U.S. patent application number 10/716168 was filed with the patent office on 2004-07-15 for high yield process for producing theaflavins and products of such process.
Invention is credited to Fields, Chris, Slaga, Thomas J., Zhao, Jifu.
Application Number | 20040137129 10/716168 |
Document ID | / |
Family ID | 27623404 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040137129 |
Kind Code |
A1 |
Zhao, Jifu ; et al. |
July 15, 2004 |
High yield process for producing theaflavins and products of such
process
Abstract
A method for the production of black tea theaflavins from green
tea polyphenols through use of mushroom tyrosinase, and therapeutic
agents of such process.
Inventors: |
Zhao, Jifu; (Littleton,
CO) ; Slaga, Thomas J.; (Golden, CO) ; Fields,
Chris; (Lake Zurich, IL) |
Correspondence
Address: |
DAVID G. HENRY
900 Washington Avenue
P.O. Box 1470
Waco
TX
76703-1470
US
|
Family ID: |
27623404 |
Appl. No.: |
10/716168 |
Filed: |
November 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10716168 |
Nov 18, 2003 |
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10625500 |
Jul 22, 2003 |
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10625500 |
Jul 22, 2003 |
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09721438 |
Nov 22, 2000 |
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6602527 |
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10716168 |
Nov 18, 2003 |
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10654867 |
Sep 4, 2003 |
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10654867 |
Sep 4, 2003 |
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10625500 |
Jul 22, 2003 |
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Current U.S.
Class: |
426/597 |
Current CPC
Class: |
A23F 3/10 20130101; C12Y
110/03001 20130101 |
Class at
Publication: |
426/597 |
International
Class: |
A23F 003/00 |
Claims
We claim:
1. A therapeutic agent produced by a process comprising the steps
of: selecting a first measure of green tea polyphenols; and mixing
said green tea polyphenols with a second measure of tyrosinase.
2. The product of claim 1 wherein the process further comprises the
step of extracting the mixture of said green tea polyphenols and
tyrosinase after a period of oxidation reaction there between which
produces theaflavins, but before the substantial formation
compounds other than theaflavins.
3. A sunburn preventative agent produced by a process comprising
the steps of: selecting a first measure of green tea polyphenols;
and mixing said green tea polyphenols with a second measure of
tyrosinase.
4. The product of claim 3 wherein the process further comprises the
step of extracting the mixture of said green tea polyphenols and
tyrosinase after a period of oxidation reaction there between which
produces theaflavins, but before the substantial formation
compounds other than theaflavins.
5. A sunburn therapeutic agent produced by a process comprising the
steps of: selecting a first measure of green tea polyphenols; and
mixing said green tea polyphenols with a second measure of
tyrosinase.
6. The product of claim 5 wherein the process further comprises the
step of extracting the mixture of said green tea polyphenols and
tyrosinase after a period of oxidation reaction there between which
produces theaflavins, but before the substantial formation
compounds other than theaflavins.
7. A nutritional supplement produced by a process comprising the
steps of: selecting a first measure of green tea polyphenols; and
mixing said green tea polyphenols with a second measure of
tyrosinase.
8. The product of claim 7 wherein the process further comprises the
step of extracting the mixture of said green tea polyphenols and
tyrosinase after a period of oxidation reaction there between which
produces theaflavins, but before the substantial formation
compounds other than theaflavins.
9. A chemopreventative agent produced by a process comprising the
steps of: selecting a first measure of green tea polyphenols; and
mixing said green tea polyphenols with a second measure of
tyrosinase.
10. The product of claim 9 wherein the process further comprises
the step of extracting the mixture of said green tea polyphenols
and tyrosinase after a period of oxidation reaction there between
which produces theaflavins, but before the substantial formation
compounds other than theaflavins.
11. A process for producing a disease preventative and therapeutic
agent comprising the steps of: selecting a first measure of green
tea polyphenols; and mixing said green tea polyphenols with a
second measure of tyrosinase.
12. The product of claim 10 wherein the process further comprises
the step of extracting the mixture of said green tea polyphenols
and tyrosinase after a period of oxidation reaction there between
which produces theaflavins, but before the substantial formation
compounds other than theaflavins.
Description
CITATION TO PARENT APPLICATION(S)
[0001] This application is a continuation-in-part of co-pending
U.S. patent application Ser. No. 10/625,500, which was a
continuation-in-part of U.S. application Ser. No. 09/721,438 (now
U.S. Pat. No. 6,602,527), from which priority is claimed pursuant
to 35 U.S.C. 120.
BACKGROUND OF THE INVENTION
[0002] 1. Field of The Invention
[0003] The present invention relates to the synthesis of highly
beneficial theaflavins from substrates yielded from green tea
leaves, and to the products of such process.
