Critical Reviews ill Plant Sciences, 16(5):415480 (1997)

Tea Chemistry

I
Ii Matthew . Harbowy and Douglas A. Balentine
Lipton. 800 Sylvan Avenue. Englewood Cliffs. NJ 07660

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

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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 s 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.

EY WORDS: tea chemistry, polyphenol complexation, antioxidant properties.

I. INTRODUCTION approximately 3 5 to 40 I of beverage. India

has the largest total consumption of tea
The real voyage of discovery consists (540,000 metric tons, 620 g per capita) and
not in seeking flew landscapes but in Ireland has the largest per capita consump­
having new eyes. tion, at 3220 g (Anon., 1 994). Camellia
·Proust sinensis is a very i mportant agricultural and
commercial product with a unique horticul­
The universe is 1I0t only queerer than ture and manufacturing process.
we suppose, but queerer thall we can Chinese mythology teaches that i n the
suppose. year 2737 B.C. Emperor Shen Nung discov­
-J. B. S. Haldane ered tea, according to the Chinese medical
book, the Pen T'sao, written during the Han
It is a picture-perfect image of serenity: Dynasty, circa 25 to 22 1 A.D. The first men­
relaxing with a good book, sipping a cup of tion of tea is believed to occur i n the Erh Ya,
hot tea. For thousands of years, the harvest­ a Chinese dicti onary circa 400 B.C., but the
ing, processing, and packaging of the leaf of modern character ch'a, signifying tea, was
Camellia sinensis, known worldwide as tea, not popularized until the writing of The Clas­
has developed as an integral part of society sic of Tea, Ch'a Ching, by Lu Yu i n 780
and culture. With the advent of the post­ A.D. The history of tea is extremely relevant
colonial scientific era, the perspective of because before the Tang dynasty (6 1 8 to 906
scientific investigation was added to the tra­ A.D.) tea was probably only considered as a
dition and mythology of tea cultivation. medicinal, but then became popular as a
Today, we can look at a cup of tea and beverage. As the method of brewing and
admire the complexities of this beverage and consuming tea varied as tea moved from
the plant that makes it possible. Tea chemis­ culture to culture, the chemical aspects driv­
try has led both consumers and researchers ing acceptability to the consumer varied.
to debate numerous issues and to probe for a It is likely that tea was consumed as a
deeper understanding of the nature of this vegetable i n a soup through the Tang dy­
beverage. With the growing popularity of nasty, often mixed with onions, salt, orange
tea and i ncreased awareness of the potential peel, and/or ginger. Brick tea, still popular
health benefits associated with tea consump­ among the modern Mongolians and moun­
tion, tea chemi stry promises to endure as a tainous people of the Himalayas, was pre­
growing and vibrant field. pared by steaming and compressing the leaf
After water, tea is the most widely con­ into bricks. During the Song Dynasty (960
sumed beverage in the world today. Cur­ to 1279 A.D.), however, this practice fel l
rently in the U.S., per capita consumption of from favor and was replaced by a powdered
tea is approximately 340 g, which produces form of the tea, which was whipped into a

417
froth. A bright green color and low astrin­ mon practice. This involves planting leaf
gency (derived from careful shading of the cuttings in nurseries where they develop into
plant) and a delicate aroma accompany pow­ seedings within 6 months. The seedings are
dered green tea. This preparation method of then transplanted to the fields. The tea plant,
tea is a custom that survives today in Japan once established, will be economically vi­
as Mattcha. The modem custom of brewed able for decades barring disease, infestation,
tea leaves arose during the Ming dynasty or other destructi ve forces.
( 1 368 to 1 644), coinciding with the West's The tea tree is maintained as a shrub
arrival i n China. Although there are many during the growing season through frequent
variations on brewing technique, which can manual harvesting, about every 8 to 12 d
i mpact flavor and chemistry of the brew sig­ during prime growing season. Tea is an ev­
nificantly, it is the basic custom of brewing ergreen tree, but it is largely dormant through
the dried tea leaves in hot water that has the winter season. Mechanical harvesting
been popularized and spread throughout most methods have been developed, but these are
of the English-speaking world. only popular where labor is expensive and
The modern tea industry has its origins where tea is not grown on steep mountain
in the spread of tea cultivation into India slopes. Plantations also maintain unharvested
between 1 8 1 8 and 1 834, derived either tea trees for seed production.
through i mport of the tea plant from China Immediately after harvest, the tea leaves
or through discovery of native Indian tea (usually the flush, or first two leaves and the
var. Assamica, a varietal better adapted to bud of the growing tea shoot) are brought to
tropical production and having a larger leaf factories si tuated close to the tea gardens for
style. Development of tea plantations, and manufacturing. It is the manufacturing pro­
migration of the technology of plantation cess that determines the type of tea pro­
operation from India to tropical areas in duced. There are three general types of manu­
Africa, South America, and Russia (Geor­ factured tea: Green (unfermented), Oolong
gia), has established a variety of localized (partially fermented), and Black (fully fer­
practices and tea products (Eden, 1 976). mented). The manufacturing processes used
Excellent reviews on the culture and produc­ to produce each type of tea differ in the
tion of tea are available (Wilson and Clifford, degree of enzymatic oxidation or "fermenta­
1 992). Figure I illustrates the major histori­ tion". Fermentation refers not to an exog­
cal periods in the development of tea culti­ enous, microbial process, as with beer or
vation and processing worldwide. wine, but the natural browning reaction cata­
Through cultivation, tea has become an lyzed by enzymes endogenous to the plant.
important agricultural product throughout the The green tea manufacturing process
world, particularly in regions lying close to involves the rapid steaming or pan firing of
the equator. Geographical areas that receive the freshly harvested leaves to inactivate
annual rainfall of at least 50 inches per year enzymes, preventing fermentation, produc­
and have a mean average temperature of ing a dry, stable product. Green teas are
30°C are the most favorable for growth and typically produced i n two categories: "White
agriculture of tea (Eden, 1 976). tea" and "Yellow tea", the latter is withered
Traditionally, . sinensis has been propa­ (wilted), resulting in a small degree of fer­
gated, hybridized, and bred through seeds. mentation (Bokuchava and Skobeleva, 1980).
To maintain clonal purity and to accelerate There is some variety of terminology be­
establishment of new productive stands of tween Chinese and Japanese green tea manu­
tea, vegetative propagation is now a com- facture, and with the increase in popularity

418
 �Wr'
�->
-.,,

A spreads to
�:it
Japan
600-800 AD?

Te tullivation spreads

-t. ;� tu.�t.
pacific islands

.
India 9QgAD
1818-1834 AD

f:
f
')
\"

FIGURE 1_ The cultivation of tea worldwide .

..
�
"
of green tea worldwide, pilot production of fermentation of the dhool largely determines
green teas in other regions, such as Darjeeling the flavor charateristics of the finished prod­
in India, from traditionally black tea variet­ uct. Fermented tea leaves are then fired to
ies have led to a wide variety of green tea inactivate the enzymes and dry the leaves.
products on the market. This procedure is accompanied by the final
When oolong and black teas are to be chemical transformations to the product re­
produced, the fresh leaves are allowed to sulting from the high temperatures involved
wither until the moisture content of the leaves i n firing. Figure 2 highlights the differences
is reduced to 55 to 72% of the leaf weight. in manufacture of the most common tea
This causes a concentration of polyphenols products.
in the leaves and deterioration of leaf struc­ The manufactured teas are then sized,
tural integrity. Withering is important for graded, and evaluated for flavor and infu­
aroma development. The withered leaves are sion color by professional tea tasters. The
rolled and crushed, initiating fermentation teas are packaged into sacks or wooden chests
of the tea polyphenols. The fermenting mass and are sold at the world tea auctions. Total
formed from rolling and used in black tea world production of tea in 1 993 was 2.58
manufacture i s referred to as dhool. The pro­ million metric tons, of which 0.59 million
cess used to macerate the leaf plays an metric tons was green tea and 1 .89 million
important role in the final grade of tea. metric tons was black tea. India and China
Two common methods are Orthodox and are the major tea-producing countries, manu­
CTC. Orthodox rolling of the leaf is per­ facturing 53% of tea produced (Anon., 1 994).
formed by mechanically applying weight or Figure 3 illustrates available consumption
compression to the leaves. Orthodox pro­ data of tea worldwide.
cessing is typically used for production of There are numerous polyphenolic com­
large-leaf finished tea products. CTC (crush, pounds produced by the growing tea plant,
tear, curl) processing i s a significant modern and the pathways for in vivo biosynthesis of
improvement of this procedure that minces these phytochemicals have been elucidated
the leaf in a continuous, high-yielding pro­ generally. The chemistry of green and black
cess and produces smaller-leaf teas. tea, therefore, typically centers on the
Oolong teas are prepared by firing the polyphenolic composition of these teas, with
leaves shortly after rolling to terminate the polyphenols being the major proportion of
oxidation process and dry the leaves. The extracted solids in black teas. Although this
rolling process for oolong teas is only de­ is a Western bias, both i n terms of chemistry
signed to slightly damage the leaf and impart of production and beverage as well as the
'twist' to the finished product. Black teas are recent interest in the health-promoting as­
prepared through a separate fermentation pects of tea, it is important that the overall
process in which cooled air is circulated composition be given careful consideration.
through the rolled and crushed leaves to The organization of this review attempts to
moderate the reaction, as the onset of fer­ reemphasize these areas, in contrast to pre­
mentation i s accompanied by a rise in tem­ vious reviewers.
perature from the exothermic fermentation One of the most comprehensive reviews
process. This fermentation process results in of tea chemistry available is that of Sanderson
the oxidation of simple polyphenols to more (Sanderson, 1972). Complementary to
complex condensed polyphenols that give Sanderson's review is that by the Russian
oolong and black teas their bright red colors tea chemists Bokuchava and Skobeleva
and brisk astringent flavor. The degree of (Bokuchava and Skobeleva, 1 980), which

420
 T he Tea Plant

'-- - -- - - - -- �
/ PIUCking

I T he �ea F IUS h 1
�
degree of 'fermentation'

Withering
Firing "White" tea
(green tea)
Withered
Flush

Rolling Firing "Yellow" tea

(green tea)

Firing "Red" tea

'Fermentation'
(aalang tea)

Fermented

---.
Dhool

Firing Black Tea

FIGURE 2. The tea manufacturing process.

421
 India 560

USA 91

CIS 132

UK 151

China 408

Other 221

Morocco 30
Sy r i a 22
Sri lanka 22
Japan 130 Poland 31
Egypt 64

Iran 85
Turkey 124
Pakistan 117

FIGURE 3. Consumption of tea, 1993 (10' metric tons).

provides a variant perspective and insight erts, 1 942; Roberts, 1 962) and Graham's
into Russian-language literature sources, (Graham, 1983; Graham, 1 984 ) reviews of
which are typically not cited in English-lan­ tea chemistry, a review by Stahl (Stahl, 1962),
guage publications. HPLC techniques, now Wickremasinghe (Wickremasinghe, 1 978),
available for rapidly measuring a number of Balentine (Balentine, 1 992), and a review by
important tea polyphenols, including the cat­ Robertson (Robertson, 1 992). An excellent
echins, f1avonols, flavonol glycosides, and comprehensi ve review of the work on tea
the theaflavins, have dramatically changed aroma to the early 1 980s i s also available
the way tea chemistry i s studied, and these (Bokuchava and Skobeleva, 1 986), as well
methods have been reviewed in detail (Fin­ as a more recent review (Robinson and
ger et aI., 1 992). Owuor, 1 992).
There are a variety of other reviews on Reviews published subsequent to that of
the subject of tea, including Roberts' (Rob- Sanderson appear to attempt an update of the

422
subject, but none have critically reassessed tion, there are differences between the com­
the field. It is hoped that this review can take position of the tea leaf and other phytological
some of the historical material, combined components, as well as differences between
with a comprehensive and critical account of the leaf and the "brew". It is therefore diffi­
recent research, and synthesize a review of cult to synthesize an overall picture of the
tea chemi stry as it is currently viewed and chemistry of tea without resorting to a sub­
applied in our laboratory. An overemphasis division of the problem.
on polyphenols, compounds that are signifi­ With this in mind, we have attempted to
cant to color and astringency but less rel­ focus on the "extract solids", the component
evant to teas such as shaded green tea pro­ of tea leaves that is extracted by boiling
duction, dominates the l i terature. Low water. Table I gives an approximate compo­
polyphenol content and high chlorophyll sition of black and green tea beverage solids
content are important for producing an em­ by chemical class. Although the length of
erald-green and smooth finished green tea time for steeping and the amount of water in
product that lacks astringency, and the brothy which the leaves are steeped can vary widely,
character of the amino acids, and flowery these factors generally control the amount of
character of the aroma constituents, play a solids extracted, and to a lesser extent influ­
much more significant role. In addition to ence the composition. A typical brew of one
the nonpolyphenolic consti tuents, the tea bag in one cup of water produces a solu­
polyphenols of black tea are a poorly under­ tion of 0.35% wtlwt solids, and from this
stood but seemingly well-defined group of value the dose expected from consumption
compounds. The use of "thearubigen" in the of one cup of tea can be calculated. This is
literature signifies a wide group of com­ typically how tea phytochemicals are con­
pounds whose chemical identity has not been sumed. Notes on the composition of the
traced to any identifiable chemical group. Its 'flush' or the fresh tea leaves, or other parts
use was most significant as a colorimetric of the tea plant, are added for completeness
indicator for plantation production, but as a
chemical identifier its use has seemed to
hamper true identiication of the mass balance TABLE 1
The Composition of a Typical Tea
of black tea polyphenols. Work at identifying
Beverage, %w/wt Solids
these compounds in a systematic fasion has
begun, and it is hoped that future research Green tea Black tea
will deemphasize the term ' thearubigen' in
30% 9%
4%
favor of more chemically accurate des­ Catechins

2% 3%
Theaflavins
criptors.
2% 1%
Simple polyphenols

6% 23%
Flavonols

3% 3%
Other polyphenols
II. CHEMICAL COMPOSITION, AN
3% 3%
Theanine

6% 6%
OVERVIEW Aminoacids

2% 2%
Peptides/Protein

7% 7%
Organic acids
In order to divide the subject of tea chem­
4% 4%
Sugars
istry into well-ordered i ssues, it is most con­
3% 3%
Other carbohydrates

3% 3%
venient to do so on the basis of chemical lipids

<1% <1%
Caffeine
composition. Frequently, researchers' inter­
5% 5%
Other methylxanthines
est in specific compounds or a class of com­
5% 5%
Potassium
pounds induces a focusing of attention on a Other minerals/ash
small fraction of the total tea mass. In addi- Aroma Trace Trace

423
within the review. We have also subdivided B. Cafeine, Methylxanthines, and
the issue of tea compounds by chemical class. Related Compounds
This is done to focus on specific aspects of
tea chemistry, such as aroma or polyphe­ Tea has been valued historically for
nols, and because the methods used for de­ its caffeine content. Caffeine [ 1 ] i s viewed
termination of the various chemical classes as an important constituent of tea, bestow­
are quite different. ing mood and cognitive-enhancing proper­
The strict division of tea chemistry by ties (Bokuchava and Skobeleva, 1 980).
fu nctional chemical identity has its uses, but Figure 4 illustrates the methylxanthines of
begins to lose value i n discussing subjects tea. Tea leaves contain between 2 and 5%
that cross the boundaries of chemical class wt/wt caffeine depending on the variety.
or those that have no distinctive chemical
identity, such as discussions concerning the
'thearubigens'. Modern tea research has
reached the point where simple chemical
subdivision has been refined with a fair de­
gree of accuracy, and determination of the
synergies and 'boundary violations' present

[1]: Caffeine
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

[2]: Theobromine
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.
NH2
�N�COOH
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 o

[3]: Theanine
the plant as food (Beart et aI., 1 985). The
evolution of salivary proline-rich proteins,
which bind polyphenols effecti vely, has
ameliorated this defense mechanism, con­
verting it to 'astringency' (Luck et aI., 1 994). FIGURE 4. Nitrogenous tea phytochemicals.

