U.S. patent application number 14/027235 was filed with the patent office on 2014-03-13 for therapeutic composition produced using chinese gall and hydrogen peroxide.
This patent application is currently assigned to LiveLeaf, Inc.. The applicant listed for this patent is LiveLeaf, Inc.. Invention is credited to ALEXANDER L. HUANG, GIN WU.
Application Number | 20140072655 14/027235 |
Document ID | / |
Family ID | 42709967 |
Filed Date | 2014-03-13 |
United States Patent
Application |
20140072655 |
Kind Code |
A1 |
HUANG; ALEXANDER L. ; et
al. |
March 13, 2014 |
THERAPEUTIC COMPOSITION PRODUCED USING CHINESE GALL AND HYDROGEN
PEROXIDE
Abstract
Methods of and compositions for producing and using plant-based
materials are provided. The methods include using biopolymers or
their synthetic equivalents combined with a stable source of
reactive oxygen species that when applied to or combined with a
separate source of oxido-reducing enzyme or catalyst will cause the
formation of an activated biopolymer with increased protein binding
affinity and microbial control activities.
Inventors: |
HUANG; ALEXANDER L.; (Menlo
Park, CA) ; WU; GIN; (San Rafael, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LiveLeaf, Inc. |
San Carlos |
CA |
US |
|
|
Assignee: |
LiveLeaf, Inc.
San Carlos
CA
|
Family ID: |
42709967 |
Appl. No.: |
14/027235 |
Filed: |
September 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13680007 |
Nov 16, 2012 |
8586110 |
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14027235 |
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12715270 |
Mar 1, 2010 |
8343552 |
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13680007 |
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12317638 |
Dec 23, 2008 |
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12715270 |
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61209260 |
Mar 4, 2009 |
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61009484 |
Dec 28, 2007 |
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Current U.S.
Class: |
424/616 |
Current CPC
Class: |
A61K 31/122 20130101;
A61K 36/22 20130101; A61K 38/44 20130101; A01N 59/00 20130101; A61K
33/40 20130101; A61P 31/00 20180101; A61P 31/04 20180101; A23L
33/105 20160801; A61P 29/00 20180101; A61K 36/82 20130101; A01N
65/08 20130101; C07K 1/1072 20130101; A23K 20/111 20160501; A61P
1/12 20180101; A61P 31/12 20180101; A61P 43/00 20180101; A61K 31/05
20130101; A61P 17/02 20180101; C07G 1/00 20130101; A61P 1/00
20180101; A61K 36/185 20130101; Y02A 50/30 20180101; A61K 38/44
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/616 |
International
Class: |
A61K 33/40 20060101
A61K033/40; A61K 31/122 20060101 A61K031/122; A01N 59/00 20060101
A01N059/00; A01N 65/08 20060101 A01N065/08; A61K 36/22 20060101
A61K036/22; A61K 31/05 20060101 A61K031/05 |
Claims
1. An anti-bacterial composition prepared by: a) combining Chinese
gall with water and heating at 80.degree. C. to 150.degree. C. to
substantially denature enzymes endogenous to said pomegranate rind
and b) adding from 1% to less than 10% of hydrogen peroxide to the
product obtained from part (a).
2. The anti-bacterial composition of claim 1, wherein the
composition endogenously contains a component selected from the
group consisting of a tannin, a lignin, a flavonoid, a
hydroxycoumarin and an alkaloid.
3. The anti-bacterial composition of claim 1, wherein said
composition is in a dose ranging from 5 .mu.g to 2500 .mu.g.
4. The anti-bacterial composition of claim 1, wherein the heating
in part (a) is carried out from 80.degree. C. to 110.degree. C.
5. The anti-bacterial composition of claim 1, wherein 1% to 2% of
hydrogen peroxide is added to part (b).
6. The anti-bacterial composition of claim 1, wherein the process
further comprises diluting to a hydrogen peroxide concentration of
25 ppm.
7. A kit comprising a dry form of the anti-bacterial composition of
claim 1.
8. A method for treating a bacterial infection, comprising
administering to a subject in need thereof, an effective amount of
the anti-bacterial composition of claim 1.
9. The method of claim 8, wherein the administering comprises
administering orally in a dose ranging from 5.0 .mu.g to 2500 .mu.g
per day based on dry weight equivalent.
10. A method of treating bacteria-infected water, comprising
administering to said water an effective amount of the
anti-bacterial composition of claim 1.
11. A method of treating or preventing a bacterial infection in a
wounded tissue, comprising administering to said tissue, an
effective amount of the anti-bacterial composition of claim 1.
12. A method for producing an anti-bacterial composition, wherein
the method comprises; a) combining Chinese gall with water and
heating at 80.degree. C. to 150.degree. C. to substantially
denature enzymes endogenous to said pomegranate rind and b) adding
from 1% to less than 10% of hydrogen peroxide to the product
obtained from part (a).
13. The method of claim 12, wherein said heating in part (a) is
carried out from 80.degree. C. to 110.degree. C.
14. The method of claim 12, wherein 1% to 2% of hydrogen peroxide
is added in part (b).
15. The method of claim 12, The anti-bacterial composition of claim
1, further comprising diluting to a hydrogen peroxide concentration
of 25 ppm.
16. An anti-bacterial composition prepared by: a) obtaining a water
soluble extract of Chinese gall having at least substantially no
active endogeneous enzymes; b) combining the water soluble extract
from part (a) with water to create a solution; c) adding hydrogen
peroxide to the solution from part (b) at a concentration of less
than 10%; and,
17. The anti-bacterial composition of claim 16, further comprising
diluting the solution to a hydrogen peroxide concentration of 25
ppm.
18. A dosage form of an anti-bacterial solution comprising at least
5 ug of a water soluble extract of Chinese gall and at least 25 ppm
hydrogen peroxide.
19. The dosage form of claim 18 in the form of an aqueous
solution.
20. The dosage form of claim 18 in a dry form.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/680,007, filed Nov. 16, 2012, which is a continuation of
U.S. application Ser. No. 12/715,270, filed Mar. 1, 2010, now U.S.
Pat. No. 8,343,552, which claims the benefit of U.S. Provisional
Application No. 61/209,260, filed Mar. 4, 2009, and is a
continuation-in-part of U.S. application Ser. No. 12/317,638, filed
Dec. 23, 2008, which claims the benefit of U.S. Provisional
Application No. 61/009,484, filed Dec. 28, 2007; each application
of which is hereby incorporated herein by reference in its
entirety.
FIELD OF INVENTION
[0002] The present invention generally relates to controlled
enhancement of protein binding affinity of biomaterials. More in
particular, the invention relates to stabilization of and
controlled activation of plant biopolymers by enzymes of animals,
plants, bacteria, and/or other catalysts to cause locally enhanced
oxidation and/or cross-linking of proteins, micro-organisms and
biologic tissues.
BACKGROUND OF THE INVENTION
[0003] Despite hundreds of millions of years of divergent
evolution, almost all plants, animals and pathogens share some
common biochemical fundamentals and strategies for environmental
defense. This makes botany a rich source of useful and compatible
compounds for the control of pathogens in animals. The use of
functional biochemistry from the plants has long been the basis for
traditional and herbal medicines and often considered less likely
to trigger unwanted immunological responses between less
genetically distant species within the same phyla than highly
purified large complicated proteins or polymers.
[0004] Immune systems of most of the higher organisms protect from
infection with defenses of increasing specificity. The simplest is
a physical barrier that prevents pathogens, such as bacteria and
viruses, from entering the organism. Plants and animals also have
innate immune systems that are either genetically coded responses
to specific pathogens, or various non-specific responses to
pathogen chemistries.
[0005] Plants typically have a two branched immune system. The
first recognizes and responds to molecules common to many classes
of microbes, including non-pathogens, by increased expression of
ROS (Reactive Oxygen Species) generating enzymes capable of
initiating oxidative bursts, but such direct oxidative response is
energy costly and must be strictly regulated to prevent
autotoxicity. Many pathogenic microorganisms (bacteria, fungi,
protozoa) are equipped with peroxidases or catalases as
countermeasures against such ROS bursts. The second branch of the
innate immune system is the multi-component wound response as
described above initiated by the reaction between quinonic
compounds and amino acids when cells are damaged. These compounds
are usually compartmentally separated and do not cooperate in
living systems. In plants, cellular disruption causes various
phenol compounds and reactive oxygen species to come into contact
with polyphenol oxidases (PPO), oxidizing the phenol compounds to
form quinonic compounds that aggressively associate with each other
and amino acids of the cells or any microorganisms present. This
effects many physiologic phenomena, such as browning or discoloring
of foods, precipitation of proteins, germicidal activity,
astringency, changes in food digestibility and more.
[0006] Polyphenol oxidation in plant systems generates
oxidized-polyphenols (also referred to as o-polyphenols, oxidized
biopolymers, polyquinones and quinonic compounds) with a
multiplicity of quinonic groups that are capable of covalent
bonding. Once formed, the high affinity o-polyphenols spontaneously
form covalent intra- and inter-chain cross-links that condense
proteins far more aggressively than hydrogen bonds characteristic
of non-oxidized polyphenols. In plant systems, o-polyphenols cross
link damaged cell proteins to form a refractory shield between the
healthy tissues and further assault. They also prevent pathogen
propagation by aggressively binding to their metabolic pathways,
disabling virulence enzymes and arresting pathogen motility.
[0007] Higher vertebrates possess an additional layer of
protection, the adaptive immune system, which allows for a stronger
immediate immune response to previously encountered pathogens. The
aggregation of smaller molecules on the pathogen creates large
complexes with an increased antigenicity of the pathogen to the
host immune system. Each pathogen is "remembered" by a signature
antigen. Should a pathogen infect the body more than once, these
specific memory cells are used to quickly and efficiently eliminate
it; however, these tailored responses can take many days to
develop. In the interim, primary defense against newly encountered
pathogens, especially in infection of immunologically deficient or
immature animals relies solely on the innate immune systems and
often is associated with negative physiologic responses such as
diarrhea, vomiting, fever, inflammation, etc. Such systemic
responses to infection are the expression of the very large numbers
of immune effectors that can be extremely metabolically expensive,
even fatal to the host.
[0008] One of the most common dangers associated with an unchecked
systemic response by the innate immune system is diarrheal
dehydration triggered by infectious diseases or parasites.
Diarrheal dehydration affects over 2 billion people each year and
is the most common cause of death for Third World infants,
responsible for over 1.5 million deaths per year. Besides
re-hydration, most efforts to treat diarrhea have focused on
increasing human mucosal immunity by modulating systemic immune
responses, such as by using intestinal motility reducing drugs,
mucous permeability modifiers or antibiotic therapies. These
approaches have limited success but introduce undesirable risks of
side effects, pathogen resistance, or physiologic senescence.
