Title: Black tea extract for
prevention of disease
United States Patent: 7,238,376
Issued: July 3, 2007
Inventors: Chen; Kuang Yu
(Belle Mead, NJ), Ho; Chi-Tang (East Brunswick, NJ), Rosen; Robert T.
(Monroe Township, NJ), Ghai; Geetha (Murray Hill, NJ)
Assignee: Rutgers, The
State University (New Brunswick, NJ)
Appl. No.: 10/870,187
Filed: June 17, 2004
Compositions and methods for preventing
and treating disease are provided. The compositions are extracts of black
tea that include a mixture of theaflavin-3-gallate and
Description of the Invention
BACKGROUND OF THE
Epidemiological studies have suggested
that tea may have a protective role in disease, including certain human
cancers. Catechin polyphenols isolated from green tea have been shown to
inhibit proliferation of cultured mammalian cells including colon
carcinoma, lung carcinoma, breast carcinoma, melanoma, and leukemic cells
(Lea, M. A. et al. 1993. Cancer Lett. 68:231-236; Valcic, S. et al. 1996.
Anticancer Drugs 7:461-468). It has been reported that a major green tea
catechin polyphenol, (-)epigallocatechin gallate (EGCG), inhibits growth
of human tumor cells, including Caco-2 colorectal cancer cells, Hs578T
breast cancer cells, and SV40-transformed W138 cells, but has little or no
inhibitory effect on the growth of their normal counterparts (Chen, Z. P.
et al. 1998. Cancer Lett. 129:173-179).
Black tea extracts have also been shown to be potent in inhibiting
tumorigenesis in several animal model systems, including skin (Javed, S.
et al. 1998. Biomed. Environ. Sci. 11:30-7-313), lung (Yang, G. Y. et al.
1997. Carcinogenesis 18:2361-2365), colon (Weisburger, J. H. et al. 1998.
Carcinogenesis 19:229-232), esophagus (Morse, M. A. et al. 1997. Nutr.
Cancer 29:7-12), and mammary gland (Rogers, A. E. et al. 1998.
Carcinogenesis 19:1269-1273). The major black tea polyphenols have been
characterized to be theaflavin (TF-1), theaflavin-3-gallate and
theaflavin-3'-gallate mixture (TF-2), and theaflavin-3,3'-digallate
(TF-3). These theaflavin polyphenols are fermentation products derived
from green tea polyphenols and are responsible for the characteristic
color, fragrance and taste of black tea.
The biological effects of each individual black tea polyphenol have not
been well-studied in terms of their molecular mechanisms. TF-3 has been
shown to be as potent as EGCG from green tea inhibiting the growth of
human A431 carcinoma cells and in reducing autophosphorylation of EGF and
PDGF receptors (Liang, Y. C. et al. 1999. Carcinogenesis 20:733-736).
There are no reports of the activity of any other black tea polyphenols.
SUMMARY OF THE
An object of the present invention is an
extract of black tea which comprises a theaflavin-3-gallate and a
theaflavin-3'-gallate mixture. Also included in the present invention is a
composition which comprises the black tea extract in combination with at
least one other compound selected from a rosemary extract, a Mexican
Bamboo extract, a Huzhang extract, resveratrol, a green tea extract, an
orange peel extract, and a hydroxylated or methoxylated resveratrol
Another object of the present invention is a method for inhibiting tumor
cell growth in an animal which comprises administering to an animal the
extract of black tea.
Yet another object of the present invention is a method for preventing or
treating disease associated with Cox-2 gene expression in an animal which
comprises administering to an animal an effective amount of an extract of
black tea and wherein the disease is selected from the group consisting of
cancer, inflammation, and arthritis. Also included in the present
invention is a method wherein the black tea extract is administered in
combination with at least one additional compound selected from a rosemary
extract, a Mexican Bamboo extract, a Huzhang extract, resveratrol, a
hydroxylated resveratrol analog, a green tea extract, an orange peel
extract or a methoxylated resveratrol analog.
