US 20090 185990A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0185990 A1 KOGANOV (43) Pub. Date: Jul. 23, 2009
(54) BOACTIVE COMPOSITIONS FROM (60) Provisional application No. 60/535,861, filed on Jan. THEACEA PLANTS AND PROCESSES FOR 12, 2004. THER PRODUCTION AND USE Publication Classification (75) Inventor: Michael KOGANOV, White (51) Int. Cl. Plains, NY (US) A6IR 8/49 (2006.01) A63L/353 (2006.01) Correspondence Address: A61O 1704 (2006.01) HESLN ROTHENBERG EARLEY & MEST A6IP 700 (2006.01) PC (52) U.S. Cl...... 424/59:514/456 S COLUMBIA. CIRCLE ALBANY, NY 12203 (US) (57) ABSTRACT The present invention relates to isolated bioactive composi (73) Assignee: Integrated Botanical tions containing bioactive fractions derived from Theacea Technologies, LLC, Ossining, NY plants. The present invention also relates to bioactive topical (US) formulations containing the bioactive compositions. The present invention further relates to methods of using the bio (21) Appl. No.: 12/335,534 active compositions of the present invention, including, for example, methods for inhibiting inflammatory activity in skin Filed: Dec. 15, 2008 tissue of a mammal, for protecting skin tissue of a mammal (22) from ultraviolet light-induced damage, and for normalizing skin disorders in skin tissue of a mammal. The present inven Related U.S. Application Data tion also relates to methods for isolating bioactive fractions (62) Division of application No. 11/033.925, filed on Jan. derived from cell juice or a cell walls component a Theacea 12, 2005, now Pat. No. 7,473,435. plant. Patent Application Publication Jul. 23, 2009 Sheet 1 of 40 US 2009/0185990 A1
10 20 3. 40 42
Grinding, Cell Coagulation Cooling
Maceration e (Microwave &
& Treatment) Centrifugation Pressing
sk
2 ; Conventional Menbaue S ea Fraction Processing (Tccipitate)
!------is a so - - - r a kraatas: fixxarkR&r Erwist's “st &
36
Water Prying Coagulation
------85°C (Microwave (Isoelectric Stirring Treatment) Precipitation)
150 - of our Positive ition A
Control Cytoplasm (White, Green, (Extract of Fraction Centrifugation Centrifugation Oolong, Black Ce Wals (Precipitate) Tca) Fraction)
ess cars
56
Cell Juice Serunn Supernatant (Supernatant)
rivals carrivia's O
Qomposition Composition D Cornpgsition B
Filtration & (Extract of (Cell Juice (Extract of Tests Cytoplasm Serum) Mctmbrane
Fraction) Fraction)
ski, erett's as
Fig. 1 Patent Application Publication Jul. 23, 2009 Sheet 2 of 40 US 2009/0185990 A1
200 250 300 350 4OO 450 Wavelength, nm
Black Tea Extract u v are v White Tea Extract - - - Cell Walls Fraction Extract Fig. 2 Patent Application Publication Jul. 23, 2009 Sheet 3 of 40 US 2009/0185990 A1
200 250 3OO 350 400 450 Wavelength, nm
Cell Walls Faction Extract u b ur t 8 Membrane Fraction Extract - - - Cell Juice Serum Fig. 3 Patent Application Publication Jul. 23, 2009 Sheet 4 of 40 US 2009/0185990 A1
250 300 350 400 450 Wavelength, nm
- White Tea Extract Black Tea Extract a- - - Cell Walls Fraction Extract Fig. 4 Patent Application Publication Jul. 23, 2009 Sheet 5 of 40 US 2009/0185990 A1
250 3OO 350 4OO 450 Wavelength, nm
- Cell Walls Fraction Extract - a Membrane Fraction Extract - - - Cell Juice Serum Fig. 5 Patent Application Publication Jul. 23, 2009 Sheet 6 of 40 US 2009/0185990 A1
Wavelength, nm
White Tea Extract Cell Walls Fraction Extract ------Membrane Fraction Extract ------Cell Juice Serum
Fig. 6 Patent Application Publication Jul. 23, 2009 Sheet 7 of 40 US 2009/0185990 A1
250 300 350 400 450 Wavelength, nm
- Applied on Vitro-Skin - Solution Fig. 7A Patent Application Publication Jul. 23, 2009 Sheet 8 of 40 US 2009/0185990 A1
250 300 350 . . . . 400 450 Wavelength, nm
- Applied on Vitro-Skin Solution Fig. 7B Patent Application Publication Jul. 23, 2009 Sheet 9 of 40 US 2009/0185990 A1
250 3OO 35O 400 450 Wavelength, nm
- Applied on Vitro-Skin - Solution Fig. 8A Patent Application Publication Jul. 23, 2009 Sheet 10 of 40 US 2009/0185990 A1
250 300 350 400 450 Wavelength, nm
- Applied on Vitro-Skin - Solution Fig. 8B Patent Application Publication Jul. 23, 2009 Sheet 11 of 40 US 2009/0185990 A1
0.05
O.OO
-0.05
-0.10
-0.15
-0.20
-0.25
-0.30 - -o- 250 3OO 350 4OO 450 Wavelength, nm
XV-XVXSWX Vitro-Skin after Irradiation (60J/sq.cm) Fig. 9 Patent Application Publication Jul. 23, 2009 Sheet 12 of 40 US 2009/0185990 A1
O.2
O. s A------V : N fifty 2 0.04 s C C CC -0.1
-0.2
--- 250 300 350 400 450 Wavelength, nm
- White Tea Extract White Tea Extract after irradiation (60J/sq.cm) ---. White Tea Extract after irradiation (Substrate Contribution Eliminated) Fig. 10 Patent Application Publication Jul. 23, 2009 Sheet 13 of 40 US 2009/0185990 A1
250 300 350 400 450 Wavelength, nm.
- Cell Walls Fraction Extract . . . . . Cell Walls Fraction Extract. After irradiation (60J/sq.cm) --- Cell Walls Fraction Extract. After Irradiation (Substrate Contribution Eliminated) Fig.11 Patent Application Publication Jul. 23, 2009 Sheet 14 of 40 US 2009/0185990 A1
250 300 350 400 450 Wavelength, nm
- Membrane Fraction Extract Membrane Fraction Extract after irradiation (60J/sq.cm) Fig. 12 Patent Application Publication Jul. 23, 2009 Sheet 15 of 40 US 2009/0185990 A1
250 300 350 AOO 450 Wavelength, nm
- Cell Juice Serum Cell Juice Serum After Irradiation (60J/sq.cm) --- Cell Juice Serum. After irradiation (Substrate Contribution Eliminated Fig. 13 Patent Application Publication Jul. 23, 2009 Sheet 16 of 40 US 2009/0185990 A1
16O 140 120 100
40
O : X - - - Control 10-5 10-4 10-3 10-2 Concentration, % Dry Matter
-o-, 24 hours - O - 48 hours
Fig. 14 Patent Application Publication Jul. 23, 2009 Sheet 17 of 40 US 2009/0185990 A1
160 140 120 100
60
40 20
Control 10-5 10-4 10-3 10-2 Concentration, % Dry Matter
-e- 24 hours --O. 24 hours + TGF --v- 24 hours + TGF
Fig. 15 Patent Application Publication Jul. 23, 2009 Sheet 18 of 40 US 2009/0185990 A1
160 - - - -
140
12O
1OO
80 . .
20
O --- ot Control 10-5 104 10-3 10-2 Concentration, % Dry Matter
-8- 24 hours ---O 48 hours
Fig. 16 Patent Application Publication Jul. 23, 2009 Sheet 19 of 40 US 2009/0185990 A1
16O 140
120
1OO 80 60 40
20
-- re- -- Control 105 104 10-3 102 Concentration, % Dry Matter
-o-, 24 hours O 24 hours + TGF --v- 24 hours + TGF
Fig. 17 Patent Application Publication Jul. 23, 2009 Sheet 20 of 40 US 2009/0185990 A1
160
140
120
1OO
60 40 -
20 -
r
Control 10-4 1O-3 Concentration, % Dry Matter
-o- 24 hours --O 48 hours
Fig. 18 Patent Application Publication Jul. 23, 2009 Sheet 21 of 40 US 2009/0185990 A1
Control 10-5 104 10-3 10-2 Concentration, % Dry Matter
-o- 24 hours - O - 24 hours + TGF --v- 24 hours + TGF
Fig. 19 Patent Application Publication Jul. 23, 2009 Sheet 22 of 40 US 2009/0185990 A1
oil- Y. & -- Control 105 104 10-3 10-2 Concentration, %Dry Matter
-8-24 hours -O 48 hours
Fig. 20 Patent Application Publication Jul. 23, 2009 Sheet 23 of 40 US 2009/0185990 A1
160
40
120
1OO
80 - 60 - 40 - 20 O ------control 10° 10' 10- 10-2 Concentration, % Dry Matter
-0- 24 hours O - 24 hours + TGF --v- 24 hours + TGF
Fig. 21 Patent Application Publication Jul. 23, 2009 Sheet 24 of 40 US 2009/0185990 A1
160
140 - 120
100 80 -
6O -
40 20
O unxWu X------Control 10-5 10-4 103 Concentration, % Dry Matter
-o- 24 hours O 48 hours
Fig. 22 Patent Application Publication Jul. 23, 2009 Sheet 25 of 40 US 2009/0185990 A1
160
140 120 -
100
80
60
40 20
Control 10-5 104 103 10-2 Concentration, % Dry Matter
-o- 24 hours O. 48 hours
Fig. 23 Patent Application Publication Jul. 23, 2009 Sheet 26 of 40 US 2009/0185990 A1
160 140 120
1OO 80
60
40 20
Control 10-5 10-4 103 102 Concentration, % Dry Matter
-0- 24 hours -O 48 hours
Fig. 24 Patent Application Publication Jul. 23, 2009 Sheet 27 of 40 US 2009/0185990 A1
16O. 140 - 120 100 80 - 60 -
0 ---- - Control 10-5 1O-4 10-3 102 Concentration, % Dry Matter
-o- 24 hours ... O 48 hours Fig. 25 Patent Application Publication Jul. 23, 2009 Sheet 28 of 40 US 2009/0185990 A1
160 - — 140 - 120 - 100 80 -
60 -
40 20 -
Control 10-5 10-4 103 10-2 Concentration, %Dry Matter
-O- 24 hours O 48 hours
Fig. 26 Patent Application Publication Jul. 23, 2009 Sheet 29 of 40 US 2009/0185990 A1
160
140 120
1OO
80
60
40
O - s - To Control 105 10-4 10-3 Concentration, % Dry Matter
-- 24 hours - O - 48 hours
Fig. 27 Patent Application Publication Jul. 23, 2009 Sheet 30 of 40 US 2009/0185990 A1
160 - — 140
120 100
60
40
20 -
Control 105 1O-4 10-3 102 Concentration, % Dry Matter
-O- 24 hours - O - 48 hours
Fig. 28 Patent Application Publication Jul. 23, 2009 Sheet 31 of 40 US 2009/0185990 A1
160 140
120 100 80 -
60
AO 20
Control 10-5 104 10-3 102 Concentration, % Dry Matter
---, 24 hours ...... - O - 48 hours
Fig. 29 Patent Application Publication Jul. 23, 2009 Sheet 32 of 40 US 2009/0185990 A1
O - r - t Control 10-5 10-4 1O-3. 102 Concentration, % Dry Matter
-- MVP-2 ... O MVP-9 Fig. 30 Patent Application Publication Jul. 23, 2009 Sheet 33 of 40 US 2009/0185990 A1
O ------Control 10-5 10-4 103 10-2 Concentration, % Dry Matter
-- MMP-2 ...O. MMP-9 Fig. 31 Patent Application Publication Jul. 23, 2009 Sheet 34 of 40 US 2009/0185990 A1
Control 105 10-4 10-3 10-2 Concentration, % Dry Matter
-o- MMP-2 O. MMP-9 Fig. 32 Patent Application Publication Jul. 23, 2009 Sheet 35 of 40 US 2009/0185990 A1
------Control 10° 10' 10-3 10-2 Concentration, % Dry Matter
-- MMP-2 ...O. MMP-9
Fig. 33 Patent Application Publication Jul. 23, 2009 Sheet 36 of 40 US 2009/0185990 A1
Fig. 34 Patent Application Publication Jul. 23, 2009 Sheet 37 of 40 US 2009/0185990 A1
Fig. 35 Patent Application Publication Jul. 23, 2009 Sheet 38 of 40 US 2009/0185990 A1
Fig. 36 Patent Application Publication Jul. 23, 2009 Sheet 39 of 40 US 2009/0185990 A1
S 3 low 3 met 3 high
Fig. 37 Patent Application Publication Jul. 23, 2009 Sheet 40 of 40 US 2009/0185990 A1
1 O
a. is 8 s O as S. 6 E us
E 4 - O Šese C E 92 2 -
C O
O WTE CWF E
( -)-epigallocatechin ( +)-catechin ( -)-epigallocatechin-3-gallate ( -)-epicatechin ( -)-gallocatechin gallate ( -)-epicatechin gallate
Fig. 38 US 2009/O 185990 A1 Jul. 23, 2009
BOACTIVE COMPOSITIONS FROM 0008. In particular, fresh leaves of Camelia sinensis have THEACEA PLANTS AND PROCESSES FOR been reported to contain 22.2% polyphenols, 17.2% protein, THER PRODUCTION AND USE 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. 0001. This application claims the benefit of U.S. Provi purdue.edu/newcrop? duke energy/Camelia sinensis.html). sional Patent Application Ser. No. 60/535,861, filed Jan. 12, Per 100 g, the leaf is reported to contain 8.0 g H2O, 24.5 g. 2004. protein, 2.8 g fat, 58.8g total carbohydrate, 8.7 g fiber, 5.9 g, ash, 327 mg Ca. 313 mg P. 24.3 mg Fe, 50 mg Na, 2700 ug FIELD OF THE INVENTION B-carotene equivalent, 0.07 mg thiamine, 0.8 mg riboflavin, 7.6 mg niacin, and 9 mg ascorbic acid. Another report tallies 0002 The present invention relates to bioactive composi 8.0 g H2O, 28.3 g protein, 4.8g fat, 53.6 g total carbohydrate, tions, processes for their production from Theacea plants, and 9.6 g fiber, 5.6 gash, 245 mg Ca. 415 mg. P. 18.9 mg Fe, 60 mg uses of these compositions. Na, 8400 ug B-carotene equivalent, 0.38 mg thiamine, 1.24 mg riboflavin, 4.6 mg niacin, and 230 mg ascorbic acid. Yet BACKGROUND OF THE INVENTION another gives 8.1 g HO, 24.1 g protein, 3.5g fat, 59.0 g total carbohydrate, 9.7 g fiber, 5.3 gash, 320 mg Ca, 185 mg. P. 31.6 0003. The Theacea (tea plants) family includes trees or mg Fe, 8400 ug B-carotene equivalent, 0.07 mg thiamine, shrubs comprising about 40 genera and 600 species. Camelia 0.79 mg riboflavin, 7.3 mg niacin, and 85 mgascorbic acid (J. Sinensis occupies a unique position in the Theacea family, A. Duke and A. A. Atchley, “Proximate Analysis.” In: because this particular species of plant is predominantly used Christie, B. R. (ed.), The Handbook of Plant Science in Agri as a single raw material source to produce all three basic kinds culture, CRC Press, Inc., Boca Raton, Fla. (1984)). of tea: green tea, oolong tea, and black tea (collectively 0009 Leaves also contain carotene, riboflavin, nicotinic referred to herein as the “tea plant”). According to some acid, pantothenic acid, and ascorbic acid. Caffeine and tannin sources, there is a fourth type of tea, i.e., the so-called “white are among the more active constituents (Council for Scientific tea, which is produced exclusively from the buds or tips of and Industrial Research, 1948-1976). Ascorbic acid, present the tea plant. in the fresh leaf, is destroyed in making black tea. Malic and 0004. The three basic forms of tea are determined by the Oxatic acids occur, along with kaempferol, quercitrin, theo degree of processing, which involves the identical tender phylline, theobromine, Xanthine, hypoxanthine, adenine, young tea leaves. The leaves are plucked, sorted, cleaned, and gums, dextrins, and inositol. Chief components of the Volatile variously oxidized before steaming or drying. The term “fer oil (0.007-0.014% fresh weight of leaves) are hexenal, hex mentation' is frequently used to describe the processing of enol, and lower aldehydes, butyraldehyde, isobuteraldehyde, tea, but the term “oxidation' is a much more accurate descrip isovaleraldehyde, as well as n-hexyl, benzyl and phenylethyl tion of the chemical transformations which take place. alcohols, phenols, cresol, hexoic acid, n-octyl alcohol, 0005. Although there are some variations in the process geraniol, linalool, acetophenone, benzyl alcohol, and citral. ing, it is generally agreed that green tea has the lowest degree 0010. It was found that the fresh tea leaf has an unusually of oxidation and that black tea has the highest. Oolong tea is high level of flavanol group of polyphenols (catechins), considered to be partially oxidized, and thus occupies the which may reach up to 30% of leaf dry matter. Catechins place between green and black tea. With respect to process include predominantly (-)-epicatechin, (-)-epicatechingal ing, there is very little difference (or no difference at all) late, (-)-epigallocatechin and (-)-epigallocatechin gallate. between green and white tea. Additionally there are unique to tea 3-galloylquinic acid 0006 Green tea is made from fresh leaves that are steamed (theogallin) and unique amino acid theanine (5-N-ethyl and wilted, and then immediately dried. Black tea is made glutamine) (Duke, J. A., Handbook of Energy Crops (1983), from leaves that are wilted and crushed in rollers, then see www.hort.purdue.edu/newcrop? duke energy/Camellia allowed to oxidize for several hours before they are dried. sinensis.html). Oolong tea comes from leaves that are only partially oxidized 0011 Tea leaves contain high levels of polyphenol-oxi before drying. dase and peroxidase. The first enzyme catalyzes the aerobic 0007 Worldwide, tea is the second (after water) most oxidation of the catechins and this process is initiated when commonly consumed liquid, and is the sixth (after water, soft the integrity of the leaf cell structure is disrupted. Phenol drinks, coffee, beer, and milk) most commonly consumed oxidase is responsible for generation of bisflavanols, theafla liquid in the United States. Tea consumption continues to Vins, epitheaflavic acids, and thearubigens, which constitute increase worldwide, especially due to the growing public the largest mass of the extractable matter in black tea. Most of awareness concerning health benefits of this liquid. There is a these compounds readily form complexes with caffeine, growing number of publications suggesting anti-angiogenic, which has significant level (2-4% of dry matter) in fresh anti-bacterial, anti-cancerogenic, anti-inflammatory, anti leaves. Peroxidase plays important role in generation of the mutagenic, anti-oxidant, anti-septic, and detoxifying proper above complexes with proanthocyanidins. The catechin ties of teas and their ingredients. The list of tea benefits also quinones also initiate the formation of many of the hundreds includes reduction of the risk of rheumatoid arthritis, lower of Volatile compounds found in the black tea aroma fraction. ing cholesterol levels, and anti-diabetic properties. Not all of Additionally, the transformation of relatively soluble glyco these benefits have been proven to be statistically significant. sides to lower solubility aglycones takes place. Nevertheless, the very broad spectrum of tea benefits reflects 0012 All complex cascades of the above processes are the unique composition of the very powerful biologically initiated by disruption of the leaf cell structure and are inten active Substances, which exist in fresh plant leaves and Sur sified with the time of oxidation. As result, the composition of Vive conventional tea processing. black tea, which is usually processed with intensive rolling or US 2009/O 185990 A1 Jul. 23, 2009 cutting and relatively long time oxidation, is much more tive composition of the present invention. The method further different than that of the fresh leaf. Although green tea (and involves applying the bioactive composition to the skin tissue white tea) is processed with minimum oxidation, and its in an amount effective to reduce ultraviolet light-induced composition more similar to that of fresh leaves, there are damage of the skin tissue and to prevent oxidative damage of non-enzymatic and enzymatically catalyzed changes, which the skin tissue. occur extremely rapidly following plucking, and new volatile 0019. The present invention also relates to a method for Substances that are produced during the drying stage. Thus, normalizing skin disorders in skin tissue of a mammal. This even relatively gentle green tea processing initiates certain method involves providing the bioactive composition of the departure from original fresh plant composition and can present invention. The method further involves applying the diminish the therapeutic value and other potential benefits of bioactive composition to the skin tissue in an amount effec fresh tea plant leaves. tive to normalize a cell disorder in the skin tissue. 