[0009] There is constant commercial demand for botanical
alternatives to antibiotics and synthetic chemical disinfectants
for the control of disease associated with water, surface, and food
borne pathogens. The explosive rise in antibiotic resistant
diseases has been associated with the overuse of antibiotics in
both humans and livestock. Many regional governments and
international health organizations have called for phase out of
unnecessary antibiotic use, especially in livestock feeds where
they are used sub-therapeutically to enhance growth. To date, it is
widely recognized that there are few cost effective and
environmentally sound alternatives for the safe control of
pathogens. Decades of research on plants as sources of new
antimicrobials has primarily focused on mechanical or solvent
extraction of specific plant compounds and has not been successful
in generating compositions with potency, safety, user preference
and environmental profile necessary to match the performance of
current antibiotics and germicides.
SUMMARY OF THE INVENTION
[0010] In an aspect, a biochemical composition comprises a
processed fluid containing a molecule having a hydroxyl group is
combined with an activating mechanism to activate the molecule by
oxidizing the hydroxyl group with an oxidizing agent and a
catalyst. Activating the molecule increases the binding affinity of
the molecule.
[0011] In one embodiment, the molecule comprises a polyphenol. In
an alternative embodiment, the molecule comprises a polymeric
carbohydrate molecule or polysaccharide derivatives. In another
embodiment, the polyphenol is derived from a plant. In an
embodiment, the plant comprises camellia sinensis, or punica
granatum or other polyphenol bearing plants. In another embodiment,
the polyphenol is derived from the root, leaves, sterns, bark,
fruit or other tissues of polyphenol bearing plants.
[0012] In an alternative embodiment, the molecule comprises tannin,
lignin, flavonoid, hydroxycoumarin, or alkaloids. In another
embodiment, the molecule comprises at least an artificial synthetic
section. In an embodiment, the catalyst comprises a catalase, a
peroxidase, a phenoloxidases, a tyrosinase, or a metal catalyst. In
an alternative embodiment, the catalyst is located at an animal
cell. In another embodiment, the catalyst is generated by a
pathogen. In an embodiment, the pathogen comprises virus, bacteria,
fungi, an eukaryotic organism, or prionic. In an alternative
embodiment, the oxidizing agent comprises reactive oxygen species
(ROS).
[0013] In another embodiment, the reactive oxygen species comprises
hydrogen peroxide. In another embodiment, the reactive oxygen
species comprises inorganic or organic peroxides. In an embodiment,
the reactive oxygen species comprises a product of ozone reduction
by superoxide dismutase, glucose oxidase, hydration of a
percarbonate, or hydration of carbamide peroxide (urea peroxide) or
other indirect method of generating stable reactive oxygen species.
In an alternative embodiment, the processed fluid is prepared from
a dry mixture containing a polyphenol or a polysaccharide
derivative. In another embodiment, the processed fluid is prepared
from intact plant material containing a polyphenol. In another
embodiment, the activating mechanism is initiated when the
polyphenol or the polysaccharide derivative is in contact with the
enzyme and the oxidizing agent in a solution. In an embodiment, the
hydroxyl group becomes a carbonyl group after the activation of the
hydroxyl group. In an alternative embodiment, the molecule
comprises a quinonic group after the activation of the hydroxyl
group. In another embodiment, the molecule has an effect in
inactivating a pathogen after the activation of the hydroxyl group.
In an embodiment, the oxidizing agent provides free radicals. In
alternative embodiments, the activated molecule provides free
radicals.
[0014] In a second aspect, a method of preparing a composition
comprises obtaining a biopolymer from a plant and activating a
hydroxyl group on the biopolymer, so that a molecular binding
affinity of the biopolymer is increased.
[0015] In an embodiment, the biopolymer comprises a polyphenol, a
polysaccharide derivative, or a polymeric molecule. In alternative
embodiments, the activating is performed by placing the biopolymer
in contact with an enzyme or an oxidizing agent. In another
embodiment, the method further comprises cross-linking the
activated biopolymer with a protein of an animal. In an embodiment,
the method further comprises a plurality of biopolymers forming
cross-linking structures among themselves. In an alternative
embodiment, the method further comprises binding the biopolymer
with a pathogen. In an alternative embodiment, the method comprises
binding the biopolymer with a cell. In another embodiment, the
method comprises binding the biopolymer with a virus. In a further
embodiment, the binding of the polymer to multiple pathogens causes
agglomeration or removal of microorganisms, proteins or
proteinaceous structures from solution. In another embodiment, the
binding ceases the propagation of the pathogen. In some
embodiments, the activated biopolymer is capable of blocking a
metabolic pathway of a pathogen. In an alternative embodiment, the
method further comprises treating a diarrhea symptom of an animal
using the activated biopolymer. In a further embodiment, the method
comprises treating any damaged tissue of an animal using the
activated biopolymer. In another embodiment, the activating the
hydroxyl group is triggered by an enzyme on a site of an animal. In
an embodiment, the activating the hydroxyl group is achieved by
exogenous addition of an enzyme. In an alternative embodiment, the
method further comprises removing or inactivating an oxidoreductase
or a reducing compound that reacts with an oxidizer. In another
embodiment, the method further comprises forming a barrier by
cross-linking the biopolymer with a protein of an animal, so an
invasion of a pathogen is prevented. In a further embodiment, the
method comprises cross-linking the biopolymer with a protein of an
animal, so as to promote accelerated wound healing.
[0016] In a third aspect, a method of facilitating a localized
reaction comprises localizing an added reactive oxygen species in a
reactive proximity of a hydroxyl group on a biopolymer, activating
the hydroxyl group of the biopolymer, and applying the biopolymer
to a target site.
[0017] In an embodiment, the biopolymer comprises a polyphenol or a
polysaccharide derivative. In an alternative embodiment, the
activating is achieved by causing an encountering of an enzyme. In
another embodiment, the reactive oxygen species comprises hydrogen
peroxide. In an embodiment, the method further comprises increasing
the density of the hydrogen peroxide in the reactive proximity of a
hydroxyl group by adding hydrogen peroxide to a solution containing
the biopolymer. In an alternative embodiment, the biopolymer
comprises multiple hydroxyl groups.
[0018] In a fourth aspect, a controlled chemical delivery method
comprises preparing a bio-molecule containing a pre-selected
chemical substance and selecting a size, a weight, or a combination
thereof to control a penetration rate of the bio-molecule through
an animal tissue.
[0019] In an embodiment, the chemical substance comprises a
phenolic compound. In an alternative embodiment, the chemical
substance comprises quinonic compound. In another embodiment, the
bio-molecule comprises some amount of an extract from a plant. In
an embodiment, preparing the molecule comprises isolating the
molecule. In an alternative embodiment, selecting comprises
extracting. In another embodiment, the penetration rate is
moderate, so that an application of the bio-molecule to an animal
does not cause a toxic response from the tissue of the animal. In a
further embodiment, an application of the bio-molecule to an animal
reduces the external stimulation of immune response from the animal
system. In an embodiment, the ingestion of the bio-molecule
improves the utilization of nutrition for growth in animal
systems.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 illustrates several reaction pathways for phenol unit
conversion into quinones in accordance with an embodiment of the
present application.
[0021] FIG. 2 illustrates a composition prepared in accordance with
one embodiment of the present application.
[0022] FIG. 3 illustrates a flowchart of a method of preparing a
plant-based composition in accordance with one embodiment of the
present application.
[0023] FIG. 4 illustrates a flowchart of a method of inactivating
reducing agents/enzymes using a solvent in accordance with one
embodiment of the present application.
[0024] FIG. 5 illustrates a flowchart of a method of heat
inactivating reducing agents/enzymes in accordance with one
embodiment of the present application.
[0025] FIG. 6 illustrates applications of the plant-based
composition in accordance with some embodiments.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention comprises novel bioactive
compositions. In an aspect, a biochemical system comprises a
biopolymer or the synthetic equivalent combined with a stable
source of reactive oxygen species (ROS) and a separate source of an
oxido-reducing enzyme or catalyst. The combinations of the
substances are able to cause the formation of an oxidized
biopolymer with increased protein binding affinity and microbial
control activity.
[0027] Some embodiments of present invention contain a mixture of
plant based material with astringent and/or germicidal properties
and an activation precursor in stable solution. The solution is
substantially free of enzymes and catalytic substances that can
cause the other components to react in a manner that causes
degradation. The mixture of this plant based germicidal material
and activation precursor is catalyzed by various enzymes of animal,
plant or microbial cells to release oxidative radicals and to form
an activated plant based material with significantly enhanced
astringent and germicidal properties. The release of oxidative
radicals and the formation of activated plant material are
generally localized to the catalyzing biologic enzyme source, thus
concentrating such activity in the proximity of the triggering
biologic entity or material for maximum effect. The triggering
catalyst can also be of non-biologic origins such as a metal that
causes reduction of the activation precursor to cause
auto-oxidation of the plant based material into its activated
form.
[0028] In alternative embodiments, the bioactive and germicidal
system contains a mixture of a plant based material, such as
polyphenols. A person of ordinary skill in the art would appreciate
that any macromolecules, polymers, aggregate of small molecules,
cellular membrane fragments, cross linked compounds containing a
multiplicity of exposed phenolic units and an activation precursor
(e.g., an oxidizer, such as hydrogen peroxide), or combinations
thereof are applicable.
[0029] In other embodiments, activator materials contain ozone or
peroxone. In some embodiments, the plant material can be a source
of naturally occurring hydrogen peroxide. However, an endogenous
concentration of H.sub.2O.sub.2 is highly variable and can
generally be lost in the processing of the plant material. One of
the advantageous aspects of some embodiments is that the source of
the oxidizing agent can come from exogenously added hydrogen
peroxide, ozone, peroxone or other commercial oxidizer as the
activation precursor.
[0030] In some embodiments, a phenol unit can carry 1 to 3 hydroxyl
groups (OH) that can react with the oxidizer. Therefore, a phenolic
groups contained macromolecule composed substantially of can carry
hundreds to thousands or more hydroxy groups. This high density
molecular cluster of hydroxy groups provides various other
non-covalent-bond opportunities, charge attraction, and physical
sequestration for hydrogen peroxide molecules within close and
reactive proximity to the phenolic units. Sequestration in
accordance with embodiments of the present invention provides a
novel method of maintaining stability and enhanced saturation of
hydrogen peroxide within the reactive proximity of the polyphenolic
substrate at lower concentration solutions than the equivalent
non-sequestered components. Sequestration can be the result of
combining a sufficiently high concentration of hydrogen peroxide in
water to establish intermolecular attractive forces between a
substantial portion of the polyphenol hydroxyl functional groups
and the hydrogen peroxide molecules. Increasing sequestration can
significantly reduce oxidizer loss from heating, ultraviolet
exposure, reducing contaminants, and spontaneous degradation while
increasing the potential number of high affinity binding sites on
the polymer upon encountering the appropriate enzymes.
