U.S. patent application number 11/894541 was filed with the patent office on 2007-12-20 for anti-inflammatory cranberry flavonol extract preparations.
Invention is credited to Mou-Tuan Huang, Robert T. Rosen, Sharon L. Rosen, Nicholi Vorsa, Irina O. Vvedenskaya.
Application Number | 20070292539 11/894541 |
Document ID | / |
Family ID | 36206471 |
Filed Date | 2007-12-20 |
United States Patent
Application |
20070292539 |
Kind Code |
A1 |
Vorsa; Nicholi ; et
al. |
December 20, 2007 |
Anti-inflammatory cranberry flavonol extract preparations
Abstract
The present invention is directed to extracts of cranberries
(Vaccinium macrocarpon) comprising either mixed flavonols that are
substantially free of anthocyanins and proanthocyanidins or a
purified cranberry flavonol compound, including
myricetin-3-.beta.-xylopyranoside, quercetin-3-.beta.-glucoside,
quercetin-3-.alpha.-arabinopyranoside,
3'-methoxyquercetin-3-.alpha.-xylopyranoside,
quercetin-3-O-(6''-p-coumaroyl)-.beta.-galactoside, and
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside. The present
invention also embodies the use of those extracts, as well as
extracts comprising the cranberry flavonol compound
quercetin-3-.alpha.-arabinofuranoside, for the treatment of
inflammatory disorders. Pharmaceutical, food, dietary supplement,
and cosmetic compositions utilizing the extracts or compounds of
the present invention are also recited.
Inventors: |
Vorsa; Nicholi; (Atco,
NJ) ; Vvedenskaya; Irina O.; (Edison, NJ) ;
Huang; Mou-Tuan; (Englewood Cliffs, NJ) ; Rosen;
Robert T.; (Monroe Township, NJ) ; Rosen; Sharon
L.; (Monroe Township, NJ) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Family ID: |
36206471 |
Appl. No.: |
11/894541 |
Filed: |
August 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10985471 |
Nov 10, 2004 |
7270837 |
|
|
11894541 |
Aug 20, 2007 |
|
|
|
60518294 |
Nov 10, 2003 |
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Current U.S.
Class: |
424/732 ;
426/531; 426/599; 426/615; 426/655; 514/456; 514/738; 549/400 |
Current CPC
Class: |
A23L 33/11 20160801;
A61P 29/00 20180101; A61K 31/7048 20130101; A61P 43/00 20180101;
A61K 31/353 20130101; A61K 36/45 20130101 |
Class at
Publication: |
424/732 ;
426/531; 426/599; 426/615; 426/655; 514/456; 514/738; 549/400 |
International
Class: |
A61K 36/45 20060101
A61K036/45; A23L 1/30 20060101 A23L001/30; A23L 2/02 20060101
A23L002/02; A61P 43/00 20060101 A61P043/00; C07D 311/00 20060101
C07D311/00; A61P 29/00 20060101 A61P029/00; A61K 31/352 20060101
A61K031/352 |
Claims
1. A flavonol composition comprising an extract of cranberry
flavonols, the extract being substantially free of anthocyanins and
proanthocyanidins, wherein each of the cranberry flavonols includes
pharmaceutically acceptable salts thereof.
2. A method for the prevention and treatment of an inflammatory
condition comprising administration of a therapeutically effective
dose of the flavonol composition of claim 1.
3. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier in admixture with the flavonol composition of
claim 1.
4. A method for the prevention and treatment of an inflammatory
condition comprising administration of a therapeutically effective
dose of the pharmaceutical composition of claim 3.
5. A dietary supplement composition comprising a consumable carrier
in admixture with the flavonol composition of claim 1.
6. A method for the prevention and treatment of an inflammatory
condition comprising administration of a therapeutically effective
dose of the dietary supplement composition of claim 5.
7. A food composition comprising a consumable carrier in admixture
with the flavonol composition of claim 1.
8. A method for the prevention and treatment of an inflammatory
condition comprising administration of a therapeutically effective
dose of the food composition of claim 7.
9. The food composition of claim 7 wherein the consumable carrier
is a cranberry-containing food product.
10. The food composition of claim 9 wherein the
cranberry-containing food product is a dried cranberry, a sweetened
and dried cranberry, a powdered cranberry, a flavored fruit piece,
a sauce, a jelly, a relish, a juice, a wine or a cranberry
juice-containing product.
11. The food composition of claim 7 wherein the consumable carrier
is a beverage.
12.-23. (canceled)
24. A compound having the formula as shown, including
pharmaceutically acceptable salts thereof: ##STR1##
25. A method for the prevention and treatment of an inflammatory
condition comprising administration of a therapeutically effective
dose of the compound of claim 24, including pharmaceutically
acceptable salts thereof.
26. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier in admixture with the compound of claim 24.
27. A method for the prevention and treatment of an inflammatory
condition comprising administration of a therapeutically effective
dose of the pharmaceutical composition of claim 26.
28. A dietary supplement composition comprising a consumable
carrier in admixture with the compound of claim 24.
29. A method for the prevention and treatment of an inflammatory
condition comprising administration of a therapeutically effective
dose of the dietary supplement composition of claim 28.
30. A food composition comprising a consumable carrier in admixture
with the compound of claim 24.
31. A method for the prevention and treatment of an inflammatory
condition comprising administration of a therapeutically effective
dose of the food composition of claim 30.
32. The food composition of claim 30 wherein the consumable carrier
is a cranberry-containing food product.
33. The food composition of claim 32, wherein the
cranberry-containing food product is a dried cranberry, a sweetened
and dried cranberry, a powdered cranberry, a flavored fruit piece,
a sauce, a jelly, a relish, a wine or a cranberry juice-containing
product.
34. The food composition of claim 30, wherein the consumable
carrier is a beverage.
35. A cosmetic composition comprising a cosmetically acceptable
carrier in admixture with the flavonol composition of claim 1.
36. A method for the prevention and treatment of an inflammatory
condition comprising administration of a therapeutically effective
dose of the cosmetic composition of claim 35.
37.-38. (canceled)
39. A cosmetic composition comprising a cosmetically acceptable
carrier in admixture with the compound of claim 24.
40. A method for the prevention and treatment of an inflammatory
condition comprising administration of a therapeutically effective
dose of the cosmetic composition of claim 39.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority under 35 USC
.sctn.119(e) to U.S. Provisional Application Ser. No. 60/518,294,
filed on Nov. 10, 2003, which is incorporated by reference in its
entirety herein.
FIELD OF THE INVENTION
[0002] The present invention is directed to extracts of cranberries
(Vaccinium macrocarpon) comprising either mixed flavonols that are
substantially free of anthocyanins and proanthocyanidins or a
purified cranberry flavonol compound, including
myricetin-3-.beta.-xylopyranoside, quercetin-3-.beta.-glucoside,
quercetin-3-.alpha.-arabinopyranoside,
3'-methoxyquercetin-3-.alpha.-xylopyranoside,
quercetin-3-O-(6''-p-coumaroyl)-.beta.-galactoside, and
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside. The present
invention also embodies the use of those extracts, as well as
extracts comprising the cranberry flavonol compound
quercetin-3-.alpha.-arabinofuranoside, for the treatment of
inflammatory disorders, particularly irritation of the urinary
tract due to both bacterial and non-bacterial causes.
BACKGROUND OF THE INVENTION
[0003] Millions of women are diagnosed with urinary tract
infections each year. Countless numbers of dogs and cats also
suffer from chronic urinary infections and die from renal
infections. E. coli is the most common pathogen associated with
these infections, causing over 80% of urinary tract infections.
Over 30% of women suffer recurrent infections within a 6 to
12-month period and are forced to resort to extended use of
antibiotics to treat these infections. Recurrent use of antibiotics
can lead to pathogen resistance and result in deleterious side
effects and toxicity reactions. Consequently there exists a need
for safe alternative medications (e.g., non-antibiotics) that can
be used to prevent or treat urinary tract infections in both
animals and humans. Moreover, urinary tract inflammation is a
painful and often debilitating symptom of bacterial infection as
well as many diseases of both known and unknown etiology. Thus,
there is currently a need for new treatments to effectively
mitigate the pain arising from inflammation of the urinary
tract.
[0004] The two main forms of urinary tract infection are the renal
infection known as pyelonephritis and the bladder infection
referred to as bacterial cystitis. As the bladder is closer to the
anus, the site of entry for bacteria giving rise to urinary tract
infections, cystitis is far more common than pyelonephritis.