0013. Numerous recent studies clearly demonstrate that 0020. The present invention also relates to a method for therapeutic benefits of tea are decreased in the following isolating a bioactive fraction derived from cell juice of a sequence: white tead green tead oolong tead black tea. Thus, Theacea plant. This method involves providing a Theacea exploration of fresh tea plants may prevent the degradation of plant. The Theacea plant is then separated into cell juice and specific activities, which are observed as a result of conven a cell walls component. The cell juice is then treated under tional tea processing. Fresh, tender Camelia leaves contain conditions effective to yield a bioactive fraction. Suitable approximately 80% water. Swelling and dehydration of the bioactive fractions include, without limitation, a membrane cells is prevented by the cells' rigid cell walls. The disruption fraction, a membrane fraction extract, a cytoplasm fraction, a of the cell wall structure triggers the dehydration of fresh cytoplasm fraction extract, and/or a cell juice serum. The plant tissue followed by the sequence of unwanted physico bioactive fraction is then isolated from the treated cell juice. chemical and biochemical processes: osmotic shock, decom The present invention further relates to an isolated bioactive partmentalization and disruption of enzymes, hydrolysis and composition produced by this method. oxidation, polymerization of phenols, transformation of gly 0021. The present invention also relates to a method for cosides to aglycones, generation of products of Maillard reac isolating a bioactive fraction derived from a cell walls com tion, isomerization, and microbial contamination. Therefore, ponent of a Theacea plant. This method involves providing a fresh Camellia contains very broad spectrum of biologically Theacea plant. The Theacea plant is then separated into cell active Substances and only part of them became available juice and a cell walls component. The cell walls component is during conventional extraction processes. Thus, only cell treated under conditions effective to yield a bioactive frac walls, catabolites, and stable metabolites can be extracted tion. The bioactive fraction is then isolated from the treated with boiled water to obtain tea drink or for extraction with cell walls component. The present invention further relates to different solvents to obtain limited parts of biologically active an isolated bioactive fraction produced by this method. components (predominantly polyphenols and flavonoids). 0022. The present invention is useful in addressing the 0014. In light of the potential of fresh tea leaves as sources deficiencies of conventional tea processing methods, particu of valuable therapeutic and other potentially beneficial bio larly the inability of conventional tea processing to preserve a active compositions, exploration of fresh tea plants is needed broad spectrum of potent bioactive compositions. As pro to determine how to maximize their therapeutic and other vided by the present invention, processing of fresh Camellia potentially beneficial bioactive properties. biomass without fermentation and excessive heat treatment can yield more powerful and diversified bioactive composi SUMMARY OF THE INVENTION tions than products of conventional tea processing. 0015 The present invention relates to a bioactive compo BRIEF DESCRIPTION OF DRAWINGS sition. In one embodiment, the bioactive composition includes an isolated bioactive fraction derived from a Theacea 0023 FIG. 1 is a schematic drawing demonstrating one plant. Suitable bioactive fractions can include, without limi embodiment of the process for preparing the bioactive com tation, a cell walls fraction, a cell walls fraction extract, a positions of the present invention. membrane fraction, a membrane fraction extract, a cytoplasm 0024 FIG. 2 is a graph showing the UV/VIS spectra of fraction, a cytoplasm fraction extract, a cell juice serum, extracts of cell walls fraction and conventional teas (Dilution and/or combinations thereof. 1:1000). 0016. The present invention also relates to a bioactive (0025 FIG. 3 is a graph showing the UV/VIS spectra of topical formulation Suitable for topical application to a mam Camellia bioactive compositions (Dilution 1:4000). mal. In one embodiment, the bioactive topical formulation 0026 FIG. 4 is a graph showing the absorbance spectra of includes a topically effective amount of the bioactive compo extracts of cell walls fraction and conventional teas applied on sition of the present invention. The bioactive topical formu Vitro-Skin R testing substrate (IMS Testing Group, Milford, lation can further include a topically acceptable carrier. Conn.). Dry matter levels are equalized. 0017. The present invention also relates to a method for 0027 FIG. 5 is a graph showing the absorbance spectra of inhibiting inflammatory activity in skin tissue of a mammal. Camellia bioactive compositions applied on Vitro-Skin R This method involves providing the bioactive composition testing substrate (IMS Testing Group, Milford, Conn.). Dry according to the present invention. The method further matter levels are equalized. involves applying the bioactive composition to the skin tissue 0028 FIG. 6 is a graph showing the absorbance spectra of in an amount effective to inhibit inflammatory activity in the Camellia bioactive compositions and white tea extract skin tissue. applied on Vitro-Skin R testing substrate (IMS Testing Group, 0018. The present invention also relates to a method of Milford, Conn.). protecting skin tissue of a mammal from ultraviolet light 0029 FIG. 7A is a graph showing the absorbance spectra induced damage. This method involves providing the bioac of Camelia membrane fraction extract in diluted solution US 2009/O 185990 A1 Jul. 23, 2009
(1:200) and applied on Vitro-Skin R testing substrate (IMS 0046 FIG. 24 is a graph showing the effect of cell walls Testing Group, Milford, Conn.). FIG. 7B is a graph showing fraction extract on Mono Mac 6 cells cultivated for 24 hours the absorbance spectra of Camelia cell juice serum in diluted and 48 hours. solution (1:200) and applied on Vitro-Skin R testing substrate 0047 FIG. 25 is a graph showing the effect of cell walls (IMS Testing Group, Milford, Conn.). fraction extract on Mono Mac 6 cells cultivated for 24 hours 0030 FIG. 8A is a graph showing the absorbance spectra and 48 hours in the presence of 10 nM PMA. of Barley (Hordeum vulgare) cell juice serum in diluted solu 0048 FIG. 26 is a graph showing the effect of membrane tion (1:200) and applied on Vitro-Skin Rg testing substrate fraction extract on Mono Mac 6 cells cultivated for 24 hours (IMS Testing Group, Milford, Conn.). FIG. 8B is a graph and 48 hours. showing the absorbance spectra of Sage (Salvia officinalis) 0049 FIG. 27 is a graph showing the effect of membrane cell juice serum in diluted solution (1:200) and applied on fraction extract on Mono Mac 6 cells cultivated for 24 hours Vitro-Skin R testing substrate (IMS Testing Group, Milford, and 48 hours in the presence of 10 nM PMA. Conn.). 0050 FIG. 28 is a graph showing the effect of cell juice 0031 FIG. 9 is a graph showing the effect of broad spec serum on Mono Mac 6 cells cultivated for 24 hours and 48 trum UV irradiation on Vitro-Skin R) testing substrate (IMS hours. Testing Group, Milford, Conn.). 0051 FIG. 29 is a graph showing the effect of cell juice 0032 FIG. 10 is a graph showing the effect of broad spec serum on Mono Mac 6 cells cultivated for 24 hours and 48 trum UV irradiation on white tea extract applied on Vitro hours in the presence of 10 nM PMA. Skin R testing substrate (IMS Testing Group, Milford, 0052 FIG. 30 is a graph showing the effect of white tea Conn.). extract on level of MMPs secreted by PMA stimulated Mono 0033 FIG. 11 is a graph showing the effect of broad spec Mac 6 cells. trum UV irradiation on cell walls fraction extract applied on 0053 FIG. 31 is a graph showing the effect of cell walls Vitro-Skin R testing substrate (IMS Testing Group, Milford, fraction extract on level of MMPs secreted by PMA stimu Conn.). lated Mono Mac 6 cells. 0034 FIG. 12 is a graph showing the effect of broad spec 0054 FIG. 32 is a graph showing the effect of membrane trum UV radiation on Camelia membrane fraction extract fraction extract on level of MMPs secreted by PMA stimu lated Mono Mac 6 cells. applied on Vitro-Skin R testing substrate (IMS Testing Group, 0055 FIG. 33 is a graph showing the effect of cell juice Milford, Conn.). serum on level of MMPs secreted by PMA stimulated Mono 0035 FIG. 13 is a graph showing the effect of broad spec Mac 6 cells. trum UV radiation on Camellia cell juice serum applied on 0056 FIG. 34 is a gelatin Zymogram of culture media Vitro-Skin R testing substrate (IMS Testing Group, Milford, collected after 48 hours exposure of Mono Mac 6 cells to Conn.). white tea extract, along with culture media collected from 0036 FIG. 14 is a graph showing the effect of white tea cells cultured in the absence (U) or presence (S) of 10 nM extract on MDA-MB-435S cells cultivated for 24 hours and PMA, but in the absence of the Camellia compositions. 48 hours. 0057 FIG. 35 is a gelatin Zymogram of culture media 0037 FIG. 15 is a graph showing the effect of white tea collected after 48 hours exposure of Mono Mac 6 cells to cell extract on MCF-7 cells cultivated for 24 hours (control) and walls fraction extract, along with culture media collected for 24 hours and 48 hours in the presence of 5 ng/ml TGF-B. from cells cultured in the absence (U) or presence (S) of 10 0038 FIG. 16 is a graph showing the effect of cell walls nM PMA, but in the absence of the Camellia compositions. fraction extract on MDA-MB-435S cells cultivated for 24 0.058 FIG. 36 is a gelatin Zymogram of culture media hours and 48 hours. collected after 48 hours exposure of Mono Mac 6 cells to 0039 FIG. 17 is a graph showing the effect of cell walls membrane fraction extract, along with culture media col fraction extract on MCF-7 cells cultivated for 24 hours (con lected from cells cultured in the absence (U) or presence (S) trol) and for 24 hours and 48 hours in the presence of 5 ng/ml of 10 nM PMA, but in the absence of the Camellia composi TGF-B. tions. 0040 FIG. 18 is a graph showing the effect of membrane 0059 FIG. 37 is a gelatin Zymogram of culture media fraction extract on MDA-MB-435S cells cultivated for 24 collected after 48 hours exposure of Mono Mac 6 cells to cell hours and 48 hours. juice serum, along with culture media collected from cells 0041 FIG. 19 is a graph showing the effect of membrane cultured in the absence (U) or presence (S) of 10 nM PMA, fraction extract on MCF-7 cells cultivated for 24 hours (con but in the absence of the Camelia compositions. trol) and for 24 hours and 48 hours in the presence of 5 ng/ml 0060 FIG. 38 is a bar graph comparing the content of TGF-B. various catechins in the white tea extract (“WTE) and in the 0042 FIG. 20 is a graph showing the effect of cell juice cell walls fraction extract (“CWFE), the membrane fraction serum on MDA-MB-435S cells cultivated for 24 hours and 48 extract (“MFE), and the cell juice serum (“CJS) of the hours. present invention. 0043 FIG. 21 is a graph showing the effect of cell juice serum on MCF-7 cells cultivated for 24 hours (control) and DETAILED DESCRIPTION OF THE INVENTION for 24 hours and 48 hours in the presence of 5 ng/ml TGF-B. 0061 The present invention relates to a bioactive compo 0044 FIG. 22 is a graph showing the effect of white tea sition. In one embodiment, the bioactive composition extract on Mono Mac 6 cells cultivated for 24 and 48 hours. includes an isolated bioactive fraction derived from a Theacea 0045 FIG. 23 is a graph showing the effect of white tea plant. As used herein, the term "isolated bioactive fraction” is extract on Mono Mac 6 cells cultivated for 24 hours and 48 meant to include fractions that are isolated from a Theacea hours in the presence of 10 nM PMA. plant (e.g., fresh biomass of a Theacea plant) that has not US 2009/O 185990 A1 Jul. 23, 2009 undergone any conventional tea processing (e.g., heat treat about 0.005 and about 0.007 milligrams of (-)-epicatechin ment, oxidation, fermentation, drying). Suitable isolated bio gallate per gram of dry matter of the cell walls fraction active fractions can include, without limitation, a cell walls eXtract. fraction, a cell walls fraction extract, a membrane fraction, a 0065. In one embodiment of the bioactive composition of membrane fraction extract, a cytoplasm fraction, a cytoplasm the present invention, the bioactive fraction can be a mem fraction extract, a cell juice serum, and/or combinations brane fraction. thereof. 