[0031] In some embodiments, some amount of crude extracts of plant
material, including the fragments of plant cells populated with
partially denatured reactive oxygen species (ROS) processing
enzymes like catalase, peroxidase, dismutase, glucose oxidase, or a
combination thereof, can function as a hydrogen peroxide
sequestration structure. These ROS include hydrogen peroxide
(H.sub.2O.sub.2), superoxide (O.sub.2.sup.-), singlet oxygen
(.sup.1O.sub.2*), and hydroxyl radical (.OH). In alternative
embodiments, the activation precursor can be generated by the
degradation of dissolved ozone (O.sub.3) or by its conversion by
active dismutases into H.sub.2O.sub.2. In other embodiments, the
activation precursor can also be generated by the enzymatic
conversion of any superoxide, such as a fatty acid peroxide.
[0032] The component of the composition that contributes the phenol
units can contain a polyphenol and/or any heterogeneous or
homogenous macromolecule that is synthetic, plant, or animal
derived. In some embodiments, the polyphenol contains more than two
phenol groups. In alternative embodiments, the polyphenol contains
more than 30 phenol groups. In other embodiments, the polyphenol
contains from 100 to 10,000 phenol groups. A person of ordinary
skill in the art would appreciate that any number of phenol groups
can be contained in the composition so long as the compositions
contain the desired features described herein, such as the rate of
absorption is moderate.
[0033] Plant based materials described herein include, but not
limited to, polyphenols, lignins, polysaccharides and other large
molecule materials or structures that are predominantly terminated
by carbonyl groups that are available for quinone transformation.
Effective solutions can be processed from a vast variety of
different plant tissues of different species due the ubiquitous
nature of appropriate materials.
[0034] The typical art for obtaining antimicrobial compounds from
botanical sources relies on mechanical or solvent extraction of
organic molecules in manners that do not provide practical means
for harnessing the wound response chemistry of living plant tissues
for commercial application, especially in animal or other plant
systems. Cellular disruption, whether from commercial processing,
pathogen attack, herbivores, environmental damage or natural
decomposition, triggers the reactions and makes the antimicrobial
compounds useless in a very short period of time.
[0035] Polyphenols constitute a vast range of organic polymers
produced by plants and are important substrates in the wound
response of plants. These polymers are able to undergo some
oxidative conversion to form antimicrobial compounds, but may
produce slightly different behaviors. Polysaccharides may also
undergo some oxidative transformation, forming bioadhesives with
antimicrobial and wound sealing potential and can be effective
quinone analogues.
[0036] The underlying processes are rooted in a complex series of
chemical reactions that may involve multiple direct and indirect
enzymatic formations of highly reactive intermediates.
oxidoreductases can mediate this chemical cascade, typically in the
presence of a source of reactive oxygen species, with several
possible modalities converting hydroxyl (OH) functional groups of
aromatic polymers into carbonyl groups (.dbd.O) that form covalent
bonds responsible for high strength biologic structures and
antimicrobial defenses. Carbonyl groups are functional groups
contain a carbon atom double-bonded to an oxygen atom: C.dbd.O.
Ketone groups contain carbonyl groups (C--C(.dbd.O)--C) that each
of the carbonyl groups bonds to two other carbon atoms with the
carbon atom of the carbonyl. Some of the particular interested
substrates include compounds with multiple aromatic subunits
forming organic molecules with fully conjugated cyclic dione
structures with >C(.dbd.O) groups in any arrangement of double
bonds, including polycyclic and heterocyclic analogues, that form
covalent bonds to nucleophilic amino acids and proteins. The term
"quinonic" used in this document includes any compounds containing
subunits with any number of carbonyl groups.
[0037] The converted or oxidized hydroxyl subunits exhibit
increased binding affinity characteristic of multiple quinones and
semiquinones in a heteropolymer or homopolymer configuration. The
oxidation is able to only take place on a portion of the functional
groups of a polyphenol. The typical heterogeneous character of
these oxidized polyphenols is able to present quinonic activity
while retaining some basic polyphenol behavior. Differences in
gross structure, tertiary form, and molecular weight will cause
different affinities for different proteins with increased gross
mixture or intramolecular heterogenicity providing broader spectrum
potential.
[0038] Besides having been identified as a transient cofactor in a
number of biologic cross-linking activities, quinone monomers have
long been known for a very potent germicidal effect. In addition to
provide a source of stable free radicals, quinones are able to
irreversibly complex with nucleophilic amino acids in proteins,
often leading to inactivation of the protein and loss of associated
biologic function. For that reason, the potential antimicrobial
effect of quinonic compounds is great. Probable targets in the
microbial cell are surface-exposed adhesions, cell wall
polypeptides, motility effectors and membrane-bound enzymes.
Quinones can also render substrates unavailable to the
microorganism.
[0039] As with all plant-derived antimicrobials, the possible toxic
effects of quinones must be thoroughly examined. Some quinones have
demonstrated antimicrobial effectiveness at 5 to 6 log dilution.
Quinone monomers are small molecules that easily penetrate tissues
and exhibit toxicity that can limit their medicinal use, whereas
polyquinonic compounds contain a multitude of quinonic segments in
a biopolymer, compound molecule, or synthetic analog. Polyquinonic
compounds of sufficient molecular weight and size can have reduced
systemic absorption, corresponding to reduced toxicity potential to
higher organisms.
[0040] The study of germicidal activity of quinone compounds
started as early as early 1900s but was not well understood until
the 1940s. Formalin inhibition of the color reaction between
quinone and many different proteins, such as egg albumin, casein,
horse serum, and peptone, indicates that the reaction is
principally between quinones and the amino group of proteins. The
germicidal mechanism of a polyquinone alone can take three primary
forms: the covalent binding reaction with bacterial proteins,
cross-linking of ruptured cell cyto-proteins to astringently form a
barrier refractory to pathogens, and a REDOX cycling mode that
generates peroxides and free radicals that cause oxidative damage
to the pathogen envelope.
[0041] The major product of phenol oxidation was identified by
Pryor in 1940 as o-diphenols. This production can result from
auto-oxidation in the presence of oxygen radicals or from enzyme
conversion by phenoloxidases. Phenoloxidases (e.g., L-dopa: oxygen
oxidoreductase; EC 1.14.18.1), also known as polyphenol oxidases
and tyrosinases (e.g., lysyl oxidase; EC 1.4.3.13), are
copper-containing proteins that catalyze the oxidation of
monophenols into o-diphenols and the subsequent oxidation of
o-diphenols to the corresponding o-quinones. Phenoloxidases are
widespread in the animal kingdom, as well as in plants, fungi, and
prokaryotes. Insects also use them in sclerotization in rapid
formation of high strength egg casings, cocoons and silk
structures.
[0042] Even though they are the result of hundreds of millions of
years of divergent evolution, the very close structural similarity
of oxidative enzymes including peroxidase, polyphenol oxidase,
laccase, etc., found in plant, fungi, bacteria and peroxidases such
as myeloperoxidase, lactoperoxidase and all the peroxidases from
different animal tissues, indicates the need to achieve the same
basic biologic ends. This functional similarity can be utilized as
a novel method for triggering controlled oxidation of polyphenols
across biologic kingdoms or phyla.
[0043] For example, tyrosinase is the core enzyme of the
phenoloxidase family along with several other oxidoreductases that
catalyze a step in the formation of melanin pigments. The
tyrosinase in mammals is functionally similar to phenoloxidase in
the chemical cascade that causes sclerotization, melanization, and
production of antimicrobial peptides in insects.
[0044] Hydrogen peroxide is one of the most common biologic sources
of reactive oxygen species involved in the enzymatic creation of
polyquinones. It is expressed in substantial quantity in live
tissues of many plants. It is a ubiquitous metabolic product and a
key initiator that is consumed in the polyphenol oxidation process
that occurs in damaged tissues. Both the oxidizer (H.sub.2O.sub.2)
itself and its enzyme mediated reaction product, polyquinones, are
antimicrobial with the latter also having strong astringent
properties. These are the primary compounds that enable the
traditional medicinal use of many fresh plant materials as wound
dressings. However, antimicrobial potential within the vicinity of
a fresh cut plant tissue is largely degraded within minutes due to
the transient nature of these compounds in wounded plant
tissue.
[0045] The mixture of plant materials, oxidizers and enzymes have
been used to generate oxidized polyphenols and carbohydrates for a
variety of industrial and commercial uses, but the composition of
plant material with enzymes denatured or removed to allow stable
combination with oxidizer for the purpose of applying to a target
that provides a separate source of catalyst or enzyme to affect
biologic systems is novel.
[0046] References describing compositions of plant materials and
oxidizers for use on biologic systems is limited. US 2002/0034553
describes an aloe vera gel and Irish moss as a thickened passive
carrier for delivering hydrogen or zinc peroxide as a source of
oxygenation to create unfavorable conditions for anaerobic bacteria
on dermal wounds. U.S. Pat. No. 5,260,021 discloses a hydrogen
peroxide-containing gel ointment as a vehicle for carrying oxygen
for use only as a disinfectant for contact lenses or the like. U.S.
Pat. No. 4,696,757 describes a hydrogen peroxide carrying gel for
treating surface cuts and for bleaching hair. None of these patents
make reference to combining an oxidizer, such as hydrogen peroxide,
and a polyphenol component with the intent of causing or enabling a
reaction between the two.
[0047] In higher plant physiology, hydrogen peroxide, polyphenols,
proteins and oxidoreductases are segregated in the structured
cytoplasm, organelles and membrane structures of the living cell.
Disruption of the cell by infection, injury, crushing, pulverizing,
desiccating, ensiling or other physically damaging processes result
in the mixing and exhausting of the useful reactive potential of
these components. The current art of botanical extraction offers no
obvious means to capture a stable combination of these components.
It is therefore not surprising that despite over 50 years since the
discovery and documentation of the function of oxidized polyphenol
systems within plants, the botanical health and agriculture
industries have not successfully commercialized this
multi-molecular chemistry, instead focusing on capture of
inherently stable nutritional and pharmacologic molecules that can
be simply packaged or extracted.
