing conditions present
during tea fermentation (Bokuchava and
Skobeleva, 1 986). Figure 6 illustrates one
such unique aroma constituent, theaspirone
[9], which i s produced from oxidation of
p-carotene [7].
III. POL YPHENOLS IN TEA
The term 'polyphenoJ' 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
[7]: p-Carotene
/

[8]: p-Ionone
many other aroma constituents
[9]: Theaspirone
FIGURE 6. Uniquelea aroma cons||luenls.
429
treated i n greater depth i n 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. i t is natural that tea chemistry is often
considered to be synonymous wi th 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
beverage. it is no surprise that this review
and much of the tea li terature 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 thar
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 1 5 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 [C30 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. i ncl udi ng
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 Galocatechins
The catechins [ 1 0-1 3] represent the ma
jor polyphenolic constituent of green tea and
are i l lustrated in Figure 7. Catechins are
members of a more general class of fla
vonoid. the flavan-3-0Is (also referred to as
flavanol s). Three subgroupings of the
flavanols [afzelechin [ 1 4]. catechin [ 15] .
gallocatechin [ 1 6]. shown i n Figure 8]. rep
resenting varying degrees ofB-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% wtiwt of
dissolved solids.
A large percentage of the catechins
present in tea exist as gallic acid esters. While
|1
'z
[10]: Epicatechin EC H
H
[11]: Epicatechin Gal l ate ECG Gal l ate
H
[12]: Epigail ocatechin EGC H
H
[13]: Epigail ocatechin Gal l ate EGCG Gal l ate
H
FIGURE 7. Thepr|nc|pa|lea calech|ns.
_OH
HO' _

OH
OH
|
1 |
2
[14] : Afzel echi n H
H
[15]: Catechin C H
H
[16] : Gal l ocatechin GC H
H
FIGURE 8. Tne I|avan-3-o|s.
gallation i s found to occur principally at the
3-position, various other gallated species
have been isolated, including the epigallo
catechin digallates (Nonaka et aI., 1983), and
epicatechin digallate (Coxon et aI., 1 972;
Hashimoto et aI., 1 987). 3-Methyl gallates
of EC and EGC have also been reported
(Saijo, 1 982). EGCG was once thought to be
unique to the tea plant (Graham, 1983) but
now has been isolated from other sources
(Danne et aI., 1 994).
The four most common catechins are
epigallocatechin gallate (EGCG, [ 1 3] ) ,
epigallocatechin (EGC, [ 1 2]), epicatechin
gallate (ECG, [ 1 1 D, and epicatechin (EC,
[ 1 0]). Catechin (C, [ 1 5]) and gallocatechin
(GC, [ 1 6]) are also present i n smaller quan
tities. While gallocatechin gallate (GCG) and
431
catechin gallate (CG) have also been ob
served, i t is likely that these are products of
racemization and not 'native' to the tea plant
(Roberts, 1 962; Robertson, 1 992).
2. Flavonols
The flavonols [kaempferol, quercitin, and
myricitin] and their glycosides [ 1 7-1 9] 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 i n
Figure 9 Analyses of the flavonol glycosides
in general (McDowell et aI., 1 990) and of
the flavonol diglycosides (Finger et aI. ,
1 99 1 a) and triglycosides (Finger et aI. ,
1 99 1 b) in tea leaf and of flavonol glycosides
in tea seed (Sekine et aI., 1 99 1 ; Sekine et al
1 993) have been performed.
Use of hydrolysis to determine flavonol
and flavonol glycoside content as their agly
cones (Hertog et aI., 1 993) has proven to be
useful i n determining overall flavonol con
tent (about 0.5 to 2.5% wtiwt extract, as
aglycone) of tea infusions.
HO
OH 0
[1 7]: Kaempferol Gl ycoside
[18]: Quercitin Gl ycoside
[19]: Myricitin Gl ycoside
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,
1 954; Cartwright and Roberts, 1 955) and
have been detected by HPLC (Bailey et aI.,
1 990; Hashi moto et aI., 1 992). The simple
polyphenols and their depsides are shown i n
Figure 10.
Cinnamic acid derivatives of quinic acid,
the coumaryl and carrtoyl- quinic acids(ia-
cluding chlorogenic acid or 5-caffeoylquinic
acid [23]) have also been identified in tea
(Cartwright et aI., 1 955). Chi orogenic acid
and 4-coumarylquinic acid [22] have been
detected by HPLC (Bailey et aI., 1 990).
4. Other Polyphenols
Flavones and their glycosides (Engel
hardt et aI., 1 993), such as apigenin [24],
have been detected in tea but represent a
very small fraction of the polyphenols
present. Flavone glycosides can potentially
R,
OH
OGlycoside
KaG
QuG
MyG
H
OH
OH
H
H
OH
FIGURE 9. TneI|avooo|g|ycos|des.
432
OH
HoocOH
OH
[20]: Gal l i c Acid
OH
OH
HOOC OH
OHOH
[21 ] :
Theogal l i n
OH
R
oOH
o
HOOC
OH
OH
OH
[22] : Coumaryl qui nic acid,
|
=H
[23]: Chl orogeni c aci d,
|
=H
FIGURE 10. Ga|||c acid and lhedeps|des
be measured quantitatively as aglycones
(Hertog et ai., 1 993). Careful resolution of
individual flavone glycosides (Engelhardt
et ai., 1 993) has confirmed their presence in
tea. Apigenin is illustrated in Figure I I .
Flavan-3,4-diols such as leucocyanidin [27]
have also been reported (Roberts et ai., 1 956).
These are ill ustrated i n Figure 1 2.
A number of proanthocyanidin species,
such as prodelphinidin B2 [25] gallates, have
been isolated and are present in green tea
(Non aka et ai., 1 983; Nonaka et ai , 1 984).
The assamicains, such as assamicain A [26]
isolated from C sinensis var. assamica, ap
pear to be ring-opened products of the
proanthocyanidins (Hashimoto et ai., 1 989a).
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
OH
HO
OH 0
[24] : Fl avone (Apigenin)
OH
HO
OH
" " OH
OH
OH
HO
" '
O
R1 OH
o
"
" OH
"
'OR1
[25] : Proanthocyanidin
(Epigal l ocatechin-4-a- Epigal locatechin,
or Prodel phinidi n B2)
FIGURE 1 1. V| sce|laneouspo|yphenols.
precipitate proteins), there is also a small
quantity of hydrolyzable tannin (Nonaka
et aI., 1 984; Yoshida et aI., 1 990; Hatano
et aI., 1 99 1 ; Han et aI., 1 994). The unique
hydrolyzable tannins in tea are typically
"hybrid" tannins such as camelliatannin A
[29], which i s a galloylglucose derivative
with pendant catechins, The tannic acid de
rivatives common to gall-nuts and tree bark
are not present i n significant quantities in tea
infusions. The tea tannins are illustrated i n
Figure 1 3,
434
C. Black Tea Polyphenols
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 whi ch 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
OH
HO OH
OH
o . . ,
" OGa
OH Ga=Galiate
[26]: Assamicain A
OH
OH
HO
OH
OH OH
[27] : Flavan-3, 4-diol
(Leucocyanidin)
FIGURE 12. Visce||aneouspo|yphenols.
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-
pending on the degree of fermentation, how
ever, some green tea polyphenols remain
unconverted. This is parti cularly true in the
case of oolong teas and some Darjeeling
teas, which have been known to rcscmblc
green tea both in chemical constitution (Ding
et a!., 1 992a) 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

0
0
0

0
0
0

q [
"
0

[28] : Hydrolyzable Tanni n

(Pentagal l oyl gl ucose)

[29]: Camel i iatanni n A
FIGURE 1 3. Tann|ns.