0066. In one embodiment of the bioactive composition of 0062. The bioactive compositions and bioactive fractions the present invention, the bioactive fraction can be a mem of the present invention can have various catechin profiles and brane fraction extract. In a specific embodiment of the present total catechin content amounts, as defined below, and as invention, the membrane fraction extract can have a total determined using conventional catechin diagnostic methods catechin content of between about 15.0 and about 30.5 mil well known in the art. As used herein, the term “catechin' ligrams per gram of dry matter, particularly between about generally refers to all catechins, including, but not limited to, 18.0 and about 27.5 milligrams per gram of dry matter, and the following specific types of catechins: (i) (-)-epigallocat more particularly between about 21.0 and about 24.5 milli echin (see CAS No. 970-74-1, which is hereby incorporated grams per gram of dry matter. In another specific embodi by reference in its entirety); (ii) (+)-catechin (see CAS No. ment, the membrane fraction extract can have a catechin 7295-85-4, which is hereby incorporated by reference in its content profile as follows: (i) between about 1.7 and about 3.3 entirety); (iii) (-)-epicatechin (see CAS No. 490-46-0, which milligrams of (-)-epigallocatechin per gram of dry matter of is hereby incorporated by reference in its entirety); (iv) (-)- the membrane fraction extract; (ii) between about 6.1 and epigallocatechin gallate (see CAS No. 989-51-5, which is about 10.2 milligrams of (+)-catechin per gram of dry matter hereby incorporated by reference in its entirety); (v) (-)- of the membrane fraction extract; (iii) between about 0.3 and gallocatechin gallate (see CAS No. 4233-96-9, which is about 1.1 milligrams of (-)-epicatechin per gram of dry mat hereby incorporated by reference in its entirety); and (vi) ter of the membrane fraction extract; (iv) between about 6.2 (-)-epicatechin gallate (see CAS No. 1257-08-5, which is and about 12.5 milligrams of (-)-epigallocatechingallate per hereby incorporated by reference in its entirety). “Total cat gram of dry matter of the membrane fraction extract; (v) echin content” (as used herein) refers to the combined content between about 0.007 and about 0.03 milligrams of (-)-gallo level of all catechins contained in a particular bioactive com catechin gallate per gram of dry matter of the membrane position or bioactive fraction of the present invention, and is fraction extract; and (vi) between about 1.3 and about 3.3 not meant to be limited to the content levels of just the specific milligrams of (-)-epicatechin gallate per gram of dry matter types of catechins listed herein above. As used herein, the of the membrane fraction extract. More particularly, the term “catechin content profile' is used to describe the membrane fraction extract can have a catechin content profile amounts of selected catechins contained in a particular bio as follows: (i) between about 2.0 and about 3.0 milligrams of active composition or bioactive fraction of the present inven (-)-epigallocatechin per gram of dry matter of the membrane tion. fraction extract; (ii) between about 7.0 and about 9.0 milli 0063. In one embodiment of the bioactive composition of grams of (+)-catechinpergram of dry matter of the membrane the present invention, the bioactive fraction can be a cell walls fraction extract; (iii) between about 0.5 and about 0.9 milli fraction. grams of (-)-epicatechin per gram of dry matter of the mem 0064. In one embodiment of the bioactive composition of brane fraction extract; (iv) between about 8.0 and about 10.0 the present invention, the bioactive fraction can be a cell walls milligrams of (-)-epigallocatechin gallate per gram of dry fraction extract. In a specific embodiment of the present matter of the membrane fraction extract; (v) between about invention, the cell walls fraction extract can have a total 0.01 and about 0.02 milligrams of (-)-gallocatechin gallate catechin content of between about 2.1 and about 4.5 milli per gram of dry matter of the membrane fraction extract; and grams per gram of dry matter, particularly between about 2.6 (vi) between about 1.8 and about 2.8 milligrams of (-)-epi and about 4.0 milligrams per gram of dry matter, and more catechin gallate per gram of dry matter of the membrane particularly between about 3.0 and about 3.6 milligrams per fraction extract. gram of dry matter. In another specific embodiment, the cell 0067. In one embodiment of the bioactive composition of walls fraction extract can have a catechin content profile as the present invention, the bioactive fraction can be a cyto follows: (i) between about 2.0 and about 3.0 milligrams of plasm fraction. (+)-catechin per gram of dry matter of the cell walls fraction 0068. In one embodiment of the bioactive composition of extract; (ii) between about 0.005 and about 0.02 milligrams of the present invention, the bioactive fraction can be a cyto (-)-epicatechin per gram of dry matter of the cell walls frac plasm fraction extract. tion extract; (iii) between about 0.005 and about 0.02 milli 0069. In one embodiment of the bioactive composition of grams of (-)-epigallocatechin gallate per gram of dry matter the present invention, the bioactive fraction can be a cell juice of the cell walls fraction extract; and (iv) between about 0.003 serum. In a specific embodiment, the cell juice serum can and about 0.01 milligrams of (-)-epicatechingallate per gram have a total catechin content of between about 8.0 and about of dry matter of the cell walls fraction extract. More particu 20.0 milligrams per gram of dry matter, particularly between larly, the cell walls fraction extract can have a catechin con about 10.0 and about 18.0 milligrams per gram of dry matter, tent profile as follows: (i) between about 2.2 and about 2.7 and more particularly between about 12.0 and about 16.0 milligrams of (+)-catechin per gram of dry matter of the cell milligrams per gram of dry matter. In another specific walls fraction extract; (ii) between about 0.01 and about 0.015 embodiment, the cell juice serum can have a catechin content milligrams of (-)-epicatechin per gram of dry matter of the profile as follows: (i) between about 2.1 and about 4.4 milli cell walls fraction extract; (iii) between about 0.01 and about grams of (-)-epigallocatechin per gram of dry matter of the 0.015 milligrams of (-)-epigallocatechin gallate per gram of cell juice serum; (ii) between about 4.2 and about 8.6 milli dry matter of the cell walls fraction extract; and (iv) between grams of (+)-catechin per gram of dry matter of the cell juice US 2009/O 185990 A1 Jul. 23, 2009
serum; (iii) between about 0.2 and about 2.0 milligrams of 0075. The present invention also relates to a bioactive (-)-epicatechin per gram of dry matter of the cell juice serum; topical formulation Suitable for topical application to a mam (iv) between about 1.2 and about 3.2 milligrams of (-)-epi mal. In one embodiment, the bioactive topical formulation gallocatechin gallate per gram of dry matter of the cell juice includes a topically effective amount of the bioactive compo serum; (v) between about 0.01 and about 0.1 milligrams of sition of the present invention. The bioactive topical formu (-)-gallocatechin gallate per gram of dry matter of the cell lation can further include a topically acceptable carrier. Suit juice serum; and (vi) between about 0.2 and about 1.3 milli able topically acceptable carriers can include, without grams of (-)-epicatechingallate per gram of dry matter of the limitation, a hydrophilic cream base, a hydrophilic lotion cell juice serum. More particularly, the cell juice serum can base, a hydrophilic Surfactant base, a hydrophilic gel base, a have a catechin content profile as follows: (i) between about hydrophilic solution base, a hydrophobic cream base, a 3.0 and about 3.5 milligrams of(-)-epigallocatechin per gram hydrophobic lotion base, a hydrophobic Surfactant base, a of dry matter of the cell juice serum; (ii) between about 5.0 hydrophobic gelbase, and/or a hydrophobic solution base. In and about 7.0 milligrams of (+)-catechin per gram of dry one embodiment, the bioactive composition can be present in matter of the cell juice serum; (iii) between about 0.7 and an amount ranging from between about 0.001 percent and about 1.5 milligrams of (-)-epicatechin per gram of dry mat about 90 percent of the total weight of the bioactive topical ter of the cell juice serum; (iv) between about 1.7 and about formulation. 2.7 milligrams of (-)-epigallocatechingallate per gram of dry 0076. The present invention also relates to a method for matter of the cell juice serum; (v) between about 0.03 and inhibiting inflammatory activity in skin tissue of a mammal. about 0.07 milligrams of (-)-gallocatechin gallate per gram This method involves providing the bioactive composition of dry matter of the cell juice serum; and (vi) between about according to the present invention. The method further 0.5 and about 1.0 milligrams of (-)-epicatechin gallate per involves applying the bioactive composition to the skin tissue gram of dry matter of the cell juice serum. in an amount effective to inhibit inflammatory activity in the 0070. In one embodiment, fresh biomass of Theacea skin tissue. In one embodiment of this method, the bioactive plants can be used to isolate the bioactive compositions of the composition can further include a stabilizing agent (Suitable present invention. The fresh biomass can be taken from Thea examples of which are as described herein). In another cea plants that are of the Camellia and/or Eurya genera. embodiment of this method, the bioactive composition can Suitable species of the Camelia genus for use in the present further include a topically acceptable carrier (suitable invention can include, without limitation, Camelia sinensis, examples of which are as described herein). Camellia japonica, Camelia reticulate, and Camelia Sasan 0077. The present invention also relates to a method of qua. Suitable species of the Eurya genus for use in the present protecting skin tissue of a mammal from ultraviolet light invention can include, without limitation, Eurya sandwicen induced damage. This method involves providing the bioac SS tive composition of the present invention. The method further 0071. The bioactive composition of the present invention involves applying the bioactive composition to the skin tissue can further include a stabilizing agent. Suitable stabilizing in an amount effective to reduce ultraviolet light-induced agents are those that are commonly used in the art. Particular damage of the skin tissue and to prevent oxidative damage of Suitable stabilizing agents can include, without limitation, an the skin tissue. In one embodiment, the method is useful in emulsifier, a preservative, an anti-oxidant, a polymer matrix, protecting skin tissue from ultraviolet light-induced damage and/or mixtures thereof. caused by ultraviolet light in a range of between about 320 0072. In one aspect of the present invention, the bioactive and about 400 nanometers. In another embodiment of this fraction can have modulatory activity on at least one mammal method, the bioactive composition can further include a sta cell function. Such modulatory activity can include, for bilizing agent (Suitable examples of which are as described example, cell growth inhibition activity, cell growth stimula herein). In another embodiment of this method, the bioactive tion activity, enzyme secretion activity, enzyme inhibition composition can further include a topically acceptable carrier activity, anti-oxidant activity, UV-protection activity, anti (suitable examples of which are as described herein). inflammatory activity, wound healing activity, and/or combi 0078. The present invention also relates to a method for nations of these activities. With respect to cell growth inhibi normalizing skin disorders in skin tissue of a mammal. This tion activity, such activity can involve growth inhibition of method involves providing the bioactive composition of the cancer cells. Suitable cancer cells that can be inhibited to present invention. The method further involves applying the grow by the bioactive fractions of the present invention can bioactive composition to the skin tissue in an amount effec include, without limitation, breast cancer cells and/or colon tive to normalize a cell disorder in the skin tissue. In one cancer cells. The described cell growth inhibition activity can embodiment of this method, the bioactive composition can also include growth inhibition of leukemia cells. Suitable further include a stabilizing agent (Suitable examples of leukemia cells that can be inhibited to grow by the bioactive which areas described herein). In another embodiment of this fractions of the present invention can include, without limi method, the bioactive composition can further include a topi tation, monocytic leukemia cells. cally acceptable carrier (Suitable examples of which are as 0073. In another embodiment, the bioactive composition described herein). can be effective in inhibiting unwanted hyper-proliferation or 007.9 The present invention also relates to a method for hypo-proliferation of skin cells and/or inhibiting unwanted isolating a bioactive fraction derived from cell juice of a uncoordinated enzyme activities or enzyme secretion pro Theacea plant. This method involves providing a Theacea cesses in the skin cells. plant (e.g., in the form of fresh biomass). Suitable Theacea 0074. In another embodiment, the bioactive composition plants for use in this method are as described herein, Supra. of the present invention can further include a delivery system The Theacea plant (e.g., fresh biomass) is then separated into for systemic or topical administration that are commonly cell juice and a cell walls component. The cell juice is then used in the art. treated under conditions effective to yield a bioactive frac US 2009/O 185990 A1 Jul. 23, 2009
tion. Suitable bioactive fractions include, without limitation, I0085 Conventional tea processing 22 of fresh biomass 10 a membrane fraction, a membrane fraction extract, a cyto is used to produce, for example, positive control 150 (of plasm fraction, a cytoplasm fraction extract, and/or a cell various teas, including, for example, white, green, oolong, juice serum. The bioactive fraction is then isolated from the and black teas). treated cell juice. In one embodiment, the various suitable I0086 Composition A110. Composition B120, Composi bioactive fractions produced by this method are as described tion C130, Composition D 140, and positive control 150 can herein. The present invention further relates to an isolated then be used for filtration and tests 80. I0087. The present invention also relates to a device for bioactive composition produced by this method. selectively dispersing into a liquid low molecular weight and 0080. The present invention also relates to a method for reduced, non-oxidized components of a bioactive composi isolating a bioactive fraction derived from a cell walls com tion. In one embodiment, the device includes a bioactive ponent of a Theacea plant. This method involves providing a composition of the present invention. The bioactive compo Theacea plant (e.g., in the form of fresh biomass). The Thea sition can be enclosed in a filtering pouch. A Suitable filtering cea plant (e.g., fresh biomass) is then separated into cell juice pouch can be one that is effective in selectively dispersing into and a cell walls component. The cell walls component is a liquid the low molecular weight and reduced, non-oxidized treated under conditions effective to yield a bioactive frac components of the bioactive composition. In one embodi ment, the pouch includes a selective membrane that allows tion. The bioactive fraction is then isolated from the treated dispersal of the low molecular weight and reduced, non cell walls component. In one embodiment, the various Suit oxidized components of bioactive compositions from within able bioactive fractions produced by this method are as the pouch into the liquid, but where the membrane inhibits described herein. The present invention further relates to an dispersal of high molecular weight and oxidized components isolated bioactive fraction produced by this method. from within the pouch into the liquid. As used herein, the term 0081. By way of example, the overall process for prepar “low molecular weight and reduced, non-oxidized compo ing the bioactive fractions of the present invention (as nents' include components of the bioactive composition of described herein above) is schematically shown in FIG. 1. the present invention that are less than or equal to about 5,000 Daltons. In one embodiment of this method, the bioactive Details of the processing steps are further described in the composition can further include a stabilizing agent (Suitable Examples (infra). As depicted in FIG. 1, fresh biomass 10 examples of which are as described herein). In another (e.g., fresh plant biomass) of Theacea plants is subjected to embodiment of this method, the bioactive composition can grinding, maceration, and pressing 20 under conditions effec further include a topically acceptable carrier (suitable tive to destroy rigid cell walls, and thereby to yield plant cell examples of which are as described herein). juice 30 and cell walls 32. The fresh biomass 10 is also used I0088. The present invention also relates to a method of for conventional tea processing 22 to produce positive control making a therapeutic beverage containing low molecular 150 for comparative testing and evaluation. Cell juice 30 is weight and reduced, non-oxidized bioactive compositions. Subjected to coagulation 40 (e.g., microwave treatment) to This method involves providing a device produced according achieve quantitative coagulation of membrane fraction com to the method of the present invention. The device is con tacted with a liquid under conditions effective to cause the ponents of fresh plant biomass 10. Coagulation 40 is suffi low molecular weight and reduced, non-oxidized compo cient to enable Subsequent separation of the coagulated mem nents of the bioactive compositions to disperse into the liquid. brane fraction from other non-coagulated components of cell In one embodiment of this method, the bioactive composition juice 30. As shown in FIG. 1, one embodiment of such sepa can further include a stabilizing agent (Suitable examples of ration is achieved by cooling and centrifugation 42 to yield which areas described herein). In another embodiment of this membrane fraction (precipitate) 50 and supernatant 60, method, the bioactive composition can further include a topi which is free from specific chloroplast membrane compo cally acceptable carrier (Suitable examples of which are as nents such as chlorophyll and phospholipids. described herein). A suitable liquid for use in this method can 0082 To produce the cell walls fraction extract (i.e., Com include, without limitation, water. The water can be hot or position A110), cell walls 32 are subjected to drying 34 (e.g., cold. The present invention further relates to a therapeutic several Subsequent microwave treatments) and then mixing beverage produced according to this method. the dried material with water 36 under conditions commonly used to prepare conventional teas (e.g., mixing in water at 85° EXAMPLES C.). To produce the membrane fraction extract (i.e., Compo Example 1 sition B120), membrane fraction 50 is subjected to mixing with solvent 52 and then centrifugation 54 to yield superna Preparation of Bioactive Compositions Derived from tant 56 and Composition B120. Camelia sinensis Plants 0083) To produce the cytoplasm fraction extract (i.e., Composition C130), supernatant 60 is subjected to coagula 0089. A schematic of one embodiment of the method of tion 62 (e.g., isoelectric precipitation) and centrifugation 64 preparing the bioactive compositions of the present invention to yield cytoplasm fraction (precipitate) 70 containing most is shown in FIG.1. Below is a description of relevant aspects of the Soluble cytoplasm proteins. Cytoplasm fraction (pre of one embodiment of the method of the present invention. 0090 Biomass Preparation. Sufficient amounts of fresh cipitate) 70 is then subjected to mixing with solvent 72, fol Camellia (Camelia sinensis) plant biomass (only top tender lowed by centrifugation 74 to yield supernatant 76 and then young leaf tissue with buds) were harvested to yield approxi Composition C 130. mately 100 kg of dry matter. The level of dry matter in the 0084. To produce cell juice serum (i.e., Composition D fresh biomass was calculated to be 21.70%, requiring harvest 140), Supernatant 60 is Subjected coagulation 62 (e.g., iso ing of approximately 461 kg of fresh plant biomass to yield electric precipitation) and centrifugation 64 to yield cell juice 100 kg of dry matter. Care was taken to preserve the inherent serum (supernatant) 66 and then Composition D 140. moisture content of the plant biomass and to avoid wilting due US 2009/O 185990 A1 Jul. 23, 2009
