U.S. patent application number 13/918070 was filed with the patent office on 2014-01-02 for methods to isolate anti-microbials from fruit or seed extracts.
This patent application is currently assigned to Wisconsin Alumni Research Foundation. The applicant listed for this patent is Wisconsin Alumni Research Foundation. Invention is credited to Brant Lawrence Kedrowski, Teri Shors.
Application Number | 20140004214 13/918070 |
Document ID | / |
Family ID | 49778423 |
Filed Date | 2014-01-02 |
United States Patent
Application |
20140004214 |
Kind Code |
A1 |
Kedrowski; Brant Lawrence ;
et al. |
January 2, 2014 |
METHODS TO ISOLATE ANTI-MICROBIALS FROM FRUIT OR SEED EXTRACTS
Abstract
The invention provides a method of isolating one or more
compounds present in fruit juice or seed extracts that have
anti-microbial activity.
Inventors: |
Kedrowski; Brant Lawrence;
(Oshkosh, WI) ; Shors; Teri; (Madison,
WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wisconsin Alumni Research Foundation |
Madison |
WI |
US |
|
|
Assignee: |
Wisconsin Alumni Research
Foundation
Madison
WI
|
Family ID: |
49778423 |
Appl. No.: |
13/918070 |
Filed: |
June 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61660332 |
Jun 15, 2012 |
|
|
|
Current U.S.
Class: |
424/732 ;
549/382 |
Current CPC
Class: |
A61K 31/353 20130101;
Y02A 50/411 20180101; C07D 493/14 20130101; A61K 2236/00 20130101;
A61K 36/45 20130101 |
Class at
Publication: |
424/732 ;
549/382 |
International
Class: |
A61K 36/45 20060101
A61K036/45; C07D 493/14 20060101 C07D493/14 |
Claims
1. A method to isolate compounds having anti-microbial activity,
comprising: a) providing a fruit or fruit seed extract dissolved or
suspended in water, alcohol or in a water alcohol mixture; b)
separating the extract on a C8 column into a plurality of
fractions; and c) isolating at least one fraction that is at least
80% by weight of a compound or a plurality of compounds with
anti-microbial activity.
2. The method of claim 1 wherein the at least one fraction
comprises about 1 mg to about 100 mg of the compound.
3. The method of claim 1 further comprising subjecting the at least
one fraction to rotavapping at 40.degree. C.
4. The method of claim 1 further comprising lyophilizing the at
least one fraction.
5. The method of claim 1 wherein the extract is a filtered
extract.
6. The method of claim 1 wherein prior to separation on the C8
column, the extract was subjected to separation on a C18 solid
phase extraction column and ion exchange chromatography column.
7. The method of claim 1 wherein high pressure liquid
chromatography is employed to separate the extract.
8. The method of claim 1 wherein the at least one fraction has at
least about 95% of the compound,
9. The method of claim 1 wherein the fruit is a cranberry.
10. The method of claim 1 wherein the seed extract is a cranberry
seed extract.
11. The method of claim 1 wherein a plurality of chromatographic
separation techniques are employed to isolate the fraction having
the substantially pure compound.
12. The method of claim 111 wherein the separation includes solid
phase exchange, ion exchange chromatography, high performance
liquid chromatography or simulated moving bed chromatography.
13. The method of claim 1 wherein at least one compound is a
flavonol, proanthocyanin, diterpenoid epoxide, or
phenylpropanoid.
14. An antimicrobial compound isolated by: a) separating a fruit or
fruit seed extract dissolved or suspended in water or in a water
alcohol mixture into fractions; b) separating the fractions with
anti-microbial activity into subtractions; c) isolating a
subfraction with anti-microbial activity that is stable to
rotavapping at 40.degree. C. and is resistant to protease
activity.
15. The compound of claim 14 wherein the fruit is a cranberry.
16. The compound of claim 14 wherein the seed extract is a
cranberry seed extract.
17. The compound of claim 14 wherein solid phase exchange, ion
exchange chromatography, high performance liquid chromatography or
simulated moving bed chromatography, or a combination thereof, are
employed for the separation.
18. The compound of claim 43 wherein a plurality of different
separation techniques are employed.
19. A method to prevent, inhibit or treat a microbial infection
comprising: administering to a mammal an anti-microbial amount of a
composition comprising an isolated fraction of a fruit or fruit
seed extract having at least two proanthocyanins, or one or more of
a flavonol, diterpenoid epoxide, or phenylpropanoid.
20. The method of claim 19 wherein the composition is orally or
intravenously administered.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date under
35 U.S.C. .sctn.119(e) of U.S. application Ser. No. 61/660,332,
tiled Jun. 15, 2012, the disclosure of which is incorporated by
reference herein.
BACKGROUND
[0002] Cranberries are rich in phytonutrients including polyphenols
and have among the highest antioxidant content of any food.
Numerous health benefits have been associated with cranberry
consumption, e.g., anti-bacterial properties, especially for
preventing urinary tract infections, anti-aging properties,
anti-cancer properties, anti-ulcer properties, dental-related
benefits, and heart-related benefits. There are also a few reports
of anti-viral properties (Konowalchuck et al., Appl. and Environ.
Microbiol., 35:12119 (1978); Weiss et al., Antiviral Res., 66:9
(2005); Lipson et al., Phytomedicine, 14:23 (2007); and Shots et
al., 24.sup.th Annual American Soc. For Virology Meeting (2005).
However, the compound(s) associated with those properties have yet
to be identified.
SUMMARY OF THE INVENTION
[0003] The invention provides a method to identify compounds in
fruit or fruit seed extracts, such as cranberry extracts, that have
anti-microbial activity. The method includes subjecting extracts,
e.g., fruit extracts including fruit juice and seed extracts or
reconstituted fruit extracts or juice such as those from
cranberries, raspberries or grapes, to one or more separation
techniques, identifying and isolating subtractions with
anti-microbial activity and optionally repeating separation and
identification/isolation steps using different parameters, prior to
isolating fractions having a plurality of individual compounds, or
substantially pure fractions having individual chemical compounds,
responsible for anti-microbial, e.g., anti-viral, activity.
Generally, a "substantially pure" composition will comprise more
than about 80% of a single macromolecular species present in the
composition, e.g., more than about 85%, about 90%, about 95%, and
about 99%, and in one embodiment, the object species is purified to
essential homogeneity (contaminant species cannot be detected in
the composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species. In one embodiment, a substantially pure fraction is one
where a single compound represents at least 95% all macromolecular
species present in the composition. In one embodiment, an isolated
fraction is one where a single (first) compound represents at least
50%, or at least 60%, 70%, 80%, 90%, or any integer between 60 and
100, of all macromolecular species in the fraction but other
compounds in the fraction individually or in combination with the
first compound provide for anti-microbial activity. In one
embodiment, chromatographic methods were used to separate the
multitude of compounds present in cranberry extracts into several
fractions, each containing a simpler mixture of compounds. For
example, high performance liquid chromatography (HPLC) and/or solid
phase extraction (SPE) were used to separate these complex
mixtures. In one embodiment, simulated moving bed chromatography
(SMBC) may be employed to separate mixtures. Thus, one type of
separation method may be employed once, or two or more different
types of separation methods may be employed in succession. In one
embodiment, one type of chromatography column may be employed
individually or in tandem, or two or more different types of
columns may be employed sequentially or in tandem. In one
embodiment, to detect activity, biological assays are employed,
e.g., assays that include one or more different viruses, e.g.,
vaccinia virus (a poxvirus), human parainfluenza virus type 3
(PIV3), influenza. A virus (e.g., H1N1 and H2N3 strains) and/or
poliovirus (e.g., Sabin Strain, Sero Type 1), one or more different
bacterial strains or species, and/or one or more different fungi,
to focus further separation on active fractions. Through this
iterative approach of bioassay-guided chemical separations, the
complex mixture of compounds in cranberries was narrowed down to
one or a few substantially pure compounds. Once separated as those
pure substances, analytical methods were used to elucidate the
chemical structures of these substances. The compounds displaying
signs of anti-microbial activity may then be purified in larger
quantities, e.g., using the methods to identify those compounds
which may include eliminating one or more steps, or through SMBC
technology, and the resulting compositions may be employed as a
nutraceutical or a pharmaceutical in compositions, e.g., in
powdered, tablet or liquid formulations.
[0004] As described herein below, the compounds
myricetin-beta-3-galactoside, sinapinic acid, triptolide, and
members of the proanthocyanidin class were isolated from Ocean
Spray 90MX spray-dried cranberry powder through a separation
sequence that included filtration, solid phase extraction, ion
exchange chromatography, and HPLC. The structure of the compounds
was determined using proton nuclear magnetic resonance spectroscopy
(.sup.1H NMR), MALDI-TOF mass spectrometry, Ultra High Performance
Liquid Chromatography (UPLC)-Quadrupole/Time of Flight (Q-TOF) mass
spectrometry, and/or ultraviolet (IN) spectroscopy. Thus, fractions
having myricetin-beta-3-galactoside, sinapinic acid, triptolide,
type A proanthocyanidin compounds, or combinations thereof, may be
employed as anti-microbials.
[0005] Thus, the invention provides a compound of formula (I),
e.g., myricetin-beta-3-galactoside, isolated from fruit or seed
extracts, as well as compositions having that compound, for use in
prophylactic or therapeutic methods. In one embodiment, a compound
of formula (I) has the following structure:
##STR00001##
Wherein each R is independently selected from the group consisting
of H, (C.sub.1-C.sub.6)alkyl, and (C.sub.1-C.sub.6)alkanoyl; each
R.sup.2 is independently selected from the group consisting of
halo, cyano, nitro, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkoxyl, (C.sub.1-C.sub.6)alkanoyl,
(C.sub.1-C.sub.6)alkanoyloxy, and
(C.sub.1-C.sub.5)alkylcarboxamido; n=0, 1, or 2; glyc is a
glycoside unit selected from the group consisting of a
monosaccharide, a disaccharide, and a polysaccharide having about
three to about six monosaccharide units, wherein the glycoside can
be unsubstituted, or can comprise one or more acyl groups, one or
more halo groups, one or more carboxylic acid groups, one or more
carboxester acid ester groups, or one or more carboxamido groups.
In one embodiment, both R.sup.1 groups are hydrogen. In one
embodiment, n=0. In one embodiment, glyc is a monosaccharide unit.
In one embodiment, glyc is a disaccharide unit. In one embodiment,
glyc is a galactoside. In one embodiment, give is a
3-galactoside.
[0006] The invention also provides a compound of formula (II),
e.g., sinapinic acid, isolated from fruit or seed extracts, as well
as compositions having that compound, for use in prophylactic or
therapeutic methods. A compound of formula (II) has the following
structure:
##STR00002##
wherein each of R.sup.1, R.sup.2, and R.sup.3 is independently
selected from the group consisting of hydroxyl and
(C.sub.1-C.sub.6)alkoxyl; R.sup.4 is CO.sub.2H, CO.sub.2M wherein M
is a cation, or is CO.sub.2(C.sub.1-C.sub.6)alkyl.
[0007] Further provided is a compound of formula e.g., triptolide,
isolated from fruit or seed extracts, as well as compositions
having that compound, for use in prophylactic or therapeutic
methods. A compound of formula MO has the following structure:
##STR00003##
wherein R.sup.1 is H, (C.sub.1-C.sub.6) alkyl or
(C.sub.1-C.sub.6)alkoxyl; R.sup.2 is H, OH,
(C.sub.1-C.sub.6)alkoxyl, or O(CH.sub.2).sub.nOR.sup.3, wherein
n=1, 2, or 3, and R.sup.3 is H, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)acyl, --(CH.sub.2).sub.mCO.sub.2H wherein m=1, 2,
or 3, or --P(O)(OH).sub.2, or a salt thereof.
[0008] Also provided is a compound of formula (IV), e.g., a
proanthocyanidin, isolated from fruit or seed extracts, as well as
compositions having that compound, for use in prophylactic or
therapeutic methods. A compound of formula (IV) has the following
structure:
##STR00004##
wherein * is a stereogenic carbon atom. Each stereogenic atom may
be of either (R)- or (S)-configuration.
[0009] In one embodiment, the invention provides a composition
comprising a substantially pure preparation of compound of formula
(I), (II), (III), or (IV), or a combination thereof, and optionally
a pharmaceutically acceptable carrier. In one embodiment, the
composition is in oral dosage form. In one embodiment, the
composition comprises two or more distinct compounds having formula
(IV). In one embodiment, the composition comprises two or more
distinct compounds selected from formula (I), (II) or (III).