cess and are detected in black tea (Bailey

et a!., 1 990). Due to the oxidation reactions
and thermal conditions experienced by the
tea leaf during black tea production, it i s
hypothesized some of the catechins are also
epimerized and/or degallated, which explains
the appearance of free gallic acid as well as
i ncreased levels of non-epi isomers of the
catechins (Coggon et a!., 1 973).
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 aI., 1 990).
Despite this, in a recent analysis of fla
vonols and their glycosides (Hertog et aI.,
1 993), 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
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 i s the production of
a new type of polyphenol, the theaflavins.
The fermentation of green tea leaf also re
sults in the development of characteri stic
aroma components, a darkening of color of
the leaf and extracts, and a decreasing astrin
gency with increased fermentation time.
a. Theafavins
Best known of the fermentation prod
ucts is the class of compounds known as the
theaflavins [30-33), comprising about 3 to
5% wtlwt of the extract solids. They are
i l lustrated i n 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,
1 958). 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 aI., 1 986) 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, i ncl udi ng the i sotheaflav i ns,
neotheaflavins, and theaflavic acids, which
also possess a similar benzotropolone unit.
The benzotropolone ring provides the red
color and makes the theafavi ns easily dis
tinguishable from other components.
Analysis of theaflavins began with ex
traction of water extracts (Roberts, 1 958;
Spiro et aI., 1 987; Robertson and Hall, 1 989)
into isobutyl methyl ketone (Roberts and
Smith, 1 963) or ethyl acetate (Ullah, 1 972),
followed by spectrophotometric measure
ments. Later spectrophotometric methods
improved on this technique (Xiao and Li,
1 992). The Flavognost method (Hilton, 1 973)
uses diphenyl boric acid ethanolamine to
induce a spectrophotometric shift i n the
benzotropolone ring for better accuracy.
Recent improvements in analytical technol
ogy include the use of GC (Collier and
Mallows, 1 971 ), and HPLC (Robertson and
Bendall, 1 983; Steinhaus and Engelhardt,
1 989), as well as NIR measurements on the
leaf (Hall et aI., 1 988) and absorption onto
cartridge columns (Whitehead and Temple,
1 992).
The mechanism of theaflavin formation
was fairly well defined in early papers on
purpurogallin formation and related experi
ments (Horner et aI., 1 961 ; Critchlow et aI.,
1 967; Takino and Imagawa. 1 964b). 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. 1 963),
categal l i n. and pyrogallin (Taki no and
437

|
_

[30] : Theafl avi n

[31 ] : Theafl avi n 3-Gal late
[32] : Theafl avi n 3' -Gal l ate
[33] : Theafl avi n 3, 3' -Di gal l ate
TF
TF3G
TF3' G
TFDG
H
Gal l ate
H
Gal l ate
H
H
Gal l ate
Gal l ate
FIGURE 14. ThetneaI|av|ns.
lmagawa, 1 964a), Oxidations involving na
tive tea enzymes (Roberts, 1 958), bicarbon
ate/ferric ammonium sulfate (Takino and
Imagawa, 1 964a), potassium iodate (Takino
et aI., 1 964), and peroxidase (Takino et aI.,
1 967; Finger, 1 994) have all produced
theaflavins and theaflavin-like compounds
in varying yields, Precise NMR analyses of
the theaflavins are available (Bryce et aI.,
1 970; Cai et aI., 1 995),
b. Theafavic Acids
Theaflavic acids such as epitheaflavic
acid [34], shown in Figure 1 5, are formed
from oxidative condensation of a gallic acid
molecule and a catechin (Coxon et aI . ,
1 970b). In the production of epitheaflavic
acids, gallic acid provides the tri-hydroxy
structure and thus mimics the role of a
gallocatechin i n the mechanism of theaflavin
formation. Theaflavic acids are formed from
438
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 aI. ,
1 986), where the carboxylic acid moiety
becomes a leaving group and mimics the
catechi n group i n t he mechani sm of
theaflavin formation.
c. Other Related Structures
Isotheaflavins (Coxon et aI., I 970a) and
neotheaflavins (Bryce et aI., 1 972; Robertson,
1 992) 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 i n the green tea leaf i s small, and
therefore these black tea components are
present in signifcantly lower concentration
compared with the theaflavins.
OH
OH
OH
OH
Hooe
OH
[34]: Epitheaflavic Acid
H0_0 " '
|
.
::
"
'OH
OH
OH
OH
[35]: Epitheaflagal l i n
FIGURE 15. TheepilheaI|avicacidsandre|aled
compounds
3. Further Oxidized Products: The
"Thearubigens"
From the early stages of development,
simple solvent extractions attemptedtu quan
tify the amount of colored tea compounds
present i n the black tea brew (Roberts et aI.,
1 957). The theaflavins, a group of bright
orange-red compounds, were quickly sepa
rated from the remainder. Paper chromatog
raphy experiments confirmed 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- 1 8 cartridge columns (Whitehead and
Temple, 1 992) 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
"thearubigens" 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 aI., 1 957).
Later HPLC techniques reinterpreted the is
sue with division of the thearubigens into
groups I, n, and III (Bailey et aI., 1 99 1 ; Bailey
et aI. , 1 994a). In both papers, the presence of
an unresolved mass (as illustrated i n Figure
16) is taken to represent the same compounds,
the "unresolved thearubigens". This i s based
on loose similarities of HPLC with paper
chromatography as well as more recent work
with cartridge column fractions (Wellum and
Kirby, 1 981 ).
The arrival of HPLC chromatography
led to the belief that for the first time indi
vidual thearubigens would be separated and
i sol ated (Hoefler and Coggon, 1 976;
Robertson and Bendall, 1 983). However, it
439
. .. ' = Area of well resolved spols
= Diffuse, unresolved spots
Ci nnami c Acids
....
..... -.-.
.
.
.
...
.
... _.":' .::.:.:. r.:.-::
:.......
.
Flavonol Glycosides
Catechins
Theafavins
'.
Butanol- etic Acid-Water
225
200
1 75
1 50
.. .
'Thearubigens'
Difuse spot on bo h paper
chromatography a d on
HPLC chromatogr m taken
to be synonymous
Both identified as
1 26 , 'unresolved thearu igens'
100 atechins
75
^
50
25
1 0
20
40
FIGURE 1 6. |apercnromalcgraphyvs. HF|C
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
440
(Wedzicha and Donovan, 1 989) and using
the technique of counter-current chromatog
raphy (Okuda et aI ., 1 988; Wedzicha et aI.,
1 990) 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,
due i n part to the extreme simplicity of re
versed-phase techniques. Use of reverse
phase colums with a step-gradient (Putman
and Butler, 1 989) might present a potential
useful technique for these compounds a well.
Use of reverse-phase technology has been
expanded systematically by a series of re
cent papers (Opie et aI., 1990; Bai ley et aI.,
1990; Bailey et aI., 1 99 1 ; Bailey et aI., 1 994b)
and from thesis work (Opie, 1 992). 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.
Investigations into the thearubigen frac
tions identified by early techniques revealed
that part of the thearubigens was the fla
vonol glycosides (McDowell et aI., 1 990).
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
cessful l y identified the paired role of
gallocatechins and catechins in theaflavin
formation (Sanderson et aI., 1 972). The
model fermentation approach was expanded
to investigate the role of purified tea PPO
(Coggon et aI., 1 973) and peroxidase (Dix
et aI., 1 981 ) 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, 1 983; Robertson,
1 983). Model fermentation of individual
catechins was performed (Opie et aI., 1 990),
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 oftheaflavins
and epicatechin in the presence of polyphe
nol oxidase showed that epicatechin and the
theaflavins, but not the theaflavins alone,
resulted in the degradation of the theaflavins
(Opie et aI., 1 993). 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 aI., 1 995). This approach,
however, seems limited, as epicatechin is a
minor constituent i n 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 elimi nates some of
the confusion concerning the origin of
thearubigen-like compounds that may have
been "left over" from the green tea polyp he
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 ultrafi ltration to separate a high
molecular-wei ght 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 a!., 1 994).
The crude hi storical defi ni ti on of
thearubigens on the basis of poorly resolved
paper chromatograms woul d 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 i s 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 ai., 1 957) i s used successfully to
measure thearubigens, the thearubigens mea
sured are, i n part, composed of flavonol
glycosides (McDowell et ai., 1 990). Thus,
flavonol glycosides are i n some sense
thearubigens. This places the definition of
thearubigens as oxidation products of cat
echins in an awkward position.
Some of the color attributed to the
thearubigens may in fact not be flavonoid in
nature at ali. Figure 1 7 illustrates some pos
sible alternative explanations for the brown
coloration and "acidi ty" as described i n the
historical reports. They may be highly rear
ranged compounds such as catechinic acid
[36] (Sears et ai., 1 974). 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 s i mi l ar to muconi c aci d [ 37, 38]
(Hayaishi and Hashimoto, 1 950; Speier et ai.,
1 993) or derivatives thereof (Critchlow et ai.,
1 967). The brown color attributed to the
thearubigens may also be attributed i n part
to pheophorbi de ( e. g. , [ 40] ) and the
pheophytins (Sanderson, 1 972), or polysac
charides such as [4] and polymers thereof
(Millin et ai . , 1 969). None of these conjec
tures as shown in Figure 1 7 have been i nves
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 requi re 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 aci d hydrol ysi s, produced
anthocyanidins (Brown et ai. , 1 969). 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 gi ve 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 ai.,
1 992). The buf-tan appearance resembled
that of the condensed tannins, and the mate
rials gave simi lar behavior on C- 1 8 chroma
tography when compared with cider and wine
proanthocyani dins. 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-3-0I s. 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
anthocyani di n and that linkage occurred
through an alternate location, such as the 3
'

position (Bailey et ai . , I 994a).