424
. irrawadensis, a member of the Camellia a significant impact on caffeine content
family, lacks caffeine (Roberts et ai., 1 958) (Sanderson, 1 972).
but is not processed commercially because it Caffeine is one of the most comprehen­
produces a poor finished tea product. The sively studied ingredients in the food sup­
quantity of caffeine that i nfuses into a tea ply. Extensive research does not link moder­
brew i s determined by infusion time and by ate caffeine intake to any health risks. Studies
leaf style. Longer infusion times lead to are needed to better understand the physi­
greater quantities of caffeine in a tea bever­ ological role of tea caffeine and its associa­
age. Smaller sized tea leaves give a more tion with the popularity of tea beverages.
rapid and stronger infusion, whereas larger Those individuals who are especially sensi­
leaves and uncut leaves lead to weaker infu­ tive to caffeine can find decaffeinated teas
sions. This results in more or less caffeine readily available.
extraction, respectively. The caffeif, :on­
tent of a typical tea beverage will range from
20 to 70 mg per 1 70 ml of infusion, with a 1. Theobromine
typical infusion being prepared from about 2
to 2.5 g of tea leaves. Coffee brews typically Theobromine [2] is present in tea i n much
contain from 40 to 1 55 mg caffeine per lower quantities than caffeine. Theobromine
170 ml beverage. is formed as a consequence of the biosynthe­
There has been little research done on sis of caffeine (Negishi et ai., 1 9 85a) and is
the pharmacology of tea-beverage caffeine. produced in abundance if the methylation
One study suggests a dose of caffeine from path to caffeine i s absent, such as i n
tea has a different physiological effect than . irrawadensis (Roberts e t ai., 1 958). Theo­
a pure dose of caffeine (Das et ai., 1 965). phylline, a similar di-methylxanthine, has
This has been attributed to the amino acid been reported in trace quantities in tea leaves
theanine, which is unique to tea. However, (Michl and Haberler, 1 954; Sanderson, 1972).
there are no well-designed clinical studies to Recent reports contradict as to the the exist­
support this position. The consensus among ence of this compound in tea, some failing to
scientists today is that caffeine from all bev­ detect these compounds (Hicks et ai., 1 996)
erage sources has a similar physiological and others (Meyer et ai., 1996) reporting
effect. The actual content of caffeine de­ small quantities. The xanthine content of teas
pends on many factors, particularly the is clearly an area that requires further, more
method of brewing. A brew prepared by the careful research.
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 2. Other Derivatives of Nucleic
tea bag (Hicks et ai., 1 996). Some reports Acids
have suggested that green tea contains sig­
nificantly less caffeine than black tea. This The RNA and DNA in tea leaves are
may be influenced by the clone of leaf used metabolized naturally as well as digested
to produce the tea or by the impact of differ­ under the conditions of withering and fer­
ent brewing techniques. No significant dif­ mentation by tea nucleases, nucleosidases
ferences have been found when brewing (Imagawa et ai ., 1 982), and a specific ad­
green and black teas under similar condi­ enine nucleosidase (Imagawa et ai ., 1 979).
tions (Hicks et ai., 1 996), discrediting the These catabolic reactions produce purines,
theory that withering and fermentation have which have been detected in very small quan-

425
tities i n tea (Michl and Haberler, 1 954; Optimum production of theanine i n cell
Sanderson, 1 972; Hicks et aI., 1 996). culture has been investigated (Furuya et aI.,
1 990; Matsuura et ai., 1 992). Theanine
C. Proteins and Amino Acids can also be made synthetically on a com­
mercial scale in good yield (Kawagishi
While caffeine is the most well-known and Sugiyama, 1 992).
ni trogenous component of tea, tea proteins/ Tea leaves subjected to anaerobic condi­
pep tides and amino acids contribute signifi­ tions are found to produce excess amounts
cantly to the composition of both the leaf of GABA, ory-amino butyric acid (Tsushida,
and the tea extract. Figure 4 illustrates some 1 987), derived from glutamic acid due to the
of the other important miscellaneous nitrog­ action of an endogenous glutamate decar­
enous and non nitrogenous components of boxylase in tea (Tsushida and Murai, 1 987).
tea. Recent measurement of the amino acids
in two green teas (Liang et aI., 1 990) con­ D. Carbohydrates, Pectins, and
firms the presence of 1 8 amino acids, a re­ Fiber
sult that is mirrored for black tea. Amino
acids contribute about 6% wt/wt of the ex­ Tea leaves have been shown to contain
tract solids. Tea also contains a significant free sugar residues in addition to pectic
amount of peptidic material (protein), ap­ subtances, poly saccharides, a n d fiber
proximately 6% wt/wt of extract solids. Ni­ ( M i zu n o et a i . , 1 964; Sanderson and
trogenous materials therefore comprise about Perera, 1 965). Carbohydrates contribute ap­
1 5 % wt/wt of extract solids. The free amino proximately I 1 % w/wt of extract solids
acid content of tea seems to increase during (Sanderson et aI., 1 976; Graham, 1 984). High
withering of the fresh tea leaves but de­ molecular weight pectins (polygalacturonic
creases duri ng fermentation to black tea acid) and other polysaccharides ha ve been
(Roberts and Sanderson, 1 966), as it i s analyzed on Sephadex G- 100 (Millin et aI.,
likely consumed during aroma biogenesis 1 969). As the tea plant matures, increases in
and through other routes. These pathways the content of lignin and cellulose have been
have a strong impact on the aroma of the observed (Selvendran et aI., 1 972), which is
finished product and require more detailed consistent with their role in providing struc­
investigation. tural integrity to the growing plant.
In addition to the common amino acids, A significant portion of the carbohydrate
there is a unique amino acid known only to fraction in tea extract has been found to
be present in tea. This amino acid, theanine comprise the disaccharide 2-0-(�-L-Arabino­
(y-N-ethyl glutamine, [3]), is believed to be pyranosy l)-myo-inositol [4] (Sakata et ai.,
the major amino acid present in tea, com­ 1 9 87), as shown in Figure 5. It was detected
prising about 3% wt/wt of extract solids. by NMR techniques in an aqueous fraction
Theanine is a significant component of both subjected to consecutive extraction with ethyl
green tea (Sakato, 1 950) and black tea acetate and butanol (Sakata et aI., 1 989).
(Feldheim et aI., 1 986). Theanine has been
associated with improved flavor and a modu­ E. Organic Acids and Vitam i n C
lation of the stimulative effects of caffeine
(Kimura and Mu rata, 1 97 1 ). Recent studies Tea is a significant source of oxalic acid
on the antihypertensive effect of theanine (Sanderson and Selvendran, 1 965) and malic
found that a large quantity of this compound acid (Jayman and Sivasubramanian, 1 975),
was required to exhibit an effect i n rats along with citric, isocitric, and succinic ac­
(Yokogoshi et aI., 1 995). ids (Sanderson and Sel vendran, 1 965). Tea

426
 �� O

�
H
OH
HO 0
HO 0
0H [4]: 2-0-(p-L-Arabinopyranosyl)-myo-inositol
OH
HO
OH

HO [5]: Spinasterol

FIGURE 5. Miscellaneous tea phytochemicals.

also contains shikimic and qUIntC acids, (Taylor and McDowell, 199 1 ). The compo­
which are important to the biosynthesis of sition of these components in the green tea
the polyphenols (Zaprometov, 1 9 6 1 ) . Vita­ leaf has been demonstrated to have a strong
min C (ascorbic acid) has also been detected impact on the quality of the beverage as
in green tea (Liang et aI., 1 990) and black perceived by tea tasters (Taylor et aI., 1 992).
tea (Sanderson, 1 972). Tea grown in the shade has been found to
have a lower quantity of catechins (resulting
in a less astringent beverage) and increased
6. Lipids, Chlorophylls, levels of carotenoids and chlorophyll (which
Carotenoids, and Related may assist in aroma production). This chemi­
Compounds cal balance is thought to contribute favor­
ably to taste (Mahanta and Baruah, 1 992).
The main pigments in the fresh tea leaf The carotenoids play a significant role in the
are chlorophylls and carotenoids. Chloro­ formation of aroma characteristic to black
phylls (e.g., [39]) are oxidized during the tea (Bokuchava and Skobeleva, 1 986). High
course of black tea manufacture to the chlorophyll levels and low astringency are
pheophytins and pheophorbides (e.g., [40]), important to some green tea manufacture,
which give the fermented leaf its character­ particularly Mattcha, for which a brothy,
i stic brown-black color. Some of the emerald-colored brew i s very important.
pheophytins and pheophorbides are extracted Lipids, terpenoids, and saponins make
into the black tea beverage (Sanderson, up a large portion of the fresh tea leaf, yet
1 972). An efficient HPLC method has been because of their low water solubility, are
developed for analysis of these pigments generally thought to be a minor portion of

427
the water extract solids. However, plant ste­ erals, and may be excellent chelating agents.
roids such as spinasterol [5) or lipids such as The large number of phenolic hydroxyl
the plant cuticle wax triacontanol [6) have groups provides a great number of potential
been shown to comprise an important frac­ active complexation sites.
tion of tea cream, the precipitate that forms Tea beverages are also a significant
after cooling of concentrated tea extracts source of fluoride (Elivin-Lewis et aI., 1980).
(Seshadri and Dhanaraj, 1 988). It is hypoth­ This is due, in part, to the uptake of alumi­
esized that the hydrophobic environment num fluoride (Yamada and Hattori, 1977;
presented by the tea polyphenols and caf­ Yamada and Hattori, 1 980).
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 H. Aroma
been analyzed in detail (Anan, 1 983; Bhuyan
and Mahanta, 1 984). The role of hydropho­ The essential oil or aroma of tea pro­
bic plant materials in the appearance and vides much of the pleasing flavor as well as
organoleptic properties of the brew has been scent of green and black tea beverages, yet
typically disregarded in favor of the polyphe­ comprises only a minor fraction of the total
nols, and this is an important area for which mass of the tea plant or the extracts. Tea
future research is necessary. aroma contains hundreds of compounds i n
trace quantities, the composition and mecha­
nism of production of which has been re­
G. Viti mans and Minerals viewed (Choudhury, 1982; Bokuchava and
Skobeleva, 1 9 86; Robinson and Owuor,
The tea plant has been shown to be rich 1992).
in potassium (Sanderson ct aI., 1 976) and Withcring has been determined to play
contains significant quantities of calcium and an important role in aroma development, as
magnesium, as well as small amounts of i n oolong teas (Takeo, 1 984; Kharebava,
manganese, iron, and phosphorus (Kalita 1 9 86) and black teas (Owuor et aI., 1 987).
and Mahanta, 1 993), copper and nickel Many of the aroma components of tea can be
(Burke and Albright, 1 970), and sodium, found as glycoside derivatives, which are
boron, and molybdenum (Hasselo, 1 965). freed during the fermentation process due to
Zinc (Tolhurst, 1 962) and sulfur (Pethiyagoda the action of glycosidases. Fresh tea enzymes
and Krishnapillai, 1 970) are essential ele­ permit the release of additional aroma con­
ments for healthy maturation of the tea plant stituents in the leaf and extract, restoring
as well. Cobalt, lead, and cadmium have fresh aroma from stale tea (Guo et aI., 1992).
been detected i n the plant, and concentra­ Geranyl, linolyl, terpinyl, and neryl glyco­
tions depend principally on soil concentra­ sides can be found in fresh tea extracts (Guo
tions of these minerals (Ramakrishna et aI., et aI., 1 993). �-Glucosidase is the enzyme
1 9 87). most likely to be responsible for formation
The tea plant is known to accumulate of tea aroma from these glycosides (Morita
aluminum (Chenery, 1 955). Aluminum lev­ et aI., 1994). The exploitation of bound gly­
els can be traced by NMR techniques (Nagata cosides of aroma components by glycosi­
et aI., 1 9 9 1 ) and have been found i n com­ dase treatement offers the possibility of fu­
plexes with fluoride and catechins in the tea ture improvements i n tea quality.
plant (Nagata et aI., 1 993). Tea polyphenols Part of the aldehyde fraction may be
are commonly thought to complex with min- generated from a unique tea leaf amine oxi-

428
dase (Tsushida and Takeo, 1985). Tea also III. POLYPHENOLS IN TEA
contains a fatty acid hydroperoxide lyase,
which forms volatile aldehydes from the lipid The term 'polyphenoJ' is an inclusive
constituents of the tea leaf (Matsui et a!., descriptor referring to the millions of natural
199 1 ) . There are also many products that are and synthetic aromatic molecules that are
derived from the oxidizing conditions present substituted with multiple hydroxyl groups.
during tea fermentation (B okuchava and The polyphenols comprise one of the most
Skobeleva, 1 9 86). Figure 6 illustrates one distingishing characteristics of the tea plant
such unique aroma constituent, theaspirone and have been more thoroughly investigated
[9], which i s produced from oxidation of than any other class of compounds in tea.
p-carotene [7]. For this reason, the polyphenols in tea are

[7]: p-Ca rotene

/
�o
[8]: p-Ionone
many other aroma constituents

[9]: Theaspirone

FIGURE 6. Unique tea aroma constituents.

429
treated i n greater depth i n this section and All too frequently. popular writing on
separate from the overall review of the chemi­ tea compounds confuse the nearly hom­
cal constituents of tea. onymic flavonoid classifications. Flavonoids
Because of the abundance of polyphe­ are the widest subgrouping. i ncluding
nols present in tea leaves and in tea bever­ flavanols. which have a saturated central (C)
ages. i t is natural that tea chemistry is often ring and include the catechins. the major
considered to be synonymous with tea green tea polyphenols. and flavonols. which
polyphenol chemistry. The polyphenols are have an unsaturated central (C) ring and a
principally responsible for the color and as­ ketone group. Careful attention must be paid
tringency and partially responsible for the when reading any of these three classifica­
flavor of the tea beverage. The compounds tions. These classifications are all inclusive
are known antioxidants and are being stud­ under the broader term polyphenol. which
ied as agents that might reduce risk factors refers to any compound that contains aro­
associated with cancer and heart disease. matic rings with multiple pendant phenolic
While careful attention needs to be paid to OH groups or derivatives thereof.
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 B. Green Tea Polyphenols
by the discussion of the tea polyphenols.
Green tea polyphenols consist of both
simple and complex polyphenols. The large
A. Chemical Classification majority of polyphenols in green tea are fla­
vonoid monomers called catechins and fla­
The polyphenols in tea may be subdi­ vonols.
vided by several chemical backbone struc­
tures. Simple tea polyphenols are those thar
are synthesized during the early stages of 1. Catechins and Ga/ocatechins
polyphenol biosynthesis. whereas the degree
of complexity of the polyphenols increases The catechins [ 1 0- 1 3] represent the ma­
as one progresses down the biosynthetic jor polyphenolic constituent of green tea and
pathway. The flavonoids. a subgrouping of are illustrated in Figure 7. Catechins are
polyphenols and the dominant class of green members of a more general class of fla­
tea polyphenols. are synthesized in part from vonoid. the flavan-3-0Is (also referred to as
the simple polyphenols and represent com­ flavanols). Three subgroupings of the
pounds with 1 5 or greater carbon atoms [C" flavanols [afzelechin [ 1 4]. catechin [ 15 ] .
stage] in the basic framework. The polyphe­ gallocatechin [ 1 6]. shown i n Figure 8]. rep­
nols of black tea represent further chemical resenting varying degrees ofB-ring hydroxy­
transformations of the green tea polyphenols lation. are the dominant forms. of which the
and therefore comprise a third level of com­ epi-isomers of the catechins and gallo­
plexity. Unique black tea polyphenols are catechins are the principal components found
commonly thought to be polymers of the in tea. The tea catechins. a term commonly
green tea polyphenols and therefore are used to refer to both catechins and gallo­
thought to be comprised of molecules of catechins. make up as much as 30% wtiwt of
approximately 30 carbon atoms [C30 stage] dissolved solids.
or greater. as the simplest polymer would be A large percentage of the catechins
a dimer such as procyanidin. present in tea exist as gallic acid esters. While

430
 R1 R2
H H
H
[10]: Epicatechin EC

EGC H OH
[11]: Epicatechin Gal late ECG Gallate

OH
[12]: Epigailocatechin
[13]: Epigailocatechin Gallate EGCG Gallate

FIGURE 7. The principal tea catechins.

