U.S. patent application number 11/179771 was filed with the patent office on 2005-12-01 for efficient method for producing compositions enriched in total phenols.
This patent application is currently assigned to PHENOLICS, LLC. Invention is credited to Bailey, David T., Daugherty, F. Joseph, Freeberg, Delano R., Gourdin, Gerald T., Nichols, Rebecca L., Richheimer, Steven L., Tempesta, Michael S..
Application Number | 20050266104 11/179771 |
Document ID | / |
Family ID | 32392402 |
Filed Date | 2005-12-01 |
United States Patent
Application |
20050266104 |
Kind Code |
A1 |
Gourdin, Gerald T. ; et
al. |
December 1, 2005 |
Efficient method for producing compositions enriched in total
phenols
Abstract
This invention provides a process for the preparation of
compositions enriched in total phenols from a crude plant extract.
The process includes a novel column purification step using a
brominated polystyrene resin. This invention also includes
compositions enriched in total phenols. The enriched compositions
are characterized as containing monomeric, oligomeric and polymeric
phenols and having HPLC chromatograms substantially as set forth in
FIGS. 10-13. This invention encompasses methods of using the total
phenol-enriched compositions for treating warm-blooded animals,
including humans, infected with paramyxovaridae such as respiratory
syncytial virus, orthomyoxovaridae such as influenza A, B, and C,
parainfluenza, Herpes viruses such as HSV-1 and HSV-2, and
Flaviviruses such as West Nile Virus, and for treating inflammation
such as caused by arthritis, stress and digestive disease.
Inventors: |
Gourdin, Gerald T.;
(Boulder, CO) ; Richheimer, Steven L.;
(Westminster, CO) ; Tempesta, Michael S.; (El
Granada, CA) ; Bailey, David T.; (Boulder, CO)
; Nichols, Rebecca L.; (Broomfield, CO) ;
Daugherty, F. Joseph; (Omaha, NE) ; Freeberg, Delano
R.; (Algonquin, IL) |
Correspondence
Address: |
HOGAN & HARTSON LLP
ONE TABOR CENTER, SUITE 1500
1200 SEVENTEENTH ST
DENVER
CO
80202
US
|
Assignee: |
PHENOLICS, LLC
|
Family ID: |
32392402 |
Appl. No.: |
11/179771 |
Filed: |
July 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11179771 |
Jul 12, 2005 |
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10302264 |
Nov 22, 2002 |
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10302264 |
Nov 22, 2002 |
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09943158 |
Aug 30, 2001 |
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6780442 |
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60229205 |
Aug 31, 2000 |
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Current U.S.
Class: |
424/732 ;
424/735; 424/765 |
Current CPC
Class: |
Y02A 50/393 20180101;
Y02A 50/30 20180101; A61P 31/14 20180101; A61K 36/73 20130101; A61K
36/185 20130101; A61K 36/736 20130101; A61K 31/352 20130101; A61P
29/00 20180101; A61P 31/00 20180101; A61P 31/16 20180101; A61K
31/70 20130101; A61P 31/22 20180101; C07D 311/62 20130101; A61K
36/35 20130101; A61K 36/45 20130101; A61K 36/87 20130101; A61K
36/45 20130101; A61K 2300/00 20130101; A61K 36/73 20130101; A61K
2300/00 20130101; A61K 36/736 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/732 ;
424/735; 424/765 |
International
Class: |
A61K 035/78 |
Claims
We claim:
1. A composition enriched in total phenols prepared without the
addition of bisulfite ions from plant material comprising fruits
and berries selected from the group consisting of blueberries,
blackberries, strawberries, cranberries, raspberries, red currants,
black currants and cherries, wherein said composition has an
enriched total phenol concentration greater than 12% and said
composition exhibits in vitro COX-2 activity enhanced by a factor
of at least 2.5 over a standard aspirin dose of 660 .mu.g/mL.
2. The composition of claim 1, wherein the enriched total phenol
concentration is greater than 25%.
3. A composition enriched in total phenols prepared from plant
material comprising fruits and berries selected from the group
consisting of blueberries and cranberries, wherein said composition
has an enriched total phenol concentration greater than 12% and
said composition exhibits in vitro COX-2 activity enhanced by a
factor of at least 2.5 over a standard aspirin dose of 660
.mu.g/mL.
4. The composition of claim 3, wherein the enriched total phenol
concentration is greater than 25%.
5. A composition enriched in total phenols prepared without the
addition of bisulfite ions from plant material comprising fruits
and berries selected from the group consisting of blueberries,
blackberries, strawberries, cranberries, raspberries, plums, red
currants, black currants and cherries, wherein said composition has
an enriched total phenol concentration greater than 12.5% and said
composition exhibits in vitro antiviral activity against a virus
which is more potent than an ED.sub.50 value of 100 .mu.g/ML.
6. The composition of claim 5, wherein the enriched total phenol
concentration is greater than 25%.
7. The composition of claim 5, wherein the virus is selected from
the group consisting of influenza virus type A, influenza virus
type B, rhinovirus type 2, Herpes simplex virus 1, Herpes simplex
virus 2, parainfluenza virus, West Nile virus, Varicella-zoster
virus, Rhinovirus Type 2, Adenovirus Type I, and Punta Toro A
virus.
8. The composition of claim 7, wherein the enriched total phenol
concentration is greater than 25%.
9. The composition of claim 5, wherein the selected berries are
cranberries and/or blueberries.
10. The composition of claim 9, wherein the virus is selected from
the group consisting of influenza virus type A, influenza virus
type B, rhinovirus type 2, Herpes simplex virus 1, Herpes simplex
virus 2, parainfluenza virus, West Nile virus, Varicella-zoster
virus, Rhinovirus Type 2, Adenovirus Type I, and Punta Toro A
virus.
11. The composition of claim 10, wherein the enriched total phenol
concentration is greater than 25%
Description
RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 10/302,264, filed Nov. 22, 2002 and entitled
"Efficient Method for Producing Compositions Enriched in Total
Phenols", which is a Continuation-in-Part of, and claims priority
to, U.S. patent application Ser. No. 09/943,158, filed Aug. 30,
2001, and entitled "Efficient Method for Producing Compositions
Enriched in Anthocyanins," which claims priority to U.S.
Provisional Application No. 60/229,205, filed Aug. 31, 2000, and
entitled "Efficient Method for Producing Compositions Enriched in
Anthocyanins," all of said application being incorporated in their
entireties herein, by this reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the extraction and
purification of flavonoid compounds from plant material, and more
specifically to the production of compositions enriched in total
phenols.
[0004] 2. Description of the Prior Art
[0005] Flavonoid compounds are present in all aerial parts of
plants, with high concentrations found in the skin, bark, and
seeds. Such compounds are also found in numerous beverages of
botanical origin, such as tea, cocoa, and wine. The flavonoids are
a member of a larger family of compounds called polyphenols. That
is, these compounds contain more than one hydroxyl group (OH) on
one or more aromatic rings. The physical and chemical properties,
analysis, and biological activities of polyphenols and particularly
flavonoids have been studied for many years.
[0006] Anthocyanins are a particular class of naturally occurring
flavonoid compounds that are responsible for the red, purple, and
blue colors of many fruits, vegetables, cereal grains, and flowers.
For example, the colors of fruits such as blueberries, bilberries,
strawberries, raspberries, boysenberries, marionberries,
cranberries, elderberries, etc. are due to many different
anthocyanins. Over 300 structurally distinct anthocyanins have been
identified in nature. Because anthocyanins are naturally occurring,
they have attracted much interest for use as colorants for foods
and beverages.
[0007] Recently, the interest in anthocyanin pigments has
intensified because of their possible health benefits as dietary
antioxidants. For example, anthocyanin pigments of bilberries
(Vaccinium myrtillus) have long been used for improving visual
acuity and treating circulatory disorders. There is experimental
evidence that certain anthocyanins and other flavonoids have
anti-inflammatory properties. In addition, there are reports that
orally administered anthocyanins are beneficial for treating
diabetes and ulcers and may have antiviral and antimicrobial
activities. The chemical basis for these desirable properties of
flavonoids is believed to be related to their antioxidant capacity.
Thus, the antioxidant characteristics associated with berries and
other fruits and vegetables have been attributed to their
anthocyanin content.
[0008] Proanthocyanidins, also known as "oligomeric
proanthocyanidins," "OPCs," or "procyanidins," are another class of
naturally occurring flavonoid compounds widely available in fruits,
vegetables, nuts, seeds, flowers, and barks. Proanthocyanidins
belong to the category known as condensed tannins. They are the
most common type of tannins found in fruits and vegetables, and are
present in large quantities in the seeds and skins. In nature,
mixtures of different proanthocyanidins are commonly found
together, ranging from individual units to complex molecules
(oligomers or polymers) of many linked units. The general chemical
structure of a polymeric proanthocyanidin comprises linear chains
of flavonoid 3-ol units linked together through common C(4)-C(6)
and/or C(4)-C(8) bonds. .sup.13C NMR has been useful in identifying
the structures of polymeric proanthocyanidins, and recent work has
elucidated the chemistry of di-, tri-, and tetrameric
proanthocyanidins. Larger oligomers of the flavonoid 3-ol units are
predominant in most plants and are found with average molecular
weights above 2,000 Daltons and containing 6 or more monomer units
(Newman, et al., Mag. Res. Chem., 25:118 (1987)).
[0009] Considerable recent research has explored the therapeutic
applications of proanthocyanidins, which are primarily known for
their antioxidant activity. However, these compounds have also been
reported to demonstrate antibacterial, antiviral, anticarcinogenic,
anti-inflammatory, anti-allergic, and vasodilatory actions. In
addition, they have been found to inhibit lipid peroxidation,
platelet aggregation, capillary permeability and fragility, and to
affect enzyme systems including phospholipase A2, cyclooxygenase,
and lipoxygenase. For example, proanthocyanidin monomers (i.e.,
anthocyanins) and dimers have been used in the treatment of
diseases associated with increased capillary fragility and have
also been shown to have anti-inflammatory effects in animals
(Beladi, et al., Ann. N.Y. Acad. Sci., 284:358 (1977)). Based on
these reported findings, oligomeric proanthocyanidins (OPCs) may be
useful components in the treatment of a number of conditions
(Altern. Med. Rev. 5(2):144-151 (2000)).
[0010] Proanthocyanidins may also protect against viruses. In in
vitro studies, proanthocyanidins from witch hazel (Hamamelis
virginiana) killed the Herpes simplex 1 (HSV-1) virus (Erdelmeier,
C. A., Cinatl, J., Plant Med. June: 62(3):241-5 (1996); DeBruyne,
T., Pieters, L., J. Nat. Prod. July: 62(7):954-8 (1999)). Another
study was carried out to determine the structure-activity
relationships of the antiviral activity of various tannins. It was
found that the more condensed the chemical structure, the greater
the antiviral effect (Takechi, M., et al., Phytochemistry,
24:2245-50 (1985)). In another study, proanthocyanidins were shown
to have anti-Herpes simplex activity in which the 50 percent
effective doses needed to reduce herpes simplex plaque formation
were two to three orders of magnitude less than the 50 percent
cytotoxic doses (Fukuchi, K., et al., Antiviral Res., 11:285-298
(1989)).
[0011] Cyclooxygenase (COX-1, COX-2) or prostaglandin endoperoxide
H synthase (PGHS-1, PGHS-2) enzymes are widely used to measure the
anti-inflammatory effects of plant products (Bayer, T., et al.,
Phytochemistry, 28:2373-2378 (1989); and Goda, Y., et al., Chem.
Pharm. Bull., 40:2452-2457 (1992)). COX enzymes are the
pharmacological target sites for nonsteroidal anti-inflammatory
drugs (Humes, J. L., et al., Proc. Natl. Acad. Sci. U.S.A.,
78:2053-2056 (1981); and Rome, L. H., et al., Proc. Natl. Acad.
Sci. U.S.A., 72:4863-4865 (1975)). Two isozymes of cyclooxygenase
involved in prostaglandin synthesis are cyclooxygenase-1 (COX-1)
and cyclooxygenase-2 (COX-2) (Hemler, M., et al., J. Biol. Chem.,
25:251, 5575-5579 (1976)). It is hypothesized that selective COX-2
inhibitors are mainly responsible for anti-inflammatory activity
(Masferrer, J. L., et al., Proc. Natl. Acad. Sci. U.S.A.,
91:3228-3232 (1994)). Flavonoids are now being investigated as
anti-inflammatory substances, as well as for their structural
features for cyclooxygenase (COX) inhibition activity.
[0012] Due to the above characteristics and benefits of
anthocyanins and proanthocyanidins, much effort has been put forth
toward extracting these compounds from fruits, vegetables, and
other plant sources. In addition to proanthocyanidins and
anthocyanins, plants, fruits, and vegetables also contain other
compounds such as mineral salts, common organic acids such as
citric or tartaric acid, carbohydrates, flavonoid glycosides and
catechins. It is often desirable to separate the anthocyanins and
proanthocyanidins from other naturally occurring compounds.