to moisture loss. The harvesting was conducted in Such a low-speed centrifugation biomass. The filtered plant cell manner as to avoid or minimize chopping, mashing, and juice was exposed to microwave treatment using a tempera crushing of the collected biomass to avoid the disruption of ture probe control. This treatment continued until the tem the leaf cell structure, which triggers the endogenous enzy perature of the cell juice reached 60°C. Once coagulation was matic reactions catalized by phenol-oxidase and peroxidase. induced, the treated cell juice was immediately cooled to 40° Because these reactions are intensified with the time of oxi C. Separation of the membrane fraction from the coagulated dation, all steps were completed in the shortest possible cell juice was achieved using centrifugation at greater than or period of time. For example, the harvested biomass was deliv ered for processing not more than 10 minutes after cutting. equal to 3,000 g for greater than or equal to 20 minutes. This This was done to minimize exposure of the plant biomass to yielded a membrane fraction (precipitate) and a cell juice Sun, high temperature, and other negative environmental fac Supernatant, which contained a cytoplasm fraction and a cell tors. A washing step was performed to remove Soil particles serum fraction (i.e., low molecular weight soluble compo and other debris from the plants prior to further processing. nents). The membrane fraction having dry mater level This washing was accomplished by washing the harvested 32.89% was used in preparing the extract of membrane-de plants for s5 minutes in s1 kg/cm water pressure. The rived bioactive composition. The cell juice Supernatant was residual water wash did not contain any green or brown pig used for further processing to yield cytoplasm fraction and ments, indicating proper water pressure and washing dura cell juice serum. tion. The excess water was removed from the washed plant (0094 Preparation of the Membrane Fraction Extract biomass. (Composition B). One part of membrane fraction (10.0 kg) 0091 Grinding, Maceration, and Pressing of Plant Biom and two parts of Dimethyl Sulfoxide—(20.0 kg) were mixed ass. After harvesting, collecting, and washing the plant bio at room temperature for 1 hour with permanent stirring. Then mass, the plants then underwent grinding, maceration, and material was centrifuged at greater than or equal to 4,000 g for pressing to extract the intracellular content (i.e., the plant cell greater than or equal to 45 minutes. The precipitate was juice) and to separate it from the fiber-enriched cell walls discarded and supernatant was filtered through the filter hav fraction (cell walls fraction). A hammer mill (Model VS 35, ing 0.8 um porous. This filtrate having dry meter level Vincent Corporation, FL) having 10 HP engine and set of 6.83% membrane fraction extract (composition B) was screens was used to grind the biomass to yield plant tissue used for further tests of its activities. particles of Suitably Small size in a shortest amount of time (0095 Separation of the Cytoplasm Fraction from the Cell and without significant increase of biomass temperature. The Juice Supernatant. In order to separate out the cytoplasm hammer mill was set to produce the maximum size of mac fraction, the cell juice Supernatant was subjected to isoelectric erated plant particles of s().5 centimeters during s 10 sec precipitation. Precipitation of the cytoplasm fraction was onds of treatment. The biomass temperature was increased induced using a titration method utilizing 5.0N Hydrochloric only si5° C. A horizontal continuous screw press (Compact Acid (HCl) to bring the pH of the cell juice supernatant to 4.0. Press “CP-6, Vincent Corporation, FL) was immediately The separation of precipitated cytoplasm fraction having dry used to extract the plant cell juice from the plant. The pressure matter level 14.5% from supernatant was achieved by cen on the cone of the screw press was maintained at a level of 24 trifugation at greater than or equal to 3,000 g for greater than kg/cm, with a screw speed of 12 rpm and only a temperature or equal to 20 minutes. increase of s5°C. This treatment yielded the 185 kg of cell 0096 Preparation of the Extract of Cytoplasm Fraction walls fraction having dry matter level 41.39% and 276 kg of (Composition C). One part of cytoplasm fraction (10.0 kg) plant cell juice having dry mater level 8.49%. and two parts of Dimethyl Sulfoxide—(20.0 kg) were mixed 0092 Preparation of Cell Walls Fraction Extract (Compo at room temperature for 1 hour with permanent stirring. Then sition A). The aliquot of cell walls fraction having initial dry material was centrifuged at greater than or equal to 4,000 g for matter level 41.39% was dried in microwave hood combina greater than or equal to 45 minutes. The precipitate was tion (Model GH9115XE, Whirlpool) during 30 sec and then discarded and supernatant was filtered through the filter hav cooled during 30 sec. This treatment was repeated several ing 0.8 um porous. This filtrate having dry meter level times till dry matter level in cell walls fraction reached 3.50%—extract of cytoplasm fraction (composition C) can be 96.52%. The 66.01 of deionized water having temperature used for further tests of its activities. 85°C. were added to 4.0 kg of dry cell walls fraction and steer (0097. Preparation of Cell Juice Serum (Composition D). with high agitation for 5 min. These conditions are in agree After Separation of cytoplasm fraction the Supernatant con ment with tea preparation procedure, which is described in tained suspended particles. In order to separate out these D'Amelio, F. S. Botanicals. A Phytocosmetic Desk Refer particles, the Supernatant was centrifuged at greater than or ence, Boca Raton, London, New York, Washington, D.C.: equal to 7,500 g for greater than or equal to 30 minutes. The CRC Press, p. 361 (1999), which is hereby incorporated by transparent Supernatant—cell juice serum was filtered reference in its entirety (see also the discussions at www. through the filter having 0.8 um porous. This filtrate (compo leaftea.com; www.divinitea.com; www.equatorcoffee.com, sition D) having dry matter level 5.69% was used for further which are hereby incorporated herein in their entirety). The tests of its activities. mixture was filtered through 4-layers of nylon fabric and then 0.098 Preparation of Conventional Tea Extracts—Con through the filter having 0.8 um porous. The pH of obtained trols. The same lot of fresh Camelia leaves, which was used cell walls extract was equal 5.24 and dry matter level was to preparation of compositions A, B, C and D was used to equal 0.84%. This extract was further used for tests of its produce conventional white and black tea. activities. 0099. The following procedure was used to produce white 0093 Separation of the Membrane Fraction from the Cell tea. The fresh biomass contained 21.70% dry matter was Juice. The initial plant cell juice having dry matter level placed for 20 sec in boiling water to inactivate endogenous 8.49% contained small fiber particles, which were removed enzymes phenol-oxidase and peroxidase. During this pro by filtration through four layers of nylon fabric or by using cedure the leaves were kept in the nylon screen bag. Then US 2009/O 185990 A1 Jul. 23, 2009
treated leaves were dried in microwave during 30 sec and then used as the Sources for preparation of their extracts, which are cooled during 30 sec. This treatment was repeated several categorized as bioactive compositions. The cell juice serum times until dry matter level in biomass reached 93.74%. Then was directly used “as is as Subsequent bioactive composition 66.01 of deionized water having temperature 85°C. were having no exogenous solvents. added to 4.0 kg of dry leaves and steer with high agitation for 5 min. These conditions are in agreement with tea preparation TABLE 1 procedure, which is described in D'Amelio, F. S. Botanicals. A Phytocosmetic Desk Reference, Boca Raton, London, New Distribution of 100 kg Dry Matter Among Products of Fractionation York, Washington, D.C.: CRC Press, p. 361 (1999), which is of Fresh Biomass hereby incorporated by reference in its entirety (see also the Plant Source discussions at www.leaftea.com; www.divinitea.com; and Product Cameia sinensis www.equatorcoffee.com, which are hereby incorporated by Initial Biomass 1OO.O reference in their entirety). The mixture was filtered through Cell Walls Fraction 76.6 4-layers of nylon fabric and filtered through the filter having Cell Juice 23.4 Membrane Fraction 6.5 0.8 um porous. The pH of obtained cell walls fraction extract Cytoplasm Fraction O.6 was equal 5.52 and dry matter level was equal 1.10%. This Cell Juice Serum 16.3 extract was further used for tests of its activities. 0100. The following procedure was used to produce black tea. The fresh biomass contained 21.70% dry matter was kept 0102. It should be noted that the three selected materials at 25° C. with periodical (1 hour “on” and 1 hour “off”) are the most diversified representation of all functional struc aeration until dry matter level reached 35%. Then leaves were tures, which exists in fresh plant tissue. Only soluble cell juice ground (crushed) to the particles having size 2-3 mm. This serum has physico-chemical properties, which allows the procedure leads to increase of biomass temperature to direct administration to the commonly used in vitro testing approximately 30°C. The ground biomass was placed in the systems. The cell walls fraction, membrane fraction and cyto form of layer (2" high) on plastic conveyor belt for fermen plasm fraction were used as raw materials for extraction with tation (oxidation) during 90 min at 25°C. The fermented solvents. Because cell walls fraction is structurally similar to biomass, which acquired the brown color. was dried at 130° conventional tea plant products, this fraction was extracted C. for 30 minto reach the dry matter level 97.5%. Then 66.0 with water to provide the best comparison with conventional 1 of deionized water having temperature 85°C. were added to teas. The membrane fraction was extracted with Dimethyl 4.0 kg of dry leaves and steer with high agitation for 5 min. Sulfoxide, which facilitates the effective solubilization of These conditions are in agreement with tea preparation pro both hydrophobic and hydrophilic components integrated in cedure which is described in D'Amelio, F. S., Botanicals. A chloroplast and mitochondria structures. The cytoplasm frac Phytocosmetic Desk Reference, Boca Raton, London, New tion was extracted with water. The cell juice serum was used York, Washington, D.C.: CRC Press, p. 361 (1999), which is “as is.’ hereby incorporated by reference in its entirety (see the dis (0103 Table 2 shows the yield of all four tested bioactive cussions at www.leaftea.com; www.divinitea.com; www. compositions: cell walls fraction extract (composition A), equatorcoffee.com, which are hereby incorporated by refer membrane fraction extracts (composition B), extract of cyto ence in their entirety). The mixture was filtered through plasm fraction (composition C), cell juice serum (composi 4-layers of nylon fabric and filtered through the filter having tion D) and controls white tea extract or black tea extract 0.8 um porous. The pH of obtained cell walls fraction extract from 100 kg of initial biomass dry mater. was equal 4.96 and dry matter level was equal 1.38%. This extract was further used for tests of its activities. TABLE 2 Example 2 Yield of Bioactive Compositions from 100 kg of Initial Biomass Plant Source Distribution of Dry Matter Regarding Preparation of Product Camelia sinensis Bioactive Compositions from Camelia sinensis, Initial Biomass 1OO.O Camellia japonica, Camelia reticulate, Camellia Composition A (Cell Walls Fraction Extract) 14.35 Sasanqua, and Eurya Sandwicensis Composition B (Membrane Fraction Extract) 2.37 Composition C (Cytoplasm Fraction Extract) O.2 0101 Various fractions collected during the production of Composition D (Cell Juice Serum) 16.3 bioactive compositions were analyzed and compared for dry Control (Extract of White Tea or Black Tea) 1514. . . 19.36 matter distribution. Table 1 shows the distribution of 100 kg dry matter among products of fractionation of tea plants. It 0104 Table 2 shows that the total yield of bioactive com was determined that the process of the present invention positions A, B, C and D from 100 kg of dry matter of Camellia permits extracted yield conversion into plant cell juices in the Sinensis equals 33.3%, which very significantly exceeds the range of from about 20 to 30% of initial biomass dry matter. yield of conventional tea process—15.14. . . 19.36%. The yield of membrane fractions dry matter was in the range from 5% to 10% of initial biomass dry matter and from 25% Example 3 to 35% of cell juice dry matter. Table 1 shows that the yields of cytoplasm fractions dry matter did not exceed 1.0% of Comparison of Composition A (Cell Walls Fraction initial biomasses dry matter and subsequently 2.5% of cell Extract) and Conventional Tea Extracts juice Supernatant dry matter. Most of cell juice Supernatant dry matter was concentrated in cell juice serum. The cell walls 0105 Various parameters of bioactive composition A and fraction, membrane fraction and cytoplasm fraction were conventional white and black tea extracts obtained from the US 2009/O 185990 A1 Jul. 23, 2009
same batch of fresh Camelia sinensis were measured and new tea category having fundamental differences compared subsequent results of these are presented in Table 3 (The used with white, green, oolong, and black teas. This novel cell experimental methods are described in Examples 9 and 20, walls fraction tea can be used in either loose or bag form or and in the U.S. Patent Application Publication No. 2003/ other manifestations to prepare a broad spectrum of drinks, 0.175235, which is hereby incorporated by reference in its beverages, and additives to nutriceutical and functional food entirety). products.