HO ¢ & '� I"

OH

�
OH
OH

R1 R2
H H
OH H
[14]: Afzelechin

OH OH
[15]: C atechin C
[16] : Gallocatechin GC

FIGURE 8. The flavan·3·ols.

gallation i s found to occur principally at the now has been isolated from other sources
3-position, various other gallated species (Danne et aI., 1 994).
have been isolated, including the epigallo­ The four most common catechins are
catechin digallates (Nonaka et aI., 1983), and epigallocatechin gallate (EGCG, [ 1 3 ] ) ,
epicatechin digallate (Coxon et aI., 1 972; epigallocatechin (EGC, [ 1 2]), epicatechin
Hashimoto et aI., 1 987). 3-Methyl gallates gallate (ECG, [ 1 1 D, and epicatechin (EC,
of EC and EGC have also been reported [ 1 0]). Catechin (C, [ 1 5]) and gallocatechin
(Saijo, 1 982). EGCG was once thought to be (GC, [ 1 6]) are also present i n smaller quan­
unique to the tea plant (Graham, 1983) but tities. While gallocatechin gallate (GCG) and

431
catechin gallate (CG) have also been ob­ 3. Simple Polyphenols
served, i t is likely that these are products of
racemization and not 'native' to the tea plant Gallic acid [20] and its quinic acid ester
(Roberts, 1 962; Robertson, 1 992). (or depside, as quinic acid esters are com­
monly referred), theogallin [2 1 ], have been
identified in tea (Cartwright and Roberts,
2. Flavonols 1 954; Cartwright and Roberts, 1 955) and
have been detected by HPLC (Bailey et aI.,
The flavonols [kaempferol, quercitin, and 1 990; Hashi moto et aI., 1 992). The simple
myricitin] and their glycosides [ 1 7- 1 9] have polyphenols and their depsides are shown i n
only been recognized recently as significant Figure 10.
components in tea, although their presence Cinnamic acid derivatives of quinic acid,
as trace constituents has always been ac­ the coumaryl and carr:oy l- quinic acids (in­
knowledged. The flavonols are illustrated i n cluding chlorogenic acid or 5-caffeoylquinic
Figure 9 Analyses of the flavonol glycosides acid [23]) have also been identified in tea
in general (McDowell et aI., 1 990) and of (Cartwright et aI., 1 955). Chi orogenic acid
the flavonol diglycosides (Finger et aI., and 4-coumarylquinic acid [22] have been
1 99 1 a) and triglycosides (Finger et aI., detected by HPLC (Bailey et aI., 1 990).
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. 4. Other Polyphenols
Use of hydrolysis to determine flavonol
and flavonol glycoside content as their agly­ Flavones and their glycosides (Engel­
cones (Hertog et aI., 1 993) has proven to be hardt et aI., 1 993), such as apigenin [24],
useful i n determining overall flavonol con­ have been detected in tea but represent a
tent (about 0.5 to 2.5% wtiwt extract, as very small frac tion of the polyphenols
aglycone) of tea infusions. present. Flavone glycosides can potentially

R,
OH

HO

OGlycoside
OH 0

[1 7]: Kaempferol Glycoside KaG H H

[18]: Quercitin Glycoside QuG OH H
[19]: M yricitin Glycoside MyG OH OH

FIGURE 9. The lIavonol glycosides.

432
 -
OH

Hooc OH

OH

[20]: Gallic Acid

:�
OH

OH

HOOC n OH

OH � OH
OH
[2 1 ] : Th eo ga llin

-
R

o OH

n [22] : Coumarylquinic acid, R =H

o

[23]: Chlorogenic acid , R =OH

HOOC

OH� OH
OH

FIGURE 10. Gallic acid and the depsides.

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

(Hertog et ai., 1 993). Careful resolution of have also been reported (Roberts et ai., 1 956).
individual flavone glycosides (Engelhardt These are ill ustra ted in Figure 1 2.
et ai., 1 993) has confirmed their presence in
tea. Apigenin is illustrated in Figure I I . 5. Tannins

.
A number of proanthocyanidin species,
such as prodelphinidin B 2 [25] gallates, have Although it is commonly stated that there
been isolated and are present in green tea are no tannins (meaning hydrolyzable tannins
(Non aka et ai., 1 983; Nonaka et ai 1 984).
, such as pentagalloylglucose [28]) in tea, this
The assamicains, such as assamicain A [26] statement is not strictly true. In addition to
isolated from . sinensis var. assamica, ap­ the gallic acid esters of the catechins and
pear to be ring-opened products of the their oxidation products (which can be hy­
proanthocyanidins (Hashimoto et ai., 1 989a). drolyzed to produce gallic acid readily and

433
 OH
HO

OH 0

[24]: Flavone (Apigenin)

�OH
OH

HO " " �OH

OH
" 'OR1 y OH

lOH
OH
HO o
""
" 'OR1

[25] : Proanthocyanidin
(Epigallocatechin-4-a-Epigal locatechin ,
or Prodelphinidin B2)

FIGURE 1 1 . Miscellaneous polyphenols,

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

quantity of hydrolyzable tannin (Nonaka
et aI., 1 984; Yoshida et aI., 1 990; Hatano B lack tea polyphenols are produced
et aI., 1 99 1 ; Han et aI., 1 994). The unique from the controlled enzymatic reactions
hydrolyzable tannins in tea are typically involved in the fermentation of green leaf
"hybrid" tannins such as camelliatannin A during commercial and model black tea
[29], which i s a galloylglucose derivative production, The extent and conditions un­
with pendant catechins, The tannic acid de­ der which fermentation occurs determines
rivatives common to gall-nuts and tree bark the degree to which the polyphenols of green
are not present i n significant quantities in tea tea are transformed to those unique to black
infusions. The tea tannins are illustrated in tea. It is reasonable to expect that black tea
Figure 1 3 , should contain an amount of polyphenols

434
 OH
HO OH

��c::
OH

o . .

" OGa
OH Ga=Galiate

[26]: Assamicain A

OH
OH

HO

OH
OH OH

[27]: Flavan-3,4-diol
(Leucocyanidin)

FIGURE 12. Miscellaneous polyphenols.

similar to green tea. However, the complex pending on the degree of fermentation, how­
nature of these polyphenols, some of which ever, some green tea polyphenols remain
are "polymeric" in nature, has largely re­ unconverted. This is parti cularly true in the
sisted chemical identification. The uniden­ case of oolong teas and some Darjeeling
tified polyphenolic constituents are often teas, which have been known tv resem bl e
referred to as thearubigens. Despite their green tea both in chemical constitution (Ding
complexity, some of the unique black tea et a!., 1 992a) and astringency.
polyphenols have been identified and char­
acterized. a. Catechins

1. Residual Green Tea Polyphenols The catechins represent the major por­
tion of green tea polyphenols and conse­
During the course of fermentation, the quently are thought to be the building blocks
polyphenols of green tea are rapidly con­ of black tea polyphenols. Some of the green
verted to the polyphenols of black tea. De- tea catechins survive the fermentation pro-

435
 HO �OH
� �)��-OH OXO OH
O
HO I
q �4
HO :"
0 0
0
0

OH
0 0

OH OH 1 &
OH HO� , OH
�
0
"

HO
OH
[28]: Hydrolyzable Tannin
(Pentagalloylglucose)

OH HO
HO HOi >
HO
HO
HO OH
HO
OH HO
[29]: Cameliiatannin A

FIGURE 1 3. Tannins.

cess and are detected in black tea (Bailey i ncreased levels of non-epi isomers of the
et a!., 1 990). Due to the oxidation reactions catechins (Coggon et a!., 1 973).
and thermal conditions experienced by the
tea leaf during black tea production, it i s b. Flavonols
hypothesized some o f the catechins are also
epimerized and/or degallated, which explains It is likely that the majority of flavonols
the appearance of free gallic acid as well as (free as well as glycosides) present in the

436
initial fresh green leaf remain unoxidized 1 958). Market value for tea is also influ­
and are likewise present in black tea in simi­ enced by secondary factors such as aroma,
lar quantities. There is some evidence that as observed with Kenyan teas that normally
some of the flavonols are oxidized during contain higher theaflavin contents (Owuor
fermentation. It has been suggested that et aI., 1 986) and therefore not a distinctive
myricitin and myricitin glycosides are the characteristic.
most likely oxidized of the three flavonols While characterized by a unique benzo­
(kaempferol, quercitin, myricitin) (McDowell tropolone ring structure resulting from the
et aI., 1 990). dimerization of a catechin and a gallo­
Despite this, in a recent analysis of fla­ catechin, there are a series of related com­
vonols and their glycosides (Hertog et aI., pounds, i n c l u d i n g the i soth eaflav i n s ,
1 993), no significant difference between the neotheaflavins, and theaflavic acids, which
green teas and the black teas was found in also possess a similar benzotropolone unit.
terms of total flavonol as aglycone, except The benzotropolone ring provides the red
for myricitin, which was found to be slightly color and makes the thealavins easily dis­
reduced in the black tea samples compared tinguishable from other components.
with green teas. Differences between these Analysis of theaflavins began with ex­
samples might have also been derived from traction of water extracts (Roberts, 1 95 8 ;
different origins for the two analyzed mate­ Spiro et aI., 1 987; Robertson and Hall, 1 989)
rials, however, owing to the variations in into isobutyl methyl ketone (Roberts and
flavonol content of teas grown from differ­ Smith, 1963) or ethyl acetate (Ullah, 1 972),
ent clones and from different regions of the followed by spectrophotometric measure­
world. ments. Later spectrophotometric methods
improved on this technique (Xiao and Li,
1 992). The Flavognost method (Hilton , 1 973)
2. Theaflavins and Related Products uses diphenyl boric acid ethanolamine to
induce a spectrophotometric shift i n the
One of the key distinctions of black teas benzotropolone ring for better accuracy.
compared with green tea i s the production of Recent improvements in analytical technol­
a new type of polyphenol, the theaflavins. ogy include the use of GC (Collier and
The fermentation of green tea leaf also re­ Mallows, 1 97 1 ), and HPLC (Robertson and
sults in the development of characteri stic Bendall, 1 983; Steinhaus and Engelhardt,
aroma components, a darkening of color of 1 989), as well as NIR measurements on the
the leaf and extracts, and a decreasing astrin­ leaf (Hall et aI., 1 988) and absorption onto
gency with increased fermentation time. cartridge columns (Whitehead and Temple,
1 992).
The mechanism of theaflavin formation
a. Thealavins was fairly well defined in early papers on
purpurogallin formation and related experi­
Best known of the fermentation prod­ ments (Horner et aI., 1 96 1 ; Critchlow et aI.,
ucts is the class of compounds known as the 1 967; Takino and Imagawa. 1 964b). Papers
theaflavins [30-33), comprising about 3 to investigating the fermentation reaction lead­
5% wtlwt of the extract solids. They are ing to the characteristic benzotropolone ring
illustrated i n Figure 14. Theaflavin provides reported the production of a series of
a bright, red-orange appearance to the tea theaflavin-like compounds, most notably
beverage and has long been positively corre­ erycitrin (Takino and Imagawa. 1 963),
lated with market value of tea (Roberts, categal lin. and pyrogallin (Takino and

437
 OH
HO OH
Hoyoi, " " 7 ° OH _

� ""ORI OH
OH
[30]: Theaflavin TF H H

H
[31 ] : Theaflavin 3-Gal late TF3G Gallate H
[32] : Theaflavin 3'-Gallate TF3'G Gallate
[33] : Theaflavin 3 , 3'-Digallate TFDG Gallate Gal late

FIGURE 14. The theallavins,

lmagawa, 1964a), Oxidations involving na­ condensation of the non-epi forms of the
tive tea enzymes (Roberts, 1 958), bicarbon­ catechins with gallic acid through an identi­
ate/ferric ammonium su lfate (Takino and cal mechanism. Similarly, theaflagallins such
Imagawa, 1 964a), potassium iodate (Takino as epitheaflagallin [35] arise from gallo­
et aI., 1 964), and peroxidase (Takino et aI., catechins and gallic acid (Nonaka et aI.,
1 967; Finger, 1 994) have all produced 1 986), where the carboxylic acid moiety
theaflavins and theaflavin-like compounds becomes a leaving group and mimics the
in varying yields, Precise NMR analyses of catec h i n group i n the m e c h a n i s m of
the theaflavins are available (Bryce et aI., theaflavin formation.
1 970; Cai et aI., 1 995),

c. Other Related Structures

b. Thealavic Acids
Isotheaflavins (Coxon et aI., I 970a) and
Theaflavic acids such as epitheaflavic neotheaflavins (Bryce et aI., 1 972; Robertson,
acid [34], shown in Figure 1 5 , are formed 1 992) are formed in the same manner as the
from oxidative condensation of a gallic acid theaflavins, except that they arise in part
molecule and a catechin (Coxon et aI., from the non-epi forms of the pairs of cat­
1 970b). In the production of epitheaflavic echins. The abundance of non-epi forms of
acids, gallic acid provides the tri-hydroxy catechins i n the green tea leaf i s small, and
structure and thus mimics the role of a therefore these black tea components are
gallocatechin i n the mechanism of theaflavin present in signiicantly lower concentration
formation. Theaflavic acids are formed from compared with the theaflavins.

438
 OH
chromatography, but were roughly quantifi­
OH able by simple spectrophotometric tech­
niques. These compounds were given the
OH
label of thearubigens. However, subsequent
research began to compromise the under­
OH
standing of the thearubigens as a well-de­
Hooe fined group of compounds.
Refinements on the procedure for quan­
OH
tifying thearubigens were made beyond the
early paper chromatography techniques.
Solvent extraction methods, employing ethyl
[34]: Epitheaflavic Acid 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

0
OH

/-
use C- 1 8 cartridge columns (Whitehead and
Temple, 1 992) and a series of solvents to

I
elute the appropriate fractions and measure
H0 0 " '
thearubigens and theaflavins by colorimetry.
"
The significant limitation of use of this
:.. OH
'OH method is that there is no clear evidence,
OH other than a correlation, that these measured­
"thearubigens" and the "thearubigens" iden­
tified by Roberts are one and the same. This
[35]: Epitheaflagallin becomes more evident in later methods. The
thearubigens were divided into three sub­
classes on the basis of paper chromatogra­
FIGURE 1 5 . The epitheallavic acids a n d related
compounds
phy: SI, SIIa, and SIIb (Roberts et aI., 1 957).
Later HPLC techniques reinterpreted the is­
sue with division of the thearubigens into
3. Further Oxidized Products: The groups I, n, and III (Bailey et aI., 1 99 1 ; Bailey
"Thearubigens" et aI., 1 994a). In both papers, the presence of
an unresolved mass (as illustrated i n Figure
From the early stages of development, 16) is taken to represent the same compounds,
simple solvent extractions attempted tu quan­ the "unresolved thearubigens". This i s based
tify the amount of colored tea compounds on loose similarities of HPLC with paper
present i n the black tea brew (Roberts et aI., chromatography as well as more recent work
1 957). The theaflavins, a group of bright with cartridge column fractions (Wellum and
orange-red compounds, were quickly sepa­ Kirby, 1 9 8 1 ).
rated from the remainder. Paper chromatog­ The arrival of HPLC chromatography
raphy experiments confirmed that subsequent led to the belief that for the first time indi­
to solvent extraction, a series of brown-red vidual thearubigens would be separated and
compounds remained in the aqueous phase, i s o l ated (Hoefler and Coggon, 1 97 6 ;
which were not well resolved by 2-D paper Robertson and Bendall, 1 983). However, it

439
 �
�.�..' = Area of well resolved spols

= Diffuse, unresolved spots Cinnamic Acids

.
Flavonol Glycosides

.
.
�....•.... .-.-. .
Catechins

(
..._.::':::.:.:. r.:.-::-... . .

...
. ..

.
..
..
.
TheaJavins
.