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 ai., 1 995). Both the theafulvin
fraction, as well as the caffeine-precipitable
X
HOO
,
X

"
'
OH
OH
[1 0] : Epi catechi n
(colorless)
COOH
COOH
c
X
OH
OH
x
HO
[36] : Catechi ni c Aci d
(brown)
OH
|
CH

00H
-
OH
OH
X 0
OH
[37]
[30] : Theaflavin
[38]
(brown, acidic)
[39]: Chlorophyll B
CHO
(red)
"
"CH
2
(brown, hydrophobic)
H3C
"
HO
o
(Brown, acidic)
CHO
CH3
(green)
CH3
[40]: Pheophorbi de
FIGURE 17. A|lernalivebrcwn pigments,
fraction, were analyzed by this method. In
both cases, the proanthocyanidin content of
these fractions seemed to be explainable on
the basis of proanthocyanidins observed in
green tea polyphenols (Hashimoto et aI.,
1 987), and the gallate content of these
443
proanthocyanidins would explain most of
the hydrolyzed gallate i n 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 1 0% is proanthocyanidin in
nature and 3% is derived from gallate esters.
The low color i mpact this fraction is likely
to have on the overall beverage should be
consi dered as small evidence for support of
this fraction as a thearubigen. The thearubi
gens have been suggested to be dark brown
and strongly influencing the color of the
beverage, i n addition to being -20% of the
total extractable solids. Also, gallate esters
are the predominant form of catechins in
green tea, comprising -1 5% wtlwt 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
identifed conclusively as a major thearubigen
until stronger, direct evidence exists.
Attempts at isolation of the thearubigens
ha ve generated at least two other new frac
tions: the theacitrins (Powell et aI., 1 994)
and a caffeine precipitable thearubigen frac
tion (Powell et aI., 1 993).
b. Gallic Acid Production
One of the products of the fermentation
process is the appearance of gallic acid [ 1 2],
constituting approxi mately 1 % wtlwt of the
444
extract solids (Graham, 1 984). 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 uf
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 pol yphenols such a 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 gal lated 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 li terature,
and this fai lure might explain why true iden
tification of the thearubigens has eluded sci
entific inquiry for so long.
c. Bisfavanols and Proanthocyanidins
The bisflavanols, such as bisflavanol A
[41 l , arise from paired condensation of two
gallocatechins (Vuataz and Brandenberger,
1 96 1 ). It was initially expected that these
would be i ntermediates i n the formation of
theaflavins. The extra OH substitution from
the use of two gallocatechins, as opposed to
a catechin/gallocatechi n pair, provides a
mechanistic barrier by replacing a hydrogen
atom, which is lost easily i n tautomerization,
with an OH group (see Figure 21 ). This does
not rule out the possibility of further rear
rangement or condensation, possibly to
thearubigins.
theasinensins. They were found to be present
i n green tea leaf (Nonaka et aI., 1 983) and
oolong tea (Hashimoto et aI., 1 988). As well
as the gallocatechin-gallocatechin dimer
products, the theasinensins include catechin
gall ocatechi n di mer products such as
theasinensin F [42]. The theasinensin family
is depicted in Figure 1 8.
The bisfavanols were later rediscovered
and reclassified under the wider name of
Basic work on the separation, character
ization, and chemical identification of black
tea constituents has been well advanced in
recent l iterature. In a massive compendium
of work on tannins and related compounds,
_
'

...

'
.

'

'
|
...

[41] : Bi sfl avanol A, or Theasi nensi n A

uO

uO__
. .
9
'
...

Ou Ou
[42] : Theasi nensi n F
FIGURE 18. The lheas|nens|sns,
445
best summarized i n brief by one member of
this series (Hashimoto et aI., 1 992), 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 i t is anticipated that a good HPLC tech
nique that quantitatively establishes a good
mass balance of the unknown tea polyp he
nols wi l l require many more years of dedi
cated work unless a workng hypothesis for
their formation can be established.
The presence of proanthocyanidins and
theasinensins in green tea as well as black
tea indicates that great care must be taken to
exclude the nascent green polyphenols in the
quantitation of thearubigens. Furthermore,
these compounds do not possess significant
absorption in the visible region of the spec
trum, which rules them out as contributors to
mass of the brown thearubigens.
d. Mixed Oxidation Products of
Po/ypheno/s and Other Compounds
While the most widely known coupled
oxidation products of green tea fermentation
are the theaflavins, resulting exclusively from
the reaction of catechins and gallocatechins,
there are other possible oxidation products
i n the fermentation system not arising solely
from these materials.
The a-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 a-quinone (Van
Sumere et aI., 1 975).
Theogallinin [43] and the theaflavonins
such as [44] (Hashimoto et aI., 1 992) repre
sent two recently identifed species that con
tribute to the balance of polyphenolic mate-
446
rial present i n black tea. They are the prod
uct of the condensation of catechins (which
are the l i kel y oxi di zed species) wi th
theogallin and myricitrin, respecti vely.
Erycitrin [45] (Takino and Imagawa, 1 963)
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 1 9.
OH
H
o
HOOe
OHOH
OH
OH
OH
OH
OH
[43] : Theogal l i ni n
OH
OH
HO
OH 0
[44] : Theaflavoni n, R=Gal l oyl
[45] : Erycitri n, R=H
FIGURE 19. V|xedcx|dalicn prcducts.
e. Aroma Formation from Po/ypheno/
Oxidation
The aldehyde aroma components arising
in black tea aroma have been attributed to
condensation of corresponding amino acids
with an a-quinone to form an amine-substi
tuted derivative (Bokuchava and Popov,
1 954) during polyphenol oxidation. The ci
tation of this mechanism is i nteresting as
some products (aldehydes, carbon dioxide,
and ammonia) have been observed (Popov,
1 956; Skobeleva and Popov, 1 962). Tea leaf
amine oxi dase produces aldehydes and am
monia as byproducts (Tsushida and Takeo,
1 985). Detection of these byproducts i n fer
mentation studies does not constitute proof
that polyphenol oxidation is involved i n their
production, because these studies do not ex
clude the possibility of the presence of amine
oxidase as a contaminant. Nonetheless, there
i s 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 simi lar
nitrogen-substituted catechin is formed by
heating epicatechin with alanine i n a model
system produci ng 7-C(N-ethyl ami no)
epicatechin [47] (Anan et a! . , 1 987), which
is a brown compound suggestive of the
thearubigens. This is consistent with obser
vations of nitrogen incorporation into cat
echins (Kito et a!., 1 968; Konishi, 1 969).
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 2 1 i l lustrates the most plausible
mechanism of theasinensin and theaflavin
formation. It is likely that the quinone groups
are present i n 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 theaflavi n. Theaflavins may be
formed directly from the quinones provided
they are present in suffi ciently large quanti
ties but might be interrupted at an intermedi
ate stage. Thus, Figure 2 1 also shows how
the theasinensins may arise during their for
mation.
a. O% ngtheanin and Theasinensins
Oolongtheanin and theasinensins F [42]
and G (Hashimoto et a!., 1988) may be unique
products of the oolong tea fermentation
system and are shown i n Figure 22.
Theasinensins AlB and DIE are atropisomers
about the biphenyl l i nk 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
HOO
"
,
"
HCHRCOOH
OH
[46] : Ni trogen Substituted Catechi n,
R=derived from ami no acid NH2CHRCOOH
[47]: 7 -C(N-ethyl ami no)-epicatechi n
FIGURE 20. W|lrogen-suosliluled catech|ns.
is expected to produce theaflavin digallate,
as epicatechin [ ! O] and epigallocatechin [ 1 2]
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
(Hashi moto et aI., 1 989b). 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-
448
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
OH