[0048] There is significant understanding of enzymatically oxidized
polyphenols within the botanical sciences, but there is no known
reference for a stable ex-vivo method and composition for
restoring, duplicating, or enhancing the capability of this
polyphenol oxidation system for use with animal physiology or other
biologic applications outside the context of in-vivo plant
biochemistry.
[0049] The environmental interfaces and immunologic needs of the
plant kingdom are in many ways similar to those of animals.
External plant tissues, such as leaf cuticles, fruit rinds, and
seed husks, are living tissues adapted to defend against similar
pathogens and physical stresses as human derma and mucosa. Animals
and plants also have some analogous mechanisms for coping with
wounding. As such, the biochemical mechanisms used in the plants
are able to be applied to animals.
[0050] In another aspect, a method of producing stable biochemical
systems comprises extracting a stable polyphenol substrate in a
composition free of active reducing agents, enzymes or catalysts
and combining the polyphenol substrate with a concentrated source
of reactive oxygen species that promote initiation and propagation
of oxidation reactions when applied to or combined with a separate
source of appropriate oxidoreductases or catalysts.
[0051] Compositions in accordance with some embodiments of the
invention introduce hydrogen peroxide in sufficiently high
concentrations into an aqueous plant biopolymer solution to
establish sequestering or stabilizing of concentrated oxidizers
within intimate reactive proximity of the hydroxy functional groups
of the polyphenols. The sequestered oxidizer resists diffusion away
from the biopolymer when subjected to dilution. Reducing agents are
removed or denatured in formulations containing the
oxidizer-polyphenol combination to render the oxidizer-polyphenol
combination substantially unreactive to proteins until brought in
contact with a surface, tissue, organism, coating or solution that
provides a source of oxidoreductase enzymes or other catalytic
agents that directly or indirectly mediate conversion of the
biopolymer into an activated form.
[0052] In another aspect, the compositions contain an efficient
target-specific formation of oxidized biopolymers mediated by
enzymes of animal origin. This mechanism enables the delivery of
medically and commercially useful effects to animal body tracts or
tissues. These effects are analogous to basic plant wound
responses. Many of the biochemical reactions involved are well
studied but still not fully understood. Nonetheless, there is no
prior art for stabilizing and applying the stabilized combination
of oxidizers and polyphenols to animal physiology or pathogens as
the source of enzymatic activation. Common phenol complexing acting
enzymes in many plants, bacteria, and fungi such as laccases or
phenol oxidases are functionally similar to common animal enzymes,
such as catalases, peroxidases, that can also cause the formation
of quinonic groups with enhanced affinity for amino acids of
proteins.
[0053] In some embodiments, the liquid solution prepared has many
significant practical advantages over conventional typical
antimicrobial botanical extracts. These include increased water
solubility and deliverability, broader pH stability, higher
temperature stability, predictable potency, low animal toxicity,
low minimum inhibitory concentrations on a broad spectrum of gram
negative and gram positive bacteria, low odor and taste, efficiency
in use of raw materials, and lack of harmful environmental
breakdown products.
[0054] Some embodiments of the present invention mimic plant
defense mechanisms that have co-evolved with environmental
pathogens over many millions of years. The non-specific protein
binding action of the activated biopolymers contain multiple
activities that can cause microbial inactivation. All are radically
different than conventional germicidal or antibiotic actions and
are far less likely to promote resistance in a manner that has been
associated with subtherapeutic use of and environmental
contamination with, antibiotics.
[0055] The oxidized polyphenols can have several biologic
mechanisms of action. A single such polyquinonic molecule can
contain several to thousands of binding sites in a dense and
non-diluting population, so that the compound can cross-link and
condense amino acids and proteins of damaged organisms to form a
refractory barrier against pathogens and irritants. The compound is
able to bind to and cause significant distortion and inactivation
of the metabolic pathways and virulence effects of the pathogens.
They are able to physically agglomerate, entrain or immobilize
pathogens to prevent the pathogens from propagating. Quinonic or
phenolic forms also can cause oxidative radical release to cause
pathogen membrane damage and lysis. They are also able to block
receptors responsible for pain and inflammatory responses.
[0056] Polyphenols generated in accordance with embodiments of the
invention can be individual soluble polymers, compound molecules,
macromolecular conglomerates, or aggregates with proteins or
cellular fragments. Stabilizing sequestration of the oxidizers can
result from hydrogen affinities, electron sharing, or from
capillary effects on a larger scale.
[0057] In many situations, it can be beneficial to promote
heterogeneous mixtures of activated biopolymers to maximize
bio-activity/bioavailability against a broader range of pathogens.
The combination of polyphenols extracted from multiple plant
materials can serve to further broaden the binding spectrum. In
some cases, not all phenol subunits undergo quinone conversion and
the substrate can retain characteristics and behaviors of the
unoxidized compounds in addition to the quinonic behavior.
[0058] Those of ordinary skill in the art will understand that many
other natural or synthesized organic polymers can also be oxidized
to form a multiplicity of carbonyl or quinonic functional groups
that can be used in place of or combined with polyphenols.
[0059] In some aspects, non-botanical enzymes are used to trigger
the oxidation of polyphenols. This allows introduction of a stable
polyphenol-oxidizer solution to passively traverse animal body
tracts to deliver unreacted materials to compromised tissues that
cannot be practically accessed by other means, thereby increasing
the effective bioavailability of the useful composition. Target
catalyzed quinone formation constrains the non-specific biochemical
reactions to the immediate proximity of compromised tissue, thereby
reducing the risk of undesirable effects of gross tissue or
systemic interaction.
[0060] The existence of functionally identical enzymes in the
animal and plant kingdoms enable the novel transference of the
enzyme mediated polyphenol reactions to non-botanical applications.
For example, the G.I. tract of higher vertebrates has 2 different
types of peroxidases. The first type comprises a soluble peroxidase
found in both rat and pig digestive mucus that is secreted from the
immune system eosinophil. The second type comprises an insoluble
peroxidase found in G. I. mucosal cells that are only released in
wounding. The second type of peroxidase is basically the functional
equivalent to peroxidases involved in polyquinone formation of
plant wound response and can site-specifically trigger quinonic
activity in the direct proximity of damaged, vulnerable, or
infected cells while remaining passive to healthy tissue.
[0061] Such a stable polyphenol-oxidizer composition has
particularly useful biochemical effects on a tissue lesion or an
irritated mucus membrane of the digestive tract, respiratory tract,
urinary tract, reproductive tract, or other mucosal interfaces
within higher animals. Ingestion of the composition in liquid
solution will passively deliver it to the site of damaged cells and
infection where oxidoreductase enzymes will catalyze polyquinonic
activity. The polyquinones will cross-link and condense cellular
proteins of damaged host cells into a protective barrier. The
non-specific high-affinity binding to surface proteins, enzymes,
receptors, and structures critical to metabolism, virulence, and
motility immobilize and inactivate a very broad spectrum of
pathogens. The agglomeration of pathogens can also increase the
potential antigenicity to evoke host immune responses.
[0062] In an aspect of the present invention, the strong but
localized astringent effect on the affected tissue can also reduce
fluid exudates. Non-localized gross astringency from high
concentrations of tannins and other polyphenols in the digestive
system are known to interfere with nutritional absorptions and can
cause mucosal damages; whereas, target-localized activation of
quinonic behavior minimizes these concerns.
[0063] In some aspects, the compositions comprise novel bioactive
microbial control and tissue protective systems. The mixture of the
bioactive material and activation precursors are catalyzed by
various enzymes of animal, plant, or microbial cells. If the
activating enzyme is associated with tissue of interest such as
wounded or infected tissue, the ingestion or application of the not
activated composition will allow passage to compromised tissue even
deep in a body tract. Release of oxidative radicals and formation
of activated plant material site-specifically on target tissue
significantly increases bioavailability and constrains the enhanced
activity to the immediate proximity of the enzyme source for
reduced collateral interaction with other tissues.
[0064] In some of the embodiments, the bioactive plant-based
material can be polyphenols or any bio-molecules, synthetic
polymers, aggregates of small molecules, cellular fragments, or
cross-linked groups of compounds comprising a multiplicity to tens
of thousands of exposed reacting sites
[0065] Many plants can be used as an inexpensive source of
appropriate polyphenols. Camilla Sinensis leaf is an example that
can be a good source of a botanical raw material because of common
availability of cultivated sources, documented low natural toxicity
and high water soluble polyphenol content. The flavonol group of
polyphenols (non-oxidized catechins) constitutes up to 30% of the
dry leaf weight of camellia sinensis, making it an economical
source. Effective antimicrobial astringent compositions have also
been produced from many different plant species and structures
including rye seeds, mung beans, daikon skin, pomegranate rinds,
bearberries, aloe vera skin, organ pipe cactus, Chinese gall,
oregano leaves, persimmon fruit, wheat germ, barley seeds, and
coffee beans, demonstrating the ubiquitous nature of this plant
defense mechanism in the plant kingdom.
[0066] In some aspects, methods of extracting a polyphenol
substrate from plant materials enable stable formulation, storage,
and delivery by having extracts that contain substantially free
active oxidoreductases or other reducing agents. Some aspects of
the invention involve thermal or solvent denaturing of plant raw
materials to obtain plant biopolymer component free of active
polyphenol oxidizing enzymes. Raw material supply availability and
different plant tissue types can possibly dictate different
processing. For instance, desiccated or dried plant materials are
already devoid of hydrogen peroxide and therefore can undergo
enzymatic denaturing processes after polyphenol extraction.
[0067] An example of an efficient process for producing an
economical polyphenol raw material source free of degrading enzymes
comprises desiccating freshly harvested whole camellia sinensis
leaves rapidly in high temperature air to denature the polyphenol
oxidases that can cause oxidation of green leaf polyphenols. This
process maintains a very close composition to live tea leaves with
the exception of the loss of hydrogen peroxide, water, and a few
enzymatic changes that typically occur extremely rapidly upon
harvest. The leaves are then pulverized to facilitate handling and
extraction efficiency.
[0068] In contrast, black tea undergoes an example of an
alternative manufacturing process. In the manufacturing process,
Camilla sinensis leaves are crushed and their cellular structures
are disrupted while still containing active polyphenol oxidase.
This initiates enzymatic aerobic oxidation of catechins into
quinones that spontaneously condense to form volatile compounds.
Plant material so processed is still able to be a useful source of
polyphenols, but the plant material will have lower content of
polyphenols and will require additional enzyme denaturing by
heating the plant material or its extract to a temperature
sufficient (preferably 80.degree. C. to 110.degree. C.) to blanch
or denature the enzymes. A protein denaturing solvent, such as
ethanol, is able to be alternately used in the plant material
extraction process to destroy or remove cellular enzymes.