TABLE 3 Example 4 Various Parameters of Bioactive Composition A and Extracts of Preparation of Bioactive Compositions for Different White and Black Teas. Applications Extract of 0.108 All bioactive compositions can be used as solutions, Cell Walls Controls Suspensions, dispersions, pastes or dried powders incorpo Fraction Extract of Extract of rated into a variety of formulations for systemic or topical Parameter (Composition A) White Tea Black Tea administrations. The Solubilized forms of compositions can be filtrated through filters having the 0.2 um porous to com Dry Matter, 9% O.84 1.10 1.38 pH 5.24 5.52 4.96 pletely remove non-completely solubilized small particles Conductivity, mS/cm 1.57 2.23 5.32 and endogenous microorganisms. The dry matter level in Total Dissolved Solids, g/L O.78 1.23 2.71 bioactive compositions before and after sterilized filtration is Redox Potential, mV 123 159 188 presented in Table 4. Area under the Spectra 5.727 6.604 4.616 Curve (ASC) 200-450 nm, Abs' nm. TABLE 4 ASC: Dry Matter 6.818 6.004 3.345 Superoxide Scavenging 26.3 114.5 227.1 Level of Dry Matter in Bioactive Compositions Before (numerator) Activity (ICRso), and After (denominator) Sterilized Filtration g DMIml Color (1 ... 10 scale) 9.6 4.7 7.5 Plant Source Flavor (1 ... 10 scale) 9.3 6.1 6.6 Product Cameia sinensis Mouthfeel (1 ... 10 scale) 9.4 6.5 5.3 Composition A O.84 (Extract of Cell Walls Fraction) 0.72 Composition B 6.83 0106 Table 3 shows that cell walls fraction extract has (Extract of Membrane Fraction) 6.12 lower levels of dry matter, electrolytes and dissolved solids Composition D 5.69 compare to conventional white and black tea extracts. The (Cell Juice Serum) 5.59 UV/VIS spectral data show that cell walls fraction extract has Control 1.10 the highest specific value of the area under the spectra curve, (Extract of White Tea) 1.03 i.e. this particular Camellia product (composition A) has the highest level of optically active constituents per unit of dry 0109 Table 4 shows that dry matter levels in all bioactive matter. Additionally, the cell walls fraction extract has the compositions were decreased after sterilizing filtration. How lower amount of redox potential, which indicates that this ever this decrease did not lead to any loss or significant composition is less oxidized then conventional white and reduction of their biological activities and was in the range black tea extracts. The cell walls fraction extract demon 2-14%. Therefore major part of compositions is presented by strated Superoxide Scavenging activity, resulting in 50% inhi soluble bioactive ingredients. bition of cytochrome c reduction (ICRs) at a much lower 0110 Fresh Camellia leaves contain relatively low concentration then white and black tea extracts. A Qualitative molecular weight (reduced, non-oxidized) ingredients. As a Descriptive Analysis ("ODA) test method was selected to result of oxidation and polymerization processes in the manu systematically characterize and quantify teas based on color, facturing of conventional teas the above potent ingredients flavor, and mouthfeel, which govern acceptability of tea bev are transformed into the parts of high molecular weight Sub erages. The QDA method employs a trained panel of expert stances having relatively low activity. tasters to quantify the above attributes of tea beverages rela 0111. The bioactive compositions of the present invention tive to defined reference standards. The comparative evalua are obtained without fermentation (oxidation) and excessive tion of the color, flavor and mouthfeel of teas demonstrated heat treatment. This in turn prevents the irreversible loses of that cell walls fraction extract significantly exceed the same fresh plant activities, which can be delivered with maximum characteristics of conventional teas. potency using, for example, novel tea bag or analogous deliv ery systems. Instead of conventional paper tea bags, which 0107 Thus, cell walls fraction, which was obtained from allow all soluble tea ingredients to move through large pores fresh Camelia biomass without any fermentation (oxidation) to the Surrounding water, the novel tea bag is made from and heat treatment, is distinct from all other teas (Wilson et semi-permeable membrane. This bag contains bioactive com al., eds. Tea. Cultivation to Consumption, London: Chapman position inside and allows only ingredients having molecular Hall (1992), which is hereby incorporated by reference in its weight below certain membrane cut off (for example, 5,000 entirety). Additionally, the key Camelia enzymes (phenol Dalton) to penetrate to the Surrounding water phase. The oxidase and peroxidase) always remain within conventional ingredients having higher molecular weight remain inside the teas. Instead, the present invention includes separation of bag and thus are not included in the beverage. fresh Camellia leaves to cell walls fraction and cell juice, 0112 Therefore, the cut off of ingredients having molecu which is enriched by these enzymes and thus cell walls frac lar weight above a certain level allows the production of a tion does not contain endogenous phenol-oxidase and peroxi beverage having no oxidized ingredients because all oxidized dase. Therefore, cell walls fraction must be categorized as a ingredients of bioactive compositions having molecular US 2009/O 185990 A1 Jul. 23, 2009
weight above a certain level remain inside the bag. The novel light-characteristic odor. No Solvent (i.e. glycol, oil, or water) tea bag design can be based on pyramidal tea bag construc was added to the carrier medium. Table 5 summarizes the tion, which utilizes dialysis membrane tube. The design of Physical and Chemical data of topical ingredient SF. novel tea bag can also include thin plastic frame having bio active composition inside and two transparent surfaces built TABLE 5 from semi-permeable membrane. The selection of particular membrane cut off value is determined by the type of bioactive Physical and Chemical Parameters of Topical Ingredient SF composition, but in general higher cut off enabling release into Surrounding water phase of a higher percent of compo Parameter Method Results sition's dry matter lower amount of the redox potential is Solid Content, % See Example 20, “Method 1 S.04 preferable. Specific Gravity, g/cm USP &841> 101S Color Gardner Scale S-6 Refractive Index USP 831 1312 Example 5 PH USP 791. 4.0 Redox Potential, mV See reference 1 75 Preparation of Topical Ingredient SF Derived from Conductivity, Sim See reference 2 1.02 Cell Juice Serum References: 0113 Cell juice serum (composition D) cannot be used as 1 Handbook of Chemistry and Physics, 80" Edition, CRC Press, 1999 an active ingredient of topical products due to the lack of 2000, 5-90; stability and deterioration of color and odor. The described 2) Handbook of Chemistry and Physics, 80" Edition, CRC Press, 1999 procedure allows for the refinement of cell juice serum frac 2000, 8-21, which are hereby incorporated by reference in their entirety. tion to yield a stable and active topical ingredient SF (this procedure is similar to previously described in U.S. Patent 0115 Table 6 describes the UV-Spectra data regarding Application Publication No. 2003/0175235, which is hereby topical ingredient SF. incorporated by reference in its entirety). The refinement of the cell juice serum involved the following steps: heat treat TABLE 6 ment, cooling, filtration, and stabilization. Refinement was performed immediately after separation of the cell juice UV-Spectra of Topical Ingredient SF (1:500 Dilution serum from the cytoplasm fraction as described in Example 1. The cell juice serum was exposed to microwave treatment Peak Parameter Method Results using a temperature probe control. This treatment continued #1 Start, nm USP 197s 450 until the temperature of the cell juice serum reached 99 C. Apex, nm 266.5 (90° C. was required as was previously described in U.S. End, nm 247 Patent Application Publication No. 2003/0175235, which is Height, Abs O.231 hereby incorporated by reference in its entirety). Once coagul Area, Absx nm 13.676 #2 Start, nm. USP 197s 247 lation was induced the treated cell juice serum was immedi Apex, nm 204 ately cooled to 10°C. The coagulated cell juice serum was End, nm 2OO vacuum filtrated through filter having porous 0.8 um (double Height, Abs 1.396 layers of Whatman No. 2 filters were used in U.S. Patent Area, Absx nm 32.413 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 0116. The microbial analysis conducted in accordance for further processing (i.e., stabilization). Stabilization of the with the following procedure (USP <61>) demonstrated that cell juice serum filtrate was achieved by adding preservatives (no exogenous anti-oxidant was required as was previously topical ingredient SF contains less then 100 colony forming described in U.S. Patent Application Publication No. 2003/ units per gram of sample and has no pathogens (E. coli, 0.175235, which is hereby incorporated by reference in its Candida albicans, Pseudomonas sp. and Staphylococcus entirety) and incubating the mixture until complete solubili aureus). This data demonstrates that topical ingredient SF zation was achieved. The preservatives used included the satisfies the industry requirements for ingredients of topical following: 0.1% potassium sorbate, 0.1% sodium benzoate, products. 0.1% sodium methyl paraben, and 0.1% citric acid. This preparation resulted in the production of 16.3 kg of dry matter 0117 Topical ingredient SF was determined to be stable yield (or approximately 286 Liters) of the topical ingredient (i.e., maintaining physical and chemical integrity) for at least SF, which was used for characterization of its physico-chemi 12-18 months while storedata temperature of between 15 and cal and bioactive qualities. The recommended storage condi tions for topical ingredient SF include storage in a closed 25° C. in a closed container protected from light. Topical container protected from light at a temperature of between ingredient SF is a biodegradable product. In a controlled 15° C. and 25° C. clinical evaluation, topical ingredient demonstrated the bio logical activities, which are summarized in Table 7. Example 6 Product Specifications of Topical Ingredient SF TABLE 7 Derived from Cell Juice Serum Fraction Biological Activities of Topical Ingredient SF 0114 Topical Ingredient SF was prepared according to the Activity Method g DM/ml process described above in Example 5. Analyses of topical Superoxide Scavenging Activity (ICRs) See Example 20, 69.5 ingredient SF were conducted to determine its various Method 7 physico-chemical, microbial, cytotoxicity, and bioactivity Elastase Inhibitory (IC.so) See Example 20, 32.3 characteristics, as described below. Topical ingredient SF is a Method 5' clear liquid, which has a light-yellow-brown color and a US 2009/O 185990 A1 Jul. 23, 2009
istic odor. As result, Such material cannot be used as a topical TABLE 7-continued ingredient. The procedure described below allows for trans formation of freshly obtained membrane fractions into stable Biological Activities of Topical Ingredient SF and active topical ingredients (this procedure is similar to Activity Method g DM/ml previously described in the U.S. Patent Application Publica tion No. 2003/0175235, which is hereby incorporated by MMP-9 Inhibitory (IC.so) See Example 20, 14.6 reference in its entirety). Method 6’ Trypsin Inhibitory (IC.so) See reference 1 7.8 0.124 Immediately after separation of the membrane frac tion from cell juice according to the process described above Reference: in Example 1, the membrane fraction was stabilized and 1 Cannel et al., Planta Medica. 54: 10-14 (1988), which is hereby incorpo incorporated into a polymer matrix. To prepare approxi rated by reference in its entirety. mately 100 grams oftopical ingredient MF the cell membrane 0118 Table 7 shown that topical ingredient SF demon fraction was stabilized by mixing it with non-ionic emulsifier strated Superoxide Scavenging ability. In a controlled clinical Polysorbate 80 (Tween 80) and antioxidants (Tenox 4). Spe evaluation, topical ingredient SF demonstrated a 50% inhibi cifically, 20 grams of fresh membrane fraction was mixed tion of cytochrome c reduction (ICRs) at a concentration vigorously with 3.5 grams of Tween 80 and 0.1 gram of Tenox 69.5 lug dry matter per ml. The ICRs of positive control 4 (solution of Butylated Hydroxyanisole and Butylated (roSmarinic acid)–26.5 g/ml. In addition to anti-oxidant Hydroxytoluene in oil) until homogeneous, while avoiding properties, topical ingredient SF demonstrated antipro aeration during mixing. teolytic activities against peptide hydrolases, for example 0.125. Once stabilized, the membrane fraction was incor elastase, gelatinase B or so-called matrix metalloproteinase 9 porated into a polymer matrix (i.e., a dispersion of polymeric (MMP-9), and trypsin. Among these enzymes the unique emulsifier, acrylates/C10-C30 acrylate crosspolymer). The position belongs to elastase and MMP-9, which act synergis polymer matrix was prepared by dispersing 0.9 grams of tically and play an extremely important role in skin inflam Pemulen TR-2 in 69.2 grams of warm deionized water and mation. It should be noted, that both MMP-9 and elastase are mixing until uniform using moderate agitation, while avoid secreted by white blood cells (neutrophils) and these enzymes ing aeration. In parallel, 5 grams of Glycerin and 1.0 gram of are the key enzymes in the final pathway leading to inflam Phenonip (mixture of Phenoxyethanol (and) Methylparaben mation. It is generally agreed that if preparation can inhibit (and) Butylparaben (and) Ethylparaben (and) Propylparaben) both enzymes (elastase and MMP-9), such preparation is were combined in a separate vessel and mixed until uniform. considered to be very effective to treat inflammatory pro With moderate agitation, the phases containing Pemulen and CCSSCS. Glycerin with Phenonip were combined and mixed until uni 0119. It should be noted that skin aging processes, sun form. To incorporate the membrane fraction into the polymer burns, formation of wounds and Scars have the very same matrix, the phase containing the membrane fraction, Tween inflammation mechanism, which involves both MMP-9 and 80, and TenoX 4 was added to the phase containing the elastase. Thus, topical ingredient SF capable of inhibiting Pemulen, Glycerin, and Phenonip, and then mixed with vig both of the above enzymes has very wide spectrum of appli orous agitation while avoiding aeration. Stabilization of the cations, among which are inflammatory injury because the membrane fraction mixture was achieved by neutralizing it following reasons: with 18% aqueous solution of sodium hydroxide (NaOH) and 0120 a. These two enzymes can synergize to degrade mixed vigorously to produce a uniform system having a pH of all the components of extracellular matrix of human 5.0+0.4. This preparation, which started from 100 kg of fresh tissue; Camellia biomass (approximately 461 kg of fresh leaves hav I0121 b. Elastase can inactivate the body's own inhibi ing 21.7% dry matter), resulted in the production of 11.85 kg tory defense against MMP-9; and of Dry Matter yield (or approximately 172 liters) of topical 0.122 c. MMP-9 can inactivate the body's own inhibi ingredient MF, which was used for characterization of its tory defense against elastase. physico-chemical and bioactive qualities. The recommended The combination of anti-inflammatory and anti-oxidant prop storage conditions for topical ingredient MF include storage erties of topical ingredient SF suggests that this hydrophilic in a closed container protected from light at a temperature preparation based on bioactive composition D is capable to between 2 and 8°C. act systemically on very fundamental skin disorder problems. Example 8 Example 7 Preparation of Topical Ingredient MF Derived from Product Specifications of Topical Ingredient MF Membrane Fraction Derived from Membrane Fraction 0123. The freshly obtained membrane fraction is a paste 0.126 Topical ingredient MF was prepared according to having intensive color and specific odor. This fraction is rep the process described above in Example 7. Analyses of topi resented predominantly by chloroplasts and its composition cal ingredient MF were conducted to determine its various includes predominantly phospholipids, membrane proteins, physico-chemical, microbial, cytotoxicity, and bioactivity chlorophyll, and carotenoids. The drying of membrane frac characteristics, as described below. Topical ingredient MF is tion results in irreversible loses of many valuable properties an opaque gel, which has a green-brown color and light required for the exploration of membrane fraction as a topical characteristic odor. Topical ingredient MF was formulated ingredient. Without drying, the unstable membrane fraction is utilizing the natural cell juice constituents gelled with a poly quickly transformed into the dark color un-dispersible and mer to assure the highest level of purity uniformity, compat insoluble conglomerates having a strong and non-character ibility, stability, safety and efficacy. US 2009/O 185990 A1 Jul. 23, 2009
0127 Table 8 describes the physical and chemical data of Example 9 topical ingredient MF. Spectral Analyses of the Bioactive Compositions TABLE 8 Derived from Camelia sinensis Plants Physical and Chemical Parameters of Topical Ingredient MF I0132) Introduction to Spectral Analyses. Ultraviolet (UV) Parameter Method Results radiation has damaging effects on human skin. Short-term effects include tanning and Sunburn, while the long-term Non-Volatile Residue See Example 20, “Method 2 6.9 effects of cumulative UV exposure include photoaging of the (NVR),9% Specific Gravity, g/cm USP &841> 1.035 skin and increased risk of skin cancer. Ultraviolet skin injury Viscosity, cps USP 911 18,700 is mediated by oxidative damage, and a number of plant pH USP 791. 4.6 extracts with antioxidant activity are showing promise as Total Carotenoids, 9% NVR See Example 20, “Method 4 O.36 protective agents: grape seed extract (Carini et al., “Protective Lutein, % NVR See Example 20, “Method 4 O.34 Effect of Procyanidines from Vitis vinifera Seeds on UV Induced Photodamage: In vitro and In vivo Studies.” Pro ceedings of the 19th IFSCC Congress 3:55-63 (1996), which 0128. Table 9 summarizes the L*a*b* values data regard is hereby incorporated by reference in its entirety), lycopene ing topical ingredient MF. (Di Mascio et al., “Lycopene as the Most Efficient Biological Carotenoid Singlet Oxygen Quencher.” Archives of Biochem TABLE 9 istry and Biophysics 274:532-8 (1989); and Ribaya-Mercado et al., “Skin Lycopene is Destroyed Preferentially Over L*a*b* Values of Topical Ingredient MF B-Carotene During Ultraviolet Irradiation in Humans.” Jour Parameter Method Results nal of Nutrition 125:1854-9 (1995), which are hereby incor L* See Example 20, “Method 3 30.27 porated by reference in their entirety), silymarin (Morazzoni a: 27.36 et al., “Silybum marianum (Carduus marianus). Fitoterapia b* 42.56 66:3-42 (1995); Katiyar et al., “Protective Effects of Sily marin Against Photocarcinogenesis in a Mouse Skin Model.” Journal of the National Cancer Institute 89:556-66 (1997), 0129 Microbial analyses demonstrated that topical ingre which are hereby incorporated by reference in their entirety), dient MF satisfies the industry requirements for topical ingre and especially green tea extract which has higher efficacy dients with regard to CFUs and absence of pathogens (USP compared with extracts produced from other plant sources <61>). (Katiyar et al., “Protection Against Ultraviolet-B Radiation Induced Local and Systemic Suppression of Contact Hyper 0130 Topical ingredient MF was determined to be stable sensitivity and Edema Responses in C3H/HeN Mice by (i.e., maintaining physical and chemical integrity) for at least Green Tea Polyphenols. Photochemistry and Photobiology 12-18 months while stored at a temperature of between 2 and 62:855-61 (1995); Ruch et al., “Prevention of Cytotoxicity 8° C. in a closed container protected from light. Topical and Inhibition of Intercellular Communication by Antioxi ingredient MF is a biodegradable product. In a controlled dant Catechins Isolated from Chinese Green Tea, Carcino clinical evaluation, topical ingredient MF demonstrates genesis 10:1003-8 (1989); Wang et al., “Protection Against elastase inhibitory activity and trypsin inhibitory activity. Ultraviolet B Radiation-Induced Photocarcinogenesis in Table 10 summarizes certain bioactivity results for topical Hairless Mice by Green Tea Polyphenols. Carcinogenesis ingredient MF. 12:1527-30 (1991), which is hereby incorporated by refer ence in its entirety). TABLE 10 I0133. It was found that the leaves of the tea plant (Camel lia sinensis) have a high content of polyphenols with antioxi Bioactivity Results of Topical Ingredient MF dant activity including (-)-epicatechin, (-)-epicatechin-3- Activity Method ICso (Lig/ml) gallate, (-)-epigallocatechin, and (-) epigallocatechin-3- gallate. Green tea extract has shown an antioxidant activity Elastase Inhibitory (ICs) See Example 20, “Method 5' 12.3 against hydrogen peroxide and the Superoxide radicals and MMP-9 Inhibitory (ICs) See Example 20, “Method 6' S.6 prevention of oxidative cytotoxicity. The extract can also Trypsin Inhibitory (ICso) See reference 1 3.8 prevent the inhibition of intercellular communication, a pos Reference: sible mechanism of tumor promotion. There is a close asso 1 Cannel et al., Planta Medica. 54: 10-14 (1988), which is hereby incorpo ciation between UV-induced immune Suppression and the rated by reference in its entirety. development of skin cancer, and green tea extract has been 0131 Table 10 shown that topical ingredient MF demon found to protect against inflammation and immune Suppres sion caused by UV-B radiation. Green tea extract given orally strated properties similar to topical ingredient SF (see in drinking water or applied topically protects against UV-B- Example 6). Although topical ingredient MF has no SuperoX induced skin carcinogenesis in animal models. These results ide scavenging activity, it demonstrates higher specific indicate that green tea extract taken orally may help to prevent enzyme inhibition activities than topical ingredient SF. Thus skin cancer. topical ingredient MF, which is based on bioactive composi I0134. Although the UV protection properties of Camellia tion B, should be considered as a potent multiphase anti products have been established, the greater potential of tea inflammatory ingredient having broad applications for treat plant as a source for effective protection of the skin against ment of skin disorders. Sun damage has not fully explored due to the limitations of US 2009/O 185990 A1 Jul. 23, 2009 conventional technology, which is driven towards focusing on taken in (5) five replications. The absorbance spectra of a limited to relatively narrow band of active ingredients: samples before and after irradiation were used for statistical predominately cathechins. analysis. 0135 Comparative UV protection properties studies 0140 Samples. The following bioactive compositions, described herein between “novel' and “conventional which were prepared according to the process described Camellia products have now demonstrated that the technol above in Example 1 were evaluated: composition A (cell ogy of “fresh Camellia fractionation' is capable of yielding walls fraction extract having 0.84% dry matter), composition more potent products. The comparison was made by utilizing B (membrane fraction extract having 6.83% dry matter), the methods commonly used to determine spectral properties composition D (cell juice serum having 5.69% dry matter). of solutions and in-vitro sun protection factor (SPF). Extract of conventional white tea having 1.10% dry matter 0.136 Methodology A: UV/VIS Spectra. UV/VIS spectra and extract of conventional black tea having 1.38% dry matter of Camellia products in 200-450 nm region were obtained were used as controls. All samples were obtained from the using pharmacopoeia compliant Spectrophotometer Ultro same batch of fresh Camelia, which was collected at spec 4300 Pro (Amersham Biosciences Ltd., Buckingham Charleston Tea Plantation, SC. These samples did not contain shire, England). The spectral parameters of diluted in distilled any additives. water Camelia products were determined according to the 0141 Analyses. It was found that all Camelia samples procedure described in USP <197>. have high UV absorbance values and thus they were diluted 0.137 Methodology B: Absorbance Spectra. Absorbance with distilled water. The UV-VIS spectra of diluted Camellia spectra of Camelia products in 250-450 nm region were products are presented on FIGS. 2 and 3. obtained using UV-1000S Transmittance Analyzer (Lab 0142. The spectra of all liquid samples have certain simi sphere, Inc., North Sutton, N.H.) and Vitro-Skin R testing larities. For example, positions of peaks are varied in rela substrate (IMS Testing Group, Milford, Conn.), which mim tively narrow ranges of w=269-274 nm and W-205 ics the Surface properties of human skin. It contains both 208 nm, which indicate the presence of aromatic rings and optimized protein and lipid components and is designed to conjugated systems of O-TC bonds in all tested samples. How have topography, pH, critical Surface tension and ionic ever, the apex value of peaks, area under each peak and total strength similar to human skin. area under integral spectral curves are different (Table 11), 0.138. The Camellia samples were uniformly spread on a which Suggest that tested Samples have different composi Surface of pre-hydrated Substrate (application dose-2.0 ul/sq. tions of optically active constituents.