Anthocyanins have been extracted from plants and fruits by various
procedures. One method of extracting anthocyanins employs the
addition of bisulfate to form zwitterionic species. The extract is
passed through an ion exchange column which adsorbs the
zwitterionic anthocyanin adducts, and the adsorbed anthocyanins are
eluted from the resin with acetone, alkali, or dimethylformamide
(DMF). Disadvantages of this process include the presence of
bisulfate, which interferes with adsorption of anthocyanins,
thereby requiring multiple column adsorptions. Elution with alkali
degrades the anthocyanins considerably, while DMF is not a
recognized food additive and therefore must be completely removed
before the anthocyanins can be added to any food products.
[0013] In order to capture these flavonoid compounds, well-defined
and precise processing and separation techniques are needed.
Nafisi-Movaghar, et al. in U.S. Pat. No. 5,912,363 describe a
method for the extraction and purification of proanthocyanidins
from plant material comprising heating an aqueous mixture of plant
material, filtering the aqueous solution through an ultrafiltration
membrane to remove larger molecular weight polymers and
particulates to produce a permeate containing extracted
proanthocyanidins, separating the proanthocyanidins from the liquid
by contacting the permeate with an adsorbent material which is
capable of releasably retaining the proanthocyanidins, and eluting
the retained proanthocyanidins with a polar solvent. However, this
method uses a very hot extraction temperature, which can cause
degradation of the proanthocyanidins. Further, the ultrafiltration
removes some of the low molecular weight polyphenolic material from
the final product.
[0014] Many processes known in the art for extracting and isolating
proanthocyanidins and/or anthocyanins from various plant materials
use toxic and/or environmentally hazardous materials. Consequently,
the current methods available for isolating and purifying
proanthocyanidins are not easily scaled up to an efficient
commercial process where disposal considerations of various
chemicals and solvents play an important role in the overall
feasibility of the process. Further, proanthocyanidins and
anthocyanins must be isolated in a manner that minimizes their
natural tendency toward degradation.
[0015] There is still a need, therefore, for an efficient process
for isolating and purifying compositions containing phenolic
compounds such as proanthocyanidins for uses in nutraceuticals and
pharmaceuticals that is cost-effective, scalable, economically
sound, does not require the use of toxic solvents or reagents, and
isolates the phenolic compounds from plant material in a manner
that minimizes their tendency toward degradation.
SUMMARY OF THE INVENTION
[0016] The present invention provides simplified and economic
methods for the extraction, isolation, and purification of
compositions enriched in total phenols. More specifically, one
aspect of this invention provides a method of preparing
compositions enriched in total phenols comprising: (a) providing a
crude extract of one or more plant materials that contain phenolic
compounds, said extract comprising proanthocyanidins, anthocyanins,
other small phenolics and non-phenolic compounds; (b) filtering the
crude extract; (c) contacting the crude extract with a brominated
polystyrene resin which releasably adsorbs said phenols but does
not substantially retain the non-phenolic compounds; (d) washing
said resin with a wash eluent to elute said non-phenolic compounds;
(e) eluting the resin with a first eluent and collecting a first
fraction containing phenols; (f) eluting the resin with a second
eluent and collecting a second fraction containing phenols; and (g)
isolating the fractions from step (e) or from step (f) or combining
said fractions from steps (e) and (f) to obtain a composition
enriched in total phenols and substantially depleted of said
non-phenolic compounds. This invention further provides total
phenol-enriched compositions isolated by the methods of this
invention.
[0017] This invention further provides methods of fractionating the
total phenol-enriched compositions to separate polar
proanthocyanidins from non-polar proanthocyanidins. This invention
further provides compositions enriched in polar proanthocyanidins
and compositions enriched in non-polar proanthocyanidins. The polar
proanthocyanidins were found to have biological activities that are
different than the non-polar proanthocyanidins.
[0018] When the total phenol-enriched compositions of this
invention are analyzed by reversed-phase HPLC on a C-18 lipophilic
column, characteristic sets of elution peaks of compounds absorbing
at 280 nm and 510 nm are observed. More specifically, the total
phenol-enriched compositions of this invention are characterized as
having a characteristic set of elution peaks in the region between
60 and 75 minutes in an HPLC trace substantially as illustrated in
FIGS. 10-13 when the HPLC analysis is performed as described
herein.
[0019] When the total phenol-enriched compositions of this
invention are analyzed by IR spectrometry, characteristic
absorption peaks of compounds substantially as shown in FIGS. 33-40
are observed. The compositions of this invention are useful as
nutraceuticals and pharmaceuticals. For example, the compositions
of this invention are useful as anti-infective (e.g., antiviral,
anti-UTI and antimicrobial) agents and as anti-inflammatory
agents.
[0020] The foregoing and other features, utilities and advantages
of the invention will be apparent from the following more
particular descriptions of preferred embodiments of the invention
and as illustrated in the accompanying drawings and as particularly
pointed out in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The accompanying drawings, which are incorporated herein and
form a part of the specification, illustrate non-limiting
embodiments of the present invention, and together with the
description, serve to explain the principles of the invention.
[0022] In the Drawings:
[0023] FIG. 1 is a flow chart of a method for preparing a total
phenol-enriched composition according to the method of this
invention.
[0024] FIG. 2 is an HPLC chromatogram at 510 nm of a total
phenol-enriched composition ("fraction 3") prepared from
bilberries.
[0025] FIG. 3 is an HPLC chromatogram at 280 nm of a total
phenol-enriched composition ("fraction 3") prepared from
bilberries.
[0026] FIG. 4 is an HPLC chromatogram at 510 nm of a total
phenol-enriched composition ("fraction 3") prepared from
blueberries.
[0027] FIG. 5 is an HPLC chromatogram at 280 nm of a total
phenol-enriched composition ("fraction 3") prepared from
blueberries.
[0028] FIG. 6 is an HPLC chromatogram at 280 nm of a filtered
elderberry extract.
[0029] FIG. 7 is an HPLC chromatogram at 510 nm of a filtered
elderberry extract.
[0030] FIG. 8 is an HPLC chromatogram at 280 nm of a first fraction
eluted during column loading of a filtered elderberry extract.
[0031] FIG. 9 is an HPLC chromatogram at 510 nm of a first fraction
eluted during column loading of a filtered elderberry extract.
[0032] FIG. 10 is an HPLC chromatogram at 280 nm of a third
fraction eluted with 70% ethanol during column purification of an
elderberry extract on a brominated polystyrene resin.
[0033] FIG. 11 is an HPLC chromatogram at 510 nm of a third
fraction eluted with 70% ethanol during column purification of an
elderberry extract on a brominated polystyrene resin.
[0034] FIG. 12 is an HPLC chromatogram at 280 nm of a fourth
fraction eluted with 90% ethanol during column purification of an
elderberry extract on a brominated polystyrene resin.
[0035] FIG. 13 is an HPLC chromatogram at 510 nm of a fourth
fraction eluted with 90% ethanol during column purification of an
elderberry extract on a brominated polystyrene resin.
[0036] FIG. 14 is an HPLC chromatogram using an alternate HPLC
method of the proanthocyanidins standard prepared as described in
Example 10.
[0037] FIG. 15 is a flow chart of a method for separating polar
proanthocyanidins from non-polar proanthocyanidins.
[0038] FIG. 16 is an HPLC chromatogram at 280 nm of a filtered
elderberry extract.
[0039] FIG. 17 is an HPLC chromatogram at 280 nm of an elderberry
polar proanthocyanidin composition ("fraction 5") isolated from the
combined flow-through and wash fractions from a VLC C-18
column.
[0040] FIG. 18 is an HPLC chromatogram at 280 nm of an elderberry
non-polar proanthocyanidin composition ("fraction 6") isolated in
the 60% methanol eluent from a VLC C-18 column.
[0041] FIG. 19 is an HPLC chromatogram at 280 nm of an elderberry
polar proanthocyanidin composition ("fraction 7") isolated after
semi-preparative HPLC purification.
[0042] FIG. 20 is a .sup.13C NMR spectrum of an elderberry polar
proanthocyanidin composition ("fraction 7") after purification by
semi-preparative HPLC.
[0043] FIG. 21 is an HPLC chromatogram at 280 nm of an elderberry
non-polar proanthocyanidin composition ("fraction 6") isolated
during VLC chromatography on C-18 media and before purification on
a Sephadex LH-20 column, in which the non-proanthocyanidin peaks
are marked with an asterisk.
[0044] FIG. 22 is an HPLC chromatogram at 280 nm of the elderberry
non-polar proanthocyanidin composition ("fraction 8") after
purification on a Sephadex LH-20 column.
[0045] FIG. 23 is an HPLC chromatogram at 368 nm of an elderberry
non-polar proanthocyanidin composition ("fraction 6") isolated
during VLC chromatography on C-18 media and before purification on
a Sephadex LH-20 column.
[0046] FIG. 24 is an HPLC chromatogram at 368 nm of an elderberry
non-polar proanthocyanidin composition ("fraction 8") after
purification on a Sephadex LH-20 column.
[0047] FIG. 25 is a .sup.13C NMR spectrum of an elderberry
non-polar proanthocyanidin composition ("fraction 8") after
purification on a Sephadex LH-20 column.
[0048] FIG. 26 is an HPLC chromatogram at 280 nm of a blueberry
polar proanthocyanidin composition ("fraction 5") isolated during
VLC chromatography on C-18 media and before semi-preparative HPLC
purification.
[0049] FIG. 27 is an HPLC chromatogram at 280 nm of a blueberry
polar proanthocyanidin composition ("fraction 7") after
purification by semi-preparative HPLC.
[0050] FIG. 28 is an HPLC chromatogram at 280 nm of a blueberry
non-polar proanthocyanidin composition ("fraction 6") isolated
during VLC chromatography on C-18 media and before semi-preparative
HPLC purification.
[0051] FIG. 29 is an HPLC chromatogram at 280 nm of a blueberry
non-polar proanthocyanidin composition ("fraction 8") after
purification by semi-preparative HPLC.
[0052] FIG. 30 is an HPLC chromatogram at 280 nm of a plum polar
proanthocyanidin composition ("fraction 5") isolated during VLC
chromatography on C-18 media and before semi-preparative HPLC
purification.
[0053] FIG. 31 is an HPLC chromatogram at 280 nm of a plum polar
proanthocyanidin composition ("fraction 7") after purification by
semi-preparative HPLC.
[0054] FIG. 32 is an HPLC chromatogram at 280 nm of a plum
non-polar proanthocyanidin composition ("fraction 6") isolated
during the 40% and 70% methanol elution from a VLC C-18 column.
[0055] FIG. 33 is an IR spectrum of a purified elderberry polar
proanthocyanidin composition ("fraction 7").
[0056] FIG. 34 is an IR spectrum of a purified elderberry non-polar
proanthocyanidin composition ("fraction 8").
[0057] FIG. 35 is an IR spectrum of a purified cranberry non-polar
proanthocyanidin composition ("fraction 8").
[0058] FIG. 36 is an IR spectrum of a purified cranberry polar
proanthocyanidin composition ("fraction 7").
[0059] FIG. 37 is an IR spectrum of a purified blueberry polar
proanthocyanidin composition ("fraction 7").
[0060] FIG. 38 is an IR spectrum of a purified blueberry non-polar
proanthocyanidin composition ("fraction 8").
[0061] FIG. 39 is an IR spectrum of a purified plum polar
proanthocyanidin composition ("fraction 7").
[0062] FIG. 40 is an IR spectrum of a purified plum non-polar
proanthocyanidin composition ("fraction 6").
[0063] FIG. 41 is an HPLC chromatogram at 280 nm of a cranberry
polar proanthocyanidin composition ("fraction 5") before
semi-preparative HPLC purification.
[0064] FIG. 42 is an HPLC chromatogram at 280 nm of a cranberry
polar proanthocyanidin composition ("fraction 7") after
semi-preparative HPLC purification.
[0065] FIG. 43 is an HPLC chromatogram at 280 nm of a cranberry
non-polar proanthocyanidin composition ("fraction 6").
DETAILED DESCRIPTION OF THE INVENTION
[0066] This invention provides methods for preparing compositions
enriched in total phenols from plant materials that naturally
contain phenolic compounds such as anthocyanins and
proanthocyanidins. The method of this invention further provides
compositions enriched in total phenols.
[0067] As used herein, the term "extract" refers to a substance
derived from a plant source that naturally contains phenolic
compounds, including extracts prepared from the whole plant or from
various parts of the plant, such as the fruits, leaves, stems,
roots, bark, etc. Thus, the method of this invention is not limited
to the particular part of the plant used to prepare the extract.
The present method can use any source of anthocyanins and
proanthocyanidins, most typically from botanically derived plant
material such as seeds, fruits, skins, vegetables, nuts, tree
barks, and other plant materials that contain phenolic compounds.