[0010] In one embodiment, the invention provides a composition
comprising an isolated fraction of a fruit or fruit seed extract
having a compound of formula (I), (II), (III), or (IV), or a
combination thereof, and optionally a pharmaceutically acceptable
carrier. In one embodiment, the composition is in oral dosage form.
In one embodiment, the composition comprises two or more distinct
compounds having formula (IV). In one embodiment, the composition
comprises two or more distinct compounds selected from formula (I),
(II) or (III).
[0011] In one embodiment, a composition useful in the prophylactic
or therapeutic methods of the invention, is prepared by the
separation and isolation methods of invention, which yield a
separated and isolated fraction of a fruit or fruit seed extracts
that has anti-microbial activity and is characterized by the
presence of one or more of the following:
myricetin-beta-3-galactoside, sinapinic acid, triptolide,
proanthocyanidin A, e.g., where one or more of those compounds
represents at least 50% of the population of compounds in the
preparation.
[0012] The compositions may be in dry (e.g., powder or crystal)
form, or in liquid form, e.g., dissolved in an aqueous liquid, such
as those suitable for consumption or administration.
[0013] Further provided is a method to prevent, inhibit or treat a
microbial infection in an animal, e.g., an avian or mammal. The
method includes administering a composition that includes one or
more of proanthocyanin, or one or more of a flavonol, diterpenoid
epoxide, or phenylpropanoid, e.g., a hydroxycinnamic acid, or
combinations thereof, to the animal in an effective amount. In one
embodiment, the compound in the composition prevents or inhibits
microbial infection and/or eliminates or inhibits microbial growth,
e.g., replication. In one embodiment, the mammal is human. In one
embodiment, the composition is orally or intravenously
administered. In one embodiment, the composition is a tablet. In
one embodiment, the microbe is a virus, e.g., a poxvirus or an
influenza virus.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1. Schematic of an exemplary separation protocol using
cranberry juice fractions. Boxes represent cranberry juice
fractions. Red borders indicate fractions with strong activity
against vaccinia virus. Black borders indicate fractions with weak
or no antiviral activity. Boxes shaded with yellow indicate
fractions that have good water solubility and purity. Fractions
shaded pink include those with low water solubility or low purity
levels. Arrows represent separation methods applied to purify a
fraction further.
[0015] FIG. 2. Schematic of an exemplary separation protocol with
cranberry seed powder fractions. Boxes represent cranberry seed
fractions. Red borders indicate fractions with strong activity
against vaccinia virus. Black borders indicate fractions with weak
or no antiviral activity. Arrows represent separation methods
applied to purify a fraction further.
[0016] FIGS. 3-6 show the progression of purification of compounds
F2a2 and F2a3 from 90MX cranberry powder at each stage by HPLC. The
column used for analytical HPLC was a Waters Symmetry brand C8
column, 3.9.times.150 mm, with a 5 .mu.m particle size and a flow
rate 0.5 mL/min. A binary solvent gradient was used for these
analytical HPLC separations with solvent A=water+0.1% formic acid
and solvent B=methanol+0.1% formic acid. Solvent composition at
various times was as follows, time (percent B): initial (10% B), 50
min. (35% B), 80 min. (60% B), 85 min. (80% B), 90 min. (80%
B).
[0017] FIG. 3. Overlaid HPLC chromatograms are shown of the initial
separation of 90MX into fractions F1, F2, and F3 by C18 solid phase
extraction. The column contained C18 media from Grace-Davison
Discovery Sciences, using an Octave 100 chromatograph (Semba
Biosciences, Middleton, Wis.).
[0018] FIG. 4. Overlaid HPLC chromatograms are shown before and
after the second purification step using strong cation exchange
chromatography. The column was a Redi-Sep Rf SCX brand, 5 g, from
Isco/Teledyne. Both traces were recorded over a range of
wavelengths from 250 to 600 nm. The red trace shows the starting F2
material while the green trace shows the purified F2a material.
[0019] FIG. 5. Overlaid HPLC chromatograms are shown of fractions
F2a2, F2a3, F2a4, and F2a5 separated in the third step by
prep-scale C8 HPLC. A Waters Symmetry C8 column was used for prep
HPLC, 19.times.150 mm, 7 .mu.m, with a flow rate of 12 mL/min. The
same solvent program as was used in C8 analytical HPLC was used for
prep HPLC. All traces in FIG. 5 were recorded over a range of
wavelengths from 250 to 600 nm.
[0020] FIGS. 6A and 6B. These show analytical HPLC chromatographs
that were used to carry out final purification of fractions F2a2
and F2a3 by collecting the peaks eluting at 56 min. and 50 min.,
respectively. Fraction F2a2 was purified using a Luna brand
pentafluorophenyl (2) phase column from Phenomenex of dimensions
4.6.times.150 mm, with a 5 .mu.m particle size and a flow rate 0.5
mL/min. The same gradient solvent program as was used as before for
the C8 column. Peaks in FIGS. 6A and 6B were acquired at 277 nm and
356 nm, respectively.
[0021] FIGS. 7-9 show the anti-viral activity against vaccinia
virus (FIG. 7), poliovirus 1 (FIG. 8), and influenza A/H1N1 (FIG.
9), respectively, of powder fractions F2a2 and F2a3 after
separation and removal of solvent.
[0022] FIG. 10. Overlaid HPLC chromatograms are shown of fractions
E1, E2, and E3 separated by prep-scale C8 HPLC. A Waters Symmetry
C8 column was used for prep HPLC, 19.times.150 mm, 7 .mu.m, with a
flow rate of 12 mL/min. The same solvent program as was used in C8
analytical HPLC was used for prep HPLC. All traces in FIG. 10 were
recorded over a range of wavelengths from 250 to 600 nm.
[0023] FIG. 11 shows the anti-viral activity against vaccinia virus
of seed powder fractions of E0, E1, E2, and E3 after separation and
removal of solvent.
[0024] FIGS. 12-14 show examples of antiviral (vaccinia virus, FIG.
12; poliovirus, FIG. 13; and influenza virus, FIG. 14) testing data
in tests. To prepare samples for testing, they were diluted in
MEM/EBSS cell culture medium (HyClone catalog #SH30024.01) to test
at concentrations of 100 mg/L, 10 mg/L, 1 mg/L and 0.1 mg/L.
Samples that were not soluble in the MEM/EBSS were first diluted to
10 mg/L in DMSO (Fisher BioReagents catalog #BP231-100). A control
experiment was performed to demonstrate that the DMSO did not
affect viral activity or cell growth.
[0025] FIG. 12. A vaccinia virus plaque reduction assay is shown.
The photograph of the plate on the left is that of a control plate,
and the photograph of the plate on the right is that of a test
plate. Methods were adapted from Current Protocols in Molecular
Biology (1998), Unit 16.16 contributed by Bernard Moss and Patricia
L. Earl. The titer of the vaccinia virus stock was determined by
plaque assay prior to the inhibition assay. Virus was added to each
sample dilution and the 1% DMSO control to yield between 20 and 80
plaque-forming units (PFU) of vaccinia virus. Each sample dilution
plus virus was inoculated into duplicate wells of a 12-well plate
containing BS-C-1 cells. Each sample dilution without virus was
inoculated to a single well to test for cytotoxicity. Four wells
were inoculated with virus only to confirm the virus concentration
of the working dilution of vaccinia virus at the time of the test.
The plates were incubated for 45 minutes at 37.degree. C. with 5%
CO.sub.2. The medium was aspirated from each well and 1 mL of EMEM5
growth medium was added to each well. The plates were incubated for
two days at 37.degree. C. with 5% CO.sub.2. The plates were stained
with 0.25% crystal violet in 20% ethanol and the plaques were
counted. An example of this testing protocol is shown in FIG. 10.
In this test, healthy BS-C-1 cells take up the stain and appear
purple while cells killed by vaccinia infection appear as colorless
spots (plaques). Counting the number of plaques present in each
test well (right sample plate) compared to control wells (left
plate) provides a measure of viral inhibition for juice
samples.
[0026] FIG. 13 shows an example of a poliovirus 1 inhibition assay.
The photograph of the plate on the left is that of a back titer
plate, and the photograph of the plate on the right is that of a
test plate. Methods were adapted form The World Health
Organizations Polio Laboratory Manual 4th edition (2004).
Poliovirus 1 did not show good plaque formation in BS-C-1 cells
when tested by plaque reduction assay as described for the vaccinia
virus. The procedures were developed to use neutralization testing
with cranberry extracts in place of poliovirus antiserum. The
TCID.sub.50 titer of the Poliovirus 1 stock was determined prior to
the inhibition assay. Virus was added to each sample dilution and
the 1% DMSO control to yield concentrations of 100 TCID.sub.50 and
10 TCID.sub.50 of poliovirus 1. Each sample dilution plus virus was
inoculated into four wells of a 24-well plate containing BS-C-1
cells. Each sample dilution without virus was inoculated to a
single well to test for cytotoxicity. A back titer was performed to
confirm the virus concentration of the working dilution to be 100
TCID.sub.50 of poliovirus 1 at the time of the test. The plates
were incubated for one hour at 37.degree. C. with 5% CO.sub.2. The
medium was aspirated from each well and 1 mL of EMEM5 growth medium
was added to each well. The plates were incubated for seven days at
37.degree. C. with 5% CO.sub.2 with the medium being replaced on
day three or four. The plates were stained with 0.25% crystal
violet in 20% ethanol. The left plate shows back titer results for
poliovirus at varying concentrations of virus after staining. Wells
containing healthy BS-C-1 cells stain purple while those exhibiting
a cytopathic effect appear colorless. The test plate at right shows
inhibition at sample concentrations greater than or equal to 1
mg/mL.
[0027] FIG. 14 shows an example of an influenza A/H1N1 inhibition
assay using the known inhibitor Tamiflu. Methods were adapted from
the World Health Organizations Global Influenza Surveillance
Network's Manual for the Laboratory Diagnosis and Virological
Surveillance of Influenza (2011). Influenza A viruses do not form
plaques in MDCK cells and may not show cytopathic effect even when
the cells are infected. The methods were developed to use cranberry
extracts as viral inhibitor in place of influenza-specific
antiserum. Therefore, inhibition of viral cytopathic effect (CPE)
and detection of the presence of virus by hemagglutination (HA)
were tested. The TCID.sub.50 of the influenza A/H1N1 stock virus
was determined prior to the inhibition assay. Virus was added to
each sample dilution to yield concentrations of 100 TCID.sub.50 and
10 TCID.sub.50 of influenza A/H1N1. Each sample dilution plus virus
was inoculated into duplicate wells of a 12-well plate containing
MDCK cells. Each sample dilution without virus was inoculated to a
single well to test for cytotoxicity. A back titer was performed to
confirm the virus concentration of the working dilution to be 100
TCID.sub.50 of influenza at the time of the test. The plates were
incubated fir one hour at 34.degree. C. with 5% CO.sub.2. The
medium was aspirated from each well and replaced with 1 mL of EMEM5
growth medium. The plates were incubated for five days at
34.degree. C. with 5% CO.sub.2 with the medium being replaced on
day three or four. The plates were observed microscopically for
CPE. Influenza infection was confirmed by testing samples from each
well for hemagglutination of guinea pig red blood cells in 96-well
V-bottom plates. The presence of influenza virus will cause the
formation of a lattice with a suspension of 0.75% guinea pig red
blood cells in 1.times. PBS with 0.4% bovine serum albumin. This is
referred to as hemagglutination. Wells in which the virus has been
inhibited will have no hemagglutination and the red blood cells
will settle to the bottom forming a red button. The left portion of
the plate shows back titer results for H1N1. The right portion
shows test results with a known inhibitor of influenza, the
antiviral drug Tamiflu. Red buttons in each test well indicate
effective viral inhibition.
[0028] FIGS. 15A-I show results for anti-PIV3 activity of fractions
F2a2 and F2a3. A) Back titers results. B) CPE and C) percent
inhibition, respectively, of PIV3 with F2a2. D) CPE and E) percent
inhibition, respectively, of PIV3 with F2a3, F) Percent inhibition
of [[with]] 100 or 10 TCID.sub.50 of PIV3 by F2a2. G) Percent
inhibition of 100 or 10 TCID.sub.50of PIV3 by F2a3. H) Graph of
percent inhibition of 100 TCID.sub.50 HPIV3 with various
concentrations of F2a2 and F2a3. I) Graph of percent inhibition of
10 TCID.sub.50 HPIV3 with various concentrations of F2a2 and
F2a3.
[0029] FIGS. 16A-D show results for anti-influenza virus activity
of fractions F2a2 and F2a3. A) Percent inhibition by F2a2 of 100 or
10 TCID.sub.50 of H3N2. B) Percent inhibition of 100 or 10
TCID.sub.50 H3N2 by F2a3. C) Comparison of percent inhibition of
100 TCID.sub.50 of H3N2 with various concentrations of F2a2 or
F2a3. D) Comparison of percent inhibition of 10 TCID.sub.50 of H3N2
with various concentrations of F2a2 or F2a3.