'Thearubigens'

Butanol- etic Acid-Water

225

200
Difuse spot on bo h paper
chromatography a d on
1 75 H PLC chromatogr m taken

1 50
to be synonymous

,
Both identified as
1 26 'unresolved thearu igens'

"
100 atechins

75

50

25

10 20 40

FIGURE 1 6. Paper chromatography vs. HPLC.

became clear early on that chromatography (Wedzicha and Donovan, 1 989) and using
using reversed-phase materials was not an the technique of counter-current chromatog­
ideal solution, due in part to the observation raphy (Okuda et aI ., 1 988; Wedzicha et aI.,
that some tea components were not eluted 1 990) have shown some promise in separa­
from cartridge columns. tion and identification. The absence of re­
Strategies for separation of the thearu­ cent reports suggests that use of such "alter­
bigens on normal-phase chromatography native" technologies has fallen into disfavor,

440
due i n part to the extreme simplicity of re­ diffuse rising baseline. A study of a model
versed-phase techniques. Use of reverse fermentation of a mixed system of theaflavins
phase colums with a step-gradient (Putman and epicatechin in the presence of polyphe­
and Butler, 1 989) might present a potential nol oxidase showed that epicatechin and the
useful technique for these compounds s well. theaflavins, but not the theaflavins alone,
Use of reverse-phase technology has been resulted in the degradation of the theaflavins
expanded systematically by a series of re­ (Opie et aI., 1 993). In another study, the pres­
cent papers (Opie et aI., 1990; Bai ley et aI., ence of a diffuse peak on reverse-phase chro­
1990; Bailey et aI., 1 99 1 ; Bailey et aI., 1 994b) matography (having become a de-facto quali­
and from thesis work (Opie, 1 992). The re­ fier for thearubigens) was most significantly
verse-phase technique seemed to point to the formed during model oxidation of epicatechin
thearubigens in one of two classes: a series alone, and such a fermentation brew has been
of red (-450 nm) compounds eluting dis­ suggested as a strategy for thearubigen iso­
cretely and a diffuse peak appearing as a lation (Opie et aI., 1 995). This approach,
'rising baseline' across the same chromato­ however, seems limited, as epicatechin is a
gram. minor constituent i n tea relative to other
Investigations into the thearubigen frac­ catechins, and to suggest that studies of its
tions identified by early techniques revealed fermentation products will lead to identifi­
that part of the thearubigens was the fla­ cation of thearubigens can only result in a
vonol glycosides (McDowell et aI., 1 990). very minor portion of the said thearubigens
Being present in green tea, these compounds being identified.
are clearly not thearubigens. They contrib­ Model fermentation systems seem to be
ute significantly to the absorbance around the most reasonable approach for determin­
-350 nm in both the solvent partitioning and ing the origin of individual thearubigens as
cartridge column techniques and must be discrete chemical identities. The use of model
excluded from these measurements if the fermentation systems elimi nates some of
term thearubigen is to retain its original the confusion concerning the origin of
meaning, that is, as a product of oxidation of thearubigen-like compounds that may have
green tea polyphenols. been "left over" from the green tea polyp he­
Early model fermentation systems suc­ nols. However, until better techniques for
cessfu l l y identified the paired role of analysis are discovered, little information can
gallocatechins and catechins in theaflavin be gleaned from model fermentation studies.
formation (Sanderson et aI., 1 972). The In addition, model fermentation systems will
model fermentation approach was expanded always suffer the question of whether the
to investigate the role of purified tea PPO model is accurately representing thearubigen
(Coggon et aI., 1 973) and peroxidase (Dix production.
et aI., 1 9 8 1 ) on formation of the theaflavins Use of ultrafi ltration to separate a high­
and thearubigens. After the arrival of HPLC, molecular-weight thearubigen frac tion
this technique was used in the attempt to confirmed the presence of a high-molecular­
justify the individual thearubigin peaks' ori­ weight "polymer", but these high-molecular­
gin (Robertson and Bendall, 1 983; Robertson, weight polymers represent at most only 2%
1 983). Model fermentation of individual of the total brew solids (Kuhr et a!., 1 994).
catechins was performed (Opie et aI., 1 990), The crude h i s torical defi n ition of
and peaks were identified that are potential thearubigens on the basis of poorly resolved
thearubigens, but attention was drawn away paper chromatograms would seem to
from individual analysis and focused on a represent a stumbling block for systematic

441
chemical identification of tea compounds, volved and not a simple, single central struc­
because no one unique chemical structure ture as exhibited by the theaflavins.
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 a. Theafulvins and Theacitrins
"thearubigens" except as a historical artifact
and as a semiquantitative analytical number Early reports suggested that the thearubi­
used by tea tasters. One reason is that if the gens, after aci d hydrol ysis, produced
solvent extraction/colorimetry technique anthocyanidins (Brown et ai., 1 969). This
(Roberts et ai., 1 957) i s used successfully to would lead one to believe that at least some
measure thearubigens, the thearubigens mea­ of the thearubigens are a class of condensed
sured are, i n part, composed of flavonol tannin, possessing linkages at the 4-position,
glycosides (McDowell et ai., 1 990). Thus, which may have benzotropolone units or
flavonol glycosides are i n some sense other chromophores to gi ve the characteristic
thearubigens. This places the definition of dark brown appearance of the thearubigens.
thearubigens as oxidation products of cat­ Recent work on this approach has iso­
echins in an awkward position. lated a fraction believed to be part of the
Some of the color attributed to the thearubigens, termed theafulvin (Bailey et ai.,
thearubigens may in fact not be flavonoid in 1 992). The buf-tan appearance resembled
nature at ali. Figure 1 7 illustrates some pos­ that of the condensed tannins, and the mate­
sible alternative explanations for the brown rials gave simi lar behavior on C- 1 8 chroma­
coloration and "acidity" as described i n the tography when compared with cider and wine
historical reports. They may be highly rear­ proanthocyani dins. However, acid hydroly­
ranged compounds such as catechinic acid sis of the two materials gave widely differ­
[36] (Sears et ai., 1 974). Another explana­ ing results. Condensed tannins gave reason­
tion is that thearubigens are overoxidation able yields of anthocyanidins derived from
products of theaflavins or direct products of the hydrolysis products, a behavior that is
peroxidase, a theory that is well supported typical of polymeric flavan-3-0Is. The
by model studies of PPO and peroxidase. In anthocyanidins are readily identified by their
this respect, they may be ring-opened prod­ bright color and characteristic absorption
u c ts s i m i lar to m uc o n i c acid [ 3 7 , 3 8 ] spectrum and behavior on PRP-phase chro­
(Hayaishi and Hashimoto, 1 950; Speier et ai., matography. The products of hydrolysis of
1 993) or derivatives thereof (Critchlow et ai., the theafulvin fraction, on the other hand,
1 967). The brown color attributed to the yielded similar colored materials, but which
thearubigens may also be attributed in part were unretained by PRP chromatography.
to pheophorb i de ( e . g . , [40]) and the This led the authors to believe that only the
pheophytins (Sanderson, 1972), or polysac­ end groups of the polymer were converted to
charides such as [4] and polymers thereof anthocyanidin and that linkage occurred
(Millin et ai. , 1 969). None of these conjec­ through an alternate location, such as the 3 '­
tures as shown in Figure 1 7 have been i nves­ position (Bailey et ai., I 994a).
tigated. However, the presence of brown­ In another report, Porter's reagents were
colored products that partition into all of the used to achieve hydrolysis of the pro­
phases of solvent extraction, including the anthocyanidin fraction, as well as analyze
aqueous phase, butanol phase, and ethyl ac­ gallate and flavonol content by HPLC
etate phase, seems to require that there be a (Powell et ai., 1 995). Both the theafulvin
number of possible chemical moieties in- fraction, as well as the caffeine-precipitable

442
 HO y
�( O
,
X
x
" '
OH
OH HO

[1 0]: Epicatechi n [36] : Catechinic Acid

(colorless) (brown)

800H
I
OH CH
COOH
COOH -
c
OH � OH
X X 0
X
OH OH
OH

[37] [30] : Theaflavin [38]

(red) (Brown, acidic)
(brown, acidic)

""CH2

"
CHO
[39]: Chlorophyll B (brown, hydrophobic)

;
CHO H3C

HO

CH 3 o

(green)
CH 3
[40]: Pheophorbide

FIGURE 1 7 . Alternative brown pigments.

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

both cases, the proanthocyanidin content of green tea polyphenols (Hashimoto et aI.,
these fractions seemed to be explainable on 1 9 87), and the gallate content of these

443
proanthocyanidins would explain most of extract solids (Graham, 1 984). This simple
the hydrolyzed gallate i n the fractions. This polyphenol is thought to be a product of
result weighs heavily against the possibility degallation of the 3-galloyl substituted cat­
that these fractions are thearubigens derived echins and gallocatechins that are abundant
solely from catechin or theaflavin precur­ in the natural beverage. Although the pro­
sors, unless the chemical characteristics of duction of gallic acid is not well understood,
these "thearubigens" had been transformed either native esterase (tannase) activity or
drastically in chemical nature. oxidative degallation during the fermenta­
It should be noted that the theafulvin tion is a likely pathway to its formation.
fraction, a buff-tan solid, is isolated in ap­ A possible method of investigation of
proximately 3% yield from tea extract sol­ thearubigens is to use the presence of gallated
ids, of which 1 0% is proanthocyanidin in polyphenols to establish mass balance of the
nature and 3% is derived from gallate esters. polyphenolic fraction. Tannase treatment uf
The low color i mpact this fraction is likely both green and black tea produced from the
to have on the overall beverage should be same clone along with measurement of gal­
consi dered as small evidence for support of lic acid content should establish the amount
this fraction as a thearubigen. The thearubi­ of gallic acid oxidized into polymers. Sub­
gens have been suggested to be dark brown tracting the free gallic acid (released in the
and strongly influencing the color of the original black tea extract) and the consumed
beverage, i n addition to being -20% of the gallic acid (computed from the gallic acid
total extractable solids. Also, gallate esters released by tannase treatment) from the gal­
are the predominant form of catechins in lic acid released from tannase treatment, one
green tea, comprising - 1 5 % wtlwt of ex­ can establish the molar concentration of cat­
tract, of which -35% of the mass of these is echins involved in black tea polyphenols.
gallic acid. Therefore, it is reasonable, to By then subtracting the molar quantity of
expect -5% of the mass of black tea extract known gallated pol yphenols such s catechins
to be gallate, as confirmed by tannase hy­ and theaflavins, one can establish a molecu­
drolysis (unpublished results). Therefore, one lar basis for the thearubigens and better hy­
would expect to find much greater than pothesize on the true chemical constitution
-0.3% of the extract from which a thearu­ of the thearubigens. Further, the difference
bigen fraction was derived to be present as in mass balance between recovered gallic
thearubigen gallate esters. Given this criti­ acid and original gal lated species in the green
cism, the theafulvin fraction should not be leaf would establish the amount of gallic
identiied conclusively as a major thearubigen acid polymerized into the "thearubigen
until stronger, direct evidence exists. mass". Good establishment of cycles of mass
Attempts at isolation of the thearubigens balance such as this are notoriously absent
ha ve generated at least two other new frac­ from much of the tea chemistry li terature,
tions: the theacitrins (Powell et aI., 1 994) and this fai lure might explain why true iden­
and a caffeine precipitable thearubigen frac­ tification of the thearubigens has eluded sci­
tion (Powell et aI., 1 993). entific inquiry for so long.

b. Gallic Acid Production c. Bislavanols and Proanthocyanidins

One of the products of the fermentation The bisflavanols, such as bisflavanol A

process is the appearance of gallic acid [ 1 2], [4 1 l , arise from paired condensation of two
constituting approxi mately 1 % wtlwt of the gallocatechins (Vuataz and Brandenberger,

444
1 96 1 ). It was initially expected that these theasinensins. They were found to be present
would be i ntermediates i n the formation of i n green tea leaf (Nonaka et aI., 1 983) and
theaflavins. The extra OH substitution from oolong tea (Hashimoto et aI., 1 988). As well
the use of two gallocatechins, as opposed to as the gallocatech in-gallocatechin dimer
a catechin/gallocatechin pair, provides a products, the theasinensins include catechin­
mechanistic barrier by replacing a hydrogen gall ocate c h i n dimer products such as
atom, which is lost easily i n tautomerization, theasinensin F [42]. The theasinensin family
with an OH group (see Figure 2 1 ). This does is depicted in Figure 1 8.
not rule out the possibility of further rear­ Basic work on the separation, character­
rangement or condensation, possibly to ization, and chemical identification of black
thearubigins. tea constituents has been well advanced in
The bislavanols were later rediscovered recent l iterature. In a massive compendium
and reclassified under the wider name of of work on tannins and related compounds,

d
°H OGallaleOH OH "
�
' I I
HO ° OH ?

H° OH
r°i., ""· : I OH
�
OH OGallaleOH
[41]: Bi sflavanol A, or Theasinensin A

OH
OH
HO
HO q.. ....
�
l ° .. '
I
OH

OH
OGalialeOH OH
�

[42]: Theasinensin F

FIGURE 18. The theasinensisns.

445
best summarized i n brief by one member of rial present i n black tea. They are the prod­
this series (Hashimoto et aI., 1 992), a great uct of the condensation of catechins (which
number of oxidation products from both are the l i ke l y oxi dized species) w i th
oolong and black tea sources have been char­ theogallin and myricitrin, respecti vely.
acterized, as well as novel polyphenols from Erycitrin [45] (Takino and Imagawa, 1 963)
green tea. This approach is immensely use­ is a theaflavonin, the discovery of which
ful, but it is a tedious and expensive route, predates that of the theaflavonins, lending
and i t is anticipated that a good HPLC tech­ futher confusion to the terminology. These
nique that quantitatively establishes a good oxidation products are shown in Figure 1 9 .
mass balance of the unknown tea polyp he­
nols will require many more years of dedi­ OH
cated work unless a woring hypothesis for OH
their formation can be established.
The presence of proanthocyanidins and OH
H
theasinensins in green tea as well as black
tea indicates that great care must be taken to OH
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 HOOe n o
OH

trum, which rules them out as contributors to

mass of the brown thearubigens. OH� OH
OH

d. Mixed Oxidation Products of

[43] : Theogallinin
Po/ypheno/s and Other Compounds

While the most widely known coupled

oxidation products of green tea fermentation
are the theaflavins, resulting exclusively from OH
the reaction of catechins and gallocatechins,
there are other possible oxidation products
OH
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 HO
selectivity for condensation. It is likely there­
fore that other nucleophilic species, includ­
ing active thiols and amine groups, as well
OH 0
as other unoxidized polyphenolic species,
can condense with the a-quinone (Van
Sumere et aI., 1 975). [44]: Theaflavonin, R=Galloyl
Theogallinin [43] and the theaflavonins
[45] : Erycitrin , R=H
such as [44] (Hashimoto et aI., 1 992) repre­
sent two recently identiied species that con­
tribute to the balance of polyphenolic mate- FIGURE 19. Mixed oxidation products.