OH
`

'OH
OH [T ]. EC- H=H
j
J 2]. EGC- H=OH

|

OH
jT

|

OH
XOH
Low qulnOn cOncnI|aI|On
O
jd
O

j
XO
H|h qu|nOncOncnI|aI|On
X
H
ThaIav|n
OH
x
.
HO
X
X
|']
||O

OH
X
OH
OH
OH
|J
O
j=]
HO
O

'OH
OH
jdj' EC- H=H
j]. EGC- H=OH
0
||O
.
OH
HO
.
j]
H
FIGURE 21. Mechanism of theaflavin formation.
449
OH

OH
HO
h

'- ' " OH
HO_O , .
7
OH
'OGa|late
OH
[bb]. O| OnglMaan| n
HO
HO
~
OH
OH
, . OH
. OH
'OGal|atc
[bb] d-U-AsOO|0y| Lp| ga| | OOalaOM|nLa| | ala
FIGURE 22. Oc|cngteapclypnenc|s
the enzymes that activate these mechanisms,
provide their own set of challenges to tea
chemistry.
A. Cafeine Formation
Caffeine had been thought to he princi
pally synthesized during the withering stage
of freshly plucked tea leaves (Roberts and
Sanderson, 1 966), although is probably syn
thesized throughout the life of the plant.
Caffeine is most l i kely synthesized from
adenine nucleotides, the dominant free pu
rine forms in tea (Takino et aI. , 1 972). Ad
enosine is a major product of RNA metabo
lism in tea (Imagawa et aI., 1 976). Adenosine
[57] is converted to adenine [58] (Imagawa
et aI. , 1 979), and through hypoxanthine [59]
450
(or inosine) to xanthine [60] (or xanthosine),
from which xanthosine [6 1 ] is the starting
branch of caffeine biosynthesis (Negishi
et aI., 1 992). Guanosine is also converted to
xanthosine, but plays an apparently minor
role in caffeine biosynthesis (Negishi et aI.,
1 992). Xanthosine is methylated at the 7-po
sition (Negishi et aI., 1 985) to 7-methyl xan
thosine [62]. 7-Methyl xanthosine is then
hydrolyzed to 7-methyl xanthi ne [63]
(Negishi et aI., 1 988), which is subsequently
methylated to theobromine [2] and caffeine
[ 1 ] (Suzuki and Takahashi, 1 975; Suzuki
and Takahashi, 1 976). The final methylation
can be terminated, as found in the variant
C irrawadensis (Roberts et aI., 1 958). Fig
ure 23 illustrates the mechanism of forma
tion of theobromine and caffeine i n the tea
plant.
NH
NH

_
N
O
N

N N
N
H
N
_ ee
OH
`
N
_ OH [58
)
N
N ON
N

.
OH

OH
N
N

Hyxanlh ne
O
OH

N
HO
Adenoslne
|
[
5
7
)
O
`
N
N
anthosine(61)
Xanlh|ne(60)
CH_
Oj

.
OH
,N

HO
7 -Melhy|-xan!hcsine(62)

CH
H

z`
O

N
N
I
H
7-Melhy|xanlhine(63)

CH
O
CH
H

CH
-

O`N
N
O`N
N
I I
C C
Thecbrcmlne(2) Caeine( 1)
FtGURE 23. 6iosynlhesis oIcaIIe|ne.
B. Theanine Formation
Theanine [ 3] (n-ethyl glutamine) i s
fonned from the action of thea nine synthetase
[L-Glutamate: Ethylamine ligase] (Sasaoka,
1 965; Sasaoka et a!., 1 965) on ethylamine
derived from alanine (Takeo, 1 974), and
glutamic acid. Theanine degradation prod
ucts may serve as precursors for synthesis of
the catechin A ring, apparently from utiliza
tion of the N-ethyl group (Kito et a!., 1 968;
Feldheim et a!., 1 986). It is likely that the
N-ethyl group enters the general glycolyte
pathway, forming the catechin precursor ma-
451
10nyl-CoA. Theanine formation is a light
dependent process requiring the presence of
ATP as a cofactor (Sasaoka, 1 965). Theanine
synthesis and accumulation is inhibited by
production of glutamine (Matsuura and
Kakuda, 1 990).
C. Biochemistry of Flavonoid
Compound Formation
The pathways for the de novo biosynthe
sis of f1avonoids i n both soft and woody
plants have been generally elucidated and
reviewed i n detail elsewhere (Jain and Takeo,
1 984; Heller and Forkmann, 1 994). Simi lar
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 li terature
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
(Ni kolaeva et aI., 1 982); however, tyrosine
may also participate i n polyphenol produc
tion through a l i ght-dependent pathway
(Zaprometov and Bukhlaeva, 1 97 1 ) . Deami
nation of phenylalanine [64] forms cinnamic
acid [65] by the action of phenylalanine
ammonia lyase (Iwasa, 1 976; Shiplova and
Zaprometov, 1 977; Zagoskina et aI., 1 990).
Hydroxylation of ci nnami c acid forms
coumaric acid [66], which is generated from
an enzyme complex referred to as ci nnamate
452
4-hydroxylate or ci nnamate 4-monooxy
genase. Hydroxylation is achieved typically
by a cytochrome P-450 enzyme activity as
sociated with the enzyme complex (Heller
and Forkmann, 1 988), and a light-indepen
dent cinnamate 4-monooxygenase activity
has been reported from tea shoots (Saijo,
1 980). The coumaric acid aromatic ring forms
the ' B' ring of the f1avonoids. Figure 24
il lustrates the biosynthesis of coumaric acid
and the various functions this compound
performs in the tea plant.
The gallic and quinic acids origi nate via
the shiki mate/arogenate pathway. The key
enzymes in shikimic acid biosynthesis have
been detected in tea (Saijo and Takeo, 1 979).
Carbohydrates play an important role, pre
sumably as a precursor to shikimic acid,
because radiolabels from both myo-inositol
and glucose are incorporated into catechi ns
(Wang and Huang, 1 987). Gallic acid and
quinic acid play key roles in forming esters
with various polyphenols. Gallic acid i s 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
i ng agents such as NADPH, all of which
play important roles as various stages of the
biosynthesis (Zaprometov, 1 987). Light i s
very i mportant for catechin production, and
shaded tea plants produce finished tea
products with reduced astringency due to
the lower concentrations of polyphenols
(Mahanta and Baruah, 1 992).
Coumaric acid, along with other ci nnaric
acid derivatives and phenylalanine, is also a
precursor to tea plant lignins (Zagoskina and
Zaprometov, 1 976; Strekova et aI., 1980).
Lignins are ubiquitous throughout the plant
kingdom. Tea lignins are methylated (rein
forcing low solubility) by the action of
oo