Most colored fruits, berries, and vegetables are known to contain
phenolic compounds. Examples of plants, fruits, berries, and
vegetables that contain phenolic compounds include, but are not
limited to, blueberries, bilberries, elderberries, plums,
blackberries, strawberries, red currants, black currants,
cranberries, cherries, raspberries, grapes, currants, hibiscus
flowers, bell peppers, beans, peas, red cabbage, purple corn, and
violet sweet potatoes. The raw plant material may be used either as
is (wet) or may be dried prior to extraction. Optionally, the raw
plant material may be presorted by separating and removing the
components low in anthocyanins and proanthocyanidins prior to
extraction.
[0068] In one embodiment, the phenolic-enriched compositions of the
present invention are obtained by extracting and purifying one or
more berries and/or fruits containing phenolic compounds including,
but not limited to, blueberries, bilberries, elderberries, plums,
blackberries, strawberries, red currants, black currants,
cranberries, cherries, raspberries, and grapes.
[0069] As used herein, the terms "phenols" and "phenolic compounds"
are used interchangeably and include monomeric, oligomeric and
polymeric compounds having one or more phenolic groups, and
include, but are not limited to, anthocyanins, proanthocyanidins,
and flavonoids.
[0070] As used herein, the term "total phenol-enriched composition"
refers to a composition enriched in one or more phenolic compounds
and having substantially depleted levels of non-phenolic compounds
present in crude extracts of plants, fruits, berries, and
vegetables. Examples of such non-phenolic compounds include, but
are not limited to, sugars, cellulose, pectin, amino acids,
proteins, nucleic acids, plant sterols, fatty acids, and
triglycerides.
[0071] The method of this invention is based on the discovery that
purifying an extract containing phenols on a brominated polystyrene
resin rather than on a conventional polystyrene resin or other
resins used in the art provides total phenol-enriched compositions
having higher purities, as discussed below in detail.
[0072] FIG. 1 is a flowchart showing the steps of one embodiment of
the process of this invention in which a composition enriched in
total phenols may be prepared. The process of this invention
provides an economical and efficient method of obtaining
compositions enriched in total phenols by eliminating several
process steps and by reducing the amount of reagents needed in the
process, thereby reducing production costs and waste disposal
issues.
[0073] In one embodiment of the process of this invention, as
illustrated in steps 10-70 in FIG. 1, phenolic compounds (e.g.,
proanthocyanidins and anthocyanins) and non-phenolic compounds are
extracted from a fresh or dried plant material (step 10). Those
skilled in the art will recognize that a variety of extraction
methods are available in the literature, such as vat extraction,
percolation, countercurrent extraction, etc. The particular method
of extraction employed is not essential to the process of the
present invention. The degree of comminutation of the plant
material prior to the extraction process should provide sufficient
particulate surface area for the extraction solvent to contact.
[0074] In one embodiment of the process shown in FIG. 1, the
extraction step (step 10) is accomplished by placing fresh or dried
plant material in an appropriate amount of extraction solvent. In
one embodiment, the extraction solvent comprises an acidified
alcohol solution having about 0-95% ethanol in water and a suitable
acid in an amount of about 0-3%, more preferably about 0.006-0.012%
by weight. In another embodiment, the extraction solvent comprises
an acidified alcohol solution having between about 0-100% methanol
in water and between about 0-3% by weight of a suitable acid.
Suitable acids that may be used in the extraction step include, but
are not limited to, sulfuric acid (H.sub.2SO.sub.4), acetic acid
(HOAc) or hydrochloric acid (HCl). The addition of an acid to the
extraction solvent prevents degradation of the proanthocyanidins
and anthocyanins. Thus, in one embodiment the acidic conditions are
maintained throughout most of the steps of the process of this
invention as illustrated in FIG. 1. The plant material is contacted
with the extraction solution for an appropriate amount of time at a
temperature between about room temperature and 75.degree. C.,
preferably at 40.degree. C., to form the crude extract. The amount
of plant material to extraction solvent used in the extraction
process varies between about 2:1 to about 1:20 on a gram to
milliliter basis. In one embodiment, the ratio of plant material to
extraction solvent is between about 1:4 and 1:8.
[0075] The crude extract contains phenolic compounds such as
proanthocyanidins, anthocyanins and other phenolics, as well as
undesired non-phenolic materials such as sugars, pectin, plant
sterols, fatty acids, triglycerides, and other compounds. Solid
residue contained in the crude extract is separated from the liquid
portion, and the solids are either re-extracted as described above
or discarded.
[0076] In one embodiment of step 10 (FIG. 1), pectinase is added
either to the plant material or to the extraction solvent before or
during the extraction process. Alternatively, the pectinase can be
added to the crude extract after the extraction process is
complete. The pectinase serves to prevent the extract from gelling
at any point during or after the extraction process so that it will
remain flowable during the column purification. The amount of
pectinase added will depend, of course, on the amount of plant
material used to prepare the extract. Typically, the pectinase is
added in an amount between about 0 and 0.12% by weight of the plant
material.
[0077] With continued reference to FIG. 1, if either an ethanolic
or methanolic extraction solvent was used to prepare the crude
extract in step 10, the crude extract is concentrated (step 20)
until the crude extract contains less than 6% ethanol or methanol,
preferably maintaining a temperature of 40.degree. C. or less
during concentration. Water is added to dilute the concentrated
crude extract, and the diluted crude extract is either concentrated
and diluted again with water prior to step 30, or is carried on
directly to step 30 without performing a second dilution. Of
course, if water was used as the extraction solution in the
preparation of the crude extract, step 20 is not necessary, and in
this case the crude extract from step 10 is taken directly on to
step 30 as shown by the dashed arrow in FIG. 1.
[0078] Step 30 of the process shown in FIG. 1 comprises filtering
the crude extract from step 10 or 20 to remove solids that may have
precipitated from the crude extract. The inventors discovered that
by adjusting the extraction conditions as described for step 10,
the amount of undesirable non-phenolic compounds that precipitate
from the crude extract by filtration in step 30 is increased.
Various filtration methods may be employed in filtration step 30 of
the process of this invention. One filtration method that may be
employed in step 30 comprises adding a measured amount of a filter
aid such as diatomaceous earth or cellulose to the crude extract.
The mixture of crude extract and filter aid is preferably shaken or
stirred until homogeneous and filtered through a bed of filter aid.
The bed is washed with an aqueous acidic solution, preferably about
0.006% aqueous sulfuric acid.
[0079] Other filtration methods that may be used in step 30 of FIG.
1 include filtering the crude extract through a bed of sand or a 30
micron polypropylene filter that is preferably covered with glass
wool. Yet another filtration method comprises using a bag filter (a
bag-shaped cloth filter composed of polyethylene or polypropylene),
which may advantageously be placed in-line with the purification
column of step 40 described below. The filters described above are
used to remove precipitated solids and are not size exclusion
filters.
[0080] To isolate the phenolic compounds according to the method
shown in FIG. 1, the filtered extract isolated in step 30 is
contacted with a brominated polystyrene adsorbent material capable
of releasably adsorbing the phenolic compounds such as
proanthocyanidins and anthocyanins, but which retains less of the
undesired non-phenolic materials that were present in the filtered
extract. The present inventors discovered that a high purity
composition enriched in total phenols could be obtained by
purifying the filtered extract isolated in step 30 on a brominated
polystyrene resin, such as SP-207 (Supelco; Bellafonte, Pa.),
manufactured by Mitsubishi Chemical America. SP-207 resin is a
macroporous, brominated styrenic polymeric bead type resin designed
for reversed-phase chromatographic applications, and has a particle
size distribution between about 250-600 microns and a pore size
range between about 100-300 Angstroms. The bromination of the
aromatic rings provides increased hydrophobicity to the polystyrene
resin, and is designed to provide a resin having increased
selectivity for hydrophobic molecules relative to conventional
styrene-divinylbenzene polymeric reversed-phase supports. Because
of its tight binding properties, brominated polystyrene resin is
not typically used in the purification of natural products.
[0081] Thus, since it was known that conventional polystyrene
resins tend to bind phenolic compounds such as proanthocyanidins
and anthocyanins so tightly that it is very difficult to elute such
compounds from the polystyrene resin, it was expected that the
brominated polystyrene resin would bind phenolic compounds even
tighter. Therefore, it was not expected that a brominated
polystyrene resin would be suitable for the purification of
phenolic compounds. However, the inventors surprisingly and
unexpectedly discovered that the brominated polystyrene resin binds
phenolic compounds such as proanthocyanidins and anthocyanins less
tightly than non-brominated polystyrene resins, but still allows
for the separation of phenolic compounds from undesired
non-phenolic compounds.
[0082] In one embodiment of the method shown in FIG. 1, the
filtered extract isolated in step 30 is loaded onto a column packed
with brominated polystyrene resin having a particle size
distribution between about 250-600 microns and a pore size range
between about 100-300 Angstroms (step 40). However, while step 40
is described herein in terms of contacting the extract with a resin
packed into a column, such a description is merely for ease of
explanation. Thus, the resin need not be packed into a column in
order to perform the method of this invention. The amount of
filtered extract that is loaded onto the column depends on the
plant material used to prepare the crude extract. For example, when
the crude extract is prepared from bilberries, about 16-30 grams of
total phenols may be loaded per liter of resin. As another example,
when the crude extract is prepared from blueberries, about 15-45
grams of total phenols may be loaded per liter of resin. When the
crude extract is prepared from elderberries, about 15-40 grams of
total phenols may be loaded per liter of resin. The filtered
extract may be diluted with water prior to loading if the solids
concentration in the concentrated crude extract exceeds 200 grams
per liter. The fractions eluting during column loading in step 40
(FIG. 1) are collected as "fraction 1."
[0083] Subsequent to loading the filtered crude extract onto the
resin, undesired non-phenolic materials (e.g., sugars, salts,
organic acids, etc.) which have little or no affinity for the
adsorbent are eluted from the resin with an aqueous wash solvent
comprising at least 0.003% acid such as aqueous sulfuric acid,
aqueous acetic acid or aqueous hydrochloric acid (FIG. 1, step 50).
For example, about three column volumes of 0.006% aqueous sulfuric
acid or 0.1% aqueous acetic acid can be used to elute the
extraneous materials. The eluent is collected as "fraction 2."
[0084] With continued reference to FIG. 1, the column is next
eluted with a first eluent comprising a polar organic solvent such
as about 50 to 70% ethanol/water or about 50 to 90% methanol/water
(step 60). Typically about 2 to 12 column volumes of eluting
solvent are used in Step 60. In one embodiment, the first eluent
contains about 0.003% of an acid such as sulfuric acid,
hydrochloric acid or acetic acid. The fraction(s) collected during
elution step 60 are collected as "fraction 3." "Fraction 3"
contains a portion of the phenolic compounds contained in the crude
extract and is particularly enriched in anthocyanins and contains
proanthocyanidins.
[0085] After the majority of the anthocyanins have been eluted from
the column, as determined by UV-VIS spectroscopy, the column is
eluted with a second eluent (step 70; FIG. 1) comprising a polar
organic solvent comprising a greater percentage of ethanol or
methanol than the solvent used to elute the anthocyanins (step 60).
For example, the second eluent may comprise about 50 to 90%
ethanol/water or about 75 to 100% methanol/water. The fraction(s)
collected during elution step 70 are collected as "fraction 4."
"Fraction 4" contains an additional portion of the phenolic
compounds originally contained in the crude extract and is
typically enriched in proanthocyanidins. "Fraction 4" may also
contain anthocyanins not isolated during elution step 60.
[0086] Recovery of the phenolic compounds in "fraction 3" and
"fraction 4" can be accomplished in any convenient manner such as
by evaporation, distillation, freeze-drying, and the like, to
provide a total phenol-enriched composition of this invention.
[0087] The above-described process is suitable for preparing
compositions sufficiently enriched in total phenols for use as
nutraceuticals from a variety of plant materials that contain
phenolic compounds including, but not limited to, elderberries,
plums, blueberries, bilberries, blackberries, strawberries, red
currants, black currants, cranberries, cherries, raspberries,
grapes, hibiscus flowers, bell peppers, beans, peas, red cabbage,
purple corn, and violet sweet potatoes. In one embodiment, the
enriched compositions of this invention contain at least 10-80%
total phenols. In another embodiment, the compositions contain at
least 12% total phenols. In yet another embodiment, the
compositions contain at least 25% total phenols.
[0088] It was discovered that the total phenol-enriched
compositions, and in particular the compositions isolated from
"fraction 3," "fraction 4," or a combination thereof, prepared from
fruits and berries in particular produce similar HPLC chromatograms
having the characteristic peaks such as those shown in FIGS. 12 and
13 that are not contained in HPLC chromatograms of compositions
prepared from plant material other than fruits and berries. For
example, the HPLC chromatograms of all total phenol-enriched
compositions prepared from fruits and berries according to the
method illustrated in FIG. 1 and isolated from "fraction 4" were
found to contain characteristic peaks between 60 and 75 minutes
similar to peaks in the chromatogram shown in FIGS. 12 and 13 for a
"fraction 4" composition isolated from elderberries. The total
phenol-enriched compositions of this invention, isolated either
from "fraction 3," "fraction 4," or a combination thereof, and
prepared specifically from fruits and berries have anti-infective
(e.g., antiviral) and anti-inflammatory activity, as described
below in detail.
