U.S. patent application number 13/762647 was filed with the patent office on 2013-06-13 for bioactive compositions from theacea plants and processes for their production and use.
This patent application is currently assigned to AKZO NOBEL SURFACE CHEMISTRY LLC. The applicant listed for this patent is AKZO NOBEL SURFACE CHEMISTRY LLC. Invention is credited to Michael KOGANOV.
Application Number | 20130146481 13/762647 |
Document ID | / |
Family ID | 34806966 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130146481 |
Kind Code |
A1 |
KOGANOV; Michael |
June 13, 2013 |
BIOACTIVE COMPOSITIONS FROM THEACEA PLANTS AND PROCESSES FOR THEIR
PRODUCTION AND USE
Abstract
The present invention relates to isolated bioactive compositions
containing bioactive fractions derived from Theacea plants. The
present invention also relates to bioactive topical formulations
containing the bioactive compositions. The present invention
further relates to methods of using the bioactive compositions of
the present invention, including, for example, methods for
inhibiting inflammatory activity in skin tissue of a mammal, for
protecting skin tissue of a mammal from ultraviolet light-induced
damage, and for normalizing skin disorders in skin tissue of a
mammal. The present invention also relates to methods for isolating
bioactive fractions derived from cell juice or a cell walls
component a Theacea plant.
Inventors: |
KOGANOV; Michael; (White
Plains, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SURFACE CHEMISTRY LLC; AKZO NOBEL |
Chicago |
IL |
US |
|
|
Assignee: |
AKZO NOBEL SURFACE CHEMISTRY
LLC
Chicago
IL
|
Family ID: |
34806966 |
Appl. No.: |
13/762647 |
Filed: |
February 8, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13279901 |
Oct 24, 2011 |
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13762647 |
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12335534 |
Dec 15, 2008 |
8043635 |
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13279901 |
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11033925 |
Jan 12, 2005 |
7473435 |
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12335534 |
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60535861 |
Jan 12, 2004 |
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Current U.S.
Class: |
206/.5 ;
514/456 |
Current CPC
Class: |
A61P 35/02 20180101;
A61K 31/353 20130101; A61P 17/18 20180101; A61P 39/06 20180101;
A61K 8/9789 20170801; A61P 17/16 20180101; A61P 17/00 20180101;
A61P 35/00 20180101; A61Q 17/04 20130101; A61P 29/00 20180101; A61K
36/82 20130101 |
Class at
Publication: |
206/5 ;
514/456 |
International
Class: |
A61K 36/82 20060101
A61K036/82; A61K 31/353 20060101 A61K031/353 |
Claims
1. A bioactive composition comprising: an isolated bioactive
fraction derived from a Theacea plant, wherein said bioactive
fraction is selected from the group consisting of a cell walls
fraction, a cell walls fraction extract, a membrane fraction, a
membrane fraction extract, a cytoplasm fraction, a cytoplasm
fraction extract, a cell juice serum, and combinations thereof.
2. The bioactive composition according to claim 1, wherein said
bioactive fraction is a cell walls fraction.
3. The bioactive composition according to claim 1, wherein said
bioactive fraction is a cell walls fraction extract.
4. The bioactive composition according to claim 3, wherein said
cell walls fraction extract has a total catechin content of between
about 2.1 and about 4.5 milligrams per gram of dry matter.
5. The bioactive composition according to claim 3, wherein said
cell walls fraction extract has a catechin content profile
comprising: between about 2.0 and about 3.0 milligrams of
(+)-catechin per gram of dry matter of the cell walls fraction
extract, between about 0.005 and about 0.02 milligrams of
(-)-epicatechin per gram of dry matter of the cell walls fraction
extract, between about 0.005 and about 0.02 milligrams of
(-)-epigallocatechin gallate per gram of dry matter of the cell
walls fraction extract, and between about 0.003 and about 0.01
milligrams of (-)-epicatechin gallate per gram of dry matter of the
cell walls fraction extract.
6. The bioactive composition according to claim 1, wherein said
bioactive fraction is a membrane fraction.
7. The bioactive composition according to claim 1, wherein said
bioactive fraction is a membrane fraction extract.
8. The bioactive composition according to claim 7, wherein said
membrane fraction extract has a total catechin content of between
about 15.0 and about 30.5 milligrams per gram of dry matter.
9. The bioactive composition according to claim 7, wherein said
membrane fraction extract has a catechin content profile
comprising: between about 1.7 and about 3.3 milligrams of
(-)-epigallocatechin per gram of dry matter of the membrane
fraction extract, between about 6.1 and about 10.2 milligrams of
(+)-catechin per gram of dry matter of the membrane fraction
extract, between about 0.3 and about 1.1 milligrams of
(-)-epicatechin per gram of dry matter of the membrane fraction
extract, between about 6.2 and about 12.5 milligrams of
(-)-epigallocatechin gallate per gram of dry matter of the membrane
fraction extract, between about 0.007 and about 0.03 milligrams of
(-)-gallocatechin gallate per gram of dry matter of the membrane
fraction extract, and between about 1.3 and about 3.3 milligrams of
(-)-epicatechin gallate per gram of dry matter of the membrane
fraction extract.
10. The bioactive composition according to claim 1, wherein said
bioactive fraction is a cytoplasm fraction.
11. The bioactive composition according to claim 1, wherein said
bioactive fraction is a cytoplasm fraction extract.
12. The bioactive composition according to claim 1, wherein said
bioactive fraction is a cell juice serum.
13. The bioactive composition according to claim 12, wherein said
cell juice serum has a total catechin content of between about 8.0
and about 20.0 milligrams per gram of dry matter.
14. The bioactive composition according to claim 12, wherein said
cell juice serum has a catechin content profile comprising: between
about 2.1 and about 4.4 milligrams of (-)-epigallocatechin per gram
of dry matter of the cell juice serum, between about 4.2 and about
8.6 milligrams of (+)-catechin per gram of dry matter of the cell
juice serum, between about 0.2 and about 2.0 milligrams of
(-)-epicatechin per gram of dry matter of the cell juice serum,
between about 1.2 and about 3.2 milligrams of (-)-epigallocatechin
gallate per gram of dry matter of the cell juice serum, between
about 0.01 and about 0.1 milligrams of (-)-gallocatechin gallate
per gram of dry matter of the cell juice serum, and between about
0.2 and about 1.3 milligrams of (-)-epicatechin gallate per gram of
dry matter of the cell juice serum.
15. The bioactive composition according to claim 1, wherein said
Theacea plant is a Camellia plant or a Eurya plant.
16. The bioactive composition according to claim 15, wherein said
Camellia plant is selected from the group consisting of Camellia
sinensis, Camellia japonica, Camellia reticulate, and Camellia
sasanqua.
17. The bioactive composition according to claim 15, wherein said
Eurya plant is Eurya sandwicensis.
18. The bioactive composition according to claim 1 further
comprising a stabilizing agent.
19. The bioactive composition according to claim 18, wherein said
stabilizing agent is selected from the group consisting of an
emulsifier, a preservative, an antioxidant, a polymer matrix, and
mixtures thereof.
20. A bioactive topical formulation suitable for topical
application to a mammal, said bioactive topical formulation
comprising: a topically effective amount of the bioactive
composition according to claim 1 and a topically acceptable
carrier.
21. The bioactive topical formulation according to claim 20,
wherein the topically acceptable carrier is selected from the group
consisting of a hydrophilic cream base, a hydrophilic lotion base,
a hydrophilic surfactant base, a hydrophilic gel base, a
hydrophilic solution base, a hydrophobic cream base, a hydrophobic
lotion base, a hydrophobic surfactant base, a hydrophobic gel base,
and a hydrophobic solution base.
22. The bioactive topical formulation according to claim 20,
wherein the bioactive composition is present in an amount ranging
from between about 0.001 percent and about 90 percent of the total
weight of the bioactive topical formulation.
23-58. (canceled)
59. A beverage comprising at least one bioactive composition
according to claim 1.
60. The beverage according to claim 59, wherein said beverage
comprises the at least one bioactive composition dispersed in a
liquid.
61. The beverage according to claim 60, wherein said liquid is
selected from the group consisting of water, green tea, oolong tea,
black tea, white tea, flavored tea, soft drink, coffee, milk,
shake, alcoholic drink, non-alcoholic drink, sports drink, fruit
juice, vegetable juice, artificially sweetened juice, sparkling
water, punch, cider, and nutritional supplement drink.
62. The beverage according to claim 59, wherein said Theacea plant
is a Camellia plant or a Eurya plant.
63. The beverage according to claim 62, wherein said Camellia plant
is selected from the group consisting of Camellia sinensis,
Camellia japonica, Camellia reticulate, and Camellia sasanqua, and
wherein said Eurya plant is Eurya sandwicensis.
64. A therapeutic beverage comprising at least one bioactive
composition according to claim 1.
65. The therapeutic beverage according to claim 64, wherein said
beverage comprises the at least one bioactive composition dispersed
in a liquid.
66. The therapeutic beverage according to claim 65, wherein said
liquid is selected from the group consisting of water, green tea,
oolong tea, black tea, white tea, flavored tea, soft drink, coffee,
milk, shake, alcoholic drink, non-alcoholic drink, sports drink,
fruit juice, vegetable juice, artificially sweetened juice,
sparkling water, punch, cider, and nutritional supplement
drink.
67. The therapeutic beverage according to claim 64, wherein said
Theacea plant is a Camellia plant or a Eurya plant.
68. The therapeutic beverage according to claim 67, wherein said
Camellia plant is selected from the group consisting of Camellia
sinensis, Camellia japonica, Camellia reticulate, and Camellia
sasanqua, and wherein said Eurya plant is Eurya sandwicensis.
69. A nutriceutical product comprising at least one bioactive
composition according to claim 1.
70. The nutriceutical product according to claim 69, wherein said
Theacea plant is a Camellia plant or a Eurya plant.
71. The nutriceutical product according to claim 70, wherein said
Camellia plant is selected from the group consisting of Camellia
sinensis, Camellia japonica, Camellia reticulate, and Camellia
sasanqua, and wherein said Eurya plant is Eurya sandwicensis.
72. A functional food product comprising at least one bioactive
composition according to claim 1.
73. The functional food product according to claim 72, wherein said
Theacea plant is a Camellia plant or a Eurya plant.
74. The functional food product according to claim 73, wherein said
Camellia plant is selected from the group consisting of Camellia
sinensis, Camellia japonica, Camellia reticulate, and Camellia
sasanqua, and wherein said Eurya plant is Eurya sandwicensis.
75. A device for dispersing at least one bioactive composition from
a Theacea plant into a liquid in order to prepare a beverage, said
device comprising: a filtering pouch or bag comprising a
semi-permeable membrane; and at least one bioactive composition
according to claim 1 contained in the filtering pouch or bag.
76. The device according to claim 75, wherein said liquid is
selected from the group consisting of water, green tea, oolong tea,
black tea, white tea, flavored tea, soft drink, coffee, milk,
shake, alcoholic drink, non-alcoholic drink, sports drink, fruit
juice, vegetable juice, artificially sweetened juice, sparkling
water, punch, cider, and nutritional supplement drink.
77. The device according to claim 75, wherein said beverage is a
therapeutic beverage.
78. The device according to claim 75, wherein said Theacea plant is
a Camellia plant or a Eurya plant.
79. The device according to claim 78, wherein said Camellia plant
is selected from the group consisting of Camellia sinensis,
Camellia japonica, Camellia reticulate, and Camellia sasanqua, and
wherein said Eurya plant is Eurya sandwicensis.
80. A method of preparing a beverage, said method comprising the
steps of: providing a device according to claim 75; contacting the
device with a liquid under conditions effective to disperse the at
least one bioactive composition of the device into the liquid,
thereby yielding a beverage comprising the at least one bioactive
composition.
81. The method according to claim 80, wherein said device is
contacted with the liquid under conditions effective to cause the
low molecular weight and reduced, non-oxidized components of the at
least one bioactive composition to disperse in the liquid.
82. The method according to claim 80, wherein said liquid is
selected from the group consisting of water, green tea, oolong tea,
black tea, white tea, flavored tea, soft drink, coffee, milk,
shake, alcoholic drink, non-alcoholic drink, sports drink, fruit
juice, vegetable juice, artificially sweetened juice, sparkling
water, punch, cider, and nutritional supplement drink.
83. The method according to claim 80, wherein said beverage is a
therapeutic beverage.
84. The method according to claim 80, wherein said Theacea plant is
a Camellia plant or a Eurya plant.
85. The method according to claim 84, wherein said Camellia plant
is selected from the group consisting of Camellia sinensis,
Camellia japonica, Camellia reticulate, and Camellia sasanqua, and
wherein said Eurya plant is Eurya sandwicensis.
86. A formulation for systemic or topical administration of at
least one bioactive composition from a Theacea plant, said
formulation comprising: a solution, suspension, dispersion, paste,
or dried powder comprising at least one bioactive composition
according to claim 1.
87. The formulation according to claim 86, wherein said Theacea
plant is a Camellia plant or a Eurya plant.
88. The formulation according to claim 87, wherein said Camellia
plant is selected from the group consisting of Camellia sinensis,
Camellia japonica, Camellia reticulate, and Camellia sasanqua, and
wherein said Eurya plant is Eurya sandwicensis.
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/535,861, filed Jan. 12, 2004.
FIELD OF THE INVENTION
[0002] The present invention relates to bioactive compositions,
processes for their production from Theacea plants, and uses of
these compositions.
BACKGROUND OF THE INVENTION
[0003] The Theacea (tea plants) family includes trees or shrubs
comprising about 40 genera and 600 species. Camellia sinensis
occupies a unique position in the Theacea family, because this
particular species of plant is predominantly used as a single raw
material source to produce all three basic kinds of tea: green tea,
oolong tea, and black tea (collectively referred to herein as the
"tea plant"). According to some sources, there is a fourth type of
tea, i.e., the so-called "white tea," which is produced exclusively
from the buds or tips of the tea plant.
[0004] The three basic forms of tea are determined by the degree of
processing, which involves the identical tender young tea leaves.
The leaves are plucked, sorted, cleaned, and variously oxidized
before steaming or drying. The term "fermentation" is frequently
used to describe the processing of tea, but the term "oxidation" is
a much more accurate description of the chemical transformations
which take place.
[0005] Although there are some variations in the processing, it is
generally agreed that green tea has the lowest degree of oxidation
and that black tea has the highest. Oolong tea is considered to be
partially oxidized, and thus occupies the place between green and
black tea. With respect to processing, there is very little
difference (or no difference at all) between green and white
tea.
[0006] Green tea is made from fresh leaves that are steamed and
wilted, and then immediately dried. Black tea is made from leaves
that are wilted and crushed in rollers, then allowed to oxidize for
several hours before they are dried. Oolong tea comes from leaves
that are only partially oxidized before drying.
[0007] Worldwide, tea is the second (after water) most commonly
consumed liquid, and is the sixth (after water, soft drinks,
coffee, beer, and milk) most commonly consumed liquid in the United
States. Tea consumption continues to increase worldwide, especially
due to the growing public awareness concerning health benefits of
this liquid. There is a growing number of publications suggesting
anti-angiogenic, anti-bacterial, anti-cancerogenic,
anti-inflammatory, anti-mutagenic, anti-oxidant, anti-septic, and
detoxifying properties of teas and their ingredients. The list of
tea benefits also includes reduction of the risk of rheumatoid
arthritis, lowering cholesterol levels, and anti-diabetic
properties. Not all of these benefits have been proven to be
statistically significant. Nevertheless, the very broad spectrum of
tea benefits reflects the unique composition of the very powerful
biologically active substances, which exist in fresh plant leaves
and survive conventional tea processing.
[0008] In particular, fresh leaves of Camellia sinensis have been
reported to contain 22.2% polyphenols, 17.2% protein, 4.3%
caffeine, 27.0% crude fiber, 0.5% starch, 3.5% reducing sugars,
6.5% pectins, 2.0% ether extract, and 5.6% ash (Duke, J. A.,
Handbook of Energy Crops (1983), see
www.hort.purdue.edu/newcrop/duke_energy/Camellia_sinensis.html).
Per 100 g, the leaf is reported to contain 8.0 g H.sub.2O, 24.5 g
protein, 2.8 g fat, 58.8 g total carbohydrate, 8.7 g fiber, 5.9 g
ash, 327 mg Ca, 313 mg P, 24.3 mg Fe, 50 mg Na, 2700 .mu.g
.beta.-carotene equivalent, 0.07 mg thiamine, 0.8 mg riboflavin,
7.6 mg niacin, and 9 mg ascorbic acid. Another report tallies 8.0 g
H.sub.2O, 28.3 g protein, 4.8 g fat, 53.6 g total carbohydrate, 9.6
g fiber, 5.6 g ash, 245 mg Ca, 415 mg P, 18.9 mg Fe, 60 mg Na, 8400
.mu.g .beta.-carotene equivalent, 0.38 mg thiamine, 1.24 mg
riboflavin, 4.6 mg niacin, and 230 mg ascorbic acid. Yet another
gives 8.1 g H.sub.2O, 24.1 g protein, 3.5 g fat, 59.0 g total
carbohydrate, 9.7 g fiber, 5.3 g ash, 320 mg Ca, 185 mg P, 31.6 mg
Fe, 8400 .mu.g .beta.-carotene equivalent, 0.07 mg thiamine, 0.79
mg riboflavin, 7.3 mg niacin, and 85 mg ascorbic acid (J. A. Duke
and A. A. Atchley, "Proximate Analysis," In: Christie, B. R. (ed.),
The Handbook of Plant Science in Agriculture, CRC Press, Inc., Boca
Raton, Fla. (1984)).
[0009] Leaves also contain carotene, riboflavin, nicotinic acid,
pantothenic acid, and ascorbic acid. Caffeine and tannin are among
the more active constituents (Council for Scientific and Industrial
Research, 1948-1976). Ascorbic acid, present in the fresh leaf, is
destroyed in making black tea. Malic and oxatic acids occur, along
with kaempferol, quercitrin, theophylline, theobromine, xanthine,
hypoxanthine, adenine, gums, dextrins, and inositol. Chief
components of the volatile oil (0.007-0.014% fresh weight of
leaves) are hexenal, hexenol, and lower aldehydes, butyraldehyde,
isobuteraldehyde, isovaleraldehyde, as well as n-hexyl, benzyl and
phenylethyl alcohols, phenols, cresol, hexoic acid, n-octyl
alcohol, geraniol, linalool, acetophenone, benzyl alcohol, and
citral.