[0069] Most plants produce hydrogen peroxide as part of routine
biologic activities as well as in response to stress. The
concentration of H.sub.2O.sub.2 in plant tissue varies tremendously
by species, tissue type, environmental stress and seasons. It is
lost or consumed in typical post harvest processing and is
generally impractical to capture from natural sources, especially
given the low cost of synthetic equivalent oxidizers.
[0070] In some embodiments, the methods and compositions in
accordance with some embodiments of the present invention comprise
the exogenous addition of hydrogen peroxide, which can be a
commercially practical and stable source of reactive oxygen species
for improved generation of quinonic subunits within the
polyphenols. A person of ordinary skill in the art will also
understand that other direct sources of reactive oxygen species can
be used for various applications, such as ozone, zinc peroxide,
peroxidases, carbamide peroxide, sodium percarbonate, calcium
peroxide, magnesium peroxide, sodium perborate monohydrate, ozonide
(O.sub.3.sup.-), superoxide (O.sub.2.sup.-), oxide (O.sup.2-),
dioxygenyl (O.sub.2.sup.+) or indirect source(s) of reactive oxygen
species, such as oxygen gas, disassociated water, catalytic
decomposed fatty acids, glucose, and polyphenols are able to be
used.
[0071] In some aspects, embodiments of the present invention
include combinations of high concentration reactive oxygen species,
such as hydrogen peroxide, with a polyphenol substrate to provide a
stabilizing environment that resists diffusion of hydrogen peroxide
molecules away from intimate reactive proximity to the phenol
subunits. Polyphenol structures provide many non-covalent bond
opportunities and charge attraction to hydrogen peroxide molecules.
Hydrogen peroxide is also a product of the auto-oxidation of
polyphenols, helping to maintain gross equilibrium in solution.
Stable sequestrations are able to shield the hydrogen peroxide from
heat, ultraviolet exposure, reducing contaminants, and spontaneous
degradation.
[0072] FIG. 1 illustrates several reaction pathways for phenol unit
102 conversion into quinones 104 or semi-quinones 106 in accordance
with one embodiments of the invention. As used herein "quinone"
refers to all quinonic compounds, such as quinones and
semi-quinones. Hydrogen peroxide 108 can be both a source of
reactive oxygen species for initiating oxidation, and can also be a
product of polyphenol oxidation. Stable equilibrium in a solution
with polyphenols can therefore be established. Once started, the
source of reactive oxygen species (ROS) facilitates efficient
propagation of quinone generation through polyphenol substrates
even without direct enzymatic mediation. Concentrated
H.sub.2O.sub.2 in water is therefore a good oxidizer component.
However, hydrogen peroxide can be indirectly achieved through other
reactions, such as decomposition of ozone, fatty acids, or
percarbonates to name only a few such reactions. Cellular
oxidoreductases can also be involved in the indirect generation of
the oxygen species involved in the initiation or propagation of the
oxidation reaction cascade. For example, the catalase that defends
animal cells and many pathogens from ROS damage will disassociate
H.sub.2O.sub.2 into water and reactive oxygen species.
[0073] Hydrogen peroxide is naturally produced in plant and animal
cells but its concentration can vary tremendously depending on
species, season, stress, and tissue type. Although certain plant
types, such as succulents, can store significant quantities of
hydrogen peroxide in their tissues, it is generally impractical to
extract it from plant sources due to the presence of reducing
enzymes segregated from the hydrogen peroxide and/or polyphenols
only by delicate subcellular divisions that are inevitably breached
by typical commercial extraction processes. Mixing triggers the
oxidative wound response and rapidly consumes the hydrogen
peroxide, leaving excess polyphenols, enzymes, and other
non-involved botanical compounds.
[0074] Some aspects of the present application include the use of a
separately manufactured or generated source of reactive oxygen
species in combination with the polyphenol substrate substantially
free of active reducing agents or enzymes.
[0075] In some embodiments, the oxidizer-biopolymer compositions
are aqueous solutions. In alternative embodiments, fibers,
hydrogels, microporous media, micelles, emulsions, and other
structures physically encapsulate the biopolymer-oxidizer
composition. In still other embodiments, mixtures of dry powders,
granules, or other non-liquid polyphenol bearing materials combined
with a dry oxidizer, such as potassium percarbonate, are used as a
kit to be hydrated to produce a useful polyphenol-oxidizer
solution.
[0076] In some embodiments, the catalyst is delivered to the target
site separately as a liquid, aerosol, or as a surface coating on an
applicator, dressing, or cleaning implement. An example of this is
an absorbent sponge infused with a reducing agent, such as catalase
or copper, that will cause rapid release of oxygen radicals and
quinone formation when brought in contact with a
polyphenol-oxidizer composition. This can be used to generate a
strong germicidal action, particularly to destroy viruses on
non-biologic surfaces or to sanitize healthy tissues in the absence
of exposed catalyzing enzymes. For example, viral envelopes
generally do not have enzymes, but are made up of proteins that can
be bound by polyquinones. Thus, a separately delivered catalyst or
enzyme can be used to initiate the viral/germicidal reactions.
[0077] In some aspects, polyquinonic compounds in accordance with
the embodiments comprise utilities such as a microbial flocculant.
Adding these polyquinonic compounds to a contaminated water source
can cause aggregation of the microorganisms into masses that either
precipitate out or can be more easily filtered out by mechanical
means. The deposition of polyquinonic compounds on mechanical
filter media can trap proteins and microorganisms while imparting
germicidal characteristics. This can be accomplished by applying an
activated polyquinone to a filter media or circulating polyquinone
forming compositions through a filter media that has a catalytic
aspect to its surface such as a bacterial biofilm. This can find
application in many recirculating and single-pass filtration
applications.
[0078] FIG. 2 illustrates a composition 200 prepared in accordance
with some embodiments of the present invention. The composition 200
contains biopolymers 202 containing hydroxyl group contained
molecules 204. The hydroxyl group contained molecules 204 are
illustrated as polyphenols, but can be phenols, polyphenols,
polysaccharides, or combinations thereof. In alternative
embodiments, the hydroxyl group containing molecules 204 are
tannins, lignins, and flavonoids. A person of ordinary skill in the
art would appreciate that the biopolymers 202 are able to be any
short linkage molecules (such as 2 to 100 repeating units or 100 to
1000 repeating units), macromolecules, long chain molecules, ring
structured molecules, .pi. electron stacking and/or structural
stacking molecules. Further, the biopolymers 202 are also able to
be any substance that can be derived or obtained from plants or
artificial synthetic molecules. Moreover, the biopolymers 202 are
able to be obtained from combinations of plants. For example, the
extract of plant A contains a high ratio of polyphenols and the
extract of plant B contains a high ratio of polysaccharides. The
biopolymer 202 can be obtained from a mixture of the extracts of
both plant A and plant B. In such a case, 70% of the biopolymer 202
can come from plant A and 30% of the biopolymer 202 can come from
plant B, so that the composition 200 can have chemical properties
closer to polyphenol than to the chemical properties of
polysaccharides. Thus, desired reactive properties of the
composition 200, such as a desired reactivity and reaction rate,
can be designed by using different combinations of the plants A and
B.
[0079] The composition 200 is also able to contain an oxidizing
reagent 206 and/or an enzyme 208. In some embodiments, the
oxidizing agent 206 comprises reactive oxygen species. In some
embodiments, the oxidizing agent 206 comes from commercially
available hydrogen peroxide 210, such as >60%, 20%-60%, 35%, and
8%-20% of H.sub.2O.sub.2 in water. In alternative embodiments, the
oxidizing agent 206 comprises 1-2% or less than 10% H.sub.2O.sub.2
in water. In some embodiments, the oxidizing agent 206 comes from a
reaction of ozone 212, fatty acid 214, or percarbonate 216.
[0080] In some embodiments, the oxidizing agent 206, such as
hydrogen peroxide, is endogenously produced by the biopolymer or
the plants. In alternative embodiments, the oxidizing agent 206 is
exogenously added to the system, such as by adding commercially
available hydrogen peroxide to a solution of the biopolymer 202 and
the enzyme 208.
[0081] In some embodiments, the enzyme 208 is endogenously
generated or exogenously added. For example, the enzyme 208 used to
activate or facilitate the reaction is generated by pathogens 230
on the tissue of an animal 218. Alternatively, the enzyme 218 is
generated at the cells/tissues of animals 218 and/or plants 232.
Moreover, the enzyme can be added to a solution containing the
biopolymer 202 and the oxidizing agent 206 before applying the
composition 200 to an animal or a plant. In some embodiments, the
hydroxy group contained molecule 204 forms quinonic compounds 234
and/or 246. The quinonic compound 234, 242, 244, 246, hydroxy group
236, 238, 240, and the hydroxyl group contained molecule 204 can
provide interactions, such as covalent bonding forces, hydrogen
bond interactions, or electron stacking interactions, to keep the
reactive oxygen species (ROS) localized in the reactive proximity.
The reactive oxygen species can be oxidizing reagents.
[0082] FIG. 3 shows the step 300 of a process for preparing a
plant-based composition in accordance with one embodiment of the
present invention. At Step 302, active reducing agents or catalysts
are inactivated or removed. At Step 304, polyphenol substrates
substantially free of active reducing agents or catalysts are
extracted. At Step 306, the polyphenol substrates are mixed with a
source of reactive oxygen species and/or catalysts for initiating
and propagating of oxidation reactions. As described above, the
source of reactive oxygen species, such as hydrogen peroxide, can
be exogenously added or endogenously generated by a plant extract.
Similarly, the catalysts for initiating the oxidation reactions can
be exogenously added or endogenously generated at an applied site,
such as a pathogen-infected site or a tissue wounded area. At Step
308, the mixture is applied to a target site, such as a wounded
area of an animal. The method 300 described above is one
embodiment. All the steps are optional, and additional steps are
able to be added. The sequence of the steps can be in any order.
Other variations are applicable. For example, a solution,
substantially free of reducing catalysts and containing polyphenols
extracted from a plant, is mixed with hydrogen peroxide. The
solution is able to be stored in a container for a later activation
process. The solution is activated through an activation mechanism
after being delivered to a pathogen-infected site of an animal,
which generate catalysts for activating the reactions. In other
examples, a solution containing polyphenol and hydrogen peroxide is
activated by exogenous addition of catalysts before the application
to a target, such as a site of an animal or a plant.
[0083] FIG. 4 shows the step 400 of a process for inactivating
reducing agents/enzymes by solvent in accordance with an embodiment
of the invention. At Step 402, the plants that are used to make the
plant-based composition are chosen. At Step 404, polyphenol
compositions are extracted from the plant using a solution. At Step
406, the solution is heated to between 80.degree. C. to 110.degree.