[0030] FIG. 17. Analysis of fraction F2a2. A) TOF MS ES+
chromatogram of major components in F2a2. B) TOF MS ES-
chromatogram of major components F2a2. C) Photo diode array
chromatogram of major components in F2a2. D) TOF MS ES+
chromatogram of minor components in F2a2. E) TOE MS ES-
chromatogram of minor components in F2a2. F) Photo diode array
chromatogram of minor components in F2a2. G) Structures for
molecules (or related compounds) corresponding to the major
components.
[0031] FIG. 18. Analysis of fraction F2a3. A) TOF MS ES+
chromatogram of major components in F2a3. B) TOF MS ES-
chromatogram of major components in F2a3. C) Photo diode array
chromatogram of major components in F2a3. D) TOF MS ES+
chromatogram of minor components in F2a3. E) TOF MS ES-
chromatogram of minor components in F2a3. F) Photo diode array
chromatogram of minor components in F2a3. G) Structures consistent
with major components.
[0032] FIG. 19. Temperature stability study of F2 (panel A) and
F2a2 (panel B).
[0033] FIG. 20. Anti-viral activity of cranberry seed powder when
applied subsequent to (panel A) or before (panel B) vaccinia virus
infection.
[0034] FIG. 21. Comparison of anti-viral activity (panel A,
vaccinia virus; panel B, influenza virus) of cranberry seed powder
prepared by different methods. Material labeled as JAM or JAM1-91
refers to cranberry seed powder extracts produced by extraction
using 70% methanol/30% water. C) Inhibition of 100 TCID.sub.50 of
H1N1 with cranberry seed extract prepared by different methods. D)
Inhibition of 10 TCID.sub.50 of H1N1 with cranberry seed extract
prepared by different methods.
DETAILED DESCRIPTION
[0035] Cranberry juice is an enormously complex mixture containing
hundreds of compounds in varying amounts. In order to study the
anti-microbial effects of cranberries or other fruits, the
bioactive agents in juices or extracts thereof are isolated in
sufficient quantities to permit chemical characterization and
anti-microbial testing. In one embodiment, this separation is
accomplished using chromatographic techniques including but not
limited to Solid Phase Extraction (SPE) and preparatory-scale High
Performance Liquid Chromatography (HPLC), either alone or in
combination, e.g., sequentially.
Exemplary Separation Methods
[0036] Solid-phase extraction (SPE) is a separation process by
which compounds that are dissolved or suspended in a liquid mixture
are separated from other compounds in the mixture according to
their physical and chemical properties. Analytical laboratories use
SPE to concentrate and purify samples for analysis. SPE uses the
affinity of solutes dissolved or suspended in a liquid (known as
the mobile phase) for a solid through which the sample is passed
(known as the stationary phase) to separate a mixture into desired
and undesired components. The result is that either the desired
analytes of interest or undesired impurities in the sample are
retained on the stationary phase. The portion that passes through
the stationary phase is collected or discarded, depending on
whether it contains the desired analytes or undesired impurities.
If the portion retained on the stationary phase includes the
desired analytes, they can then be removed from the stationary
phase for collection in an additional step, in which the stationary
phase is rinsed with an appropriate eluent. The stationary phase
comes in the form of, for example, a packed syringe-shaped
cartridge, a 96 well plate, a 47- or 90-mm flat disk, or a MEPS
device, each of which can be mounted on its specific type of
extraction manifold. The manifold allows multiple samples to be
processed by holding several SPE media in place and allowing for an
equal number of samples to pass through them simultaneously. A
typical cartridge SPE manifold can accommodate up to 24 cartridges,
while a typical disk SPE manifold can accommodate 6 disks. Most SPE
manifolds are equipped with a vacuum port. Application of vacuum
speeds up the extraction process by pulling the liquid sample
through the stationary phase. The analytes are collected in sample
tubes inside or below the manifold after they pass through the
stationary phase. Solid phase extraction cartridges and disks are
available with a variety of stationary phases, each of which can
separate analytes according to different chemical properties. Most
stationary phases are based on silica that has been bonded to a
specific functional group. Some of these functional groups include
hydrocarbon chains of variable length (for reversed phase SPE),
quaternary ammonium or amino groups (for anion exchange), and
sulfonic acid or carboxyl groups (for cation exchange).
Normal Phase SPE Procedure
[0037] A typical solid phase extraction involves four basic steps.
First, the cartridge is equilibrated with a non-polar solvent or
slightly polar, which wets the surface and penetrates the bonded
phase. Then water, or buffer of the same composition as the sample,
is typically washed through the column to wet the silica surface.
The sample is then added to the cartridge. As the sample passes
through the stationary phase, the analytes in the sample will
interact and retain on the sorbent while the solvent, salts, and
other impurities pass through the cartridge. After the sample is
loaded, the cartridge is washed with buffer or solvent to remove
further impurities. Then, the analyte is eluted with a non-polar
solvent or a buffer of the appropriate pH.
Reversed Phase SPE
[0038] Reversed phase SPE separates analytes based on their
polarity. The stationary phase of a reversed phase SPE cartridge is
derivatized with hydrocarbon chains, which retain compounds of mid
to low polarity due to the hydrophobic effect. The analyte can be
eluted by washing the cartridge with a non-polar solvent, which
disrupts the interaction of the analyte and the stationary
phase.
Ion Exchange SPE
[0039] Ion exchange sorbents separate analytes based on
electrostatic interactions between the analyte of interest and the
charged groups on the stationary phase. For ion exchange to occur,
both the stationary phase and sample must be at a pH where ion-ion
interactions may occur.
Anion Exchange
[0040] Anion exchange sorbents are derivatized with positively
charged functional groups that interact and retain negatively
charged anions, such as deprotonated acids. Strong anion exchange
sorbents contain quaternary ammonium groups that have a permanent
positive charge in aqueous solutions, and weak anion exchange
sorbents use amine groups which are charged when the pH is below
about 9. Strong anion exchange sorbents are useful because any
strongly acidic impurities in the sample will bind to the sorbent
and usually will not be eluted with the analyte of interest; to
recover a strong acid a weak anion exchange cartridge should be
used. To elute the analyte from either the strong or weak sorbent,
the stationary phase is washed with a solvent that neutralizes the
charge of either the analyte, the stationary phase, or both. Once
the charge is neutralized, the electrostatic interaction between
the analyte and the stationary phase no longer exists and the
analyte will elute from the cartridge.
Cation Exchange
[0041] Cation exchange sorbents are derivatized with functional
groups that interact and retain positively charged cations, such as
protonated amines. Strong cation exchange sorbents contain
aliphatic sulfonic acid groups that are always negatively charged
in aqueous solution, and weak cation exchange sorbents contain
aliphatic carboxylic acids, which are charged when the pH is above
about 5. Strong cation exchange sorbents are useful because any
strongly basic impurities in the sample will bind to the sorbent
and usually will not be eluted with the analyte of interest; to
recover a strong base a weak cation exchange cartridge should be
used. To elute the analyte from either the strong or weak sorbent,
the stationary phase is washed with a solvent that neutralizes
ionic interaction between the analyte and the stationary phase.
HPLC
[0042] High-performance liquid chromatography (sometimes referred
to as high-pressure liquid chromatography), HPLC, is a
chromatographic technique used to separate a mixture of compounds
in analytical chemistry and biochemistry with the purpose of
identifying, quantifying and purifying the individual components of
the mixture. Some common examples are the separation and
quantitation of performance enhancement drugs (e.g. steroids) in
urine samples, or of vitamin D levels in serum. HPLC typically
utilizes different types of stationary phases (i.e. sorbents)
contained in columns, a pump that moves the mobile phase and sample
components through the column, and a detector capable of providing
characteristic retention times for the sample components and area
counts reflecting the amount of each analyte passing through the
detector. The detector may also provide additional information
related to the analyte, (e.g., UV/Vis spectroscopic data, if so
equipped). Analyte retention time varies depending on the strength
of its interactions with the stationary phase, the composition and
flow rate of mobile phase used, and on the column dimensions. HPLC
is a form of liquid chromatography that utilizes small size columns
(typically 250 mm or shorter and 4.6 mm i.d. or smaller; packed
with smaller particles), and higher mobile phase pressures compared
to ordinary liquid chromatography. With HPLC, a pump (rather than
gravity) provides the higher pressure required to move the mobile
phase and sample components through the densely packed column. The
increased density arises from the use of smaller sorbent particles.
Such particles are capable of providing better separation on
columns of shorter length when compared to ordinary column
chromatography.
[0043] The sample to be separated and analyzed is introduced, in a
discrete small volume, into the stream of mobile phase percolating
through the column. The components of the sample move through the
column at different velocities, which are functions of specific
physical or chemical interactions with the stationary phase. The
velocity of each component depends on its chemical nature, on the
nature of the stationary phase (column) and on the composition of
the mobile phase. The time at which a specific analyte elutes
(emerges from the column) is called the retention time. The
retention time measured under particular conditions is considered
an identifying characteristic of a given analyte. The use of
smaller particle size packing materials require the use of higher
operational pressure ("backpressure") and typically improves
chromatographic resolution (i.e. the degree of separation between
consecutive analytes emerging from the column). Common mobile
phases used include any miscible combination of water with various
organic solvents (the most common are acetonitrile and methanol).
Some HPLC techniques use water free mobile phases (see Normal Phase
HPLC below). The aqueous component of the mobile phase may contain
buffers, acids (such as formic, phosphoric or trifluoroacetic acid)
or salts to assist in the separation of the sample components. The
composition of the mobile phase may be kept constant ("isocratic
elution mode") or varied ("gradient elution mode") during the
chromatographic analysis. Isocratic elution is typically effective
in the separation of sample components that are not very dissimilar
in their affinity for the stationary phase.
[0044] In gradient elution the composition of the mobile phase is
varied typically from low to high eluting strength. The eluting
strength of the mobile phase is reflected by analyte retention
times with high eluting strength producing fast elution (=short
retention times). A typical gradient profile in reversed phase
chromatography might start at 5% acetonitrile (in water or aqueous
buffer) and progress linearly to 95% acetonitrile over 5-25
minutes. Period of constant mobile phase composition may be part of
any gradient profile. For example, the mobile phase composition may
be kept constant at 5% acetonitrile for 1-3 minutes, followed by a
linear change up to 95% acetonitrile.
[0045] The composition of the mobile phase depends on the intensity
of interactions between analytes and stationary phase (e.g.
hydrophobic interactions in reversed-phase HPLC). Depending on
their affinity for the stationary and mobile phases analytes
partition between the two during the separation process taking
place in the column. This partitioning process is similar to that
which occurs during a liquid-liquid extraction but is continuous,
not step-wise. In this example, using a water/acetonitrile
gradient, more hydrophobic components will elute (come off the
column) late, once the mobile phase gets more concentrated in
acetonitrile (i.e. in a mobile phase of higher eluting
strength).
[0046] The choice of mobile phase components, additives (such as
salts or acids) and gradient conditions depend on the nature of the
column and sample components. Often a series of trial runs are
performed with the sample in order to find the HPLC method that
gives the best separation.
Partition Chromatography
[0047] The partition coefficient principle has been applied in
paper chromatography, thin layer chromatography, gas phase and
liquid-liquid applications. Partition chromatography uses a
retained solvent, on the surface or within the grains or fibers of
an "inert" solid supporting matrix as with paper chromatography; or
takes advantage of some coulombic and/or hydrogen donor interaction
with the solid support. Molecules equilibrate (partition) between a
liquid stationary phase and the eluent. Known as Hydrophilic
Interaction Chromatography (HILIC) in HPLC, this method separates
analytes based on polar differences. HILIC most often uses a bonded
polar stationary phase and water miscible, high organic
concentration, mobile phases. Partition HPLC has been used
historically on unbonded silica or alumina supports. Each works
effectively for separating analytes by relative polar differences.
HILIC bonded phases have the advantage of separating acidic, basic
and neutral solutes in a single chromatogram.
[0048] The polar analytes diffuse into a stationary water layer
associated with the polar stationary phase and are thus retained.
Retention strengths increase with increased analyte polarity, and
the interaction between the polar analyte and the polar stationary
phase (relative to the mobile phase) increases the elution time.
The interaction strength depends on the functional groups in the
analyte molecule which promote partitioning but can also include
coulombic (electrostatic) interaction and hydrogen donor
capability.
[0049] Use of more polar solvents in the mobile phase will decrease
the retention time of the analytes, whereas more hydrophobic
solvents tend to increase retention times.