Moreover, although cystitis is not as deadly as its renal
counterpart, it is associated with pervasive inflammation giving
rise to severe bladder pain as well as frequent, urgent, and
painful urination. Cystitis is also often a recurrent condition,
resulting in long term and often debilitating discomfort for those
afflicted.
[0005] Unfortunately, the causes of the battery of adverse
conditions affecting the urinary tract are only partially
understood. Thus, inflammatory conditions of the bladder of both
known and unknown etiology are commonly referred to as cystitis. As
noted above, cystitis may be bacterial in nature, arising from
infection of the bladder by E. coli. Cystitis may also embody
urinary tract inflammation due to a number of non-bacterial
sources, for example, allergic responses to food or insufficient
water intake, which allows the bladder and urethral tissues to
become dry, thus leading to deposition of crystallized uric acid on
the tissues and associated irritation. Interstitial cystitis is a
recurrent condition of uncertain etiology. It is a source of
frustration for both doctors and patients alike, as it has no
apparent infective cause and is frequently debilitating. Indeed,
according to the first epidemiological study of interstitial
cystitis in the U.S. (Held, et al. 1990), 50% of patients could not
maintain full-time employment due to the painful effects of the
disease.
[0006] Treatments for cystitis include antimicrobials,
anti-inflammatory agents, buffering agents, muscle relaxants, mast
cell stabilizers, painkillers including tricyclic antidepressants
and transcutaneous electrical nerve stimulator (TENS) units,
catheterization and intravesicular instillation of heparin, the
anti-inflammatory agent dimethyl sulfoxide, the detergent sodium
oxychlorosene, the caustic agent silver nitrate, or chromolyn
sodium, and surgical techniques. Unfortunately, many of these
treatments are themselves painful, and many are also unsuitable for
personal treatment in the home.
[0007] The fruit of the American cranberry (Vaccinium macrocarpon)
has received considerable attention for its putative human health
benefits. Most of the focus is on the flavonoid constituents due to
their relatively high biological activity in various assays. In
vitro chemical assays have rated cranberries as having some of the
highest antioxidant values of over 21 fruits (Vinson et al. 2001;
Sun et al. 2002), and the overall phenolic content appears to
correlate with the level of antioxidant activity. The
`Folin-Ciocalteu` colorimetric test has found cranberry to have one
of the highest phenolic contents of a number of fruit species
tested (Vinson et al. 2001; Sun et al. 2002). The phenolic classes
identified in cranberry include phenolic acids (Marwan et al. 1982;
Heimhuber et al. 1990; Zheng et al. 2000; Zuo et al. 2002),
anthocyanins (Hong et al. 1986; Hong et al. 1990), flavonols (Puski
et al. 1967; Yan et al. 2002), and flavan-3-ols, which consist of
both monomers and the polymer classes procyanidins and
proanthocyanidins (Foo et al. 1981; Foo et al. 2000a; Foo et al.
2000b; Cunningham et al. 2002). As described more fully below, we
have previously identified cranberry A-type proanthocyanidins as
possessing anti-adherence activities against uropathogenic type P
E. coli (Foo et al. 2000a; Foo et al. 2000b; U.S. Pat. No.
6,608,102).
[0008] Cranberry juice has been shown to reduce bacteriuria
associated with urinary tract infections in humans (Avorn et al.
1994). One mechanism implicated in yielding this beneficial effect
is the ability of certain compounds present in cranberries to
inhibit the adhesion of type I and type P fimbriated E. coli to
human epithelial cells (Sobota 1984; Schmidt et al. 1988; Zafriri
et al. 1989). Type P fimbriated E. coli have been implicated as the
main cause of pyelonephritis, while type I fimbriated E. coli are
the predominant causative agent of bacterial cystitis.
[0009] Zafriri et al. (1989) reported that fructose present in
cranberry juice inhibited the adherence of type 1 E. coli to
uroepithelial cells, and that cranberry juice also contained one or
more non-dialyzable substances that inhibited binding of type P E.
coli but failed to define the chemical nature of those
substances.
[0010] U.S. Pat. Nos. 5,474,774, 5,525,341, and 5,646,178 to Walker
et al. disclose cranberry extracts having the ability to inhibit
the adherence of E. coli to uroepithelial cells. This activity was
obtained by extracting whole cranberries with acidified alcohol
followed by separation of the activity from simple sugars by
precipitation with a metal acetate or sulfate. Upon further
manipulation, the reported activity consisted of a fraction
enriched in polyphenol and flavonoid compounds that contained as
much as 10% anthocyanins. The specificity of this anti-adherence
activity for type 1 or type P E. coli was, however, not
determined.
[0011] WO 96/30033 and U.S. Pat. Nos. 5,646,178 and 5,650,432 to
Walker et al. disclose a series of proanthocyanidin monomers,
dimers, and polymers as well as flavonoid derivatives thereof and
related compounds purported to have the ability to interfere with
bacterial adherence to a surface. The dimers and polymers of Walker
were limited to compounds having B-type interflavanoid linkages.
However, Walker failed to provide any experimental data correlating
biological activity with a specifically identified compound. The
extraction method involved alkalinizing a plant material homogenate
to a pH greater than 10 (a treatment which causes degradation of
proanthocyanidins) and precipitating the polyphenolic compounds
(together with other materials) by addition of alcohol. This
precipitate contained the proposed anti-adherence activity and was
further fractionated to yield the purified active compound. Using
this process with an aqueous solution of commercially available
Ocean Spray cranberry powder, Walker reported obtaining a single
active compound and partially characterized the compound but failed
to provide its chemical structure. The Walker assay methods could
not distinguish between anti-adherence activities with respect to
type 1 or type P E. coli, thus Walker was also unable to
characterize the biological activity of this compound.
[0012] Commonly assigned U.S. Pat. No. 6,608,102 to Howell et al.
discloses plant proanthocyanidin extracts that are substantially
free of anthocyanins and flavonols and specifically inhibit the
adherence of type P E. coli to epithelial cells. The Howell '102
patent represents the culmination of our work that conclusively
demonstrated that proanthocyanidins are the chemical agents in
cranberries and other plants that are responsible for this
anti-adherence activity, and that proanthocyanidins having at least
one A-type interflavanoid linkage are particularly potent agents
for this activity. Later U.S. Pat. Nos. 6,210,681 and 6,440,471 to
Walker et al. disclose similar proanthocyanidin extracts.
[0013] Thus, we have previously determined that proanthocyanidins
present in cranberries exhibit anti-adherence activity with respect
to the binding of uroepithelial cells solely by type P fimbriated
E. coli. However, as bladder infections are associated with type 1
fimbriated E. coli, the anti-adherence activity of these compounds
is ineffective for the treatment of bacterial cystitis. Although
fructose in cranberry juice has been found to possess an analogous
anti-adherence activity with respect to the binding of type 1
fimbriated E. coli to uroepithelial cells and is therefore
theoretically effective at hindering bladder infections, this
discovery was made well over a decade ago, yet there remains a
substantial need for efficacious treatments for cystitis.
[0014] Cranberries and cranberry products have been used in the
treatment of urinary tract infections both alone and in conjunction
with other therapies. The basis for such treatments has
traditionally implicated the antimicrobial properties associated
with cranberries. Originally, this antimicrobial activity was
thought to arise from acidification of the urine due to the intake
of cranberry juice, resulting in an unfavorable environment for
bacterial survival. Later, the focus shifted to the bacterial
anti-adherence activity of fructose and proanthocyanidins that has
been elucidated in the aforementioned literature. For example,
Cystopurin, a Roche product for the treatment of cystitis that is
available in the UK, incorporates a cranberry juice extract as an
adjunctive therapy in a potassium citrate buffer designed to
alleviate painful urination by neutralizing urinary acidity.
However, as noted above, the only constituent of cranberries known
to possess anti-adherence activity against the type 1 fimbriated E.
coli commonly involved in bacterial cystitis is fructose. Moreover,
inflammation of the urinary tract may also arise from non-bacterial
sources, in which case such anti-adherence activities are
therapeutically ineffective. Finally, individuals having
non-bacterial cystitis must exercise care in the process of
self-medication with cranberry products, as, depending on the
nature of the preparation, acids and other bladder irritants may be
present that could result in aggravation of the inflammatory
condition.