[0089] When the total phenol-enriched compositions of this
invention are analyzed by IR spectrometry, characteristic peaks
from the phenolic compounds are also observed. More specifically,
the total phenol-enriched compositions of this invention are
characterized as having IR absorption peaks substantially as
illustrated in FIGS. 33-40.
[0090] It was also discovered that the total phenol-enriched
compositions (e.g., "fraction 3," "fraction 4," or a combination
thereof) could be further partitioned into a "polar"
proanthocyanidin-enriched fraction and a "non-polar"
proanthocyanidin-enriched fraction using low pressure Vacuum Liquid
Chromatography (VLC) on a reversed-phase lipophilic column, such as
a C-18 column as described in detail in Example 11 and as shown in
FIG. 15. For example, a "fraction 3" composition isolated from an
elderberry extract was dissolved in water and loaded onto a C-18
column. The column was washed with 100% water to collect materials
that are not strongly retained by the C-18 media. The flow through
and wash fractions were combined as "fraction 5" and contained the
more polar proanthocyanidins. Thus, "fraction 5" is referred to
herein as the "polar" proanthocyanidin-enriched fraction (FIG. 15).
The polar proanthocyanidin-enriched "fraction 5" from elderberry
typically has some purple color, suggesting that the polymers in
this fraction contain at least one or more cationic anthocyanidin
subunits within the oligomeric proanthocyanidin chains. The VLC
column was then eluted with 30 to 100% methanol to collect the
proanthocyanidins that are more strongly retained by the C-18 media
used in the low-pressure column. The methanol fractions were
combined as "fraction 6" and contained proanthocyanidins that are
less polar than those collected in "fraction 5." Thus, "fraction 6"
is referred to herein as the "non-polar" proanthocyanidin-enriched
fraction (FIG. 15). The non-polar proanthocyanidin-enriched
"fraction 6" has little if any color, suggesting that the
oligomeric proanthocyanidin chains in this fraction do not contain
cationic anthocyanidin subunits.
[0091] Thus, the present invention provides a method of
conveniently separating the polar proanthocyanidins from the
non-polar proanthocyanidins contained in either "fraction 3,"
"fraction 4," or a combination thereof. It was also found that a
polar proanthocyanidin-enriched "fraction 5" and non-polar
proanthocyanidin-enriched "fraction 6" could be isolated directly
by loading a crude filtered aqueous extract (FIG. 1, step 30) onto
a C-18 VLC column. It is to be understood that the terms "polar"
and "non-polar" when used to describe the isolated
proanthocyanidin-enriched fractions 5 and 6, respectively, refer to
the polarity of the proanthocyanidins in fractions 5 and 6 relative
to one another, that is, how the particular fractions behave on a
C-18 VLC column. The polar proanthocyanidin-enriche- d compositions
(fraction 5) and the non-polar proanthocyanidin-enriched
compositions ("fraction 6") of this invention have substantially
reduced levels of anthocyanins, as discussed in the Examples.
[0092] The polar and non-polar proanthocyanidin-enriched fractions
("fraction 5" and "fraction 6," respectively) were found to have
different biological activities, and the non-polar fraction was
found to have greater antiviral activity than the polar fraction in
certain assays as described in Example 17.
[0093] Each of the polar and non-polar proanthocyanidin-enriched
fractions 5 and 6, respectively, can be purified further as shown
in FIG. 15 and as described in Examples 12-14. For example, the
polar proanthocyanidin-enriched "fraction 5" isolated during the
VLC separation can be loaded onto a semi-preparative C-18 HPLC
column that releasably retains the polar proanthocyanidins. The
column is then washed with a solvent gradient comprising increasing
percentages of acetonitrile, methanol or ethanol to elute most of
the anthocyanins and other polar compounds, and then with at least
60% acetonitrile, methanol or ethanol to elute "fraction 7"
containing the purified polar proanthocyanidins (FIG. 15).
Additionally, the non-polar proanthocyanidin-enriched "fraction 6"
isolated during the VLC separation can be further purified by gel
filtration or reversed-phase semi-preparative HPLC. Gel filtration,
also called size exclusion or gel permeation chromatography, is a
liquid chromatography technique that separates molecules according
to their size. This type of media retains smaller compounds, while
the larger non-polar proanthocyanidin-enriched "fraction 8" (FIG.
15) elute with the flow-through eluent. The purified polar and
non-polar proanthocyanidin-enriched fractions 7 and 8,
respectively, of this invention have substantially reduced levels
of anthocyanins and flavonoids, and also have substantially reduced
levels of non-phenolic compounds. It was further observed that the
purified polar and non-polar proanthocyanidin-enriched "fraction 7"
and "fraction 8", respectively, have different biological
activities.
[0094] The total phenol-enriched compositions ("fraction 3,"
"fraction 4," or a combination thereof), polar
proanthocyanidin-enriched compositions (fractions 5 and 7), and
non-polar proanthocyanidin-enriched compositions (fractions 6 and
8) of this invention possess a range of biological activities. For
example, the compositions of this invention were found to have
antiviral activities, as described in Examples 15 and 16. The
compositions of this invention can be used either alone or in
combination with other antiviral agents to prevent and/or treat
diseases induced by or complicated with viral infections from
viruses including, but not limited to, influenza A, B, and C,
parainfluenza virus, adenovirus type 1, Punta Toro Virus A, Herpes
simplex virus I and II, rhinovirus, West Nile virus,
Varicella-zoster virus and measles virus. Accordingly, the total
phenol-enriched compositions, polar proanthocyanidin-enriched
compositions, and non-polar proanthocyanidin-enriched compositions
of this invention can be advantageously used in prophylactic and
therapeutic applications against diseases induced by such viruses
by administering a therapeutically effective amount of a
composition of this invention.
[0095] Proanthocyanidins have also been investigated as
anti-inflammatory substances due to their inhibition of
cyclooxygenase (COX) activity. It has been shown that it is
desirable for anti-inflammatory substances to be selective for
COX-2 inhibition rather than COX-1 inhibition. Accordingly, another
aspect of this invention comprises a method of treating
inflammatory diseases in mammals comprising administering a
therapeutically effective amount of a total phenol-enriched
composition, polar proanthocyanidin-enriched composition, or a
non-polar proanthocyanidin-enriched composition of this invention.
For example, total phenol-enriched compositions isolated as
fractions 3 and 4 during purification of a blueberry extract were
found to have high COX-2/COX-1 inhibition selectivity and an
IC.sub.50 of 108 .mu.g/mL (Example 17). The compositions of this
invention can be used either alone or in combination with other
anti-inflammatory agents to prevent or inhibit inflammatory
responses. Such responses may be caused by conditions or diseases
including, but not limited to, osteoarthritis, allergenic rhinitis,
cardiovascular disease, upper respiratory diseases, wound
infections, neuritis and hepatitis.
[0096] It is known that proanthocyanidins isolated from cranberries
and blueberries inhibit bacteria from attaching to the bladder
wall, thereby reducing the potential for maladies such as urinary
tract infections (Howell, et al., New England J. Medicine,
339:1085-1086 (1998)). It has been postulated that
proanthocyanidins exert their effect by inhibiting the adhesion of
bacteria. Accordingly, another aspect of this invention comprises a
method of preventing or treating urogenital infections in a mammal
comprising administering an effective amount of a total
phenol-enriched composition, polar proanthocyanidin-enriched
composition, or a non-polar proanthocyanidin-enriched composition
of this invention in an amount sufficient to prevent, reduce, or
eliminate the symptoms associated with such infections. The
compositions of this invention can be used either alone or in
combination with other antimicrobial agents.
[0097] It is further known that proanthocyanidins are potent
antioxidants. For example, the antioxidant effects of
proanthocyanidins are presumed to account for many of their
benefits on the cardiovascular and immune systems. Accordingly, the
total phenol-enriched compositions, polar proanthocyanidin-enriched
compositions, and non-polar proanthocyanidin-enriched compositions
of this invention may be used as dietary supplements (e.g., dietary
antioxidants) and for the treatment of disorders in humans and
mammals. For example, the compositions of this invention may be
used for improving visual acuity and for treating circulatory
disorders, diabetes, and ulcers.
[0098] The total phenol-enriched compositions, polar
proanthocyanidin-enriched compositions, and non-polar
proanthocyanidin-enriched compositions of this invention can also
be combined with immunoactive agents, including but not limited to,
arabinogalactan, species of Echinacea, vitamins, minerals,
polysaccharides and astragalus.
[0099] The total phenol-enriched compositions, polar
proanthocyanidin-enriched compositions, and non-polar
proanthocyanidin-enriched compositions of this invention can also
be combined with antimutagenic agents including, but not limited
to, green tea extracts, catechins, epicatechins, epigallocatechins,
gallocatechins, and flavonoids.
[0100] The total phenol-enriched compositions, polar
proanthocyanidin-enriched compositions, and non-polar
proanthocyanidin-enriched compositions of this invention may be
formulated as pills, capsules, liquids, or tinctures. In
formulating compositions according to this invention, a wide range
of excipients may be used, the nature of which will depend, of
course, on the intended mode of application of the composition.
Examples of excipients include preservatives, carriers, and
buffering, thickening, suspending, stabilizing, wetting,
emulsifying, coloring and flavoring agents, and in particular
carboxy vinyl polymers, propylene glycol, ethyl alcohol, water,
cetyl alcohol, saturated vegetable triglycerides, fatty acid esters
or propylene glycol, triethanolamine, glycerol, starch, sorbitol,
carboxymethyl cellulose, lauryl sulphate, dicalcium phosphate,
lecithin, etc.
[0101] The foregoing description is considered as illustrative only
of the principles of the invention. Further, since numerous
modifications and changes will readily occur to those skilled in
the art, it is not desired to limit the invention to the exact
construction and process shown as described above. Accordingly, all
suitable modifications and equivalents may be resorted to falling
within the scope of the invention as defined by the claims that
follow.
[0102] The words "comprise," "comprising," "include," "including,"
and "includes" when used in this specification and in the following
claims are intended to specify the presence of stated features,
integers, components, or steps, but they do not preclude the
presence or addition of one or more other features, integers,
components, steps, or groups thereof.
EXAMPLES
Example 1
Purification of Bilberry Using a Water Extraction
[0103] Three extractions were performed on 1 kg of dried bilberry
raw material. The first extraction used 6 L of water and the other
two extractions used 4 L of water. All extractions were acidified
with concentrated sulfuric acid to an acid concentration of 5 g/L.
There was approximately an 88% recovery of anthocyanins into the
crude extract. Exactly 2.3 L of the crude extract were filtered
through a 30 micron polypropylene filter with a layer of glass wool
over the filter. The glass wool was changed once and the filter
rinsed with deionized water. The final volume of the filtrate was
2.43 L with a 90.9% recovery of anthocyanins in the filtrate.
[0104] A column was packed with brominated polystyrene resin SP-207
(Supelco; Belefonte, Pa.) and equilibrated with 0.1% acetic acid.
The column was loaded with 2.24 L of the filtrate at a solids
concentration of 29.8 g/L using a flow rate of 2.2 mL/min. The
loading bleed was less than 0.9% of the loaded anthocyanins with an
overall loss of 4.07% of the anthocyanins in the loading and first
two column washes. There was an 88.4% recovery of the anthocyanins
in the elution step and an anthocyanins mass balance of 92.5%. A
few hundred milliliters of elution product were evaporated to
dryness on a rotary evaporator and then lyophilized. Final assay of
the dried product was by standard spectrophotometric determination
of absorbance at 535 nm against a delphinidin chloride standard
(102 absorbance units/g/L at 1.0 cm). The enriched composition
contained 43% total anthocyanins by weight.
Example 2
Purification of Bilberry Using a 70% Ethanol Extraction
[0105] Dried bilberry raw material (667 g), assayed at 2.0%
anthocyanins, was extracted by percolation using 70% ethanol/water
containing 3% sulfuric acid by volume. The solids in the crude
extract contained 3.9% by weight total anthocyanins. One liter of
the first extraction volume was mixed with 100 mL deionized water
and evaporated in vacuo to about 460 mL to remove the alcohol.
Deionized water (300 mL) was added to the mixture, and an
additional 170 mL of liquid were evaporated. Deionized water (210
mL) was added to make the final volume 800 mL. To the aqueous
mixture was added 150 g of Celite 512 (0.5 to 0.9 g of Celite per
gram of solids). The mixture was shaken until homogeneous. The
Celite/extract mixture was poured over a 30 g bed of damp Celite
512 under vacuum. Upon completion of filtration, the bed was washed
with 1.20 L of 1% aqueous sulfuric acid in 200 mL increments. The
filtrate volume was 1855 mL. To the filtrate was added 145 mL of
deionized water to give a final volume of 2.0 L.