Normal-Phase Chromatography
[0050] Normal-phase HPLC (NP-HPLC), or adsorption chromatography,
separates analytes based on their affinity for a polar stationary
surface such as silica, hence it is based on analyte ability to
engage in polar interactions (such as hydrogen-bonding or
dipole-dipole type of interactions) with the sorbent surface.
NP-HPLC uses a non-polar, non-aqueous mobile phase, and works
effectively for separating analytes readily soluble in non-polar
solvents. The analyte associates with and is retained by the polar
stationary phase. Adsorption strengths increase with increased
analyte polarity. The interaction strength depends not only on the
functional groups present in the structure of the analyte molecule,
but also on steric factors. The effect of steric hindrance on
interaction strength allows this method to resolve (separate)
structural isomers.
[0051] The use of more polar solvents in the mobile phase will
decrease the retention time of analytes, whereas more hydrophobic
solvents tend to induce slower elution (increased retention times).
Very polar solvents such as traces of water in the mobile phase
tend to adsorb to the solid surface of the stationary phase forming
a stationary bound (water) layer which is considered to play an
active role in retention. This behavior is somewhat peculiar to
normal phase chromatography because it is governed almost
exclusively by an adsorptive mechanism (i.e., analytes interact
with a solid surface rather than with the solvated layer of a
ligand attached to the sorbent surface; see also reversed-phase
HPLC below). Adsorption chromatography is still widely used for
structural isomer separations in both column and thin-layer
chromatography formats on activated (dried) silica or alumina
supports.
Displacement Chromatography
[0052] The basic principle of displacement chromatography is: A
molecule with a high affinity for the chromatography matrix (the
displacer) will compete effectively for binding sites, and thus
displace all molecules with lesser affinities. There are distinct
differences between displacement and elution chromatography. In
elution mode, substances typically emerge from a column in narrow,
Gaussian peaks. Wide separation of peaks, e.g., to baseline, is
desired in order to achieve maximum purification. The speed at
which any component of a mixture travels down the column in elution
mode depends on many factors. But for two substances to travel at
different speeds, and thereby be resolved, there must be
substantial differences in some interaction between the
biomolecules and the chromatography matrix. Operating parameters
are adjusted to maximize the effect of this difference. In many
cases, baseline separation of the peaks can be achieved only with
gradient elution and low column loadings. Thus, two drawbacks to
elution mode chromatography, especially at the preparative scale,
are operational complexity, due to gradient solvent pumping, and
low throughput, due to low column loadings. Displacement
chromatography has advantages over elution chromatography in that
components are resolved into consecutive zones of pure substances
rather than "peaks". Because the process takes advantage of the
nonlinearity of the isotherms, a larger column feed can be
separated on a given column with the purified components recovered
at significantly higher concentrations.
Reversed-Phase Chromatography (RPC)
[0053] Reversed phase HPLC (RP-HPLC) has a non-polar stationary
phase and an aqueous, moderately polar mobile phase. One common
stationary phase is a silica which has been surface-modified with
RMe.sub.2SiCl, where R is a straight chain alkyl group such as
C.sub.18H.sub.37 or C.sub.8H.sub.17. With such stationary phases,
retention time is longer for molecules that are less polar, while
polar molecules elute more readily (early in the analysis). An
investigator can increase retention times by adding more water to
the mobile phase; thereby making the affinity of the hydrophobic
analyte for the hydrophobic stationary phase stronger relative to
the now more hydrophilic mobile phase. Similarly, an investigator
can decrease retention time by adding more organic solvent to the
eluent. RP-HPLC is so commonly used that it is often incorrectly
referred to as "HPLC" without further specification. The
pharmaceutical industry regularly employs RP-HPLC to qualify drugs
before their release.
[0054] RP-HPLC operates on the principle of hydrophobic
interactions, which originate from the high symmetry in the dipolar
water structure and play the most important role in all processes
in life science. RP-HPLC allows the measurement of these
interactive forces. The binding of the analyte to the stationary
phase is proportional to the contact surface area around the
non-polar segment of the analyte molecule upon association with the
ligand on the stationary phase. This solvophobic effect is
dominated by the force of water for "cavity-reduction" around the
analyte and the C.sub.18-chain versus the complex of both. The
energy released in this process is proportional to the surface
tension of the eluent (water: 7.3.times.10.sup.-6 J/cm.sup.2,
methanol: 2.2.times.10.sup.-6 J/cm.sup.2) and to the hydrophobic
surface of the analyte and the ligand respectively. The retention
can be decreased by adding a less polar solvent (methanol,
acetonitrile) into the mobile phase to reduce the surface tension
of water. Gradient elution uses this effect by automatically
reducing the polarity and the surface tension of the aqueous mobile
phase during the course of the analysis.
[0055] Structural properties of the analyte molecule play an
important role in its retention characteristics. In general, an
analyte with a larger hydrophobic surface area (C--H, C--C, and
generally non-polar bonds, such as S--S and others) is retained
longer because it is non-interacting with the water structure. On
the other hand, analytes with higher polar surface area (conferred
by the presence of polar groups, such as --OH, --NH.sub.2,
COO.sup.- or --NH.sub.3.sup.- in their structure)are less retained
as they are better integrated into water. Such interactions are
subject to steric effects in that very large molecules may have
only restricted access to the pores of the stationary phase, where
the interactions with surface ligands (alkyl chains) take place.
Such surface hindrance typically results in less retention.
[0056] Retention time increases with hydrophobic (non-polar)
surface area. Branched chain compounds elute more rapidly than
their corresponding linear isomers because the overall surface area
is decreased. Similarly organic compounds with single C--C-bonds
elute later than those with a C.dbd.C or C--C-triple bond, as the
double or triple bond is shorter than a single C--C-bond.
[0057] Aside from mobile phase surface tension (organizational
strength in eluent structure), other mobile phase modifiers can
affect analyte retention. For example, the addition of inorganic
salts causes a moderate linear increase in the surface tension of
aqueous solutions (ca. 1.5.times.10.sup.-7 J/cm.sup.2 per Mol for
NaCl, 2.5.times.10.sup.-7 J/cm.sup.2 per Mol for
(NH.sub.4).sub.2SO.sub.4), and because the entropy of the
analyte-solvent interface is controlled by surface tension, the
addition of salts tend to increase the retention time. This
technique is used for mild separation and recovery of proteins and
protection of their biological activity in protein analysis
(hydrophobic interaction chromatography, HIC).
[0058] Another important factor is the mobile phase pH since it can
change the hydrophobic character of the analyte. For this reason
most methods use a buffering agent, such as sodium phosphate, to
control the pH. Buffers serve multiple purposes: control of pH,
neutralize the charge on the silica surface of the stationary phase
and act as ion pairing agents to neutralize analyte charge.
Ammonium formate is commonly added in mass spectrometry to improve
detection of certain analytes by the formation of analyte-ammonium
adducts. A volatile organic acid such as acetic acid, or most
commonly formic acid, is often added to the mobile phase if mass
spectrometry is used to analyze the column effluent.
Trifluoroacetic acid is used infrequently in mass spectrometry
applications due to its persistence in the detector and solvent
delivery system, but can be effective in improving retention of
analytes such as carboxylic acids in applications utilizing other
detectors, as it is a fairly strong organic acid. The effects of
acids and buffers vary by application but generally improve
chromatographic resolution.
Size-Exclusion Chromatography
[0059] Size-exclusion chromatography (SEC), also known as gel
permeation chromatography or gel filtration chromatography,
separates particles on the basis of size. It is generally a
low-resolution chromatography and thus it is often reserved for the
final, "polishing" step of a purification. It is also useful for
determining the tertiary structure and quaternary structure of
purified proteins. SEC is used primarily for the analysis of large
molecules such as proteins or polymers. SEC works by trapping these
smaller molecules in the pores of a particle. The larger molecules
simply pass by the pores as they are too large to enter the pores.
Larger molecules therefore flow through the column quicker than
smaller molecules, that is, the smaller the molecule, the longer
the retention time.
[0060] This technique is widely used for the molecular weight
determination of polysaccharides. SEC is the official technique
(suggested by European pharmacopeia) for the molecular weight
comparison of different commercially available low-molecular weight
heparins.
Ion-Exchange Chromatography
[0061] In ion-exchange chromatography (IC), retention is based on
the attraction between solute ions and charged sites bound to the
stationary phase. Ions of the same charge are excluded. Types of
ion exchangers include: polystyrene resins which allow cross
linkage which increases the stability of the chain. Higher cross
linkage reduces resin swelling, which increases the equilibration
time and ultimately improves selectivity; cellulose and dextran ion
exchangers (gels) which possess larger pore sizes and low charge
densities making them suitable for protein separation; and
controlled-pore glass or porous silica. In general, ion exchangers
favor the binding of ions of higher charge and smaller radius.
[0062] An increase in counter ion (with respect to the functional
groups in resins) concentration reduces the retention time. A
decrease in pH reduces the retention time in cation exchange while
an increase in pH reduces the retention time in anion exchange. By
lowering the pH of the solvent in a cation exchange column, for
instance, more hydrogen ions are available to compete for positions
on the anionic stationary phase, thereby eluting weakly bound
cations.
[0063] This form of chromatography is widely used in the following
applications: water purification, preconcentration of trace
components, ligand-exchange chromatography, ion-exchange
chromatography of proteins, high-pH anion-exchange chromatography
of carbohydrates and oligosaccharides, and others.
Bioaffinity Chromatography
[0064] This chromatographic process relies on the property of
biologically active substances to form stable, specific, and
reversible complexes. The formation of these complexes involves the
participation of common molecular forces such as Van der Waals
interaction, electrostatic interaction, dipole-dipole interaction,
hydrophobic interaction, and the hydrogen bond. An efficient,
biospecific bond is formed by a simultaneous and concerted action
of several of these forces in the complementary binding sites.
Aqueous Normal-Phase Chromatography
[0065] Aqueous normal-phase chromatography (ANP) is a
chromatographic technique which encompasses the mobile phase region
between reversed-phase chromatography (RP) and organic normal phase
chromatography (ONP). This technique is used to achieve unique
selectivity for hydrophilic compounds, showing normal phase elution
using reverse-phase solvents.
Isocratic Flow and Gradient Elution
[0066] A separation in which the mobile phase composition remains
constant throughout the procedure is termed isocratic (meaning
constant composition). The mobile phase composition does not have
to remain constant. A separation in which the mobile phase
composition is changed during the separation process is described
as a gradient elution. One example is a gradient starting at 10%
methanol and ending at 90% methanol after 20 minutes. The two
components of the mobile phase are typically termed "A" and "B"; A
is the "weak" solvent which allows the solute to elute only slowly,
while B is the "strong" solvent which rapidly elutes the solutes
from the column. In reverse-phase chromatography, solvent A is
often water or an aqueous buffer, while B is an organic solvent
miscible with water, such as acetonitrile, methanol, THF, or
isopropanol.
[0067] In isocratic elution, peak width increases with retention
time linearly according to the equation for N, the number of
theoretical plates. This leads to the disadvantage that
late-eluting peaks get very flat and broad. Their shape and width
may keep them from being recognized as peaks.
[0068] Gradient elution decreases the retention of the
later-eluting components an that they elute faster, giving narrower
(and taller) peaks for most components. This also improves the peak
shape for tailed peaks, as the increasing concentration of the
organic eluent pushes the tailing part of a peak forward. This also
increases the peak height (the peak looks "sharper"), which is
important in trace analysis. The gradient program may include
sudden "step" increases in the percentage of the organic component,
or different slopes at different times--all according to the desire
for optimum separation in minimum time.
[0069] In isocratic elution, the selectivity does not change if the
column dimensions (length and inner diameter) change--that is, the
peaks elute in the same order. In gradient elution, the elution
order may change as the dimensions or flow rate change.
[0070] The driving force in reversed phase chromatography
originates in the high order of the water structure. The role of
the organic component of the mobile phase is to reduce this high
order and thus reduce the retarding strength of the aqueous
component.
Parameters
Internal Diameter
[0071] The internal diameter (ID) of an HPLC column is one
parameter that influences the detection sensitivity and separation
selectivity in gradient elution. It also determines the quantity of
analyte that can be loaded onto the column. Larger columns are
usually seen in industrial applications, such as the purification
of a drug product for later use. Low-ID columns have improved
sensitivity and lower solvent consumption at the expense of loading
capacity. Larger ID columns (over 10 mm) are used to purify usable
amounts of material because of their large loading capacity.