[b4] . |hany| al an| na
Phenylalanine
ammonia lyase

oo

[bb]. C| nna0| OAOlO

Cinnamate
4-monooxgenase
Ooo
NI
o '
, Ooo
|
b
) 1y|03|D
Tyrosine Amonia-Lyase?
o

[bb]. COu0a|| OAOlO

to catechins and favonols to lignins
+ quinic acid
(through navonoid biosynthesis) (through lignin condensation)
O
oo
o
o o
o
[22]. Coumaql qu| ni cacid
FIGURE 24. Cinnamate biosynthesis.
methyl transferase enzymes (Nikolaeva and
Zaprometov, 1 990).
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, 1 988).
Malonyl-CoA is produced by carboxyla
tion of the glycolysis product acetyl-CoA
via acetyl-CoA carboxylase (Lowenstein,
1 981 ) . A hydroxycinnaric acid-CoA deriva
tive typically combines with three molecules
of malonyl-CoA to form chaIcones via chal-
453
cone synthase (Ebel and Hahlbrock, 1 982).
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, 1 994).
Theanine has also been demonstrated to
be a significant factor i n the synthesis of the
phloroglucinol nucleus (Kito et aI., 1 968;
Feldheim et aI. , 1 986). Incorporation of the
ethyl group of theanine into the catechins
has been demonstrated by radiolabel ing
ethylamine, a theanine precursor (Sasaoka
et aI . , 1 962; Kito et aI., 1 968). Ethylamine is
the best demonstrated substrate of tea amine
oxidase (Tsushida and Takeo, 1 985), which
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, 1 975).
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' -hydroxy lases (Heller
and Forkmann, 1 988), accounting for the
presence of caffeic acids, quercitin, and cat
echin derivatives; a much more specific 3',5'
hydroxylase (Heller and Forkmann, 1 988)
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 i n
this step i s chalcone isomerase, which usu
ally bypasses the natural, racemic, anti-ad
dition (Heller and Forkmann, 1 988) to form
a characteristic 2S-stereochemistry (Heller
et aI. , 1 979) of the resulting flavone by syn
addition (Ebel and Hahlbrock, 1 982).
Ring closure of chalcones followed by
hydroxyl ati on resul ts i n formation of
dihydroflavonols. The flavanone is typically
hydroxylated by the enzyme flavanone 3-
hydroxylase, an oxoglutarate-dependent
dioxygenase (Heller and Forkmann, 1 988).
The formation of the 2R,3R dihydroflavonol
has been shown to form either the f1avonols
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 aI., 1 987).
4. Flavanols/Flavonols
The dihydroflavonols are enzymatically
transformed to the f1avonols through action
of a 2-oxoglutarate-dependent dioxygenase
(Heller and Forkmann, 1 994), forming a
double bond through abstraction of vicinal
hydrogens on the 2R,3R dihydroflavonols as
proposed for flavone synthase (Britsch,
1 990). This activity is referred to as flavonol
synthase.
Formation of the 2R, 3S dihydroflavonols
is likely to be preferred in the tea plant, as
formation of the epi-forms of the f1avan-3-
ols is the preferred biosynthesis product.
However, while the route to formation of the
f1avonols and non-epi-flavan-3-0Is is well
defined, the formation of the epi-flavan-3-
ols remains conjecture (Stafford, 1 988). Fig
ure 25 illustrates the proposed biosynthetic
pathway from cinnamic acids to flavonols
and flavan-3-ols.
The formation of proanthocyanidin poly
mers has also been considered, although i t is
unclear if epicatechin [ 1 0] or leucocyanidin
[27] is the significant precursor (Nikolaeva
HO
_.-CH,_

Jx Acelyl CcA
CH,
SCcA
CAS

CH CcAS
1661: Ccumar|cacid
OH
OH

(as CoA ester)

Chalcone
synlhase
OH
Chalcone
isomerase
-
[
68}: chalcone
OH
OO

OH
1
6
91:
2R-Flavanone
hydroxylase
F,avnone
OH
_

. .
'

'OH
Dihyrnavonol
OH
reduase?
[0): dydrcf|acnc|
j

OH

OH
OH j|ycosyla!icn
| /l 0avoocl
( 1 7) Kaemplerol glycoside
HOO.
.