[0004] 2. Background Information
[0005] Theaflavins (TFs) are colorless catechin dimers that
significantly contribute to the brown-orange color and astringency
of black tea liquors when oxidized. Generally, TFs may be formed
through the polyphenol oxidase (PPO)-dependent oxidative
polymerization of green tea polyphenols (GTP) or flavanoids during
fermentation of green tea to black tea. TFs are primarily composed
of a mixture of theaflavin, theaflavin-3'-O-gallate,
theaflavin-3-3'-di-O-gallate and theaflavin-3-gallate.
[0006] TFs have highly significant anti-oxidant properties, as well
as significant anti-inflammatory antimicrobial and antiviral
activity. Specifically, TFs are reported effective against various
diseases, including cancer, cardiovascular and cerebrovascular
diseases, diabetes, hypercholesterolemia, etc. as a cancer
chemopreventive agent, as a photoprotective agent, as a protective
agent against UVB-induced skin damage, as an inhibitor of cell
transformation and PhIPO-induced mutagenicity.
[0007] The present invention provides a uniquely effective, high
yield process for specifically producing theaflavins extracts from
green tea polyphenols (GTP), a product which can then be
incorporated into nutritional supplements and even topical
preparations. In the latter case, such would be useful in the
prevention of UV-related skin damage and ultimately of skin
cancer.
[0008] The present method results from research, one product of
which was the realization that any presumption of an equivalence in
beneficial characteristics of green and black tea polyphenols
(based on studies that find the benefits of the two teas to be
roughly equivalent), and a resulting belief, among some, that black
tea presents nothing more beneficial than green tea, is merely the
result of greatly differing concentrations of beneficial compounds
respectively green and black tea.
[0009] The theaflavins of black tea are demonstrably of greater
value in the maintenance of health, but appear in conventionally
produced black tea in substantially lower concentrations than the
corresponding, and differently structured polyphenols in green tea
(principally epigallocatechin gallate ("EGCG")). When equal
quantities are compared, black tea theaflavins (produced through
oxidation of green tea polyphenols) are considerably more
beneficial than the green tea polyphenols. Yet, as alluded to
above, conventionally produced black tea contains far lower levels
of theaflavins than would be desirable, else the benefits of black
tea theaflavins, compared to those of green tea polyphenols, would
have been more clearly evident from prior studies.
[0010] Presently, black tea theaflavins are produced when tea
leaves are rolled such that phenolase in the tea leaves (remaining
separate from the green tea polyphenols in the unadulterated tea
leaf) is forcibly mixed with the green tea polyphenols, by which
the latter is oxidized, in part, to form black tea theaflavins.
However, the present production of black tea favors over-oxidation
to form highly condensed tannins, rather than the more desirable,
beneficial theaflavins.
[0011] It naturally follows, that it would be highly desirable to
provide some methodology by which optimally high yields of black
tea theaflavins may be produced from green tea polyphenols
["GTP"]).
SUMMARY OF THE INVENTION
[0012] It is an object of the present invention to provide a
health-promoting, antioxidant composition produced through a novel
and unobvious oxidative processing of green tea polyphenols.
[0013] It is an object of the present invention to provide a
health-promoting composition produced through a novel and unobvious
oxidative processing of green tea polyphenols.
[0014] It is an object of the present invention to provide a
chemopreventive composition produced through a novel and unobvious
oxidative processing of green tea polyphenols.
[0015] It is an object of the present invention to provide a
photoprotective composition produced through a novel and unobvious
oxidative processing of green tea polyphenols.
[0016] It is an object of the present invention to provide a
composition which is inhibitive of PhIPO-induced mutagenicity and
which is produced through a novel and unobvious oxidative
processing of green tea polyphenols.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0017] The present invention is of the high yield production of
beneficial, theaflavins, the natural counterpart of which are
characteristic constituents of black tea, yet appearing in black
tea (when conventionally produced) in far lower levels than would
be desired. The practice of such invention involves the use of
mushroom.
[0018] Throughout this discussion, the words "synthetic" or
"synthesized" are used. It is to be understood that this should not
be interpreted as indicating a chemical difference between
theaflavins produced by conventional means, versus the presently
disclosed method, but merely that which is produced is
"synthesized" using the new method of the present invention.