446
e. Aroma Formation from Po/ypheno/ partial oxidation, rather than total fermenta­
Oxidation tion, occurs. This characterization is also
representative of some Darjeeling teas, whose
The aldehyde aroma components arising oxidase activity is not permitted to come to
in black tea aroma have been attributed to full expression. Gentler oxidation apparently
condensation of corresponding amino acids creates its own unique set of aroma and
with an a-quinone to form an amine-substi­ polyphenolic compounds.
tuted derivative (Bokuchava and Popov, One residual question concerning the
1 954) during polyphenol oxidation. The ci­ mechanism of theaflavin formation is the
tation of this mechanism is i nteresting as concerted oxidation of the catechins. Com­
some products (aldehydes, carbon dioxide, pounds such as the bisflavonols, also known
and ammonia) have been observed (Popov, as theasinensins A-E, represent "dead end"
1 956; Skobeleva and Popov, 1 962). Tea leaf condensation products because they cannot
amine oxi dase produces aldehydes and am­ proceed mechanistically to the theaflavins.
monia as byproducts (Tsushida and Takeo, Figure 2 1 i l lustrates the most plausible
1 985). Detection of these byproducts i n fer­ mechanism of theasinensin and theaflavin
mentation studies does not constitute proof formation. It is likely that the quinone groups
that polyphenol oxidation is involved i n their are present i n low concentrations at any one
production, because these studies do not ex­ given time due to their high reactivity. It
clude the possibility of the presence of amine seems more reasonable that the quinone re­
oxidase as a contaminant. Nonetheless, there acts with an unoxidized polyphenol at low
i s compelling evidence for the role of cat­ quinone concentrations, and is then oxidized
echins in aroma generation in black tea. The further to theaflavin. Theaflavins may be
mechanism also suggests the formation of a formed directly from the quinones provided
nitrogen-substituted catechin [46]. A simi lar they are present in suffi ciently large quanti­
nitrogen-substituted catechin is formed by ties but might be interrupted at an intermedi­
heating epicatechin with alanine i n a model ate stage. Thus, Figure 2 1 also shows how
system produ c i n g 7-C(N-ethy lamino)­ the theasinensins may arise during their for­
epicatechin [47] (Anan et a!., 1 987), which mation.
is a brown compound suggestive of the
thearubigens. This is consistent with obser­
vations of nitrogen incorporation into cat­ a. O% ngtheanin and Theasinensins
echins (Kito et a!., 1 968; Konishi, 1 969).
These studies lead one to believe that a small Oolongtheanin and theasinensins F [42]
fraction of the thearubigen fraction may and G (Hashimoto et a!., 1988) may be unique
possess nitrogen content derived from these products of the oolong tea fermentation
compounds. The ni trogen substituted cat­ system a n d are s h o w n i n F igure 2 2 .
echins are depicted in Figure 20. Theasinensins AlB and DIE are atropisomers
about the biphenyl link of the same conden­
sation products (the gallocatechins). Oolong­
4. Oolong Tea Polyphenols theanin is likely to be a further oxidized
form of one of these theasinensins. Intrigu­
Typically understood as an intermediate ing to note, however, is the detection of
between green and black teas, oolong tea is theasinensins F and G, atropisomers of a
characterized by a much shorter fermenta­ mixed condensation of epicatechin gallate
tion time under gentler conditions such that and epigallocatechin gallate. Such a mixture

447
 �
yi Y
OH

�4
HO O " ,

" HCHRCOOH

OH

[46] : N itrogen Substituted Catechin,

R=derived from amino acid N H2CH RCOOH

[47]: 7 -C(N-ethylamino)-epicatechin

FIGURE 20. Nitrogen-substituted catechins.

is expected to produce theaflavin digallate, diates on the path between green and black
as epicatechin [ ! O] and epigallocatechin [ 1 2] tea, with green representing the unfermented
will produce theaflavin [30]. However, the leaf most closely and black tea representing
gentler oxidation present in oolong tea sys­ either complete or near-complete fermenta­
tems may have permitted the detection of tion.
this intermediate.
In addition to oolongtheanin [55], two
oolonghomobisflavans, and a vitamin C IV. BIOCHEMISTRY OF TEA
derivative of EGCG, 8-ascorbyl EGCG
[56], were also isolated from oolong tea and Tea is the subject of many biochemical
may be unique oolong tea polyphenols investigations because it produces a number
(Hashi moto et aI., 1 989b). It is difficult at of unique natural products. In contrast to the
this time to place oolong tea polyphenols as isolation and quantification of the various
either unique polyphenols of the divergent components of tea, the mechanisms by which
path of oolong tea production or as interme- the tea plant forms these compounds, and

448
 OH o
' OH

PPO
•

:.. " �
'OH " 'OH
OH [1 0]: EC- R=H OH [48]: EC- R=H
[ 1 2]: EGC- R=OH [49]: EGC- R=OH

° c
[4 � ) HO
OH
0

Gy
.
X PPO

OH X OH
.

OH

�OH
[1 2 ]9 OH OH
X OH [50]
Low quinone concentration

a
o
o
[48

�)0
x-� o
HO

�
[49�
X O X
[52]

/
High quinone concentration

OH
HO
[0]
[54]
x
X H H
Thealavin

FIGURE 2 1 . Mechanism of theaflavin formation.

449
 OH
. . ,OH

HO h °

) �
J
H0 0 '" OH

-- I OH
'OGallale
"

OH

[55]: Oolongtheanin

HO

OH
HO _J....

"
. ., Q OH
OH

'OGallale
OH

[56]: 8-C-Ascorbyl Epigallocatechin Gallate

FIGURE 22. Oolong lea polyphenols.

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

450
 NH,

t=
N

"� '�
NH,
�
� 0

,J-)
9, ( 1 ,
N N N"

9,'"·
N H N
Adenine " OH
N
" OH [58) � N O'N
8
N

" ,�
N
· OH

�/
�
" 'OH "N N
O OH
Hypoxanthine "
�
Adenosine

[5
7)
�
' N
I
O""'N
1

Xanthine (60)
N

N
/
�
HO
xanthOSJne (61)

CH3 , 0]

Wf
, ·,OH

� ;o
/N N'
H ) HO
7-Methyl-xanthosine (62)

ol
� CH3

1 I)
H'

�
N
I
O""' N N
H
I

7-Methylxanthine (63)

o�
' �
I I )
C H3 0 CH3

--
H � CH 3-. �
N - N
1 1 )
O"'N N O"'N N
I I
C� C�
Theobromine (2) Cafeine ( 1 )

FtGURE 23. Biosynthesis of caffeine,

B. Theanine Formation glutamic acid. Theanine degradation prod­

ucts may serve as precursors for synthesis of
Theanine [3] (n-ethyl glutamine) i s the catechin A ring, apparently from utiliza­
fonned from the action of thea nine synthetase tion of the N-ethyl group (Kito et a!., 1 968;
[L-Glutamate:Ethylamine ligase] (Sasaoka, Feldheim et a!., 1 986). It is likely that the
1 965; Sasaoka et a!., 1 965) on ethylamine N-ethyl group enters the general glycolyte
derived from alanine (Takeo, 1 974), and pathway, forming the catechin precursor ma-

451
10nyl-CoA. Theanine formation is a light­ 4-hydroxylate or cinnamate 4-monooxy­
dependent process requiring the presence of genase. Hydroxylation is achieved typically
ATP as a cofactor (Sasaoka, 1 965). Theanine by a cytochrome P-450 enzyme activity as­
synthesis and accumulation is inhibited by sociated with the enzyme complex (Heller
production of glutamine (Matsuura and and Forkmann, 1 988), and a light-indepen­
Kakuda, 1 990). dent cinnamate 4-monooxygenase activity
has been reported from tea shoots (Saijo,
1 980). The coumaric acid aromatic ring forms
C. Biochem istry of Flavonoid the 'B' ring of the f1avonoids. Figure 24
Compound Formation il lustrates the biosynthesis of coumaric acid
and the various functions this compound
The pathways for the de novo biosynthe­ performs in the tea plant.
sis of f1avonoids i n both soft and woody The gallic and quinic acids origi nate via
plants have been generally elucidated and the shiki mate/arogenate pathway. The key
reviewed i n detail elsewhere (Jain and Takeo, enzymes in shikimic acid biosynthesis have
1 984; Heller and Forkmann, 1 994). Simi lar been detected in tea (Saijo and Takeo, 1 979).
pathways for biosynthesis are used by a wide Carbohydrates play an important role, pre­
variety of plant species. The regulation and sumably as a precursor to shikimic acid,
control of these pathways in tea and the because radiolabels from both myo-inositol
nature of the enzymes involved in synthesis and glucose are incorporated into catechins
in tea have not been studied exhaustively. (Wang and Huang, 1 9 87). Gallic acid and
The following discussion extracts critical quinic acid play key roles in forming esters
information from the pertinent tea literature with various polyphenols. Gallic acid i s a
and otherwise assumes that flavonoid pro­ key component of tannins and gives the cat­
duction by Camellia sinensis generally fol­ echins their tannin-like qualities.
lows the pathways used in other plant spe­ Light has been shown to strongly induce
cies. biosynthesis of the phenolics by stimulation
of the various enzymes along the biosyn­
thetic path. Photosynthesis is associated with
1. Phenylalanine and the Shikimate production of acetyl-CoA, ATP, and reduc­
Pathway ing agents such as NADPH, all of which
play important roles as various stages of the
Phenylalanine is thought to be the direct biosynthesis (Zaprometov, 1 987). Light is
precursor of polyphenols in tea plants very i mportant for catechin production, and
(Nikolaeva et aI., 1 982); however, tyrosine shaded tea plants produce finished tea
may also participate in polyphenol produc­ products with reduced astringency due to
tion through a l i ght-dependent pathway the lower concentrations of polyphenols
(Zaprometov and Bukhlaeva, 1 97 1 ) . Deami­ (Mahanta and Baruah, 1992).
nation of phenylalanine [64] forms cinnamic Coumaric acid, along with other ci nnanic
acid [65] by the action of phenylalanine acid derivatives and phenylalanine, is also a
ammonia lyase (Iwasa, 1 976; Shiplova and precursor to tea plant lignins (Zagoskina and
Zaprometov, 1 977; Zagoskina et aI., 1990). Zaprometov, 1 976; Strekova et aI., 1980).
Hydroxylation of cinnamic acid forms Lignins are ubiquitous throughout the plant
coumaric acid [66], which is generated from kingdom. Tea lignins are methylated (rein­
an enzyme complex referred to as cinnamate forcing low solubility) by the action of

452
 �COOH
V NH,

[64] : Phenylalanine

l Phenylalanine
ammo nia·lyase

COOH
o
�

:COOH
I
[65]: Cinnamic Acid
! Cinnamate
4-monooygenase
HO �
NL
"

/ [67]: Tyrosi ne
�
v �COOH
J Tyrosine mmonia-Lyase?
HO
[66]: Coumaric Acid

/ \
+ quinic acid

o
to catechins and lavonols
(through navonoid biosynthesis)
to lignins
(through lignin condensation)
Hooc Z�OH
OH-'OH
OH
[22]: Coumaylquinic acid

FIGURE 24. Cinnamate biosynthesis.

methyl transferase enzymes (Nikolaeva and preferred substrate for chain extension in
Zaprometov, 1 990). most plant species, typically formed by a
hydroxycinnamate :CoA-ligase (Heller and
Forkmann, 1 988).
2. Chain Extension, Hydroxylation Malonyl-CoA is produced by carboxyla­
tion of the glycolysis product acetyl-CoA
After formation of the B ring moiety, the via acetyl-CoA carboxylase (Lowenstein,
biosynthesis of catechins proceeds with a 1 9 8 1 ) . A hydroxycinnanic acid-CoA deriva­
series of chain extensions to form the C and tive typically combines with three molecules
A ring backbone. 4-Coumaroyl-CoA is the of malonyl-CoA to form chaIcones via chal-

453
cone synthase (Ebel and Hahlbrock, 1 982). basic structure is tautomerization of the chal­
This enzyme apparently requires no cofac­ cone structure to a flavanone. The enzyme i n
tors and performs the condensation of the this step i s chalcone isomerase, which usu­
malonyl units and the cyclization to form ally bypasses the natural, racemic, anti-ad­
the phloroglucinol ' A ' ring (Heller and dition (Heller and Forkmann, 1 988) to form
Forkmann, 1 994). a characteristic 2S-stereochemistry (Heller
Theanine has also been demonstrated to et aI., 1 979) of the resulting flavone by syn
be a significant factor i n the synthesis of the addition (Ebel and Hahlbrock, 1 982).
phloroglucinol nucleus (Kito et aI., 1 968; Ring closure of chalcones followed by
Feldheim et aI., 1 986). Incorporation of the hydroxylation results i n formation of
ethyl group of theanine into the catechins dihydroflavonols. The flavanone is typically
has been demonstrated by radiolabeling hydroxylated by the enzyme flavanone 3-
ethylamine, a theanine precursor (Sasaoka hydroxylase, an oxoglutarate-dependent
et aI ., 1 962; Kito et aI., 1 968). Ethylamine is dioxygenase (Heller and Forkmann, 1 988).
the best demonstrated substrate of tea amine The formation of the 2R,3R dihydroflavonol
oxidase (Tsushida and Takeo, 1 985), which has been shown to form either the f1avonols
is converted to acetaldehyde. Theanine may or the non-epi flavan-3-ols, whereas it is
be a storage mechanism for this amine, with believed that a competing hydroxylase forms
acetaldehyde being the first intermediate on the 2R, 3S dihydroflavonol [70], which has
the path to catechin B-ring synthesis, fol­ been isolated (Nonaka et aI., 1 987).
lowed by conversion to acetyl-CoA and/or
malonyl-CoA. 4. Flavanols/Flavonols
Camellia sinensis produces flavonoids
in which the tri-hydroxy B ring species pre­ The dihydroflavonols are enzymatically
dominates; however, within the flavonols the transformed to the f1avonols through action
di-hydroxy B ring is more common. It is of a 2-oxoglutarate-dependent dioxygenase
unclear whether 3 ' and/or 5' hydroxylation (Heller and Forkmann, 1 994), forming a
is occurring before or after chalcone syn­ double bond through abstraction of vicinal
thase (Hahlbrock and Grisebach, 1 975). hydrogens on the 2R,3R dihydroflavonols as
Because 3 ',4'-hydroxylated forms of all the proposed for flavone synthase (Britsch,
species can be found throughout, and 4' ­ 1 990). This activity is referred to as flavonol
mono hydroxylated species are found more synthase.
commonly in the earlier branches of biosyn­ Formation of the 2R, 3S dihydroflavonols
thesis, it is possible that tea contains nonspe­ is likely to be preferred in the tea plant, as
cific or numerous 3' -hydroxy lases (Heller formation of the epi-forms of the f1avan-3-
and Forkmann, 1 988), accounting for the ols is the preferred biosynthesis product.
presence of caffeic acids, quercitin, and cat­ However, while the route to formation of the
echin derivatives; a much more specific 3',5'­ f1avonols and non-epi-flavan-3-0Is is well
hydroxylase (Heller and Forkmann, 1 9 88) defined, the formation of the epi-flavan-3-
accounts for the gallocatechins and the ols remains conjecture (Stafford, 1 988). Fig­
smaller quantity of myricetin derivatives. ure 25 illustrates the proposed biosynthetic
pathway from cinnamic acids to flavonols
3. Chalcone/Flavone and flavan-3-ols.
Tautomerization The formation of proanthocyanidin poly­
mers has also been considered, although i t is
Subsequent to formation of the chalcone, unclear if epicatechin [ 1 0] or leucocyanidin
the final step in formation of the catechin [27] is the significant precursor (Nikolaeva

454
 0ySCOA C H, yO i OH
Jx Acelyl CoA CH,
COAS
'
8
CH, CoAS
SCoA

Y
P I
"

1661: Coumaric acid

(as CoA ester)

: Chalcone
ynlha se

"y0H
s

OH
HO OH
Chalcone
isomerase HO yj V O "

7 Y
-

1691: 2R-Flavanone
OH ° [ 68}: chalcone OH °

�OH
F,av none
hydroxylase

Howo 0 .
1 & '.
..

��
'OH
Dihdonavonol
OH ° redtase?
70): 2R.3S-dlhydroflavonol

�OH
·V
������� j �OH
HO yj
Y"'! OH
O ..

[7 l}. fl avan-3.4-diol

Hoyoil · ·0 OH 6H

Y0H
Flavan-3.4-diol
re ductase?