[0010] It was found that the fresh tea leaf has an unusually high
level of flavanol group of polyphenols (catechins), which may reach
up to 30% of leaf dry matter. Catechins include predominantly
(-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin and
(-)-epigallocatechin gallate. Additionally there are unique to tea
3-galloylquinic acid (theogallin) and unique amino acid theanine
(5-N-ethylglutamine) (Duke, J. A., Handbook of Energy Crops (1983),
see
www.hort.purdue.edu/newcrop/duke_energy/Camellia_sinensis.html).
[0011] Tea leaves contain high levels of polyphenol-oxidase and
peroxidase. The first enzyme catalyzes the aerobic oxidation of the
catechins and this process is initiated when the integrity of the
leaf cell structure is disrupted. Phenol-oxidase is responsible for
generation of bisflavanols, theaflavins, epitheaflavic acids, and
thearubigens, which constitute the largest mass of the extractable
matter in black tea. Most of these compounds readily form complexes
with caffeine, which has significant level (2-4% of dry matter) in
fresh leaves. Peroxidase plays important role in generation of the
above complexes with proanthocyanidins. The catechin quinones also
initiate the formation of many of the hundreds of volatile
compounds found in the black tea aroma fraction. Additionally, the
transformation of relatively soluble glycosides to lower solubility
aglycones takes place.
[0012] All complex cascades of the above processes are initiated by
disruption of the leaf cell structure and are intensified with the
time of oxidation. As result, the composition of black tea, which
is usually processed with intensive rolling or cutting and
relatively long time oxidation, is much more different than that of
the fresh leaf. Although green tea (and white tea) is processed
with minimum oxidation, and its composition more similar to that of
fresh leaves, there are non-enzymatic and enzymatically catalyzed
changes, which occur extremely rapidly following plucking, and new
volatile substances that are produced during the drying stage.
Thus, even relatively gentle green tea processing initiates certain
departure from original fresh plant composition and can diminish
the therapeutic value and other potential benefits of fresh tea
plant leaves.
[0013] Numerous recent studies clearly demonstrate that therapeutic
benefits of tea are decreased in the following sequence: white
tea>green tea>oolong tea>black tea. Thus, exploration of
fresh tea plants may prevent the degradation of specific
activities, which are observed as a result of conventional tea
processing. Fresh, tender Camellia leaves contain approximately 80%
water. Swelling and dehydration of the cells is prevented by the
cells' rigid cell walls. The disruption of the cell wall structure
triggers the dehydration of fresh plant tissue followed by the
sequence of unwanted physico-chemical and biochemical processes:
osmotic shock, decompartmentalization and disruption of enzymes,
hydrolysis and oxidation, polymerization of phenols, transformation
of glycosides to aglycones, generation of products of Maillard
reaction, isomerization, and microbial contamination. Therefore,
fresh Camellia contains very broad spectrum of biologically active
substances and only part of them became available during
conventional extraction processes. Thus, only cell walls,
catabolites, and stable metabolites can be extracted with boiled
water to obtain tea drink or for extraction with different solvents
to obtain limited parts of biologically active components
(predominantly polyphenols and flavonoids).
[0014] In light of the potential of fresh tea leaves as sources of
valuable therapeutic and other potentially beneficial bioactive
compositions, exploration of fresh tea plants is needed to
determine how to maximize their therapeutic and other potentially
beneficial bioactive properties.
SUMMARY OF THE INVENTION
[0015] The present invention relates to a bioactive composition. In
one embodiment, the bioactive composition includes an isolated
bioactive fraction derived from a Theacea plant. Suitable bioactive
fractions can include, without limitation, a cell walls fraction, a
cell walls fraction extract, a membrane fraction, a membrane
fraction extract, a cytoplasm fraction, a cytoplasm fraction
extract, a cell juice serum, and/or combinations thereof.
[0016] The present invention also relates to a bioactive topical
formulation suitable for topical application to a mammal. In one
embodiment, the bioactive topical formulation includes a topically
effective amount of the bioactive composition of the present
invention. The bioactive topical formulation can further include a
topically acceptable carrier.
[0017] The present invention also relates to a method for
inhibiting inflammatory activity in skin tissue of a mammal. This
method involves providing the bioactive composition according to
the present invention. The method further involves applying the
bioactive composition to the skin tissue in an amount effective to
inhibit inflammatory activity in the skin tissue.
[0018] The present invention also relates to a method of protecting
skin tissue of a mammal from ultraviolet light-induced damage. This
method involves providing the bioactive composition of the present
invention. The method further involves applying the bioactive
composition to the skin tissue in an amount effective to reduce
ultraviolet light-induced damage of the skin tissue and to prevent
oxidative damage of the skin tissue.
[0019] The present invention also relates to a method for
normalizing skin disorders in skin tissue of a mammal. This method
involves providing the bioactive composition of the present
invention. The method further involves applying the bioactive
composition to the skin tissue in an amount effective to normalize
a cell disorder in the skin tissue.
[0020] The present invention also relates to a method for isolating
a bioactive fraction derived from cell juice of a Theacea plant.
This method involves providing a Theacea plant. The Theacea plant
is then separated into cell juice and a cell walls component. The
cell juice is then treated under conditions effective to yield a
bioactive fraction. Suitable bioactive fractions include, without
limitation, a membrane fraction, a membrane fraction extract, a
cytoplasm fraction, a cytoplasm fraction extract, and/or a cell
juice serum. The bioactive fraction is then isolated from the
treated cell juice. The present invention further relates to an
isolated bioactive composition produced by this method.
[0021] The present invention also relates to a method for isolating
a bioactive fraction derived from a cell walls component of a
Theacea plant. This method involves providing a Theacea plant. The
Theacea plant is then separated into cell juice and a cell walls
component. The cell walls component is treated under conditions
effective to yield a bioactive fraction. The bioactive fraction is
then isolated from the treated cell walls component. The present
invention further relates to an isolated bioactive fraction
produced by this method.
[0022] The present invention is useful in addressing the
deficiencies of conventional tea processing methods, particularly
the inability of conventional tea processing to preserve a broad
spectrum of potent bioactive compositions. As provided by the
present invention, processing of fresh Camellia biomass without
fermentation and excessive heat treatment can yield more powerful
and diversified bioactive compositions than products of
conventional tea processing.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1 is a schematic drawing demonstrating one embodiment
of the process for preparing the bioactive compositions of the
present invention.
[0024] FIG. 2 is a graph showing the UV/VIS spectra of extracts of
cell walls fraction and conventional teas (Dilution 1:1000).
[0025] FIG. 3 is a graph showing the UV/VIS spectra of Camellia
bioactive compositions (Dilution 1:4000).
[0026] FIG. 4 is a graph showing the absorbance spectra of extracts
of cell walls fraction and conventional teas applied on
Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.). Dry matter levels are equalized.
[0027] FIG. 5 is a graph showing the absorbance spectra of Camellia
bioactive compositions applied on Vitro-Skin.RTM. testing substrate
(IMS Testing Group, Milford, Conn.). Dry matter levels are
equalized.
[0028] FIG. 6 is a graph showing the absorbance spectra of Camellia
bioactive compositions and white tea extract applied on
Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.).
[0029] FIG. 7A is a graph showing the absorbance spectra of
Camellia membrane fraction extract in diluted solution (1:200) and
applied on Vitro-Skin.RTM. testing substrate (IMS Testing Group,
Milford, Conn.). FIG. 7B is a graph showing the absorbance spectra
of Camellia cell juice serum in diluted solution (1:200) and
applied on Vitro-Skin.RTM. testing substrate (IMS Testing Group,
Milford, Conn.).
[0030] FIG. 8A is a graph showing the absorbance spectra of Barley
(Hordeum vulgare) cell juice serum in diluted solution (1:200) and
applied on Vitro-Skin.RTM. testing substrate (IMS Testing Group,
Milford, Conn.). FIG. 8B is a graph showing the absorbance spectra
of Sage (Salvia officinalis) cell juice serum in diluted solution
(1:200) and applied on Vitro-Skin.RTM. testing substrate (IMS
Testing Group, Milford, Conn.).
[0031] FIG. 9 is a graph showing the effect of broad spectrum UV
irradiation on Vitro-Skin.RTM. testing substrate (IMS Testing
Group, Milford, Conn.).
[0032] FIG. 10 is a graph showing the effect of broad spectrum UV
irradiation on white tea extract applied on Vitro-Skin.RTM. testing
substrate (IMS Testing Group, Milford, Conn.).
[0033] FIG. 11 is a graph showing the effect of broad spectrum UV
irradiation on cell walls fraction extract applied on
Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.).
[0034] FIG. 12 is a graph showing the effect of broad spectrum UV
radiation on Camellia membrane fraction extract applied on
Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.).
[0035] FIG. 13 is a graph showing the effect of broad spectrum UV
radiation on Camellia cell juice serum applied on Vitro-Skin.RTM.
testing substrate (IMS Testing Group, Milford, Conn.).
[0036] FIG. 14 is a graph showing the effect of white tea extract
on MDA-MB-435S cells cultivated for 24 hours and 48 hours.
[0037] FIG. 15 is a graph showing the effect of white tea extract
on MCF-7 cells cultivated for 24 hours (control) and for 24 hours
and 48 hours in the presence of 5 ng/ml TGF-.beta..
[0038] FIG. 16 is a graph showing the effect of cell walls fraction
extract on MDA-MB-435S cells cultivated for 24 hours and 48
hours.
[0039] FIG. 17 is a graph showing the effect of cell walls fraction
extract on MCF-7 cells cultivated for 24 hours (control) and for 24
hours and 48 hours in the presence of 5 ng/ml TGF-.beta..
[0040] FIG. 18 is a graph showing the effect of membrane fraction
extract on MDA-MB-435S cells cultivated for 24 hours and 48
hours.
[0041] FIG. 19 is a graph showing the effect of membrane fraction
extract on MCF-7 cells cultivated for 24 hours (control) and for 24
hours and 48 hours in the presence of 5 ng/ml TGF-.beta..
[0042] FIG. 20 is a graph showing the effect of cell juice serum on
MDA-MB-435S cells cultivated for 24 hours and 48 hours.
[0043] FIG. 21 is a graph showing the effect of cell juice serum on
MCF-7 cells cultivated for 24 hours (control) and for 24 hours and
48 hours in the presence of 5 ng/ml TGF-.beta..
[0044] FIG. 22 is a graph showing the effect of white tea extract
on Mono Mac 6 cells cultivated for 24 and 48 hours.
[0045] FIG. 23 is a graph showing the effect of white tea extract
on Mono Mac 6 cells cultivated for 24 hours and 48 hours in the
presence of 10 nM PMA.
[0046] FIG. 24 is a graph showing the effect of cell walls fraction
extract on Mono Mac 6 cells cultivated for 24 hours and 48
hours.
[0047] FIG. 25 is a graph showing the effect of cell walls fraction
extract on Mono Mac 6 cells cultivated for 24 hours and 48 hours in
the presence of 10 nM PMA.
[0048] FIG. 26 is a graph showing the effect of membrane fraction
extract on Mono Mac 6 cells cultivated for 24 hours and 48
hours.
[0049] FIG. 27 is a graph showing the effect of membrane fraction
extract on Mono Mac 6 cells cultivated for 24 hours and 48 hours in
the presence of 10 nM PMA.
[0050] FIG. 28 is a graph showing the effect of cell juice serum on
Mono Mac 6 cells cultivated for 24 hours and 48 hours.
[0051] FIG. 29 is a graph showing the effect of cell juice serum on
Mono Mac 6 cells cultivated for 24 hours and 48 hours in the
presence of 10 nM PMA.
[0052] FIG. 30 is a graph showing the effect of white tea extract
on level of MMPs secreted by PMA stimulated Mono Mac 6 cells.
[0053] FIG. 31 is a graph showing the effect of cell walls fraction
extract on level of MMPs secreted by PMA stimulated Mono Mac 6
cells.
[0054] FIG. 32 is a graph showing the effect of membrane fraction
extract on level of MMPs secreted by PMA stimulated Mono Mac 6
cells.
[0055] FIG. 33 is a graph showing the effect of cell juice serum on
level of MMPs secreted by PMA stimulated Mono Mac 6 cells.
[0056] FIG. 34 is a gelatin zymogram of culture media collected
after 48 hours exposure of Mono Mac 6 cells to white tea extract,
along with culture media collected from cells cultured in the
absence (U) or presence (S) of 10 nM PMA, but in the absence of the
Camellia compositions.
[0057] FIG. 35 is a gelatin zymogram of culture media collected
after 48 hours exposure of Mono Mac 6 cells to cell walls fraction
extract, along with culture media collected from cells cultured in
the absence (U) or presence (S) of 10 nM PMA, but in the absence of
the Camellia compositions.
[0058] FIG. 36 is a gelatin zymogram of culture media collected
after 48 hours exposure of Mono Mac 6 cells to membrane fraction
extract, along with culture media collected from cells cultured in
the absence (U) or presence (S) of 10 nM PMA, but in the absence of
the Camellia compositions.
[0059] FIG. 37 is a gelatin zymogram of culture media collected
after 48 hours exposure of Mono Mac 6 cells to cell juice serum,
along with culture media collected from cells cultured in the
absence (U) or presence (S) of 10 nM PMA, but in the absence of the
Camellia compositions.
[0060] FIG. 38 is a bar graph comparing the content of various
catechins in the white tea extract ("WTE") and in the cell walls
fraction extract ("CWFE"), the membrane fraction extract ("MFE"),
and the cell juice serum ("CJS") of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0061] The present invention relates to a bioactive composition. In
one embodiment, the bioactive composition includes an isolated
bioactive fraction derived from a Theacea plant. As used herein,
the term "isolated bioactive fraction" is meant to include
fractions that are isolated from a Theacea plant (e.g., fresh
biomass of a Theacea plant) that has not undergone any conventional
tea processing (e.g., heat treatment, oxidation, fermentation,
drying). Suitable isolated bioactive fractions can include, without
limitation, a cell walls fraction, a cell walls fraction extract, a
membrane fraction, a membrane fraction extract, a cytoplasm
fraction, a cytoplasm fraction extract, a cell juice serum, and/or
combinations thereof.
[0062] The bioactive compositions and bioactive fractions of the
present invention can have various catechin profiles and total
catechin content amounts, as defined below, and as determined using
conventional catechin diagnostic methods well known in the art. As
used herein, the term "catechin" generally refers to all catechins,
including, but not limited to, the following specific types of
catechins: (i) (-)-epigallocatechin (see CAS No. 970-74-1, which is
hereby incorporated by reference in its entirety); (ii)
(+)-catechin (see CAS No. 7295-85-4, which is hereby incorporated
by reference in its entirety); (iii) (-)-epicatechin (see CAS No.
490-46-0, which is hereby incorporated by reference in its
entirety); (iv) (-)-epigallocatechin gallate (see CAS No. 989-51-5,
which is hereby incorporated by reference in its entirety); (v)
(-)-gallocatechin gallate (see CAS No. 4233-96-9, which is hereby
incorporated by reference in its entirety); and (vi)
(-)-epicatechin gallate (see CAS No. 1257-08-5, which is hereby
incorporated by reference in its entirety). "Total catechin
content" (as used herein) refers to the combined content level of
all catechins contained in a particular bioactive composition or
bioactive fraction of the present invention, and is not meant to be
limited to the content levels of just the specific types of
catechins listed herein above. As used herein, the term "catechin
content profile" is used to describe the amounts of selected
catechins contained in a particular bioactive composition or
bioactive fraction of the present invention.
[0063] In one embodiment of the bioactive composition of the
present invention, the bioactive fraction can be a cell walls
fraction.
[0064] In one embodiment of the bioactive composition of the
present invention, the bioactive fraction can be a cell walls
fraction extract. In a specific embodiment of the present
invention, the cell walls fraction extract can have a total
catechin content of between about 2.1 and about 4.5 milligrams per
gram of dry matter, particularly between about 2.6 and about 4.0
milligrams per gram of dry matter, and more particularly between
about 3.0 and about 3.6 milligrams per gram of dry matter. In
another specific embodiment, the cell walls fraction extract can
have a catechin content profile as follows: (i) between about 2.0
and about 3.0 milligrams of (+)-catechin per gram of dry matter of
the cell walls fraction extract; (ii) between about 0.005 and about
0.02 milligrams of (-)-epicatechin per gram of dry matter of the
cell walls fraction extract; (iii) between about 0.005 and about
0.02 milligrams of (-)-epigallocatechin gallate per gram of dry
matter of the cell walls fraction extract; and (iv) between about
0.003 and about 0.01 milligrams of (-)-epicatechin gallate per gram
of dry matter of the cell walls fraction extract. More
particularly, the cell walls fraction extract can have a catechin
content profile as follows: (i) between about 2.2 and about 2.7
milligrams of (+)-catechin per gram of dry matter of the cell walls
fraction extract; (ii) between about 0.01 and about 0.015
milligrams of (-)-epicatechin per gram of dry matter of the cell
walls fraction extract; (iii) between about 0.01 and about 0.015
milligrams of (-)-epigallocatechin gallate per gram of dry matter
of the cell walls fraction extract; and (iv) between about 0.005
and about 0.007 milligrams of (-)-epicatechin gallate per gram of
dry matter of the cell walls fraction extract.
[0065] In one embodiment of the bioactive composition of the
present invention, the bioactive fraction can be a membrane
fraction.