[0015] After bacterial adherence, internalization of type 1 E. coli
by bladder epithelial cells represents another potential target for
the treatment of bacterial cystitis. Traditional thinking had
considered uropathogenic E. coli to exist as extracellular
pathogens within the urinary tract, despite the fact that
transmission electron microscopy studies of infected rat and mouse
bladders indicated that bladder epithelial cells could internalize
the pathogens in vivo (Fukushi et al. 1979; Mc Taggart et al.
1990). Although such internalization was initially regarded as a
host defense mechanism, Mulvey et al. (1998) suggested that it
inured beneficially to the survival of the bacteria. Specifically,
they determined that type 1 E. coli induced programmed cell death
and exfoliation of bladder epithelial cells, however, pathogens
could avoid expulsion by such mechanisms by invading into deeper
tissue. They also speculated that the frequency of recurrence of
infection despite antibiotic treatment could be linked to the
persistence of bacteria within the cells of the bladder long after
the death of extracellular pathogens by such treatments. This
speculation was later confirmed by their discovery that a
persistent reservoir of E. coli could be established by bacterial
invasion of bladder epithelial cells, followed by intracellular
replication and reemergence of the pathogens (Mulvey et al. 2001).
Upon reemergence, the pathogens evade clearance via exfoliation of
the infected bladder cells by anchoring themselves and invading
into deeper, healthy cells that become exposed to the bladder lumen
as a result of the exfoliation of cells from more superficial
layers. These internalized pathogens can persist in a latent state
and, as the result of an as yet undetermined trigger, reemerge to
cause recurrent infections.
[0016] Martinez et al. (2000) reported that the FimH element, an
adhesin located on the tip of the type 1 pilus, mediates the
bacterial invasion of human bladder epithelial cells. Specifically,
they determined that the FimH element induces rearrangements of the
host cell cytoskeleton resulting in a zippering effect by which the
host cell engulfs the pathogen. Martinez also reported that these
cytoskeletal rearrangements required protein tyrosine
phosphorylation and phosphoinositide 3-kinase activation, and that
a quercetin derivative, LY294002 (Vlahos et al. 1994), was a potent
inhibitor of both of these activities and effectively inhibited
type 1 pilus-mediated bacterial invasion of bladder epithelial
cells in urinary tract infections. Tyrosine kinase inhibitors,
including both quercetin and myricetin, directly inhibit enzymes
such as the hexose transporter GLUT1 via specific competition for
the ATP binding site (Vera et al. 2001). Myricetin has been found
to be the most potent flavonol for inhibiting transport of
methylglucose and deoxyglucose, while iso-rhamnetin
(3'-methoxyquercetin) was the most potent at inhibiting transport
of dehydroascorbic acid (Hera et al. 2001).
[0017] Quercetin has attracted much attention for its potential
health benefits, and has been associated with numerous biological
activities, including anti-inflammatory activities (Formica et al.
1995). Quercetin and related compounds inhibit a number of the
processes associated with inflammation including lipopolysaccharide
induced production of nitric oxide and tumor necrosis factor
.alpha. (TNF-.alpha.) (Kawada et al. 1998; Wadsworth et al. 1999)
and cytokine production (Xagorari et al. 2001). The invasion of
bladder cells by type 1 E. coli has been shown to induce cytokine
production by a lipopolysaccharide dependent mechanism (Schilling
et al. 2001). Among fruit species, the cranberry contains one of
the highest concentrations of quercetin, ranging from 11 to 25
mg/100 g of fresh fruit (Bilyk et al. 1986; Hakkinen et al. 1999).
Quercetin is predominantly found in a conjugated form with various
sugars, and the sugar moiety may significantly influence its
bioavailability and adsorption (Hollman et al. 1995; Hollman et al.
1999; Woffram et al. 2002).
[0018] Quercetin has been used in the treatment of inflammatory
conditions associated with the urinary tract. U.S. patent
application Ser. Nos. 757,358 and 848,187 to Katske, et al.
disclose compositions and methods for the treatment of
non-bacterial prostatitis and non-bacterial cystitis, respectively.
These compositions comprise bioflavonoids having a substantial
percentage of quercetin that exhibit both anti-oxidative and
anti-inflammatory properties and a digestive enzyme such as
bromelin or papain to increase the intestinal absorption of the
bioflavonoid component. The compositions are directed towards
treating the pain associated with cystitis and prostatitis by an
anti-inflammatory mechanism. However, substances such as papain are
allergenic to many individuals, and these enzymes have caustic and
corrosive effects on the digestive mucous membranes.
[0019] Thus, there is presently a need for non-antibiotic
treatments for urinary tract infections. There is also a need for a
generalized therapy for cystitis, whether bacterial or
non-bacterial in origin, that is innocuous and can be used for
self-medication in the home. Accordingly, applicants have
discovered that the cranberry flavonol compositions of the present
invention advantageously possess superior anti-inflammatory
activity. These compositions are amenable to use in the treatment
of inflammatory disorders, particularly those of the urinary tract
arising from both bacterial and non-bacterial sources. Moreover,
these compositions can be formulated to contain one or more
flavonols that can inhibit the invasion of uroepithelial cells by
type 1 fimbriated E. coli. Advantageously, these compositions are
also free from irritants of the bladder as well as the digestive
mucous membranes. Thus, the cranberry flavonol compositions of the
present invention embody natural, innocuous treatments that can be
used beneficially for self-medication by those afflicted with
inflammatory conditions of the urinary tract generally, and
cystitis particularly. The cranberry flavonol compositions of the
present invention are effective for and amenable to the treatment
of such inflammatory conditions regardless of whether such
conditions originate from bacterial or non-bacterial sources.
SUMMARY OF THE INVENTION
[0020] The present invention is directed to cranberry flavonol
extracts that are substantially free of anthocyanins and
proanthocyanidins. These flavonol extracts have marked
anti-inflammatory activity, and are thus useful in the treatment of
inflammatory conditions generally, and particularly in the
treatment of urinary tract inflammation. The invention also
provides a method of obtaining these flavonol extracts from
cranberries (Vaccinium macrocarpon). The present invention is also
directed to purified cranberry flavonol compounds, including
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside, which we have found
to possess marked anti-inflammatory activity, as well as methods to
obtain these compounds from cranberries. Additionally, the present
invention embodies a processing method to increase the yield of
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside in cranberry powder.
Finally, the invention relates to methods of preventing or treating
inflammatory conditions, particularly urinary tract inflammation
from either bacterial or non-bacterial sources, in a mammal by
administering a composition comprising a flavonol extract of
cranberries or a cranberry flavonol compound such as
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside to the mammal in an
amount and for a time sufficient to prevent, reduce or eliminate
inflammation and thereby lead to an amelioration or curing of the
condition. Preferably the mammal undergoing treatment is a human,
but the method is also applicable to animals, especially
domesticated animals, such as cats and dogs, and livestock.
[0021] Pharmaceutical compositions are provided which comprise a
flavonol composition, including pharmaceutically acceptable salts
of any of the flavonol compounds, and a pharmaceutically acceptable
carrier. Also provided are pharmaceutical compositions comprising
at least one cranberry flavonol compound, such as
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside, including
pharmaceutically acceptable salts thereof, and a pharmaceutically
acceptable carrier. In some instances, it may be preferable to
provide the therapeutic dosage in the form of a food additive in a
beverage such as a cranberry juice-based beverage containing
additional flavonols or
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside. The invention also
provides food and dietary supplement compositions comprising a
flavonol composition or at least one cranberry flavonol compound,
such as quercetin-3-O-(6''-benzoyl)-.beta.-galactoside, including
pharmaceutically acceptable salts of any of these compounds, mixed
with a consumable carrier. Consumable carriers include, but are not
limited to, livestock feed, domestic animal feed and consumable
food products, especially cranberry containing food products, and
are defined more fully in the detailed description of the invention
herein. Finally, the present invention also provides cosmetic
compositions incorporating the active agents disclosed herein.
These pharmaceutical, food, dietary supplement, and cosmetic
compositions are useful to prevent or treat inflammatory conditions
generally, and particularly urinary tract inflammation, and more
particularly cystitis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Table 1 provides chromatographic and mass spectral data for
individual components of Fraction 1 of the 60% methanol eluate from
Sephadex LH-20 chromatography of cranberry extract.