[0106] A portion of the filtrate (695 mL) was loaded at 2.2
mL/minute (1.3 mL/min/cm.sup.2) onto a column loaded with 170 mL
brominated polystyrene resin (SP-207). This gave a load value of 17
g of anthocyanins per liter of column media. The column was washed
with one column volume of 0.1% aqueous acetic acid followed by 2.5
column volumes of 0.1% HOAc/10% ethanol/90% water. The column was
then eluted with 10 column volumes of 70% ethanol/water, and the
70% ethanol fractions were combined and concentrated in vacuo at
60.degree. C. and 50 mbar to provide a dark, dry, shiny amorphous
solid ("fraction 3"). Final assay of the dried product was by
standard spectrophotometric determination of absorbance at 535 nm
against a delphinidin chloride standard (102 absorbance units/g/L
at 1.0 cm). The enriched composition contained 32% total
anthocyanins by weight.
[0107] FIGS. 2 and 3 are HPLC chromatograms at 510 nm and 280 nm,
respectively, of a total phenol-enriched composition ("fraction 3")
prepared from bilberries according to the process of this
invention.
[0108] Table 1 summarizes the percent of each anthocyanin in a
typical anthocyanin-enriched composition ("fraction 3").
1TABLE 1 Identification and content of each anthocyanin present in
a bilberry "fraction 3" Elution Name order % composition
Delphinidin-3-O-galactosi- de 1 3.3 Delphinidin-3-O-glucoside 2 3.9
Cyanidin-3-O-galactoside 3 2.1 Delphinidin-3-O-arabinoside 4 2.6
Cyanidin-3-O-glucoside 5 2.8 Petunidin-3-O-galactoside 6 1.0
Petunidin-3-O-glucoside 7 2.5 Cyanidin-3-O-arabinosid- e 8 1.7
Peonidin-3-O-galactoside 9 0.3 Petunidin-3-O-arabinoside 10 0.8
Malvidin-3-O-galactoside 11 2.1 Peonidin-3-O-glucoside (co-elute)
Malvidin-3-O-glucoside 12 2.5 Peonidin-3-O-arabinose 13 0.1
Malvidin-3-O-arabinose 14 0.6 Total 26.3
Example 3
Total Phenol-Enriched Compositions from Blueberries
[0109] To 940 g of dried and ground blueberry (Van Drunen
FutureCeuticals; Momence, Ill.) were added 4.0 liters of extraction
solvent (1.0% w/v sulfuric acid in 70% ethanol) in a 10 L round
bottom flask. The flask was rotated in a constant temperature water
bath held at 40.degree. C. for two hours. The mixture was swirled
and filtered through a 150 g bed of Celite 512 under vacuum. The
blueberry biomass cake was washed with 500 mL of extraction
solvent. The cake was carefully scraped away from the Celite bed,
poured into a round bottom flask, and re-extracted following the
above-described procedure. A third extraction was then performed.
The three crude extracts were combined.
[0110] A portion of the combined extracts (2.00 L) was concentrated
in vacuo to 175 mL at a water bath temperature of 40.degree. C. The
evaporated extract was diluted with deionized water to give 675 mL
of crude blueberry extract. The crude extract was loaded without
filtration onto a previously conditioned (i.e., washed with
acetone) and equilibrated column loaded with 170 mL of brominated
polystyrene resin (SP-207). The column was washed with 0.1% acetic
acid and with 0.1% HOAc/10% ethanol. The anthocyanins were eluted
with 70% ethanol. The product pool was evaporated in vacuo at
60.degree. C. and 50 mbar. Final product assay was by standard
spectrophotometric determination of absorbance at 535 nm against a
delphinidin chloride standard (102 absorbance units/g/L at 1.0 cm).
The purified blueberry composition ("fraction 3") contained 18%
total anthocyanins by weight, with an overall recovery of
anthocyanins of 95%.
[0111] FIGS. 4 and 5 are HPLC chromatograms at 510 nm and 280 nm,
respectively, of a total phenol-enriched composition ("fraction 3")
prepared from blueberries according to the method of this
invention.
Example 4
Higher Purity Total Phenol-Enriched Composition from
Blueberries
[0112] In this example, a portion of a total phenol-enriched
composition prepared from blueberries and having 18% total
anthocyanins by weight, prepared as described in Example 3, was
passed through either a strong or a weak anion exchange resin to
remove residual acids in order to increase the purity of the
enriched composition.
[0113] Approximately 1.0 g of the total phenol-enriched blueberry
composition was dissolved in 50 mL of water and passed through a 9
mL column containing either a strong anion exchange resin (Super
Q-650 M; TosoHaas; Montgomery, Pa.) or a weak anion exchange resin
(DEM-63; Whatman). The column was washed with 30-35 mL of water. In
the case of the strong anion exchange resin column, the resin was
further washed with 25 mL of 20% ethanol, followed by 40% ethanol.
The composition isolated from the strong anion exchange column
contained 28.3% total anthocyanins by weight, and the recovery was
88%. The composition isolated from the weak anion exchange column
contained 30.6% total anthocyanins by weight, and the recovery was
88%.
Example 5
Total Phenol-Enriched Compositions from Bilberry Using Pectinase
Treatment
[0114] Warm water (548 g) was added to 1024 g of frozen bilberries.
The mixture was pureed in a blender and then heated to 40.degree.
C. Next, 150 .mu.L of pectinase (Quest Super 7.times.; Quest
International, Norwich, N.Y.) were added for a 30 minute treatment
at 40.degree. C. with stirring. Approximately 4 mL of concentrated
sulfuric acid were added to the slurry to achieve an acid
concentration of 0.5% (w/w). The mixture was then heated to
45.degree. C. and extracted for 15 minutes with very slow stirring.
Dicalite (164 g) was added to the extracted mixture, which was then
filtered over a 26 g Dicalite bed. The resulting cake was washed
three times with 400 mL of warm 0.1% aqueous sulfuric acid. This
extract was filtered through a 25 .mu.m pressure filter. All of the
filtered extract (2.4 L) was loaded onto a 170 mL SP-207 column.
After loading, the column was washed with 0.1% aqueous acetic acid
and eluted with 70% aqueous ethanol to provide "fraction 3".
"Fraction 3" was evaporated to dryness and then placed on a
lyophilizer for 48 hours. The final product was assayed for total
anthocyanins by standard spectrophotometric determination of
absorbance at 535 nm. The total phenol-enriched composition
contained 40% total anthocyanins by weight. The overall recovery of
anthocyanins was approximately 79%.
Example 6
Enriched Compositions from Elderberry Biomass Powder
[0115] Approximately 190 g of dried elderberry biomass powder (BI
Nutraceuticals, Long Beach, Calif.) assayed at 1.88% anthocyanins
and 5.31% total phenols were added to 1000 g of warm water. The
solution was mixed thoroughly and transferred to a hot water bath
at 45.degree. C. To the solution was added 190 .mu.L of pectinase
(Super 7.times., Quest), and then the mixture was allowed to sit
for 30 minutes. The mixture was acidified to a pH of 2.5 using 2.5
mL of concentrated H.sub.2SO.sub.4 and gently mixed for ten
minutes. To this acidified mixture was added 164 g of Celite, and
then the acidified mixture was filtered over a 26 g Celite bed. The
filter cake was washed three times with 400 mL of acidified warm
water, for a total of 1200 mL. The filtrate was then filtered
through a 25 .mu.m pressure filter to provide an elderberry
extract.
[0116] The elderberry extract was loaded onto 170 mL of SP-207
(Mitsubishi Chemical) brominated polystyrene column at a rate of
2.3 mL/min (1.3 mL/min/cm.sup.2). The eluent collected off the
column during loading was collected as "fraction 1." After loading,
the column was washed with 3 column volumes (3.times.170 mL) of
0.006% aqueous sulfuric acid. The eluent from this wash was
collected as "fraction 2." The column was then eluted with 8-10
column volumes of 70% aqueous ethanol, which were collected as
"fraction 3." The column was then washed with 3 column volumes of
90% aqueous ethanol, which were collected as "fraction 4." The
column was re-equilibrated with 8 column volumes of 0.006% aqueous
sulfuric acid. Fractions 3 and 4 were evaporated to dryness and
then lyophilized until dry. Several of the fractions isolated
during elution from the brominated polystyrene resin were analyzed
for anthocyanins and total phenols as described in Examples 7 and
8. Table 2 summarizes the column data.
2TABLE 2 Analysis and recovery of anthocyanins and polyphenols in
elderberry fractions Anthocyanins Polyphenols % Purity % Recovery %
Purity % Recovery "fraction 1" 0.05 2.79 1.37 26.4 "fraction 2"
1.68 7.79 5.57 8.5 "fraction 3" 18.7 99.4 42.8 74.7 "fraction 4"
0.67 0.49 2.61 0.6
[0117] FIGS. 6-13 show the HPLC chromatograms of the filtered
elderberry extract and of certain fractions isolated during column
purification. The HPLC conditions used are those described in
Example 9.
[0118] FIGS. 6 and 7 show the HPLC chromatograms at 280 nm and 510
nm, respectively, of the filtered elderberry extract.
[0119] FIGS. 8 and 9 show the HPLC chromatograms at 280 nm and 510
nm, respectively, of "fraction 1" collected during column loading
of the filtered elderberry extract onto the brominated polystyrene
resin.
[0120] FIGS. 10 and 11 show the HPLC chromatograms at 280 nm and
510 mm, respectively, of "fraction 3" collected during column
elution of the filtered elderberry extract using 70% ethanol from
the brominated polystyrene resin.
[0121] FIGS. 12 and 13 show the HPLC chromatograms at 280 nm and
510 nm, respectively, of "fraction 4" collected during column
elution of the filtered elderberry extract using 90% ethanol from
the brominated polystyrene resin.
[0122] The total phenol-enriched compositions of this invention
comprise the compounds showing peaks in the region between 60 and
75 minutes in the standard HPLC chromatograms substantially as
shown in FIGS. 10-13.
Example 7
Quantitative Determination of Anthocyanins
[0123] This method is used to determine the total anthocyanins in
various biomass samples and dried purified total phenol-enriched
compositions by UV-VIS spectrophotometry, using an external
standard. Each sample tested (e.g., a concentrated total
phenol-enriched composition, dried biomass, or fresh/frozen
biomass) requires a different preparation procedure as described
below.
[0124] Total phenol-enriched compositions--Accurately weigh 75-100
mg of the purified total phenol-enriched composition into a 100 mL
volumetric flask and dilute to volume with 2% HCl/MeOH. Mix well
and dilute 0.40-1.6 mL of this sample to 10.0 mL with 2%
HCl/MeOH.
[0125] Dry Biomass--Into a coffee grinder place an amount of dry
biomass sufficient to cover the blades of the grinder. Grind for
about 1 minute or until finely ground. Alternatively use a mortar
and pestle to finely grind the raw material. Accurately weigh about
50-100 mg of finely ground biomass into a 100 mL volumetric flask
and then add about 80 mL of 2% HCl/MeOH and cap. Place the flask
into a 50.degree. C. oil bath or forced air oven for 30-60 minutes,
shake gently for 30 seconds, and sonicate for 5 minutes. Allow the
solution to cool to room temperature. Add 2% HCl/MeOH to the mark
and mix. Filter a portion of the sample through a 0.45 .mu.m PTFE
syringe filter into a vial. Dilute 1.0 mL of the filtrate to 10.0
mL with 2% HCl/MeOH. The dilution factor would be 10 mL/1 mL or
10.
[0126] Frozen/Fresh Biomass--Weigh 400.0 g frozen/fresh biomass
into a 1000 mL polypropylene beaker. Add 400 g of near boiling
water into the beaker. Puree using a mechanical blender (Waring or
other). Using a wide-bore polyethylene dropper, remove a
representative 0.5-1.5 g sample and transfer into a tared 100 mL
volumetric flask. Add 80 mL of 2% HCl/MeOH and cap. Place the flask
into a 50.degree. C. oil bath or forced air oven for 60-120
minutes, shake gently for 30 seconds and then sonicate for 5
minutes. Allow the solution to cool to room temperature. Add 2%
HCl/MeOH to the mark and mix. Filter a portion through a 0.45 .mu.m
PTFE syringe filter into a vial. The dilution factor would be the
total weight of the biomass and water divided by the weight of the
biomass [e.g., (400 g+400 g)/400 g=2].
[0127] Loss on Drying--The calculation to obtain the total
anthocyanins content in the above samples requires the
determination of the moisture content, or % LOD (loss on drying),
of the material. To determine the % LOD, transfer and distribute
evenly 0.5-3.0 g of sample into an accurately weighed aluminum
weigh pan, and record the weight to the nearest 0.1 mg. Place the
sample in an oven at 105.degree. C..+-.3.degree. C. for 2 hours (do
not exceed 2 hrs 15 min). After the sample has cooled to room
temperature (a dessicator may be used), weigh the sample and record
the weight to the nearest 0.1 mg. The % LOD is determined to the
nearest 0.1% using Equation 1: 1 % LOD = 1 - W D - W P W SP - W P
.times. 100 Eq . 1
[0128] where % LOD=percentage loss on drying; W.sub.D=dry weight of
the pan and sample (g); W.sub.P=weight of the pan (g); and
W.sub.SP=initial weight of the pan and sample (g).