[
7
l}. fl
avan-3.4-diol
OH H
Flavan-3.4-diol
reductase?
OH

1 1 4) novan-J,ol a|ze|ech|n,
FIGURE 25. 6|csynlhesiscIf|avcncids.
5_ Esterase et aI. , 1 982). Proanthocyanidin polymer for
mation has been attributed to polyphenol
oxidase and the formation of the thearubigens
(Brown et aI., 1 969). 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, 1 962). Gallic acid,
found principally in its esterified form i n
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, 1 983).
A significant quantity of the small
polyphenols exists as quinic acid esters
(depsides), particularly the depside of gallic
acid, theogallin (Stagg and Swaine, 1 971 ).
Quinic acid is derived from the shikimic
acid pathway via dehydroquinic acid, an in
termediate on the pathway to shikimic acid
(Sprinson, 1 960; Neish, 1 964). Quinic acid
esters of coumaric and caffeic acid (chloro
genic and caffeoylquinic acid) have also been
detected i n tea (Sanderson, 1 972).
Gallated f1avan-3-0Is (catechins) are the
major flavonoids produced by Camellia
sinensis (Sanderson, 1 972). It is of interest
that Camellia sinensis forms gallated esters
rather than sugar esters, or glycosides, of
catechins. Glycosides are the typical plant
strategy for rendering the major f1avonoids
water soluble (Heller and Forkmann, 1 994),
although proanthocyanidin and catechin gly
cosides, while not rare, are not known to be
common (Achmadi et aI., 1 994). 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 aI. , 1 980;
Heller and Forkmann, 1 988). Gallic acid es
terifcation 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, 1 983).
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 acti vity 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 oxi dase (EC 1 . 1 4. 1 8. 1 ;
monophenol monooxygenase [tyrosinase] or
EC 1 . 1 0.3.2; 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 i s likely to be dependent on
solubilization of the protein (Tolbert, 1 973).
PPO is distributed throughout the plant
(Durmishidzern and Puridze, 1 980) and has
been identified and analyzed in the floral
organs (Singh and Ravindranath, 1 994). PPO
i s localized within plant cells i n the mito
chondria (Bokuchava et aI., 1 970), the chlo
roplasts (Roberts, 1 941 ), and the peroxisomes
(Kato et aI., 1 976). Using antibody tech
niques, polyphenol oxidase activity has also
been localized in the epidermis palisade cells
(Wickremasinghe et aI., 1 967). Recent re
views on the subject of PPO are available
(Zawistowski et aI., 1 99 1 ; Whi taker, 1 994;
Steffens et aI. , 1 994).
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 i nfection
(Zawistowski et aI., 1 99 1 ). In tea, key reac
tions occurring during "fermentation" are
catalyzed by PPO. These reactions are initi
ated by crushing and/or 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, 1 99 1 ). 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
been determined; however, the reaction
mechanisms of PPO from other plants have
been characterized (Zawistowski et aI., 1 99 1 ;
Whitaker, 1 994; Steffens et aI. , 1 994). PPO
activity i s dependent on oxygen concentra
tion (Robertson, 1 983). The o-quinone spe
cies from tea has been looked for in a model
system (Korver et aI. , 1 973).
Polyphenol oxidase is diffi cult 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, 1 987). 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 1 30 to 1 60
kDa (Gregory and Bendall, 1 966). 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 oftypi
cal PPO has a molecular weight of 45 kDa
(Steffens et aI., 1 994). 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 I g to 20 kDa. Mushroom tyrosinase has
been found to have a quarternary structure
comprised of two different subunits of 43
kDa and 1 3.5 kDa, with a total molecular
weight of 1 20 kDa and the formula LH2
(Zawistowski et aI., 1 991 ). 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 -1 44
kDa. This is consistent with a report of
isoforms of 1 1 7, 56, 41 .5, and 35 kDa (Buzun
et aI., 1 974) and another report with isoforms
al 1 1 8 and 4 1 kDa (Durmishidzern and
Puridze, 1 980). The copper content has been
measured at 0.26 wt/wt%, or about 4 to 5
copper atoms for the complex, which i s con
sistent with other literature (Vamos-Vigyazo,
1 98 1 ; Interesse et aI., 1 983; Zawistowski
et aI., 1 99 1 ) . This number is probably low
because copper diffuses readily away from
the enzyme, resulting in a loss of activity
(Mayer, 1 987; Zawistowski et aI. , 1 99 1 ) .
Consistent with observed sigmoidal activity
(Pruidze, 1 975), 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 i s
evidence that PPOs from different plant spe
cies have preferential substrates (Whi taker,
1 994; Steffens et aI., 1 994). Studies of PPO
from tea have mainly been based on model
fermentation studies that utilize purified or
semipurified substrates (Dix et aI. , 1 98 1 ;
Robertson and Bendall, 1 983; Opie et aI.,
1 990; Finger, 1 994). It is clear from these
reports that PPO from tea reacts effecti vely
with both di- and tri-hydroxylated catechins,
reacting with specificity for the o-dihydroxy
moiety (Gregory and Bendall, 1 966; Coggon
et aI., 1 973). 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 aI., 1 99 1 ). 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 i s evi dence of theafl avi ns and
thearubigens as feedback inhibitors (Pruidze
and Grigorashvili, 1 975). 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
PPO has an observed optimum pH in the
range 4.6 to 5.6 (Takeo, 1 965; Takeo and
Uritani, 1 966; Gregory and Bendall, 1 966;
Perera and Wickremasinghe, 1 972; Coggon
et aI., 1 975; Robertson, 1 983).
2. Peroxidase
Peroxidase (EC 1 . 1 1 . 1 .7) is thought to
play an i ntegral role in the fermentation pro
cess and i s found i n fresh green leaf
(Bokuchava and Popov, 1 948; Gregory and
Bendall, 1 966; Bokuchava and Skobeleva,
1 969). Coupled with PPO, which is thought
to produce the peroxide, an acti vator 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 aI . , 1 981 ) . Reactions of
epicatechin and PPO have been proposed as
a model for thearubigen formation (Opie
et aI . , 1 993) and epicatechin oxidation prod
ucts by this system have been suggested as a
good model of thearubigens (Opie et aI. ,
1 995). This is likely due to the fact that the
isolation procedure used for purification of
PPO (Opie et aI., 1 990) was found to contain
peroxidase activity in an earlier report
(Robertson and Bendall, 1 983). Model fer
mentations using PPO and peroxidase, both
separately and in conjunction, show the
coupled nature of these enzymes (Finger,
1 994) and point to the need for further inves
tigation of the role of both of these enzymes
i n the development of the unique black tea
polyphenols and other compounds present
in black tea extracts.
E. Extracel l ul ar 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
ofKombu-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 i s
produced by burying green tea leaves i n a
confined space, thereby allowing mi crobio
logical fermentation to blacken the leaves
(Shao et aI . , 1 995). 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. I . I .20) such as those iso
lated from fermentation broths of Aspergil
lus Flavus, A. Niger, or A. Orzae 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, 1 936; Roberts and Wood,
1 95 1 ; Roberts and Myers, 1 959) and its ben
eficial impact on manufacture of cold water
soluble instant teas (Coggun et ai., 1 975).
The use of tannase significantly inhibits the
ability of tea polyphenols to complex and
precipi tate, and reduces the ability of black
tea to form tea cream (Nagalakshmi et ai. ,
1 985). Tannase activity and thermal stability
in the presence of tea extracts has been de
termined (Thomas and Murtagh, 1 985).
V. CHEMICAL PROPERTIES OF TEA
COMPOUNDS
Beyond the i nherent ecological proper
ties of tea compounds i n the tea plant, tea
phytochemicals exhibit unusual properties
that make them interesting subjects for
chemical study.
A. Formation of Cream and Haze i n
Black Tea
The onset of cooling in a freshly brewed
cup of black tea is accompanied by produc
tion of a dark red/brown 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 mi lky-red color of tea to which
cream has been added and thus was dubbed
' tea cream' (Bradfield and Penney, 1 944;
Roberts, 1 963; Smith, 1 968).
Tea cream can be separated from the
brew most conveniently by centrifugation of
a concentrated beverage (Smith, 1 968).
Tea cream was demonstrated by early
researchers to contain caffeine, theaflavin,
and thearubigens, as well as traces of a
number of other substances (Roberts, 1 963;
Smith, 1 968). Studies on cream composi
tion (Nagalakshmi and Seshadri, 1 983)
and decreaming (Nagalakshmi et ai., 1 985)
show that protein and caffeine contribute
significantly to cream formation. Addition
al contributions from lipids in tea such as
triacontanol and spi nasterol have been
shown (Seshadri and Dhanaraj, 1 988). Simple
carbohydrates have been shown to assist i n
solubilization of cream (Nagalakshmi et ai.,
1 984) but do not appear to complex signifi
cantly with polyphenols in model studies
(Williamson et ai., 1 995). Hi gh-molecular
weight thearubigens have been associated
with cream formati on (Hazarika et ai., 1 984).
HPLC analysis of the phenolic portion of tea
cream (Powell et ai. , 1 993) revealed 86%
'thcarubigens', 1 2% theafluvins, and 2% fla
vonol glycosides.
Whi l e the pol yphenol i c consti tuents'
rol e i n cream formation i s wel l under
stood, the presence of proteins/peptides,
carbohydrates, leaf fines (fragments of
leaves), and other materials needs to be
studied further to ful l y determine the
mechani sm behi nd cream formati on.
Analysis of the pH optimum for cream
formation (Smith, 1 968), whi ch closely
mi rrors tannin-BSA pH curves (Hagerman
and Klucher, 1 986), combined with knowl
edge of the significant contribution of pro
tein to the mass of cream (Nagalakshmi
and Seshadri, 1 983), 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, 1 97 1 )
and in the literature (Rutter and Stainsby,
459
1 975; Bee et aI., 1 987). Tea cream has been
shown to be comprised of ultra fine spherical
colloidal droplets that are clearly visualized
in a range of concentrations of tea in water,
increasing i n 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 aI., 1 987). Tea cream formation has been
interpreted i n terms of phase separation and
is likened to two liquids miscible at high
temperatures but immiscible when cooled
(Harbron et aI., 1 989).
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 aI., 1 984; Ozawa et aI., 1 987).
Immobilized polyphenols bind proteins re
versibly, with restoration of enzyme activity
after elution (Oh et aI., 1 985). Dissolved ash
