U.S. patent application number 13/372034 was filed with the patent office on 2012-06-07 for stabilized 3-hydroxyflavan compositions and methods therefor.
Invention is credited to Yukihiko Hara, Hiroshi Hojo, Slobodan Jovanovic.
Application Number | 20120142947 13/372034 |
Document ID | / |
Family ID | 37727597 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120142947 |
Kind Code |
A1 |
Hara; Yukihiko ; et
al. |
June 7, 2012 |
Stabilized 3-Hydroxyflavan Compositions and Methods Therefor
Abstract
Compositions and methods are directed to covalent adducts
between reducing agents and optionally substituted
3-hydroxyflavans, wherein the reducing agent is covalently bound to
the B-ring of the 3-hydroxyflavan. Such adducts exhibit markedly
increased stability towards oxidation as compared to the unmodified
3-hydroxyflavan. Particularly preferred 3-hydroxyflavans include
green tea catechins, and especially EGCG, while especially
preferred reducing agents include NAC and glutathione.
Inventors: |
Hara; Yukihiko; (Tokyo,
JP) ; Hojo; Hiroshi; (Shizuoka, JP) ;
Jovanovic; Slobodan; (Ontario, CA) |
Family ID: |
37727597 |
Appl. No.: |
13/372034 |
Filed: |
February 13, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11996933 |
Jul 21, 2008 |
8138359 |
|
|
PCT/US05/26521 |
Jul 26, 2005 |
|
|
|
13372034 |
|
|
|
|
Current U.S.
Class: |
549/399 |
Current CPC
Class: |
C07D 311/62
20130101 |
Class at
Publication: |
549/399 |
International
Class: |
C07D 311/62 20060101
C07D311/62 |
Claims
1. A composition comprising: a reducing agent in a first quantity,
and a 3-hydroxyflavan in a second quantity, wherein the
3-hydroxyflavan has an A-Ring and a B-ring; wherein the reducing
agent is selected from the group consisting of an N-substituted
cysteine, glutathione, dithiothreitol, dithioerythrol,
mercaptoethanol, and Tris(2-carboxyethyl)phosphine; wherein the
first quantity is a predetermined function of the second quantity
such that the reducing agent is present in an amount effective to
reduce discoloration of the composition; wherein the discoloration
is due to at least one of a non-enzymatic oxidation, a
non-enzymatic dimerization, a non-enzymatic oligomerization, and a
non-enzymatic polymerization of the 3-hydroxyflavan; wherein the
reducing agent has a structure to allow reaction with an atom in
the B-ring of the 3-hydroxyflavan to form a covalent adduct to
thereby reduce the discoloration, with the proviso that (a) where
the 3-hydroxyflavan is catechin, then the reducing agent is not
glutathione or glutamine-cysteine dipeptide; (b) where the
3-hydroxyflavan is epicatechin, then the reducing agent is not
dithiothreitol or glutamine-cysteine dipeptide.
2. The composition of claim 1 wherein the reducing agent is present
in at least stoichiometric quantity relative to the
3-hydroxyflavan.
3. The composition of claim 1 wherein the composition is a cosmetic
formulation for topical administration to skin.
4. The composition of claim 1 wherein the composition is a
nutritional formulation for enteral administration to a mammal.
5. The composition of claim 1 wherein the composition is an
intermediate in a process for isolation of a 3-hydroxyflavan.
6. The composition of claim 1 wherein the 3-hydroxyflavan is
selected from the group consisting of (-)-Epigallocatechin gallate,
(-)-Gallocatechin gallate, (+)-Epigallocatechin gallate,
(+)-Gallocatechin gallate, (-)-Epigallocatechin, (-)-Gallocatechin,
(+)-Epigallocatechin, (+)-Gallocatechin, (-)-Epicatechin gallate,
(-)-Catechin gallate, (+)-Epicatechin gallate, (+)-Catechin
gallate, (-)-Epicatechin, (-)-Catechin, (+)-Epicatechin, and
(+)-Catechin.