[0129] Assay Procedures--The UV/VIS spectrophotometer is set to
read in photometry mode with the visible lamp on. The instrument is
zeroed at 535 nm using 2% HCl/MeOH in a 1 cm pathlength glass,
quartz, or disposable polystyrene cuvette. The absorbance of the
prepared sample is measured at 535 nm in the same or matched 1 cm
cuvettes.
[0130] Calculations--The concentration of total anthocyanins is
calculated as shown in Equation 2: 2 C ANTHOS = ABS SAMP .times. DF
E S Eq . 2
[0131] where C.sub.ANTHOS=concentration of the total anthocyanins
in the sample (mg/mL); ABS.sub.SAMP=absorbance of the sample at 535
nm; DF=dilution factor, as described below; and
E.sub.S=absorptivity (absorbance of a 1 mg/mL solution at 535 nm in
2% HCl/MeOH using a 1 cm cuvette) of the appropriate external
standard, either cyanidin chloride (101.1; for cherry, cranberry,
elderberry, and plum) or delphinidin chloride (102.0; for bilberry
and blueberry). The dilution factor (DF) for a dry biomass is 1,
and the dilution factor for fresh/frozen biomass is the total
weight of the biomass and water divided by the weight of the
biomass (e.g., (400 g+400 g)/400 g). The dilution factor for a
purified extract is the final dilution volume divided by the volume
of the extract solution (e.g., 10 mL/0.40 mL).
[0132] The percent total anthocyanins is calculated as shown in
Equation 3: 3 % Anthos = C ANTHOS .times. Volume .times. 100 W S
.times. S LOD Eq . 3
[0133] where % Anthos=percentage of total anthocyanins in the
sample; C.sub.ANTHOS=concentration of total anthocyanins (mg/mL);
Volume=initial volume of the sample preparation (usually 100 mL);
W.sub.S=weight of the biomass or total phenol-enriched compositions
used in the preparation (usually 50-100 mg for dry biomass,
500-1500 mg for fresh/frozen biomass, or 75-100 mg for purified
extracts); and S.sub.LOD=[(100-% LOD)/100] for dry or fresh biomass
or purified extract (for fresh or frozen biomass this factor does
not apply).
Example 8
Quantitative Determination of Total Polyphenols
[0134] This method is used to quantitatively determine the total
polyphenols in various biomass samples and dried purified enriched
compositions by UV-VIS spectrophotometry, using gallic acid as the
external standard.
[0135] The procedure requires a 20% Na.sub.2CO.sub.3 solution and
2% HCl/MeOH. To prepare the Na.sub.2CO.sub.3 solution, weigh
approximately 100 g of Na.sub.2CO.sub.3 into a 500 mL volumetric
flask containing about 350 mL deionized water. Sonicate for 10
minutes; shake to mix. Dilute to volume using deionized water and
agitate until homogeneous. To prepare the 2% HCl/MeOH, transfer
about 350 mL of methanol into a 500 mL volumetric flask. Pipet into
the flask 10.0 mL of HCl. Dilute to volume using methanol and mix
until homogeneous.
[0136] To prepare the gallic acid stock standard, accurately weigh
100 mg of gallic acid (Sigma; St. Louis, Mo.) into a 100 mL
volumetric flask. Add 70 mL of deionized water and sonicate for 5
minutes until dissolved. Dilute to volume using deionized water,
cap, and mix until homogeneous.
[0137] Each sample tested (e.g., total phenol-enriched composition,
dry biomass, or fresh/frozen biomass) requires a different
preparation procedure and was prepared as described in Example
7.
[0138] Loss on Drying--The calculation to obtain the total
polyphenols content in the above samples requires the determination
of the moisture content, or % LOD, of the material. To determine
the % LOD, transfer and distribute evenly 0.5-3.0 g of sample into
an accurately weighed aluminum weigh pan, and record the weight to
the nearest 0.1 mg. Place the sample in an oven at 105.degree.
C..+-.3.degree. C. for 2 hours (do not exceed 2 hrs 15 min). After
the sample has cooled to room temperature (a dessicator may be
used), weigh the sample and record the weight to the nearest 0.1
mg. The % LOD is determined to the nearest 0.1% using Equation 1
above.
[0139] Colorimetric Development Procedures--A clean 100 mL
volumetric flask is set aside to serve as the reagent blank. Two
100 mL volumetric flasks are labeled "high" standard and "low"
standard. Using the gallic acid stock solution, pipet 800 .mu.L
into the "high" standard flask and 200 .mu.L into the "low"
standard flask. For dry biomass samples, pipet 20 mL of the
filtered solution into a 100 mL volumetric flask. For fresh/frozen
biomass samples, pipet 10 mL of the filtered solution into a 100 mL
volumetric flask. For purified samples, pipet 0.80-2.0 mL into a
100 mL volumetric flask. The following are added to each of the
volumetric flasks (including the reagent blank) prepared above:
[0140] 1. Add sufficient deionized water to each flask to bring the
total volume to approximately 65 mL.
[0141] 2. Pipet 5.0 mL of the FC Phenol Reagent (Sigma) into each
flask, agitate gently.
[0142] 3. Pipet 15.+-.2 mL of the 20% Na.sub.2CO.sub.3 solution
into each flask.
[0143] 4. Mix the solutions in each flask with gentle swirling,
dilute to volume with deionized water, cap, and invert.
[0144] 5. Allow the solutions to develop for at least three but not
more than four hours.
[0145] 6. Filter 10 mL aliquots of samples requiring filtration
through 0.45 .mu.m PVDF syringe filters into suitable
containers.
[0146] Assay Procedure--The UV-VIS spectrophotometer is set to read
in photometry mode with the visible lamp on. The analysis is
carried out in 1 cm pathlength glass, quartz, or disposable
polystyrene cuvettes. The instrument is zeroed at 760 nm using the
reagent blank. The absorbance of each solution is measured at 760
nm in the same or matched 1 cm cuvettes.
[0147] Calculations--To calculate the concentration of total
polyphenols the absorptivity of gallic acid must first be
determined. This value is obtained as described in Equation 4: 4 E
R = A R .times. D R C R .times. ( 1 - E LOD ) Eq . 4
[0148] where E.sub.R=absorptivity of the reference standard (gallic
acid) at 760 nm in absorbance units/g/L; A.sub.R=absorbance of the
reference standard solution; C.sub.R=concentration of gallic acid
in the stock standard solution, D.sub.R=dilution factor for the
gallic acid standard (125 for "high" standard or 500 for "low"
standard); and E.sub.LOD=loss on drying of the gallic acid solids
as a percent.
[0149] The absorptivities for the "high" and "low" standards are
averaged for use in Equation 5 below. The concentration of total
polyphenols in the color development sample preparations is
calculated as shown in Equation 5: 5 C P = A S .times. D FC E R Eq
. 5
[0150] where C.sub.P=concentration of total polyphenols in the FC
sample preparation (mg/mL); A.sub.S=absorbance of the FC sample
preparation; D.sub.FC=sample dilution factor, where DF is typically
5 for dry biomass, 10 for fresh/frozen biomass, and 50-125 for
purified enriched composition; and E.sub.R=average absorptivity of
the gallic acid standards.
[0151] The percent total polyphenols is calculated as shown in
Equation 6: 6 % P = C P .times. V S .times. D S .times. 100 W S
.times. S LOD Eq . 6
[0152] where % P=percentage of total polyphenols in the sample;
C.sub.P=the concentration of total polyphenols (mg/mL);
V.sub.s=volume of original sample preparation (usually 100 mL);
W.sub.S=weight of the biomass or purified composition used in the
original sample preparation (usually 50-100 mg for dry biomass,
500-1500 mg for fresh/frozen biomass, and 75-100 mg for purified
extracts); D.sub.S=original sample dilution factor, where D.sub.S
is 1 for dry biomass, 2 for fresh/frozen biomass, or 1 for purified
extract; and S.sub.LOD=[(100-% LOD)/100] for biomass or purified
extracts. For fresh or frozen biomass this factor does not
apply.
Example 9
HPLC Qualitative Assay
[0153] This method is used to qualify compounds in various
biomasses and purified enriched compositions by high performance
liquid chromatography (HPLC). Each type of sample requires a
different preparation procedure as described below.
[0154] Dry Biomass: The dry biomass, if not already powdered, is
ground through a 1 mm screen using the Wiley mill. Using an
appropriately sized extraction thimble and a soxhlet extraction
apparatus, weigh out approximately 12 g of powdered biomass into
the thimble and extract using 200 mL of methanol. Extract through
at least 20 cycles or until the extraction solvent is clear.
Transfer the extract quantitatively to a 250 mL volumetric flask
using methanol, dilute to volume and mix. Filter the extract
through a 0.45 .mu.m PTFE syringe filter into an HPLC vial.
[0155] Frozen/Fresh Biomass: Weigh 400 g frozen/fresh biomass into
a 1000 mL polypropylene beaker. Add 400 g of near boiling water
into the beaker. Puree using a mechanical blender (Waring or
other). Using a wide-bore polyethylene dropper, remove a
representative 0.5-1.5 g sample and transfer into a tared 100 mL
volumetric flask. Add 80 mL MeOH, cap, and heat at 50.degree. C.
for 30 minutes. Allow the solution to cool to room temperature,
adjust to volume with methanol, and then sonicate until
homogeneous. Filter a portion through a 0.45 .mu.m PTFE syringe
filter into an HPLC vial.
[0156] Purified Enriched Composition: Accurately weigh 50-100 mg of
the enriched composition into a glass scintillation vial and add
10.0 mL of 50% MeOH/H.sub.2O. Sonicate for 5 minutes. Filter
through a 0.45 .mu.m PTFE syringe filter into an HPLC vial.
[0157] The HPLC is set up as required. In one embodiment of this
invention, the aqueous mobile phase was prepared by mixing 5 mL of
trifluoroacetic acid (TFA) into 1000 mL of high purity, Type 1
water. A 20 .mu.L sample was injected at ambient temperature. A 280
nm wavelength was used for detection, the flow rate was 1.0 mL/min,
and the run time was 105 minutes. A Zorbax column was packed with 5
.mu.m SBC-18 in a 150.times.4.6 mm ID column. In this embodiment,
the mobile phase was set up as follows: channel A: 100%
acetonitrile; channel B: 0.5% TFA in H.sub.2O; and channel C: 100%
methanol. Table 3 summarizes the HPLC gradient for this embodiment
of the invention.
[0158] If available, standard preparations of compounds known to
exist in the sample may be prepared at concentrations of
approximately 1 mg/mL. These standard preparations can be used to
determine the approximate retention times and thus identify those
compounds in the sample chromatograms. As this method is used for
qualification purposes only, no calculations are required.
3TABLE 3 HPLC gradient for qualitative analysis Time (min) % A % B
% C 0.0 0 95 5 7.0 5 90 5 32.1 8 84 8 33.0 9 83 8 63.0 14 78 8 91.5
27 65 8 99.0 72 20 8 104.0 72 20 8 104.1 0 95 5 112.0 0 95 5
Example 10
Quantitative HPLC Method for Determination of Percent
Proanthocyanidins
[0159] This HPLC method is used to determine the amount of
proanthocyanidins in various fractions and enriched compositions.
Each type of sample requires a different preparation and is
prepared as described in Example 9. The method uses a 5 .mu.m
Zorbax column packed with Stablebond SBC-18 in a 150.times.4.6 mm
column. The flow rate was 1.5 mL/min, the detector was set at 280
nm, the injection volume was 10 .mu.L, and the run time was 24 min.
The mobile phase was: channel A=100% acetonitrile; channel B=0.1%
trifluoroacetic acid in water; channel C=100% methanol. The
gradient employed is provided in Table 4. The proanthocyanidins
typically eluted as a group of broad peaks in the HPLC chromatogram
at elution times between 11-22 minutes.
4TABLE 4 HPLC gradient for % analysis for proanthocyanidins Time
(min.) % A % B % C 0 14 78 8 9 14 78 8 17 34 58 8 22 34 58 8 22.1
14 78 8 26 14 78 8
[0160] To quantitate the proanthocyanidins, a previously prepared
in-house proanthocyanidin standard is utilized with a purity
greater than 90%. A sample of this is prepared at 5.5 mg/mL in 70%
ethanol and analyzed using the HPLC method described in this
Example. The chromatogram for this standard includes a large, broad
peak in the 11-22 minute retention time range (as seen in FIG. 14)
which is due to the proanthocyanidins. Manually integrate the
entire 11-22 minute peak. The peak area response factor for the
standard is then determined by dividing the entire 11-22 minute
peak area by the product of the standard's concentration and its
purity as shown in Equation 7: 7 RF = PA C std .times. P std Eq .