[0023] Table 2 provides NMR data for eight components of Fraction 1
of the 60% methanol eluate from Sephadex LH-20 chromatography of
cranberry extract.
[0024] Table 3 provides data on the inhibitory effect of cranberry
fractions from Sephadex LH-20 chromatography on TPA-induced edema
of mouse ears.
[0025] Table 4 provides data on the inhibitory effect of
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside (Cranberry peak 19
from Fraction 1 of the 60% methanol eluate from Sephadex LH-20
chromatography) on TPA-induced edema of mouse ears.
[0026] FIG. 1 shows HPLC chromatograms of the flavonol glycosides
extracted from fresh cranberries (60% methanol Fraction 1 after
Sephadex LH-20 column chromatography) and a cranberry solids powder
(90MX) showing an increase in the concentration of the compound
corresponding to Peak 19 (having retention time of approximately 45
minutes) upon processing to form the cranberry solids powder, under
the following chromatographic conditions: Sorbax SB-C18 250
mm.times.4.6 mm i.d. column; eluate, 2% formic acid in methanol;
flow rate, 1 ml min.sup.-1; absorbance, 340 nm.
[0027] FIG. 2 is the HPLC chromatogram of Fraction 1 of the 60%
methanol eluate from Sephadex LH-20 chromatography, with peaks
numbered corresponding to the compound numbers presented in Table
2, under the following chromatographic conditions: Sorbax SB-C18
250 mm.times.4.6 mm i.d. column; eluate, 2% formic acid in
methanol; flow rate, 1 ml min.sup.-1; absorbance, 340 nm.
[0028] FIG. 3 is the HPLC chromatogram of Fraction 2 of the 60%
methanol eluate from Sephadex LH-20 chromatography under the
following chromatographic conditions: Sorbax SB-C18 250
mm.times.4.6 mm i.d. column; eluate, 2% formic acid in methanol;
flow rate, 1 ml min.sup.-1; absorbance, 340 nm, wherein the peak
labeled M is myricetin and the peak labeled Q is quercetin.
[0029] FIG. 4 shows the chemical structures of eight flavonol
glycosides isolated from Fraction 1 of the 60% methanol eluate from
Sephadex LH-20 chromatography, specifically:
myricetin-3-.beta.-xylopyranoside (2),
quercetin-3-.beta.-galactoside (5), quercetin-3-.beta.-glucoside
(6), quercetin-3-.beta.-arabinopyranoside (9),
quercetin-3-.alpha.-arabinofuranoside (10),
3'-methoxyquercetin-3-.alpha.-xylopyranoside (15),
quercetin-3-O-(6''-.beta.-coumaroyl)-.beta.-galactoside (16a), and
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside (19).
[0030] FIG. 5 shows the proton NMR spectrum (bottom) of Cranberry
peak 16a from Fraction 1 of the 60% methanol eluate from Sephadex
LH-20 chromatography and the trace extracted from the 6 Hz
optimized IMPRESS-HMBC data set at 166.7 ppm showing 2- and 3-bond
coupling responses from the olefin protons of the coumaroyl moiety
and the galactose C-6'' protons to the same ester carbonyl
resonance.
DETAILED DESCRIPTION OF THE INVENTION
[0031] We have characterized the flavonol glycosides in spray-dried
cranberry powder and have isolated a cranberry extract containing
mixed flavonols that is substantially free of both anthocyanins and
proanthocyanidins. This extract exhibits a high degree of
anti-inflammatory activity in the TPA-induced mouse ear edema assay
and is rich in glycosylated quercetin derivatives. We have also
isolated and characterized six flavonols not previously reported in
cranberry, including the novel compound
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside, which we have found
to be a potent anti-inflammatory agent in the TPA-induced mouse ear
edema assay. Interestingly, we have also discovered that this
compound is more abundant in processed cranberry powder than in the
fresh fruit itself (FIG. 1), and thus have determined means to
generate the purified compound in high yield.
[0032] The cranberry flavonol extracts and compounds of the present
invention can be used in various food, dietary supplement,
pharmaceutical, and, cosmetic formulations for the treatment and
prevention of inflammatory conditions generally. Moreover, as many
of the extracts are both rich in glycosylated quercetin derivatives
and free from bladder irritants, they can be used advantageously
for the treatment and prevention of cystitis arising from either
bacterial or non-bacterial origins. An "inflammatory condition" is
defined herein as any condition or disease in which inflammation is
either symptomatic or plays a prominent role in the condition or
disease, including but not limited to: urinary tract infection
and/or inflammation, osteoarthritis, rheumatoid arthritis,
cardiovascular diseases, dermatitis, cancer, diabetes, obesity,
asthma, multiple sclerosis, and other diseases in which
inflammation is involved.
[0033] Compositions comprising the extracts or compounds of the
present invention can be formulated for administration as foods or
dietary supplements using one or more consumable carriers. A
"consumable carrier" is herein defined as any food, food
ingredient, or food additive, or any excipient utilized for
tabletting, encapsulation, or other formulation of an active agent
for oral administration, whether for human or animal use. Specific
additives are well known to those of skill and are listed in places
such as the U.S. Pharmacopeia. For dietary supplements, the extract
can be mixed according to methods routine in the art. Dietary
supplements can be prepared in a variety of forms including, but
not limited to, liquid, powder, or solid pill forms. In the present
invention, the active agents can be administered either alone or in
combination with other phytochemicals where combining compounds or
extracts would lead to synergistic effects. Examples of other
phytochemicals which can be used in combination with the active
agents of the present invention 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. The active agents of the present invention
can also be 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 such agents into foods.
[0034] Alternatively, compositions comprising these extracts can be
administered as conventional pharmaceuticals using one or more
physiologically acceptable carriers or excipients, referred to
herein as "pharmaceutically acceptable carriers." Pharmaceutical
compositions can be formulated for administration by any route
including, but not limited to, inhalation or insufflation (through
mouth or nose), oral, buccal, parenteral, topical, vaginal, or
rectal administration. The active agents of the present invention
can be applied topically to treat and prevent inflammatory
conditions in either pharmaceutical or cosmetic applications.
Compositions for use in the present invention can also be
administered in the form of 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. The compositions of the
present invention can be formulated to provide controlled time
release of the active agents. For buccal administration the extract
can be formulated as a tablet or lozenge.
[0035] 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. For rectal administration or vaginal administration,
compositions for use in of the present invention can be formulated
as suppositories, creams, gels, or retention enemas.
[0036] 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.
[0037] Freeze dried cranberry powder extracted with 80% aqueous
acetone followed by ethyl acetate yielded a phenolic fraction with
a high degree of anti-inflammatory activity in the TPA-induced
mouse ear edema assay. In order to optimize the resolution of
compounds within the main phenolic classes (phenolic acids,
anthocyanins, flavonols, and proanthocyanidins), we assayed a
number of analytical C18 reverse-phase columns, eluates and
gradients to determine which of these combinations provided the
best HPLC profile. As a result, we determined that upon Sephadex
LH-20 column chromatography of the ethyl acetate extract with
successive volumes of water, 60% aqueous methanol (v/v) (collected
in two fractions referred to herein as Fraction 1 and Fraction 2),
100% methanol, and 70% aqueous acetone (v/v) to separate the
cranberry components generally into the main phenolic classes, HPLC
separation of these eluates on a Zorbax SB-C18 reversed phase
column with a binary solvent system (Solvent A, 2% aqueous formic
acid; Solvent B, 20% formic acid in methanol) and gradient elution
(Linear gradient of 5-25% B from 0 to 5 min, linear gradient of
25-40% B from 5 to 25 min, isocratic elution with 40% B from 25 to
30 min, linear gradient of 40-95% B from 30 to 45 min, and
isocratic elution with 95% B from 45 to 50 min) provides optimal
simultaneous resolution of cranberry phenolic acids, anthocyarins
and flavonols.
[0038] After Sephadex LH-20 column chromatography of the ethyl
acetate extract, we assayed the 60% methanol (Fraction 1 and
Fraction 2 combined), 100% methanol, and 70% acetone eluates for
anti-inflammatory activity via the TPA-induced mouse ear edema
assay as described in Example 2 herein. Of these three, the 60%
methanol eluate exhibited a significant dose response effect in
reducing the weight of mouse ear edema by 34.3% and 78.6% at 166
.mu.g and 500 .mu.g, respectively, as compared to the estimated
mean inflammation of the acetone and TPA control. HPLC analysis
with PDA detection indicated this fraction was composed primarily
of phenolics with maximum absorbance near 350-360 nm. We determined
that Fraction 1 of the 60% methanol eluate consisted of flavonol
glycosides, while the later-eluting Fraction 2 consisted of
flavonol aglycones, predominantly myricetin and quercetin. Further
isolation and determination of the components was undertaken as
described herein. As expected, the 70% acetone eluate tested
positive for proanthocyanidins with the HCl-vanillin assay.