[0066] In one embodiment of the bioactive composition of the
present invention, the bioactive fraction can be a membrane
fraction extract. In a specific embodiment of the present
invention, the membrane fraction extract can have a total catechin
content of between about 15.0 and about 30.5 milligrams per gram of
dry matter, particularly between about 18.0 and about 27.5
milligrams per gram of dry matter, and more particularly between
about 21.0 and about 24.5 milligrams per gram of dry matter. In
another specific embodiment, the membrane fraction extract can have
a catechin content profile as follows: (i) between about 1.7 and
about 3.3 milligrams of (-)-epigallocatechin per gram of dry matter
of the membrane fraction extract; (ii) between about 6.1 and about
10.2 milligrams of (+)-catechin per gram of dry matter of the
membrane fraction extract; (iii) between about 0.3 and about 1.1
milligrams of (-)-epicatechin per gram of dry matter of the
membrane fraction extract; (iv) between about 6.2 and about 12.5
milligrams of (-)-epigallocatechin gallate per gram of dry matter
of the membrane fraction extract; (v) between about 0.007 and about
0.03 milligrams of (-)-gallocatechin gallate per gram of dry matter
of the membrane fraction extract; and (vi) between about 1.3 and
about 3.3 milligrams of (-)-epicatechin gallate per gram of dry
matter of the membrane fraction extract. More particularly, the
membrane fraction extract can have a catechin content profile as
follows: (i) between about 2.0 and about 3.0 milligrams of
(-)-epigallocatechin per gram of dry matter of the membrane
fraction extract; (ii) between about 7.0 and about 9.0 milligrams
of (+)-catechin per gram of dry matter of the membrane fraction
extract; (iii) between about 0.5 and about 0.9 milligrams of
(-)-epicatechin per gram of dry matter of the membrane fraction
extract; (iv) between about 8.0 and about 10.0 milligrams of
(-)-epigallocatechin gallate per gram of dry matter of the membrane
fraction extract; (v) between about 0.01 and about 0.02 milligrams
of (-)-gallocatechin gallate per gram of dry matter of the membrane
fraction extract; and (vi) between about 1.8 and about 2.8
milligrams of (-)-epicatechin gallate per gram of dry matter of the
membrane fraction extract.
[0067] In one embodiment of the bioactive composition of the
present invention, the bioactive fraction can be a cytoplasm
fraction.
[0068] In one embodiment of the bioactive composition of the
present invention, the bioactive fraction can be a cytoplasm
fraction extract.
[0069] In one embodiment of the bioactive composition of the
present invention, the bioactive fraction can be a cell juice
serum. In a specific embodiment, the cell juice serum can have a
total catechin content of between about 8.0 and about 20.0
milligrams per gram of dry matter, particularly between about 10.0
and about 18.0 milligrams per gram of dry matter, and more
particularly between about 12.0 and about 16.0 milligrams per gram
of dry matter. In another specific embodiment, the cell juice serum
can have a catechin content profile as follows: (i) between about
2.1 and about 4.4 milligrams of (-)-epigallocatechin per gram of
dry matter of the cell juice serum; (ii) between about 4.2 and
about 8.6 milligrams of (+)-catechin per gram of dry matter of the
cell juice serum; (iii) between about 0.2 and about 2.0 milligrams
of (-)-epicatechin per gram of dry matter of the cell juice serum;
(iv) between about 1.2 and about 3.2 milligrams of
(-)-epigallocatechin gallate per gram of dry matter of the cell
juice serum; (v) between about 0.01 and about 0.1 milligrams of
(-)-gallocatechin gallate per gram of dry matter of the cell juice
serum; and (vi) between about 0.2 and about 1.3 milligrams of
(-)-epicatechin gallate per gram of dry matter of the cell juice
serum. More particularly, the cell juice serum can have a catechin
content profile as follows: (i) between about 3.0 and about 3.5
milligrams of (-)-epigallocatechin per gram of dry matter of the
cell juice serum; (ii) between about 5.0 and about 7.0 milligrams
of (+)-catechin per gram of dry matter of the cell juice serum;
(iii) between about 0.7 and about 1.5 milligrams of (-)-epicatechin
per gram of dry matter of the cell juice serum; (iv) between about
1.7 and about 2.7 milligrams of (-)-epigallocatechin gallate per
gram of dry matter of the cell juice serum; (v) between about 0.03
and about 0.07 milligrams of (-)-gallocatechin gallate per gram of
dry matter of the cell juice serum; and (vi) between about 0.5 and
about 1.0 milligrams of (-)-epicatechin gallate per gram of dry
matter of the cell juice serum.
[0070] In one embodiment, fresh biomass of Theacea plants can be
used to isolate the bioactive compositions of the present
invention. The fresh biomass can be taken from Theacea plants that
are of the Camellia and/or Eurya genera. Suitable species of the
Camellia genus for use in the present invention can include,
without limitation, Camellia sinensis, Camellia japonica, Camellia
reticulate, and Camellia sasanqua. Suitable species of the Eurya
genus for use in the present invention can include, without
limitation, Eurya sandwicensis.
[0071] The bioactive composition of the present invention can
further include a stabilizing agent. Suitable stabilizing agents
are those that are commonly used in the art. Particular suitable
stabilizing agents can include, without limitation, an emulsifier,
a preservative, an anti-oxidant, a polymer matrix, and/or mixtures
thereof.
[0072] In one aspect of the present invention, the bioactive
fraction can have modulatory activity on at least one mammal cell
function. Such modulatory activity can include, for example, cell
growth inhibition activity, cell growth stimulation activity,
enzyme secretion activity, enzyme inhibition activity, anti-oxidant
activity, UV-protection activity, anti-inflammatory activity, wound
healing activity, and/or combinations of these activities. With
respect to cell growth inhibition activity, such activity can
involve growth inhibition of cancer cells. Suitable cancer cells
that can be inhibited to grow by the bioactive fractions of the
present invention can include, without limitation, breast cancer
cells and/or colon cancer cells. The described cell growth
inhibition activity can also include growth inhibition of leukemia
cells. Suitable leukemia cells that can be inhibited to grow by the
bioactive fractions of the present invention can include, without
limitation, monocytic leukemia cells.
[0073] In another embodiment, the bioactive composition can be
effective in inhibiting unwanted hyper-proliferation or
hypo-proliferation of skin cells and/or inhibiting unwanted
uncoordinated enzyme activities or enzyme secretion processes in
the skin cells.
[0074] In another embodiment, the bioactive composition of the
present invention can further include a delivery system for
systemic or topical administration that are commonly used in the
art.
[0075] The present invention also relates to a bioactive topical
formulation suitable for topical application to a mammal. In one
embodiment, the bioactive topical formulation includes a topically
effective amount of the bioactive composition of the present
invention. The bioactive topical formulation can further include a
topically acceptable carrier. Suitable topically acceptable
carriers can include, without limitation, a hydrophilic cream base,
a hydrophilic lotion base, a hydrophilic surfactant base, a
hydrophilic gel base, a hydrophilic solution base, a hydrophobic
cream base, a hydrophobic lotion base, a hydrophobic surfactant
base, a hydrophobic gel base, and/or a hydrophobic solution base.
In one embodiment, the bioactive composition can be present in an
amount ranging from between about 0.001 percent and about 90
percent of the total weight of the bioactive topical
formulation.
[0076] The present invention also relates to a method for
inhibiting inflammatory activity in skin tissue of a mammal. This
method involves providing the bioactive composition according to
the present invention. The method further involves applying the
bioactive composition to the skin tissue in an amount effective to
inhibit inflammatory activity in the skin tissue. In one embodiment
of this method, the bioactive composition can further include a
stabilizing agent (suitable examples of which are as described
herein). In another embodiment of this method, the bioactive
composition can further include a topically acceptable carrier
(suitable examples of which are as described herein).
[0077] The present invention also relates to a method of protecting
skin tissue of a mammal from ultraviolet light-induced damage. This
method involves providing the bioactive composition of the present
invention. The method further involves applying the bioactive
composition to the skin tissue in an amount effective to reduce
ultraviolet light-induced damage of the skin tissue and to prevent
oxidative damage of the skin tissue. In one embodiment, the method
is useful in protecting skin tissue from ultraviolet light-induced
damage caused by ultraviolet light in a range of between about 320
and about 400 nanometers. In another embodiment of this method, the
bioactive composition can further include a stabilizing agent
(suitable examples of which are as described herein). In another
embodiment of this method, the bioactive composition can further
include a topically acceptable carrier (suitable examples of which
are as described herein).
[0078] The present invention also relates to a method for
normalizing skin disorders in skin tissue of a mammal. This method
involves providing the bioactive composition of the present
invention. The method further involves applying the bioactive
composition to the skin tissue in an amount effective to normalize
a cell disorder in the skin tissue. In one embodiment of this
method, the bioactive composition can further include a stabilizing
agent (suitable examples of which are as described herein). In
another embodiment of this method, the bioactive composition can
further include a topically acceptable carrier (suitable examples
of which are as described herein).
[0079] The present invention also relates to a method for isolating
a bioactive fraction derived from cell juice of a Theacea plant.
This method involves providing a Theacea plant (e.g., in the form
of fresh biomass). Suitable Theacea plants for use in this method
are as described herein, supra. The Theacea plant (e.g., fresh
biomass) is then separated into cell juice and a cell walls
component. The cell juice is then treated under conditions
effective to yield a bioactive fraction. Suitable bioactive
fractions include, without limitation, a membrane fraction, a
membrane fraction extract, a cytoplasm fraction, a cytoplasm
fraction extract, and/or a cell juice serum. The bioactive fraction
is then isolated from the treated cell juice. In one embodiment,
the various suitable bioactive fractions produced by this method
are as described herein. The present invention further relates to
an isolated bioactive composition produced by this method.
[0080] The present invention also relates to a method for isolating
a bioactive fraction derived from a cell walls component of a
Theacea plant. This method involves providing a Theacea plant
(e.g., in the form of fresh biomass). The Theacea plant (e.g.,
fresh biomass) is then separated into cell juice and a cell walls
component. The cell walls component is treated under conditions
effective to yield a bioactive fraction. The bioactive fraction is
then isolated from the treated cell walls component. In one
embodiment, the various suitable bioactive fractions produced by
this method are as described herein. The present invention further
relates to an isolated bioactive fraction produced by this
method.
[0081] By way of example, the overall process for preparing the
bioactive fractions of the present invention (as described herein
above) is schematically shown in FIG. 1. Details of the processing
steps are further described in the Examples (infra). As depicted in
FIG. 1, fresh biomass 10 (e.g., fresh plant biomass) of Theacea
plants is subjected to grinding, maceration, and pressing 20 under
conditions effective to destroy rigid cell walls, and thereby to
yield plant cell juice 30 and cell walls 32. The fresh biomass 10
is also used for conventional tea processing 22 to produce positive
control 150 for comparative testing and evaluation. Cell juice 30
is subjected to coagulation 40 (e.g., microwave treatment) to
achieve quantitative coagulation of membrane fraction components of
fresh plant biomass 10. Coagulation 40 is sufficient to enable
subsequent separation of the coagulated membrane fraction from
other non-coagulated components of cell juice 30. As shown in FIG.
1, one embodiment of such separation is achieved by cooling and
centrifugation 42 to yield membrane fraction (precipitate) 50 and
supernatant 60, which is free from specific chloroplast membrane
components such as chlorophyll and phospholipids.
[0082] To produce the cell walls fraction extract (i.e.,
Composition A 110), cell walls 32 are subjected to drying 34 (e.g.,
several subsequent microwave treatments) and then mixing the dried
material with water 36 under conditions commonly used to prepare
conventional teas (e.g., mixing in water at 85.degree. C.).
[0083] To produce the membrane fraction extract (i.e., Composition
B 120), membrane fraction 50 is subjected to mixing with solvent 52
and then centrifugation 54 to yield supernatant 56 and Composition
B 120.
[0084] To produce the cytoplasm fraction extract (i.e., Composition
C 130), supernatant 60 is subjected to coagulation 62 (e.g.,
isoelectric precipitation) and centrifugation 64 to yield cytoplasm
fraction (precipitate) 70 containing most of the soluble cytoplasm
proteins. Cytoplasm fraction (precipitate) 70 is then subjected to
mixing with solvent 72, followed by centrifugation 74 to yield
supernatant 76 and then Composition C 130.
[0085] To produce cell juice serum (i.e., Composition D 140),
supernatant 60 is subjected coagulation 62 (e.g., isoelectric
precipitation) and centrifugation 64 to yield cell juice serum
(supernatant) 66 and then Composition D 140.
[0086] Conventional tea processing 22 of fresh biomass 10 is used
to produce, for example, positive control 150 (of various teas,
including, for example, white, green, oolong, and black teas).
[0087] Composition A 110, Composition B 120, Composition C 130,
Composition D 140, and positive control 150 can then be used for
filtration and tests 80.
[0088] The present invention also relates to a device for
selectively dispersing into a liquid low molecular weight and
reduced, non-oxidized components of a bioactive composition. In one
embodiment, the device includes a bioactive composition of the
present invention. The bioactive composition can be enclosed in a
filtering pouch. A suitable filtering pouch can be one that is
effective in selectively dispersing into a liquid the low molecular
weight and reduced, non-oxidized components of the bioactive
composition. In one embodiment, the pouch includes a selective
membrane that allows dispersal of the low molecular weight and
reduced, non-oxidized components of bioactive compositions from
within the pouch into the liquid, but where the membrane inhibits
dispersal of high molecular weight and oxidized components from
within the pouch into the liquid. As used herein, the term "low
molecular weight and reduced, non-oxidized components" include
components of the bioactive composition of the present invention
that are less than or equal to about 5,000 Daltons. In one
embodiment of this method, the bioactive composition can further
include a stabilizing agent (suitable examples of which are as
described herein). In another embodiment of this method, the
bioactive composition can further include a topically acceptable
carrier (suitable examples of which are as described herein).
[0089] The present invention also relates to a method of making a
therapeutic beverage containing low molecular weight and reduced,
non-oxidized bioactive compositions. This method involves providing
a device produced according to the method of the present invention.
The device is contacted with a liquid under conditions effective to
cause the low molecular weight and reduced, non-oxidized components
of the bioactive compositions to disperse into the liquid. In one
embodiment of this method, the bioactive composition can further
include a stabilizing agent (suitable examples of which are as
described herein). In another embodiment of this method, the
bioactive composition can further include a topically acceptable
carrier (suitable examples of which are as described herein). A
suitable liquid for use in this method can include, without
limitation, water. The water can be hot or cold. The present
invention further relates to a therapeutic beverage produced
according to this method.
EXAMPLES
Example 1
Preparation of Bioactive Compositions Derived from Camellia
sinensis Plants
[0090] A schematic of one embodiment of the method of preparing the
bioactive compositions of the present invention is shown in FIG. 1.
Below is a description of relevant aspects of one embodiment of the
method of the present invention.
[0091] Biomass Preparation.
[0092] Sufficient amounts of fresh Camellia (Camellia sinensis)
plant biomass (only top tender young leaf tissue with buds) were
harvested to yield approximately 100 kg of dry matter. The level of
dry matter in the fresh biomass was calculated to be 21.70%,
requiring harvesting of approximately 461 kg of fresh plant biomass
to yield 100 kg of dry matter. Care was taken to preserve the
inherent moisture content of the plant biomass and to avoid wilting
due to moisture loss. The harvesting was conducted in such a manner
as to avoid or minimize chopping, mashing, and crushing of the
collected biomass to avoid the disruption of the leaf cell
structure, which triggers the endogenous enzymatic reactions
catalized by phenol-oxidase and peroxidase. Because these reactions
are intensified with the time of oxidation, all steps were
completed in the shortest possible period of time. For example, the
harvested biomass was delivered for processing not more than 10
minutes after cutting. This was done to minimize exposure of the
plant biomass to sun, high temperature, and other negative
environmental factors. A washing step was performed to remove soil
particles and other debris from the plants prior to further
processing. This washing was accomplished by washing the harvested
plants for .ltoreq.5 minutes in .ltoreq.1 kg/cm.sup.2 water
pressure. The residual water wash did not contain any green or
brown pigments, indicating proper water pressure and washing
duration. The excess water was removed from the washed plant
biomass.
[0093] Grinding, Maceration, and Pressing of Plant Biomass.
[0094] After harvesting, collecting, and washing the plant biomass,
the plants then underwent grinding, maceration, and pressing to
extract the intracellular content (i.e., the plant cell juice) and
to separate it from the fiber-enriched cell walls fraction (cell
walls fraction). A hammer mill (Model VS 35, Vincent Corporation,
FL) having 10 HP engine and set of screens was used to grind the
biomass to yield plant tissue particles of suitably small size in a
shortest amount of time and without significant increase of biomass
temperature. The hammer mill was set to produce the maximum size of
macerated plant particles of .ltoreq.0.5 centimeters during
.ltoreq.10 seconds of treatment. The biomass temperature was
increased only .ltoreq.5.degree. C. A horizontal continuous screw
press (Compact Press "CP-6", Vincent Corporation, FL) was
immediately used to extract the plant cell juice from the plant.
The pressure on the cone of the screw press was maintained at a
level of 24 kg/cm.sup.2, with a screw speed of 12 rpm and only a
temperature increase of .ltoreq.5.degree. C. This treatment yielded
the 185 kg of cell walls fraction having dry matter level 41.39%
and 276 kg of plant cell juice having dry mater level 8.49%.
[0095] Preparation of Cell Walls Fraction Extract (Composition
A).
[0096] The aliquot of cell walls fraction having initial dry matter
level 41.39% was dried in microwave hood combination (Model
GH9115XE, Whirlpool) during 30 sec and then cooled during 30 sec.
This treatment was repeated several times till dry matter level in
cell walls fraction reached 96.52%. The 66.01 of deionized water
having temperature 85.degree. C. were added to 4.0 kg of dry cell
walls fraction and steer with high agitation for 5 min. These
conditions are in agreement with tea preparation procedure, which
is described in D'Amelio, F. S., Botanicals. A Phytocosmetic Desk
Reference, Boca Raton, London, New York, Washington, D.C.: CRC
Press, p. 361 (1999), which is hereby incorporated by reference in
its entirety (see also the discussions at www.leaftea.com;
www.divinitea.com; www.equatorcoffee.com, which are hereby
incorporated herein in their entirety). The mixture was filtered
through 4-layers of nylon fabric and then through the filter having
0.8 .mu.m porous. The pH of obtained cell walls extract was equal
5.24 and dry matter level was equal 0.84%. This extract was further
used for tests of its activities.
[0097] Separation of the Membrane Fraction from the Cell Juice.