C. to blanch for denaturing the enzymes. At Step 408, a protein
denaturing solvent, such as ethanol, is used to destroy or remove
cellular enzymes contained in the plant.
[0084] FIG. 5 shows the step 500 of a process for inactivating
reducing agents/enzymes by heat in accordance with an embodiment of
the invention. At Step 502, the plant used to make the plant-based
composition is chosen. At Step 504, the polyphenol oxidase from the
plant is denatured by desiccating whole camellia sinensis leaves
rapidly in high temperature air. At Step 506, the water cellular
enzyme is removed and/or inactivated. At Step 508, the leaves are
pulverized. At Step 510, solvent is added to extract the
polyphenols.
[0085] FIG. 6 illustrates some applications of the plant-based
composition in accordance with some embodiments. The applications
include animal nutritional supplements, animal tissue coagulation,
pathogen barrier formation, target triggering reactions, exogenous
addition of oxidative enzyme for biocidal applications, hydrogen
peroxide sequestration, timed delivery, pathogen metabolic halt,
medicinal beverages, receptor antagonists, microbial flocculants,
biological substance preservation, antimicrobial wash, agriculture
applications, pond water sanitation, medical device surface
treatments, and medicine delivery vehicles, to name only a few such
applications.
[0086] Some modes of action in accordance with embodiments include
the function of target activation of a dense population of
high-affinity, low-specificity binding sites on a relatively large
bio-molecule substrate. In some embodiments, a whole plant extract
is used. In some embodiments, a mixture of plants is blended with
different dominant phenolic species and molecular weights with
slightly different protein affinities, so the broadest possible
range of activity can be facilitated.
[0087] Typically, the concept of drug design of conventional drugs
has been focused on highly specific molecular interactions. In
comparison, some embodiments of the invention use non-selective
activity that is made highly effective and safe by site-specific
activation. In some embodiments of the present invention, the
molecular complexes traverse the digestive system in a waterborne
solution of molecules that keep the necessary multiple reactive
components sequestered in direct reactive proximity of each other
despite diffusion gradients in high levels of dilution. This
maintains full bio-availability until it encounters damaged mucosal
tissues or pathogens that present appropriate enzymes to activate
the complex. Activation at the site catalyzes a highly localized
transformation of the passive molecular complex into an aggressive
protein binder with hundreds or thousands of potential active
sites, which are far more aggressive than the two binding sites
found on antibodies. This highly site specific activation and
non-absorbed nature of the large "sticky" polymer creates a
powerful, precisely targeted action that presents minimal adverse
systemic potential. An accumulation of these "sticky" plant
biopolymers becomes firmly anchored to the target site and starts
to mimic the highly efficient mechanical immune reaction that would
normally occur at the site of a plant injury.
[0088] In some embodiments, the mechanisms in plant immunology
described above are applied to animals. The mechanisms include (1)
non-specific binding to functional pathways of infecting bacteria
or yeasts, which can kill the bacteria or yeasts by impairing their
metabolism and reproduction, (2) binding to toxins (generally
proteinaceous) present at the site and/or blocking pathogens from
expression of more enzymatic virulence factors, (3) immobilizing
and/or impairing their motility or causing agglomeration that
prevents propagation and shedding, (4) cross-linking effects of
proteins of damaged cells into a physical barrier that reduces
exposure to further infection or irritation, (5) binding to
inflammatory signal receptors functioning as antagonists for a
variety of physiologic responses, (6) disabling the cell
penetration mechanism or entrapping viruses and/or preventing
propagation and shedding, and (7) localized astringent and barrier
effect to reduce interstitial fluid loss from damaged tissues.
[0089] In some embodiments, the effective dosages are extremely
small compared to physical astringents used in the traditional
method to arrest diarrhea and believed to be insufficient to create
any physical astringent effect that has been associated with
intestinal damage or nutritional uptake impairment associated with
the use of tannins.
[0090] Some experiments are performed using the complex prepared in
accordance with the embodiments of the present invention. The
effectiveness of the complexes is supported by consistent
observation of improved growth in pigs treated with the complexes
made in accordance with some of the embodiments. The insensitivity
of the complexes to dilution, the passive nature on healthy mucosa,
and the minimal activity on non-pathogenic bacteria are some of the
factors that enable and make low concentration waterborne delivery
preferable and more effective.
[0091] The mixture prepared in accordance with some embodiments of
the present invention can be useful on a tissue lesion or an
irritated mucus membrane of the digestive tract, respiratory tract,
urinary tract, reproductive tract or other mucosal interface within
higher animals. These tissues and infecting bacteria can be a
source of oxidative enzymes that catalyze the conversion of the
polyphenol substrate into its oxidized form. In some embodiments,
the ingestion of the phenolic/oxidizer composition described above
can direct and/or indirectly convert the polyphenols into a
polyquinonic (or o-polyphenol) compound through direct enzymatic
conversion or auto-oxidation of hydroxyl units with an enzyme
decomposed hydrogen peroxide. The polyquinonic molecule can then
covalently bind to proteins of the damaged tissue cells or the
surface proteins of pathogens to immobilize, inactivate, and/or
condense them. The bound proteins can form a protective matrix,
which reduces the ability of pathogens to colonize, provides a
strong astringent effect that contracts the wound and reduces the
fluid exudate. The underlying reactions can take different
pathways, depending on the specific enzymes, the structure and type
of the polyphenol molecule, the relative concentration of the
oxidizer, the dilution environment, etc. An oxidizer that is not
directly involved in enzymatic formation of quinonic units can
release oxidative radicals that directly damage cellular structures
of the pathogens. The localized burst of such radicals in proximity
of immobilized pathogens creates a focused germicidal action that
is far more efficient than the diffuse action of germicides in free
solution. The use of more than one plant material as substrates or
combinations with plants with known germicidal activity without
oxidation as additional constituents can be desirable for
increasing the spectrum or potency of antimicrobial activity.
[0092] In other embodiments, the oxidative enzymes can be delivered
to the reaction site separately. As an example, one of the
embodiments contains fighting viruses in the absence of infected
tissue or bacteria by providing a source of catalyzing enzymes.
Viral envelopes generally do not have the enzymes, but are made up
of proteins that can be bound by the macromolecule once converted
into a polyquinone. The use of such a pre-converted polyquinone is
applicable in some embodiments.
[0093] Some embodiments of the present invention can be used in
solution as a germicidal additive. It can be triggered by the
enzymes of microorganisms, such as bacteria and fungi, in the
absence of other plant or animal tissue. The germicidal
effectiveness on surfaces or in solution can be further enhanced by
the addition of an enzyme or other catalyst to the surface or
solution to be treated or by the application of the compositions to
a surface, implement, or vessel that is treated with an activating
enzyme or catalyst.
[0094] Some embodiments of the present invention can be used as a
microbial floculant, aggregating the microorganisms into masses
that either precipitate or can be filtered out by mechanical means.
The aggregation of germs and their products can also increase the
potential antigenicity to evoke immune responses. Astringent
activity can be cytotoxic to the germs as the result of (1) cross
linking that disables the germ cells' surface proteins and enzymes
or receptors, signal transduction components, nutritional
absorption and transfer functions and (2) reducing or impairing the
mobility of germs and viruses. Both types of actions can disable
the infectivity of viruses.
[0095] The bactericidal test demonstrated that an amount of
hydrogen peroxide that can be exhausted by the bacteria in 16 hours
can be used to completely kill all bacteria, without being
consumed, when a composition made in accordance with the
embodiments of the present invention is used. The plant-based
material that did not undergo oxidization reactions did not show
any bactericide effect. This indicated that at least some of the
soluble polyphenolic compound was converted to polyquinone. In an
in-vivo environment, this plant based germicidal effect will be
further increased if the amount of catalase or peroxidase is not so
limited. A transformed tobacco plant with a chimeric tobacco
anionic peroxidase is capable of producing a high level peroxidase,
demonstrated to be 7 times faster at wound healing than a
non-transferred tobacco plant. The increased efficiency of the
polyphenol to quinone conversion is more likely due to the
significant amount of enzymes than to the amount of the
substrate.
[0096] Some aspects of the present invention include the protein
cross linking capability. Tannin has well known protein binding
ability. However, tannin-protein binding is hydrogen bond based and
the preferred binding protein is proline-rich-protein, usually in
the saliva, and mucus fluid of digestive system. The protein cross
linking and precipitation test demonstrates that the egg albumin
formed dense and complete precipitation within 10 minutes. The
precipitation with plants only takes 3 days to receive similar
results. The sample with the oxidizing agent only had no
precipitation effect at all. This indicates the embodiments of the
present invention have greatly increased protein cross linking
capacity and efficiency.
[0097] Some of the embodiments include a mixture of a plant based
germicidal and a protein cross linker composition and oxidizer. The
plant based germicidal and protein cross linker composition can
contain a plant having high phenolic compounds. The oxidizer can be
hydrogen peroxide. Some of the methods that can be used to
manufacture such compositions are disclosed in the U.S. patent
application Ser. No. 12/317,638, filed Dec. 23, 2008, and entitled
"PLANT-BASED BIOCIDAL MATERIALS AND SYSTEMS," which is hereby
incorporated by reference in its entirety.
[0098] Some embodiments of the present invention can be applied to
the oxidation-reduction and/or radical reactions of the plant
phenolic compounds. The plant phenolic compounds can include
phenols, polyphenols, hydroxycoumarins, flavonoids, alkaloids, and
any other components with chemical subunits similar to monophenols
or diphenols to oxidize, to reduce, or to crosslink other molecules
in or on microorganisms, plants, and animals, thereby, generating
antimicrobial effects, antiviral effects, astringent effects, and
wound healing effects on different target organisms or tissues. The
terms "quinone" and "polyquinone" used in the present disclosure
include any monomer or organic polymer of aromatic ring
configurations with one or more double bonded oxygen.
[0099] The compositions and methods can be utilized to facilitate
the healing of the damaged wound on the skin or apply to the
digestive system for healing of any infection of the digestive
system. The compositions and methods can also be utilized in
various targeted environmental applications, such as in-vitro and
in-vivo uses.