7. The composition of claim 1 wherein the 3-hydroxyflavan is
selected from the group consisting of (-)-Epigallocatechin gallate,
(-)-Gallocatechin gallate, (+)-Epigallocatechin gallate,
(+)-Gallocatechin gallate, (-)-Epigallocatechin, (-)-Gallocatechin,
(+)-Epigallocatechin, and (+)-Gallocatechin.
8. The composition of claim 1 wherein the 3-hydroxyflavan is
selected from the group consisting of (-)-Epigallocatechin gallate,
(-)-Gallocatechin gallate, (+)-Epigallocatechin gallate, and
(+)-Gallocatechin gallate.
9. The composition of claim 1 having a pH between 4.0 and 6.0,
inclusive
10. The composition of claim 1 wherein the reducing agent is
selected from the group consisting of glutathione and N-acetyl
cysteine.
11. A method of reducing non-enzymatic discoloration of a
3-hydroxyflavan-containing composition, wherein the non-enzymatic
discoloration is due to at least one of an oxidation, a
dimerization, a oligomerization, and a polymerization of the
3-hydroxyflavan, comprising: combining the 3-hydroxyflavan with a
reducing agent selected from the group consisting of an
N-substituted cysteine, glutathione, dithiothreitol,
dithioerythrol, mercaptoethanol, and Tris(2-carboxyethyl)phosphine
under conditions that allow reaction of the reducing agent with an
atom in the B-ring of the 3-hydroxyflavan to form a covalent adduct
to thereby reduce the discoloration; with the proviso that (a)
where the 3-hydroxyflavan is catechin, then the reducing agent is
not glutathione or glutamine-cysteine dipeptide; (b) where the
3-hydroxyflavan is epicatechin, then the reducing agent is not
dithiothreitol or glutamine-cysteine dipeptide.
12. The method of claim 11 wherein the reducing agent is present in
a stoichiometric quantity relative to the 3-hydroxyflavan.
13. The method of claim 11 wherein the 3-hydroxyflavan is selected
from the group consisting of (-)-Epigallocatechin gallate,
(-)-Gallocatechin gallate, (+)-Epigallocatechin gallate,
(+)-Gallocatechin gallate, (-)-Epigallocatechin, (-)-Gallocatechin,
(+)-Epigallocatechin, (+)-Gallocatechin, (-)-Epicatechin gallate,
(-)-Catechin gallate, (+)-Epicatechin gallate, (+)-Catechin
gallate, (-)-Epicatechin, (-)-Catechin, (+)-Epicatechin, and
(+)-Catechin.
14. The method of claim 11 wherein the 3-hydroxyflavan is selected
from the group consisting of (-)-Epigallocatechin gallate,
(-)-Gallocatechin gallate, (+)-Epigallocatechin gallate,
(+)-Gallocatechin gallate, (-)-Epigallocatechin, (-)-Gallocatechin,
(+)-Epigallocatechin, and (+)-Gallocatechin.
15. The method of claim 11 wherein the 3-hydroxyflavan is selected
from the group consisting of (-)-Epigallocatechin gallate,
(-)-Gallocatechin gallate, (+)-Epigallocatechin gallate and
(+)-Gallocatechin gallate.
16. The method of claim 11 further comprising a step of adjusting
the pH of the composition to 4.0 to 6.0, inclusive.
17. The composition of claim 11 wherein the reducing agent is
selected from the group consisting of glutathione and N-acetyl
cysteine.
Description
[0001] This application is a divisional application of allowed
application with Ser. No. 11/996,933, which was filed Jul. 21,
2008, and which is a 371 application of International patent
application having serial number PCT/US05/26521, which was filed
Jul. 26, 2005.
FIELD OF THE INVENTION
[0002] Compositions and methods for chemically modified catechins
and catechin-containing compositions, especially as they relate to
stabilized catechins and compositions.