OF THE INVENTION
The black tea polyphenol extract known as
TF-2, a mixture of theaflavin-3-gallate and theaflavin-3'-gallate, has
been found to inhibit both cancer cell growth and apoptosis in cancer
cells. None of the other black tea polyphenols tested showed this
activity. Additionally, TF-2 was shown to specifically inhibit expression
of the Cox-2 gene, a gene known to be associated with both inflammatory
reactions and carcinogenesis, at both the mRNA and protein level. The
present invention provides both compositions and methods for prevention
and treatment of disease, including cancer.
The effects of black tea polyphenols on proliferation of W138 and W138VA
cells was examined. W138 diploid fibroblasts have a finite life span
whereas the virally transformed W138VA cells are cancerous in nude mice.
Both cell types were grown in 24 well plates in the presence of increasing
doses of the theaflavin polyphenols, TF-1, TF-2 and TF-3 (0, 1, 5, 10, 25
and 50 .mu.M). Viable cells were stained with crystal violet 4 days after
plating. A decrease in staining intensity relative to the untreated
control cells were indicative of growth inhibition by the polyphenol
compound. Of the three theaflavin polyphenols tested, only TF-2 had
significant effects on cells growth. Growth of W138VA cells was inhibited
significantly by TF-2 at doses in the range of 10 to 50 .mu.M, while there
was no significant effect on cell growth in W138 cells. The other 2
polyphenols tested did not exhibit this differential growth inhibition of
W138 versus W138VA cells. The IC.sub.50 value for TF-2 and growth
inhibition of W138VA cells was estimated to be 3 .mu.M.
To determine whether the differential inhibitory effect of TF-2 is general
for other types of cancerous cells, the effects of TF-2 on Caco-2
colorectal cancer cells were examined and compared with effects of TF-2 in
their normal counterpart, CCD-33Co colorectal cells. In control cells,
there was a slight decrease in the density of cells in the CCD-33Co
culture in the presence of the highest dose of TF-2 only, 50 .mu.M. In
contrast, the presence of TF-2 at wither 10 or 50 .mu.M in Caco-2 cultures
resulted in a significant reduction of cell number as well as a dramatic
morphological change in the cells. These data demonstrate that TF-2 has
effects to inhibit cell growth in more than one type of cancer cell.
Since apoptosis is a major cause underlying growth inhibition in cells,
the effects of TF-2 on apoptosis induction in transformed cells was
examined using a TUNEL (terminal deoxynucleotidyl transferase-mediated
dUTP nick-end labeling) assay. It has been reported that theaflavin and
theaflavin digallate can induce apoptosis in human lymphoid leukemia cells
and human stomach tumor cells (Hibasami, H. et al. 1998. Int. Mol. Med.
1:725-727). Using the TUNEL assay, TF-2 treatment (100 .mu.M for 18 hours)
caused almost every cell in the W138VA culture to exhibit apoptosis, as
indicated by green fluorescence in the assay. In contrast, almost no cells
in the normal W138 culture exhibited green fluorescence after TF-2
treatment. DNA fragmentation analysis was then performed to determine the
time course of the apoptotic effect. DNA fragmentation was observed within
4 hours following TF-2 treatment in W138VA cells. There was no significant
DNA fragmentation in TF-2-treated W138 cells over a 24 hour period. These
results indicate that transformed W138VA cells had a greater propensity
for apoptosis in response to TF-2 as compared to non-transformed cells
(W138). This observation likely accounts at least in part for the
differential inhibitory effect of TF-2 in the growth of transformed
As a result of the significant effects of TF-2 on growth of Caco-2 cells
in culture, the potential role of the Cox-2 gene in the growth suppressive
effects of cancerous colon cells was examined. Cox-2 gene expression has
been shown to be linked to the development of colon cancer, with increased
expression observed in about 90% of human colorectal cancers and 40% of
premalignant colorectal adenomas (Eberhart, C. E. et al. 1994.