[0039] Assay of Fraction 1 of the 60% methanol eluate using the
chromatographic method according to the present invention afforded
22 peaks in contrast to the 9 reported previously in cranberry (Yan
et al. 2002) (FIG. 2). Additionally, the method of the present
invention is more effective for the separation of individual
flavonol glycosides within this class of flavonoids. The increased
resolution provided by our method enabled us to identify additional
flavonoids, six of which were structurally determined. Two of these
newly identified compounds, 16a and 19, are very rare acylated
quercetin-galactosides.
Quercetin-3-O-(6''-p-coumaroyl)-.beta.-galactoside (16a) has only
been reported in Ledum palustre L. (Jin et al. 1999). We have
isolated quercetin-3-O-(6''-benzoyl)-.beta.-galactoside (19) for
the first time from a natural source. The increased resolution
provided by our method enables the isolation of individual flavonol
glycosides or aglycones present in cranberries and thus also the
preparation of bioactive compositions incorporating one or more of
these compounds.
[0040] Using the analytical methods of the present invention, we
discovered some distinct differences between the flavonol glycoside
profiles of processed cranberry powder and fresh cranberries, the
most pronounced of which were the elevated contents of myricetin,
quercetin, and quercetin-3-O-(6''-benzoyl)-.beta.-galactoside after
processing. Specifically, we evaluated the content of the latter
compound in extracts of three commercial cranberry cultivars
(Stevens, Ben Lear, and Early Black) and four samples of processed
cranberry powder. We consequently determined that this component
rose from an average of 1.27% (Range 0.16-2.28%) of the total
flavonol fraction in fresh fruit to 3.44% (Range 2.50-4.02%) in
processed powder extracts, as shown in FIG. 1. As described in
Example 3 below, we found this compound to be a highly effective
inhibitor of inflammation in the TPA-induced mouse ear edema assay.
Thus, our invention also embodies a method for obtaining this
compound in high yield by isolating the compound from processed
cranberry preparations. Such processing entails the concentration
of cranberry juice by means known to those of skill in the art,
including but not limited to heating at atmospheric pressure or
under vacuum, freeze drying, or a combination of these to obtain a
cranberry preparation having a high solids content.
[0041] The invention is illustrated more fully by the following
non-limiting Examples. Moreover, applicants' article
"Characterization of Flavonols in Cranberry (Vaccinium macrocarpon)
Powder" J. Agric. Food Chem. 2004, 52, 188-195 is incorporated by
reference in its entirety herein.
EXAMPLE 1
Isolation and Characterization of Flavonol Glycosides and Aglycones
from Cranberry Powder
Reagents
[0042] All reagents were purchased from Fisher Scientific (PA) and
Sigma (MO) and were of analytical or HPLC grade. Dimethylsulfoxide
(DMSO)-d.sub.6 (99.96% D) was obtained from Cambridge Isotope
Laboratories (MA). Standards of quercetin-3-galactoside,
quercetin-3-glucoside, quercetin-3-rhamnoside, quercetin, myricetin
and kaempferol were purchased from INDOFINE Chemical Company, Inc.
(NJ). Sephadex.TM. LH-20 for column chromatography was obtained
from Amersham Pharmacia Biotech AB (Sweden).
Extraction and Fractionation of Flavonoids
[0043] Flavonoids from 1.0 kg of freeze dried cranberry powder
90-MX supplied by Ocean Spray Cranberries, Inc., (MA) were
extracted twice with 80% acetone/water (v/v) (1:10), filtered and
partially evaporated under reduced pressure at 35.degree. C. to
remove the acetone. The resultant aqueous phase was defatted by
extraction with hexane (1:1) and the aqueous layer was further
extracted with three portions of ethyl acetate (1:1). After
concentration in vacuo, the pooled ethyl acetate fraction (24.8 g)
was further fractionated using column chromatography. A portion of
ethyl acetate extract (8.0 g) was dissolved in 60% methanol and
loaded onto the 100.times.50 mm column packed with hydrated
Sephadex LH-20. Subsequent elution with water, 60% methanol/water
(v/v), 100% methanol and 70% acetone/water (v/v) was applied to
fractionate phenolic classes and remove all phenolic constituents
from the column. Fractions eluted were monitored by analytical HPLC
and tested for anti-inflammatory activity as described below. The
60% methanol fraction contained mostly constituents with maximum
absorbance at 340 nm and was eluted from the column in two
sequential 1200 ml volumes, yielding 660 mg (Fraction 1) and 600 mg
(Fraction 2) of phenolics. Fraction 1 was used for the further
isolation of individual constituents by preparative HPLC as
described below. Final purification of the compound 16a was
achieved on a Sephadex LH-20 column (20.times.40 mm) using methanol
as the eluting solvent.
HPLC Apparatus and Chromatographic Conditions
[0044] Analytical HPLC
[0045] HPLC analysis was performed on a Waters Millenium HPLC
system composed of a Water In-line Degasser, a Waters 600E
Multisolvent Delivery System, a Waters 717 plus Autosampler, and a
Waters 996 photodiode array detector. A Zorbax SB-C18 250
mm.times.4.6 mm i.d. (5 .mu.m) reversed phase column protected with
a Waters Guard-Pak Precolumn Module was used for analysis.
Separations were carried out in a binary solvent system: solvent A,
2% formic acid; solvent B, 2% formic acid in methanol. A program of
a linear gradient 5-25% B from 0 to 5 min, 25-40% B from 5 to 25
min, an isocratic elution with 40% B from 25 to 30 min, a linear
gradient 40-95% B from 30 to 45 min and an isocratic elution with
95% B from 45 to 50 min at flow rate of mL/min was used. PDA
detection was used to monitor the eluate from 210 to 700 nm.
[0046] Preparative HPLC
[0047] A Zorbax SB-C18 250 mm.times.21.2 mm i.d. (5 .mu.m) column
was used with the binary solvent system and gradient elution as
described in the preceding section and a flow rate of 15 mL/min.
The column effluents were monitored from 210 to 400 mm. Fractions
were collected using a Waters Fraction Collector II. A gradient
elution program afforded the collection of eight individual
compounds that were purified by re-chromatography under the same
conditions.
Identification of Cranberry Flavonol Glycosides and Aglycones
[0048] Individual constituents of the flavonol extract according to
the present invention were identified by comparison of
chromatographic retention time and UV spectral characteristics with
standards, as well as by MS and NMR techniques.
[0049] Comparison with Standards
[0050] Standard curves for identification of flavonols were
prepared using authentic standards dissolved in methanol at a
concentration of 1 mg/ml and stored at -20.degree. C. as stock
solutions. Identification of quercetin-3-galactoside,
quercetin-3-glucoside, quercetin-3-rhamnoside, myricetin and
quercetin was performed by matching their retention time and
spectral characteristics measured at 340 nm against those of
standards.
[0051] Mass Spectrometry
[0052] Atmospheric pressure chemical ionization (APCI) mass
spectrometry in the negative-ion detection mode was obtained on a
VG Platform mass spectrometer (Micromass, Manchester, U.K.). A
Zorbax SB-C18 250.times.4.6 mm reversed-phase column and the
methanol/formic acid/water mobile phase described above were used
with a flow rate of 1 ml/min. Typical tuning parameters were as
follows: corona, 3 kV; high voltage lens, 0.0 kV; cone 15 V; source
temperature, 150.degree. C.; and APCI probe temperature,
450.degree. C. Spectra were scanned over a mass range of m/z
150-1100 at 1.0 s per cycle.
[0053] Positive-ion electrospray ionization (ESI) mass spectrometry
was acquired on a ThermoFinnigan TSQ-Quantum mass spectrometer
using standard operating parameters. A Zorbax Eclipse XDB C18
150.times.4.6 mm reversed-phase column and a binary solvent
gradient of A (water/acetonitrile/trifluoroacetic acid (TFA)
95:5:0.025) and B (water/acetonitrile/TFA 5:95:0.025) were used.