[0098] The initial plant cell juice having dry matter level 8.49%
contained small fiber particles, which were removed by filtration
through four layers of nylon fabric or by using low-speed
centrifugation biomass. The filtered plant cell juice was exposed
to microwave treatment using a temperature probe control. This
treatment continued until the temperature of the cell juice reached
60.degree. C. Once coagulation was induced, the treated cell juice
was immediately cooled to 40.degree. C. Separation of the membrane
fraction from the coagulated cell juice was achieved using
centrifugation at greater than or equal to 3,000 g for greater than
or equal to 20 minutes. This yielded a membrane fraction
(precipitate) and a cell juice supernatant, which contained a
cytoplasm fraction and a cell serum fraction (i.e., low molecular
weight soluble components). The membrane fraction having dry mater
level 32.89% was used in preparing the extract of membrane-derived
bioactive composition. The cell juice supernatant was used for
further processing to yield cytoplasm fraction and cell juice
serum.
[0099] Preparation of the Membrane Fraction Extract (Composition
B).
[0100] One part of membrane fraction (10.0 kg) and two parts of
Dimethyl Sulfoxide--(20.0 kg) were mixed at room temperature for 1
hour with permanent stirring. Then material was centrifuged at
greater than or equal to 4,000 g for greater than or equal to 45
minutes. The precipitate was discarded and supernatant was filtered
through the filter having 0.8 .mu.m porous. This filtrate having
dry meter level 6.83%--membrane fraction extract (composition B)
was used for further tests of its activities.
[0101] Separation of the Cytoplasm Fraction from the Cell Juice
Supernatant.
[0102] In order to separate out the cytoplasm fraction, the cell
juice supernatant was subjected to isoelectric precipitation.
Precipitation of the cytoplasm fraction was induced using a
titration method utilizing 5.0 N Hydrochloric Acid (HCl) to bring
the pH of the cell juice supernatant to 4.0. The separation of
precipitated cytoplasm fraction having dry matter level 14.5% from
supernatant was achieved by centrifugation at greater than or equal
to 3,000 g for greater than or equal to 20 minutes.
[0103] Preparation of the Extract of Cytoplasm Fraction
(Composition C).
[0104] One part of cytoplasm fraction (10.0 kg) and two parts of
Dimethyl Sulfoxide--(20.0 kg) were mixed at room temperature for 1
hour with permanent stirring. Then material was centrifuged at
greater than or equal to 4,000 g for greater than or equal to 45
minutes. The precipitate was discarded and supernatant was filtered
through the filter having 0.8 .mu.m porous. This filtrate having
dry meter level 3.50%--extract of cytoplasm fraction (composition
C) can be used for further tests of its activities.
[0105] Preparation of Cell Juice Serum (Composition D).
[0106] After separation of cytoplasm fraction the supernatant
contained suspended particles. In order to separate out these
particles, the supernatant was centrifuged at greater than or equal
to 7,500 g for greater than or equal to 30 minutes. The transparent
supernatant--cell juice serum was filtered through the filter
having 0.8 .mu.m porous. This filtrate (composition D) having dry
matter level 5.69% was used for further tests of its
activities.
[0107] Preparation of Conventional Tea Extracts--Controls.
[0108] The same lot of fresh Camellia leaves, which was used to
preparation of compositions A, B, C and D was used to produce
conventional white and black tea.
[0109] The following procedure was used to produce white tea. The
fresh biomass contained 21.70% dry matter was placed for 20 sec in
boiling water to inactivate endogenous enzymes--phenol-oxidase and
peroxidase. During this procedure the leaves were kept in the nylon
screen bag. Then treated leaves were dried in microwave during 30
sec and then cooled during 30 sec. This treatment was repeated
several times until dry matter level in biomass reached 93.74%.
Then 66.01 of deionized water having temperature 85.degree. C. were
added to 4.0 kg of dry leaves and steer with high agitation for 5
min. These conditions are in agreement with tea preparation
procedure, which is described in D'Amelio, F. S., Botanicals. A
Phytocosmetic Desk Reference, Boca Raton, London, New York,
Washington, D.C.: CRC Press, p. 361 (1999), which is hereby
incorporated by reference in its entirety (see also the discussions
at www.leaftea.com; www.divinitea.com; and www.equatorcoffee.com,
which are hereby incorporated by reference in their entirety). The
mixture was filtered through 4-layers of nylon fabric and filtered
through the filter having 0.8 .mu.m porous. The pH of obtained cell
walls fraction extract was equal 5.52 and dry matter level was
equal 1.10%. This extract was further used for tests of its
activities.
[0110] The following procedure was used to produce black tea. The
fresh biomass contained 21.70% dry matter was kept at 25.degree. C.
with periodical (1 hour "on" and 1 hour "off") aeration until dry
matter level reached 35%. Then leaves were ground (crushed) to the
particles having size 2-3 mm. This procedure leads to increase of
biomass temperature to approximately 30.degree. C. The ground
biomass was placed in the form of layer (2'' high) on plastic
conveyor belt for fermentation (oxidation) during 90 min at
25.degree. C. The fermented biomass, which acquired the brown
color. was dried at 130.degree. C. for 30 min to reach the dry
matter level 97.5%. Then 66.01 of deionized water having
temperature 85.degree. C. were added to 4.0 kg of dry leaves and
steer with high agitation for 5 min. These conditions are in
agreement with tea preparation procedure which is described in
D'Amelio, F. S., Botanicals. A Phytocosmetic Desk Reference, Boca
Raton, London, New York, Washington, D.C.: CRC Press, p. 361
(1999), which is hereby incorporated by reference in its entirety
(see the discussions at www.leaftea.com; www.divinitea.com;
www.equatorcoffee.com, which are hereby incorporated by reference
in their entirety). The mixture was filtered through 4-layers of
nylon fabric and filtered through the filter having 0.8 .mu.m
porous. The pH of obtained cell walls fraction extract was equal
4.96 and dry matter level was equal 1.38%. This extract was further
used for tests of its activities.
Example 2
Distribution of Dry Matter Regarding Preparation of Bioactive
Compositions from Camellia sinensis, Camellia japonica, Camellia
reticulate, Camellia sasanqua, and Eurya sandwicensis
[0111] Various fractions collected during the production of
bioactive compositions were analyzed and compared for dry matter
distribution. Table 1 shows the distribution of 100 kg dry mater
among products of fractionation of tea plants. It was determined
that the process of the present invention permits extracted yield
conversion into plant cell juices in the range of from about 20 to
30% of initial biomass dry matter. The yield of membrane fractions'
dry matter was in the range from 5% to 10% of initial biomass dry
matter and from 25% to 35% of cell juice dry matter. Table 1 shows
that the yields of cytoplasm fractions dry matter did not exceed
1.0% of initial biomasses dry matter and subsequently 2.5% of cell
juice supernatant dry matter. Most of cell juice supernatant dry
matter was concentrated in cell juice serum. The cell walls
fraction, membrane fraction and cytoplasm fraction were used as the
sources for preparation of their extracts, which are categorized as
bioactive compositions. The cell juice serum was directly used "as
is" as subsequent bioactive composition having no exogenous
solvents.
TABLE-US-00001 TABLE 1 Distribution of 100 kg Dry Matter Among
Products of Fractionation of Fresh Biomass Plant Source Product
Camellia sinensis Initial Biomass 100.0 Cell Walls Fraction 76.6
Cell Juice 23.4 Membrane Fraction 6.5 Cytoplasm Fraction 0.6 Cell
Juice Serum 16.3
[0112] It should be noted that the three selected materials are the
most diversified representation of all functional structures, which
exists in fresh plant tissue. Only soluble cell juice serum has
physico-chemical properties, which allows the direct administration
to the commonly used in vitro testing systems. The cell walls
fraction, membrane fraction and cytoplasm fraction were used as raw
materials for extraction with solvents. Because cell walls fraction
is structurally similar to conventional tea plant products, this
fraction was extracted with water to provide the best comparison
with conventional teas. The membrane fraction was extracted with
Dimethyl Sulfoxide, which facilitates the effective solubilization
of both hydrophobic and hydrophilic components integrated in
chloroplast and mitochondria structures. The cytoplasm fraction was
extracted with water. The cell juice serum was used "as is."
[0113] Table 2 shows the yield of all four tested bioactive
compositions: cell walls fraction extract (composition A), membrane
fraction extracts (composition B), extract of cytoplasm fraction
(composition C), cell juice serum (composition D) and
controls--white tea extract or black tea extract from 100 kg of
initial biomass dry mater.
TABLE-US-00002 TABLE 2 Yield of Bioactive Compositions from 100 kg
of Initial Biomass Plant Source Product Camellia sinensis Initial
Biomass 100.0 Composition A (Cell Walls Fraction Extract) 14.35
Composition B (Membrane Fraction Extract) 2.37 Composition C
(Cytoplasm Fraction Extract) 0.2 Composition D (Cell Juice Serum)
16.3 Control (Extract of White Tea or Black Tea) 15.14 . . .
19.36
[0114] Table 2 shows that the total yield of bioactive compositions
A, B, C and D from 100 kg of dry matter of Camellia sinensis equals
33.3%, which very significantly exceeds the yield of conventional
tea process--15.14 . . . 19.36%.
Example 3
Comparison of Composition a (Cell Walls Fraction Extract) and
Conventional Tea Extracts
[0115] Various parameters of bioactive composition A and
conventional white and black tea extracts obtained from the same
batch of fresh Camellia sinensis were measured and subsequent
results of these are presented in Table 3 (The used experimental
methods are described in Examples 9 and 20, and in the U.S. Patent
Application Publication No. 2003/0175235, which is hereby
incorporated by reference in its entirety).
TABLE-US-00003 TABLE 3 Various Parameters of Bioactive Composition
A and Extracts of White and Black Teas. Extract of Controls Cell
Walls Fraction Extract of Extract of Parameter (Composition A)
White Tea Black Tea Dry Matter, % 0.84 1.10 1.38 pH 5.24 5.52 4.96
Conductivity, mS/cm 1.57 2.23 5.32 Total Dissolved Solids, g/L 0.78
1.23 2.71 Redox Potential, mV 123 159 188 Area under the Spectra
Curve (ASC) 5.727 6.604 4.616 200-450 nm, Abs nm ASC:Dry Matter
6.818 6.004 3.345 Superoxide Scavenging Activity (ICR.sub.50), 26.3
114.5 227.1 .mu.g DM/ml Color (1 . . . 10 scale) 9.6 4.7 7.5 Flavor
(1 . . . 10 scale) 9.3 6.1 6.6 Mouthfeel (1 . . . 10 scale) 9.4 6.5
5.3
[0116] Table 3 shows that cell walls fraction extract has lower
levels of dry matter, electrolytes and dissolved solids compare to
conventional white and black tea extracts. The UV/VIS spectral data
show that cell walls fraction extract has the highest specific
value of the area under the spectra curve, i.e. this particular
Camellia product (composition A) has the highest level of optically
active constituents per unit of dry matter. Additionally, the cell
walls fraction extract has the lower amount of redox potential,
which indicates that this composition is less oxidized then
conventional white and black tea extracts. The cell walls fraction
extract demonstrated superoxide scavenging activity, resulting in
50% inhibition of cytochrome c reduction (ICR.sub.50) at a much
lower concentration then white and black tea extracts. A
Qualitative Descriptive Analysis ("QDA") test method was selected
to systematically characterize and quantify teas based on color,
flavor, and mouthfeel, which govern acceptability of tea beverages.
The QDA method employs a trained panel of expert tasters to
quantify the above attributes of tea beverages relative to defined
reference standards. The comparative evaluation of the color,
flavor and mouthfeel of teas demonstrated that cell walls fraction
extract significantly exceed the same characteristics of
conventional teas.
[0117] Thus, cell walls fraction, which was obtained from fresh
Camellia biomass without any fermentation (oxidation) and heat
treatment, is distinct from all other teas (Wilson et al., eds.,
Tea: Cultivation to Consumption, London: Chapman Hall (1992), which
is hereby incorporated by reference in its entirety). Additionally,
the key Camellia enzymes (phenol-oxidase and peroxidase) always
remain within conventional teas. Instead, the present invention
includes separation of fresh Camellia leaves to cell walls fraction
and cell juice, which is enriched by these enzymes and thus cell
walls fraction does not contain endogenous phenol-oxidase and
peroxidase. Therefore, cell walls fraction must be categorized as a
new tea category having fundamental differences compared with
white, green, oolong, and black teas. This novel cell walls
fraction tea can be used in either loose or bag form or other
manifestations to prepare a broad spectrum of drinks, beverages,
and additives to nutriceutical and functional food products.
Example 4
Preparation of Bioactive Compositions for Different
Applications
[0118] All bioactive compositions can be used as solutions,
suspensions, dispersions, pastes or dried powders incorporated into
a variety of formulations for systemic or topical administrations.
The solubilized forms of compositions can be filtrated through
filters having the 0.2 .mu.m porous to completely remove
non-completely solubilized small particles and endogenous
microorganisms. The dry matter level in bioactive compositions
before and after sterilized filtration is presented in Table 4.
TABLE-US-00004 TABLE 4 Level of Dry Matter in Bioactive
Compositions Before (numerator) and After (denominator) Sterilized
Filtration Plant Source Product Camellia sinensis Composition A
0.84 (Extract of Cell Walls Fraction) 0.72 Composition B 6.83
(Extract of Membrane Fraction) 6.12 Composition D 5.69 (Cell Juice
Serum) 5.59 Control 1.10 (Extract of White Tea) 1.03
[0119] Table 4 shows that dry matter levels in all bioactive
compositions were decreased after sterilizing filtration. However
this decrease did not lead to any loss or significant reduction of
their biological activities and was in the range 2-14%. Therefore
major part of compositions is presented by soluble bioactive
ingredients.
[0120] Fresh Camellia leaves contain relatively low molecular
weight (reduced, non-oxidized) ingredients. As a result of
oxidation and polymerization processes in the manufacturing of
conventional teas the above potent ingredients are transformed into
the parts of high molecular weight substances having relatively low
activity.
[0121] The bioactive compositions of the present invention are
obtained without fermentation (oxidation) and excessive heat
treatment. This in turn prevents the irreversible loses of fresh
plant activities, which can be delivered with maximum potency
using, for example, novel tea bag or analogous delivery systems.
Instead of conventional paper tea bags, which allow all soluble tea
ingredients to move through large pores to the surrounding water,
the novel tea bag is made from semi-permeable membrane. This bag
contains bioactive composition inside and allows only ingredients
having molecular weight below certain membrane cut off (for
example, 5,000 Dalton) to penetrate to the surrounding water phase.
The ingredients having higher molecular weight remain inside the
bag and thus are not included in the beverage.
[0122] Therefore, the cut off of ingredients having molecular
weight above a certain level allows the production of a beverage
having no oxidized ingredients because all oxidized ingredients of
bioactive compositions having molecular weight above a certain
level remain inside the bag. The novel tea bag design can be based
on pyramidal tea bag construction, which utilizes dialysis membrane
tube. The design of novel tea bag can also include thin plastic
frame having bioactive composition inside and two transparent
surfaces built from semi-permeable membrane. The selection of
particular membrane cut off value is determined by the type of
bioactive composition, but in general higher cut off enabling
release into surrounding water phase of a higher percent of
composition's dry matter lower amount of the redox potential is
preferable.
Example 5
Preparation of Topical Ingredient SF Derived from Cell Juice
Serum
[0123] Cell juice serum (composition D) cannot be used as an active
ingredient of topical products due to the lack of stability and
deterioration of color and odor. The described procedure allows for
the refinement of cell juice serum fraction to yield a stable and
active topical ingredient SF (this procedure is similar to
previously described in U.S. Patent Application Publication No.
2003/0175235, which is hereby incorporated by reference in its
entirety). The refinement of the cell juice serum involved the
following steps: heat treatment, cooling, filtration, and
stabilization. Refinement was performed immediately after
separation of the cell juice serum from the cytoplasm fraction as
described in Example 1. The cell juice serum was exposed to
microwave treatment using a temperature probe control. This
treatment continued until the temperature of the cell juice serum
reached 99.degree. C. (90.degree. C. was required as was previously
described in U.S. Patent Application Publication No. 2003/0175235,
which is hereby incorporated by reference in its entirety). Once
coagulation was induced the treated cell juice serum was
immediately cooled to 10.degree. C. The coagulated cell juice serum
was vacuum filtrated through filter having porous 0.8 .mu.m (double
layers of Whatman No. 2 filters were used in U.S. Patent
Application Publication No. 2003/0175235, which is hereby
incorporated by reference in its entirety). The precipitate was
discarded and the resulting cell juice serum filtrate was used for
further processing (i.e., stabilization). Stabilization of the cell
juice serum filtrate was achieved by adding preservatives (no
exogenous anti-oxidant was required as was previously described in
U.S. Patent Application Publication No. 2003/0175235, which is
hereby incorporated by reference in its entirety) and incubating
the mixture until complete solubilization was achieved. The
preservatives used included the following: 0.1% potassium sorbate,
0.1% sodium benzoate, 0.1% sodium methyl paraben, and 0.1% citric
acid. This preparation resulted in the production of 16.3 kg of dry
matter yield (or approximately 286 Liters) of the topical
ingredient SF, which was used for characterization of its
physico-chemical and bioactive qualities. The recommended storage
conditions for topical ingredient SF include storage in a closed
container protected from light at a temperature of between
15.degree. C. and 25.degree. C.
Example 6
Product Specifications of Topical Ingredient SF Derived from Cell
Juice Serum Fraction
[0124] Topical Ingredient SF was prepared according to the process
described above in Example 5. Analyses of topical ingredient SF
were conducted to determine its various physico-chemical,
microbial, cytotoxicity, and bioactivity characteristics, as
described below. Topical ingredient SF is a clear liquid, which has
a light-yellow-brown color and a light-characteristic odor. No
solvent (i.e. glycol, oil, or water) was added to the carrier
medium. Table 5 summarizes the Physical and Chemical data of
topical ingredient SF.
TABLE-US-00005 TABLE 5 Physical and Chemical Parameters of Topical
Ingredient SF Parameter Method Results Solid Content, % See Example
20, "Method 1" 5.04 Specific Gravity, g/cm.sup.3 USP <841>
1.015 Color Gardner Scale 5-6 Refractive Index USP <831>
1.312 PH USP <791> 4.0 Redox Potential, mV See reference [1]
75 Conductivity, S/m See reference [2] 1.02 References: [1]
Handbook of Chemistry and Physics, 80.sup.th Edition, CRC Press,
1999-2000, 5-90; [2] Handbook of Chemistry and Physics, 80.sup.th
Edition, CRC Press, 1999-2000, 8-21, which are hereby incorporated
by reference in their entirety.