Analytical scale columns (4.6 mm) have been the most common type of
columns. They are used in traditional quantitative analysis of
samples and often use a UV-Vis absorbance detector. Narrow-bore
columns (1-2 mm) are used for applications when more sensitivity is
desired either with special UV-Vis detectors, fluorescence
detection or with other detection methods like liquid
chromatography-mass spectrometry Capillary columns (under 0.3 mm)
are used almost exclusively 2.0 with alternative detection means
such as mass spectrometry. They are usually made from fused silica
capillaries, rather than the stainless steel tubing that larger
columns employ.
Particle Size
[0072] Most traditional HPLC is performed with the stationary phase
attached to the outside of small spherical silica particles (very
small beads). These particles come in a variety of sizes with 5
.mu.m beads being the most common. Smaller particles generally
provide more surface area and better separations, but the pressure
required for optimum linear velocity increases by the inverse of
the particle diameter squared.
[0073] This means that changing to particles that are half as big,
keeping the size of the column the same, will double the
performance, but increase the required pressure by a factor of
four. Larger particles are used in preparative HPLC (column
diameters 5 cm up to >30 cm) and for non-HPLC applications such
as solid-phase extraction.
Pore Size
[0074] Many stationary phases are porous to provide greater surface
area. Small pores provide greater surface area while larger pore
size has better kinetics, especially for larger analytes. For
example, a protein which is only slightly smaller than a pore might
enter the pore but does not easily leave once inside.
Pump Pressure
[0075] Pumps vary in pressure capacity, but their performance is
measured on their ability to yield a consistent and reproducible
flow rate. Pressure may reach as high as 40 MPa (6000
lbf/in.sup.2), or about 400 atmospheres. Modern HPLC systems have
been improved to work at much higher pressures, and therefore are
able to use much smaller particle sizes in the columns (<2
.mu.m). These "Ultra High Performance Liquid Chromatography"
systems or RSLC/UHPLCs can work at up to 100 MPa (15,000
lbf/in.sup.2), or about 1000 atmospheres. The term "UPLC" is a
trademark of the Waters Corporation, but is sometimes used to refer
to the more general technique.
[0076] In chromatography, the simulated moving bed (SMB) technique
is a variant of high performance liquid chromatography; it is used
to separate particles and/or chemical compounds that would be
difficult or impossible to resolve otherwise. This increased
separation is brought about by a valve-and-column arrangement that
is used to lengthen the stationary phase indefinitely.
[0077] In the moving bed technique of preparative chromatography
the feed entry and the analyte recovery are simultaneous and
continuous, but because of practical difficulties with a
continuously moving bed in the simulated moving bed technique
instead of moving the bed, the sample inlet and the analyte exit
positions are moved continuously, giving the impression of a moving
bed.
[0078] True moving bed chromatography (MBC) is only a theoretical
concept. Its simulation, SMBC is achieved by the use of a
multiplicity of columns in series and a complex valve arrangement,
which provides for sample and solvent feed, and also analyte and
waste takeoff at appropriate locations of any column, whereby it
allows switching at regular intervals the sample entry in one
direction, the solvent entry in the opposite direction, whilst
changing the analyte and waste takeoff positions appropriately as
well.
[0079] One advantage of the SMBC is high speed, because a system
could be near continuous, whilst its disadvantage is that it only
separates binary mixtures. It does not say, but perhaps it can be
assumed that this is equivalent with the separation of a single
component from a group of compounds. With regard to efficiency it
compares with simple chromatography technique like continuous
distillation does with batch distillation.
[0080] Specifically, an SMB system has two or more identical
columns, which are connected to the mobile phase pump, and each
other, by a multi-port valve. The plumbing is configured in such a
way that: [0081] a) all columns will be connected in series,
regardless of the valve's position; [0082] b) each different
position of the valve will reconnect the columns to one another in
one possible sequential arrangement of the columns; and [0083] c)
all possible positions of the valve will arrange the columns in
every possible sequential order.
[0084] For example, consider a case where two HPLC columns, A and
B, are connected to one another, and the mobile-phase pump, via a
six-port, two-position valve (e.g., a Rheodyne 7000). One valve
position will distribute the flow in the manner
[0085] Pump.fwdarw.Column A.fwdarw.Column B.fwdarw.Waste,
while the other position will distribute the flow in the manner
[0086] Pump.fwdarw.Column B.fwdarw.Column A.fwdarw.Waste.
[0087] Consequently, switching of the valve will "leapfrog" the
columns over one another. If elution across two columns in series
is not adequate to resolve two compounds in a given run, the eluent
can then be made to go through 3, 4, 5 . . . columns in additional
runs by carefully timed switching. This increases the number of
theoretical plates until separation can be attained.
[0088] When affinity differences between molecules are very small,
it is sometimes not possible to improve resolution via mobile- or
stationary-phase changes. In these cases, the multi-pass approach
of SMB can separate mixtures of those compounds by allowing their
small retention time differences to accumulate.
[0089] At industrial scale an SMB chromatographic separator is
operated continuously, requiring less resin and less solvent than
batch chromatography. The continuous operation facilitates
operation control and integration into production plants.
[0090] In size exclusion chromatography, where the separation
process is driven by entropy, it is not possible to increase the
resolution attained by a column via temperature or solvent
gradients. Consequently, these separations often require SMB, to
create usable retention time differences between the molecules or
particles being resolved. SMB is also very useful in the
pharmaceutical industry, where resolution of molecules having
different chirality must be done on a very large scale.
Exemplary Methods and Microbes
[0091] Once a method is identified that results in a substantially
pure preparation of a compound with anti-microbial activity, that
method or methods that are similar thereto, e.g., methods that
eliminate or add a step and optionally omit testing for activity at
each or all separation steps, may be employed to isolate large
quantities of the bioactive compound, e.g., isolate about 1 mg to
about 1000 mg of a substantially pure preparation per run. Thus,
the invention provides a composition, e.g., a powder or liquid
composition, having a substantially pure preparation of a compound
with anti-microbial activity. That composition may be employed as a
neutraceutical or in pharmaceutical compositions.
[0092] In one embodiment, the invention provides a method of
treating, inhibiting or preventing a bacterial infection, e.g.,
infection by Listeria or a pan resistant gram-negative bacilli,
such as Pseudomonas aeruginosa, or multi-resistant gram-positive
bacteria like methicillin resistant Staphylococcus aureus, as well
as Mycobacterium tuberculosis, or nontuberculosis Mycobacterium or
Nocardia, or E. coli in an animal such as a mammal. In one
embodiment, the method comprises administering an effective amount
of a composition of the invention to a mammal after the mammal has
been infected with the bacterium. In one embodiment, the method
comprises administering an effective amount of a composition of the
invention to the mammal before the mammal is infected with the
bacterium.
[0093] In one embodiment, the present invention is directed to a
method of treating, inhibiting or preventing a viral infection in
an animal such as a mammal, e.g., viruses including but not limited
to rabies, poxviruses, herpesviruses, influenza A, influenza B,
influenza C, flaviviruses including West Nile virus and Dengue
virus, paramyxoviruses including Respiratory Syncyctial virus,
parvoviruses, retroviruses, poxviruses, hepatitis viruses, and
gastroenteroviruses including poliovirus, coxsackie virus,
rotavirus, norovirus, and astrovirus. In one embodiment, the method
comprises administering an amount of a composition of the
invention, e.g., after a mammal has been infected with or exposed
to a virus, effective to inhibit or treat the viral infection. In
one embodiment, a composition of the invention is administered to a
mammal before a mammal is exposed to the virus. In one embodiment,
the pathogen is an influenza virus, e.g., influenza A virus. In one
embodiment, the influenza A virus is a H5N1 virus strain.
[0094] In one embodiment, the invention provides a method of
treating, inhibiting or preventing a parasite infection in an
animal such as a mammal, e.g., infection by various species of
Plasmodium, such as Plasmodium berghei, and Plasmodium falciparum
and other Coccidia such as Cryptosporidium parvum, or other
protozoan parasites such as Trypanosome brucei, Entamoeba
histolytica, Leishmania species and helminth parasites such as
Schistosoma mansoni. In one embodiment, the method comprises
administering an amount of a composition of the invention to the
mammal effective to inhibit or treat the parasitic infection after
the mammal has been infected with the parasite. In one embodiment,
a composition of the invention is administered to a mammal before
the mammal is exposed to the parasite.
[0095] In one embodiment, the invention provides a method of
treating, inhibiting or preventing a fungal infection in an animal,
e.g., Cryptococcus, Aspergillus, species, Histoplasma capsulatum,
Blastomyces dermatitidis, Coccidiomycosis immitis and Penicillium
marcenscens. In one embodiment, the method comprises administering
an effective amount of a composition of the invention to the mammal
after the mammal has been infected with the fungus. In one
embodiment, the method comprises administering an effective amount
of a composition of the invention to the mammal before the mammal
is infected with the fungus.
[0096] As will be apparent to one skilled in the art, the optimal
concentration of the active agent in a composition of the invention
will necessarily depend upon the specific agent(s) used, the
characteristics of the animal, e.g., avian or mammal, and the
nature of the microbial infection. These factors can be determined
by those of skill in the medical and pharmaceutical arts in view of
the present disclosure. In general, the active agent(s) in the
composition of the invention are administered at a concentration
that either modulates anti-microbial activity against microbial
infection or modulates an immune response allowing the host to
recover from or clear a microbial infection, without significant,
harmful or adverse side effects.
[0097] Specific dosages may be adjusted depending on conditions of
disease, the age, body weight, ethnic background, general health
conditions, sex, diet, lifestyle and/or current therapeutic regimen
of the animal, as well as for intended dose intervals,
administration routes, excretion rate, and combinations of drugs.
Any of the dosage forms described herein containing effective
amounts are well within the bounds of routine experimentation and
therefore, well within the scope of the instant disclosure.
[0098] A composition may comprise a compound of the invention in an
amount of about 1 .mu.g to about 2000 mg of the compound per dose
for a mammal weighing about 20 to 25 g. In one embodiment, the
composition comprises a compound of the invention an amount of
about 1 mg to about 1000 mg, e.g., about 10 mg to about 100 mg, or
an amount of about 0.1 to about 1000 .mu.g, e.g., about 1 .mu.g to
about 10 .mu.g. In one embodiment, the composition comprises a
compound of the invention an amount of about 20 .mu.g/kg to about
2000 .mu.g/kg, e.g., about 50 .mu.g/kg to about 500 .mu.g/kg or
about 100 .mu.g/kg to about 400 .mu.g/kg.
[0099] The desired dose of the composition may be presented in a
continuous infusion, a single dose, or as divided doses
administered at appropriate intervals, for example as two, three,
four or more sub-doses per day. Optionally, a dose of composition
may be administered on one day, followed by one or more doses
spaced as desired thereinafter. In one exemplary embodiment, an
initial dose is given, followed by a dose of the same composition
approximately two to four days later. In one particular embodiment,
the mammal is administered a first dose of the composition at about
48 hours post-infection and a second dose of the composition at
about 96 hours post-infection. Other dosage schedules may also be
used.
[0100] Following an initial administration of the composition,
animals may receive one or several doses adequately spaced
thereafter. In some embodiments, the subsequent doses comprise the
same amounts and type of active agent as the initial
administration. In other embodiments, the subsequent doses may
comprise a reduced amount and/or a different type of active
agent.
[0101] In addition to the active agent(s), one or more suitable
pharmaceutically acceptable carriers may be used. As used herein,
the term "pharmaceutically acceptable carrier" refers to an
acceptable vehicle for administering a composition to mammals
comprising one or more non-toxic excipients that do not react with
or reduce the effectiveness of the pharmacologically active agents
contained therein. The proportion and type of pharmaceutically
acceptable carrier in the composition may vary, depending on the
chosen route of administration. Suitable pharmaceutically
acceptable carriers for the compositions of the present disclosure
are described in the standard pharmaceutical texts. See, e.g.,
"Remington's Pharmaceutical Sciences", 18.sup.th Ed., Mack
Publishing Company, Easton, Pa. (1990). Specific non-limiting
examples of suitable pharmaceutically acceptable carriers include
water, saline, dextrose, glycerol, ethanol, or the like and
combinations thereof.
[0102] Optionally, the composition may further comprise minor
amounts of auxiliary substances such as agents that enhance the
antimicrobial effectiveness of the preparation, stabilizers,
preservatives, and the like.
[0103] In one embodiment, the composition may also comprise a bile
acid or a derivative thereof, in particular in the form of a salt.
These include derivatives of cholic acid and salts thereof, in
particular sodium salts of cholic acid or cholic acid derivatives.
Examples of bile acids and derivatives thereof include cholic acid,
deoxycholic acid, chenodeoxycholic acid, lithocholic acid,
ursodeoxycholic acid, hyodeoxycholic acid and derivatives such as
glyco-, tauro-, amidopropyl-1-propanesulfonic-,
amidopropyl-2-hydroxy-1-propanesulfonic derivatives of the
aforementioned bile acids, or
N,N-bis(3-D-gluconoamidopropyl)deoxycholamide. A particular example
is sodium deoxycholate (NaDOC).