[0100] In some embodiments, the applications of the present
invention include antimicrobials, anthelmintics,
anti-laxatives/anti-diarrheas, analgesics, anti-inflammatories,
cosmetic ingredients, keratolytics, oxidizers for industrial
processes, metal chelating agents, and organic adhesives. In
alternative embodiments, the physiologic uses include antiseptics,
disinfectants, virucides, fungicides, astringents, tissue
adhesives, wound protectants, biofilm preventions,
anti-inflammatories, analgesics, haemostasis, product
preservatives, coagulants, flocculants, oral rinses, irrigants,
debriding agents, gastric tonics, anti-diarrheal, ulcer treatments,
sclerotizing agents, water sanitizers, water preservatives,
oxidizing cleaners, and deodorizers. In alternative embodiments,
the applications include having the humans or animals ingest the
compositions to treat or prevent pathogens or pathogenic molecules
from infecting, damaging, or being absorbed by the tissues of their
digestive systems. In other embodiments, the applications include
the prevention or treatment of animal diarrhea through the
reduction of fluid secretions through astringent,
anti-inflammatory, or anti-microbial action. In some embodiments,
the applications include the treatments of gastric reflux erosions,
peptic ulcers, or other lesions of digestive system. In alternative
embodiments, the applications include the treatments of nasal or
aural cavity irritations or infections. In other embodiments, the
applications include antimicrobial sprays to the respiratory tract
to reduce the pathogens and also to protect the respiratory tract
lining from invasion by the pathogens, such as bacteria, viruses,
and fungi. In some embodiments, the applications include
respiratory tract sprays and sinus rinses to flush the contact
allergens. In alternative embodiments, the applications include
urinary tract rinses for anti-infective or anti-inflammatory
treatments or routine antiseptic rinses for urinary tract implant
and kidney dialysis patients. In other embodiments, the
applications include antiseptic organ preservation for organ
transplantation.
[0101] In some embodiments, the applications include an
antimicrobial wash for bacteria, viruses, and yeast infections on
normal or damaged skin, surface wounds, or in any mucosal cavity.
In alternative embodiments, the applications include tissue
adhesives for accelerated healing, closure, or haemostasis of
surgical incisions or injuries. In other embodiments, the
applications include treatments of surgical incisions or topical
wounds for scar reduction. In some embodiments, the applications
include first aid treatment for topical cuts, burns, or abrasions.
In alternative embodiments, the applications include antiseptic
salves, ointment rinses, or irrigants for oral mucosal ulcer
treatment, and dental procedures. In other embodiments, the
applications include periodontitis treatments and sensitive-tooth
treatments, such as tooth micro-crack sealing. In some embodiments,
the applications include oral rinses for halitosis. In alternative
embodiments, the applications include soaks for dermatitis, jock
itch, vaginal infections, and athlete's foot. In other embodiments,
the applications include burn, chronic wound, and ulcer
antimicrobial and healing treatments. In some embodiments, the
applications include the prevention or reduction of biofilm
formation on tissues or surfaces.
[0102] In alternative embodiments, the applications include
agriculture applications. For example, the surface pathogen can be
reduced and the micro-wounds can be sealed by spraying or soaking
the plant with the polyphenol-oxidizer composition. Further, the
general health of the plants can be improved by strengthening the
surface structure or stimulating enhanced growth or development by
the cross-linking reactions.
[0103] In other embodiments, the applications include aerosol or
liquid sprays of the composition as a bio-security sanitizer for
animal farm facilities. In some embodiments, the applications
include animal feed sterilization. In alternative embodiments, the
applications include food or water additives for preservation and
prevention of disease transmission. In other embodiments, the
applications include plant, fresh fruit, and vegetable washes. The
spraying or rinsing a solution containing the composition disclosed
herein can kill or suppress surface bacteria, extend shelf life,
and protect the surface from or deter pest-invasion in live crops
or agricultural produce. In some embodiments, the applications
include plant seed disinfection for storage and sanitation before
germination. In alternative embodiments, the applications include
preservation spray or water treatment for freshly cut flowers. In
other embodiments, the applications include tissue adhesive for
plant grafting and groundwater remediation.
[0104] In some embodiments, the applications include meat and sea
food preservation spray to reduce bacteria and to form thin
anti-digestive layers to prevent a microbial invasion. The
alternative embodiments include meat processing sanitizers for
prevention of microbial contamination. In alternative embodiments,
the applications include pond water sanitation for fisheries, such
as fish, shrimp, oyster, abalone, and mussels. In other
embodiments, the applications include disease treatment for aquatic
plants and animals. In some embodiments, the applications include
aquarium sanitizers, preservative additives for liquid-containing
products, disinfectant ingredients for surface cleaners, quinone
REDOX cycling coatings for medical devices, clothing and food
preparation equipment, hospital environment and instrument
sanitization, antimicrobial hydrating solutions for hydrophilic
coated medical devices, and organic anti-corrosive treatments for
metals.
[0105] In alternative embodiments, the applications include
industrial water shock, preservatives, or antifoulants. In other
embodiments, the applications include hot tub and swimming pool
water sanitation. In some embodiments, the applications include
carriers for small molecule therapeutic compounds. In alternative
embodiments, the applications include stabilizers for oxidizers,
modification of food flavors, and injection into tumors and
cysts.
[0106] In some embodiments, the compositions are able to be in a
dry powder form. The composition is able to be fed to an animal in
a dry powder form or in a combination with at least one fluid. The
compositions in a dried form are able to contain polyphenol or
polymeric molecules, reactive oxygen species, catalysts, or a
combination thereof. The reactive oxygen species can contain sodium
percarbonate, potassium percarbonate and/or any other substance
that is capable of activating the polyphenol and/or the polymeric
molecules. The reactive oxygen species, the material containing
polyphenol or polymeric molecules, a catalyst, or a combination
thereof are able to be fed to animals concurrently or
separately.
[0107] In some embodiments, the term "polyphenols" used herein
contains more than one phenol unit or building block per molecule.
In alternative embodiments, the term "polyphenols" contains one
phenol unit per molecule. In other embodiments, the term
"polyphenols" includes hydrolysable tannins (Gallic acid esters of
glucose and other sugars) and phenylpropanoids, such as lignins,
flavonoids, and condensed tannins.
[0108] In some embodiments, the hydrogen peroxide added to a
polyphenol contained solution is 1.about.2%. In alternative
embodiments, the hydrogen peroxide added to a polyphenol contained
solution is less than 10%. A person who has ordinary skill in the
art would appreciate that any concentrations of hydrogen peroxide
are applicable so long as the concentration of the hydrogen
peroxide is not too high to overreact with the polyphenols in the
solution. The overreacting reactions include providing a
concentration of hydrogen peroxide, which makes the activated
polyphenols unable to perform the functions described in the
present application.
[0109] In some embodiments, the term "fluid" used herein includes
liquid, gas, supercritical fluid, a mobile solid form of substance,
or a combination thereof. In some embodiments, the term "pathogen"
includes any infectious agents, gems, bacteria, virus, or a
combination thereof. In some embodiments, the term "pathogen"
includes any biological substances that can potentially cause
disease, illness, damage, harm, or negative impact to a host, such
as an animal or another biological substance. In some embodiments,
the term "biopolymer" includes any substances that can be derived
or obtained from a plant, an animal, or biological substances. In
alternative embodiments, the term "biopolymer" includes any
polymeric molecules produced by a biological organism, such as a
live plant. Further, the term "biopolymer" can include cellulose
and starch, proteins and peptides, and DNA and RNA. In some
embodiments, the term "biopolymer" includes plural units of sugars,
amino acids, and nucleotides. In some embodiments, the teen
"binding affinity" includes any intermolecular or intramolecular
interactions and/or bondings. For example, covalent bonds, ionic
bonds, hydrogen bonds, dipole moments, induced dipole moments, and
electrostatic forces. In some embodiments, the term "reactive
proximity" refers to any interaction that exists between two or
more molecules/atoms, so that the two or more molecules are not
randomly freely moving in a solution.
[0110] In some embodiments, the term "effect in inactivating a
pathogen" disclosed herein includes blocking pathogens from
accessing animal/plant tissues, smothering the metabolic pathways
of the pathogens, binding viral factors, immobilizing/aggregating
the pathogens, and/or performing oxidative damages to the
pathogens. In some embodiments, the sources of reactive oxygen
species and/or hydrogen peroxide are able to be obtained from
natural and artificial sources, such as a flesh aloe and/or
cilantro. In some embodiments, the activated polyphenol include
o-polyphenol, oxidized polyphenol, polyphenone, and
polyquinone.
[0111] The term "process fluid" is able to include a fluid that is
artificially and/or biologically processed. The term "biologically
processed" is able to refer that an added composition is processed
by a biological substance, such as an animal. The term
"artificially processed" is able to include filtration,
desiccation, isolation, extraction, or any other manufacturing or
chemical/biological lab processes. For example, the term "process
fluid" is able to include the situation that a dry powder form of
substances is fed to animals and having a fluid of the animals or
added fluid to dissolve or disperse the dry powder, thereby forming
a processed fluid. The dry powder is able to contain both
polyphenols and reactive oxygen species, such as sodium
percarbonate and potassium percarbonate, to be fed to the animals.
Alternatively, the dry powder is able to contain mainly
polyphenols. The reactive oxygen species is able to be fed to the
animal in a liquid form together or separately with the dry powder.
In another alternative embodiment, the dry powder form is able to
contain mainly reactive oxygen species. The polyphenols or hydroxyl
groups contained molecules are able to be fed to the animals in a
liquid form together or separately with the dry powder.
[0112] The following experiments show the effectiveness of the
compositions prepared in accordance with some of the embodiments of
the present invention.
Experiment 1
[0113] Three samples of purified powdered bovine serum albumin
(BSA) were prepared in aqueous solution. Sample #1 contained BSA
only. Sample #2 contained BSA and polyphenol oxidase. Sample #3
contained BSA, polyphenol oxidase, and hydrogen peroxide. Each
sample showed similar steady state turbidity after 30 minutes as
measured by a spectrophotometer. An aqueous solution of polyphenols
(tannin) from Chinese Gall was added to each of the samples. After
one hour, Sample #1 showed little visible change. Sample #2
exhibited an increase in turbidity from increased particle size,
indicating minor protein coagulation. Sample #3 exhibited heavy
precipitate on the bottom of the test tube and a lack of turbidity,
demonstrating that a substantial increase in protein coagulation
can be achieved by the enzymatic reaction of polyphenols with a
source of reactive oxygen species.
Experiment 2
[0114] A solution of Chinese Gall and hydrogen peroxide was added
to (1) a tube containing powdered chicken egg white (the
desiccation processes used in manufacturing powdered egg white
denatures enzymes) reconstituted in water and (2) a tube containing
fresh chicken egg albumin in water. Much greater precipitation was
observed in the fresh chicken egg albumin sample, demonstrating
that plant polyphenols can be catalyzed by enzymes of animal origin
to increase protein binding consistent with quinone formation
exhibited in plant wounding.
Experiment 3
[0115] Formulation A was prepared using the method described below.