[0125] Table 6 describes the UV-Spectra data regarding topical
ingredient SF.
TABLE-US-00006 TABLE 6 UV-Spectra of Topical Ingredient SF (1:500
Dilution) Peak Parameter Method Results #1 Start, nm USP
<197> 450 Apex, nm '' 266.5 End, nm '' 247 Height, Abs ''
0.231 Area, Abs x nm '' 13.676 #2 Start, nm USP <197> 247
Apex, nm '' 204 End, nm '' 200 Height, Abs '' 1.396 Area, Abs x nm
'' 32.413
[0126] The microbial analysis conducted in accordance with the
following procedure (USP <61>) demonstrated that topical
ingredient SF contains less then 100 colony forming units per gram
of sample and has no pathogens (E. coli, Candida albicans,
Pseudomonas sp. and Staphylococcus aureus). This data demonstrates
that topical ingredient SF satisfies the industry requirements for
ingredients of topical products.
[0127] Topical ingredient SF was determined to be stable (i.e.,
maintaining physical and chemical integrity) for at least 12-18
months while stored at a temperature of between 15 and 25.degree.
C. in a closed container protected from light. Topical ingredient
SF is a biodegradable product. In a controlled clinical evaluation,
topical ingredient demonstrated the biological activities, which
are summarized in Table 7.
TABLE-US-00007 TABLE 7 Biological Activities of Topical Ingredient
SF Activity Method .mu.g DM/ml Superoxide Scavenging See Example
20, 69.5 Activity (ICR.sub.50) "Method 7" Elastase Inhibitory
(IC.sub.50) See Example 20, 32.3 "Method 5" MMP-9 Inhibitory
(IC.sub.50) See Example 20, 14.6 "Method 6" Trypsin Inhibitory
(IC.sub.50) See reference [1] 7.8 Reference: [1] Cannel et al.,
Planta Medica 54:10-14 (1988), which is hereby incorporated by
reference in its entirety.
[0128] Table 7 shown that topical ingredient SF demonstrated
superoxide scavenging ability. In a controlled clinical evaluation,
topical ingredient SF demonstrated a 50% inhibition of cytochrome c
reduction (ICR.sub.50) at a concentration 69.5 .mu.g dry matter per
ml. The ICR.sub.50 of positive control (rosmarinic acid)=26.5
.mu.g/ml. In addition to anti-oxidant properties, topical
ingredient SF demonstrated antiproteolytic activities against
peptide hydrolases, for example elastase, gelatinase B or so-called
matrix metalloproteinase 9 (MMP-9), and trypsin. Among these
enzymes the unique position belongs to elastase and MMP-9, which
act synergistically and play an extremely important role in skin
inflammation. It should be noted, that both MMP-9 and elastase are
secreted by white blood cells (neutrophils) and these enzymes are
the key enzymes in the final pathway leading to inflammation. It is
generally agreed that if preparation can inhibit both enzymes
(elastase and MMP-9), such preparation is considered to be very
effective to treat inflammatory processes.
[0129] It should be noted that skin aging processes, sunburns,
formation of wounds and scars have the very same inflammation
mechanism, which involves both MMP-9 and elastase. Thus, topical
ingredient SF capable of inhibiting both of the above enzymes has
very wide spectrum of applications, among which are inflammatory
injury because the following reasons: [0130] a. These two enzymes
can synergize to degrade all the components of extracellular matrix
of human tissue; [0131] b. Elastase can inactivate the body's own
inhibitory defense against MMP-9; and [0132] c. MMP-9 can
inactivate the body's own inhibitory defense against elastase. The
combination of anti-inflammatory and anti-oxidant properties of
topical ingredient SF suggests that this hydrophilic preparation
based on bioactive composition D is capable to act systemically on
very fundamental skin disorder problems.
Example 7
Preparation of Topical Ingredient MF Derived from Membrane
Fraction
[0133] The freshly obtained membrane fraction is a paste having
intensive color and specific odor. This fraction is represented
predominantly by chloroplasts and its composition includes
predominantly phospholipids, membrane proteins, chlorophyll, and
carotenoids. The drying of membrane fraction results in
irreversible loses of many valuable properties required for the
exploration of membrane fraction as a topical ingredient. Without
drying, the unstable membrane fraction is quickly transformed into
the dark color un-dispersible and insoluble conglomerates having a
strong and non-characteristic odor. As result, such material cannot
be used as a topical ingredient. The procedure described below
allows for transformation of freshly obtained membrane fractions
into stable and active topical ingredients (this procedure is
similar to previously described in the U.S. Patent Application
Publication No. 2003/0175235, which is hereby incorporated by
reference in its entirety).
[0134] Immediately after separation of the membrane fraction from
cell juice according to the process described above in Example 1,
the membrane fraction was stabilized and incorporated into a
polymer matrix. To prepare approximately 100 grams of topical
ingredient MF the cell membrane fraction was stabilized by mixing
it with non-ionic emulsifier Polysorbate 80 (Tween 80) and
antioxidants (Tenox 4). Specifically, 20 grams of fresh membrane
fraction was mixed vigorously with 3.5 grams of Tween 80 and 0.1
gram of Tenox 4 (solution of Butylated Hydroxyanisole and Butylated
Hydroxytoluene in oil) until homogeneous, while avoiding aeration
during mixing.
[0135] Once stabilized, the membrane fraction was incorporated into
a polymer matrix (i.e., a dispersion of polymeric emulsifier,
acrylates/C10-C30 acrylate crosspolymer). The polymer matrix was
prepared by dispersing 0.9 grams of Pemulen TR-2 in 69.2 grams of
warm deionized water and mixing until uniform using moderate
agitation, while avoiding aeration. In parallel, 5 grams of
Glycerin and 1.0 gram of Phenonip (mixture of Phenoxyethanol (and)
Methylparaben (and) Butylparaben (and) Ethylparaben (and)
Propylparaben) were combined in a separate vessel and mixed until
uniform. With moderate agitation, the phases containing Pemulen and
Glycerin with Phenonip were combined and mixed until uniform. To
incorporate the membrane fraction into the polymer matrix, the
phase containing the membrane fraction, Tween 80, and Tenox 4 was
added to the phase containing the Pemulen, Glycerin, and Phenonip,
and then mixed with vigorous agitation while avoiding aeration.
Stabilization of the membrane fraction mixture was achieved by
neutralizing it with 18% aqueous solution of sodium hydroxide
(NaOH) and mixed vigorously to produce a uniform system having a pH
of 5.0.+-.0.4. This preparation, which started from 100 kg of fresh
Camellia biomass (approximately 461 kg of fresh leaves having 21.7%
dry matter), resulted in the production of 11.85 kg of Dry Matter
yield (or approximately 172 liters) of topical ingredient MF, which
was used for characterization of its physico-chemical and bioactive
qualities. The recommended storage conditions for topical
ingredient MF include storage in a closed container protected from
light at a temperature between 2 and 8.degree. C.
Example 8
Product Specifications of Topical Ingredient MF Derived from
Membrane Fraction
[0136] Topical ingredient MF was prepared according to the process
described above in Example 7. Analyses of topical ingredient MF
were conducted to determine its various physico-chemical,
microbial, cytotoxicity, and bioactivity characteristics, as
described below. Topical ingredient MF is an opaque gel, which has
a green-brown color and light-characteristic odor. Topical
ingredient MF was formulated utilizing the natural cell juice
constituents gelled with a polymer to assure the highest level of
purity uniformity, compatibility, stability, safety and
efficacy.
[0137] Table 8 describes the physical and chemical data of topical
ingredient MF.
TABLE-US-00008 TABLE 8 Physical and Chemical Parameters of Topical
Ingredient MF Parameter Method Results Non-Volatile Residue (NVR),
% See Example 20, "Method 2" 6.9 Specific Gravity, g/cm.sup.3 USP
<841> 1.035 Viscosity, cps USP <911> 18,700 pH USP
<791> 4.6 Total Carotenoids, % NVR See Example 20, "Method 4"
0.36 Lutein, % NVR See Example 20, "Method 4" 0.34
[0138] Table 9 summarizes the L*a*b* values data regarding topical
ingredient MF.
TABLE-US-00009 TABLE 9 L*a*b* Values of Topical Ingredient MF
Parameter Method Results L* See Example 20, "Method 3" 30.27 a* ''
27.36 b* '' 42.56
[0139] Microbial analyses demonstrated that topical ingredient MF
satisfies the industry requirements for topical ingredients with
regard to CFUs and absence of pathogens (USP <61>).
[0140] Topical ingredient MF was determined to be stable (i.e.,
maintaining physical and chemical integrity) for at least 12-18
months while stored at a temperature of between 2 and 8.degree. C.
in a closed container protected from light. Topical ingredient MF
is a biodegradable product. In a controlled clinical evaluation,
topical ingredient MF demonstrates elastase inhibitory activity and
trypsin inhibitory activity. Table 10 summarizes certain
bioactivity results for topical ingredient MF.
TABLE-US-00010 TABLE 10 Bioactivity Results of Topical Ingredient
MF Activity Method IC.sub.50 (.mu.g/ml) Elastase Inhibitory
(IC.sub.50) See Example 20, "Method 5" 12.3 MMP-9 Inhibitory
(IC.sub.50) See Example 20, "Method 6" 5.6 Trypsin Inhibitory
(IC.sub.50) See reference [1] 3.8 Reference: [1] Cannel et al.,
Planta Medica 54:10-14 (1988), which is hereby incorporated by
reference in its entirety.
[0141] Table 10 shown that topical ingredient MF demonstrated
properties similar to topical ingredient SF (see Example 6).
Although topical ingredient MF has no superoxide scavenging
activity, it demonstrates higher specific enzyme inhibition
activities than topical ingredient SF. Thus topical ingredient MF,
which is based on bioactive composition B, should be considered as
a potent multiphase anti-inflammatory ingredient having broad
applications for treatment of skin disorders.
Example 9
Spectral Analyses of the Bioactive Compositions Derived from
Camellia sinensis Plants
[0142] Introduction to Spectral Analyses.
[0143] Ultraviolet (UV) radiation has damaging effects on human
skin. Short-term effects include tanning and sunburn, while the
long-term effects of cumulative UV exposure include photoaging of
the skin and increased risk of skin cancer. Ultraviolet skin injury
is mediated by oxidative damage, and a number of plant extracts
with antioxidant activity are showing promise as protective agents:
grape seed extract (Carini et al., "Protective Effect of
Procyanidines from Vitis vinifera Seeds on UV-Induced Photodamage:
In vitro and In vivo Studies," Proceedings of the 19th IFSCC
Congress 3:55-63 (1996), which is hereby incorporated by reference
in its entirety), lycopene (Di Mascio et al., "Lycopene as the Most
Efficient Biological Carotenoid Singlet Oxygen Quencher," Archives
of Biochemistry and Biophysics 274:532-8 (1989); and Ribaya-Mercado
et al., "Skin Lycopene is Destroyed Preferentially Over
.beta.-Carotene During Ultraviolet Irradiation in Humans," Journal
of Nutrition 125:1854-9 (1995), which are hereby incorporated by
reference in their entirety), silymarin (Morazzoni et al., "Silybum
marianum (Carduus marianus)," Fitoterapia 66:3-42 (1995); Katiyar
et al., "Protective Effects of Silymarin Against
Photocarcinogenesis in a Mouse Skin Model," Journal of the National
Cancer Institute 89:556-66 (1997), which are hereby incorporated by
reference in their entirety), and especially green tea extract
which has higher efficacy compared with extracts produced from
other plant sources (Katiyar et al., "Protection Against
Ultraviolet-B Radiation-Induced Local and Systemic Suppression of
Contact Hypersensitivity and Edema Responses in C3H/HeN Mice by
Green Tea Polyphenols," Photochemistry and Photobiology 62:855-61
(1995); Ruch et al., "Prevention of Cytotoxicity and Inhibition of
Intercellular Communication by Antioxidant Catechins Isolated from
Chinese Green Tea," Carcinogenesis 10:1003-8 (1989); Wang et al.,
"Protection Against Ultraviolet B Radiation-Induced
Photocarcinogenesis in Hairless Mice by Green Tea Polyphenols,"
Carcinogenesis 12:1527-30 (1991), which is hereby incorporated by
reference in its entirety).
[0144] It was found that the leaves of the tea plant (Camellia
sinensis) have a high content of polyphenols with antioxidant
activity including (-)-epicatechin, (-)-epicatechin-3-gallate,
(-)-epigallocatechin, and (-) epigallocatechin-3-gallate. Green tea
extract has shown an antioxidant activity against hydrogen peroxide
and the superoxide radicals and prevention of oxidative
cytotoxicity. The extract can also prevent the inhibition of
intercellular communication, a possible mechanism of tumor
promotion. There is a close association between UV-induced immune
suppression and the development of skin cancer, and green tea
extract has been found to protect against inflammation and immune
suppression caused by UV-B radiation. Green tea extract given
orally in drinking water or applied topically protects against
UV-B-induced skin carcinogenesis in animal models. These results
indicate that green tea extract taken orally may help to prevent
skin cancer.
[0145] Although the UV protection properties of Camellia products
have been established, the greater potential of tea plant as a
source for effective protection of the skin against sun damage has
not fully explored due to the limitations of conventional
technology, which is driven towards focusing on a limited to
relatively narrow band of active ingredients: predominately
cathechins
[0146] Comparative UV protection properties studies described
herein between "novel" and "conventional" Camellia products have
now demonstrated that the technology of "fresh Camellia
fractionation" is capable of yielding more potent products. The
comparison was made by utilizing the methods commonly used to
determine spectral properties of solutions and in-vitro sun
protection factor (SPF).
[0147] Methodology A:
[0148] UV/VIS Spectra. UV/VIS spectra of Camellia products in
200-450 nm region were obtained using pharmacopoeia compliant
Spectrophotometer Ultrospec 4300 Pro (Amersham Biosciences Ltd.,
Buckinghamshire, England). The spectral parameters of diluted in
distilled water Camellia products were determined according to the
procedure described in USP <197>.
[0149] Methodology B: Absorbance Spectra.
[0150] Absorbance spectra of Camellia products in 250-450 nm region
were obtained using UV-1000S Transmittance Analyzer (Labsphere,
Inc., North Sutton, N.H.) and Vitro-Skin.RTM. testing substrate
(IMS Testing Group, Milford, Conn.), which mimics the surface
properties of human skin. It contains both optimized protein and
lipid components and is designed to have topography, pH, critical
surface tension and ionic strength similar to human skin.
[0151] The Camellia samples were uniformly spread on a surface of
pre-hydrated substrate (application dose=2.0 .mu.l/sq. cm). After
15 min after application the initial absorbance spectra were taken
via five (5) replications. Then the substrates with applied
products were irradiated by broad-spectrum solar light simulator
(Model 16S-300 Single Port, Solar Light Company, Inc.,
Philadelphia, Pa.) equipped with 300-Watt xenon lamp. The dose
control system PMA 2100-DCS allowed precision control of the dose
delivered to a sample.
[0152] Immediately after irradiation (irradiation dose=60
Joules/sq. cm) absorbance spectra from the same spot were taken in
(5) five replications. The absorbance spectra of samples before and
after irradiation were used for statistical analysis.
[0153] Samples.
[0154] The following bioactive compositions, which were prepared
according to the process described above in Example 1 were
evaluated: composition A (cell walls fraction extract having 0.84%
dry matter), composition B (membrane fraction extract having 6.83%
dry matter), composition D (cell juice serum having 5.69% dry
matter). Extract of conventional white tea having 1.10% dry matter
and extract of conventional black tea having 1.38% dry matter were
used as controls. All samples were obtained from the same batch of
fresh Camellia, which was collected at Charleston Tea Plantation,
SC. These samples did not contain any additives.
[0155] Analyses.
[0156] It was found that all Camellia samples have high UV
absorbance values and thus they were diluted with distilled water.
The UV-VIS spectra of diluted Camellia products are presented on
FIGS. 2 and 3.
[0157] The spectra of all liquid samples have certain similarities.
For example, positions of peaks are varied in relatively narrow
ranges of .lamda..sub.max1=269-274 nm and .lamda..sub.max2=205-208
nm, which indicate the presence of aromatic rings and conjugated
systems of .sigma.-.pi. bonds in all tested samples. However, the
apex value of peaks, area under each peak and total area under
integral spectral curves are different (Table 11), which suggest
that tested samples have different compositions of optically active
constituents.
TABLE-US-00011 TABLE 11 Parameters of UV/VIS Spectra of Camellia
Products Area under Peak # 1 Peak # 2 the Spectra Start,
.lamda..sub.max1, End, Height, Start, .lamda..sub.max2, End,
Height, Curve*, nm nm nm Abs Area % nm nm nm Abs Area % Abs nm
White Tea 450 269 250 0.114 28.1 250 205 200 0.816 71.9 6.604
Extract Black Tea 450 272 250 0.094 37.3 250 205 200 0.452 62.7
4.616 Extract Cell Walls 450 272 248 0.134 35.8 248 205 200 0.614
64.2 5.727 Fraction Extract Membrane 450 274 251 0.944 16.2 251 208
200 11.98 83.8 80.133 Fraction Extract Cell Juice 450 271 249 0.692
26.3 249 205 200 5.228 73.7 39.599 Serum *The Area under Spectra
values were normalized based on dilution of the samples.
[0158] The comparison of areas under integral spectral curves
obtained from 200 nm to 450 nm clearly demonstrates that membrane
fraction extract (composition B) and cell juice serum (composition
D) had the higher absorption values (Table 11). The ratio "Area
under Spectra: Dry Matter" indicates that specific absorption value
of the samples is increasing in the following sequence: black tea
extract>white tea extract>cell walls fraction extract>cell
juice serum>membrane fraction extract (Table 12).