[0104] Examples of suitable stabilizers include sugars such as
sucrose and glycerol, encapsulating polymers, chelating agents such
as ethylenediaminetetracetic acid (EDTA), proteins and polypeptides
such as gelatin and polyglycine and combinations thereof.
[0105] Depending on the route of administration, the compositions
may take the form of a solution, suspension, emulsion, or the like.
A composition of the invention can be administered intranasally or
through enteral administration, such as orally, or through
subcutaneous injection, intra-muscular injection, intravenous
injection, intraperitoneal injection, or intra-dermal injection to
a mammal, e.g., humans, horses, other mammals, etc. Compositions
may be formulated for a particular route of delivery, e.g.,
formulated for oral delivery,
[0106] For parenteral administration, the composition of the
invention may be administered by intravenous, subcutaneous,
intramuscular, intraperitoneal, or intradermal injection, and may
further comprise pharmaceutically accepted carriers. For
administration by injection, the composition may be in a solution
in a sterile aqueous vehicle which may also contain other solutes
such as buffers or preservatives as well as sufficient quantities
of pharmaceutically acceptable salts or of glucose to make the
solution isotonic.
[0107] The composition may be delivered to the respiratory system,
for example to the nose, sinus cavities, sinus membranes or lungs,
in any suitable manner, such as by inhalation via the mouth or
intranasally. The composition may be dispensed as a powdered or
liquid nasal spray, suspension, nose drops, a gel or ointment,
through a tube or catheter, by syringe, by packtail, by pledget, or
by submucosal infusion. The composition may be conveniently
delivered in the form of an aerosol spray using a pressurized pack
or a nebulizer and a suitable propellant, e.g., without limitation,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be controlled by providing
a valve to deliver a metered amount. Capsules and cartridges of,
for example, gelatin for use in an inhaler or insufflator may be
formulated containing a powder mix of the composition and a
suitable powder base such as lactose or starch. Examples of
intranasal formulations and methods of administration can be found
in PCT publications WO 01/41782, WO 00/33813, and U.S. Pat. Nos.
6,180,603; 6,313,093; and 5,624,898, all of which are incorporated
herein by reference and for all purposes. A propellant for an
aerosol formulation may include compressed air, nitrogen, carbon
dioxide, or a hydrocarbon based low boiling solvent. The
composition of the invention may be conveniently delivered in the
form of an aerosol spray presentation from a nebulizer or the like.
In some aspects, the active ingredients are suitably micronized so
as to permit inhalation of substantially all of the active
ingredients into the lungs upon administration of the dry powder
formulation, thus the active ingredients will have a particle size
of less than 100 microns, desirably less than 20 microns, and
preferably in the range 1 to 10 microns. In one embodiment, the
composition is packaged into a device that can deliver a
predetermined, and generally effective, amount of the composition
via inhalation, for example a nasal spray or inhaler.
Pharmaceutical Formulations
[0108] The compositions of this invention may be formulated with
conventional carriers and excipients, which will be selected in
accord with ordinary practice. Aqueous formulations are prepared in
sterile form, and when intended for delivery by other than oral
administration, will generally be isotonic. All formulations will
optionally contain excipients such as those set forth in the
Handbook of Pharmaceutical Excipients (1986). Excipients include
ascorbic acid and other antioxidants, chelating agents such as
EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose,
hydroxyalkylmethylcellulose, stearic acid and the like. The pH of
the formulations ranges from about 3 to about 11, but is ordinarily
about 7 to 10.
[0109] While it is possible for the active ingredients to be
administered alone they may be present as pharmaceutical
formulations. The formulations, both for veterinary and for human
use, of the invention comprise at least one active ingredient, as
above defined, together with one or more acceptable carriers
therefor and optionally other therapeutic ingredients. The
carrier(s) must be "acceptable" in the sense of being compatible
with the other ingredients of the formulation and physiologically
innocuous to the recipient thereof.
[0110] The formulations include those suitable for the foregoing
administration routes. The formulations may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well known in the art of pharmacy. Techniques and
formulations generally are found in Remington's Pharmaceutical
Sciences (Mack Publishing Co., Easton, Pa.). Such methods include
the step of bringing into association the active ingredient with
the carrier which constitutes one or more accessory ingredients. In
general the formulations are prepared by uniformly and intimately
bringing into association the active ingredient with liquid
carriers or finely divided solid carriers or both, and then, if
necessary, shaping the product.
[0111] Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets or tablets each containing a predetermined amount of the
active ingredient; as a powder or granules; as a solution or a
suspension in an aqueous or non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The
active ingredient may also be administered as a bolus, electuary or
paste.
[0112] Pharmaceutical formulations according to the present
invention may include one or more pharmaceutically acceptable
carriers or excipients and optionally other therapeutic agents.
Pharmaceutical formulations containing the active ingredient may be
in any form suitable for the intended method of administration.
When used for oral use for example, tablets, troches, lozenges,
aqueous or oil suspensions, dispersible powders or granules,
emulsions, hard or soft capsules, syrups or elixirs may be
prepared. Compositions intended for oral use may be prepared
according to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions may contain one
or more agents including sweetening agents, flavoring agents,
coloring agents and preserving agents, in order to provide a
palatable preparation.
[0113] Formulations for oral use may be also presented as hard
gelatin capsules where the active ingredient is mixed with an inert
solid diluent, for example calcium phosphate or kaolin, or as soft
gelatin capsules wherein the active ingredient is mixed with water
or an oil medium, such as peanut oil, liquid paraffin or olive
oil.
[0114] Aqueous suspensions of the invention contain the active
materials in admixture with excipients suitable for the manufacture
of aqueous suspensions. Such excipients include a suspending agent,
such as sodium carboxymethylcellulose, methylcellulose,
hydroxypropyl methylcelluose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid polyoxyethylene stearate), a condensation product of ethylene
oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethyleneoxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous
suspension may also contain one or more preservatives such as ethyl
or n-propyl p-hydroxybenzoate, one or more coloring agents, one or
more flavoring agents and one or more sweetening agents, such as
sucrose or saccharin.
[0115] Oil suspensions may be formulated by suspending the active
ingredient in a vegetable oil, such as arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oral suspensions may contain a thickening agent, such
as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such
as those set forth above, and flavoring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an antioxidant such as ascorbic
acid.
[0116] The amount of active ingredient that may be combined with
the carrier material to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. For example, a time-release formulation intended
for oral administration to humans may contain approximately 1 to
1000 mg of active material compounded with an appropriate and
convenient amount of carrier material which may vary from about 5
to about 95% of the total compositions (weight:weight). The
pharmaceutical composition can be prepared to provide easily
measurable amounts for administration. For example, an aqueous
solution intended for intravenous infusion may contain from about 3
to 500 .mu.g of the active ingredient per milliliter of solution in
order that infusion of a suitable volume at a rate of about 30
mL/hr can occur.
[0117] Formulations suitable for intrapulmonary or nasal
administration may have a particle size for example in the range of
0.1 to 500 microns (including particle sizes in a range between 0.1
and 500 microns in increments microns such as 0.5, 1, 30 microns,
35 microns, etc.), which is administered by rapid inhalation
through the nasal passage or by inhalation through the mouth so as
to reach the alveolar sacs. Suitable formulations include aqueous
or oily solutions of the active ingredient. Formulations suitable
for aerosol or dry powder administration may be prepared according
to conventional methods and may be delivered with other therapeutic
agents such as compounds heretofore used in the treatment or
prophylaxis of a given condition.
[0118] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents.
[0119] The formulations may be presented in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water for
injection, immediately prior to use. Extemporaneous injection
solutions and suspensions are prepared from sterile powders,
granules and tablets of the kind previously described. Exemplary
unit dosage formulations are those containing a daily dose or unit
daily sub-dose, as herein above recited, or an appropriate fraction
thereof, of the active ingredient.
[0120] It should be understood that in addition to the ingredients
particularly mentioned above the formulations of this invention may
include other agents conventional in the art having regard to the
type of formulation in question, for example those suitable for
oral administration may include flavoring agents.
[0121] The invention further provides veterinary compositions
comprising at least one active ingredient as above defined together
with a veterinary carrier therefor.
[0122] Veterinary carriers are materials useful for the purpose of
administering the composition and may be solid, liquid or gaseous
materials that are otherwise inert or acceptable in the veterinary
art and are compatible with the active ingredient. These veterinary
compositions may be administered orally, parenterally or by any
other desired route.
[0123] Compounds of the invention can also be formulated to provide
controlled release of the active ingredient to allow less frequent
dosing or to improve the pharmacokinetic or toxicity profile of the
active ingredient. Accordingly, the invention also provided
compositions comprising one or more compounds of the invention
formulated for sustained or controlled release.
[0124] An effective dose of an active ingredient depends at least
on the nature of the condition being treated, toxicity, whether the
active ingredient is being used prophylactically (e.g., lower doses
may be employed), the method of delivery, and the pharmaceutical
formulation, and will be determined by the clinician using
conventional dose escalation studies. It can be expected to be from
about 0.0001 to about 100 mg/kg body weight per day. Typically,
from about 0.01 to about 10 mg/kg body weight per day, including
from about 0.01 to about 5 mg/kg body weight per day, or from about
0.05 to about 0.5 mg/kg body weight per day. For example, the daily
candidate dose for an adult human of approximately 70 kg body
weight will range from 1 mg to 1000 mg, e.g., from 5 mg to 500 mg,
and may take the form of single or multiple doses. For instance,
about 5 mg to about 750 mg, e.g., about 10 mg to about 70 mg, or
any integer in between, of the active ingredient may be
administered to a human.
[0125] The invention will be further described by the following
non-limiting examples.
EXAMPLE I
Example I
Separation of Compounds from 90MX Cranberry Powder
[0126] Stage I Separation: C18 Solid Phase Extraction. Ocean Spray
cranberry powder, 90MX was reconstituted in water to cranberry
juice and filtered through a 0.45 .mu.m membrane. The filtered
juice was loaded on a C18 solid phase extraction column
(Extract-Clean brand, standard C18, 50 .mu.m particle size, 60
angstrom pore size, Grace Davidson Discovery Sciences). The column
was then washed with water (to yield fraction F0) followed by
elution with 15% methanol (F1), 25% methanol (F2), and 100%
methanol (F3). Fraction F0 was evaporated yielding a white powder
that was about 94% of the total mass and was inactive in the
vaccinia anti-viral assay. Fractions F1, F2, and F3 were evaporated
yielding red powders, which accounted for about 6% of the total
mass of the 90MX powder. FIG. 3 shows analytical HPLC chromatograms
for fractions F1, F2, and F3 detected by UV at 340 nm. Fractions F2
and F3 had strong antiviral activity.
[0127] Stage 2 Separation: Strong Cation Exchange Chromatography.
Fractions F2 and F3 were each dissolved in a solution of methanol
and water and loaded onto a strong cation exchange column (Redi-Sep
Rf SCX brand, silica based, 40-63 .mu.m particle size, 100 angstrom
pore size, Isco-Teledyne) and eluted with a mixture of methanol and
water. Cationic anthocyanin components were retained, and the
eluent gave fractions F2a and F3a, respectively. Elution was
monitored by UV and proceeded until the signal at 340 nm had
decreased to zero. These fractions had strong antiviral activity.
FIG. 4 compares analytical HPLC chromatograms for fractions F2 and
F2a (before and after stage 2 separation), using diode array UV
detection from 250-600 nm. Anthocyanin fractions could be eluted
from the columns using a solution of water, methanol, hydrochloric
acid and sodium chloride, giving fractions F2b and F3b,
respectively. These fractions contained red anthocyanin pigments
and lacked antiviral activity.
[0128] Stage 3 Separation: Preparative Scale High Performance
Liquid Chromatography. To further separate components in fraction
F2a, prep HPLC was employed. The column used was a Waters Symmetry
brand, C8 phase, with 7 .mu.m particle size, and dimensions of
19.times.150 mm, operated at a flow rate of 12 mL/min. A binary
solvent gradient was used for this HPLC separation with solvent
A=water+0.1% formic acid and solvent B=methanol+0.1% formic acid.
Solvent composition at various times was as follows, time (percent
B): initial (10% B), 50 min. 80 min. (60% B), 85 min. (80% B), 90
min. (80% B). Individual runs contained about 40 mg of dry F2a
fraction dissolved in 1:1 water/methanol to a volume of 1 mL. The
progress of the separation was followed using UV detection at 255
and 340 nm. Eluent was collected in tubes at 2 minute intervals and
the contents of these tubes was pooled according to their UV
profile to yield fractions F2a1, F2a2, F2a3, F2a4, and F2a5.