Spectra were scanned over a mass range of m/z 190-800 at 1.0 s per
cycle.
[0054] NMR Spectroscopy
[0055] The NMR data were obtained on either a Varian INOVA
three-channel NMR spectrometer operating at a .sup.1H observation
frequency of 599.730 MHz and equipped with a 3 mm Nalorac
Z.cndot.SPEC MIDTG gradient inverse triple resonance NMR probe or a
Varian INOVA three-channel NMR spectrometer operating at a .sup.1H
observation frequency of 499.792 MHz and equipped with a 5 mm
Varian Chili-probe.RTM. gradient inverse triple resonance NMR probe
operating at a coil temperature of 25.degree. K. The sample
temperature was regulated at 20.degree. C. for all samples except
peak 9, for which data were acquired at 32.degree. C. Samples were
dissolved in -150 .mu.L of DMSO-d.sub.6 and transferred to a Wilmad
3 mm NMR tube for analysis. Peak 16a was dissolved in .about.150
.mu.L of 90:10 DMSO-d.sub.6:benzene-d.sub.6. Chemical shifts were
referenced relative to the residual solvent resonances at 2.49 and
39.5 ppm for .sup.1H and .sup.13C, respectively. All .sup.1H NMR
data were acquired with a spectral width of 16 ppm. Correlated
Spectroscopy (COSY) data were acquired as 256 increments with 8
transients per increment; squared sinebell apodization was used in
both dimensions. Heteronuclear Single Quantum Coherence (HSQC) and
Heteronuclear Single Quantum Coherence Total Correlation
Spectroscopy (HSQCTOCSY) data were acquired as 96 increments with
24 and 96 transients per increment, respectively. Both data sets
had an F1 spectral window of 146 ppm and were apodized with a
gaussian weighing function in both dimensions; the HSQCTOCSY mixing
time was set to 18 ms. Heteronuclear Multiple Bond Correlation
(HMBC) data were acquired as 96 increments with 320 transients per
increment. The F1 spectral width was 241 ppm. Squared sinebell
apodization was used in both dimensions.
Results
[0056] Chromatography
[0057] FIG. 2 shows the chromatogram of Fraction 1 of the 60%
methanol eluate from Sephadex LH-20 column chromatography, while
FIG. 3 shows the corresponding chromatogram of Fraction 2 of the
60% methanol eluate. Both chromatograms were acquired using UV
absorbance detection at 340 nm. Fraction 1 exhibits well-resolved
flavonoid peaks at R.sub.t 29-48 min and includes some minor
constituents of R.sub.t 15-26 min. Fraction 2 exhibits four peaks,
two of which (R.sub.t 39.4, 44.6) are predominant.
[0058] All peaks on the Fraction 1 chromatogram having retention
time between 29 and 48 min, peaks numbered from 1 to 22 as shown,
display absorbance profiles corresponding to those of flavonols
(Mabry et al. 1970; Machiex et al. 1990). Three peaks of R.sub.t
34.3, 35.1 and 40.4 min were found to be consistent with retention
times and UV-visible spectra of standards quercetin-3-galactoside
(5), quercetin-3-glucoside (6) and quercetin-3-rhamnoside (11),
respectively (FIG. 2, Table 1). The two major flavonoids that
eluted in Fraction 2 (FIG. 3) were identified as myricetin (&
39.8) and quercetin R.sub.t 44.6).
[0059] Mass Spectrometry
[0060] APCI LC-MS analysis in the negative-ion mode was used to
identify the molecular weights of constituents eluting in 60%
methanol Fraction 1 and to ascertain whether they were sugar
conjugates as evidenced by loss of 162/132 mass units from the
pseudomolecular ion. The results obtained are summarized in Table 1
together with spectral characteristics, distribution of peaks by
area percentages (at 340 nm) and retention time under
chromatographic conditions employed. APCI LC-MS of phenolics gives
intense deprotonated molecular ions [M-H].sup.- in the negative-ion
mode.
[0061] Peak 1 exhibited an intense [M-H].sup.- ion peak at m/z 479
and a fragment ion at m/z 317 [M-C.sub.6H.sub.11O.sub.5].sup.-
corresponding to a myricetin-hexoside. The spectrum of the peaks 2
and 4 both gave [M-H].sup.- ion peaks at m/z 449 and a fragment ion
at m/z 317 [M-C.sub.5H.sub.9O.sub.4].sup.- consistent with
myricetin-pentoside conjugates. Peaks 5 and 6 exhibited in the APCI
mass spectra characteristic [M-H].sup.- ions at m/z 463 and
fragment ions at m/z 301 [M-C.sub.6H.sub.11O.sub.5].sup.-
corresponding to quercetin hexosides. These peaks, when compared
with chromatographic behavior and UV-visible spectra of standards,
were identified as quercetin-3-.beta.-galactoside and
quercetin-3-.beta.-glucoside, respectively.
[0062] Three peaks of R.sub.t 36.3, 37.6, and 39.8 (8, 9, 10)
showed intense [M-H].sup.- ion peaks at m/z 433 and fragment ions
at m/z 301 [M-C.sub.5H.sub.9O.sub.4].sup.- consistent with
quercetin-pentoside structures. The exact nature of the sugar
moiety cannot be ascertained by LC-MS. The spectra released for
peak 11 exhibited a [M-H].sup.- ion at m/z 447 and a fragment ion
at m/z 301 [M-C.sub.6H.sub.11O.sub.4].sup.- which corresponds to
quercetin-3-.alpha.-rhamnoside as determined by comparison with a
standard.
[0063] Peak 12 gave a [M-H].sup.- ion peak at m/z 477 with a
fragment ion at m/z 315 [M-C.sub.6H.sub.11O.sub.5].sup.- consistent
with a possible structure containing a
monomethoxyquercetin-hexoside. The position of the methyl
substituent could not be determined. Peaks 14 and 15 exhibited
[M-H].sup.- ions at m/z 447 and had fragment ions at m/z 315
[M-C.sub.5H.sub.9O.sub.4].sup.- corresponding to possible
methoxylated quercetin-pentosides. The MS spectra of peaks 13, 16,
21 and 22 appear more complex and suggest the presence of a mixture
of components. Possible constituents of these peaks correspond to
monomethoxymyricetin-pentoside (m/z 463, 331) and
dimethoxymyricetin-hexoside (m/z 507, 345) for peak 13 and to
derivatives of methoxykaempferol (m/z 299) for peaks 21 and 22.
Deprotonated molecular ions for peak 16 indicate the possible
presence of a monomethoxyquercetin-pentoside (m/z 447, 315) and an
acylated derivative of quercetin-hexoside (m/z 609, 301).
[0064] One of the latest eluting peaks at R.sub.t 45.6, peak 19,
gave [M-H].sup.- and fragment ion peaks at m/z 567 and 301 which we
initially speculated to be a quercetin-hexose ester with benzoic
acid. Peak 19 was subsequently confirmed to be
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside by NMR
spectroscopy.
[0065] NMR Spectroscopy
[0066] For structural determination of various flavonoid
constituents by NMR spectroscopy, Fraction 1 of the 60% methanol
eluate was further fractionated by preparative HPLC. The two step
procedure applied for the elution of constituents with 60% methanol
during Sephadex LH-20 column chromatography was useful to prevent
the coelution of the abundant simple flavonols myricetin and
quercetin with the peaks corresponding to flavonol conjugates
(FIGS. 1 and 2). The NMR analysis of purified peaks was supported
by ESI LC-MS data performed in the positive-ion mode (Table 1).
[0067] Sufficient amounts of eight pure components (2, 5, 6, 9, 10,
15, 16, 19) were obtained. These compounds were conclusively
identified by NMR as myricetin-3-.beta.-xylopyranoside (2),
quercetin-3-.beta.-galactoside (5), quercetin-3-O-glucoside (6),
quercetin-3-.alpha.-arabinopyranoside (9),
quercetin-3-.alpha.-arabinofuranoside (10),
3'-methoxyquercetin-3-.alpha.-xylopyranoside (15), and
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside (19) (FIG. 4). Peak
16 represented a mixture of coeluting compounds, and was subjected
to additional purification on a Sephadex LH-20 column with 60%
methanol as the eluting solvent. The compound with [M-H].sup.- ion
peak at m/z 609 (16a) was used for structural determination by NMR
spectroscopy, and determined to be
quercetin-3-O-(6'-p-coumaroyl)-.beta.-galactoside (FIG. 4).