Y" HoYOj . 0
1
OH °
OH
glycosylalion
172): navonol
� 'OH
( 1 7) Kaemplerol glycoside OH 1 1 4) novan-J,ol (alzelechin)

FIGURE 25. Biosynthesis of flavonoids.

et aI., 1 982). Proanthocyanidin polymer for­ 5_ Esterase

mation has been attributed to polyphenol
oxidase and the formation of the thearubigens Shikimic acid has been shown to be a
(Brown et aI., 1 969). Further work is neces­ good precursor for biosynthesis of the gal­
sary to determine the origin of proantho­ late group (Zaprometov, 1 962). Gallic acid,
cyanidin polymers and their role in the tea found principally in its esterified form i n
plant and beverage. green tea, is thought to b e principally syn-

455
thesized through this pathway, although phe­ responsible for polyphenol oxidase activity
nylalanine and the cinnamic acids may also (observed in leaf browning and the forma­
be precursors (Saijo, 1 983). tion of black tea polyphenols) is unclear.
A significant quantity of the small
polyphenols exists as quinic acid esters
(depsides), particularly the depside of gallic 1. Polyphenol Oxidase
acid, theogallin (Stagg and Swaine, 1 97 1 ).
Quinic acid is derived from the shikimic Polyphenol oxidase (EC 1 . 1 4. 1 8 . 1 ;
acid pathway via dehydroquinic acid, an in­ monophenol monooxygenase [tyrosinase] or
termediate on the pathway to shikimic acid EC 1 . 1 0.3.2; o-diphenol: O2 oxidoreductase)
(Sprinson, 1 960; Neish, 1 964). Quinic acid is one of the more important enzymes in­
esters of coumaric and caffeic acid (chloro­ volved with the formation of black tea
genic and caffeoylquinic acid) have also been polyphenols. The enzyme is a metallo pro­
detected i n tea (Sanderson, 1 972). tein, thought to contain a binuclear copper
Gallated f1avan-3-0Is (catechins) are the active site. Polyphenol oxidase (PPO) is an
major flavonoids produced by Camellia oligomeric particulate protein that is thought
sinensis (Sanderson, 1 972). It is of interest to be bound to the plant membranes. The
that Camellia sinensis forms gallated esters bound form of the enzyme is latent and
rather than sugar esters, or glycosides, of activation i s likely to be dependent on
catechins. Glycosides are the typical plant solubilization of the protein (Tolbert, 1 973).
strategy for rendering the major f1avonoids PPO is distributed throughout the plant
water soluble (Heller and Forkmann, 1 994), (Durmishidzern and Puridze, 1 980) and has
although proanthocyanidin and catechin gly­ been identified and analyzed in the floral
cosides, while not rare, are not known to be organs (Singh and Ravindranath, 1 994). PPO
common (Achmadi et aI., 1 994). It is likely is localized within plant cells i n the mito­
that the catechins are acylated with an acti­ chondria (Bokuchava et aI., 1 970), the chlo­
vated form of gallic acid, presumably galloyl­ roplasts (Roberts, 1 94 1 ), and the peroxisomes
CoA, similar to the aromatic acylations oc­ (Kato et aI., 1 976). Using antibody tech­
curring with cinnamic acid acyltransferases niques, polyphenol oxidase activity has also
in other systems (Kamsteeg et aI., 1 980; been localized in the epidermis palisade cells
Heller and Forkmann, 1 988). Gallic acid es­ (Wickremasinghe et aI., 1 967). Recent re­
teriication is thought to be a slow process views on the subject of PPO are available
compared with its biosynthesis (Zaprometov (Zawistowski et aI., 1 99 1 ; Whi taker, 1 994;
and Bukhlaeva, 1963) and has been demon­ Steffens et aI., 1 994).
strated through tracer experiments in tea The biological role of PPO in plants is
shoots (Saijo, 1 983). thought to be associated with a plant defense
mechanism and root development. PPO be­
comes activated and available when plant
D. Latent Enzyme Activity and the tissue is damaged due to injury or infection,
Formation of Black Tea catalyzing the formation of insoluble phe­
nolic polymers that aid in wound healing
Conversion of tyrosine and related and help prevent the spread of i nfection
monophenols to diphenolic compounds is (Zawistowski et aI., 1 99 1 ). In tea, key reac­
typically accomplished by tyrosinase or tions occurring during "fermentation" are
monophenol monooxidase. Whether tyrosi­ catalyzed by PPO. These reactions are initi­
nase activity or a related enzyme acti vity is ated by crushing and/or tearing withered tea

456
leaves and lead to the formation of black tea of I g to 20 kDa. Mushroom tyrosinase has
polyphenols and aroma compounds charac­ been found to have a quarternary structure
teristic of black tea. comprised of two different subunits of 43
Polyphenol oxidase catalyzes two gen­ kDa and 1 3.5 kDa, with a total molecular
eral reactions: the hydroxylation of mono­ weight of 1 20 kDa and the formula ,H2
phenols to o-diphenols, and the oxidation of (Zawistowski et aI., 1 9 9 1 ) . These facts would
diphenols via dehydrogenation to o-quino­ suggest that the PPO from tea is also com­
nes, both 2-electron transfer reactions. The prised of four protein subunits and therefore
formation of o-quinones is then followed by has a possible molecular weight of - 1 44
condensation to form a wide range of com­ kDa. This is consistent with a report of
plex products (Mayer and Harel, 1 99 1 ). The isoforms of 1 17, 56, 4 1 .5 , and 35 kDa (Buzun
copper cofactor associated with the metallo et aI., 1 974) and another report with isoforms
protein is the essential component involved al 1 1 8 and 4 1 kDa (Durmishidzern and
with electron transfer and oxidation of the Puridze, 1 980). The copper content has been
substrate. The reaction mechanisms associ­ measured at 0.26 wt/wt%, or about 4 to 5
ated with PPO of Camellia sinensis have not copper atoms for the complex, which i s con­
been determined; however, the reaction sistent with other literature (Vamos-Vigyazo,
mechanisms of PPO from other plants have 1 98 1 ; Interesse et aI., 1 9 83; Zawistowski
been characterized (Zawistowski et aI., 1 99 1 ; et aI., 1 99 1 ) . This number is probably low
Whitaker, 1 994; Steffens et aI., 1 994). PPO because copper diffuses readily away from
activity i s dependent on oxygen concentra­ the enzyme, resulting in a loss of activity
tion (Robertson, 1 983). The o-quinone spe­ (Mayer, 1 987; Zawistowski et aI., 1 99 1 ) .
cies from tea has been looked for in a model Consistent with observed sigmoidal activity
system (Korver et aI., 1 973). (Pruidze, 1 975), multiple active sites in the
Polyphenol oxidase is diffi cult to purify, enzyme complex are likely to exist.
due in part to the presence of polyphenols Polyphenol oxidases act on a broad range
that have a strong affinity for proteins. Work of phenolic substrates with both mono-,
on isolation and characterization of PPO has di-, and trihydroxy substitutions. There i s
been reviewed (Mayer, 1 987). The enzyme evidence that PPOs from different plant spe­
appears to exist in multiple forms and is cies have preferential substrates (Whi taker,
comprised of a number of subunit proteins. 1 994; Steffens et aI., 1 994). Studies of PPO
Reports on the molecular weight of PPO in from tea have mainly been based on model
plants vary widely. The early work on isola­ fermentation studies that utilize purified or
tion of PPO from green tea leaves reported a semipurified substrates (Dix et aI., 1 98 1 ;
molecular weight in the range of 1 30 to 1 60 Robertson and Bendall, 1 98 3 ; Opie et aI.,
kDa (Gregory and Bendall, 1 966). Reports 1 990; Finger, 1 994). It is clear from these
on isolation of PPO from other plant species reports that PPO from tea reacts effecti vely
using modern biochemical methods state with both di- and tri-hydroxylated catechins,
molecular weights ranging from 40 to reacting with specificity for the o-dihydroxy
72 kDa, whereas the primary isoform oftypi­ moiety (Gregory and Bendall, 1 966; Coggon
cal PPO has a molecular weight of 45 kDa et aI., 1 973). Studies that define the kinet­
(Steffens et aI., 1 994). The molecular size of ics of PPO from tea in relation to substrate
PPO isoforms based on cloned genes indi­ type are needed to better define the enzy­
cates that PPO has a molecular weight of mology of tea PPO. PPO can be inhibited
between 57 and 65 kDa and includes puni­ by a range of chemical constituents that
tive transit peptides of a molecular weight include cyanide, carbon monoxide, EDTA,

457
sulfites, ascorbic acid, erythrobic acid, and Model system studies using peroxidase
thiol-containing compounds (Zawistowski and PPO for production of black tea polyphe­
et aI., 1 99 1 ) . In general, compounds that nols suggest that peroxidase activity is re­
block oxygen, act as antioxidants, and/or sponsible not only for decomposition of
bind copper are good inhibitors of PPO. theaflavins but the formation of thearubigens
The effect of natural flavonoids and end as well (Dix et aI., 1 9 8 1 ) . Reactions of
products of fermentation as inhib itors of epicatechin and PPO have been proposed as
tea PPO remains to be defined, although a model for thearubigen formation (Opie
there i s e v i dence of theaflavins and et aI . , 1 993) and epicatechin oxidation prod­
thearubigens as feedback inhibitors (Pruidze ucts by this system have been suggested as a
and Grigorashvili, 1 975). Most PPO en­ good model of thearubigens (Opie et aI.,
zymes have a pH optimum in the range of 1 995). This is likely due to the fact that the
5.0 to 7.0. Typical solutions of tea polyphe­ isolation procedure used for purification of
nols have a pH of approximately 4.5, and PPO (Opie et aI., 1 990) was found to contain
increasing the pH of tea fermentation reac­ peroxidase activity in an earlier report
tions leads to stronger PPO activity. Tea (Robertson and Bendall, 1 9 83). Model fer­
PPO has an observed optimum pH in the mentations using PPO and peroxidase, both
range 4.6 to 5.6 (Takeo, 1 965; Takeo and separately and in conjunction, show the
Uritani, 1 966; Gregory and Bendall, 1966; coupled nature of these enzymes (Finger,
Perera and Wickremasinghe, 1 972; Coggon 1 994) and point to the need for further inves­
et aI., 1 975; Robertson, 1 983). tigation of the role of both of these enzymes
i n the development of the unique black tea
polyphenols and other compounds present
2. Peroxidase in black tea extracts.

Peroxidase (EC 1 . 1 1 . 1 .7) is thought to

play an integral role in the fermentation pro­ E. Extracellular Enzymatic Activity
cess and i s found i n fresh green leaf
(Bokuchava and Popov, 1 948; Gregory and Many tea products, such as Kombu-cha
Bendall, 1 966; Bokuchava and Skobeleva, or Pu-Erh tea, take advantage of enzymatic
1 969). Coupled with PPO, which is thought systems external to the tea plant. In the case
to produce the peroxide, an acti vator of this of Kombu-cha, sweetened tea brew is seeded
enzyme system in other systems (Jiang and with a yeast that produces ethanol and acetic
Miles, 1993), peroxidase is also thought to acid. The yeast generates a mushroom-like
play a role in the oxidation and formation of mass that is typically reused as the seed for
the black tea compounds. future beverage production. Pu-Erh tea is
Peroxidase is a haem-based enzyme that produced by burying green tea leaves in a
catalyzes the reductive decomposition of confined space, thereby allowing microbio­
hydrogen peroxide to water and organic per­ logical fermentation to blacken the leaves
oxide species to the corresponding alcohol. (Shao et aI., 1995). Pu-Erh is thought to pos­
In contrast to superoxide dismutase, whose sess many health benefits and is often com­
activity is cycled by coupled oxidation and pressed into bricks and called Tuo-cha tea.
reduction of peroxide to oxygen and water, Tea, because of its unique phytochemi­
respectively, peroxidase cycles its activity cal composition, offers many opportunities
through oxidation of a wide variety of sub­ for study of exogenous enzyme systems. One
strates. enzyme that has gained a reputation in the

458
field of tea chemistry is tannase. Tannase Tea cream can be separated from the
enzymes (Gallic acid esterase, Tannin acyl brew most conveniently by centrifugation of
hydrolase, EC 3. I . I .20) such as those iso­ a concentrated beverage (Smith, 1 968).
lated from fermentation broths of Aspergil­ Tea cream was demonstrated by early
lus Flavus, A. Niger, or A. Oyzae represent researchers to contain caffeine, theaflavin,
the most significant of these extracellular and thearubigens, as well as traces of a
enzymes. Tannase is significant in terms of number of other substances (Roberts, 1 963;
its demonstrated effect of hydrolyzing the Smith, 1 968). Studies on cream composi­
gallic acid group from tea polyphenols (Deijs tion (Nagalakshmi and Seshadri, 1 983)
and Dijkman, 1 936; Roberts and Wood, and decreaming (Nagalakshmi et ai., 1 985)
1 95 1 ; Roberts and Myers, 1 959) and its ben­ show that protein and caffeine contribute
eficial impact on manufacture of cold water significantly to cream formation. Addition­
soluble instant teas (Coggun et ai., 1 975). al contributions from lipids in tea such as
The use of tannase significantly inhibits the triacontanol and spi nasterol have been
ability of tea polyphenols to complex and shown (Seshadri and Dhanaraj, 1 988). Simple
precipi tate, and reduces the ability of black carbohydrates have been shown to assist i n
tea to form tea cream (Nagalakshmi et ai., solubilization of cream (Nagalakshmi et ai.,
1 985). Tannase activity and thermal stability 1984) but do not appear to complex signifi­
in the presence of tea extracts has been de­ cantly with polyphenols in model studies
termined (Thomas and Murtagh, 1 985). (Williamson et ai., 1 995). High-molecular­
weight thearubigens have been associated
with cream formati on (Hazarika et ai., 1 984).
V. CHEMICAL PROPERTIES OF TEA HPLC analysis of the phenolic portion of tea
COMPOUNDS cream (Powell et ai., 1 993) revealed 86%
'thcarubigens', 1 2% theafluvins, and 2% fla­
Beyond the i nherent ecological proper­ vonol glycosides.
ties of tea compounds i n the tea plant, tea Whi l e the polyphenolic constituents'
phytochemicals exhibit unusual properties role i n cream formation i s well under­
that make them interesting subjects for stood, the presence of proteins/peptides,
chemical study. carbohydrates, leaf fines (fragments of
leaves), and other materials needs to be
studied further to ful l y determine the
A. Formation of Cream and Haze i n m e c h a n i s m b e h i n d cream form ati o n .
Black Tea Analysis of the p H optimum for cream
formation (Smith, 1 968), which closely
The onset of cooling in a freshly brewed mi rrors tannin-BSA pH curves (Hagerman
cup of black tea is accompanied by produc­ and Klucher, 1 986), combined with knowl­
tion of a dark red/brown cloud. This cloud is edge of the significant contribution of pro­
particularly notable in concentrated brews tein to the mass of cream (Nagalakshmi
of tea, where the beverage moves from a and Seshadri, 1 9 83), provides strong justi­
dark brown to a milky-red color. This appar­ fication for better analysis of the contribu­
ent 'lightening' in the concentrated solution tion of protein to cream formation.
resembles the milky-red color of tea to which The study of the physicochemical prop­
cream has been added and thus was dubbed erties of cream is reviewed and expanded
'tea cream' (Bradfield and Penney, 1 944; significantly in thesis work (Rutter, 1 97 1 )
Roberts, 1 963; Smith, 1 968). and in the literature (Rutter and Stainsby,

459
 1 975; Bee et aI., 1 987). Tea cream has been with caffeine (McManus et aI., 198 I ; Gaffney
shown to be comprised of ultra fine spherical et aI., 1 986; Martin et aI., 1 986), and of caf­
colloidal droplets that are clearly visualized feine with I-tryptophan (Nishijo et aI., 1 990).
in a range of concentrations of tea in water, Hydrogen bonding also plays a prominent
increasing i n size with increasing concentra­ role (Van Sumere et aI., 1 975; Borazan and
tion. At very high concentrations, the drop­ AI-Ani, 1 980). Although a full thermody­
lets begin to aggregate further, forming ir­ namic treatment of the i ssue is beyond the
regular shapes. The colloidal, spherical nature scope of this review, it is hoped that further
of the tea cream particles distinguishes this investigation of the following issues will lead
material from typical "crystalline" precipi­ to a better understanding of the mechanism
tates and is referred to as a coacervate (Bee of cream formation.
et aI., 1 987). Tea cream formation has been Polyphenols demonstrate hydrophobic
interpreted i n terms of phase separation and character by partitioning into the cyclodextrin
is likened to two liquids miscible at high cavity (Spencer et aI., 1 988; Cai et aI., 1 990)
temperatures but immiscible when cooled and by their octanol-water partition coeffi­
(Harbron et aI., 1 989). cient (Martin et aI ., 1 990). In addition to
hydrophobic stacking, there is evidence for
a coplanar-type hydrogen bond (Martin et aI.,
B. Complex Formation 1986). There is an apparent correlation be­
tween hydrophobicity and complexation for
The onset of cream formation in tea is hydrolyzable tannins such as pentagalloyl­
driven by complexation of the black tea glucose and vescalagin (Haslam, 1 993).
polyphenols. Green tea contains simple Hydrophobic association has also been used
polyphenols when compared with the com­ to explain copigmentation effects observed
plex polyphenols present in black tea. Green in solutions of anthocyanins and polyphe­
tea exhibits formation of a haze but does not nols (Mistry et aI., 1 99 I ; Liao et aI., 1992)
' cream' to the same extent as black tea. or xanth y l i u m dyes w i t h caffe i n e or
Polyphenols have been observed to have a cyclodextrins (Dangles and Brouillard, 1 993).
strong precipitating effect on proteins, dena­ Polyphenols bind significantly to proline­
turing and reducing enzymatic activity rich proteins, and this complexation i s
(Sekiya et aI., 1 984; Ozawa et aI., 1 987). thought to drive taste perception and differ­
Immobilized polyphenols bind proteins re­ ences among various methods of tea bever­
versibly, with restoration of enzyme activity age consumption (Luck et aI., 1 994).
after elution (Oh et aI., 1 985). Dissolved ash Closer inspection of data on condensed
metals such as hard water calcium may in­ tannins (Martin et aI., 1 990) reveals interest­
fluence complexation, as in the case of tea ing correlations. To illustrate, an i ncrease of
scum formation (Spiro and Jaganyi, 1994). one hydroxyl group decreases apparent hy­
The mechanism of complex formation is drophobicity yet i ncreases association with
a subject of some dispute in the literature. It caffeine for epicatechin/epigallocatechin,
has been asserted that polyphenols form a catechin/gallocatechin, and catechin gall ate/
"hydrophobic bond" between the flat aro­ epigallocatechin gallate pairs. Despite their
matic surface of the polyphenol rings and hydrophilic nature and twisted structure,
quasi-planar hydrophobic groups such as procyanidins show simi lar complexation with
proline i n pep tides and proteins. Evidence caffeine. The additional hydroxy group may
for this assertion is deri ved from crystal struc­ increase the hydrogen-bonding character,
tures of co-crystallized model polyphenols thereby inducing greater association with