TABLE-US-00012 TABLE 12 Selected Spectral Characteristics of
Camellia Products Dry Area under Ratio: Area under Ratio: Matter
Spectra, Area under Spectra.sup.(200-450 nm) Spectra.sup.(290-400
nm) Area under Spectra.sup.(290-400 nm) % Abs nm Dry Matter Abs nm
Dry Matter White Tea Extract 1.10 6.604 6.004 0.812 0.738 Black Tea
Extract 1.38 4.616 3.345 0.854 0.619 Cell Walls Fraction 0.84 5.727
6.818 0.776 0.924 Extract Membrane Fraction 6.83 80.133 11.733
4.304 0.630 Extract Cell Juice Serum 5.69 39.599 6.959 4.320
0.759
[0159] Based on the comparison of absorption values, novel
bioactive compositions appear to be more effective protectors of
the skin against sun damage than extracts of conventional white tea
and black tea. It should be pointed out, that UV protection
properties of Camellia products should be better estimated using
absorption data related to the area from 290 nm to 400 nm because
this particular part of spectra is responsible for UV induced
damage of the skin (Sayre et al., "A Method for the Determination
of UVA Protection for Normal Skin," Journal of American Academy of
Dermatology 23: 429-40 (1990), which is hereby incorporated by
reference in its entirety). Although absorption of tested liquid
samples in the area 290-400 nm contributes only .about.10% of total
UV/VIS absorption, novel Camellia compositions have higher
absorption in the above spectral area as well.
[0160] Thus the data related to the diluted solutions of Camellia
samples provided initial estimation of UV protection potency of
tested products, which was further evaluated using the
Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.). The results are presented in FIGS. 4-13. It was found that
novel Camellia compositions and extracts of conventional white tea
and black tea have different spectral characteristics even after
they were applied on substrate in concentrations, which were
equalized with respect to dry matter level (FIGS. 4 and 5).
[0161] The spectral of Camellia samples, which were applied on
Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.) included from four to two characteristics peaks, which have
different apex values (Table 13).
TABLE-US-00013 TABLE 13 Parameters of Absorbance Spectra of
Camellia Products Applied on Vitro-Skin .RTM. Testing Substrate
Peak # 1 Peak # 2 Peak # 3 Peak # 4 Height, Height, Height, Height,
.lamda..sub.max4 Abs .lamda..sub.max3 Abs .lamda..sub.max2 Abs
.lamda..sub.max1 Abs White Tea Extract 260 0.389 286 0.461 330
0.163 392 0.076 Black Tea Extract 260 0.399 286 0.373 330 0.175 390
0.105 Cell Wall Fraction 260 0.555 286 0.569 330 0.203 389 0.118
Extract Membrane Fraction 260 0.394 286 0.549 -- -- 394 0.059
Extract Cell Juice Serum 260 0.431 286 0.517 -- -- -- --
[0162] It should be pointed out, that parameters of characteristic
peaks of Camellia products in solutions (Table 11) were very
different compared with characteristic of Camellia products, which
were applied on Vitro-Skin.RTM. testing substrate (IMS Testing
Group, Milford, Conn.) (Table 13). As control experiments show, the
lower pH level (.about.5.5) on Vitro-Sin.RTM. surface can not be
responsible for the above differences, which probably are the
results of chemical interactions between Camellia products and
ingredients used for preparation of Vitro-Skin.RTM. testing
substrate (IMS Testing Group, Milford, Conn.) substrate. Thus,
Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.) contains both protein and lipid components which could
chemically interact with Camellia phenolic constituents. It should
be noted, that the shifts in spectral properties of tested products
were observed for all bioactive compositions as well as for
extracts of conventional teas.
[0163] Spectra of novel Camellia compositions and extracts of
conventional Camellia products "as is" were also compared, taking
into account the different dry matter contents of tested samples.
The absorbance spectra of Camellia products applied on
Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.) in equal volumes are presented on FIG. 6.
[0164] Although there are some similarities between spectra of cell
walls fraction extract and white tea extract, the first product has
higher absorbance in the area 250-280 nm and in near UV area. It
should be noted that higher absorbance does not correspond with dry
matter level, which is higher in white tea extract (1.10%) compared
with cell walls fraction extract (0.84%). It suggests that the
compositions of these two samples are not identical and that cell
walls fraction extract also has lower conductivity consisting of
greater non-dissociated optically active ingredients responsible
for high absorbance (see data presented in Table 3).
[0165] Additionally it was found that the spectra of membrane
fraction extract (composition B) and cell juice serum (composition
D) are different from spectra of cell walls fraction extract
(composition A) and white tea extract. Thus, membrane fraction
extract and cell juice serum spectra again indicate that these
products have different composition than white tea extract and cell
walls fraction extract. At the same time, the spectral data suggest
that the compositions of membrane fraction extract and cell juice
serum are not identical. For example, membrane fraction extract has
three characteristic peaks at 260 nm, 286 nm and 394 nm. The cell
juice serum spectra contain two peaks at 260 nm and 286 nm.
[0166] Comparison of these two spectra indicates that membrane
fraction extract has approximately two times higher extinction then
cell juice serum, although difference in dry matter levels is only
.about.1%. Thus, four tested Camellia products have significantly
different compositions of constituents, which are optically active
in the region 250-450 nm.
[0167] The data related to the quantitative comparison of Camellia
products are presented in Table 14.
TABLE-US-00014 TABLE 14 Selected Characteristics of Camellia
Products Applied of Vitro-Skin .RTM. Testing Substrate Area under
Area under Area under Spectra.sup.(290-400 nm) spectra.sup.(250-450
nm) Spectra.sup.(290-400 um) Area under Spectra.sup.(250-450 nm)
Abs nm Abs nm % White Tea Extract 15.577 9.312 59.78 Cell Walls
Fraction Extract 18.571 10.422 56.12 Membrane Fraction Extract
89.148 53.803 60.35 Cell Juice Serum 60.861 35.161 57.77
[0168] Table 14 shows that absorption of the samples in the area
250-450 nm and in the area 290-400 nm is increasing in the
following order: white tea extract>cell walls fraction
extract>cell juice serum>membrane fraction extract. This
sequence is completely in agreement with the sequence of specific
absorption values of Camellia products tested in the diluted
solutions (Table 12). The contribution of the absorbance in the
250-400 nm region to the absorbance of the spectra taken from 250
nm to 450 nm reached .about.55-60% when tested samples were applied
on Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.).
[0169] It should be noted that the spectra of novel Camellia
compositions in diluted solutions and after their application on
Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.) substrate are remarkably different. These differences are
both quantitative and qualitative for membrane fraction extract and
cell juice serum. Thus in the range from 250 nm to 450 nm membrane
fraction extract in solution peaks at 274 nm. The same membrane
fraction extract when applied on Vitro-Skin.RTM. testing substrate
(IMS Testing Group, Milford, Conn.), did not show any
characteristic peak at the above wavelength, but instead had two
characteristic peaks at 260 nm and 286 nm (FIG. 7A).
[0170] The similar pattern in spectral properties was observed for
cell juice serum, although additional absorption at .about.360 nm
was identified in the sample applied on Vitro-Skin.RTM. testing
substrate (IMS Testing Group, Milford, Conn.), but such phenomena
was not registered in UV/VIS spectra of the same composition in the
solution (FIG. 7B).
[0171] It should be pointed out that above differences between
spectra may be the results of chemical interaction between novel
Camellia compositions and surface of Vitro-Skin.RTM. testing
substrate (IMS Testing Group, Milford, Conn.) having protein and
lipid components, which mimic human skin. These interactions led to
the drastic increase of the absorbance in the spectral region
responsible for damaging effect of UV irradiation, i.e., novel
Camellia compositions have significant UV protection potencies.
Control experiments with Barley (Hordeum vulgare) (FIG. 8A) and
Sage (Salvia officinalis) (FIG. 8B) cell juice serums shown that
the differences in the spectra of the same samples in solution and
after their application on Vitro-Skin.RTM. testing substrate (IMS
Testing Group, Milford, Conn.) were not observed for plant sources
other than Camellia. Thus, the spectral shift in Camellia products
after application on Vitro-Skin.RTM. testing substrate (IMS Testing
Group, Milford, Conn.) indicates the specific interactions having
place only between novel Camellia products and substrate which
mimics human skin.
[0172] Control experiments with pre-hydrated substrate demonstrated
that after irradiation the absorbance of Vitro-Skin.RTM. testing
substrate (IMS Testing Group, Milford, Conn.) was significantly
decreased especially in the range 260-330 nm (FIG. 9). This effect
reflects relatively low photo-stability of non-protected substrate,
which was irradiated by high dose of broad-spectrum solar
light.
[0173] Irradiation of the substrate with applied white tea extract
initiated changes in absorbance spectra (FIG. 10) which is
analogous with the spectral changes of non-protected
Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.). When contribution of substrate was eliminated, the spectra
of non-irradiated and irradiated sample indicated some similarities
but did not demonstrate totally identical behavior. For example,
the absorbance in the range 290-310 nm was decreased and wide peak
at 360 nm started to form.
[0174] The irradiation of cell walls fraction extract (FIG. 11) led
to similar changes in the absorbance spectra especially with
respect to the curve obtained after elimination (subtraction) of
substrate contribution.
[0175] Although compositions of white tea extract and cell walls
fraction extract are not identical, the pattern of
irradiation-induced modifications in their spectra is quite
similar. It should be noted that both white tea extract and cell
walls fraction extract are not capable of fully protecting the
substrate against destructive action of irradiation and as result,
the absorbance of Vitro-Skin.RTM. testing substrate (IMS Testing
Group, Milford, Conn.) is decreased almost as much as at the
condition when this substrate was not protected at all (FIG. 9). It
is especially obvious in the range 250 nm-330 nm although at longer
wavelengths some increase in the absorbance is observed.
[0176] The irradiation of membrane fraction extract produced very
different effect on its absorbance spectra (FIG. 12). For example,
irradiation did not initiate any changes in spectral range 250-285
nm. As discussed, this particular range of spectra was very
significantly impacted by the destruction of irradiated
Vitro-Skin.RTM. testing substrate (IMS Testing Group, Milford,
Conn.) and therefore comparison of the spectra suggest that the
destruction of the substrate was completely prevented by the
presence of membrane fraction extract on its surface.
[0177] However, certain changes in membrane fraction extract
spectra were registered. For example, the absorbance was slightly
decreased in the range 290-320 nm and it was accompanied with small
increase of absorbance at longer wavelengths. It should be
especially pointed out, that membrane fraction extract was proven
to be very effective in the range of spectra where nucleic acids
and aromatic amino acids have characteristic peaks at 260 nm and
280 nm subsequently. Thus, effect of membrane fraction extract
allows the use of this product as a prospective UV protective
ingredient for topical applications.
[0178] After irradiation the serum fraction was demonstrating
certain changes in its absorbance spectra (FIG. 13).
[0179] Generally, these changes can be described as slight decrease
of the absorption in the range 250-340 nm and slight increase of
the absorption in the range 340-450 nm. It should be noted, that
elimination of the possible contribution provided by
photo-destruction of Vitro-Skin.RTM. testing substrate (IMS Testing
Group, Milford, Conn.) (see red curve on spectra above the initial
spectra of non-irradiated serum fraction) clearly indicated that
substrate was effectively preserved by the application of serum
fraction on its surface.
[0180] Observations and Conclusions.
[0181] The above results clearly show that the best of conventional
Camellia products--white tea extract--provides relatively weak
protection against the destructive action of UV irradiation. The
cell walls fraction extract demonstrated properties similar to
white tea extract, but membrane fraction extract and cell juice
serum have much more potent UV protective properties.
[0182] The UV protection properties of Camellia products were found
to be increasing in the following sequence: white tea extract=cell
walls fraction extract>cell juice serum>membrane fraction
extract.
[0183] It should be noted, that spectral properties of Camellia
products and pattern of the changes of these properties after UV
irradiation provide strong evidence that compositions of
constituents in white tea extract (control), cell walls fraction
extract, membrane fraction extract and cell juice serum all differ
and display unique activities. This is of particular interest in
the case of novel Camellia compositions where the UV activity
described above cannot be attributed to polyphenols as it is with
white tea extracts.
Example 10
Comparative Evaluation of Camellia Products: An Overview
[0184] Examples 10 through 19 describe methods, results, and
analyses relating to experiments conducted to evaluate the range of
biological activities related to the modulation of cell functions
by the bioactive compositions from Camellia sinensis of the present
invention. The primary objective was to evaluate the range of
biological activities of products obtained from the methods of the
present invention and compare these with activities of the best
product obtained by conventional (traditional) tea
technology--white tea extract, which was explored as a positive
control and compared with the following bioactive compositions of
the present invention: (1) cell walls fraction extract of fresh
leaves (composition A, as referenced herein); (2) membrane fraction
extract (composition B, as referenced herein); and (3) cell juice
serum (composition D, as referenced herein).
[0185] The tests were conducted to evaluate the effect of these
Camellia bioactive compositions on growth patterns of three human
cell lines: a myeloid line with characteristics of monocytic
leukemia cells (Mono Mac 6) and two breast cancer lines with
characteristics of early stages of the malignancy in vivo (MCF-7)
and a more highly invasive, metastatic and estrogen insensitive
line with characteristics of advanced cancer (MDA-MB-435S).
[0186] It was found that conventional white tea extract
demonstrated a certain inhibitory effect on metabolic activity of
some tumor cells. However, the extent of such inhibition was not
significant for all types of tested cells and even when such
inhibition was detected, it was generally not complete but rather
minimal or modest. The cell walls fraction extract demonstrated
properties similar to properties to those of white tea extract.
[0187] It is noteworthy that both of the cell juice derivatives:
membrane fraction extract and cell juice serum, were much more
potent inhibitors of metabolic functions of all tested cell lines
which were cultured in the presence and absence of different
stimuli. For example, membrane fraction extract clearly
demonstrated greater inhibition potency and its effect could be
reliably measured at a dose of 0.001%. The cell juice serum
demonstrated a complex response: stimulation at a lower dose and
inhibition at a high dose.
[0188] It should be noted that rather than inducing necrotic
cytolysis, membrane fraction extract and cell juice serum appear to
initiate a pathway of programmed, or apoptotic cell death in the
tumor cells. The experimental data indicate that this pathway is
attributed to loss of mitochondrial function and may require 24 to
48 hours of exposure to be detected.
[0189] As a consequence of exposure to bioactive compositions, the
metabolic function of all tested tumor cell lines: MCF-7, a model
of early stage human breast cancer, MDA-MB-435S, a model of
advanced breast cancer, and Mono Mac 6, a model of monocytic
leukemia, was inhibited, most effectively by the membrane fraction
extract and, less potently and more selectively, by the cell juice
serum. Remarkably, the white tea extract and cell walls fraction
extract were proven to be inactive or much less potent than the
above compositions B and D. This trend was clearly proven for the
cells tested under different conditions: MCF-7 cells in the absence
and in the presence of transforming growth factor, MDA-MB-435S
cells and both stimulated and non-stimulated monocytic Mono Mac 6
cells.
[0190] These results provide strong evidence of the ability of the
method of the present invention to drastically increase the potency
of Camellia plants and produce very impressive novel products
demonstrating activities, which were not identified for even the
best product of conventional tea technology.
[0191] Effects of the Camellia fractions of the present invention
on cell-mediated proteolytic activities have implications for
inflammatory tissue injury as well as tumor invasion and
metastasis. Thus, breast cancer cells and leukemia cells clearly
can be suggested as prospective targets for the bioactive
compositions of the present invention, most notably, the membrane
fraction extract. It should be noted that it was previously shown
that the colon carcinoma-derived cell line COLO 205 releases
significant levels of MMP-2, which is then activated by a
trypsin-like enzyme also secreted by the cells. This is also one of
potential targets for the Camellia fractions of the present
invention, based on results with Mono Mac 6 cells.
[0192] From these studies it has been concluded that the present
invention's bioactive compositions isolated from fresh Camellia
have activities result in impressive modulation of key cell
functions. The effects that have been observed could have valuable
applications ranging from personal care products to nutraceuticals
and potentially pharmaceuticals.
[0193] It should also be noted that the present invention's very
potent bioactive compositions are not single purified components,
but rather isolated complexes of constituents. Further
fractionation of membrane fraction extract (composition B) and cell
juice serum (composition D) could yield extremely potent
ingredients for the growing market of natural pharmaceuticals.
Example 11
Comparative Evaluation of Camellia Products: Tested
Compositions
[0194] The following bioactive compositions were used in the
experiments described in Examples 10 through 19: [0195] (1)
Positive Control: White tea extract which was prepared according to
the procedure described in Examples 1 and 4. [0196] (2) Composition
A: A cell walls fraction extract of fresh leaves of Camellia which
was prepared according to procedure described in Examples 1 and 4.
[0197] (3) Composition B: A membrane fraction extract obtained from
freshly processed leaves of Camellia and prepared according to
procedure described in Examples 1 and 4. [0198] (4) Composition D:
Cell juice serum of freshly processed leaves of Camellia and
prepared according to the procedure described in Examples 1 and
4.
[0199] The above products were obtained from the same lot of fresh
Camellia to prepare the conventional white tea extract and three
"parallel" products of the present invention (compositions A, B and
D).
[0200] There are a number of reports in the literature, which
suggest that extracts of Camellia leaves have a range of biological
activities, primarily attributed to the significant concentrations
of polyphenolic tannins that form during the curing process. These
polyphenols, as well as lower molecular weight precursors to the
polymeric tannins such as epigallocatechin-3-O-gallate (EGCG), have
been reported to display potent antioxidant activities. There is a
growing number of publications suggesting not only antioxidant, but
also anti-angiogenic, anti-bacterial, anti-neoplastic,
anti-inflammatory, anti-mutagenic, anti-septic, and detoxifying
properties of teas prepared from dried leaves of Camellia. Not all
of the above properties have been proven to confer statistically
significant benefits. Only some of them have been confirmed in
comprehensive studies using multiple testing systems.
[0201] As past reference, it should be pointed out that, from past
experience with bioactive compositions isolated from a number of
fresh plant sources other than Camellia using the present
invention's technology, such compositions were proven to be much
more potent than conventional products isolated from the same dried
plants using a number of parameters as was previously described in
the U.S. Patent Application Publication No. 2003/0175235, which is
hereby incorporated by reference in its entirety). For example, in
other types of plants (Medicago sativa, Hordeum vulgare, Lavandula
angustifolia, Calendula officinalis and Salvia officinalis),
several impressive biological activities of compositions prepared
using the method of the present invention have been identified and
evaluated, including high anti-elastase and anti-gelatinase B
(MMP-9) activities, novel modulation of the neutrophil respiratory
burst, and significant superoxide scavenging activity towards
reactive oxygen species. Other than scavenging activity, these
activities are not likely to be ascribed to mixtures of polyphenols
alone.