BACKGROUND OF THE INVENTION
[0003] Catechins, and particularly green tea catechins are well
known in numerous uses for improving health. For example, many
nutritional supplements and cosmetic formulations comprise
catechins and other allegedly active ingredients, including various
antioxidants, minerals, vitamins, etc. Unfortunately, most
catechins, and especially epigallocatechin gallate (EGCG) are
relatively unstable and tend to oxidize rapidly. Worse yet,
oxidation is typically exacerbated in aqueous environment in the
presence of metal ions.
[0004] Not surprisingly, numerous strategies have been reported to
reduce EGCG oxidation using various strategies. For example,
compositions may be formulated in the absence of oxygen.
Alternatively, EGCG oxidation may be delayed by providing
sacrificial compounds and/or anti-oxidants to the formulations.
Antioxidants have also been combined with EGCG for other purposes,
and especially to provide reduction of oxidative stress in patients
that are also treated with EGCG. For example, EGCG is combined with
NAC as described in U.S. Pat. App. Nos. 2003/0170319 and
2004/0063648 in formulations to treat cancer. In U.S. Pat. No.
6,299,925, EGCG is combined with NAC in an effervescent drink,
while the same combination is taught in GB2385768 for animal feed.
While such known combinations at least sometimes provide beneficial
effects, EGCG concentrations in such composition often rapidly
decline due to autoxidation or other oxidative processes. Selected
cysteine conjugates with catechins were described in WO 03/024951,
in which cysteine was covalently reacted with the C-ring in
4-position to form an antioxidant with increased antioxidative
properties. While such conjugates may provide at least some
advantages, various disadvantages nevertheless remain. Among other
things, as the adduct is formed on the C-ring in 4-position, such
adducts are generally not available for gallate esters of catechins
(e.g., EGCG). Furthermore, such modifications will introduce a net
charge into the molecule in many environments, and thereby tend to
prevent passive transmembrane transport.
[0005] Therefore, while numerous compositions and methods for
catechins and catechin-containing compositions are known in the
art, all or almost all of them, suffer from one or more
disadvantage. Thus, there is still a need for stabilized catechin
compositions and methods therefor.
SUMMARY OF THE INVENTION
[0006] The present invention is directed to compositions and
methods of reducing catechin oxidation wherein a composition
comprises an isolated 3-hydroxyflavan adduct in which a reducing
agent is covalently bound to a B-ring of the 3-hydroxyflavan. Most
preferably, the 3-hydroxyflavan is (-)-Epigallocatechin gallate
(EGCG), (-)-Gallocatechin gallate (GCG), (+)-Epigallocatechin
gallate, (+)-Gallocatechin gallate, (-)-Epigallocatechin (EGC),
(-)-Gallocatechin (GC), (+)-Epigallocatechin, (+)-Gallocatechin,
(-)-Epicatechin gallate (ECG), (-)-Catechin gallate (CG),
(+)-Epicatechin gallate, (+)-Catechin gallate, (-)-Epicatechin
(EC), (-)-Catechin(C), (+)-Epicatechin and/or (+)-Catechin. It is
also preferred that the reducing agent in such compositions is
covalently bound via a sulfur atom to a carbon atom of the
B-ring.
[0007] In further preferred aspects, the reducing agent is a
nutritionally acceptable reducing agent (N-acetyl cysteine).
Alternatively, or additionally, suitable reducing agents may also
include various N-substituted cysteines, glutathione,
dithiothreitol (DTT), dithioerythrol (DTE), mercaptoethanol, and/or
Tris(2-carboxyethyl)phosphine (TCEP). Most preferably, the pH of
contemplated compositions is neutral to acidic, and typically
between pH between 4.0 and 6.0, inclusive.
[0008] Thus, and viewed from a different perspective, preferred
3-hydroxyflavan adducts will have a structure according to Formula
I (Rings are identified with A, B, and C):
##STR00001##
[0009] in which n is independently between 0 and 3, inclusive;
R.sub.1 is selected from H, OH, an optionally substituted aryl
ester, an optionally substituted aryl thioester, and an optionally
substituted aryl amide; and in which R.sub.2 is a reducing agent
that is covalently bound via a sulfur atom to the carbon atom of
the B-ring. Consequently, the term "3-hydroxyflavan" as used herein
expressly includes compounds with a 3-OH group and a substituted
3-OH group (e.g., in which the OH group is esterified with a
substituted benzoic acid [e.g., Gallic acid]).