TABLE 11
Parameters of UV/VIS Spectra of Cameliia Products
Area under Peak # 1 Peak # 2 the Spectra
Start, v.1, End, Height, Start, W2, End, Height, Curve, ill ill ill Abs Area 96 mm ill ill Abs Area 96 Abs' nm
White Tea 450 269 2SO 0.114 28.1 250 205 200 O.816 71.9 6.604 Extract Black Tea 450 272 2SO 0.094 37.3 250 205 200 O452 62.7 4.616 Extract Cell Walls 450 272 248 0.134 35.8 248 205 200 O.614 642 5.727 Fraction Extract Membrane 450 274 251 O.944 16.2 251 208 2OO 11.98 83.8 80.133 Fraction Extract Cell Juice 450 271 249 O.692 26.3 249 205 200 S.228 73.7 39.599 Serum
*The Area under Spectra values were normalized based on dilution of the samples. cm). After 15 min after application the initial absorbance 0143. The comparison of areas under integral spectral spectra were taken via five (5) replications. Then the sub curves obtained from 200 nm to 450 nm clearly demonstrates strates with applied products were irradiated by broad-spec that membrane fraction extract (composition B) and cell juice trum solar light simulator (Model 16S-300 Single Port, Solar serum (composition D) had the higher absorption values Light Company, Inc., Philadelphia, Pa.) equipped with 300 (Table 11). The ratio Area under Spectra: Dry Matter indi Watt xenon lamp. The dose control system PMA 2100-DCS cates that specific absorption value of the samples is increas allowed precision control of the dose delivered to a sample. ing in the following sequence: black tea extract>white tea 0139 Immediately after irradiation (irradiation dose-60 extracted cell walls fraction extract>cell juice Joules/sq. cm) absorbance spectra from the same spot were serumdmembrane fraction extract (Table 12). US 2009/O 185990 A1 Jul. 23, 2009
TABLE 12 Selected Spectral Characteristics of Camelia Products Area under Ratio: Area under Ratio: Dry Spectra, Areaunder Spectra'''''" Spectra''''" Area under Spectra''''" Matter 96 Abs mm Dry Matter Abs mm Dry Matter White Tea Extract 1.10 6.604 6.004 O812 O.738 Black Tea Extract 1.38 4.616 3.345 O.854 O.619 Cell Walls Fraction O.84 5.727 6.818 0.776 O.924 Extract Membrane Fraction 6.83 80.133 11.733 4.304 O.630 Extract Cell Juice Serum S.69 39.599 6.959 4.32O 0.759
0144. Based on the comparison of absorption values, strate (IMS Testing Group, Milford, Conn.) (Table 13). As novel bioactive compositions appear to be more effective control experiments show, the lower pH level (-5.5) on Vitro protectors of the skin against Sun damage than extracts of Sin(R) surface can not be responsible for the above differences, conventional white tea and black tea. It should be pointed out, that UV protection properties of Camelia products should be which probably are the results of chemical interactions betterestimated using absorption data related to the area from between Camelia products and ingredients used for prepa 290 nm to 400 nm because this particular part of spectra is ration of Vitro-Skin R) testing substrate (IMS Testing Group, responsible for UV induced damage of the skin (Sayre et al., Milford, Conn.) Substrate. Thus, Vitro-Skin R) testing sub “A Method for the Determination of UVA Protection for strate (IMS Testing Group, Milford, Conn.) contains both Normal Skin.” Journal of American Academy of Dermatology protein and lipid components which could chemically inter 23:429-40 (1990), which is hereby incorporated by reference act with Camellia phenolic constituents. It should be noted, in its entirety). Although absorption of tested liquid samples that the shifts in spectral properties of tested products were in the area 290 (400 nm contributes only ~10% of total UV/VIS absorption, novel Camellia compositions have observed for all bioactive compositions as well as for extracts higher absorption in the above spectral area as well. of conventional teas. 0145 Thus the data related to the diluted solutions of 0148 Spectra of novel Camellia compositions and Camellia samples provided initial estimation of UV protec extracts of conventional Camellia products “as is were also tion potency of tested products, which was further evaluated compared, taking into account the different dry matter con using the Vitro-Skin R) testing substrate (IMS Testing Group, tents of tested samples. The absorbance spectra of Camellia Milford, Conn.). The results are presented in FIGS. 4-13. It products applied on Vitro-Skin R) testing substrate (IMS Test was found that novel Camellia compositions and extracts of ing Group, Milford, Conn.) in equal Volumes are presented on conventional white tea and black tea have different spectral FIG. 6. characteristics even after they were applied on substrate in 0149. Although there are some similarities between spec concentrations, which were equalized with respect to dry tra of cell walls fraction extract and white tea extract, the first matter level (FIGS. 4 and 5). product has higher absorbance in the area 250-280 nm and in 0146 The spectral of Camellia samples, which were near UV area. It should be noted that higher absorbance does applied on Vitro-Skin R testing substrate (IMS Testing Group, not correspond with dry matter level, which is higher in white Milford, Conn.) included from four to two characteristics tea extract (1.10%) compared with cell walls fraction extract peaks, which have different apex values (Table 13). (0.84%). It suggests that the compositions of these two
TABLE 13 Parameters of Absorbance Spectra of Cameliia Products Applied on Vitro-Skin (B Testing Substrate Peak # 1 Peak # 2 Peak # 3 Peak #4 Height, Height, Height, Height, WaA Abs was Abs was 2 Abs wall Abs White Tea Extract 26O O.389 286 0.461 330 0.163 392 O.O76 Black Tea Extract 26O O.399 286 0.373 330 0.17S 390 O. 105 Cell Wall Fraction 26O O.SSS 286 O.S69 330 0.2O3 389 0.118 Extract Membrane Fraction 26O O.394 286 0.549 394 O.O59 Extract Cell Juice Serum 260 0.431 286 O.S.17
0147. It should be pointed out, that parameters of charac samples are not identical and that cell walls fraction extract teristic peaks of Camellia products in solutions (Table 11) also has lower conductivity consisting of greater non-disso were very different compared with characteristic of Camelia ciated optically active ingredients responsible for high absor products, which were applied on Vitro-Skin R testing sub bance (see data presented in Table 3). US 2009/O 185990 A1 Jul. 23, 2009
0150. Additionally it was found that the spectra of mem 0156. It should be pointed out that above differences brane fraction extract (composition B) and cell juice serum between spectra may be the results of chemical interaction (composition D) are different from spectra of cell walls frac between novel Camellia compositions and surface of Vitro tion extract (composition A) and white tea extract. Thus, Skin R testing substrate (IMS Testing Group, Milford, Conn.) membrane fraction extract and cell juice serum spectra again having protein and lipid components, which mimic human indicate that these products have different composition than skin. These interactions led to the drastic increase of the white tea extract and cell walls fraction extract. At the same absorbance in the spectral region responsible for damaging effect of UV irradiation, i.e., novel Camelia compositions time, the spectral data Suggest that the compositions of mem have significant UV protection potencies. Control experi brane fraction extract and cell juice serum are not identical. ments with Barley (Hordeum vulgare) (FIG. 8A) and Sage For example, membrane fraction extract has three character (Salvia officinalis) (FIG. 8B) cell juice serums shown that the istic peaks at 260 nm, 286 nm and 394 nm. The cell juice differences in the spectra of the same samples in solution and serum spectra contain two peaks at 260 mm and 286 mm. after their application on Vitro-Skin R testing substrate (IMS 0151 Comparison of these two spectra indicates that Testing Group, Milford, Conn.) were not observed for plant membrane fraction extract has approximately two times sources other than Camellia. Thus, the spectral shift in higher extinction then cell juice serum, although difference in Camellia products after application on Vitro-Skin R) testing dry matter levels is only ~1%. Thus, four tested Camellia substrate (IMS Testing Group, Milford, Conn.) indicates the products have significantly different compositions of con specific interactions having place only between novel Camel stituents, which are optically active in the region 250-450 nm. lia products and Substrate which mimics human skin. 0152 The data related to the quantitative comparison of 0157 Control experiments with pre-hydrated substrate Camellia products are presented in Table 14. demonstrated that after irradiation the absorbance of Vitro
TABLE 1.4 Selected Characteristics of Cameliia Products Applied of Vitro Skin & Testing Substrate Area under Area under Area under Spectra(290–40Oran) Spectra 250-450 m) spectra (290-400 m) Area under Spectra (250-450 m) 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 53.8O3 60.35 Cell Juice Serum 60861 35.161 57.77
0153 Table 14 shows that absorption of the samples in the Skin R testing substrate (IMS Testing Group, Milford, Conn.) area 250-450 nm and in the area 290-400 nm is increasing in was significantly decreased especially in the range 260-330 the following order: white tea extract>cell walls fraction nm (FIG.9). This effect reflects relatively low photo-stability extract>cell juice serumamembrane fraction extract. This of non-protected substrate, which was irradiated by high dose sequence is completely in agreement with the sequence of of broad-spectrum Solar light. 0158 Irradiation of the substrate with applied white tea specific absorption values of Camelia products tested in the extract initiated changes in absorbance spectra (FIG. 10) diluted solutions (Table 12). The contribution of the absor which is analogous with the spectral changes of non-pro bance in the 250-400 nm region to the absorbance of the tected Vitro-Skin R testing substrate (IMS Testing Group, spectra taken from 250 nm to 450 nm reached ~55-60% when Milford, Conn.). When contribution of substrate was elimi tested samples were applied on Vitro-Skin R testing substrate nated, the spectra of non-irradiated and irradiated sample (IMS Testing Group, Milford, Conn.). indicated some similarities but did not demonstrate totally identical behavior. For example, the absorbance in the range 0154 It should be noted that the spectra of novel Camellia 290-310 nm was decreased and wide peak at 360 nm started compositions in diluted solutions and after their application to form. on Vitro-Skin R) testing substrate (IMS Testing Group, Mil 0159. The irradiation of cell walls fraction extract (FIG. ford, Conn.) Substrate are remarkably different. These differ 11) led to similar changes in the absorbance spectra espe ences are both quantitative and qualitative for membrane cially with respect to the curve obtained after elimination fraction extract and cell juice serum. Thus in the range from (subtraction) of substrate contribution. 250 nm to 450 nm membrane fraction extract in solution 0160 Although compositions of white tea extract and cell peaks at 274 nm. The same membrane fraction extract when walls fraction extract are not identical, the pattern of irradia applied on Vitro-Skin R testing substrate (IMS Testing Group, tion-induced modifications in their spectra is quite similar. It Milford, Conn.), did not show any characteristic peak at the should be noted that both white tea extract and cell walls above wavelength, but instead had two characteristic peaks at fraction extract are not capable of fully protecting the sub 260 nm and 286 nm (FIG. 7A). strate against destructive action of irradiation and as result, 0155 The similar pattern in spectral properties was the absorbance of Vitro-Skin R) testing substrate (IMS Testing observed for cell juice serum, although additional absorption Group, Milford, Conn.) is decreased almost as much as at the at ~360 nm was identified in the sample applied on Vitro condition when this substrate was not protected at all (FIG.9). Skin R testing substrate (IMS Testing Group, Milford, It is especially obvious in the range 250 nm-330 nm although Conn.), but such phenomena was not registered in UV/VIS at longer wavelengths some increase in the absorbance is spectra of the same composition in the solution (FIG. 7B). observed. US 2009/O 185990 A1 Jul. 23, 2009
0161 The irradiation of membrane fraction extract pro pare these with activities of the best product obtained by duced very different effect on its absorbance spectra (FIG. conventional (traditional) tea technology—white tea extract, 12). For example, irradiation did not initiate any changes in which was explored as a positive control and compared with spectral range 250-285 nm. As discussed, this particular the following bioactive compositions of the present inven range of spectra was very significantly impacted by the tion: (1) cell walls fraction extract of fresh leaves (composi destruction of irradiated Vitro-Skin R) testing substrate (IMS tion A, as referenced herein); (2) membrane fraction extract Testing Group, Milford, Conn.) and therefore comparison of (composition B, as referenced herein); and (3) cell juice the spectra Suggest that the destruction of the Substrate was serum (composition D, as referenced herein). completely prevented by the presence of membrane fraction (0168 The tests were conducted to evaluate the effect of extract on its surface. these Camelia bioactive compositions on growth patterns of 0162. However, certain changes in membrane fraction three human cell lines: a myeloid line with characteristics of extract spectra were registered. For example, the absorbance monocytic leukemia cells (Mono Mac 6) and two breast can was slightly decreased in the range 290-320 nm and it was cer lines with characteristics of early stages of the malignancy accompanied with Small increase of absorbance at longer in vivo (MCF-7) and a more highly invasive, metastatic and wavelengths. It should be especially pointed out, that mem estrogen insensitive line with characteristics of advanced can brane fraction extract was proven to be very effective in the cer (MDA-MB-435S). range of spectra where nucleic acids and aromatic amino (0169. It was found that conventional white tea extract acids have characteristic peaks at 260 nm and 280 nm subse demonstrated a certain inhibitory effect on metabolic activity quently. Thus, effect of membrane fraction extract allows the of some tumor cells. However, the extent of such inhibition use of this product as a prospective UV protective ingredient was not significant for all types of tested cells and even when for topical applications. Such inhibition was detected, it was generally not complete 0163. After irradiation the serum fraction was demonstrat but rather minimal or modest. The cell walls fraction extract ing certain changes in its absorbance spectra (FIG. 13). demonstrated properties similar to properties to those of 0164 Generally, these changes can be described as slight white tea extract. decrease of the absorption in the range 250-340 nm and slight 0170 It is noteworthy that both of the cell juice deriva increase of the absorption in the range 340-450 nm. It should tives: membrane fraction extract and cell juice serum, were be noted, that elimination of the possible contribution pro much more potent inhibitors of metabolic functions of all vided by photo-destruction of Vitro-Skin R testing substrate tested cell lines which were cultured in the presence and (IMS Testing Group, Milford, Conn.) (see red curve on spec absence of different stimuli. For example, membrane fraction tra above the initial spectra of non-irradiated serum fraction) extract clearly demonstrated greater inhibition potency and clearly indicated that substrate was effectively preserved by its effect could be reliably measured at a dose of 0.001%. The the application of serum fraction on its surface. cell juice serum demonstrated a complex response: Stimula 0165. Observations and Conclusions. The above results tion at a lower dose and inhibition at a high dose. clearly show that the best of conventional Camellia prod 0171 It should be noted that rather than inducing necrotic ucts—white tea extract provides relatively weak protection cytolysis, membrane fraction extract and cell juice serum against the destructive action of UV irradiation. The cell walls appear to initiate a pathway of programmed, or apoptotic cell fraction extract demonstrated properties similar to white tea death in the tumor cells. The experimental data indicate that extract, but membrane fraction extract and cell juice serum this pathway is attributed to loss of mitochondrial function have much more potent UV protective properties. The UV and may require 24 to 48 hours of exposure to be detected. protection properties of Camellia products were found to be 0172. As a consequence of exposure to bioactive compo increasing in the following sequence: white tea extract cell sitions, the metabolic function of all tested tumor cell lines: walls fraction extract>cell juice serumamembrane fraction MCF-7, a model of early stage human breast cancer, MDA eXtract. MB-435S, a model of advanced breast cancer, and Mono Mac 0166 It should be noted, that spectral properties of Camel 6, a model of monocytic leukemia, was inhibited, most effec lia products and pattern of the changes of these properties tively by the membrane fraction extract and, less potently and after UV irradiation provide strong evidence that composi more selectively, by the cell juice serum. Remarkably, the tions of constituents in white tea extract (control), cell walls white tea extract and cell walls fraction extract were proven to fraction extract, membrane fraction extract and cell juice be inactive or much less potent than the above compositions serum all differ and display unique activities. This is of par B and D. This trend was clearly proven for the cells tested ticular interest in the case of novel Camelia compositions under different conditions: MCF-7 cells in the absence and in where the UV activity described above cannot be attributed to the presence of transforming growth factor, MDA-MB-435S polyphenols as it is with white tea extracts. cells and both stimulated and non-stimulated monocytic Example 10 Mono Mac 6 cells. 