[0068] The previous identification of peaks 5 and 6 as
quercetin-3-.beta.-galactoside and quercetin-3-.beta.-glucoside,
respectively, were confirmed by NMR data (Table 2). For peak 6, the
anomeric proton appeared as a doublet (J=7.6 Hz) while the 2'',
3'', and 4'' proton resonances appeared as triplets (J=7.8-8.8 Hz),
consistent with a glucose moiety. The flavonoid at R.sub.t 39.8
(peak 10) was identified as quercetin-3-.alpha.-arabinofuranoside,
which is consistent with previously reported data Puski et al.
1967; Yan et al. 2002). Quercetin-3-.beta.-glucoside (6) and
structures 2, 9, 15, 16a, and 19 represent compounds not yet
reported in cranberry or cranberry products.
[0069] Myricetin-3-.beta.-xyloside (2) in the pyranose form was
apparent due to the 4'' carbon chemical shift and the strongly
anisochronous 5'' methylene responses which are consistent with
those of a pentose in the pyranose form. The anomeric signal
appeared as a J=7.43 Hz doublet, and taken along with the downfield
shift of the carbon resonance, indicates a .beta.-configuration.
The 3'' proton resonance is an apparent triplet (J=8.66 Hz),
establishing a trans-diaxial relationship for the 2''-3'' and
3''-4'' pairs.
[0070] The pyranose form of quercetin-3-.alpha.-arabinoside (9) was
indicated by the 4'' carbon chemical shift and the anisochronous
5'' methylene group. The anomeric signal appeared as a J=5.2 Hz
doublet at 5.22/102.3 ppm, implying the .alpha.-configuration. The
2''-3'' coupling constant of J=6.7 Hz indicated a trans-diaxial
configuration, while 3''-4'' coupling constant (J=3.1 Hz) implies
an axial-equatorial orientation, yielding the assignment of
arabinopyranose. Thus, the relatively major flavonol
quercetin-3-arabinoside exists in two sugar forms, as a pyranose
(.about.6.7%) and the previously reported (10) furanose form
(.about.9.7%) (Table 1).
[0071] Peak 15 presented NMR spectra indicative of the quercetin
backbone and a xylopyranose sugar moiety. An aromatic methoxy
resonance was also observed (3.71, 51.9 ppm) that yielded a 3-bond
HMBC response to the 3' carbon resonance at .about.144 ppm,
allowing assignment of the site of the methoxy substitution.
[0072] A quercetin backbone was observed in the isolate of peak 16a
by NMR spectroscopy, and resonances consistent with the presence of
a galactopyranose sugar moiety were observed. Two new AB spin
systems were observed in the downfield region; one integrating for
a total of two protons (7.58/145/5 and 6.29/114.6 ppm), the other
integrating for four protons (7.46/130.9 and 6.99/116.7 ppm). The
larger set of resonances was readily assigned as a 1,4-substituted
aromatic system based on HMBC data, while the smaller system was
assigned as a trans-olefin due to its 15.9 Hz coupling constant. An
IMPRESS-HMBC data set was used to establish an ester carbonyl
linkage between the galactopyranose and olefinic moieties (Yang et
al. 2003). These data, coupled with the molecular weight
information, indicate that this isolate is the C-6''
para-hydroxycinnamic acid ester of
quercetin-3-.beta.-galactopyranose (FIG. 5).
[0073] Interpretation of the NMR spectra obtained on peak 19
(.sup.1H reference, COSY, HSQC, HSQCTOCSY, and HMBC) revealed
several structurally significant features. The resonances
indicating a quercetin backbone were readily observed and assigned
by inspection. Resonances consistent with a sugar moiety were also
observed. The sugar resonances were somewhat obscured by the
residual water in the sample, but several key assignments were
possible. The anomeric methine doublet (J=7.6 Hz) was observed at
5.46/101.9 ppm, indicating an axial proton orientation, and
therefore a .beta.-linkage to the quercetin backbone. COSY and
HSQCTOCSY responses allowed complete, sequential assignment of the
sugar ring. A large coupling constant (J=8.3 Hz) was observed for
the 2''-3'' interaction, indicative of a trans-diaxial interaction,
while the 4'' proton resonance was observed as a broad singlet.
This result requires axial-equatorial interactions for both 3''-4''
and 4''-5'' and yields the assignment of the sugar moiety as
.beta.-galactose.
[0074] Another spin system was identified in the aromatic region of
the spectrum. This system consisted of an apparent
doublet-triplet-triplet pattern in a 2:2:1 ratio and can be readily
assigned as a phenyl group (ortho 7.621129.4; meta 7.26/129.3; para
7.49/133.9 ppm). Investigation of the HMBC data set revealed a
3-bond response between the ortho-protons of this phenyl ring with
a carbonyl resonance at 166.1 ppm. A second 3-bond response to this
same carbonyl carbon was observed from the 6'' methylene protons.
Taken with the C-6'' chemical shift of 64.9 ppm and the C-9''
chemical shift of 129.5 ppm, these data clearly indicate the
presence of a benzyl ester moiety at the 6'' position of the
galactose ring.
[0075] The cumulative data showed the presence of mostly
glycosylated forms of myricetin and quercetin in Fraction 1 of the
60% methanol Sephadex LH-20 eluate. Together with UV-spectra and
peak area percentage these results confirm the identity of peaks 4,
5, 10 and 11 as myricetin-3-.alpha.-arabinofuranoside,
quercetin-3-.beta.-galactoside,
quercetin-3-.alpha.-arabinofuranoside and
quercetin-3-.beta.-rhamnoside (Puski et al. 1967; Yan et al. 2002).
The peaks labeled 1, 8 and 12 correspond to
myricetin-3-.beta.-galactoside, quercetin-3-xyloside and
3'-methoxyquercetin-3-.beta.-galactoside
(iso-rharmnetin-galactoside), respectively. These components were
recently identified in cranberry extracts by Yan et al. (2002),
however, the presence of other methyl ethers in cranberries has not
been reported. We have fully characterized a second methyl ether
derivative in cranberries,
3'-methoxyquercetin-3-.alpha.-xylopyranoside (15), and our LC-MS
data suggest the presence of a number of other methoxylated
flavonols including derivatives of quercetin, myricetin and
kaempferol (Table 1).
EXAMPLE 2
Anti-Inflammatory Activity of Sephadex LH-20 Eluate Fractions
[0076] The anti-inflammatory activities of different classes of
cranberry phenolic compounds separated by Sephadex LH-20 column
chromatography were tested in vivo with the
12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema
assay described previously (Huang et al. 2003). Each of the
treatments was topically applied to both ears of four female CD-1
mice. Treatments included application of either acetone or curcumin
controls or the test extract in 20 .mu.l of acetone twenty minutes
prior to application of either 5 mg of acetone alone or 1 nmol of
TPA in acetone. At five hours post-treatment, mice were sacrificed
and 6 mm diameter ear punch biopsies were taken and weighed.
Curcumin, a well-known anti-inflammatory agent, was used as a
positive control (Chan et al. 1994; Chan 1995). Data were analyzed
statistically using ANOVA and the Student-Neuman-Kuels (SNK)
multiple means separation test (P.ltoreq.0.05). The ratio of the
difference in the weight of ear punches between TPA treated groups
receiving pretreatments of acetone and the test extract,
respectively, to the difference in the weight of ear punches
between acetone pretreated groups subsequently treated with TPA and
acetone, respectively, indicates the degree of anti-inflammatory
activity of the test extract. Results are summarized in Table
3.
EXAMPLE 3
Anti-Inflammatory Activity of
Quercetin-3-O-(6''-benzoyl)-.beta.-Galactoside
[0077] The anti-inflammatory activity of
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside (Peak 19) was tested
in vivo with the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced
mouse ear edema assay. Each of the treatments was topically applied
to both ears of at least five CD-1 female mice. Treatments included
application of either acetone or curcumin controls or the test
extract in 20 .mu.l of acetone twenty minutes prior to application
of 1.5 nmol of TPA in acetone. After 7 hours post-treatment, mice
were sacrificed and 6 mm diameter ear punch biopsies were taken and
weighed. The ratio of the difference in the weight of ear punches
between TPA treated groups receiving pretreatments of acetone and
the test extract, respectively, to the difference in the weight of
ear punches between acetone pretreated groups subsequently treated
with TPA and acetone, respectively, indicates the degree of
anti-inflammatory activity of the test extract. Results are
summarized in Table 4.