[0010] Most preferably, R.sub.1 and R.sub.2 have a structure
according to Formula II and Formula III, respectively,
##STR00002##
[0011] wherein n is between 0 and 3, inclusive, and wherein
R.sub.3, R.sub.4, and R.sub.5 are independently selected from the
group consisting of H, acyl, alkyl, aryl, each of which may be
substituted, and wherein m is an integer between 1 and 5.
[0012] In another aspect of the inventive subject matter, a
composition includes a reducing agent in a first quantity, and a
3-hydroxyflavan in a second quantity, wherein the 3-hydroxyflavan
has an A-ring and a B-ring, wherein the first quantity is a
predetermined function of the second quantity such that the
reducing agent is present in an amount effective to reduce
discoloration of the composition, wherein the discoloration is due
to at least one of an oxidation, a dimerization, a oligomerization,
and a polymerization of the 3-hydroxyflavan, and wherein the
reducing agent has a structure to allow reaction with an atom in
the B-ring of the 3-hydroxyflavan to form a covalent adduct to
thereby reduce the discoloration.
[0013] Most typically, the reducing agent is present in such
compositions in at least stoichiometric quantity relative to the
3-hydroxyflavan, and the composition is a cosmetic formulation for
topical administration to skin or a nutritional formulation for
enteral administration to a mammal. Alternatively, contemplated
compositions may also be an intermediate in a process for isolation
of a 3-hydroxyflavan.
[0014] In yet another aspect of the inventive subject matter, a
method of reducing discoloration of a 3-hydroxyflavan-containing
composition (wherein the discoloration is due to oxidation,
dimerization, oligomerization, and/or polymerization of the
3-hydroxyflavan) comprises one step in which the 3-hydroxyflavan is
combined with a reducing agent under conditions that allow reaction
of the reducing agent with an atom in the B-ring of the
3-hydroxyflavan to form a covalent adduct to thereby reduce the
discoloration.
[0015] Various objects, features, aspects and advantages of the
present invention will become more apparent from the following
detailed description of preferred embodiments of the invention,
along with the accompanying drawing.
BRIEF DESCRIPTION OF THE DRAWING
[0016] FIG. 1A is a graph depicting color formation of EGCG in
aqueous solutions of varying pH over time in the presence of
NAC.
[0017] FIG. 1B is a graph depicting color formation of EGCG in
aqueous solutions of varying pH over time in the absence of
NAC.
[0018] FIG. 1C is a graph depicting color formation as a function
of pH of the solvent in the presence and absence of NAC.
[0019] FIG. 2 is a graph depicting an elution profile of a solution
in which EGCG was allowed to react with NAC to form covalent
adducts.
DETAILED DESCRIPTION
[0020] The inventors discovered that oxidation of numerous
3-hydroxyflavans, and especially oxidative discoloration of
compositions comprising EGCG and other tea catechins can be
reduced, and in many instances even entirely avoided where the
3-hydroxyflavan is combined with a reducing agent at a
concentration and under conditions effective to form a covalent
adduct between the 3-hydroxyflavan and the reducing agent.
[0021] Most preferably, contemplated 3-hydroxyflavans include those
typically found in plant catechins, and especially in green tea
catechins. Therefore, suitable 3-hydroxyflavans may be present in
complex mixtures (e.g., polyphenon E or polyphenon B (produced by
Mitsui Norin Co., Ltd)), or as isolated compounds (e.g., EGCG).
With respect to preferred reducing agents, it is generally
contemplated that such agents will be suitable for nutritional,
pharmaceutical, and/or topical formulations, Thus, and among other
preferred reducing to agents, NAC is especially contemplated.