0173 These results provide strong evidence of the ability Comparative Evaluation of Camelia Products of the method of the present invention to drastically increase the potency of Camellia plants and produce very impressive An Overview novel products demonstrating activities, which were not iden 0167 Examples 10 through 19 describe methods, results, tified for even the best product of conventional tea technol and analyses relating to experiments conducted to evaluate Ogy. the range of biological activities related to the modulation of 0.174 Effects of the Camellia fractions of the present cell functions by the bioactive compositions from Camellia invention on cell-mediated proteolytic activities have impli Sinensis of the present invention. The primary objective was cations for inflammatory tissue injury as well as tumor inva to evaluate the range of biological activities of products sion and metastasis. Thus, breast cancer cells and leukemia obtained from the methods of the present invention and com cells clearly can be suggested as prospective targets for the US 2009/O 185990 A1 Jul. 23, 2009 bioactive compositions of the present invention, most nota proven to be much more potent than conventional products bly, the membrane fraction extract. It should be noted that it isolated from the same dried plants using a number of param was previously shown that the colon carcinoma-derived cell eters as was previously described in the U.S. Patent Applica line COLO 205 releases significant levels of MMP-2, which tion Publication No. 2003/0175235, which is hereby incor is then activated by a trypsin-like enzyme also secreted by the porated by reference in its entirety). For example, in other cells. This is also one of potential targets for the Camelia types of plants (Medicago sativa, Hordeum vulgare, Lavan fractions of the present invention, based on results with Mono dula angustifolia, Calendula officinalis and Salvia officina Mac 6 cells. lis), several impressive biological activities of compositions 0175 From these studies it has been concluded that the prepared using the method of the present invention have been present invention's bioactive compositions isolated from identified and evaluated, including high anti-elastase and fresh Camellia have activities result in impressive modulation anti-gelatinase B (MMP-9) activities, novel modulation of of key cell functions. The effects that have been observed the neutrophil respiratory burst, and significant Superoxide could have valuable applications ranging from personal care Scavenging activity towards reactive oxygen species. Other products to nutraceuticals and potentially pharmaceuticals. than Scavenging activity, these activities are not likely to be 0176 It should also be noted that the present invention's ascribed to mixtures of polyphenols alone. very potent bioactive compositions are not single purified 0185. Thus, it was especially interesting to explore a more components, but rather isolated complexes of constituents. comprehensive approach to compare the range of activities, Further fractionation of membrane fraction extract (compo which could be detected in the Camelia compositions of the sition B) and cell juice serum (composition D) could yield present invention with the activities present in an extract extremely potent ingredients for the growing market of natu obtained from the same dried plant using conventional (tra ral pharmaceuticals. ditional) Camelia technology. Accordingly, modulation of functions in living mammalian cells by the cell walls fraction Example 11 extract (composition A), membrane fraction extract (compo sition B) and cell juice serum (composition D) prepared from Comparative Evaluation of Camelia Products freshly collected leaves of Camellia have been assayed. Tested Compositions These compositions have been compared to extract of con 0177. The following bioactive compositions were used in ventional white tea prepared from dried Camellia leaves. the experiments described in Examples 10 through 19: 0186. It should be noted that, according to multiple studies (0178 (1) Positive Control: White tea extract which was of conventional Camelia products, the white tea extract dem prepared according to the procedure described in onstrated higher specific activities and therefore a preparation Examples 1 and 4. of this sort was selected as a representative positive reference 0179 (2) Composition A: A cell walls fraction extract of control for comparison with the novel Camelia products of fresh leaves of Camellia which was prepared according the present invention. to procedure described in Examples 1 and 4. Example 12 0180 (3) Composition B: A membrane fraction extract obtained from freshly processed leaves of Camellia and Comparative Evaluation of Camelia Products prepared according to procedure described in Examples 1 and 4. Rationale for Selection of Cell Lines 0181 (4) Composition D: Cell juice serum of freshly 0187. As a test system for modulation of cell functions, processed leaves of Camellia and prepared according to two human breast carcinoma-derived lines were used as mod the procedure described in Examples 1 and 4. els of neoplastic cells (MCF-7 and MDA-MB-435S), and a 0182. The above products were obtained from the same lot human monocytoid line (Mono Mac 6) was used as a model of offresh Camellia to prepare the conventional white tea extract inflammatory cells. The above cell lines are described in and three “parallel' products of the present invention (com Example 21. positions A, B and D). 0188 MCF-7 is considered a model of early or less de 0183 There are a number of reports in the literature, which differentiated breast cancer. The line still retains estrogen Suggest that extracts of Camelia leaves have a range of bio sensitivity and has a relatively low invasive phenotype; its logical activities, primarily attributed to the significant con capacity to metastasize in immunodeficient animal models is centrations of polyphenolic tannins that form during the cur quite modest. In previous studies, the MCF-7 cell line has ing process. These polyphenols, as well as lower molecular been shown to display a characteristic response to Transform weight precursors to the polymeric tannins such as cpigallo ing Growth Factor-fi (TGF-3): after culture for 24 hours in the catechin-3-O-gallate (EGCG), have been reported to display presence of TGF-B, the cells secrete increased levels of the potent antioxidant activities. There is a growing number of Matrix Metallo Proteinase (MMP) family of proteolytic publications suggesting not only antioxidant, but also anti enzymes and the pro-angiogenic factor Vascular Endothelial angiogenic, anti-bacterial, anti-neoplastic, anti-inflamma Growth Factor (VEGF), two different markers of enhanced tory, anti-mutagenic, anti-septic, and detoxifying properties invasiveness and metastatic potential. This response to of teas prepared from dried leaves of Camelia. Not all of the TGF-B is a mark of tumors and some tumor cell lines, in above properties have been proven to confer statistically sig contrast to growth arrest, which is induced in normal cells by nificant benefits. Only some of them have been confirmed in the growth factor. To evaluate the bioactive compositions of comprehensive studies using multiple testing systems. the present invention, MCF-7 cells cultured in the absence 0184 As past reference, it should be pointed out that, from and presence of TGF-B were used as targets. past experience with bioactive compositions isolated from a (0189 The MDA-MB-435S line is more highly invasive, number of fresh plant sources other than Camelia using the metastatic and estrogen insensitive. This human carcinoma present invention's technology, Such compositions were derived cell line also shows some sensitivity to TGF-3, but US 2009/O 185990 A1 Jul. 23, 2009 even in the absence of the growth factor, it spontaneously metastasis. It was also previously shown that Some agents releases higher levels of MMPs and VEGF than MCF-7, under development as anti-inflammatory and anti-tumor consistent with its use as a model of more advanced cancer. In drugs (the agents that have been investigated are known to present evaluation the effects of bioactive compositions on diminish inflammatory tissue destruction as well as invasion MDA-MB-435S cells cultured only in the absence of TGF-f and metastasis of tumor cell lines) appear to reduce the levels were examined. of the MMPs produced by cells in addition to any direct 0190. The human monocytoid line, Mono Mac 6, inhibition of MMP proteolytic activity. The objective in these expresses a number of biomarkers consistent with those of studies was to evaluate the possibility that the Camelia bio resting monocytes or macrophages, and responds like human active compositions of the present invention might have a monocytes and macrophages to pro-inflammatory activating similar capacity to diminish levels of MMPs released by stimuli such as Phorbol Myristate Acetate (PMA). The effects activated Mono Mac 6 cells. of bioactive compositions on Mono Mac 6 cells cultured in 0.197 Thus, the selected assays will allow one to reliably the absence and presence of PMA were examined, to serve as evaluate a broad spectrum of metabolic processes and effec models of resting and activated monocytes/macrophages. tively obtain important data, which might reveal mechanisms 0191 Thus, the selected combination of the cell lines of action triggered by certain Camelia bioactive composi described above provides a reliable foundation for evalua tions. tions of anti-tumor and anti-inflammatory potencies of Camellia bioactive compositions. Parallel testing of selected Example 14 cell lines with a number of functional probes provides the opportunity to draw more valuable conclusions concerning Comparative Evaluation of Camelia Products the activities of products and their mechanism of action-than Effects of Camelia Bioactive Compositions on investigation of the responses of a single test target or targets Breast Tumor Cell Lines having similar sensitivities or similar responses to certain stimuli. 0198 Throughout these studies, mitochondrial function was measured solely through assays of reduction of the tet Example 13 razolium salt MTS to its formazan. It should be noted that Some intrinsic capacity of higher concentrations of the Comparative Evaluation of Camelia Bioactive Com Camellia compositions of the present invention have been positions Rationale for Selection of Assays observed to reduce MTS directly in the absence of any viable cells, and in all the results reported here, such background 0.192 Initial evaluations were based on two viability formation of formazan in the absence of cells has been sub assays and a probe of cell functions (see Example 20, tracted from the levels of reductase activity observed in the “Method 8). presence of the cells. 0193 The first assay measures levels of the cytosolic (0199 FIGS. 14 through 21 illustrate the magnitude of the enzyme, lactic dehydrogenase, which is liberated into the reductase activity of MCF-7 cells, cultured in the absence and extracellular culture medium only when the cells lyse. Such presence of 5 ng/ml TGF-B, and MDA-MB-435S cells, cul loss of cell membrane integrity is traditionally considered to tured only in the absence of TGF-B, at 24 hours and 48 hours be a sign of necrotic cell death and reflects the cytotoxicity after the addition of various doses of each of the four Camel pattern. lia compositions, ranging from 0.01% or 0.02% (w/v, final 0194 The second assay measures mitochondrial dehydro concentration in the culture medium, based on dry weight of genase activity as reflected by the reduction of a tetrazolium the solids in the Camelia compositions) to 0.0001%. salt to its colored formazan. When the MTS reagent (a tetra Zolium salt) is applied to living cells, it is converted to an Example 15 intensely colored compound (formazan). Loss of mitochon drial dehydrogenase activity can also be associated with cell Comparative Evaluation of Camelia Bioactive Com death, but is typically a marker for the early steps in a pro positions grammed cell death, or apoptotic, pathway in which cell membrane integrity is generally retained well after the MCF-7 Cells nucleus has condensed and the mitochondria have ceased to 0200. In the absence of TGF-B, the highest tested concen function. tration (0.01%) of the composition A and white tea extract 0.195 The leakage of lactic dehydrogenase indicates com (positive control) had a marked effect on MTS reduction by plete loss of viability associated with cytolysis, while MCF-7 cells. At that concentration there was significant but decreased reduction of tetrazolium salts indicates loss of incomplete inhibition of reductase activity (-50-70% inhibi mitochondrial activity, but not necessarily irrevocable loss of tion) after 24 hours of exposure to the Camelia composition cell membrane integrity or viability. A. The similar inhibition of reductase activity was detected 0196. As an additional probe of cell functions, the effects after 24 hours of exposure to white tea extract. of the Camelia bioactive compositions on levels of protein 0201 In contrast, the two Camelia bioactive composi ases secreted by the Mono Mac 6 line have been examined tions (membrane fraction extract and cell juice serum) pre (see Example 20, “Method 9”). In previous studies with this pared from Camellia cell juice were more potent inhibitors of cell line, it was observed that, after incubation with PMA, reductase activity in MCF-7 cells in the absence of TGF-B. Mono Mac 6 cells secrete two so-called gelatinolytic matrix The membrane fraction extract (composition B) resembled metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (ge white tea extract in dose dependence, except for somewhat latinase B). These MMPs are also secreted by a number of greater potency, producing virtually complete inhibition at tumors and by their Surrounding stroma, and are implicated in 0.01%, the highest dose tested. The cell juice serum (compo inflammatory tissue injury as well as tumor invasion and sition D) also produced virtually complete inhibition at US 2009/O 185990 A1 Jul. 23, 2009
0.01%, but at the lower dose of 0.0025%, there was some reductase activity by -50% at 0.001% after 48 hours. The evidence of stimulation of reductase activity. Lower doses of white tea extract and cell walls fraction extract also had sig composition D were without significant effect. nificant inhibitory activity against MDA-MB-435S cells after (0202) When MCF-7 cells were cultured in the presence of 48 hours of exposure, which was actually greater than that of the growth factor TGF-B, their sensitivity to the Camellia the cell juice serum. No doses of any of the preparations compositions was significantly altered. After 24 hours or 48 induced activation of reductase activity in this cell line, hours of exposure to the cell walls fraction extract and white regardless of duration of exposure. tea extract, there was no evidence of either a stimulatory oran 0208. It should be noted that any impact on highly inva inhibitory effect on reductase activity at any dose, except for sive, metastatic and estrogen insensitive line MDA-MB-435S a modest inhibition of less than 20% at the highest dose is rare to observe after only 24 hours. Thus, the effect of (0.01%) of white tea extract. composition B after 24 and 48 hours is rather remarkable and 0203. In contrast, exposure of TGF-B-treated MCF-7 cells indicates that this preparation has significant activity. for 24 hours to the membrane fraction extract at a dose of 0.02% resulted in 70% inhibition of reductase activity, and Example 17 after 48 hours, reductase activity was virtually completely Comparative Evaluation of Camelia Bioactive Com abated. More modest inhibition could be detected at lower positions doses of membrane fraction extract after 24 hours, but after 48 hours, a marked activation of reductase activity was detected. Effects of Camellia Compositions on Monocytoid The same activation of reductase activity by low doses of the Cells cell juice serum as well as marked inhibition at the highest 0209 Mitochondrial Dehydrogenase Activity: Certain of dose (0.02%) was detected after 48 hours of exposure, but this the trends revealed by the preliminary studies on the breast composition had only minimal effect on reductase activity in tumor cell lines have proved to be consistent with the TGF-B-treated MCF-7 cells after the more limited exposure response of Mono Mac 6 cells to the four tested Camellia of 24 hours, regardless of dose. compositions. The membrane fraction extract (composition 0204. In evaluation, there was no detection of significant B) and cell juice serum (composition D) were more potent release of lactic dehydrogenase into the culture medium of inhibitors of MTS reductase activity in this inflammatory cell MCF-7 cells exposed for 24 hours to even the highest doses of line than the cell walls fraction extract (composition A) and any of the Camelia compositions. It would appear that, the white tea extract (positive control), and the membrane frac loss of mitochondrial function in these cells is not accompa tion extract (composition B) clearly had the greatest inhibi nied by a necrotic lysis of the cells. If the cells are in fact dying tory potency. The effects on MTS reductase in cells which during the first 48 hours of exposure, it is more likely that a were left unstimulated and those which were stimulated with programmed cell death, or apoptotic, pathway has been ini 10 nM PMA were examined, and reductase activity after 24 tiated. This conclusion is Supported by light microscopic and 48 hours of exposure to the Camellia compositions was observations, which reveal that there is some rounding of the evaluated. The effects of these compositions on MTS reduc cells, but no formation of debris or membrane fragments tase activity in Mono Mac 6 cells are shown in FIGS. 22 during the course of the exposures. through 33. 