7
[0161] where RF=peak area response factor for the standard (area
units/mg/mL); PA=peak area of the proanthocyanidins in the
standard; C.sub.std=concentration of the standard solution in
mg/mL; and P.sub.std=standard purity as a percent (usually
0.90).
[0162] The percent proanthocyanidins in a sample can be determined
using the sample preparation and HPLC analysis method described
above. The total peak area in the 11-22 minute retention time range
is determined for the sample in question. Before any calculation
can be made, however, the peak areas of non-proanthocyanidin
compounds in the proanthocyanidin retention time range must be
subtracted from the overall total peak area. Non-proanthocyanidin
compounds often appear as sharp peaks co-eluting with or on top of
the broad proanthocyanidins' peak, and their UV spectrum by diode
array is often different from the bulk of the proanthocyanidin
peak. To determine the peak area of non-proanthocyanidin peaks,
manually integrate these peaks, total their peak area and subtract
this area from the total 11-22 minute peak area. Once the net area
of the proanthocyanidins' peak in the sample has been determined,
divide this value by the peak area response factor for the in-house
standard to obtain the concentration of proanthocyanidins in the
sample as shown in Equation 8: 8 C proanthos = PA samp .times. DF
RF Eq . 8
[0163] where C.sub.proanthos=concentration of total
proanthocyanidins in the sample (mg/mL); PA.sub.samp=corrected
total peak area for the sample; DF=dilution factor (1 for dry
biomass, 2 for fresh/frozen biomass, and 1 for an enriched
composition); and RF=peak area response factor calculated using
Equation 7.
[0164] The percent total proanthocyanidins is calculated as shown
in Equation 9: 9 % Proanthocyanidins = C proanthos .times. V
.times. 100 W s Eq . 9
[0165] where % Proanthocyanidins=percent of total proanthocyanidins
in the sample; C.sub.proanthos=concentration of total
proanthocyanidins (mg/mL); V=volume of the sample preparation
(usually 250 mL for dry biomass, 100 mL for fresh/frozen biomass,
or 10 mL for enriched compositions); and W.sub.s=weight of the
biomass or enriched composition used in the sample preparation
(usually 12,000 mg for dry biomass, 500-1500 mg for fresh/frozen
biomass, or 50-100 mg for enriched compositions).
Example 11
Partitioning Polar and Non-Polar Proanthocyanidins Directly from a
Filtered Elderberry Extract
[0166] In this example, a filtered elderberry extract was prepared
and, rather than being purified on a brominated polystyrene resin,
was instead loaded directly onto a vacuum liquid chromatography
(VLC) column to partition polar proanthocyanidins and non-polar
proanthocyanidins directly from a filtered extract according to the
method illustrated in FIG. 15.
[0167] A 50 mL C-18 VLC column was prepared by filtering a 50 mL
slurry of Bakerbond 40 .mu.m flash chromatography C-18 media in
methanol through a 60 mL fritted glass filter. The column was
conditioned by washing with methanol and then with water. A 300 mL
portion of the filtered elderberry extract, containing 12.0 g of
solids, 74 mg of anthocyanins and about 780 mg of
proanthocyanidins, was loaded onto the column. An HPLC chromatogram
of the filtered extract using the HPLC method described in Example
10 is shown in FIG. 16. The flow-through eluent (about 300 mL) and
a 100 mL wash (0.1% trifluoroacetic acid (TFA)) were combined to
provide the polar proanthocyanidin "fraction 5." An HPLC
chromatogram at 280 nm of "fraction 5" is shown in FIG. 17. The
column was then eluted with 100 mL each of 30, 40, 50, 60, 70, and
100% methanol containing 0.1% TFA. An HPLC chromatogram at 280 nm
of the non-polar proanthocyanidin "fraction 6" isolated in the 60%
methanol eluent is shown in FIG. 18. The fractions were assayed for
anthocyanins and proanthocyanidins by the methods described in
Examples 7 and 10. Table 5 summarizes the results for this
experiment.
5TABLE 5 Partitioning of Elderberry Elution Anthocyanins
Proanthocyanidins % Proanthocyanidin Fraction (mg) (mg) purity
Flow- 54 554 4.7 Through + Wash 30% MeOH 11 10 1.1 40% MeOH 3 20 12
50% MeOH 1 92 92 60% MeOH 0.2 46 92 70% MeOH 0.1 41 100 100% MeOH
N/A 21
[0168] The results indicate that 71% (558 mg) of the
proanthocyanidins in the filtered extract were collected during the
loading and wash. These proanthocyanidins were the more polar
proanthocyanidins. The non-polar proanthocyanidins eluted when the
methanol concentration was increased to at least 40%. The purity of
the proanthocyanidins eluting in the 50-70% methanol fractions was
high due to the fact that the majority of the solids contained in
the filtered elderberry extract eluted in the loading eluent, water
wash, and 30% methanol wash.
Example 12
Partitioning Elderberry Proanthocyanidins by VLC Followed by
Purification by Gel Permeation Chromatography or Semi-Preparative
HPLC
[0169] A total phenol-enriched composition was prepared from
elderberry dried biomass (Martin Bauer; Germany) by collecting the
70% ethanol fraction ("fraction 3") during elution from a
brominated polystyrene resin using the procedure as described in
Example 6. A portion (2.00 g) of this total phenol-enriched
composition was dissolved in 50 mL of water and loaded onto a 15 mL
C-18 VLC column prepared with Bakerbond 40 .mu.m C-18 media. The
flow-through eluent and the 25 mL water wash were combined and
freeze-dried, yielding 733 mg of the polar proanthocyanidins
fraction ("fraction 5"). The column was then washed with 25 mL of
50% methanol. The non-polar proanthocyanidins ("fraction 6") were
eluted with 25 mL of 70% methanol. The methanol in this fraction
was removed and the resulting water suspension was freeze-dried,
yielding 192 mg of the non-polar proanthocyanidin fraction
("fraction 6"), which by HPLC assay was 100% proanthocyanidins.
This fraction had little if any color, suggesting that the
oligomeric proanthocyanidins chains in this fraction do not contain
cationic anthocyanin units.
[0170] The polar proanthocyanidins fraction ("fraction 5") was
further purified by semi-preparative HPLC to remove residual
anthocyanins and other more polar impurities. The conditions for
the semi-preparative HPLC purification of these solids are
described below.
[0171] The semi-preparative HPLC method used a 2.5.times.10 cm
Waters PrepPak cartridge filled with 6 .mu.m, 60 Angstrom, Nova-Pak
HR C-18 media (Waters; Milford, Mass.). The mobile phase was:
channel A=100% acetonitrile; channel B=0.1% trifluoroacetic acid;
channel C=100% methanol. The gradient employed in this embodiment
was as provided in Table 6. The flow rate was 30 mL/min, the
detector was set at 280 nm, and the injection volume was typically
3-5 mL of a solution containing 50-125 mg of solids. The run time
was 30 minutes. The proanthocyanidins were collected in a broad
peak that eluted between 13-20 minutes.
6TABLE 6 HPLC gradient for Elderberry proanthocyanidin purification
Time (min.) % A % B % C 0.0 11 81 8 11.0 11 81 8 19.0 34 58 8 24.0
34 58 8 25.0 82 10 8 30.0 82 10 8 30.1 11 81 8
[0172] About 600 mg of the polar proanthocyanidins fraction
("fraction 5") were dissolved in 25 mL of water. Approximately 3 mL
(75 mg) were injected in each of eight runs. The proanthocyanidin
peaks eluting between about 12-18 minutes in each run were
collected, pooled, and evaporated on a rotary evaporator, and the
residual aqueous solution freeze-dried. Approximately 100 mg of
purified polar elderberry proanthocyanidins ("fraction 7") were
obtained from 600 mg of the polar proanthocyanidin solids
("fraction 5") after VLC separation. An HPLC chromatogram at 280 nm
for the VLC-isolated polar proanthocyanidins after semi-preparative
HPLC purification is shown in FIG. 19. The polar front, comprising
sugars, amino acids, anthocyanins, organic acids, and small
flavonoid compounds, was removed by the semi-preparative HPLC
purification, as evidenced by the absence of these peaks in FIG.
19. A .sup.13C NMR spectrum of the purified polar proanthocyanidins
("fraction 7") is shown in FIG. 20.
[0173] The non-polar proanthocyanidins fraction ("fraction 6") was
further purified by gel filtration chromatography. A portion (48
mg) of the non-polar proanthocyanidin fraction ("fraction 6")
isolated during the VLC separation was dissolved in 20 mL of warm
water and loaded onto a 14 mL Sephadex LH-20 column that had
previously been equilibrated with water. The loading eluent was
collected and combined with a 40 mL column water wash. Most of the
non-polar proanthocyanidins eluted from the column at this point
while most of the smaller flavonoid impurities were retained. The
combined loading and wash eluents were freeze-dried to provide 32
mg of the purified non-polar proanthocyanidins "fraction 8." These
solids possessed strong antiviral activity. FIGS. 21 and 23 show
the HPLC chromatograms at 280 nm and 368 nm, respectively, of the
non-polar proanthocyanidins ("fraction 6") before the Sephadex
LH-20 column purification. FIGS. 22 and 24 show the HPLC
chromatograms at 280 nm and 368 nm, respectively, of the purified
non-polar proanthocyanidins ("fraction 8"). The peaks in FIG. 21
marked with asterisks are non-proanthocyanidin flavonoid compounds
based on their UV spectra. These compounds are reduced in the
purified non-polar product ("fraction 8") isolated after Sephadex
LH-20 column as shown HPLC chromatogram at 280 nm in FIG. 22. The
effect of the gel purification can be better seen by comparing the
HPLC chromatogram at 368 nm of the non-polar proanthocyanidins
before purification (FIG. 23). The non-proanthocyanidin impurities
appear in FIG. 23 at 4-6 minutes and 15-17 minutes. Except for a
small amount of the flavonoid compound eluting at 5.8 minutes,
there is no trace of flavonoid compounds in the purified sample as
shown in FIG. 24. A .sup.13C NMR spectrum of the purified non-polar
proanthocyanidin "fraction 8" is shown in FIG. 25. FIG. 33 shows an
IR spectrum of fraction 7, and FIG. 34 shows an IR spectrum of
"fraction 8."
Example 13
Purification of Blueberry Polar and Non-Polar Proanthocyanidins by
VLC Followed by Semi-Preparative HPLC
[0174] The starting material for this example was a total
phenol-enriched "fraction 3" prepared from blueberries and isolated
during the 70% ethanol elution from a brominated polystyrene resin.
A portion (6.00 g) of "fraction 3" was dissolved in 80 mL of water
and loaded onto a 30 mL C-18 VLC column as described previously.
The loading eluent was collected and combined with 100 mL of a 0.1%
TFA wash eluent ("fraction 5"). Next, the column was washed with 80
mL of 40% methanol to remove residual polar compounds ("fraction
5") and then with 80 mL of 70% methanol to give the non-polar
proanthocyanidin fraction ("fraction 6"). Table 7 summarizes the
results of this experiment.
7TABLE 7 Purification of blueberry proanthocyanidins % Solids
Proanthocyanidins Proanthocyanidins Sample (g) (mg) purity
"fraction 3" 6.00 1614 27 Loading 2.11 899 43 Eluent + Wash 40%
MeOH fraction 2.46 580 24 70% MeOH fraction 0.67 323 48
[0175] The polar proanthocyanidins "fraction 5" (loading
eluent+wash) and the non-polar proanthocyanidins fraction 6 (70%
methanol elution) were each further purified by semi-preparative
HPLC by the method described in Example 12 to provide "fraction 7"
and "fraction 8", respectively. The HPLC chromatograms at 280 nm of
the blueberry polar proanthocyanidins fraction before and after the
semi-preparative purification (i.e., "fraction 5" and "fraction 7")
are shown in FIGS. 26 and 27, respectively. The HPLC chromatograms
at 280 nm of the blueberry non-polar proanthocyanidins fraction
before and after the semi-preparative purification (i.e., "fraction
6" and "fraction 8") are shown in FIGS. 28 and 29, respectively.
The semi-preparative purifications of both the polar and non-polar
fractions removed undesired anthocyanins and polar flavonoid
compounds from the proanthocyanidins, as evidenced by the absence
of peaks between about 0 and 8 minutes in FIGS. 27 and 29. FIG. 37
shows an IR spectrum of "fraction 7," and FIG. 38 shows an IR
spectrum of "fraction 8."
Example 14
Purification of Plum Polar and Non-Polar Proanthocyanidins by VLC
Followed by Semi-Preparative HPLC
[0176] The starting material for this example was a combination of
"fraction 3" and "fraction 4" isolated from plums and containing
approximately 17% total proanthocyanidins, of which 61% were
designated as polar and 39% as non-polar. A portion (8.00 g) of
this composition was dissolved in 100 mL of water containing 0.5%
TFA and loaded onto a 45 mL C-18 VLC column as described
previously. The loading eluent was collected, and the column was
washed with 50 mL of 0.1% TFA. The loading eluent and wash
fractions were combined to provide the polar proanthocyanidins
fraction ("fraction 5"). An HPLC of the polar proanthocyanidin
"fraction 5" is shown in FIG. 30. The column was eluted with 100 mL
of 40% methanol containing 0.5% TFA followed by 100 mL of 70%
methanol containing 0.5% TFA. All methanol fractions were combined
to provide the non-polar proanthocyanidin fraction ("fraction 6").