[0022] In one exemplary aspect of the inventive subject matter,
EGCG is combined with N-acetyl cysteine (NAC) in an aqueous
solvent, wherein NAC is present in two-fold molar excess relative
to EGCG, which is present at a concentration of about 2 mM. The
aqueous solution is buffered with 100 mM phosphate buffer at a pH
of 6.5 and the reaction is allowed to proceed for 24 hours at
elevated temperature (typically 55-60.degree. C.) to form the
products (-)-EGCG-NAC and (-)-GCG-NAC as shown below.
##STR00003##
[0023] It should be appreciated, however, that numerous alternative
3-hydroxyflavans are also deemed suitable for use herein, and
especially preferred 3-hydroxyflavans include (-)-Epigallocatechin
gallate, (-)-Gallocatechin gallate, (+)-Epigallocatechin gallate,
(+)-Gallocatechin gallate, (-)-Epigallocatechin, (-)-Gallocatechin,
(+)-Epigallocatechin, (+)-Gallocatechin, (-)-Epicatechin gallate,
(-)-Catechin gallate, (+)-Epicatechin gallate, (+)-Catechin
gallate, (-)-Epicatechin, (-)-Catechin, (+)-Epicatechin, and/or
(+)-Catechin. Still further preferred 3-hydroxyflavans will
generally have a structure according to Formula IV
##STR00004##
[0024] in which R.sub.1 and R.sub.2 are independently H, OH, SH,
halogen, alkyl, alkenyl, alkynyl, aryl, O-alkyl, amino radical,
amido radical, imino radical, each of which may be optionally
substituted, in which R.sub.3 is H, optionally substituted aryl
ester, an optionally substituted aryl thioester, and an optionally
substituted aryl amide, and in which n is independently an integer
between 0 and 5. Furthermore, it should be appreciated that
contemplated compounds may have one or more chiral centers, and it
is noted that all stereochemical configurations and conformations
are contemplated herein.
[0025] Therefore, in alternative aspects of the inventive subject
matter, the 3-hydroxyflavans may be reacted as individual
compounds, or in mixtures of two or more 3-hydroxyflavans. For
example, where EGCG oxidation is to be prevented, EGCG may be
reacted as the sole reactant. On the other hand, where catechin
extracts are reacted with the reducing agent(s), complex mixtures
(e.g., polyphenon E (produced by Mitsui Norin Co., Ltd)) may be
combined with the reducing agent(s). Adducts are expected to be
formed depending on the reactivity of the particular
3-hydroxyflavan and reaction conditions chosen.
[0026] Preferably, the adduct forming reaction is carried out in an
aqueous solvent, which may or may not further include an organic
co-solvent (e.g., to increase solubility of one or more
components). Alternatively, reactions may also be carried out in an
organic solvent, and most typically in an organic solvent that is
used to isolate and/or concentrate the catechins (e.g., ethanol,
methanol, acetone, DMF, DMSO, carbon dioxide, etc). Typically the
reaction is performed at neutral (e.g., pH 6.5 to 7.5) to slightly
acidic pH (e.g., pH 4.5 to 6.4), but may also be performed at
acidic (e.g., pH of less than 4.5) or basic conditions (e.g., pH of
greater than 7.5). Still further, it should be appreciated that
suitable reaction conditions may include use of a catalyst,
pre-activation of one or more reactants (e.g., deprotonation of
thiol group, addition of leaving group into 3-hydroxyflavan, etc.),
or other manner that increases yield of the adduct.
[0027] With respect to appropriate reaction temperature and
duration, it should be recognized that a person of ordinary skill
in the art will be readily able to determine optimum conditions
(e.g., based on chromatographic analysis of reaction products).