Evaporation of the solvent from each of these fractions yielded dry
powders. FIG. 5 shows overlaid analytical HPLC chromatograms for
fractions F2a2, F2a4, and F2a5 detected by diode array UV from
250-600 nm.
[0129] Stage 4 Separation: Analytical Scale High Performance Liquid
Chromatography. As a final purification step, fractions F2a2 and
F2a3 were separated by analytical HPLC. For F2a2, the following
column was used: Phenomenex Luna brand pentafluorophenyl 2 phase
(PFP 2) of dimensions 4.6.times.150 mm, 5 .mu.m particle size, and
operated at a flow rate of 0.5 mL/min. For F2a3, the following
column was used: Waters Symmetry brand C8 phase of dimensions
3.9.times.150 mm, 5 .mu.m particle size, operated at a flow rate of
0.5 mL/min. In both cases the same binary solvent gradient was used
for HPLC separation with solvent A=water+0.1% formic acid and
solvent B=methanol+0.1% formic acid. Solvent composition at various
times was as follows, time (percent B): initial (10% B), 50 min.
(35% B), 80 min. (60% B), 85 min. (80% B), 90 min. (80% B). FIGS.
6A and 6B show analytical RPLC chromatograms for fractions F2a2
detected by UV at 277 nm and F2a3 detected by UV at 356 nm,
respectively.
[0130] Antiviral Results for F2a2 aNd F2a3 Against Vaccinia
Virus:
[0131] FIG. 7 shows the anti-vaccinia -virus activities of
compounds F2a2 and F2a3 at solution concentrations ranging from 100
mg/L to 0.1 mg/L. Activity is reported as a percentage vaccinia
infection inhibition in treated cells compared to untreated
controls. Both materials completely inhibited vaccinia at
concentrations of 10 mg/L and higher. At the more dilute
concentration of 1 mg/L, both compounds retained greater than 90%
inhibition potency. At the even more dilute concentration of 0.1
mg/L activity dropped off to 45% and 6% for F2a2 and F2a3,
respectively.
[0132] Antiviral Results for F2a2 and F2a3 Against 100 TCID50
Poliovirus 1:
[0133] FIG. 8 shows the anti-poliovirus 1 activities of compounds
F2a2 and F2a3 at solution concentrations ranging from 100 mg/L to
0.1 mg/L. Activity is reported as a percentage poliovirus 1
infection inhibition in treated cells compared to untreated
controls. Both materials completely inhibited vaccinia at
concentrations of 10 mg/L and higher. At the more dilute
concentration of 1 mg/L, both compounds retained greater than 90%
inhibition potency. At the even more dilute concentration of 0.1
mg/L activity dropped off to 45% and 6% for F2a2 and F2a3,
respectively.
[0134] Antiviral results for F2a2 and F2a3 against 10 TCID50
influenza A/H1N1: FIG. 9 shows the anti-influenza A/H1N1 activities
of compounds F2a2 and F2a3 at solution concentrations ranging from
100 mg/L to 0.1 mg/L. Activity is reported as a percentage
influenza A/H1N1 infection inhibition in treated cells compared to
untreated controls. Both materials completely inhibited vaccinia at
concentrations of 10 mg/L and higher. At the more dilute
concentration of 1 mg/L, F2a3 maintained 100% inhibition while the
activity of F2a2 fell to 50%. Both compounds were inactive against
influenza A/H1N1 at the more dilute concentration of 0.1 mg/L.
EXAMPLE II
Compounds from Cranberry Seed Powder
[0135] Separation of cranberry seed powder: FIG. 2 shows a
separation protocol that was employed with a cranberry seed powder.
Botanic Oil Innovations Cranberry Seed Nutri--Powder was extracted
according to the following protocol. The powder (5 g) was mixed
with 70% methanol/30% water solution (100 mL) for 30 minutes. Then
Celite (1 g) was stirred in and the mixture was vacuum filtered
through a pad of Celite. Solvents were removed under vacuum to give
a red-brown powder (0.74 g). This material was separated using
preparative HPLC using the following column: Waters Symmetry brand,
C8 phase, with 7 .mu.m particle size, and dimensions of
19.times.150 mm, operated at a flow rate of 12 mL/min. A binary
solvent gradient was used for this HPLC separation with solvent
A=water+0.1% formic acid and solvent B=methanol+0.1% formic acid.
Solvent composition at various times was as follows, time (percent
B): initial (10% B), 50 min, (35% B), 80 min. (60% B), 85 min. (80%
B), 90 min. (80% B). Individual runs contained about 40 mg of dry
seed extract dissolved as a solution of water/methanol to a volume
of 1 mL. The progress of the separation was followed using UV
detector at 255 and 340 nm. Eluent was collected in tubes at 2
minute intervals and the contents of these tubes was pooled
according to their UV profile to yield fractions labeled as E0-E3.
Analytical HPLC chromatograms of fractions E1, E2, and E3 are
overlaid in FIG. 10.
[0136] Antiviral activity of fractions E0, E1, E2 and E3 against
vaccinia virus: FIG. 11 shows the anti-vaccinia virus activities of
fractions E0, E1, E2 and E3 at solution concentrations ranging from
100 mg/L to 0.1 mg/L. Activity is reported as a percentage vaccinia
infection inhibition in treated cells compared to untreated
controls. Fraction E2 was the most active, followed by E3 and then
E1. Fraction E0 was inactive at all concentrations. Fractions E2
and E3 both inhibited vaccinia at 100% at a concentration of 100
trig/mL, while E1 inhibited vaccinia at 59% at this concentration.
At the more dilute concentration of 10 mg/L, fractions E2 and E3
exhibited 100% and 76% vaccina inhibition, respectively, while E1
was essentially inactive at this concentration. Upon further
dilution to a concentration of 1 mg/L, E2 maintained 88% vaccinia
inhibition while E3 was essentially inactive at this concentration.
At a concentration of 0.1 mg/L the anti-vaccinia activity of E2 was
43% inhibition.
TABLE-US-00001 TABLE 1 Anti-viral Drug Candidate Compounds Starting
Material Name of Compound Characteristics F2a3 Ocean Spray Crude
See FIG. 1. Identified to be myricetin .beta.-3 90MX Powder
galactoside using proton nuclear magnetic resonance spectroscopy
(.sup.1H NMR), MALDI-TOF mass spectrometry. Water soluble, stable,
flavonol. Absorbs at 340 nm. It has anti-viral activity against
vaccinia virus. One structural analog,
quercetin-.beta.-3-galactoside (which has the same sugar pendent
group, but a different core) was found to be inactive. F2a2* Ocean
Spray Crude See FIG. 1. Isolated and purified. It is water 90MX
Powder soluble. It is not a flavonol and absorbs at 277 nm. It is
stable and has strong anti-viral activity against vaccinia virus.
E2 Botanic Oil This material is very easy to purify because it is
Innovations Pressed extremely stable, polar and water-soluble (FIG.
Cranberry Seed 2). It quickly flows through the first extraction
Powder C18 column. It has strong anti-viral activity against
vaccinia virus. *Preliminary data suggests it may be the strongest
inhibitor of the three.
[0137] All of the compounds described in Table 1 are water-soluble
making them easy to purify. They are all very stable compounds,
e.g., stable during a drying procedure, e.g., rotavapping at
40.degree. C. (body temperature is 37.degree. C.) and stable at
room temperature in powder form. These small molecule compounds are
not susceptible to protease activity. No added chemicals are
required to solubilize them for mass production or therapeutic
applications. The compounds became more potent at each purification
step.
EXAMPLE III
[0138] SMBC technology allows complex mixtures such as cranberry
juice to be simplified into two fractions with each separation run,
which are defined as "raffinate" and "extract" fractions, and may
increase productivity up to 2.0 fold and reduce costs, e.g., up to
90%, relative to other methods. There are a number of parameters
that are optimized for each mixture separated in the process
including mobile phase composition, flow rates and valve switching
times. When adjusted, the raffinate and extract fractions each
contain roughly half of the compounds in the original mixture.
These fractions are then tested for anti-microbial activity, e,g.,
in plaque reduction assays against vaccinia virus. Fractions that
display activity are then separated via a second round of SMBC
chromatography with a new chromatographic method with modified
parameters, which will produce two new fractions. Each of these
fractions are in turn tested for activity, and the process repeated
in an iterative fashion until pure active compounds have been
isolated. Using this strategy a very complex mixture can be
separated in a minimal number of chromatographic cycles. It is
estimated each pure active compound may be produced in as few as
eight cycles. Each chromatographic cycle, including development
time, separation run time, solvent removal, and associated
biological testing, may be completed in about 1 to 2 weeks. Once
lead compounds have been thoroughly purified through
chromatography, their chemical structures are determined by proton
and carbon NMR spectroscopy, infrared spectroscopy, mass
spectrometry, and elemental analyses.
C8 Column Runs to Separate Fraction F2a
[0139] F2 was dissolved in 50% H.sub.2O/MeOH+0.1% formic acid at 10
.mu.g/mL and 0.5 mL was injected onto a C8 column at a flowrate of
5 mL/min. Isocratic runs were done at 40%, 45%, 50% and 55% MeOH
and output was monitored at 255 nm (red) and 52.0 nm (blue). The
peaks in the blue trace were from anthocyanins. The 50% and 55%
MeOH runs looked the same and 50% MeOH was used for subsequent
runs.
[0140] The Octave 100 was set to run in SMBC mode, with each of the
systems 8 C8 88.2 mL volume columns connected to the valve block
and flushed with the mobile phase. Pump 1 was designated as the
feed pump. Pump 2 was the desorbant pump, moving the elution
solvent 50/50 H.sub.2O/MeOH with 0.1% formic acid. Pump 3 was not
used, and pump 4 was attached to outlet "E" to meter the extract
flow rate. A script was developed using an online calculator to
optimize flow rates in the different zones. The following
parameters were entered into the 3-zone calculator.
Column Measurements
[0141] Column Volume ml, 19.6 "a" [0142] Flow Rate for t.sub.0,
ml/min, 5.00 "b" [0143] t.sub.0 min., 1.60 "a" [0144] Void Fraction
of Adsorbent, 0.40 "a" [0145] Fast Peak 1 t.sub.R min, 3.10 "b"
[0146] Slow Peak 2 t.sub.R min, 5.60 "b"
Henry Constants
[0146] [0147] H1 is 0.65 [0148] H2 is 1.72 [0149] Selectivity is
2.66
SMBC System Measurements
[0149] [0150] Column Volume ml, 88.2 "a" [0151] Extra Column Volume
ml, 3.90 "a" [0152] Switch Time seconds, 600 "c" [0153] Q.sub.feed
ml/min, 5 "c" [0154] Q.sub.desorbent ml/min, 18 c [0155]
Q.sub.extract ml/min, 10 [0156] Q.sub.raffinate ml/min, 13.00
[0157] Q.sub.1 ml/min, 18.00 [0158] Q.sub.2 ml/min, 8.00 [0159]
Q.sub.3 ml/min, 13.00 [0160] m.sub.1, 2.68 [0161] m.sub.2, 0.76
[0162] m.sub.3, 1.72 [0163] Mass Balance ml/min, 23.00 [0164]
m1>H2>m3>m2>H1 [0165] a=constants [0166] b=conditions
determined, first experiment [0167] c=conditions varied in the
calculator. Q.sub.feed and Q.sub.desorb are solvent inputs,
Q.sub.extract and Q.sub.raffinate are solvent outputs, and
Q.sub.feed+Q.sub.desorb=Q.sub.extract+Q.sub.raffinate [0168]
Q.sub.feed usually about 1/4 to 1/5 of Q.sub.desorbent [0169] If
Q.sub.desorb is decreased, switch time usually needs to be
increased [0170] Some combinations don't work in practice due to
back pressure limitations. [0171] These include [0172] The pressure
at any point can't exceed 295 psi. Pressure is usually greatest
within the loop of columns at the desorbant pump. [0173] The
pressure at the end of the extract line must exceed the pressure at
the desorbant pump by about 40 psi or more, or the flowrates out of
raffinate and extract lines may not be correct. This can be
achieved by placing a back pressure regulator after the extract, to
increase pressure at pump 4 (extract) vs. pump 2 (desorbant).
[0174] The configuration that was used placed a 250 psi BPR on the
extract output and a 40 psi BPR on the raffinate output. The
raffinate BPR may have been optimal, but helped maintain at least
some pressure on the feed pump, which otherwise operated near 0
psi. They also helped keep gas bubbles out of the detector flow
cells that were hooked to raffinate and extract lines.