460
caffeine. Those polyphenols should also ex­ protein complexation, precipitation, and
hibit less hydrophobicity because the extra cream formation. A simpl ified model for the
hydroxy group should prefer a hydrophilic complexation of polyphenols and caffeine
environment. Hydrogen bonding has been (Figure 26) demonstrates that association of
demonstrated to be a strong driving force for the two hydrophobic surfaces results in a net
association, particularly i n systems such as decrease of hydrophobicity because the
cyanuric acid with melamine (Mathias et aI., polyphenols have a hydrophobic core sur­
1 993) and p-cresol with caffeine (Borazan rounded at its edge by hydrophilic groups.
and AI An i , 1 980). The hydrogen-bonding
- A l l (or most) of the hydrophilic surface is
character of catechin/polyproline association available for solvation, which should result
has been demonstrated in model systems (Sun in a soluble, not precipitated, complex. This
and Mattice, 1996). mi rrors the formation of micelles, which are
Simple hydrophobic association would commonly understuud tu furm a dispersed
seem to be insufficient for explanation of '·pseudophase". It also mirrors the observed

"
h;dmphobi, '"""/
association of two

key: '" results in form ing a

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

J
hydrogen
bonding
However, hydrogen bonding reduces
suface
hydrophilic su rface area, which should
drive separation of 'cream' phase

1· Cafeine o1 Caffeine O
1POIYPhenoj 1poIYPhenolo

FIGURE 26. The hydrophobic association model.

461
self-stacking of cafeine aggregates (Thakkar manner of phase separation. Proteins are most
et aI., 1970; Shestopa et aI., 1985), which easily precipitable at their isoelectric point
attempt to compensate for unfavorable hy­ (Oh and Hoff, 1 987), when the ionic charge
drophobic association of the plane by aggre­ on a protein is lowest and the least number
gating in planar stacks of higher solubility. of 'water-solu bilizing' ionic factors are
Cream formation, however, requires the for­ present. The loose backbone of a protein can
mation of a di screte precipitate analogous to hydrogen-bond with the polyphenol, particu­
phase separation. larly at secondary ami des such as those found
Existence of hydrophobic bonds (Xianqi in proline residues, because secondary amides
and Haslam, 1 994) is supported only by cir­ have been shown to form stronger hydrogen
cumstantial evidence. Hydrophobicity i s bonds than primary ami des (Cannon, 1 955;
driven by the loss of mutual electrostatic Hagerman and Butler, 1 98 1 ). After binding,
stabilization of water molecules (or similar there is a reduction in hydrophilic surface
hydrophilic groups). Hydrophilic groups are area, and the hydrophobic sidechains are
forced to form ordered cages around a hy­ reoriented outward into aqueous solution
drophobic surface, placing the system at an resulting in unfavorable interactions and pre­
entropic disadvantage. To overcome this dis­ cipitation. For larger proteins, binding of a
advantage, the hydrophobic groups aggre­ polyphenol might occur as the hydrophobic
gate and separate from the hydrophilic sur­ core of the protein associates with the hy­
face in an attempt to minimize the surface drophobic surface of the polyphenol. The
area of this ordered cage. The hydrophobic strong hydrogen bonding interactions will
surfaces are held together very loosely by then cause hydrophilic groups to turn in­
van der Waals' forces. Aromatic surfaces ward, denaturing the protein and causing
have also been described as hydrophobic, precipitation. Hydrophobic effects must also
but the mode of hydrophobicity uf aromatic play an important role because detergents
molecules is apparently very different than such as �-octyl-D-glucopyranoside can be
that of aliphatic hydrophobic surfaces used to solubilize the precipitated polyphe­
(Makhatadze and Privalov, 1 994). In addi­ nol complex (Martin et aI., 1 990). Surfac­
tion, aromatic and other unsaturated mol­ tants are known to form ordered layers at
ecules can form hydrogen bonds with the It­ liquid-solid interfaces (Bigelow et aI., 1 946),
electrons, stabilizing the aromatic group in and the increase in entropy associated with
the presence of water (Rzepa et aI., 1 994; forming ordered monolayers may represent
Ernst et aI., 1995). There are also indications a driving force for reversal of the hydrogen
that aromatic stacking interactions in aque­ bond complex and aggregate association,
ous systems have little to do with dispersion resulting in increased water solubility of the
forces or hydrophobic interaction (Newcomb polyphenols and their complexes.
and Gellman, 1 994). Within this model, hydrophobic associa­
A more satisfying model is one in which tion and hydrogen bonding interactions are
hydrogen bonding of polyphenols to caf­ given similar importance, as both are neces­
feine (or hydrophobically associated yet sary for driving the complex interactions
soluble complexes to one another) causes a i nvolved in the model. That the proposed
net reduction of hydrophilic surface area with model does not currently explain apparent
little concomitant reduction of hydrophobic di fferences between the condensed and hy­
surface area. This places the complex at an dro l yzable t a n n i n s i s one l i m i tation.
electrostatic and entropic disadvantage, caus­ Vescalagin, castagalin, and pentagalloyl­
i n g aggregation and precipitation in the glucose have simi lar hydrogen bonding sites,

462
although pentagalloylglucose precipitates systems. Green tea polyphenols have been
proteins much more readily (Haslam, 1 993). shown to be more effective than traditional
This may be related to steric restraints, but antioxidants such as BHA, BHT, ascorbic
different tannins can have different mecha­ acid, and vitamin E (Tanizawa et aI., 1 983;
nisms of precipitation and thus no single Tanizawa et aI., 1 984; Namiki and Ozake,
model should be force-fitted to explain all 1 986; Zhao et aI., 1 989).
polyphenol interactions, unless the general­
ity can be rigorously proven and the excep­
tions well defined. 1. Chemical Antioxidant Systems

The effectiveness of purified tea polyphe­

C. Polyphenols as Antioxidants nols and crude tea extracts as antioxidants
against the autooxidation of fats has been
Research has progressed extensively on studied using the Rancimat system. I n one
the chemical and biological properties and study, purified green tea catechins were
functionality of tea polyphenols. Charcter­ evaluated against dl-a-tocopherol and BHA
ization of the antioxidant properties of tea (Ho et aI., 1 992). On a molar basis, the anti­
flavonoids is a prime example of the newer oxidant activity of the catechins was ranked
research inititative. in the following ascending order: EC <ECG
There are numerous synthetic and natu­ <EGC <EGCG. Each of the catechins was
ral compounds that block oxidative reac­ more effective than either dl-a-tocopherol
tions in vivo and in vitro by quenching free or BHA. A positive, synergistic effect be­
radicals or by preventing free radical forma­ tween the catechins and ascorbic acid or dl­
tion. These compounds have been termed a-tocopherol has also been found (Matsuzake
"antioxidants" (Namiki, 1 990; Pokorny, and Hara, 1985). Purified catechins and crude
1 992). Vitamins A, E, and C and the mineral methanol extracts of green, oolong, and black
selenium are common antioxidants occur­ teas have been shown to have significant
ring in our foods (Pokorny, 1 992), and BHA antioxidant activity. Gallic acid was found
(butylated hydroxyanisole), BHT (butylated to be a more effective antioxidant than any
hydroxy toluene), and propyl gallate are syn­ of the tea extracts or purified compounds,
thetic antioxidants approved for food use suggesting that the gallate moeties of the tea
(Gates, 1 9 87). A broad range of flavonoid or polyphenols are an important part of their
phenolic compounds have been found to have expressed antioxidant acitivity (Ho et aI.,
antioxidant activity (Ho, 1 992). Polyphenols, 1 992). Antioxidant activity of extracts of a
both synthetic and from numerous plant variety of green teas and blends of green and
sources, have also been found to be func­ black tea has been evaluated. A direct corre­
tional antioxidants in numerous test systems lation between the antioxidant index of a tea
(Ho, 1 992; Lunder, 1 992; asawa, 1 992). extract and the concentration of EGCG in
Antioxidants are the control agents for regu­ the extract was shown (Lunder, 1 992).
lation of oxidative reactions. An alternate method to the rancimat,
The antioxidant activity of tea extracts based on measuring the oxidation of linoleic
and tea polyphenols has been studied in a acid catalyzed by bubbling air (2 h incuba­
variety of model systems. It is clear that the tion at 60°C with 500 ml/min air flow rate)
polyphenols from green and black tea are and determining the peroxide value (POV)
effective antioxidants when evaluated i n both or the thiobarbituric acid value (TBA V) has
chemically and biochemically based test been used to study tea flavonoids (Tanizawa

463
et aI., 1 983; Tanizawa et aI., 1 984). Epi­ cals and monitor their formation. These stud­
catechin, green tea extract III (n-butanol ies demonstrate tea constitutents as good
phase) and green tea extract IV (acetone radical scavengers, suggesting that tea
soluble portion of extract III) were found to polyphenols can act as antioxidants in termi­
have strong antioxidant activity equal to the nation of both the inititation and chain termi­
activity of BHA and were significantly more nation stages of lipid peroxidation.
potent than vitamin E. A similar model sys­ A classic method of generating hydroxy
tem that involves the air oxidation of linoleic radicals is the Fenton reaction, a reaction of
acid i ncorporated into cetyl trimethyl­ iron salts and hydrogen peroxide. Studies to
ammonium bromide (CTAB) micelles has determine the effect of polyphenols in scav­
also been used to study the antioxidant activ­ enging hydroxy radicals generated by the
ity of flavonoids (Wang and Zheng, 1 992). Fenton reaction resulted i n the following
The micellar system is a better model for ranking of antioxidant potency: curcumin
food emulsions and biological membrane (69% inhibition), vitamin C (56% inhibi­
systems, and micellar studies showed that tion), vitamin E (35% inhibition), rosemary
flavonoids were active as chain-termination extract ( 1 6% inhibition), green tea polyp he­
antioxidants by reacting with peroxy radi­ nol ( 1 2% inhibition), and so-called "green
cals. The flavonoids had the following de­ tea fraction 6" ( 1 3 % inhibition) (Zhao et aI.,
scending order of reactivity: a-tocopherol 1 989). Caffeine was also found to be an
>morin >rutin >quercetin. There i s a clear effective scavenger of hydroxy radicals gen­
difference between prevention of oxidation erated by the Fenton reaction, but its effec­
of free linoleic acid (the crude extracts of tive concentration was three orders of
flavonoids were much more active than vita­ magnitide higher than of green tea catech ins
min E) and linoleic acid in micelles (fla­ or vitamins (Shi and Dalal, 199 1 ) .
vonoids were less active than vitamin E). It The key active sites of flavonoids for
is likely that vitamin E i s readily incorpo­ scavenging of free radicals and for antioxi­
rated into the micelles, which assures close dant activity are the o-dihydroxy structure in
proximity of the antioxidant to the lipids. the B ring, the 2,3-double bond in conjuga­
Spin-trapping methods using ESR have tion with the 4-oxo function in the C ring in
been employed to evaluate the ability of green flavonols, and the 3- and 5-hydroxy groups
teas and other natural compounds to inter­ with the 4-oxo function in the A and C rings
cept and measure free radicals. Free radicals (Nakayama et a I . , 1 9 9 3 ; Pratt, 1 99 2 ;
were generated by numerous sytems, includ­ Jovanovic e t aI., 1 994). Catechins can react
ing stimulated polymorphonuclear leukocytes with more than one free radical to form
(Zhao et aI., 1 989); hydroxy radicals pro­ quinones (Zhao et aI., 1992) and have a
duced in reaction mixture containing hydro­ lower reduction potentials than vitamin E
gen peroxide and Fe++ (from ferrolls ammo­ (Jovanovic et al.. 1 995). This should account
nium sulfate) (Zhao et aI., 1 989; Shi and for the stronger radical-scavenging activity
Dalal, 1 99 1 ; Uchida et aI., 1 987); a reaction of catechins. Tannins with a greater degree
mixture of NADPH, potassium dichromate, of gallation were more effective i n trapping
and hydrogen peroxide (Shi and Dalal, 1 99 1 ); radicals on a molar basis than EGCG, sug­
an irradiated riboflavin system (Zhao et aI., gesting that theaflavin digallate and gallated
1 989); and superoxide generated by the hy­ thearubigens from black tea could also be
poxanthine-xanthine oxidase system (Uchida effective radical scavengers. Future research
et a I . , 1 9 8 7 ) . D M PO (5 ,6-dimethy l- l ­ i s needed to understand the potential health­
pyrroline-N-oxide) was used to trap free radi- promoting properties of antioxidants, and

464
better characterization and understanding of ger radical scavenging activity than vitamin
the polyphenols i n both green and black tea C, vitamin E, rosemary extract, or curcumin
extracts i s needed. (Zhao et aI., 1 989).
Gallic acid and gallic acid esters have
been shown to have both antioxidant and
2. Biological Antioxidant Models prooxidant activity in vitro (Aruoma et aI.,
1 993; Cao et aI., 1 996). Noticeable oxida­
Biologically based model systems for tion of deoxyribose was observed with ex­
the evaluation of antioxidants have been em­ cess concentrations of gallic acid, suggest­
ployed with tea extracts and polyphenols. ing that gallic acid may act as a prooxidant
These model systems mimic the reactions and play a role in prooxidant activity in green
believed to be linked to the pathogenesis of tea.
some chronic diseases. The cytotoxic effects of reactive oxygen
Catechin was found to act as an effective species (0,· and H,O,) on cells grown in
antioxidant in an iron·loaded rat hepatocyte culture was used as a model system for the
system and was proposed to function through evaluation of the antioxidant activity of f1a­
an ion chelation mechanism (Morel et aI., vonoids (Nakayama et aI., 1 993; Ruch et aI.,
1993). However, other studies using catechin 1 989). Chinese hamster lung fibroblast V79
as an antioxidant have suggested that its cells in culture were incubated in the pres­
antioxidant activity is due to a free radical­ ence of f1avonoids for 4 h to allow sufficient
scavenging mechanism (Fraga et aI., 1 9 87; time for binding of the polyphenols to the
Ratty and Das, 1 988). It is likely that both cell surface or cellular absorption after which
properties of tea polyphenols contribute to the cells were washed to remove free f1a­
their antioxidant activity. vonoids. The washed cells were then ex­
An erythrocyte membrane ghost system posed to reactive oxygen species (either H,O,
and a rat liver microsome system were used or superoxide produced enzymatically using
to study dietary antioxidants and oxidation a combination of hypoxanthine and xanthine
processes (Namiki and Ozake, 1 986). The oxidase). In a cytotoxicity test of the f1a­
catechins from tea were found to be very vonoids, catechin and taxifolin were not toxic
active i n both systems, with EGCG and ECG at concentrations up to 200 and 1 000 �M,
having the greatest activity in blocking lipid respectively, while quercetin and kaempferol
oxidation. These catechins were 1 0 times were toxic at concentrations above 1 00 �M.
more effective than vitamin E, but were less Quercetin and kaempferol were effective i n
active than BHA. EGCG and ECG were ef­ protecting cells from both superoxide and
fective against peroxidation initiated by ei­ peroxide at concentration of 5 � and 20
ther the chemotherapeutic agent adriamycin �M, while catechin was effective at concen­
or the perferyl ion, while EGC was effective trations of 500 �M and 1000 �M (Nakayama
only against perferyl-ion induced oxidation et ai., 1 993). A similar model system used
(Osawa et aI., 1 992). This suggests that cultured mouse hepatocytes rather than ham­
EGCG and ECG were effective as both radi­ ster lung fibroblasts (Ruch et aI., 1989). These
cal scavengers and chel ators, while EGC studies showed that a green tea polyphenol
appears to be a weak radical scavenger but a fraction (GTP) was effective in scavenging
good chelator. In the stimulated polymor­ H,O, and superoxide radicals by demonstrat­
phonuclear leukocyte system, a green tea ing the ability of GTP to inhibit cell death
polyphenol fraction and a green tea extract induced by oxygen radicals and peroxides
fraction called 6 (undefined) had much stron- produced by glucose oxidase or paraquat i n