However, it is typically preferred that the temperature is between
about 20.degree. C. and 70.degree. C., and most preferably between
about 40.degree. C. and 60.degree. C. Similarly, it should be
recognized that the duration of the reaction may vary considerably,
and that the exact duration may depend on numerous factors,
including desired yield, reaction temperature, solvents, the
reducing agent, etc. Therefore, contemplated reaction temperatures
may be between several minutes to several days, or even more. For
example, where the temperature is relatively high (e.g., greater
than 70.degree. C.) and relatively low yields (e.g., less than 20%)
of the adduct are desired, suitable reaction durations may be
between 1 and 240 minutes. On the other hand, and especially where
relatively high yields (e.g., more than 80%) of the adduct are
desired, suitable reaction durations may be between 12 hours and 96
hours (or even longer). Still further, it is contemplated that the
reducing agent and the 3-hydroxyflavan may be combined at room
temperature (typically about 20.degree. C.) or other temperature
without a predetermined time and/or temperature. In such case, it
is contemplated that the adduct formation will proceed relatively
slowly and may not reach completion over several days or even
weeks.
[0028] In further preferred aspects, the molar ratio between the
3-hydroxyflavan and the reducing agent is preferably at least
stoichiometric, and more preferably, the reducing agent is present
in a molar excess relative to the 3-hydroxyflavan. For example, it
is contemplated that the molar ratio of reducing agent to
3-hydroxyflavan is at least 1:1.0, more typically at least 1:1.5,
even more typically at least 1:2.0, and most typically at least
1:2.5 (or even higher). Consequently, yields of the so formed
adducts may vary substantially, and all yields are generally
contemplated, including 0-10%, more typically 10-30%, even more
typically 30-70%, and most typically 70-99.9%.
[0029] With respect to suitable reducing agents, it is generally
contemplated that numerous reducing agents known in the art are
suitable so long as such reducing agents can react with
contemplated 3-hydroxyflavans to form a covalent adduct. However,
most typically, reducing agents will include a thiol group,
hydroxyl group, or are a phosphine. Furthermore, it is contemplated
that the reducing agent may be a single compound, or a mixture of
two or more reducing agents. Similarly, it should be recognized
that a single (or more) reducing agent may be reacted with a
mixture of 3-hydroxyflavans to form the corresponding covalent
adducts. In most preferred aspects, the reducing agent is suitable
for pharmaceutical, nutraceutical, and/or topical use (e.g., those
having an SH group, including N-substituted cysteine, glutathione,
dithiothreitol, dithioerythrol, or mercaptoethanol). Thus, the
reducing agent is preferably isolated from natural sources. For
example, such preferred reducing agents particularly include
N-acetyl cysteine isolated from Shiitake. Where NAC is isolated
from a natural source (e.g., plant, fungus, bacteria, etc.) it is
especially preferred that the 3-hydroxyflavan is also isolated from
a natural source (typically green tea). Alternatively, suitable
reducing agents may also be synthetic (e.g.,
Tris(2-carboxyethyl)phosphine, or substituted sulfur, selenium, or
oxygen-containing amino acid). Still further contemplated reducing
agents include those that are capable of reacting as a nucleophile
in an aromatic nucleophilic reaction (e.g., addition or
substitution) on the B-ring of contemplated 3-hydroxyflavans.
[0030] So formed covalent adducts may be isolated (using at least
one step of enrichment), or may be directly used without further
workup. Thus, the adducts may be a pure preparation, which may
include selected stereoisomers, enantiomers, etc., or may be a
relatively complex mixture, which may or may not further comprise
residual reducing agent and/or 3-hydroxyflavan. Contemplated
3-hydroxyflavan adducts may therefore be isolated (i.e., enriched
in the adduct using at least one purification step), or be present
in a crude reaction mixture.