metals such as hard water calcium may in
fluence complexation, as in the case of tea
scum formation (Spiro and Jaganyi, 1 994).
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 i n pep tides and proteins. Evidence
for this assertion is deri ved from crystal struc
tures of co-crystallized model polyphenols
460
with caffeine (McManus et aI., 198 I ; Gaffney
et aI., 1 986; Martin et aI., 1 986), and of caf
feine with I-tryptophan (Nishijo et aI., 1 990).
Hydrogen bonding also plays a prominent
role (Van Sumere et aI., 1 975; Borazan and
AI-Ani, 1 980). Although a full thermody
namic treatment of the i ssue 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 aI., 1 988; Cai et aI., 1 990)
and by their octanol-water partition coeffi
cient (Martin et aI . , 1 990). In addition to
hydrophobic stacking, there is evidence for
a coplanar-type hydrogen bond (Martin et aI.,
1986). There is an apparent correlation be
tween hydrophobicity and complexation for
hydrolyzable tannins such as pentagalloyl
glucose and vescalagin (Haslam, 1 993).
Hydrophobic association has also been used
to explain copigmentation effects observed
in solutions of anthocyanins and polyphe
nols (Mistry et aI., 1 99 I ; Liao et aI., 1 992)
or xanthyl i um dyes wi t h caffei ne or
cyclodextrins (Dangles and Brouillard, 1 993).
Polyphenols bind significantly to proline
rich proteins, and this complexation i s
thought to drive taste perception and differ
ences among various methods of tea bever
age consumption (Luck et aI., 1 994).
Closer inspection of data on condensed
tannins (Martin et aI., 1 990) reveals i nterest
ing correlations. To illustrate, an i ncrease of
one hydroxyl group decreases apparent hy
drophobicity yet i ncreases association with
caffeine for epicatechin/epigallocatechin,
catechin/gallocatechin, and catechin gall ate/
epigallocatechin gallate pairs. Despite their
hydrophilic nature and twisted structure,
procyanidins show simi lar 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 hydrophi l i c
environment. Hydrogen bonding has been
demonstrated to be a strong driving force for
association, particularly i n systems such as
cyanuric acid with melamine (Mathias et aI.,
1 993) and p-cresol with caffeine (Borazan
and AI-Ani , 1 980). The hydrogen-bonding
character of catechin/polyproline association
has been demonstrated in model systems (Sun
and Mattice, 1 996).
Simple hydrophobic association would
seem to be insufficient for explanation of
protein complexation, precipitation, and
cream formation. A simpl ified model for the
complexation of polyphenols and caffeine
(Figure 26) demonstrates that association of
the two hydrophobic surfaces results i n a net
decrease of hydrophobicity because the
polyphenols have a hydrophobic core sur
rounded at its edge by hydrophi l i c groups.
Al l (or most) of the hydrophi l i c surface i s
available for solvation, which should result
i n a soluble, not precipitated, complex. This
mi rrors the formation of micelles, which are
commonly understuud tu furm a dispersed
'pseudophase". It also mi rrors the observed
association of two
"
h;dmphobi, _
|Cy.
hydrophobic
surface
hydrogen
bonding
surace
'" results i n forming a
J
complex whose
hydrophobic area is
reduced, but
hydrogen-bondi ng area is
the same
(si mi l ar to a micel le and
caffeine self-aggregation)
However, hydrogen bonding reduces
hydrophilic surface area, which shoul d
drive separation of 'cream' phase
Cafeine Caffeine
@POIYPhenol0poIYPhenol
t
FIGURE 26. The hydrophobic association model.
461
self-stacking of cafeine aggregates (Thakkar
et aI., 1970; Shestopa et aI., 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 di screte precipitate analogous to
phase separation.
Existence of hydrophobic bonds (Xianqi
and Haslam, 1 994) is supported only by cir
cumstantial evidence. Hydrophobicity i s
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
entropic di sadvantage. 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 uf aromatic
molecules is apparently very different than
that of al i phatic hydrophobic surfaces
(Makhatadze and Privalov, 1 994). In addi
tion, aromatic and other unsaturated mol
ecules can form hydrogen bonds with the It
electrons, stabilizing the aromatic group in
the presence of water (Rzepa et aI. , 1 994;
Ernst et aI., 1995). There are also i ndications
that aromatic stacking interactions in aque
ous systems have l i ttle to do with dispersion
forces or hydrophobic interaction (Newcomb
and Gellman, 1 994).
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
i ng aggregation and precipitation in the
462
manner of phase separation. Proteins are most
easily precipitable at their isoelectric point
(Oh and Hoff, 1 987), 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 ami des such as those found
in proline residues, because secondary amides
have been shown to form stronger hydrogen
bonds than primary ami des (Cannon, 1 955;
Hagerman and Butler, 1 98 1 ). 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 -octyl-D-glucopyranoside can be
used to solubilize the precipitated polyphe
nol complex (Martin et aI. , 1 990). Surfac
tants are known to form ordered layers at
liquid-solid interfaces (Bigelow et aI. , 1 946),
and the increase in entropy associated with
forming ordered mono layers 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 i nteractions are
given similar importance, as both are neces
sary for driving the complex interactions
i nvolved in the model. That the proposed
model does not currently explain apparent
di fferences between the condensed and hy
drol yzable t anni ns i s one l i mi tati on.
Vescalagin, castagalin, and pentagalloyl
glucose have simi lar hydrogen bonding sites,
although pentagalloylglucose precipitates
proteins much more readily (Haslam, 1 993).
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
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 vitro by quenching free
radicals or by preventing free radical forma
tion. These compounds have been termed
"antioxidants" (Namiki, 1 990; Pokorny,
1 992). Vitamins A, E, and C and the mineral
selenium are common antioxidants occur
ring in our foods (Pokorny, 1 992), and BHA
(butylated hydroxyanisole), BHT (butylated
hydroxy toluene), and propyl gallate are syn
thetic antioxidants approved for food use
(Gates, 1 987). A broad range of flavonoid or
phenolic compounds have been found to have
antioxidant activity (Ho, 1 992). Polyphenols,
both synthetic and from numerous plant
sources, have also been found to be func
tional antioxidants in numerous test systems
(Ho, 1 992; Lunder, 1 992; asawa, 1 992).
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 i n 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 aI. , 1 983;
Tanizawa et aI. , 1 984; Namiki and Ozake,
1 986; Zhao et aI., 1 989).
1. Chemical Antioxidant Systems
The effectiveness of purified tea polyphe
nols and crude tea extracts as antioxidants
against the autooxidation of fats has been
studied using the Rancimat system. I n one
study, purified green tea catechins were
evaluated against dl-a-tocopherol and BHA
(Ho et aI., 1 992). 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 dl-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 aI.,
1 992). 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, 1 992).
An alternate method to the rancimat,
based on measuring the oxidation of linoleic
acid catalyzed by bubbling air (2 h incuba
tion at 60C with 500 ml/min air flow rate)
and determining the peroxide value (POV)
or the thiobarbituric acid value (TBA V) has
been used to study tea flavonoids (Tanizawa
463
et aI. , 1 983; Tanizawa et aI. , 1 984). Epi
catechin, green tea extract III (n-butanol
phase) and green tea extract IV (acetone
soluble portion of extract III) 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 i ncorporated i nto cetyl trimethyl
ammonium bromide (CTAB) micelles has
also been used to study the antioxidant activ
ity of flavonoids (Wang and Zheng, 1 992).
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
antioxidants by reacting with peroxy radi
cals. The flavonoids had the following de
scending order of reactivity: a-tocopherol
>morin >rutin >quercetin. There i s a clear
difference between prevention of oxidation
of free linoleic acid (the crude extracts of
flavonoids were much more active than vita
mi n E) and linoleic acid in micelles (fla
vonoids were less active than vitamin E). It
is likely that vitamin E i s 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 stimulated polymorphonuclear leukocytes
(Zhao et aI., 1 989); hydroxy radicals pro
duced in reaction mixture containing hydro
gen eroxioeand Fe++ (fromIerrotisammo-
nium sulfate) (Zhao et aI., 1 989; Shi and
Dalal, 1 99 1 ; Uchida et aI., 1 987); a reaction
mixture of NADPH, potassium dichromate,
and hydrogen peroxide (Shi and Dalal, 1 991 );
an irradiated riboflavin system (Zhao et aI.,
1 989); and superoxide generated by the hy
poxanthine-xanthine oxidase system (Uchida
et aI . , 1 987) . DMPO (5, 6-di methyl - l
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 i n the following
ranking of antioxidant potency: curcumin
(69% inhibition), vitamin C (56% inhibi
tion), vitamin E (35% inhibition), rosemary
extract ( 1 6% inhibition), green tea polyp he
nol ( 1 2% inhibition), and so-called "green
tea fraction 6" ( 1 3% inhibition) (Zhao et aI. ,
1 989). Caffeine was also found to be an
effective scavenger of hydroxy radicals gen
erated by the Fenton reaction, but its effec
ti ve concentration was three orders of
magnitide higher than of green tea catechins
or vitamins (Shi and Dalal, 1 991 ) .
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-oxo function in the C ring in
flavonols, and the 3- and 5-hydroxy groups
with the 4-oxo function in the A and C rings
(Nakayama et aI . , 1 993 ; Pratt, 1 992;
Jovanovic et aI., 1 994). Catechins can react
with more than one free radical to form
quinones (Zhao et aI., 1992) and have a
lower reduction potentials than vitamin E
(1ovanovicet a|. , 1 995). This should account
for the stronger radical-scavenging activity
of catechins. Tannins with a greater degree
of gallation were more effective i n 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
i s needed to understand the potential health
promoting properties of antioxidants, and
better characterization and understanding of
the polyphenols i n both green and black tea
extracts i s 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
system and was proposed to function through
an ion chelation mechanism (Morel et aI.,
1 993). However, other studies using catechin
as an antioxidant have suggested that its
antioxidant activity is due to a free radical
scavenging mechanism (Fraga et aI., 1 987;