465
a dose-dependent manner. An in vivo ex­ techniques and approaches of each group of
periment was conducted to determine the researchers. In order to understand much of
effect of green tea on radiation-induced lipid the language and notation used in the current
peroxidation and lethality (Uchida et aI., literature , it becomes necessary to context­
1 992). Epigallocatechin gallate (EGCG) was ualize the development of these terms and
provided to mice in drinking water for I theories, and, in effect, deconstruct the sci­
month at 0.002% or 0.0 I % concentration ence.
providing an EGCG intake of 3 or 15 mglkgl The application of HPLC in tea research
d, respectively. The mice were then irrad i­ represents a good example of this: as a new
ated with 954 cGy X-ray and killed 3 d later. technology, it offered a radical new way of
Oral consumption of EGCG inhibited irra­ looking analytically and quantitatively at tea
diation-induced formation of hepatic perox­ components. Yet, the diffuse understanding
ides i n a dose-dependant manner, reducing of the compounds i n tea, combined with
the formation of peroxides 75% and 90% for qualitative descriptors historically available,
the 0.002% and 0.0 I % doses, respectively. has provided a challenging barrier to sur­
Oral consumption of EGCG was also fou nd mount. A thearubigen i s very clearly assumed
to si gnificantly extend the lifespan of mice to be a complex mixture of polymerized
given a lethal dose of radiation. These re­ polyphenols that appears as a diffuse spot on
sults support the conclusion that tea catechins paper chromatography. The identity and
are effective scavengers of free radicals in location of that spot becomes unclear
vivo. when using a new and different technology.
Tea extracts, tea polyphenol fractions, Without a defi nitive pure standard of a
and purified catechins have been shown to thearubigen, only a qualitative assessment
be effective antioxidants in both chemically can be made of an already qualitative de­
and biologically based model systems. In scriptor. As a new technology breaks a pre­
both types of systems, the balance between viously homogenous mass of compounds into
oxidants and antioxidants is critical. Imbal­ subdivided fractions, the relative differences
ances between free radicals and antioxidants in concentration of these subdivisions among
can occur, caused by an increased produc­ different samples can negate the idea of ho­
tion of free radicals andlor decreased effec­ mogeneity and cause dramatic reevaluations
tiveness of the antioxidants within the reac­ of theories that depended on the homogene­
tion system. These imbalances can be caused ity of the original mass. Tea research has
by the radicals overwhelming the antioxi­ brought the thearubigens very close to this
dants within the system, or by an excess of point, and the homogeneity assumed by this
antioxidants leading to a prooxidant func­ classification is beginning to show weak­
tionality (Aruoma et aI., 1 993). It is critical ness.
that a proper antioxidant to oxidant balance The advancement of tea chemi stry is
be maintained in well-controlled biological dependent on comprehension of the li tera­
studies. The balance of different types of ture in the context of the directions of indi­
antioxidants i s also important, as demon­ vidual research groups.
strated with carotenoids, vitamin E, and vi­
tamin C (Bohm et aI., 1 997).
A. Analysis by Chemical
Constitution and by Technical
VI. TRENDS IN TEA RESEARCH Innovation

Tea chemistry as a research discipline Early determinations of the chemical

has been characterized and influenced by the constitution of tea and tea beverages

466
(Kursanov, 1 956; Vuataz et aI., 1 959; Millin become strong driving influences for contin­
and Rustige, 1 967) set the stage for a gener­ ued business growth and profitability. Im­
alized interpretation of the chemistry of tea provements in production yield and quality
(Sanderson, 1 972). The subdivisions of each of Indian tea has been attributed to better
category and the analytical accuracy of new technological understanding of the underly­
methods have improved the understanding ing factors contro l l i n g tea production
of the mass balance of tea chemistry. The (Mahanta et aI., 1993). It is therefore a busi­
subdivision technique has been propagated ness necessity as much as an intellectual
i n the literature (Graham, 1 984; Balentine, pursuit to gain a technological advantage, an
1 992). effort that can only be underpinned by a firm
No single technological innovation has understanding of the chemistry of tea.
propelled the efforts of tea chemistry more Many of the efforts of tea research focus
in recent years than the introduction of the on improving the value of tea by understand­
HPLC (Hoefler and Coggon, 1 976), and the ing the chemical parameters that deter­
subsequent coupling of the technique with mine price. Brisk teas are more astringent
diode-array detection (Bailey et aI., 1 990; and are associated with earlier fermentation
Opie et aI., 1 990; Opie, 1992) and mass spec­ times and higher theaflavin content (Owuor
trometry (Lin et aI., 1 993; Bailey et aI., and McDowell, 1 994). It fol l ows that
I 994b). Despite its power, HPLC is in a thearubigens, which are a product of later
stage of relative infancy compared with the fermentation times, should be expected to be
field of tea chemistry, and i t would seem that a negative quality for production of brisk
a large fraction of the task of repeating and teas. The effect of thearubigens on cream
reinterpreting the historical results of tea formation (Hazarika et aI., 1 984) can also
chemistry within the language and context determine acceptability, especially for iced
of lhis inslrumenlal meLhod s Lil l needs Lo be tea beverages. Principal component analysis
performed. Progress and innovation in sci­ has determined that theaflavins are positively
entific research is frequently the result of correlated (and some flavonol glycosides are
dramatic breakthroughs or "revolutions" negatively correlated) with tea value
(Kuhn, 1 970). As each "revolution" propa­ (McDowell et aI., 1 99 1 ). Theaflavins are very
gates itself through a scientific discipline, important for quality and price, owing to the
the paradigms and assumptions that are as­ bright red color they impart (Hilton and
sociated with the discipline must be reevalu­ Palmer-Jones, 1975; Ellis and Cloughley,
ated, and the effect of HPLC on tea chemis­ 1 98 1 ), but among similar theaflavin con­
try needs further investigation in this vein. tents, theaflavins are not a good determining
factor (Owuor et aI., 1 986). Aroma can also
B. Bulk Properties and Correlations be expected to play an important role, espe­
to Tea Taster's Profiles cially i n lowery teas (Robinson and Owuor,
1 992).
Efforts to understand the chemistry of
tea can be seen in part as moti vated by the C. Tea as an Antioxidant and as a
desire of tea plantation owners and manu­ Healthy Beverage
facturers to understand what factors drive
acceptability and market price. Tea imports The perception of tea as a healthy bever­
into the U.S. in 1 993 totaled $ 1 24 m. (Anon., age is traced to the legendary Shen Nung,
1994), a figure that has fluctuated but essen­ who claimed in the Pen Tsao, a Chinese
tially remained flat for 5 years. Improve­ book of herbal medicine, that tea is good for
ments in the quality, marketability, or yield a variety of ailments, including tumors. Tea

467
consumption i n ancient times was regarded There is growing evidence that oxidative
as healthy, likely due to the fact that the injury is a major risk factor i n development
boiling water used to make tea kills many of of cardiovascular di sease (Marx, 1 9 87;
the water-borne pathogens that caused ill­ Grundy, 1 993). Recent reports that dietary
nesses common in those times. Vitamin P supplementation with vitamin E (at 1 00 IU
activity, a reduction of capil lary fragility, and less than 400 IU) was associated with a
has been found in tea and attributed to tea significant reduction in the risk for cardio­
polyphenols (Kursanov et aI., 1 950). Today, vascular disease (36 to 54% reduction in risk
research into tea chemi stry is a fundamental for women and a 20 to 26% reduction in risk
part of studies on the role of tea as a biologi­ for men) support this hypothesis (Brody,
cal antioxidant and in prevention of chronic 1 993).
diseases. One of the key mechanisms of the hy­
There is mounting evidence that sub­ pothesis that oxidative damage is linked to
stances called free radicals play a role in the cardiovascular disease is the theory that oxi­
development of major human diseases such dation of low·density lipoprotein (LDL) leads
as heart disease and cancer. Research sug­ to damage of the arterial wall, producing
gests that antioxidants may help protect sites for fibrous plaque formation. Antioxi­
against these di seases by minimizing the dant vitamins are believed to inhibit this
detrimental impact of free radical damage to process, and studies have shown that the
cells and tissues. Much more research is antioxidant vitamins ascorbic acid, X-toco­
needed to better identify the processes in­ pherol, and �-carotene inhibit LDL oxida­
volved in free radical-mediated diseases and tion in vitro and reduce the progression of
to help develop nutritional strategies to op­ atherosclerosis in animal models.
timize antioxidant status. Studies using a Chinese green tea polyphe­
The most commonly recognized dietary nol fraction (CGTP) were conducted to deter­
sources of antioxidants are fruits and veg­ mine the effect of tea constituents on oxida­
etables, which contain vitamins C, E, and tion of low-density lipoprotein (LDL) ill vitro
carotenoids. Preliminary research indicates in a model system thought to be representa­
that the f1avonoids, which include the cat­ tive of the effects of antioxidants in modulat­
echins and f1avonols found i n both black and ing risk factors associated with cardiovascu­
green tea, also act as antioxidants. However, lar disease (Ding et aI., 1 99 1 ; Ding et aI.,
additional research is required to determine 1 992). The antioxidant activity was deter­
whether tea antioxidants substitute for or mined based on lipid peroxide production,
complement some of the protective func­ thiobarbituric ac i d reac tive substances
tions identified with the more established (TBARS), and the mobility of LDL in an
antioxidant compounds such as vitamins C, electrophoretic gel. The results of these stud­
E, and beta-carotene. The role of antioxi­ ies showed that green tea polyphenols block
dants in heart disease is unc lear, and their LDL oxidation induced by copper, UV irra­
potential role in cancer prevention and the diation, or macrophages. The mechanisms for
aging process remains speculative. the antioxidant activity of the CGTP have not
been determined but are theorized to be metal
chelation, free radical scavenging, and an
1. Lipid Oxidation and antioxidant "vitami n-sparing" (whereby anti­
Cardiovascular Disease oxidant is preferentially consumed, 'sparing'
the vitamin) effect. A dose-dependent inhibi­
Cardiovascular disease is one of the lead­ tion of copper-induced LDL oxidation and
ing causes of death in the Western World. LDL oxidation induced by transformed

468
macrophages were also found for catechin ficient to block smoke-induced DNA dam­
(Mangiapane et ai., 1 992). age due to oxidative events.
A tobacco-specific nitrosamine, 4-(me­
thylnitrosamino)-I (3-pyridyl)-I -butanone, or
2. Studies of Tobacco Nitrosamines NNK, is an oxidative carcinogen that in­
duces lung cancer in numerous laboratory
Smoking of tobacco products i s the main animal models (Xu et ai., 1992; Wang et a!.,
risk factor for development of cardiovascu­ 1 992a). After metabolic activation, NNK
lar disease and cancer. The nitrosamines reacts with DNA to form oxidized adducts
derived from tobacco smoke are good bio­ (80HdG). When solutions of green tea, black
logical oxidants that can damage cellular tea, or the green tea catechin epigallocatechin
lipids, LDL lipids, and DNA (Yang, 1 992; gallate (EGCG) were provided to mice in
Loft et ai., 1 992; Marx, 1 9 87). One outcome place of drinking water the numbers of NNK­
of oxidative damage to DNA is the 8-hy­ induced lung tumors were significantly re­
droxylation of guanine bases producing duced (Xu et ai., 1 992; Wang et a!., 1 992a).
8-hydroxydeoxyguanosine (80HdG) DNA Black and green tea were found to be equally
adducts. Repair of these DNA adducts in effective in reducing tumor numbers when
vivo leads to the excretion of 80HdG i n provided during or after NNK treatment
urine that i s a biomarker or index of the (Wang et a!., I 992a). EGCG was found to be
current state of oxidative DNA damage and almost as active as green tea infusions (Xu
repair (Saul and Ames, 1 986; Loft et ai., et ai., 1 992). In vivo (Xu et a!., 1 992) studies
1 992). Tobacco use by men and women has using mice and in vitro (Shi et ai., 1 993)
been shown to cause a 50% increase in the experiments showed that black and green tea
excretion of 80HdG, indicating that smok­ polyphenols inhibit NNK metabolism and
ing causes a significant i ncrease in the level DNA adduct formation. EGCG was found to
of oxidative damage to DNA (Loft et ai., be the most potent inhibitor of the tea
1 992). Cigarette smoking (> I 0 per day) was polyphenols.
found to significantly increase the mutation Polyphenols containing 1 ,2- or 1 ,4-
sensitivity of chromosomes in peripheral diphenol functional groups have been found
lymphocytes (Kim et a!., 1 993). This was to be potent inducers of a group of enzymes
determined by treating the cells with mito­ involved with the metabolism of carcino­
gens and measuring the frequency of sister­ gens and other xenobiotic compounds,
chromatid exchange (SCE). The degree of termed phase II enzymes (Sohn et ai., 1 994).
SCE i s a good index of the instability of Phase II enzymes include NAD(P)H:quinone
DNA and of oxidative stress. reductase, glutathione S-transferases, and
A clinical trial was conducted to deter­ UDP-glucuronosyl transferases (Talalay,
mine if consumption of green tea or coffee 1 99 2; Prochaska and Talalay, 1 992). B e­
would alter the sensitivity of DNA to SCE i n cause tea polyphenols fit the criteria for Phase
smokers. Patients were provided three cups II enzyme inducers, it i s likely that tea
of green tea or coffee per day for 6 months. polyphenols act as potent inducers of these
Consumption of three cups of green tea per enzymes, as has been reported (Khan et aI.,
day inhibited the cigarette smoking-induced 1 992). It is likely that one mechanism for the
increase i n frequency of SCE. Coffee con­ inhibition of NNK-induced lung tumors is
sumption did not change the frequency of blocking of NNK activation. A radical scav­
SCE (Kim et a!., 1 993). This demonstrates enging mechanism might also be involved in
that normal tea consumption should be suf- the inhibition of NNK-induced DNA adduct

469
formation. The effect of tea on reduction of polyphenolic constituents of green and black
tumors after NNK treatment indicates that tea is undoubtedly a central focus of tea
tea can inhibit tumor formation through a research. Indirect correlations such as those
second mechanism that does not involve used to measure thearubigens need to be
NNK metabolism. Oxidative events have systematically justified with tangible chemi­
been l inked to the promotion and progres­ cal evidence, whether by isolation of indi­
sion of initiated cells to tumors and cancers. vidual compounds as performed by HPLC,
Tea may also act to block these events or by selective hydrolysis by which the frag­
through an antioxidant mechanism. The pro­ ments can be measured with quantitative
motion of tumor formation in mouse skin by accuracy.
TPA is partially induced by formation of Tea research is moving away from its
peroxides or free radicals (Wei and Frenkel, rudimentary origins as a qualitative out­
1 993). Tea, GTP (Huang et aI., 1992; Wang growth of tea tasting and the tea industry and
et aI., 1 992b), EGCG (Fuj iki et aI., 1 992), moving into a fully quantitative and chemi­
and antioxidants (Kozombo et aI., 1983; cally i nteresting science. The perception of
Smart et aI., 1 987) were effective in block­ tea not only as a plant and as a beverage but
ing TPA-induced tumor promotion in mouse as a healthy part of the human diet and as a
skin. Mechanistic studies are needed to es­ rich source of new chemical compounds will
tablish the role of tea in blocking these cel­ propel tea research in new directions.
lular events.

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