[0031] Consequently, the inventors contemplate a composition that
includes an isolated 3-hydroxyflavan adduct in which a reducing
agent is covalently bound to a B-ring of the 3-hydroxyflavan. The
term "reducing agent is covalently bound to a B-ring" as used
herein means that a reducing agent has reacted with an atom of the
B-ring. Therefore, in the covalently bound state, the reducing
agent may be chemically changed (as compared to the chemical
structure of the reducing agent before the reaction) and may
therefore also have altered properties with respect to the ability
to act as a reducing agent. Such adduct may be present in
contemplated composition to varying degree. For example,
contemplated compositions may include the adduct in an amount of
between about 0.1 wt % to about 2 wt %, more preferably between
about 2 wt % to 10 wt %, and most preferably between about 10 wt %
and 50 wt % (and even more). An exemplary structure of preferred
compounds is depicted in Formula I below
##STR00005##
[0032] in which n is independently between 0 and 3, inclusive;
R.sub.1 is selected from H, an optionally substituted aryl ester,
an optionally substituted aryl thioester, and an optionally
substituted aryl amide; and R.sub.2 is a reducing agent that is
covalently bound via a sulfur atom to the carbon atom of the
B-ring.
[0033] Contemplated covalent adducts and compositions comprising
same may be employed in numerous manners, however, particularly
preferred manners include incorporation into food products (e.g.,
nutritional supplements [snack bar, pill, powder, etc.], beverages
[soda, green tea, etc.], pet food, etc), cosmetic products (e.g.,
shampoo, soap, skin cream or lotion, sun screen, etc.),
pharmaceutical products (e.g., antineoplastic formulations), etc.
Most preferably, such products will have a pH that is neutral to
slightly acidic (e.g., between pH 4.0 to 6.0), but alternative pH
values are also deemed suitable herein.
[0034] Therefore, the inventors also contemplate a composition that
comprises a reducing agent in a first quantity, and a
3-hydroxyflavan (having an A-Ring and a B-ring) in a second
quantity, wherein the 3-hydroxyflavan, wherein the first quantity
is a predetermined function of the second quantity such that the
reducing agent is present in an amount effective to reduce
discoloration of the composition, wherein the discoloration is due
to oxidation, dimerization, oligomerization, and/or polymerization
of the 3-hydroxyflavan. In such compositions, it is generally
contemplated that the reducing agent has a structure to allow
reaction with an atom in the B-ring of the 3-hydroxyflavan to form
a covalent adduct to thereby reduce the discoloration. Viewed from
another perspective, the inventors contemplate a method of reducing
discoloration of a 3-hydroxyflavan-containing composition
(discoloration is due to oxidation, dimerization, oligomerization,
and/or polymerization of the 3-hydroxyflavan) that includes a step
of combining the 3-hydroxyflavan with a reducing agent under
conditions that allow reaction of the reducing agent with an atom
in the B-ring of the 3-hydroxyflavan to form a covalent adduct to
thereby reduce the discoloration.
EXPERIMENTS
Prevention of Discoloration
[0035] In a typical experiment, an aqueous solution of NAC was used
in combination with EGCG to demonstrate the stabilization of EGCG
over time. Discoloration was measured using a spectrophotometer at
a fixed wavelength of 492 nm. More specifically, solutions of EGCG
(2 mM) and NAC (4 mM) were prepared in McIlvaine buffer at various
pH levels in 1.0 increments between pH 3.0 to pH 7.0. A control
experiment was set up without NAC but otherwise identical
conditions. The so prepared solutions were incubated at 55.degree.
C. and the discoloration was measured at 492 nm over the next 15
days. FIGS. 1A and 1B depict the results in a graph in which
discoloration is plotted as a function of time, and in which each
of the individual pH values are represented by a respective curve.
As can be clearly seen, discoloration is substantially reduced at
pH levels of less than 6.0. FIG. 1C depicts the discoloration after
15 days as a function of the pH for both NAC and control.
Remarkably, while glutathione and NAC readily reacted to the
adducts and prevented discoloration at the observed pH ranges in
further experiments (data not shown), cysteine failed to provide
significant protection from discoloration.
Isolation and Structure Elucidation of Adducts from EGCG and NAC
Reaction
[0036] A reaction was prepared from EGCG (1000 mg; 2.18 mM) and NAC
(713 mg; 4.37 mM) in 0.1M phosphate buffer (pH 6.5) in a volume of
1000 ml. The solution was incubated at 60.degree. C. for 24 hours.
After completion of the reaction, HPLC analysis revealed two major
products as shown in the elution profile of FIG. 2.