Ratty and Das, 1 988). 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, 1 986). The
catechins from tea were found to be very
active i n both systems, with EGCG and ECG
having the greatest activity in blocking lipid
oxidation. These catechins were 1 0 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 aI. , 1 992). This suggests that
EGCG and ECG were effective as both radi
cal scavengers and chel ators, 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 aI., 1 989).
Gallic acid and gallic acid esters have
been shown to have both antioxidant and
prooxidant activity in vitro (Aruoma et aI.,
1 993; Cao et aI., 1 996). 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 activi ty in green
tea.
The cytotoxic effects of reactive oxygen
species (0, and H,O,) on cells grown in
culture was used as a model system for the
evaluation of the antioxidant activity of f1a
vonoids (Nakayama et aI., 1 993; Ruch et aI.,
1 989). Chinese hamster lung fibroblast V79
cells in culture were incubated in the pres
ence of f1avonoids 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 f1a
vonoids. The washed cells were then ex
posed to reactive oxygen species (either H,O,
or superoxide produced enzymatically using
a combination of hypoxanthine and xanthine
oxidase). In a cytotoxicity test of the f1a
vonoids, catechin and taxifolin were not toxic
at concentrations up to 200 and 1 000 M,
respectively, while quercetin and kaempferol
were toxic at concentrations above 1 00 M.
Quercetin and kaempferol were effective i n
protecting cells from both superoxide and
peroxide at concentration of 5 and 20
M, while catechin was effective at concen
trations of 500 M and 1000 M (Nakayama
et ai., 1 993). A similar model system used
cultured mouse hepatocytes rather than ham
ster lung fibroblasts (Ruch et aI., 1989). These
studies showed that a green tea polyphenol
fraction (GTP) was effective in scavenging
H,O, and 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 i n
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 aI.,
1 992). Epigallocatechin gallate (EGCG) was
provided to mice in drinking water for I
month at 0.002% or 0.0 I % concentration
providing an EGCG intake of 3 or 15 mglkgl
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 i n a dose-dependant manner, reducing
the formation of peroxides 75% and 90% for
the 0.002% and 0.0 I % doses, respectively.
Oral consumption of EGCG was also found
to si gnificantly 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 andlor 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 aI. , 1 993). It is critical
that a proper antioxidant to oxidant balance
be maintained in well-controlled biological
studies. The balance of different types of
antioxidants i s also important, as demon
strated with carotenoids, vitamin E, and vi
tamin C (Bohm et aI. , 1 997).
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 i n tea, combined with
qualitative descriptors historically available,
has provided a challenging barrier to sur
mount. A thearubigen i s very clearly assumed
to be a complex mi xture 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 defi nitive 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 compounds into
subdivided fractions, the relati ve 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 chemi stry i s
dependent on comprehension of the li tera
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
consti tuti on of tea and tea beverages
(Kursanov, 1 956; Vuataz et aI. , 1 959; Millin
and Rustige, 1 967) set the stage for a gener
alized interpretation of the chemistry of tea
(Sanderson, 1 972). 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
i n the literature (Graham, 1 984; Balentine,
1 992).
No single technological innovation has
propelled the efforts of tea chemistry more
in recent years than the introduction of the
HPLC (Hoefler and Coggon, 1 976), and the
subsequent coupling of the technique with
diode-array detection (Bailey et aI., 1 990;
Opie et aI. , 1 990; Opie, 1992) and mass spec
trometry (Lin et aI. , 1 993; Bailey et aI.,
I 994b). Despite its power, HPLC is in a
stage of relative infancy compared with the
field of tea chemistry, and i t 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 lhis inslrumenlal meLhod sLill needs Lo be
performed. Progress and innovation in sci
entific research is frequently the result of
dramatic breakthroughs or "revolutions"
(Kuhn, 1 970). 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. Bul k Properties and Correlations
to Tea Taster's Profiles
Efforts to understand the chemistry of
tea can be seen in part as moti vated 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 1 993 totaled $ 1 24 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
i ng factors control l i ng tea producti on
(Mahanta et aI . , 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 McDowel l , 1 994). It fol l ows 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 aI., 1 984) 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) wi th tea val ue
(McDowell et aI., 1 99 1 ). Theaflavins are very
important for quality and price, owing to the
bright red color they impart (Hilton and
Palmer-Jones, 1975; Ellis and Cloughley,
1 98 1 ), but among similar theaflavin con
tents, theaflavins are not a good determining
factor (Owuor et aI. , 1 986). Aroma can also
be expected to play an important role, espe
cially i n fowery teas (Robinson and Owuor,
1 992).
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 i n 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 capil lary fragility,
has been found in tea and attributed to tea
polyphenols (Kursanov et aI., 1 950). Today,
research into tea chemi stry 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
gests that antioxidants may help protect
against these di seases by mini mizing 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 f1avonoids, which include the cat
echins and f1avonols found i n 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 i n 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 i n development
of cardiovascular di sease (Marx, 1 987;
Grundy, 1 993). Recent reports that dietary
supplementation with vitamin E (at 1 00 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,
1 993).
One of the key mechanisms of the hy
pothesis that oxidative damage is linked to
cardiovascular di sease 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, (-toco
pherol, and -carotene inhibit LDL oxida
tion in vitro and reduce the progression of
atherosclerosis in animal models.
Studies using a Chinese green tea polyphe
nol fraction (CGTP) were conducted to deter
mine the effect of tea constituents on oxida
tion of low-density lipoprotein (LDL) ill vitro
in a model system thought to be representa
tive of the effects of antioxidants i n modulat
ing risk factors associated with cardiovascu
lar disease (Ding et aI., 1 99 1 ; Ding et aI.,
1 992). The antioxidant activity was deter
mined based on lipid peroxide production,
thiobarbituric aci d reacti ve 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 "vitami n-sparing" (whereby anti
oxidant is preferentially consumed, 'sparing'
the vitamin) effect. A dose-dependent inhibi
tion of copper-induced LDL oxidation and
LDL oxidation induced by transformed
macrophages were also found for catechin
(Mangiapane et ai., 1 992).
2. Studies of Tobacco Nitrosamines
Smoking of tobacco products i s 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, 1 992;
Loft et ai., 1 992; Marx, 1 987). One outcome
of oxidative damage to DNA is the 8-hy
droxylation of guanine bases producing
8-hydroxydeoxyguanosine (80HdG) DNA
adducts. Repair of these DNA adducts in
vivo leads to the excretion of 80HdG i n
urine that i s a biomarker or index of the
current state of oxidative DNA damage and
repair (Saul and Ames, 1 986; Loft et ai.,
1 992). 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 i ncrease in the level
of oxidative damage to DNA (Loft et ai. ,
1 992). Cigarette smoking (> I 0 per day) was
found to significantly increase the mutation
sensitivity of chromosomes in peripheral
lymphocytes (Kim et a!., 1 993). This was
determined by treating the cells with mito
gens and measuring the frequency of sister
chromatid exchange (SCE). The degree of
SCE i s 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 i n
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 i n frequency of SCE. Coffee con
sumption did not change the frequency of
SCE (Kim et a!., 1 993). 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
thylnitrosamino)-I (3-pyridyl )-I -butanone, or
NNK, is an oxidative carcinogen that in
duces lung cancer in numerous laboratory
animal models (Xu et ai., 1992; Wang et a!.,
1 992a). 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 ai. , 1 992; Wang et a!., 1 992a).
Black and green tea were found to be equally
effective in reducing tumor numbers when
provided during or after NNK treatment
(Wang et a!., I 992a). EGCG was found to be
almost as active as green tea infusions (Xu
et ai., 1 992). In vivo (Xu et a!., 1 992) studies
using mice and in vitro (Shi et ai., 1 993)
experiments showed that black and green tea
polyphenols inhibit NNK metabolism and
DNA adduct formation. EGCG was found to
be the most potent i nhi bi tor 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 II enzymes (Sohn et ai., 1 994).
Phase II enzymes include NAD(P)H:quinone
reductase, glutathione S-transferases, and
UDP-glucuronosyl transferases (Talalay,
I 992; Prochaska and Talalay, I 992). Be
cause tea polyphenols fit the criteria for Phase
II enzyme inducers, it i s likely that tea
polyphenols act as potent inducers of these
enzymes, as has been reported (Khan et aI.,
1 992). 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 l inked 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
TPA is partially induced by formation of
peroxides or free radicals (Wei and Frenkel,
1 993). Tea, GTP (Huang et aI., 1992; Wang
et aI., 1 992b), EGCG (Fuj iki et aI. , 1 992),
and antioxidants (Kozombo et aI. , 1 983;
Smart et aI . , 1 987) were effective in block
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 rol e of di etary constituents
(Stavric, 1 994; Wattenberg, 1 992) and tea
(Yang and Wang, 1 993) 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 i n humans.
VII. CONCLUSION
Understanding the chemistry of tea rep
resents a key technological underpinning 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 bl ack
tea is undoubtedly a central focus of tea
research. Indirect correlations such as those
used to measure thearubigens need to be
systematically justified 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 i nteresting 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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