One gram of commercial green tea powder was prepared in 1 liter of
deionized water in a 1 liter Pyrex beaker and allowed to extract at
room temperature for 6 hours. 35% food grade hydrogen peroxide was
added to the solution and allowed to sit for 4 hours, then filtered
through a 2 micron mesh media or filter. The resulting stock
solution was diluted 1000:1, 200:1, and 100:1 with 18 Mohan water.
15 ml of dilute solutions was added to equal part culture solutions
containing 10 e7 wild strain E. coli cultures (water controls) and
allowed to incubate at 37.degree. C. At 2 hr, 4 hr, 6 hr, and 8
hrs, a sample from each test series was flooded on agar plates and
incubated, and manual colony counts were performed. The 100:1
sample achieved 100% kill at 4 hrs, the 200:1 achieved 100% kill at
6 hrs, and the 1000:1 was only bacteriostatic. This demonstrates
the feasibility of manufacturing a botanical based composition with
high germicidal capabilities with exceptionally low raw material
and energy input.
Experiment 4
[0116] A wild strain E-Coli culture was added to three samples. The
sample A contained 25 ppm hydrogen peroxide in water. The sample B
contained a solution of Formulation A diluted to equivalent 25 ppm
hydrogen peroxide fraction. The sample C contained a solution
having a green tea extract of the same concentration of Formulation
A but without hydrogen peroxide. The bacteria's population in the
hydrogen peroxide (the sample A) was initially reduced but began to
exhibit increased visual turbidity after 16 hours, indicating the
exhaustion of antimicrobial capability. The green tea extract alone
(the sample C) exhibited little noticeable antimicrobial activity.
Formulation A (the sample B) continued to kill the bacteria and
showed no rebound after 3 days, indicating 100% kill and/or
increased antimicrobial effectiveness, which demonstrates
significantly enhanced germicidal performance resulting from the
combination of green tea extract and hydrogen peroxide.
Experiment 5
[0117] Sample #2 was a pre-prepared solution using the Formulation
A above, which was diluted to duplicate the same polyphenol
concentration as a pre-prepared Sample #1. In Sample #3, polyphenol
and dilute hydrogen peroxide were combined to achieve the same
ratio of polyphenol to hydrogen peroxide as in Sample #2. Serial
dilutions of each sample were prepared and left standing for 24
hours. A solution containing 10e5/ml wild strain E Coli was added
to each sample, which was then incubated and plated out on agar for
visual colony counts. The pre-prepared polyphenol-oxidizer
solutions of Sample #2 continued to kill bacteria effectively at
significantly lower concentrations than in the Sample #3 dilutions,
demonstrating the enhanced performance at low concentrations when a
polyphenol substrate-oxidizer composition is produced at a high
concentration (in the absence of active oxidoreductases or other
reducing agents) before dilution, supporting the concept of
intermolecular force sequestration for improved stability.
Experiment 6
[0118] The following is a method of preparing Formulation B and a
demonstration of a polyphenol substrate extraction process. 30
grams of dried pomegranate rind is used, which was dried at
15.degree. C. for 1 hr, grounded to fine powder, and combined with
10 liters of deionized water heated to 80 C for 20 minutes, then
was cooled to room temperature for 2 hours. 35% food grade hydrogen
peroxide was added and allowed to sit for 6 hrs, and then filtered
through a 5 micron media. The added hydrogen peroxide solution was
less than 10% in solution after added into the solution to prevent
overreacting with the polyphenols. Serial water dilutions of the
resultant solution were added to 10e7-10e8 liquid bacteria
cultures. The resultant solution was incubated for 24 hrs and
visually observed for turbidity. As shown in Table 1, the turbidity
(+ turbid, - not turbid) indicates viable bacteria.
TABLE-US-00001 TABLE 1 Concentration (ug/ml) Bacterium species 500
250 125 62.5 31.3 15.6 7.81 3.9 1.95 0.98 0.49 Control Escherichia
coli - - - - - - - - + + + + Salmonella enterica St. Typh. - - - -
- - - + + + + + Staphylococcus aureus - - - - - - - - + + + +
Pseudomonas aeruginosa - - - - - - - + + + + + Listeria
monocytogenes - - - - - - + + + + + + Pasteurella multocida - - - -
- - - + + + + + Proteus vulgaris - - - - - + + + + + + + Klebsiella
pneumoniae - - - - - - - - + + + + Bacillus cereus - - - - - - - -
+ + + + Bordetella brochiseptica - - - - - - - - + + + +
Experiment 7
[0119] The application of a 200:1 dilution of formulation A to
bilateral symmetric lancet wounds on laboratory mice demonstrated
wound closure in approximately one third of the wounds treated with
a saline control or antibiotic ointment.
Experiment 8
[0120] A cotton pad soaked in a 200:1 dilution of Formulation A was
applied to inflamed oral mucosa for 10 minutes. Of three
volunteers, all experienced significant reduction in pain and
swelling within one hour. Infection was completely resolved in two,
demonstrating anti-inflammatory and anti-infective potential on
mucosal tissue.
Experiment 9
[0121] Ten human volunteers with current and a history of past
symptoms of frequent or chronic diarrhea were given 5 ml of 40:1
dilution of Formulation B in 250 ml of water for 5 days. Nine
expressed significant reduction in discomfort and symptoms for 1
week or more.
Experiment 10
[0122] 0.5 ml of 100:1 dilution of Formulation A and Formulation B
were introduced into a punch well on a sheep blood infused agar
plate that was surface inoculated with e-coli. The perimeter of the
well quickly formed an opaque area of cross linked blood proteins
that were refractory to penetration of the PP--O complex and showed
no bacterial suppression zone around the well. In comparison,
minimal nutrient agar without blood proteins showed a significant
suppression zone indicative of PP--O diffusion through the medium,
demonstrating the low tissue penetration of the compositions
supporting reduced toxicity potential.
Example 11
[0123] 5-day-old Asian Landrace hybrid pigs in a commercial farm
were divided into 13 test subjects and 3 control subjects, all fed
identical quantities and types of feed from birth to 3 months. Test
subjects were given 5 micrograms (dry plant weight equivalent) of
Formulation A every third day in feed water and the same dose daily
if diarrhea was observed. Controls were given antibiotic injections
to treat diarrhea. At 21 days, the test group encountered less
diarrhea and an average were 1.0 kg heavier than the controls. At 3
months the average weight of the test subjects was between 25-30%
greater than that of the controls based on girth measure,
demonstrating Formulation A's viability as an alternative to
antibiotics as a prophylactic and growth promoter.
Experiment 12
[0124] The growth of 99 purebred Landrace piglets was tracked and
evaluated. The piglets were fed an oxidizer-polyphenol composition
of Formulation B in Swine milk replacer. 21-day-old starters tend
to be stressed by environmental transition and have increased
incidences of diarrhea for approximately one week. The experimental
group showed 18% higher average weight gain during this period than
the control group, demonstrating commercial value in growth
optimization and compatibility with milk replacer.
TABLE-US-00002 TABLE 2 Experimental Group Control Group No. of
Piglets 53 46 Dosage 7.5 .mu.g (dry plant wt) 0 .mu.g Frequency
Once per day None Testing period 8 days 8 days Avg. wt. at
beginning 7.00 kg 7.225 kg Avg. Wt. at the end 8.81 kg 8.76 kg
Average weight gain 1.81 kg 1.535 kg
Experiment 13
[0125] Toxicological safety was tested using 10 specific
pathogen-free purebred landrace pigs, each 23 days old. The pigs
were administered one 250 .mu.g or one 2500 .mu.g dose daily for 45
days. Hematology and growth were monitored and histology performed,
showing no negative effect on tissue or organs.
Experiment 14
[0126] 50 weaned starter piglets were divided into 5 groups and
each given 12 .mu.g dose once every third day for 5 weeks.
Statistical analysis shows superior weight gain in experimental
groups. The above experiments are evidence of the effective uses of
polyphenol-oxidizer compositions in the control of pathogens and
show measurable value in numerous commercial and medically useful
applications. Although the experiments demonstrate direct growth
promotion benefits in agricultural animal production, the use of
swine are known to have close physiologic and immunologic
similarities to humans and are commonly used as predictors of
performance and safety in humans. Potential effects can therefore
be projected and claimed for humans. This is supported by direct
experience in the rapid quelling of occasional digestive discomfort
and diarrhea of non-specific causes. Single doses ranging from 20
.mu.g to 250 .mu.g (dry wt. equiv.) have been observed to be
effective in resolving diarrhea in humans, with symptomatic relief
typically noticed within one hour.
[0127] Production
[0128] Some of the embodiments have been produced according to the
methods described herein. The manufacturing process can use organic
production materials and procedures using National Organic Program
(NOP) approved and Generally recognized as Safe (GRAS) food-grade
materials. The manufacturing process can be carried out in a
clean-room laboratory under Good Manufacturing Practice (GMP).
[0129] Some methods of production include botanical pre-process,
protein denaturization and extraction, intramolecular sequestration
of reactants into meta-stable phenolic complex, post processing,
and dilution and formulation with additional ingredients.
[0130] The process disclosed herein is applicable to a wide range
of plant species and tissue types due to the ubiquitous nature of
the chemistry of interest. The sources of the plant can be chosen
based on the availability of cultivation sources, key fraction
content and secondary constituents that can potentially impart
desirable or undesirable characteristic effects, such as toxicity
and auto-degradation.
[0131] The manufacturing of the composition disclosed herein can
incorporate several standardized quality control methods that
directly measure substrate contents, initiator potency,
microbiologic performance, and molecular binding capacity. The
manufacturing of the composition had been tested in room
temperature for over a year. The result shows accelerated stability
testing with retained samples. The microbiologic stability dilution
threshold of the compound has been determined to be above that
which makes it is self-preserving.
[0132] Some of the embodiments have been successfully produced from
a variety different plant bases. Preferred results are obtained
when non-controversial well characterized food plants with a long
history of use in complementary and alternative medicines are used.
A person of ordinary skill in the art would appreciate that many
plants, plant tissues or combinations can be used to make different
formulations for different markets, as long as the working
mechanism is functionally, structurally, chemically, biologically,
or physically equivalent to the embodiments described herein.
[0133] The present invention has been described in teinis of
specific embodiments incorporating details to facilitate the
understanding of principles of construction and operation of the
invention. Such reference herein to specific embodiments and
details thereof is not intended to limit the scope of the claims
appended hereto. It will be readily apparent to one skilled in the
art that various modifications may be made to the embodiments
chosen for illustration without departing from the spirit and scope
of the invention as defined by the appended claims.
* * * * *