[0019] Mushroom tyrosinase is a type of polyphenol oxidase and,
most notably, will oxidize green tea polyphenols to produce
theaflavins. Green tea leaves or the polyphenols contained in such
are exposed to a mushroom tyrosinase enzyme that readily converts
the catechins into theaflavins under typical fermentation
conditions. The ideal parameters are characterized in that
polyphenolic substances contained in moist fresh tea leaves contain
a moisture content of at least 20% by weight based upon the dry
weight of tea solids contained in the moist leaves. The temperature
is maintained between about 20-50 .degrees C. at a pH between about
4.0-6.0. The reaction is executed by distributing a mushroom
tyrosinase or some form thereof, such as finely ground mushrooms,
on the tea leaves in a fluidized bed or similar unit operation to
achieve even distribution of the mushroom tyronsiane upon the
active sites of the tea leaves or tea polyphenols. For dry green
tea, a mushroom slurry can be used to soak the leaves. The
oxidation duration is 10 to 360 minutes supplying oxygen through a
number of means either naturally or in some mechanical form. The
fermentation end result is a highly concentrated theaflavin
slurry.
[0020] The fermentation step will not necessarily have converted
all the tea polyphenols into theaflavins and the final
concentration of such will be dependent on the time or duration of
the reaction along with the temperature and pH conditions as well
as the amount of mushroom tyrosinase employed. It is the intent of
the present invention to achieve a high theaflavin concentration
and allow other polyphenols or catechins to remain present within
the final slurry so as to attain a number of different slurry
profiles as feedstock into the extraction steps and ultimately into
the final end product.
[0021] The slurry can be dried and stored for future use or
filtered and send directly to solvent extraction where a typical
hydrocarbon based solvent such as ethyl acetate is used to extract
the theaflavins other solvents or alcohols can be employed if one
may desire to remove the caffeine or other sensitive compounds for
example. The extract is then dried via vacuum evaporation or other
suitable drying techniques. Further extraction purification can
also be employed by conventional chromatographic techniques with an
alcohol solvent to achieve a highly purified theaflavin/polyphenol
extract. Other conventional techniques to those skilled in the art
may also be employed to achieve a highly purified extract.
[0022] The method of the present invention uses mushroom tyrosinase
in a manner which produces optimum yields of TFs through controlled
oxidation of green tea polyphenols. While examples that follow
utilize commercially available, extracted mushroom tyrosinase and
green tea polyphenols, it should be understood that the present
invention encompasses the use of such substances from any source.
In fact, outside of the analytical proof-of-concept context in
which precise quantity control and related product measurements are
required, the mere, temporary mixing of mushrooms (source of
mushroom tyrosinase) and green tea leaves (source of green tea
polyphenols) in a suitable buffering solution will achieve the
intended results. Such an approach is the most likely, commercially
viable mode of practicing the present invention, in view of the
relative economies of using extracts verus mixing of the natural
sources for the reagents. Upon review of the present disclosure,
suitable scale-up in a commercial context will be apparent to
persons skilled in relevant arts.
EXAMPLES
[0023] The following examples are illustrative of the present
invention and parts and percentages are by dry weight unless
otherwise indicated. Theaflavins were quantified by HPLC, and used
mushroom tyrosinase and standard theaflavins from SIGMA-ALDRICH
CORPORATION. It should be noted that these examples are only
that--examples. A wide range of conditions, such as time and units
of enzyme, can be used because more time in process will offset a
lesser enzyme presence, and vice versa.
Example I
[0024] 100 mg green tea polyphenols in 50 ml acetate buffer (100
mM, pH6.5) was mixed with 25,000 units of tyrosinase and the
mixture was kept at room temperature in dark for 30 minutes. The
reaction mixture was extracted with 100 ml ethyl acetate 4 times.
The combined extracts were evaporated under reduced pressure with a
rotary evaporator to remove ethyl acetate. The residue was
dissolved in water and applied to HPLC separation. Waterman ODS
semipreparative column was employed.
[0025] HPLC mobile phase contained solvent A (5% methanol in 100 mM
acetate buffer, pH4.8) and solvent B (70% methanol in 100 mM
acetate buffer, pH4.8). Gradient method at 25.degree. C. as
follows: 0-5 min (100% A), 5-15 min (100% A to 75% A and 25% B),
15-40 min (75% A and 25% B to 50% A and 50% B), 40-50 min (50% A
and 50% B to 30% A and 70% B), 50-55 min (30% A and 70% B to 100%
A), 55-60 min (100% A) and 61 min stop of run. The theaflavins
fraction was collected and identified by their retention time and
co-chromatography with authentic TFs.
[0026] From 100 mg of crude green tea polyphenols, 41 mg of TFs was
obtained.
Example II
[0027] 100 mg green tea polyphenols in 50 ml acetate buffer (100
mM, pH6.0) was mixed with 20,000 units of tyrosinase and the
mixture was kept at room temperature in dark for 25 minutes. The
reaction mixture was extracted with 100 ml ethyl acetate 4 times.