Table 8 summarizes the results of this experiment.
8TABLE 8 Purification of plum proanthocyanidins Proanthocyanidins %
Proanthocyanidins Sample Solids (g) (mg) purity Plum fractions 8.00
1328 17 3 and 4 Loading 4.32 651 15 Eluent + Wash 40% MeOH 3.76 486
13 fraction 70% MeOH 0.45 300 67 fraction
[0177] The polar proanthocyanidin "fraction 5" (combined loading
eluent and wash eluent) was further purified by semi-preparative
HPLC by the method described in Example 12 to provide "fraction 7."
Removal of anthocyanins and other more polar impurities increased
the proanthocyanidin purity of the sample from 15% to 100%. The
HPLC chromatogram at 280 nm of the purified polar proanthocyanidin
"fraction 7" is shown in FIG. 31. The non-polar "fraction 6"
(combined 40% and 70% methanol washes) was not purified further.
The HPLC chromatogram at 280 nm of the non-polar proanthocyanidin
"fraction 6" is shown in FIG. 32. FIG. 39 is an IR spectrum of
"fraction 7" and FIG. 40 is an IR spectrum of "fraction 6".
Example 15
Purification of Proanthocyanidins Fraction from Elderberry VLC
Fraction
[0178] A VLC column was prepared using Amberchrom CG-71cd resin
(80-160 .mu.m particle size, TosoHaas; Philadelphia, Pa.). A water
extract of elderberry was prepared and a portion of this extract
was loaded onto the VLC column. The column was then washed with
water and eluted using 30%, 40%, 50%, 60%, 70%, and 100% methanol.
All fractions eluted with methanol were retained separately. The
VLC fraction eluted with 50% methanol was evaporated on a rotary
evaporator to remove the methanol and then lyophilized to remove
the water. The dried material was ground to a powder using a mortar
and pestle. The dried sample was assayed by HPLC using the method
as described in Example 10. Using the results of this assay, a
semi-preparative HPLC method was derived from the analytical HPLC
method to isolate the proanthocyanidins. The mobile phase was:
channel A=100% acetonitrile; channel B=0.5% trifluoroacetic acid in
water; channel C=100% methanol. The flow rate was set at 30 mL/min.
The gradient employed is provided in Table 9.
9TABLE 9 HPLC gradient for purification of elderberry
proanthocyanidins Time (min) % A % B % C 0.0 11.0 81.0 8.0 9.0 11.0
81.0 8.0 17.0 34.0 58.0 8.0 22.0 34.0 58.0 8.0 23.0 92.0 0.0 8.0
28.0 92.0 0.0 8.0 28.1 11.0 81.0 8.0 36.0 11.0 81.0 8.0
[0179] Approximately 500 mg of the dried material was dissolved in
water at a solids concentration of approximately 50 mg/mL. A very
small injection was made to determine the retention time of the
relevant peaks. Based on this initial injection, two peaks were
collected: Peak A, which eluted between 14 and 22 minutes and Peak
B, which eluted between 26 and 28 minutes. Five injections of the
concentrated solution were made, and the appropriate collections of
each peak were pooled from each injection. The sample obtained by
the collection of Peak A was determined to contain the
proanthocyanidins and was evaporated to remove the organic solvents
and a portion of the water. The concentrated sample was assayed
using the HPLC method as described in Example 10. The
chromatographic purity of the sample was determined to be 93.9%.
The sample was then lyophilized to obtain the dry material. Once
dry, a small portion of the sample was brought up in 70% ethanol at
a concentration of 1.918 mg/mL and re-assayed by the same HPLC
method. Using the results of this analysis and the previously
obtained chromatographic purity, a peak area response factor was
determined. This information was used to determine the
proanthocyanidins concentration in other purified fractions. The
HPLC chromatogram at 280 nm of the proanthocyanidin "standard" is
shown in FIG. 14.
Example 16
Purification of Cranberry Proanthocyanidins by VLC Followed by
Semi-Preparative HPLC
[0180] The starting material for this example was 8.00 g of
purified cranberry extract ("fraction 3"+"fraction 4") comprising
14% total proanthocyanidins. This material was dissolved in 100 mL
of water containing 1 mL of trifluoroacetic acid and loaded onto a
50 mL C-18 VLC column as described previously. The loading eluent
(100 mL) was collected and combined with 50 mL of 0.1% TFA wash
eluent to obtain "fraction 5". Next the column was washed with 100
mL of 40% methanol to remove residual polar compounds and eluted
with 100 mL of 70% methanol to give the non-polar proanthocyanidins
"fraction 6". Table 10 summarizes the results of this
experiment.
10TABLE 10 Purification of cranberry proanthocyanidins Solids
Proanthocyanidins % Proanthocyanidins Sample (g) (mg) purity
Cranberry 8.00 1514 14.3 fractions 3 + 4 Loading Eluent + 3.60 748
20.8 Wash 40% MeOH 3.39 677 20.0 fraction 70% MeOH 0.44 93 21.1
fraction
[0181] The polar proanthocyanidins fraction (loading eluent+wash)
was further purified by semi-preparative HPLC by the method
described in Example 12 to obtain "fraction 7". FIG. 41 is an HPLC
chromatogram of the polar proanthocyanidins fraction before
semi-preparative purification, and FIG. 42 is an HPLC chromatogram
of the polar proanthocyanidins fraction after purification. FIG. 43
is an HPLC chromatogram of the non-polar proanthocyanidins
fraction. Polar non-proanthocyanidin compounds such as anthocyanins
that eluted before the proanthocyanidins were removed in this
process.
Example 17
Herpes Simplex Virus 2 Assay of Elderberry Fractions
[0182] The antiviral activities of a crude elderberry extract and
fractions 1, 3 and 4 isolated as described in Example 6 were
determined using the viral cytopathic effect (CPE) assay. This
assay has previously been described (Wyde, et al., Drug Develp.
Res. 28:467-472 (1993)). All antiviral activities are reported as
50% effective dose (ED.sub.50).
[0183] Table 11 summarizes the ED.sub.50 for CPE inhibition for the
four compositions tested.
11TABLE 11 ED.sub.50 for CPE inhibition of elderberry fractions CPE
Inhibition Composition (ED.sub.50) Crude extract >100 .mu.g/mL
"fraction 1" >100 .mu.g/mL "fraction 3" >100 .mu.g/mL
"fraction 4" <0.03 .mu.g/mL
Example 18
Viral Assays
[0184] Total phenol-enriched compositions of this invention
prepared from fruits and berries have demonstrated broad activity
against a variety of DNA and RNA viruses and are suitable as active
ingredients useful in treating inflammation in humans and animals.
In cell culture, the enriched compositions exhibit potent activity
against isolates and laboratory strains of respiratory syncytial
virus (RSV), influenza A and B virus, parainfluenza virus (PIV), as
well as other respiratory and Herpes simplex viruses. Total
phenol-enriched compositions are suitable as active ingredients
useful in treating a wide range of viral infections in humans and
animals.
[0185] Assays used to measure activity against each virus are well
known to those skilled in the art. Minced specific target tissue
was exposed to the desired virus and the rate of growth of the
virus was measured in the presence and in the absence of the test
materials. The antiviral activities of purified
proanthocyanidin-enriched compositions prepared from various fruits
and berries were determined.
[0186] Cell lines: The viral assays used the following cells/cell
lines in determining relative ED.sub.50 (50% effective dose) or 50%
inhibitory endpoints: RSV (respiratory syncytial virus) and PIV
(parainfluenza virus) assays used MA-104 cells originating from
African green monkey kidneys; Influenza A and B assays used MDCK
cells originating from canine kidneys; Rhinovirus assays used HeLa
and KB cells; Herpes simplex viruses 1 and 2 used HHF cells taken
from human foreskin fibroblasts; West Nile viral assays used Vero
cells taken from African green monkey kidneys; Adenovirus type 1
assays used A549 cells originating from human lung carcinoma; and
Punta Toro A assays used LLC-MK2 cells originating from Rhesus
monkey kidneys.
[0187] The assays used known drug standards (ribivarin or
acyclovir) as positive controls. The ED.sub.50s for ribivarin in
the assays used in this Example are as follows: RSV (respiratory
syncytial virus) assay ED.sub.50=20 .mu.g/mL; PVI (parainfluenza
virus) assay ED.sub.50=20 .mu.g/mL; Influenza A and B assays
ED.sub.50=2-3 .mu.g/mL; Rhinovirus assay ED.sub.50<1 .mu.g/mL;
West Nile viral assay ED.sub.50=20 .mu.g/mL; Adenovirus type 1
assay ED.sub.50=10 .mu.g/mL; and Punta Toro A assay ED.sub.50=20
.mu.g/mL. Herpes simplex 1 and 2 assays used acyclovir as a
positive control, which has an ED.sub.50 of 1-2 .mu.g/mL in the
HSV1 and HSV2 assays.
[0188] The data obtained in the viral assays for certain
compositions of this invention are provided in Table 12. In cell
cultures, the compositions exhibited potent activity against
isolates and laboratory strains of influenza A virus (strains H1N1
and H3N3), influenza B virus, adenovirus type 1, Punta Toro A
virus, and Rhinovirus type 2. Comparison of the bioactivity data in
Table 12 to acyclovir and ribavirin in the antiviral screenings
clearly shows that the compositions of this invention are
biologically active in these assays and compete favorably with the
well-established pharmaceuticals used to treat these viral
diseases.
12TABLE 12 ED.sub.50's (.mu.g/mL) of various fractions in a COX-2
assay Virus Frac- Influenza Influenza Influenza Adenovirus Punta
Rhinovirus West Nile Varicella- Source tion A (H1N1) A (H3N2) B
Type 1 Toro A Type 2 Virus zoster virus HSV-1 HSV-2 Cranberry 4 3.2
3.2 3.2 20 5.6 61 15 Plum 4 32 32 32 25 70 70 32 Blueberry 4 32 32
32 30 25 30 31 45 11.4 Elderberry 4 28-55 28-55 88 45 11 Elderberry
6 15 Elderberry 7 inactive inactive inactive Elderberry 8 28 35
Elderberry FIG. inactive 0.08 67.7 49 14 Grape* NA 4 6 3.2 20 6.8
*(Nature's Plus; Melville, NY)
Example 19
Evaluation of COX-2 Activity of Total Phenol-Enriched
Compositions
[0189] Cyclooxygenase enzymes (COX-1 and COX-2) catalyze the
conversion of arachidonic acid and other essential fatty acids into
various prostaglandins. Prostaglandins are hormone-like substances
responsible for inflammation in mammals. Inhibition of the COX-2
enzymes can reduce inflammation in tissue with minimal side
effects. On the other hand, inhibition of COX-1 causes gastric
ulceration and other undesirable side effects in the body. Complete
inhibition of the COX-1 enzyme is not desirable. Compounds that
selectively inhibit COX-2 enzyme are better anti-inflammatory
agents. Total phenol-enriched compositions of this invention
prepared from fruits and berries have been shown to inhibit the
COX-2 enzyme, and are suitable as active ingredients useful in
treating inflammation in humans and animals.
[0190] In this assay the material to be assayed was mixed with
minced specific murine or bovine organ tissues that are known to
contain the desired enzyme. Arachidonic acid was added to this
mixture. The rate of uptake of oxygen is measured and compared with
the rate of uptake observed with known COX inhibitors. The COX-2
assay is based on quantitative production of PGE.sub.2 from
arachidonic acid using human recombinant COX-2 positive cells.
[0191] The results for several compositions are shown in Table 13.
Comparison of the data for the compositions shown in Table 13 with
determined COX-2 bioactivities of known drug standards (aspirin and
indomethacin) clearly shows that the purified
proanthocyanidin-enriched compositions of this invention are
biologically active in the COX-2 assay. Aspirin is active against
COX-2 at 660 .mu.g/mL and against COX-1 at 240 .mu.g/mL.
Indomethacin is active against COX-2 at 10 .mu.g/mL. The
compositions in Table 13 therefore show a 2.5 to 6 fold increase in
potency in the COX-2 assay over the most commonly used treatment
for inflammation (i.e., aspirin), and are indicative of the utility
of the purified proanthocyanidin-enriched compositions of this
invention in treating inflammation in mammals.
13TABLE 13 COX-2 activities for proanthocyanidin enriched
compositions Source Fraction IC.sub.50 (.mu.g/mL) Blueberry 3 108
Cranberry 4 218 Grape* N/A 275 Elderberry 4 >1000 Plum 4
>1000 *(Nature's Plus; Melville, NY)
* * * * *