Gastroenterology 107:1183-1188). A direct link between Cox-2 expression
level and polyps formation has been demonstrated in APC knockout mice,
indicating a direct role for this gene in colon cancer formation (Oshima,
M. et al. 1996. Cell 87:803-809). The Cox-2 gene encodes an inducible form
of cyclooxygenase that has been shown to be a key enzyme in prostaglandin
biosynthesis. Therefore, the effect of black tea polyphenols on Cox-2
expression was examined.
All three polyphenols were tested, TF-1, TF-2 and TF-3. Of the three
compounds tested, only TF-2 significantly suppressed Cox-2 gene expression
in Caco-2 cells. Effects were seen at doses of 50 to 100 .mu.M TF-2.
Despite their structural similarity, both TF-1 and TF-3 failed to affect
Cox-2 gene expression. The green tea polyphenol EGCG was also tested and
although it suppressed Cox-2 gene expression it was not as potent as TF-2.
The effect of TF-2 on the time course of Cox-2 gene expression in Caco-2
cells was then examined. Cox-2 mRNA was detectable in quiescent Caco-2
cells, consistent with the finding that colon cancer cells have elevated
Cox-2 gene expression. Fresh serum induced a 2- to 4-fold increase in
Cox-2 expression in Caco-2 cells as early as 4 hours after stimulation;
this was expected because of the known stimulatory effects of serum
factors. The presence of TF-2 during serum stimulation, however, not only
completely blocked the serum-induced increase in Cox-2 gene expression but
also abolished the basal level of Cox-2 mRNA.
The effect of TF-2 on Cox-2 gene expression was also examined in W138 and
W138VA cells following serum stimulation. Unlike Caco-2 cells, no Cox-2
mRNA was detectable in quiescent W138 or W138VA cells. However, serum
stimulation significantly induced the production of a 4.5 kb Cox-2
transcript in both W138 and W138VA cells within 4 hours of stimulation.
The levels of induced Cox-2 mRNA in W138VA cells were higher and more
sustained than that seen in W138 cells, suggesting that Cox-2 may be more
stable in transformed cells. In both transformed and non-transformed
cells, TF-2 blocked the serum-induced increase in Cox-2 gene expression.
To demonstrate that the inhibition of expression resulted in a decrease in
Cox-2 protein product, the relative levels of Cox-2 protein was measured
in W138 and W138VA cells. TF-2 treatment produced a substantial decrease
ion the levels of Cox-2 protein in both W138 and W138VA cells. TF-2 at a
concentration of 40 .mu.M reduced the Cox-2 protein level in W138 cells by
about 50% and completely eliminated Cox-2 protein levels in W138VA cells.
To determine whether the effect of TF-2 on Cox-2 gene expression was
specific for this gene or part of a global suppression of serum inducible
genes, the effects of TF-2 on the expression of several other genes were
determined (e.g., c-fos, c-myc, thymidine kinase, BRCA1, BRCA2, PCNA,
Cox-1). Of the genes examined, the only one affected significantly by TF-2
was Cox-2. Expression of the Cox-1 gene was completely unaffected by TF-2,
indicating that the effects of TF-2 were Cox-2-specific.
The data provided herein for black tea extracts, specifically TF-2,
support the development of foods and dietary supplements which comprise
TF-2 for animal consumption. For purposes of the present invention by
"animal" it is meant to include humans. These foods and supplements are
referred to by those of skill in the art as "nutraceuticals". Based upon
the experiments described herein, it is expected that compositions
comprising TF-2 will be useful as nutraceuticals for prevention or
treatment of cancer, as well as for other diseases associated with Cox-2
including but not limited to inflammation and arthritis. One of skill can
use the results of experiments in cells and animals described herein to
determine effective amounts to be administered to other animals, including
humans. By "effective amount" it is meant a concentration that inhibits
tumor growth, or another appropriate pharmacological endpoint, either in
vitro in cells or in vivo in animals. For example, human test doses can be
extrapolated from effective doses in cell studies, such as IC.sub.50
values, or from effective doses in vivo by extrapolating on a body weight
or surface area basis. Such extrapolations are routine in the art.