[0202] Thus, it was especially interesting to explore a more
comprehensive approach to compare the range of activities, which
could be detected in the Camellia compositions of the present
invention with the activities present in an extract obtained from
the same dried plant using conventional (traditional) Camellia
technology. Accordingly, modulation of functions in living
mammalian cells by the cell walls fraction extract (composition A),
membrane fraction extract (composition B) and cell juice serum
(composition D) prepared from freshly collected leaves of Camellia
have been assayed. These compositions have been compared to extract
of conventional white tea prepared from dried Camellia leaves.
[0203] It should be noted that, according to multiple studies of
conventional Camellia products, the white tea extract demonstrated
higher specific activities and therefore a preparation of this sort
was selected as a representative positive reference control for
comparison with the novel Camellia products of the present
invention.
Example 12
Comparative Evaluation of Camellia Products: Rationale for
Selection of Cell Lines
[0204] As a test system for modulation of cell functions, two human
breast carcinoma-derived lines were used as models of neoplastic
cells (MCF-7 and MDA-MB-435S), and a human monocytoid line (Mono
Mac 6) was used as a model of inflammatory cells. The above cell
lines are described in Example 21.
[0205] MCF-7 is considered a model of early or less
de-differentiated breast cancer. The line still retains estrogen
sensitivity and has a relatively low invasive phenotype; its
capacity to metastasize in immunodeficient animal models is quite
modest. In previous studies, the MCF-7 cell line has been shown to
display a characteristic response to Transforming Growth
Factor-.beta. (TGF-.beta.): after culture for 24 hours in the
presence of TGF-.beta., the cells secrete increased levels of the
Matrix Metallo Proteinase (MMP) family of proteolytic enzymes and
the pro-angiogenic factor Vascular Endothelial Growth Factor
(VEGF), two different markers of enhanced invasiveness and
metastatic potential. This response to TGF-.beta. is a mark of
tumors and some tumor cell lines, in contrast to growth arrest,
which is induced in normal cells by the growth factor. To evaluate
the bioactive compositions of the present invention, MCF-7 cells
cultured in the absence and presence of TGF-.beta. were used as
targets.
[0206] The MDA-MB-435S line is more highly invasive, metastatic and
estrogen insensitive. This human carcinoma-derived cell line also
shows some sensitivity to TGF-.beta., but even in the absence of
the growth factor, it spontaneously releases higher levels of MMPs
and VEGF than MCF-7, consistent with its use as a model of more
advanced cancer. In present evaluation the effects of bioactive
compositions on MDA-MB-435S cells cultured only in the absence of
TGF-.beta. were examined.
[0207] The human monocytoid line, Mono Mac 6, expresses a number of
biomarkers consistent with those of resting monocytes or
macrophages, and responds like human monocytes and macrophages to
pro-inflammatory activating stimuli such as Phorbol Myristate
Acetate (PMA). The effects of bioactive compositions on Mono Mac 6
cells cultured in the absence and presence of PMA were examined, to
serve as models of resting and activated monocytes/macrophages.
[0208] Thus, the selected combination of the cell lines described
above provides a reliable foundation for evaluations of anti-tumor
and anti-inflammatory potencies of Camellia bioactive compositions.
Parallel testing of selected cell lines with a number of functional
probes provides the opportunity to draw more valuable conclusions
concerning the activities of products and their mechanism of
action-than investigation of the responses of a single test target
or targets having similar sensitivities or similar responses to
certain stimuli.
Example 13
Comparative Evaluation of Camellia Bioactive Compositions:
Rationale for Selection of Assays
[0209] Initial evaluations were based on two viability assays and a
probe of cell functions (see Example 20, "Method 8").
[0210] The first assay measures levels of the cytosolic enzyme,
lactic dehydrogenase, which is liberated into the extracellular
culture medium only when the cells lyse. Such loss of cell membrane
integrity is traditionally considered to be a sign of necrotic cell
death and reflects the cytotoxicity pattern.
[0211] The second assay measures mitochondrial dehydrogenase
activity as reflected by the reduction of a tetrazolium salt to its
colored formazan. When the MTS reagent (a tetrazolium salt) is
applied to living cells, it is converted to an intensely colored
compound (formazan). Loss of mitochondrial dehydrogenase activity
can also be associated with cell death, but is typically a marker
for the early steps in a programmed cell death, or apoptotic,
pathway in which cell membrane integrity is generally retained well
after the nucleus has condensed and the mitochondria have ceased to
function.
[0212] The leakage of lactic dehydrogenase indicates complete loss
of viability associated with cytolysis, while decreased reduction
of tetrazolium salts indicates loss of mitochondrial activity, but
not necessarily irrevocable loss of cell membrane integrity or
viability.
[0213] As an additional probe of cell functions, the effects of the
Camellia bioactive compositions on levels of proteinases secreted
by the Mono Mac 6 line have been examined (see Example 20, "Method
9"). In previous studies with this cell line, it was observed that,
after incubation with PMA, Mono Mac 6 cells secrete two so-called
gelatinolytic matrix metalloproteinases, MMP-2 (gelatinase A) and
MMP-9 (gelatinase B). These MMPs are also secreted by a number of
tumors and by their surrounding stroma, and are implicated in
inflammatory tissue injury as well as tumor invasion and
metastasis. It was also previously shown that some agents under
development as anti-inflammatory and anti-tumor drugs (the agents
that have been investigated are known to diminish inflammatory
tissue destruction as well as invasion and metastasis of tumor cell
lines) appear to reduce the levels of the MMPs produced by cells in
addition to any direct inhibition of MMP proteolytic activity. The
objective in these studies was to evaluate the possibility that the
Camellia bioactive compositions of the present invention might have
a similar capacity to diminish levels of MMPs released by activated
Mono Mac 6 cells.
[0214] Thus, the selected assays will allow one to reliably
evaluate a broad spectrum of metabolic processes and effectively
obtain important data, which might reveal mechanisms of action
triggered by certain Camellia bioactive compositions.
Example 14
Comparative Evaluation of Camellia Products: Effects of Camellia
Bioactive Compositions on Breast Tumor Cell Lines
[0215] Throughout these studies, mitochondrial function was
measured solely through assays of reduction of the tetrazolium salt
MTS to its formazan. It should be noted that some intrinsic
capacity of higher concentrations of the Camellia compositions of
the present invention have been observed to reduce MTS directly in
the absence of any viable cells, and in all the results reported
here, such background formation of formazan in the absence of cells
has been subtracted from the levels of reductase activity observed
in the presence of the cells.
[0216] FIGS. 14 through 21 illustrate the magnitude of the
reductase activity of MCF-7 cells, cultured in the absence and
presence of 5 ng/ml TGF-.beta., and MDA-MB-435S cells, cultured
only in the absence of TGF-.beta., at 24 hours and 48 hours after
the addition of various doses of each of the four Camellia
compositions, ranging from 0.01% or 0.02% (w/v, final concentration
in the culture medium, based on dry weight of the solids in the
Camellia compositions) to 0.0001%.
Example 15
Comparative Evaluation of Camellia Bioactive Compositions: MCF-7
Cells
[0217] In the absence of TGF-.beta., the highest tested
concentration (0.01%) of the composition A and white tea extract
(positive control) had a marked effect on MTS reduction by MCF-7
cells. At that concentration there was significant but incomplete
inhibition of reductase activity (.about.50-70% inhibition) after
24 hours of exposure to the Camellia composition A. The similar
inhibition of reductase activity was detected after 24 hours of
exposure to white tea extract.
[0218] In contrast, the two Camellia bioactive compositions
(membrane fraction extract and cell juice serum) prepared from
Camellia cell juice were more potent inhibitors of reductase
activity in MCF-7 cells in the absence of TGF-.beta.. The membrane
fraction extract (composition B) resembled white tea extract in
dose dependence, except for somewhat greater potency, producing
virtually complete inhibition at 0.01%, the highest dose tested.
The cell juice serum (composition D) also produced virtually
complete inhibition at 0.01%, but at the lower dose of 0.0025%,
there was some evidence of stimulation of reductase activity. Lower
doses of composition D were without significant effect.
[0219] When MCF-7 cells were cultured in the presence of the growth
factor TGF-.beta., their sensitivity to the Camellia compositions
was significantly altered. After 24 hours or 48 hours of exposure
to the cell walls fraction extract and white tea extract, there was
no evidence of either a stimulatory or an inhibitory effect on
reductase activity at any dose, except for a modest inhibition of
less than 20% at the highest dose (0.01%) of white tea extract.
[0220] In contrast, exposure of TGF-.beta.-treated MCF-7 cells for
24 hours to the membrane fraction extract at a dose of 0.02%
resulted in 70% inhibition of reductase activity, and after 48
hours, reductase activity was virtually completely abated. More
modest inhibition could be detected at lower doses of membrane
fraction extract after 24 hours, but after 48 hours, a marked
activation of reductase activity was detected. The same activation
of reductase activity by low doses of the cell juice serum as well
as marked inhibition at the highest dose (0.02%) was detected after
48 hours of exposure, but this composition had only minimal effect
on reductase activity in TGF-.beta.-treated MCF-7 cells after the
more limited exposure of 24 hours, regardless of dose.
[0221] In evaluation, there was no detection of significant release
of lactic dehydrogenase into the culture medium of MCF-7 cells
exposed for 24 hours to even the highest doses of any of the
Camellia compositions. It would appear that, the loss of
mitochondrial function in these cells is not accompanied by a
necrotic lysis of the cells. If the cells are in fact dying during
the first 48 hours of exposure, it is more likely that a programmed
cell death, or apoptotic, pathway has been initiated. This
conclusion is supported by light microscopic observations, which
reveal that there is some rounding of the cells, but no formation
of debris or membrane fragments during the course of the
exposures.
[0222] Thus, inhibition of mitochondrial function appears to be a
predominant mode of action of all tested Camellia products, which
did not demonstrate cytotoxity or necrosis as indicated by levels
of released lactic dehydrogehase.
Example 16
Comparative Evaluation of Camellia Bioactive Compositions:
MDA-MB-435S Cells
[0223] The pattern of response of MDA-MB-435S cells to the Camellia
compositions was similar to that of MCF-7 cells in that the most
potent compositions were composition B and D, with the membrane
fraction extract (composition B) showing somewhat greater potency
than the cell juice serum (composition D). Only the membrane
fraction extract produced marked inhibition of reductase activity
after only 24 hours of exposure Inhibition reached -70% of control
reductase values at the highest dose of 0.01%, but a modest
inhibition of -10% could be reliably detected at even the lowest
dose of remarkable concentration -0.0001%. The other tested
compositions had only modest inhibitory effects at 24 hours of
exposure, and only at the higher doses tested.
[0224] After 48 hours of exposure, reductase activity was inhibited
in a dose-dependent fashion in the presence of each of the
compositions, but the potency at the highest dose of the
compositions did not reach near 100% inhibition, except for the
membrane fraction extract. This composition inhibited reductase
activity by -50% at 0.001% after 48 hours. The white tea extract
and cell walls fraction extract also had significant inhibitory
activity against MDA-MB-435S cells after 48 hours of exposure,
which was actually greater than that of the cell juice serum. No
doses of any of the preparations induced activation of reductase
activity in this cell line, regardless of duration of exposure.
[0225] It should be noted that any impact on highly invasive,
metastatic and estrogen insensitive line MDA-MB-435S is rare to
observe after only 24 hours. Thus, the effect of composition B
after 24 and 48 hours is rather remarkable and indicates that this
preparation has significant activity.
Example 17
Comparative Evaluation of Camellia Bioactive Compositions: Effects
of Camellia Compositions on Monocytoid Cells
[0226] Mitochondrial Dehydrogenase Activity:
[0227] Certain of the trends revealed by the preliminary studies on
the breast tumor cell lines have proved to be consistent with the
response of Mono Mac 6 cells to the four tested Camellia
compositions. The membrane fraction extract (composition B) and
cell juice serum (composition D) were more potent inhibitors of MTS
reductase activity in this inflammatory cell line than the cell
walls fraction extract (composition A) and white tea extract
(positive control), and the membrane fraction extract (composition
B) clearly had the greatest inhibitory potency. The effects on MTS
reductase in cells which were left unstimulated and those which
were stimulated with 10 nM PMA were examined, and reductase
activity after 24 and 48 hours of exposure to the Camellia
compositions was evaluated. The effects of these compositions on
MTS reductase activity in Mono Mac 6 cells are shown in FIGS. 22
through 33.
[0228] The white tea extract showed a weak but dose-dependent
inhibition of reductase activity after 48 hours of exposure to
PMA-stimulated cells; there was no significant loss of reductase
activity regardless of the dose or length of exposure in the
absence of PMA, nor was there any effect of any dose after 24 hours
of exposure to PMA-treated cells. The cell walls fraction extract
had no effect on Mono Mac 6 cells regardless of dose or time of
exposure and regardless of whether the cells were unstimulated or
stimulated with PMA.
[0229] The cell juice serum of fresh Camellia leaves inhibited
unstimulated Mono Mac 6 cell reductase activity modestly in a dose
dependent fashion after 24 or 48 hours of exposure. The maximum
inhibition at the highest dose of 0.01% (w/v, final concentration
in the culture medium, based on dry weight of solids in the
starting preparation) was only .about.20-30% of the control
activity. Inhibition of PMA-stimulated cells reached 50% of control
activity but only at the highest dose of composition D (0.01%), and
only after 48 hours of exposure.
[0230] The membrane fraction extract of freshly harvested Camellia
(composition B) proved to be the most potent of the tested
preparations in inhibiting Mono Mac 6 cell reductase activity as it
had toward the breast tumor cell lines. Effects of PMA stimulation
or duration of exposure to the composition had little effect on
inhibition, which was dose-dependent in the presence or absence of
PMA and was roughly the same after 24 hour or 48 hour exposure.
Reductase activity was inhibited by -70% in the presence of PMA and
by -80-90% in the absence of PMA at the highest dose of 0.02%, but
lower levels of inhibition (-15%) could be reliably measured at a
dose of 0.001%. Measurements of release of cytosolic enzymes have
not been undertaken to confirm that the loss of reductase activity
is not associated with necrotic cytolysis, but no evidence of
membrane fragmentation could be seen by light microscopic
examination of Mono Mac 6 cells exposed to any of the bioactive
compositions at 0.02% for 48 hours. Moreover, as shown below, the
cells appear still capable of secreting at least one MMP under
conditions in which reduction of MTS is markedly diminished.
[0231] These results suggest that in these cells, as well as the
breast tumor cell lines, the loss of reductase activity is
associated with a relatively selective loss of mitochondrial
function and can reflect initiation of a pathway of programmed cell
death or apoptosis.
[0232] Secretion of MMPs.
[0233] Two different assays have been used to measure the levels of
two gelatinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B),
released by Mono Mac 6 cells. These MMPs have been implicated in
inflammatory tissue damage as well as tumor invasion and
metastasis. Employed enzyme-linked immunosorbent assays (ELISAs)
for MMP-2 and MMP-9 were first used to estimate total levels of the
two enzymes in the culture medium of Mono Mac 6 cells cultured for
48 hours in the presence of 10 nM PMA and different doses of the
three Camellia bioactive compositions and positive control.
[0234] This cell line secretes only MMP-2 when it is unstimulated,
but secretes both MMP-2 and MMP-9 when it is activated. (Levels of
MMPs released after 24 hours are usually too low to be reliably
detected).
[0235] The results of the ELISA measurements are shown in FIGS. 34
through 37. As was observed for the effects of cell walls fraction
extract and white tea extract on MTS reduction by Mono Mac 6 cells,
there was no significant change in levels of secreted MMP-2 or
MMP-9 at any dose of these extracts. At the highest dose (0.01%
w/v) of the membrane fraction extract and cell juice serum, the
levels of MMP-2 were observed to be diminished, with the membrane
fraction extract exhibiting the greatest potency at this dose. It
should be noted, that an apparent slight stimulation of MMP-2
release was observed at the next highest doses of composition B
(0.001%) and composition D (0.002%). This stimulation is
reminiscent of the stimulation of MTS reductase activity in
TGF-.beta. treated MCF-7 cells at similar doses of these
compositions.
[0236] The dose-dependent diminution of MMP-2 levels detected by
ELISA was not paralleled by the effects of membrane fraction
extract and cell juice serum on MMP-9 levels. These levels were
increased (apparently markedly so by cell juice serum) at the
highest doses, but were unchanged at the lower doses tested. The
detection of unchanged or increased levels of MMP-9 secreted by
Mono Mac 6 cells exposed for 48 hours to doses of Camellia
preparations which produced significant inhibition of MTS reductase
activity after only 24 hours, is further evidence that the loss of
mitochondrial function in Mono Mac 6 cells exposed to the membrane
fraction extract or cell juice serum of Camellia does not reflect
necrotic cytolysis, in which case MMP secretion would have abruptly
ceased.
[0237] As further evidence of the effects of the Camellia
compositions on MMP secretion by Mono Mac 6 cells, the technique of
gelatin zymography was used to examine the culture media collected
as described above for the ELISA measurements. In this method, the
culture media are first subjected to electrophoresis in
gelatin-impregnated polyacrylamide gels in the presence of Sodium
Dodecyl Sulfate (SDS-PAGE) to separate the proteins on the basis of
molecular weight. The SDS is then washed out of the gels to allow
at least a portion of any enzymes present to renature and the gels
are incubated in a medium, which maximizes MMP activity. MMPs
dissolve the gelatin wherever they may be present. After
visualizing the undigested gelatin in the bulk of the gels with a
protein stain, the gels are scanned, with the MMPs appearing as
clear zones against the stained background. Negative images have
been presented here, so that the MMPs appear as dark zones against
a light background.
[0238] It should be noted that MMPs are secreted by most cells as
inactive precursors, which are then activated extracellularly.
However, because of the denaturing and renaturing sequence employed
in zymography, even the so-called inactive pro-forms of the MMPs
acquire gelatinolytic activity and produce clear zones. FIGS. 34
through 37 illustrate the negative images of gelatin zymograms of
culture media collected after 48 hour exposure of Mono Mac 6 cells
to the different Camellia bioactive compositions, along with
culture medium collected from cells cultured in the absence (U) or
presence (S) of 10 nM PMA but in the absence of Camellia
compositions.