[0175] The raffinate stream was monitored by UV at 520 nm and the
extract stream monitored at 255 nm. The script used was a standard
3-2-3 isocratic SMBC script with specific flow rates for this
system substituted in. After step 1 in an eight step script,
subsequent steps move all inlets and outlets one column position to
the right. The feed solution for this separation was F2 dissolved
in 50/50 H.sub.2O/MeOH+0.1% formic acid at 10 mg/.mu.L.
[0176] Pressures while operating this script. [0177] Pump 2=205 psi
[0178] Pump 1=23 psi [0179] Pump 4=247 psi
[0180] Column switching occurred every 600 sec. At column switches
the extract signal was maximal and decreased over time as that
batch moves past the extract outlet. The raffinate came out bright
red while the extract was pale yellow. Solvent was removed from
each solution by rotovapping and then lyophilization. This worked
well for the raffinate, which gave a red solid. HPLC results at 340
nm and 521 nm, as well as spectral plots, showed two strongly
absorbing bands between 22-34 minutes at 521 nm indicating
anthrocyanins.
EXAMPLE IV
[0181] Antiviral testing of F2a2 and F2a3 against human
parainfluenza 3 virus (HPIV3) (FIG. 15) and influenza A/H3N2 (FIG.
16) was conducted. Influenza A/Sydney/05/97(H3N2) was used as a
representative H3N2 strain. For F2a2 and HPIV3 (100 TCID.sub.50),
there was 100% inhibition at 100 mg/L, 75% inhibition at 10 mg/L,
and no inhibition 1 mg/L and 0.1 mg/L. For F2a3 and HPIV3 (100
TCID.sub.50), there was 100% inhibition at 100 mg/L, 75% inhibition
at 10 mg/L, and no inhibition 1 mg/L and 0.1 mg/L. For F2a2 with
H3N2 (100 TCID.sub.50), there was 100% inhibition at 100 mg/L, 33%
inhibition at 10 mg/L, and no inhibition 1 mg/L and 0.1 mg/L. For
F2a3 with H3N2 (100 TCID.sub.50), there was 100% inhibition at 100
mg/L, and there was no inhibition 10 mg/L, 1 mg/L and 0.1 mg/L.
EXAMPLE V
[0182] Characterization of F2a2 and F2a3 by UPLC-MS
[0183] In an attempt to identify the molecular source(s) of the
anti-viral activity observed in cranberry juice extract fractions
F2a2 and F2a3, these fractions were analyzed by Ultra High
Performance Liquid Chromatography (UPLC)-Quadrupole/Time Of Flight
(Q-TOF) mass spectrometry (MS) or UPLC/Q-TOF MS for short. Diode
array UV detection was also used in addition to MS.
UPLC Experimental Conditions
TABLE-US-00002 [0184] Column: Waters Acquity UPLC BEH C18 Gradient
Method 2.1 .times. 100 mm 1.7 .mu.m @40.degree. C. Time A % B % 5
.mu.L Injections 0.0 min 95 5 Gradient LC Method: 12.0 min 50 50
Flow = 0.45 mL/min 14.0 min 5 95 15.0 min 5 95 15.2 min 95 5 17.2
min 95 5 A = 0.1% Acetic Add in Water B = Acetonitrile Purge
Solution = 90/10 Water/Methanol Wash Solution = 90/10
Methanol/Water
MS Experimental Conditions
[0185] Positive and Negative Ion Electrospray
[0186] MS Spectra from m/z=100 to 1200
[0187] Capillary Voltage=2 kV
[0188] Cone Voltage=30 V
[0189] Collision Energy=15-30 eV (Negative Ion)
[0190] Collision Energy=15-35 eV (Positive Ion)
[0191] (Ramp used in MS E Analyses)
[0192] Source=120.degree. C.
[0193] Desolvation Temperature=450.degree. C.
[0194] Desolvation Gas Flow=800 L/hr
[0195] Nebulizer Gas Pressure=6.0 Bar
[0196] Cone Gas Flow=100 L/hr
Analysis of F2a2
[0197] UPLC chromatograms of fraction F2a2 using positive ion MS
detection (red), negative ion MS detection (green), and UV
detection (purple) are shown in FIGS. 17A-C, respectively. In all
cases, two major peaks were observed at 3.68 min. and 4.22 min. The
negative ion and UV traces also showed some additional minor
components. These are highlighted in FIGS. 17D-F, which is blowup
of the baseline regions in each chromatogram. There are two main
components of fraction F2a2 and one or both of these could be a
source of the anti-viral activity observed in the material. It is
also possible that one or more of the minor components of the
material could be responsible for the antiviral activity. It is
also possible that this particular combination of compounds is
required for activity.
[0198] The molecular formulas of the peaks at 3.68 min. and 4.22
min. were determined by accurate mass analysis of molecular ion
peaks. Both peaks were found to have molecular formula
C.sub.30H.sub.24O.sub.12 and have an average molecular weight of
576.50 amu. Hence, they are isomers of one another. Molecular
fragmentation analysis was carried out on each of the two major
components using tandem mass spectrometry. This data, combined with
the molecular formula data gave structures for the peaks at 3.68
min. and 4.22 min., which are consistent with molecules from the
proanthocyanidin A class of compounds. Examples of specific members
of this class include proanthocyanidin A2 (also known as
procyanidin A2) and proanthocyanidin A5' (also known as procyanidin
A5'). Structures for these molecules are shown in FIG. 17G.
[0199] A general structure for the proanthocyanidin A class is
shown below.
##STR00005##
[0200] Members of the proanthocyanidin A class contain five
stereogenic carbon atoms indicated by stars (*) in the figure
above. Each stereogenic atom could be of either (R)- or
(S)-configuration, leading to 2.sup.5=32 possible stereoisomers
with varying combinations of (R)- and (S)-configurations at the
starred atoms Those having common names are listed as follows, with
Chemical Abstract Services (CAS) registry numbers in brackets and
include proanthocyanidin A1 [103883-03-0], proanthocyanidin A2
[41743-41-3], proanthocyanidin A4 [111466-29-6], proanthocyanidin
A5' [111466-30-9], proanthocyanidin A6 [114569-31-2], and
proanthocyanidin A7 [114569-32-3], the structures of which are
incorporated by reference herein Several other stereoisomers in the
proanthocyanidin A class that do not have common names have also
been described and are identified by their CAS numbers as
[159247-90-2], [159247-88-8], [159247-85-5], [159247-89-9],
[157086-14-1], [130853-74-6], [159247-85-5], and [135095-75-9], the
structures of which are incorporated by reference herein. There are
two known structural isomers of the proanthocyanidin A class that
are consistent in both their molecular formula and in their
fragmentation patterns with the compounds in fraction F2a2. These
include the molecule pavetanin A [132651-90-2] and the compound
with CAS number [201302-84-3], the structures of which are
incorporated by reference herein.
Analysis of F2a3
[0201] UPLC chromatograms of fraction F2a3 using positive ion MS
detection (purple), negative ion MS detection (green), and UV
detection (red) are shown in FIGS. 18A-C, respectively. Two major
peaks were observed at 3.38 min. and 3.97 min. in all
chromatograms. Minor peaks at 4.44 min. and 4.67 min. were also
observed. These are highlighted in FIGS. 18D-F, which is blowup of
the baseline regions in each chromatogram. It is also possible that
any one or more of the major or minor components of the material
could be responsible for the anti-viral activity. It is also
possible that this particular combination of compounds is required
for activity.
[0202] The molecular formulas of the peaks at 3.38 min., 3.97 min.,
4.44 min., and 4.67 min. were determined by accurate mass analysis
of molecular ion peaks. Additionally, molecular fragmentation
analysis was carried out on each component using tandem mass
spectrometry. This data, combined with the molecular formula data
gave structures for the peaks at 3.38 min., 3.97 min., and 4.67
min., which are listed in FIG. 18C. The compound at 4.44 min had a
molecular weight of 565.2287 amu. The best formula fit for this was
C.sub.28H.sub.37O.sub.12.
[0203] Commercially available preparations of
myricetin-beta-3-galactoside, procyanidin A2, sinapinic acid and
triptolide were tested at various concentrations against vaccinia
virus, herpes simplex virus-1, poliovirus Sabin-1, parainfluenza
virus-3, and influenza A/California/07/2009 (H1N1). None of the
compounds showed anti-viral activity at the concentrations tested
(100 ng/mL-100 and triptolide was toxic at 1-100 .mu.g/mL).
[0204] The most time-consuming aspect of preparing fractions F2a2
and F2a3 is removing water solvent after chromatography
separations. This has been done under reduced pressure with mild
heating at 40.degree. C. The process is significantly faster when
heating at higher temperatures. However, it was unclear that the
active compounds would be stable at higher temperatures. Therefore,
a temperature stability study was undertaken comparing the anti
vaccinia virus activities of materials prepared by evaporation at
40.degree. C., 60.degree. C., and 100.degree. C. Materials were
tested at stage one and two of the separation, leading to fractions
F2 and F2a, respectively. The last step in the separation generates
both F2a2 and F2a3.
[0205] Three samples of cranberry juice fractions F2 and F2a were
prepared. Sample one was prepared using a rotary evaporation
temperature of temperature of 40.degree. C. throughout the
procedure. The second sample was prepared using a rotary
evaporation temperature of temperature of 60.degree. C. throughout
the procedure, and a third sample was prepared using a rotary
evaporation temperature of temperature of 100.degree. C. throughout
the procedure. The samples were tested fir stability of anti-viral
activity against vaccinia virus (FIG. 19). The results are
essentially identical for all three temperature points. This
suggests that higher temperatures may be used during solvent
evaporation without impacting antiviral activity.
EXAMPLE V
Characterization of Cranberry Seed Powder
[0206] To determine if cranberry seed extract can serve as an
effective inhibitor when used to treat cells at various times after
infection with vaccinia virus, vaccinia virus was added to all test
wells of BS-C-1 cells and incubated tier one hour at 37.degree. C.,
and then inhibitor was added to duplicate wells at 0 minutes, 30
minutes, one hour, and two hours post infection. The results (FIG.
20A) show that the inhibition is decreased when the inhibitor is
added at times well after infection.
[0207] To determine if cranberry seed extract is a more or less
effective inhibitor when cells are pretreated with it for various
times prior to infection with vaccinia virus, 10 mg/L and 100 mg/L
of the cranberry seed extracts were added to the test wells of
BS-C-1 cells, and then virus was added to duplicate wells of each
extract concentration after 0 minutes, one hour, two hours, and
three hours. It appears that the most effective inhibition is
obtained when the inhibitor is added within an hour of virus
exposure.
[0208] Alternate extraction protocols were developed and tested for
cranberry seed powder in an attempt to improve extract potency.
Five different protocols were explored. In each, cranberry seed
powder from Botanic Oil Innovations (5.000 g) was placed in a
Soxhlet extraction apparatus. The following solvents were used:
pure methanol, ethanol/water (95:5 v:v), isopropyl alcohol/water
(88:12 v:v), n-propyl alcohol/water (72:28 v:v), and water. The
extractions were run overnight and each solution was concentrated
under reduced pressure, then lyophilized to a tan powder. Yields of
solid extract ranged from 0.98 g (water) to 1.29 g (methanol).
These materials were tested for anti-viral activity against
vaccinia virus and influenza A/H1N1 (influenza
A/California/07/2009).
[0209] All of the materials tested were strong inhibitors of
vaccinia virus (FIG. 21A). However, the material produced by
methanol extraction (labeled MeOH) was the strongest inhibitor, and
the material produced by water extraction (labeled as H.sub.2O) was
the weakest inhibitor.
[0210] For inhibition of 100 TCID.sub.50 of influenza A/H1N1 with
cranberry seed extracts, the methanol-derived material (MeOH) was
by far the most potent inhibitor, followed by the ethanol-derived
material (EtOH), the isopropyl alcohol/water material
(i-PrOH/H.sub.2O) and water materials. Materials derived from
n-propyl alcohol/water or the previous extraction protocol
(JAM1-91) showed no activity against 100 TCID.sub.50 of influenza
A/H1N1.
[0211] The anti-viral potency in inhibition of 10 TCID.sub.50 of
influenza A/H1N1 with cranberry seed extracts followed the same
trend as the 100 TCID.sub.50 results. In this case, the
methanol-derived material inhibited 100% of the virus at all
concentrations tested.
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[0265] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification, this invention has been described in relation to
certain preferred embodiments thereof, and many details have been
set forth for purposes of illustration, it will be apparent to
those skilled in the art that the invention is susceptible to
additional embodiments and that certain of the details herein may
be varied considerably without departing from the basic principles
of the invention.
* * * * *