[0037] More specifically, the reaction mixture was subjected to
DIAION HP-20 column chromatography using a stepped gradient of
H.sub.2O-MeOH to give adduct fractions (342.5 mg). A portion of
that fraction (100 mg) was further fractionated by preparative
reversed-phase silica gel column chromatography using a gradient of
H.sub.3PO.sub.4aq./MeCN. Compounds under peak 1 (11.3 mg) and peak
3 (12.4 mg) ware isolated, and the structure elucidated from NMR
experiments using .sup.1H, .sup.13C, H--H COSY, HMBC, NOESY, ROESY.
.sup.1H and .sup.13C-NMR spectra data are shown in the Table below.
Each compound had an N-acetyl cysteine attached to the C2'
position. The location of the B-ring was assigned as C2 position
based on NOESY and ROESY experiments, H3 signal had correlation to
H6' at peak 3, although such correlation was not observed for peak
1.
TABLE-US-00001 Peak 1 Peak 3 .sup.13C-NMR (100 MHz acetone-d 6) C2
76.4 77.1 C3 68.5 69.4 C4 27.0 26.5 C4a 99.1 99.7 C5 157.3 156.9 C6
96.5 96.5 C7 157.6 157.2 C8 95.9 95.6 C8a 157.8 158.1 C1' 134.0
133.3 C2' 108.2 111.4 C3' 148.2 148.1 C4' 133.2 133.9 C5' 147.7
147.7 C6' 108.6 107.3 C1'' 121.8 121.3 C2'' 110.0 109.9 C3'' 145.9
146.1 C4'' 138.8 139 C5'' 145.9 146.1 C6'' 110.0 109.9 galloyl-
166.0 166.1 CO CysH.alpha. 53.5 53.7 CysH.beta. 39.9 38.6 Acetyl-
22.7 22.5 CH3 Acetyl- 171.2 171.8 CO COOH 171.9 172.3 .sup.1H-NMR
(400 MHz acetone-d 6) H2 5.69 1H brs 5.91 1H d J = 8.2 H3 5.68 1H m
5.52 1H ddd J = 5.6, 8.2, 8.2 H4a 2.95 1H dd J = 1.8, 2.76 1H dd J
= 8.2, 17.5 16.0 H4b 3.06 1H dd J = 4.4, 3.18 1H dd J = 5.6, 17.5
16.0 H6 6.07 1H d J = 2.3 6.07 1H d J = 2.4 H8 6.05 1H d J = 2.3
5.9 1H d J = 2.4 H2' H6' 6.9 1H S 6.59 1H S H2'', 6'' 6.99 2H S
7.06 2H S(br) Cys-H.alpha. 4.7 1H ddd J = 4.8, 4.64 1H ddd J = 6.6,
8.0, 8.2 6.6, 7.5 Cys-H.beta. 3.16 1H dd J = 8.2, 3.2 1H dd J =
6.6, 1 13.2 14.0 Cys-H.beta. 3.23 1H dd J = 4.8, 3.28 1H dd J =
6.6, 2 13.2 14.0 N--CH3 1.95 3H S 1.91 3H S NH 7.67 1H d J = 8.0
7.81 1H d J = 7.5 Peak-1 ##STR00006## Peak-3 ##STR00007##
[0038] Thus, specific embodiments and applications of stabilized
catechin compositions and methods therefor have been disclosed. It
should be apparent, however, to those skilled in the art that many
more modifications besides those already described are possible
without departing from the inventive concepts herein. The inventive
subject matter, therefore, is not to be restricted except in the
spirit of the appended claims. Moreover, in interpreting both the
specification and the claims, all terms should be interpreted in
the broadest possible manner consistent with the context. In
particular, the terms "comprises" and "comprising" should be
interpreted as referring to elements, components, or steps in a
non-exclusive manner, indicating that the referenced elements,
components, or steps may be present, or utilized, or combined with
other elements, components, or steps that are not expressly
referenced.
* * * * *