Compositions comprising TF-2 can be formulated for administration as a
food supplement using one or more fillers. Alternatively, compositions
comprising these extracts can be administered as conventional
pharmaceuticals using one or more physiologically acceptable carriers or
excipients. Nutraceutical compositions can be formulated for
administration by any route including, but not limited to, inhalation or
insufflation (through mouth or nose), oral, buccal, parenteral, vaginal,
or rectal administration. In one embodiment, oral administration, the
compositions are added directly to foods and ingested as part of a normal
meal. Various methods are known to those skilled in the art for addition
or incorporation of nutraceuticals into foods.
Compositions for use in the present invention can also be administered in
the form or tablets or capsules prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents, fillers,
lubricants, disintegrants, or wetting agents. Examples of specific
compounds for use in formulating tablets and capsules are described in
detail in the U.S. Pharmacopeia. Tablets comprising the extract can also
be coated by methods well known in the art. Liquid preparations for oral
administration can also be used. Liquid preparations can be in the form of
solutions, syrups or suspensions, or a dry product for reconstitution with
water or another suitable vehicle before use. Such liquid preparations can
be prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents, emulsifying agents, non-aqueous
vehicles, and preservatives. Again, specific additives are well known to
those of skill and are listed in places such as the U.S. Pharmacopeia. In
one embodiment, the oral preparation is formulated to provide controlled
time release of the active nutraceutical components. For buccal
administration the extract can be formulated as a tablet or lozenge.
For administration by inhalation, compositions for use in the present
invention can be delivered in the form of an aerosol spray in a
pressurized package or as a nebulizer, with use of suitable propellants.
In the case of a pressurized aerosol, the dosage unit can be determined by
providing a valve to deliver a metered dose.
Parenterally administered compositions are formulated to allow for
injection, either as a bolus or as a continuous infusion. Formulations for
injection can be prepared in unit dosage forms, such as ampules, or in
multi-dose units, with added preservatives. The compositions for injection
can be in the form of suspensions, solutions, or emulsions, in either oily
or aqueous vehicles. They may also contain formulatory agents such as
suspending agents, stabilizing agents, and/or dispersing agents. The
active ingredient may also be presented in powder form for reconstitution
with a suitable vehicle before use. Specific examples of formulating
agents for parenteral injection are found in the U.S. Pharmacopeia.
For rectal administration or vaginal administration, compositions for use
in of the present invention can be formulated as suppositories, creams,
gels, or retention enemas.
For dietary supplements, the extract can be added in concentrations up to
5% by weight and mixed according to methods routine in the art. Dietary
supplements for animals can be prepared in a variety of forms including,
but not limited to, liquid, powder, or solid pill forms. In the present
invention, TF-2 can administered either alone or in combination with other
phytochemicals known to affect tumor cell growth, where combining
compounds or extracts would lead to synergistic effects. Examples of other
phytochemicals which can be used in combination with TF-2 include, but are
not limited to, resveratrol and its hydroxylated and methoxylated analogs,
rosemary extract, green tea extracts, orange peel extracts, Mexican
Bamboo, and Huzhang extracts.
Claim 1 of 15 Claims
1. A method of treating or
preventing inflammation in an animal comprising, administering orally to an
animal in need thereof a composition comprising an ethyl acetate extract of
black tea, said extract comprising theaflavin-3-gallate and
theaflavin-3'-gallate in an amount effective to suppress Cyclooxygenase-2
(COX-2) gene expression in the animal so as to thereby treat or prevent
inflammation in the animal.
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