[0239] The effects of composition A and positive control were
evaluated only for the lowest dose (0.0001%, "10") and the highest
dose (0.01%, "hi") of the preparations, whereas the effects of
compositions B and D were also evaluated at the intermediate dose
of 0.001% ("med"). It is apparent from the four panels that Mono
Mac 6 cells release only MMP-2 (.about.67 kD) in the absence of
PMA, and this enzyme is found predominantly in the pro-form.
Treatment with 10 nM PMA results in induction of MMP-9 secretion
(.about.0.92 kD), as well as further proteolytic activities which
convert significant levels of the pro-forms of the two MMPs to
their slightly lower molecular weight active forms.
[0240] Consistent with the ELISA results, exposure of
PMA-stimulated Mono Mac 6 cells to cell walls fraction extract and
white tea extract had no detectable effect on the levels of either
the pro- or active forms of either of the two MMPs visualized by
gelatin zymography. In contrast, exposure to the highest dose of
compositions B and D resulted in marked diminution of the levels of
MMP-2 visualized by gelatin zymography, but no apparent change in
the levels of MMP-9.
[0241] The appearance of both pro- and active forms of MMP-9, as
well as the faint, but recognizable, band corresponding to the
active form of MMP-2 seen in the media collected from cells treated
with the highest dose of compositions B and D, suggests that the
effects of these compositions are primarily on modulation of
release of MMP-2 and do not involve additional effects on the MMP
activation mechanisms in these cultured cells.
Example 18
Comparative Evaluation of Camellia Bioactive Compositions: Summary
of Results
[0242] The experimental data indicate that Camellia bioactive
compositions trigger a dose-dependent loss of MTS reductase
activity, which is generally attributed to loss of mitochondrial
function. This inhibition may require as long as 48 hours of
exposure to be detected and at least for the first 24 hours, there
is no measurable release of cytosolic enzymes, suggesting that
rather than inducing necrotic cytolysis, the bioactive compositions
initiate a pathway of programmed, or apoptotic, cell death in the
tumor cells.
[0243] The differences in the time- and dose-dependence of the
response of MCF-7 cells and MDA-MB-435S cells, and the effects of
TGF-.beta. treatment of the MCF-7 cells, all point to a somewhat
increased resistance of the more invasive and metastatic phenotypes
to white tea extract, cell walls fraction extract, and to some
degree, cell juice serum, as evidenced by the relatively modest
loss of reductase activity within the first 24 hours of exposure.
However, the trend of greater potency of the membrane fraction
extract is evidenced by its capacity to inhibit MTS reductase
activity in TGF-.beta.-treated MCF-7 cells, as well as MDA-MB-435S
cells within 24 hours.
[0244] The effects of tested Camellia bioactive compositions on the
Mono Mac 6 cell line, a model of human monocytes/macrophages, have
certain similarities to the effects observed on breast tumor cell
lines. Based on the absence of lactic dehydrogenase in the culture
medium of the breast tumor cell lines and the presence of normal to
increased levels of secreted MMP-9 in the culture medium of Mono
Mac 6 cells, it has been concluded that these compositions do not
induce necrotic cytolysis, even at the highest dose tested (0.01%
w/v).
[0245] However, the two bioactive compositions (membrane fraction
extract and cell juice serum) induce a dose-dependent inhibition of
mitochondrial reductase activity, which reflect initiation of an
apoptotic pathway of programmed cell death in Mono Mac 6 cells.
Furthermore, exposure of these inflammatory cells to the membrane
fraction extract and cell juice serum results in selective
diminution in the levels of the gelatinolytic enzyme, MMP-2
(gelatinase A). The gelatin zymography indicates that mechanisms of
"pro-form" or zymogen activation are unaffected by the Camellia
bioactive compositions, so it is highly unlikely that the
diminished levels MMP-2 in the medium reflect enhanced proteolytic
destruction.
[0246] Thus, the metabolic activity of all tested cell lines (i.e.,
a model of early stage human breast cancer, a model of advanced
breast cancer cells, and a model of monocytic leukemia) was
effectively inhibited by the membrane fraction extract (composition
B) and, in most of cases, the cell juice serum (composition D).
Remarkably, the extract of cell walls tea (Composition A) and white
tea extract (positive control) were proven to be inactive or much
less potent than the above compositions B and D.
[0247] This trend was clearly proven for all tested MCF-7 human
cancer cells in the absence and in the presence of transforming
growth factor, for MDA-MB-435S advanced human breast cancer cells,
and for stimulated and non-stimulated monocytoid Mono Mac 6 cells.
The data related to the summary of testing and evaluation of
bioactive Camellia compositions are presented in Table 15.
TABLE-US-00015 TABLE 15 Summary of Testing and Evaluation of
Bioactive Camellia Compositions Extract of White Tea Extract of
Cell Membrane Time of Extract Walls Fraction Fraction Cell Juice
Serum Cell Line and Model Stimuli Cultivation (Positive Control)
(Composition A) (Composition B) (Composition D) Human Cancer Cells
24 hours Modest Inhibition Modest Inhibition Strong Modest
Inhibition MDA-MB-435S Inhibition Advanced Breast 48 hours
Significant but Significant but Complete Significant but Cancer
Incomplete Incomplete Inhibition Incomplete Inhibition Inhibition
Inhibition Human Cancer Cells 24 hours Significant but Significant
but Complete Complete MCF-7 Incomplete Incomplete Inhibition
Inhibition Early Breast Cancer Inhibition Inhibition TGF-.beta. 24
hours Modest Inhibition No Effect Significant but Complete
Incomplete Inhibition Inhibition 48 hours Modest Inhibition No
Effect Stimulation at Stimulation at Lower Dose and Lower Dose and
Complete Significant but Inhibition at Incomplete High Dose
Inhibition at High Dose Human Leukemia 24 hours Modest Inhibition
Modest Inhibition Complete Pronounced Cells Mono Mac 6 Inhibition
Inhibition Inflammation 48 hours No Effect No Effect Significant
but Pronounced Incomplete Inhibition Inhibition PMA 24 hours No
Effect Modest Inhibition Significant but Significant but Incomplete
Incomplete Inhibition Inhibition 48 hours Modest Inhibition No
Effect Significant but Significant but Incomplete Incomplete
Inhibition Inhibition
[0248] Table 15 shows that abilities of Camellia preparation to
modulate cell functions in a dose-dependent manner is increasing in
the following order: white tea extract=cell walls fraction
extract>cell juice serum>membrane fraction extract. The
experimental data suggests that, novel bioactive Camellia
compositions prepared by processing of fresh plant tissue into cell
juice derived membrane fraction extract (composition B) and cell
juice serum (composition D) do not trigger any outright necrotic
toxicity towards the cells.
[0249] Therefore, the technology of the present invention displays
the ability to drastically increase the potency of Camellia
bioactive compositions and to produce very impressive novel
products demonstrating activities on viable human cells which were
not demonstrable in the best products (for example, white tea
extract) produced by conventional Camellia technology.
Example 19
Comparative Evaluation of Camellia Bioactive Compositions
Implications for Future Studies
[0250] Effects of the Camellia bioactive compositions of the
present invention on cell-mediated proteolytic activities have
implications for inflammatory tissue injury as well as tumor
invasion and metastasis. Thus, breast cancer cells and monocytic
leukemia cells clearly can be suggested as prospective targets for
the Camellia bioactive compositions of the present invention, most
notably, composition B (membrane fraction extract). It was
previously shown that the colon carcinoma-derived cell line COLO
205 releases significant levels of MMP-2, which is then activated
by a trypsin-like enzyme also secreted by the cells. This type of
tumor cell is one of a number of potential targets for the Camellia
bioactive compositions of the present invention, based on results
with Mono Mac 6 cells.
[0251] From these studies, one can be confident that the bioactive
compositions isolated from fresh Camellia of the present invention
have significant activities, which result in impressive modulation
of key cell functions. The effects that have been observed have
valuable applications ranging from personal care products to
nutraceuticals and potentially pharmaceuticals.
Example 20
Protocols Used for Determining Certain Characteristics of Bioactive
Compositions
[0252] The following are various methods used for determining
certain characteristics of Bioactive Compositions. These methods
are referenced throughout the above Examples. References below to
the "tested products" or the "test samples" refer to Bioactive
Compositions.
[0253] Method 1: Method for Determination of Solid Content.
[0254] The procedure for determination of solid content included
evaporation of the tested bioactive composition in the water bath
at 100.degree. C. until complete evaporation of water, oven storage
of the sample at 105.degree. C. for 3 hours, cooling to room
temperature, and immediate determination of the weight of the
container with solid matter.
[0255] Method 2: Method for Determination of Non-Volatile
Residue.
[0256] The procedure for determination of non-volatile residue
included oven storage of the tested bioactive composition at
105.degree. C. for 5 hours, cooling, and immediate determination of
the weight of the container with solid matter.
[0257] Method 3: Method for Determination of L*a*b* Values.
[0258] The procedure for determination of L*a*b* values utilized
Hunter Labscan fixed geometry colorimeter with measuring geometry
of 0.degree./45.degree.. Standard illuminant D.sub.65 with viewing
window facing upward was used. The container with tested bioactive
composition was placed on viewing window and measured through the
bottom. The following CIELAB equations were used:
C*=(a*.sup.2+b*.sup.2).sup.1/2
DE*=[(DL).sup.2+(Da*).sup.2+(Db*).sup.2].sup.1/2
DH=[(DE*).sup.2-(DL*).sup.2-(DC*).sup.2].sup.1/2.
[0259] Method 4: Method for Determination of Total Carotenoids
Content and Lutein Content.
[0260] The tested bioactive compositions were extracted with
acetone. After homogenization and vacuum filtration, all extracts
were saponified with 30% potassium hydroxide in methanol. The
carotenoids were successively extracted from bioactive compositions
with petroleum ether. After additional treatment and
re-solubilization in ethanol, all samples were measured at 446
nm.
[0261] In order to determine the lutein content, an additional
dried sample from each sample extraction was used for high
performance liquid chromatography ("HPLC") analysis. The dried
sample was re-solubilized in MTBE and methanol. The reverse phase
HPLC system with (250.times.4.60 mm I.D.) 5 .mu.m C.sub.18 column
("Vydac") was used. The identity of lutein was conformed by the
co-chromatography of an authentic standard. The molar absorptivity
coefficient for lutein in ethanol is 144,800 cm.sup.-1
mol.sup.-1.
[0262] Method 5: Method for Determination of Elastase Inhibitory
Activity.
[0263] The elastase inhibitory activity of tested bioactive
compositions was determined using the assay, which employs
neutrophil elastase (a purified enzyme preparation produced by
"Elastin Products") and synthetic peptide soluble substrate
Methoxysuccinyl-Ala-Ala-Pro-Val-p-Nitroanilide produced by "Sigma".
Enzymatic cleavage of the substrate results in generation of
increasing yellow color over time (405 nm); the rate of color
generation is diminished by increasing concentrations of tested
bioactive compositions containing inhibitory activity. Analysis of
the concentration dependence of inhibition permits quantitation of
the potency of the inhibitory activity, expressed as that
concentration of dry matter within each tested bioactive required
to achieve 50% inhibition (IC.sub.50), but also provides
information relating to the mode of inhibition.
[0264] For the determination of IC.sub.50, the concentration of
elastase was 2.5 .mu.g/ml and concentration of substrate was 150
.mu.M. For the determination of IC, the concentrations of substrate
were 100 .mu.M and 200 .mu.M.
[0265] Method 6: Method for Determination of Gelatinase B
(MMP-9)
[0266] Inhibitory Activity. The commercially distributed assay
(MMP-9 Activity ELISA produced by "Amersham Pharmacia"), which
captures Gelatinase B specifically onto multiwell microplates by
immune recognition, was used after other proteinases were washed
away. The enzymatic activity was detected at 405 nm by hydrolysis
of a low molecular weight synthetic substrate for Gelatinase B:
APMA. Analysis of the concentration dependence of inhibition was
used to determine the potency of tested bioactive composition dry
matter.
[0267] Method 7: Method for Determination of Superoxide Scavenging
Activity.
[0268] The enzymatic system, which uses xanthine oxidase (a
purified enzyme preparation produced by "Sigma"), was used to
generate superoxide anions in high yield and in a controlled
fashion. The conversion of xanthine to hydroxanthine by this enzyme
generates amounts of superoxide anions and reduction of
ferricytochrome c to ferrocytochrome c was used as a sensitive
measure of superoxide levels. The measurements of ferrocytochrome c
level (550 nm), when tested bioactive compositions were added to
the reaction system, allow for determination of their superoxide
scavenging activity. The final concentrations per well were for
cytochrome c 75 .mu.M, xanthine 425 .mu.m/L, and xanthine oxidase
10 mU/ml.
[0269] Method 8: Method for Determination of In Vitro Toxicity and
Apoptosis.
[0270] CellTiter 96 AQ.sub.ueous One Solution Cell Proliferation
Assay and CytoTox 96 Non-radioactive Cytotoxicity Assay and
subsequent protocols were explored (both assays produced by Promega
Corporation, Madison, Wis.).
[0271] The first assay is a colorimetric method for determining the
number of viable cells which explores a tetrazolium compound
(3-(4,5-dimethylthiaazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny-
l)-2H-tetrazolium, inner salt; MTS and a electron coupling reagent
(phenazine methosulfate; PMS). MTS is bioreduced by cells into a
soluble in tissue culture medium formazan product that has an
absorbance maximum at 490 nm. The conversion of MTS into aqueous,
soluble formazan is accomplished by dehydrogenase enzymes found in
metabolically active cells and the quantity of formazan product is
directly proportional to the number of living cells in culture.
[0272] The second assay quantitatively measures lactate
dehydrogenase (LDH), a stable cytosolic enzyme that is released
upon cell lysis. Released LDH in cell culture supernatant is
measured with a 30-minute coupled enzymatic assay, which results in
the conversation of a tetrazolium salt (INT) into a red formazan
product. The amount of color formed is proportional to the number
of lysed cells.
[0273] Method 9: Method for Determination of Level of Enzymes
Secreted by Stimulated Cells.
[0274] After incubartion with PMA, Mono Mac 6 cells secrete two
gelatinolytic matrix metalloproteinases, MMP-2 (gelatinase A) and
MMP-9 (gelatinase B). The levels of these enzymes in the presence
of tested bioactive compositions were determined by two-dimensional
sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Example 21
Cell Lines Used for Testing Certain Bioactive Characteristics of
the Camellia Products
[0275] The cell line MDA-MB-435S which is considered a model of
advanced breast cancer was obtained from American Type Culture
Collection (ATCC Number HTB-129). This cell line was cultivated at
37.degree. C. in the following ATCC medium: Leibovitz's L-15 medium
with 2 mM L-glutamine supplemented with 0.01 mg/ml insulin, 90%;
fetal bovine serum, 10%.
[0276] The cell line MCF-7 which is considered a model of early or
less de-differentiated breast cancer was obtained from ATCC (Number
HTB-22) was cultivated at 37.degree. C. in the following ATCC
medium: Minimum essential medium (Eagle) with 2 mM L-glutamine and
Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM
non-essential amino acids and 1 mM sodium pyruvate and supplemented
with 0.01 mg/ml bovine insulin, 90%; fetal bovine serum, 10%.
[0277] The cell line MonoMac6 (MM6, obtained from the German
Collection of Microorganisms and Cell Cultures) which closely
resembles a differentiated human monocyte (Ziegler-Heitbrock et
al., "Establishment of a Human Cell Line (Mono Mac 6) with
Characteristics of Mature Monocytes," International Journal of
Cancer 41:456-461 (1988), which is hereby incorporated by reference
in its entirety). Cells were maintained in RPMI 1640, supplemented
with 2 mM L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, 1 mM sodium pyruvate, 10% FCS, nonessential amino
acids, 9 .mu.g/ml insulin, and 1 mM oxalacetic acid. For assay
conditions, 0.2% glucose was also added.
Example 22
Catechin Analyses of the Camellia Products
[0278] The cell walls fraction extract, the membrane fraction
extract, and the cell juice serum of the present invention were
analyzed for content of various catechins. A white tea sample was
used as a control. The following catechins were assayed:
(-)-epigallocatechin; (+)-catechin; (-)-epicatechin;
(-)-epigallocatechin gallate; (-)-gallocatechin gallate; and
(-)-epicatechin gallate.
[0279] The samples were extracted using 0.1% H.sub.3PO.sub.4 and
sonication for about 15 minutes. After centrifugation, the extract
was injected on HPLC. C-18 reverse phase column was used as the
stationary phase. 0.1% phosphoric acid and acetonitrile were used
as the mobile phases. The detection was at 280 nm. The calculation
is based on comparing areas of each catechin listed with its pure
standard. The results are shown in FIG. 38 and Table 16
(below).
TABLE-US-00016 TABLE 16 Catechin Content of Bioactive Camellia
Compositions Average mg/g based Average mg/g on 100% dry based on
Sample Chemical Analyzed matter product as is White Tea
(-)-epigallocatechin 0.3 0.0033 Extract (+)-catechin 1.33 0.0146
(-)-epicatechin 0.15 0.0017 (-)-epigallocatechin gallate 0.65
0.0071 (-)-gallocatechin gallate 0.003 0.0000 (-)-epicatechin
gallate 0.212 0.0023 Cell Walls (-)-epigallocatechin 0.00 0.0000
Fraction (+)-catechin 2.39 0.0201 Extract (-)-epicatechin 0.01
0.0001 (-)-epigallocatechin gallate 0.01 0.0001 (-)-gallocatechin
gallate 0.00 0.0000 (-)-epicatechin gallate 0.006 0.0001 Membrane
(-)-epigallocatechin 2.27 0.1552 Fraction (+)-catechin 8.96 0.6121
Extract (-)-epicatechin 0.60 0.0409 (-)-epigallocatechin gallate
9.28 0.6340 (-)-gallocatechin gallate 0.01 0.0006 (-)-epicatechin
gallate 2.33 0.1589 Cell Juice (-)-epigallocatechin 3.01 0.1714
Serum (+)-catechin 6.09 0.3465 (-)-epicatechin 0.95 0.0539
(-)-epigallocatechin gallate 1.97 0.1120 (-)-gallocatechin gallate
0.04 0.0024 (-)-epicatechin gallate 0.57 0.0325
[0280] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow.
* * * * *
References