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TABLE-US-00001 TABLE 1 Retention Times, Relative Peak Areas,
.lamda..sub.max, m/z values of [M + H].sup.+, [M - H].sup.- and
Fragment Ions of Flavonoid Constituents Resolved by HPLC of 60%
Methanol Fraction 1. Retention Relative time peak [M - H].sup.- and
fragment ions [M + H].sup.+ Peak (min) area, % .lamda..sub.max, nm
in APCI MS in ESI MS Structure 1 29.0 8.3 261.5 356.5 479 (25%),
317 (100%) nd Myricetin-3-.beta.-galactoside.sup.1 2 29.8 0.9 261.5
356.5 449 (100%), 317 (90%) 451
Myricetin-3-.alpha.-xylopyranoside.sup.3 3 32.0 2.2 233.2 266.2 nd
nd nd 299.4 356.5 4 32.5 3.6 261.5 356.5 449 (65%), 317 (100%) nd
Myricetin-3-.alpha.-arabinofuranoside.sup.1,2 5 34.3 21.5 256.8
356.5 463 (55%), 301 (100%) 465
Quercetin-3-.beta.-galactoside.sup.1,2 6 35.1 0.9 256.8 356.5 463
(60%), 301 (100%) 465 Quercetin-3-.beta.-glucoside.sup.3 7 35.8 1.3
256.8 356.5 nd nd nd 8 36.3 8.2 256.8 356.5 433 (45%), 301 (100%)
nd Quercetin-3-.alpha.-xylopyranoside.sup.1 9 37.6 6.7 256.8 356.5
433 (35%), 301 (100%) 435
Quercetin-3-.alpha.-arabinopyranoside.sup.3 10 39.8 9.7 256.8 356.5
433 (35%), 301 (100%) 435
Quercetin-3-.alpha.-arabinofuranoside.sup.1,2 11 40.4 14.3 256.8
351.7 447 (55%), 301 (100%) nd Quercetin-3-rhamnopyranoside.sup.1,2
12 41.2 1.6 256.8 356.5 477 (100%), 315 (25%) nd
3'-methoxyquercetin-3-.beta.-galactoside.sup.1 13 41.7 3.9 256.8
356.5 507 (100%), 463 (48%), nd Dimethoxymyricetin-hexoside 345
(48%), 331 (46%) nd Methoxymyricetin-pentoside 14 42.9 0.6 256.8
356.5 447 (100%), 315 (55%) nd Methoxyquercetin-pentoside 15 43.3
2.3 256.8 356.5 447 (100%), 315 (32%) nd
3'-methoxyquercetin-3-.alpha.-xylopyranoside.sup.3 16 44.0 4.6
256.8 351.7 609 (35%), 447 (70%), 611.sup.4
Methoxyquercetin-pentoside; Quercetin-3-O-(6''-p- 256.8 318.4 315
(25%), 301 (100%) coumaroyl)-.beta.-galactoside.sup.3 (Compound
16a) 351.7sh 17 44.5 0.8 256.8 356.5 nd nd nd 18 44.8 1.5 256.8
313.6 nd nd nd 356.5 19 45.6 5.6 256.8 356.5 567 (100%), 301 (52%)
569 Quercetin-3-O-(6''-benzoyl)-.beta.-galactoside.sup.3 20 46.2
0.1 256.8 356.5 nd nd nd 21 46.8 0.3 256.8 356.5 581 (53%), 431
(100%), nd Methoxykaempferol derivative 299 (28%) 22 47.4 1.1 256.8
290sh 593 (50%), 431 (100%), nd Methoxykaempferol derivative 364.6
345 (68%), 299 (75%) Note: nd--not determined; .sup.1according to
Yan et al. 2002 (10); .sup.2according to Puski et al. 1967 (9);
.sup.3compounds not previously identified in cranberry; .sup.4[M +
H].sup.+ for peak 16a.
[0121] TABLE-US-00002 TABLE 2 .sup.1H and .sup.13C NMR shifts of
Cranberry Flavonol Glycosides (Parts per Million) 2 5 6 9 10 15 16a
19 Position .sup.13C .sup.1H .sup.13C .sup.1H .sup.13C .sup.1H
.sup.13C .sup.1H .sup.13C .sup.1H .sup.13C .sup.1H .sup.13C .sup.1H
.sup.13C .sup.1H 6 99.5 6.15 99.4 6.04 100.4 6.06 99.6 6.14 99.5
6.14 94.6 6.14 99.8 6.41 99.4 6.13 8 94.1 6.32 94.8 6.23 95.5 6.27
94.5 6.35 94.3 6.35 89.7 6.39 94.6 6.54 94.0 6.29 2' 109.1 7.12
117.1 7.59 116.8 7.50 116.8 7.47 116.1 7.43 108.6 7.71 117.0 7.84
116.5 7.45 5' 116.1 6.79 116.1 6.81 116.1 6.80 116.2 6.79 111.0
6.82 116.4 7.05 115.8 6.77 6' 109.1 7.12 122.7 7.39 122.7 7.44
122.7 7.60 122.4 7.50 118.2 7.46 122.7 7.85 122.8 7.58 1'' 102.5
5.31 103.1 4.97 102.2 5.06 102.3 5.22 108.6 5.53 97.5 5.20 102.9
5.66 101.9 5.45 2'' 74.3 3.34 71.8 3.59 74.3 3.26 71.7 3.73 83.0
4.11 69.5 3.17 71.8 3.58 72.1 3.58 3'' 77.0 3.14 73.3 3.43 76.2
3.25 72.6 3.48 77.6 3.67 71.3 3.14 73.9 3.41 74.4 3.42 4'' 70.2
3.31 68.6 3.69 69.9 3.15 66.9 3.62 86.8 3.51 65.1 3.21 69.1 3.64
69.3 3.68 5'' 66.9 2.91/ 75.8 3.36 77.1 3.06 65.2 3.18/ 61.6 3.25
61.7 3.54/ 73.8 3.66 74.2 3.75 3.60 3.57 2.89 6'' 60.9 3.34/ 61.1
3.34/ 64.3 4.26/ 64.9 4.23 3.44 3.47 4.35
[0122] TABLE-US-00003 TABLE 3 Inhibitory effect of cranberry
fractions after Sephadex LH-20 column chromatography on TPA-induced
edema of mouse ears. Weight data are expressed as the mean of 8 ear
punches. MEAN WEIGHT SEPARATION OF EAR (SNK PUNCHES multiple means
% TREATMENT (mg/Ear) test, P .ltoreq. 0.05) INHIBITION Acetone +
Acetone 7.4 e -- Acetone + TPA 12.4 a -- Cranberry, 60% MeOH 10.7 b
34.3 (166 .mu.g) + TPA Cranberry, 60% MeOH 8.5 c d e 78.6 (500
.mu.g) + TPA Cranberry, 100% MeOH 10.2 b c 45.3 (166 .mu.g) + TPA
Cranberry, 100% MeOH 9.8 b c d 53.3 (500 .mu.g) + TPA Cranberry,
70% Acetone 8.6 c d e 77.0 (166 .mu.g) + TPA Cranberry, 70% Acetone
8.1 d e 85.5 (500 .mu.g) + TPA Curcumin (95 .mu.g) + TPA 8.5 c d e
79.0
[0123] TABLE-US-00004 TABLE 4 Inhibitory effect of
quercetin-3-O-(6''-benzoyl)-.beta.-galactoside (Cranberry peak 19)
on TPA-induced edema of mouse ears. Weight data are expressed as
the mean of 10 ear punches (12 punches for Acetone + TPA
treatment.) .+-. the standard error. NUMBER WEIGHT OF OF MICE EAR
PUNCHES % TREATMENT PER GROUP (mg/Ear) INHIBITION Acetone + Acetone
5 7.57 .+-. 0.13 -- Acetone + TPA 6 16.12 .+-. 0.64 -- Cranberry
Peak 19 5 13.21 .+-. 0.77 34.0 (87.5 .mu.g) + TPA Cranberry Peak 19
5 11.41 .+-. 0.54 55.1 (175 .mu.g) + TPA Curcumin 5 11.22 .+-. 0.77
57.3 (135 .mu.g) + TPA
* * * * *