The combined extracts were evaporated under reduced pressure with a
rotary evaporator to remove ethyl acetate. The residue was
dissolved in water and applied to HPLC separation.
[0028] From 100 mg of crude Green tea polyphenols, 35 mg of TFs was
obtained.
Example III
[0029] 200 mg green tea polyphenols in 70 ml acetate buffer (100
mM, pH6.8) was mixed with 30,000 units of tyrosinase and the
mixture was kept at room temperature in dark for 35 minutes. The
reaction mixture was extracted with 100 ml ethyl acetate 4 times.
The combined extracts were evaporated under reduced pressure with a
rotary evaporator to remove ethyl acetate. The residue was
dissolved in water and applied to HPLC separation.
[0030] From 200 mg of crude green tea polyphenols, 92 mg of TFs was
obtained.
[0031] The theaflavins produced according to the present invention
may be incorporated into nutritional supplements and even topical
creams, the latter for preventing UV-related skin damage through
sun exposure.
[0032] In the case of nutritional supplements, the present
synthesized products may be incorporated into capsules or tablets,
and administered orally. There is no specific dosage which is
demonstrably better or worse than another. A capsule or tablet of
within conventional size ranges, if consisting solely of oxidized
extracts of the tea leaves here described, will provide a quite
therapeutic dosage of theaflavins, and a much more potent such
dosage than any extract of any tea leaves produced by any known
method or process. Clearly, however, such extracts may well be
combined with other nutritional supplement agents, in which case a
perhaps proportionately less beneficial effect may be expected. One
thing is certain, the theaflavins produced by the present process
are health-promoting compounds, and the enhancement of their
production provided by the instant process is beneficial in
enabling the efficient delivery thereof.
[0033] The synthetically produced black tea theaflavins have proven
to be as effective as theaflavins produced by conventional black
tea processing. The present inventors have evaluated the
theaflavins so produced as described hereafter.
Trials
[0034] To evaluate the preventative characteristics of the
theaflavins produced according to the present method, the lower
back of healthy adult human volunteers received a single topical
application of theaflavins produced by the present process (0.2
mg/cm.sup.3) in 5% Tween 80), and 30 minutes later, were exposed to
120 mJ/cm.sup.2 of UVB. 24 hours after UVB exposure, sunburn
severity indices were devised as area (cm.sup.2) times intensity of
red color lesion (0, no lesion; 1, barely detectable erythema; 2,
moderate erythema; 3, bright erythema).
[0035] The following demonstrate the observed UVB damage
preventative results:
1 SUNBURN TREATMENT INCIDENCE (%) SUNBURN SEVERITY TF Alone 0 0
Vehicle + UVB 100 4.8 .+-. 1.1 Synth. TFs 30 0.4 .+-. 0.4
(91%.dwnarw.) (0.2 mg) + UVB Conventional 20 0.2 .+-. 0.5
(96%.dwnarw.) TFs P < 0.01
[0036] To evaluate the therapeutic characteristics of the
theaflavins produced according to the present method, the lower
back of healthy adult human volunteers received a single dosage
(120 mJ/cm.sup.2) of UVB. Immediately, and 4 hours after UVB
exposure, the exposed sites twice received topical application of
either synthetized or conventionally-produced theaflavins (0.2
mg/cm.sup.3 in 5% Tween 80). 24 hours after UVB exposure, sunburn
severity indices were evaluated as in the first trial.
[0037] The following demonstrate the observed UVB damage
therapeutic results:
2 SUNBURN TREATMENT INCIDENCE (%) SUNBURN SEVERITY TF Alone 0 0
Vehicle + UVB 100 4.8 .+-. 1.1 Synth. TFs 80 1.2 .+-. 0.7
(75%.dwnarw.) (0.2 mg) + UVB Conventional TFs 50 1.4 .+-. 1.6
(71%.dwnarw.) P < 0.01
[0038] The above results indicate that the synthesized theaflavins
are effectively interchangeable with conventional theaflavins, with
respect to efficacy. This interchangeability presumptively extends
to other therapeutic contexts, such as in nutritional
supplements.
[0039] In view of the foregoing, it is-clear that practice of the
present invention will yield a product, the use of which will yield
a highly beneficial substance which can be used in both ingested
and topical applications. This, in turn, represents a "painless"
source of chemorepressive and sunburn resistance substances for
consumption by consumers.
[0040] Although the invention has been described with reference to
specific embodiments, this description is not meant to be construed
in a limited sense. Various modifications of the disclosed
embodiments, as well as alternative embodiments of the inventions
will become apparent to persons skilled in the art upon the
reference to the description of the invention. It is, therefore,
contemplated that the appended claims will cover such modifications
that fall within the scope of the invention.
* * * * *