0205 Thus, inhibition of mitochondrial function appears 0210. The white tea extract showed a weak but dose-de to be a predominant mode of action of all tested Camellia pendent inhibition of reductase activity after 48 hours of products, which did not demonstrate cytotoxity or necrosis as exposure to PMA-stimulated cells; there was no significant indicated by levels of released lactic dehydrogehase. loss of reductase activity regardless of the dose or length of Example 16 exposure in the absence of PMA, nor was there any effect of any dose after 24 hours of exposure to PMA-treated cells. The Comparative Evaluation of Camelia Bioactive Com cell walls fraction extract had no effect on Mono Mac 6 cells positions regardless of dose or time of exposure and regardless of MDA-MB-435S Cells whether the cells were unstimulated or stimulated with PMA. 0211. The cell juice serum of fresh Camellia leaves inhib 0206. The pattern of response of MDA-MB-435S cells to ited unstimulated Mono Mac 6 cell reductase activity mod the Camellia compositions was similar to that of MCF-7 cells estly in a dose dependent fashion after 24 or 48 hours of in that the most potent compositions were composition Band exposure. The maximum inhibition at the highest dose of D, with the membrane fraction extract (composition B) show 0.01% (w/v. final concentration in the culture medium, based ing somewhat greater potency than the cell juice serum (com on dry weight of Solids in the starting preparation) was only position D). Only the membrane fraction extract produced --20-30% of the control activity. Inhibition of PMA-stimu marked inhibition of reductase activity after only 24 hours of lated cells reached 50% of control activity but only at the exposure. Inhibition reached -70% of control reductase val highest dose of composition D (0.01%), and only after 48 ues at the highest dose of 0.01%, but a modest inhibition of hours of exposure. -10% could be reliably detected at even the lowest dose of 0212. The membrane fraction extract of freshly harvested remarkable concentration -0.0001%. The other tested com Camellia (composition B) proved to be the most potent of the positions had only modest inhibitory effects at 24 hours of tested preparations in inhibiting Mono Mac 6 cell reductase exposure, and only at the higher doses tested. activity as it had toward the breast tumor cell lines. Effects of 0207. After 48 hours of exposure, reductase activity was PMA stimulation or duration of exposure to the composition inhibited in a dose-dependent fashion in the presence of each had little effect on inhibition, which was dose-dependent in of the compositions, but the potency at the highest dose of the the presence or absence of PMA and was roughly the same compositions did not reach near 100% inhibition, except for after 24 hour or 48 hour exposure. Reductase activity was the membrane fraction extract. This composition inhibited inhibited by -70% in the presence of PMA and by -80-90% US 2009/O 185990 A1 Jul. 23, 2009 20 in the absence of PMA at the highest dose of 0.02%, but lower measurements. In this method, the culture media are first levels of inhibition (-15%) could be reliably measured at a Subjected to electrophoresis in gelatin-impregnated polyacry dose of 0.001%. Measurements of release of cytosolic lamide gels in the presence of Sodium Dodecyl Sulfate (SDS enzymes have not been undertaken to confirm that the loss of PAGE) to separate the proteins on the basis of molecular reductase activity is not associated with necrotic cytolysis, weight. The SDS is then washed out of the gels to allow at but no evidence of membrane fragmentation could be seen by least a portion of any enzymes present to renature and the gels light microscopic examination of Mono Mac 6 cells exposed are incubated in a medium, which maximizes MMP activity. to any of the bioactive compositions at 0.02% for 48 hours. MMPs dissolve the gelatin wherever they may be present. Moreover, as shown below, the cells appear still capable of After visualizing the undigested gelatin in the bulk of the gels secreting at least one MMP under conditions in which reduc with a protein stain, the gels are scanned, with the MMPs tion of MTS is markedly diminished. appearing as clear Zones against the stained background. 0213. These results suggest that in these cells, as well as Negative images have been presented here, so that the MMPs the breast tumor cell lines, the loss of reductase activity is appear as dark Zones against a light background. associated with a relatively selective loss of mitochondrial 0219. It should be noted that MMPs are secreted by most function and can reflect initiation of a pathway of pro cells as inactive precursors, which are then activated extra grammed cell death or apoptosis. cellularly. However, because of the denaturing and renaturing 0214 Secretion of MMPs. Two different assays have been sequence employed in Zymography, even the so-called inac used to measure the levels of two gelatinases, MMP-2 (gelati tive pro-forms of the MMPs acquire gelatinolytic activity and nase A) and MMP-9 (gelatinase B), released by Mono Mac 6 produce clear Zones. FIGS. 34 through 37 illustrate the nega cells. These MMPs have been implicated in inflammatory tive images of gelatin Zymograms of culture media collected tissue damage as well as tumor invasion and metastasis. after 48 hour exposure of Mono Mac 6 cells to the different Employed enzyme-linked immunosorbent assays (ELISAS) Camellia bioactive compositions, along with culture medium for MMP-2 and MMP-9 were first used to estimate total levels collected from cells cultured in the absence (U) or presence of the two enzymes in the culture medium of Mono Mac 6 (S) of 10 nM PMA but in the absence of Camelia composi cells cultured for 48 hours in the presence of 10 nM PMA and tions. different doses of the three Camellia bioactive compositions 0220. The effects of composition A and positive control and positive control. were evaluated only for the lowest dose (0.0001%, “lo') and 0215. This cell line secretes only MMP-2 when it is the highest dose (0.01%, “hi') of the preparations, whereas unstimulated, but secretes both MMP-2 and MMP-9 when it the effects of compositions B and D were also evaluated at the is activated. (Levels of MMPs released after 24 hours are intermediate dose of 0.001% (“med'). It is apparent from the usually too low to be reliably detected). four panels that Mono Mac 6 cells release only MMP-2 (-67 0216. The results of the ELISA measurements are shown kD) in the absence of PMA, and this enzyme is found pre in FIGS.34 through 37. As was observed for the effects of cell dominantly in the pro-form. Treatment with 10 nM PMA walls fraction extract and white tea extract on MTS reduction results in induction of MMP-9 secretion (-92 kD), as well as by Mono Mac 6 cells, there was no significant change in further proteolytic activities which convert significant levels levels of secreted MMP-2 or MMP-9 at any dose of these of the pro-forms of the two MMPs to their slightly lower extracts. At the highest dose (0.01% w/v) of the membrane molecular weight active forms. fraction extract and cell juice serum, the levels of MMP-2 0221 Consistent with the ELISA results, exposure of were observed to be diminished, with the membrane fraction PMA-stimulated Mono Mac 6 cells to cell walls fraction extract exhibiting the greatest potency at this dose. It should extract and white tea extract had no detectable effect on the be noted, that an apparent slight stimulation of MMP-2 levels of either the pro- or active forms of either of the two release was observed at the next highest doses of composition MMPs visualized by gelatin Zymography. In contrast, expo B (0.001%) and composition D (0.002%). This stimulation is sure to the highest dose of compositions B and D resulted in reminiscent of the stimulation of MTS reductase activity in marked diminution of the levels of MMP-2 visualized by TGF-B treated MCF-7 cells at similar doses of these compo gelatin Zymography, but no apparent change in the levels of sitions. MMP-9. 0217. The dose-dependent diminution of MMP-2 levels 0222. The appearance of both pro- and active forms of detected by ELISA was not paralleled by the effects of mem MMP-9, as well as the faint, but recognizable, band corre brane fraction extract and cell juice serum on MMP-9 levels. sponding to the active form of MMP-2 seen in the media These levels were increased (apparently markedly so by cell collected from cells treated with the highest dose of compo juice serum) at the highest doses, but were unchanged at the sitions B and D, Suggests that the effects of these composi lower doses tested. The detection of unchanged or increased tions are primarily on modulation of release of MMP-2 and do levels of MMP-9 secreted by Mono Mac 6 cells exposed for not involve additional effects on the MMP activation mecha 48 hours to doses of Camellia preparations which produced nisms in these cultured cells. significant inhibition of MTS reductase activity after only 24 Example 18 hours, is further evidence that the loss of mitochondrial func tion in Mono Mac 6 cells exposed to the membrane fraction Comparative Evaluation of Camelia Bioactive Com extract or cell juice serum of Camellia does not reflect positions necrotic cytolysis, in which case MMP secretion would have abruptly ceased. Summary of Results 0218. As further evidence of the effects of the Camelia 0223) The experimental data indicate that Camellia bioac compositions on MMP secretion by Mono Mac 6 cells, the tive compositions trigger a dose-dependent loss of MTS technique of gelatin Zymography was used to examine the reductase activity, which is generally attributed to loss of culture media collected as described above for the ELISA mitochondrial function. This inhibition may require as long US 2009/O 185990 A1 Jul. 23, 2009 21 as 48 hours of exposure to be detected and at least for the first sure of these inflammatory cells to the membrane fraction 24 hours, there is no measurable release of cytosolic enzymes, extract and cell juice serum results in selective diminution in Suggesting that rather than inducing necrotic cytolysis, the the levels of the gelatinolytic enzyme, MMP-2 (gelatinaseA). bioactive compositions initiate a pathway of programmed, or The gelatin Zymography indicates that mechanisms of “pro apoptotic, cell death in the tumor cells. form' or zymogen activation are unaffected by the Camellia 0224. The differences in the time- and dose-dependence of bioactive compositions, so it is highly unlikely that the dimin the response of MCF-7 cells and MDA-MB-435S cells, and the effects of TGF-B treatment of the MCF-7 cells, all point to ished levels MMP-2 in the medium reflect enhanced pro a somewhat increased resistance of the more invasive and teolytic destruction. metastatic phenotypes to white tea extract, cell walls fraction 0227 Thus, the metabolic activity of all tested cell lines extract, and to Some degree, cell juice serum, as evidenced by (i.e., a model of early stage human breast cancer, a model of the relatively modest loss of reductase activity within the first advanced breast cancer cells, and a model of monocytic leu 24 hours of exposure. However, the trend of greater potency kemia) was effectively inhibited by the membrane fraction of the membrane fraction extract is evidenced by its capacity extract (composition B) and, in most of cases, the cell juice to inhibit MTS reductase activity in TGF-B-treated MCF-7 serum (composition D). Remarkably, the extract of cell walls cells, as well as MDA-MB-435S cells within 24 hours. tea (Composition A) and white tea extract (positive control) 0225. The effects of tested Camelia bioactive composi were proven to be inactive or much less potent than the above tions on the Mono Mac 6 cell line, a model of human mono cytes/macrophages, have certain similarities to the effects compositions B and D. observed on breast tumor cell lines. Based on the absence of 0228. This trend was clearly proven for all tested MCF-7 lactic dehydrogenase in the culture medium of the breast human cancer cells in the absence and in the presence of tumor cell lines and the presence of normal to increased levels transforming growth factor, for MDA-MB-435S advanced of Secreted MMP-9 in the culture medium of Mono Mac 6 human breast cancer cells, and for stimulated and non-stimu cells, it has been concluded that these compositions do not lated monocytoid Mono Mac 6 cells. The data related to the induce necrotic cytolysis, even at the highest dose tested Summary of testing and evaluation of bioactive Camelia (0.01% w/v). compositions are presented in Table 15.
TABLE 1.5 Summary of Testing and Evaluation of Bioactive Camelia 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-43SS 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 Inhibition Inhibition Early Breast Cancer TGF-B 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 48 hours No Effect No Effect Significant but Pronounced incomplete inhibition inhibition Inflammation 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
0226. However, the two bioactive compositions (mem 0229. Table 15 shows that abilities of Camelia prepara brane fraction extract and cell juice serum) induce a dose tion to modulate cell functions in a dose-dependent manner is dependent inhibition of mitochondrial reductase activity, increasing in the following order: white tea extract=cell walls which reflect initiation of an apoptotic pathway of pro fraction extract>cell juice serumamembrane fraction extract. grammed cell death in Mono Mac 6 cells. Furthermore, expo The experimental data Suggests that, novel bioactive Camel US 2009/O 185990 A1 Jul. 23, 2009 22 lia compositions prepared by processing of fresh plant tissue viewing window facing upward was used. The container with into cell juice derived membrane fraction extract (composi tested bioactive composition was placed on viewing window tion B) and cell juice serum (composition D) do not trigger and measured through the bottom. The following CIELAB any outright necrotic toxicity towards the cells. equations were used: 0230. Therefore, the technology of the present invention displays the ability to drastically increase the potency of Camellia bioactive compositions and to produce very impres sive novel products demonstrating activities on viable human cells which were not demonstrable in the best products (for example, white tea extract) produced by conventional Camel 0237 Method 4: Method for Determination of Total Caro lia technology. tenoids Content and Lutein Content. The tested bioactive Example 19 compositions were extracted with acetone. After homogeni Zation and vacuum filtration, all extracts were Saponified with Comparative Evaluation of Camelia Bioactive Com 30% potassium hydroxide in methanol. The carotenoids were positions Successively extracted from bioactive compositions with petroleum ether. After additional treatment and re-solubiliza Implications for Future Studies tion in ethanol, all samples were measured at 446 nm. 0231. Effects of the Camellia bioactive compositions of 0238. In order to determine the lutein content, an addi the present invention on cell-mediated proteolytic activities tional dried sample from each sample extraction was used for have implications for inflammatory tissue injury as well as high performance liquid chromatography (“HPLC) analy tumor invasion and metastasis. Thus, breast cancer cells and sis. The dried sample was re-solubilized in MTBE and metha monocytic leukemia cells clearly can be suggested as pro nol. The reverse phase HPLC system with (250x4.60 mm spective targets for the Camelia bioactive compositions of I.D.) 5 um Cs column (“Vydac') was used. The identity of the present invention, most notably, composition B (mem lutein was conformed by the co-chromatography of an brane fraction extract). It was previously shown that the colon authentic standard. The molar absorptivity coefficient for carcinoma-derived cell line COLO 205 releases significant lutein in ethanol is 144,800 cm mol'. levels of MMP-2, which is then activated by a trypsin-like 0239 Method 5: Method for Determination of Elastase enzyme also secreted by the cells. This type of tumor cell is Inhibitory Activity. The elastase inhibitory activity of tested one of a number of potential targets for the Camelia bioactive bioactive compositions was determined using the assay, compositions of the present invention, based on results with which employs neutrophil elastase (a purified enzyme prepa Mono Mac 6 cells. ration produced by "Elastin Products”) and synthetic peptide 0232 From these studies, one can be confident that the soluble substrate Methoxysuccinyl-Ala-Ala-Pro-Val-p-Ni bioactive compositions isolated from fresh Camellia of the troanilide produced by “Sigma'. Enzymatic cleavage of the present invention have significant activities, which result in Substrate results in generation of increasingyellow color over impressive modulation of key cell functions. The effects that time (405 nm); the rate of color generation is diminished by have been observed have valuable applications ranging from increasing concentrations of tested bioactive compositions personal care products to nutraceuticals and potentially phar containing inhibitory activity. Analysis of the concentration maceuticals. dependence of inhibition permits quantitation of the potency of the inhibitory activity, expressed as that concentration of Example 20 dry matter within each tested bioactive required to achieve Protocols Used for Determining Certain Characteris 50% inhibition (ICs), but also provides information relating tics of Bioactive Compositions to the mode of inhibition. 0240 For the determination of ICs, the concentration of 0233. The following are various methods used for deter elastase was 2.5ug/mland concentration of substrate was 150 mining certain characteristics of Bioactive Compositions. uM. For the determination of K, the concentrations of sub These methods are referenced throughout the above strate were 100 uM and 200 uM. Examples. References below to the “tested products” or the 0241 Method 6: Method for Determination of Gelatinase “test samples’ refer to Bioactive Compositions. B (MMP-9) Inhibitory Activity. The commercially distrib 0234 Method 1: Method for Determination of Solid Con uted assay (MMP-9 Activity ELISA produced by Amersham tent. The procedure for determination of solid content Pharmacia'), which captures Gelatinase B specifically onto included evaporation of the tested bioactive composition in multiwell microplates by immune recognition, was used after the water bath at 100° C. until complete evaporation of water, other proteinases were washed away. The enzymatic activity oven storage of the sample at 105° C. for 3 hours, cooling to was detected at 405 nm by hydrolysis of a low molecular room temperature, and immediate determination of the weight synthetic substrate for Gelatinase B: APMA. Analysis weight of the container with solid matter. of the concentration dependence of inhibition was used to 0235 Method 2: Method for Determination of Non-Vola determine the potency of tested bioactive composition dry tile Residue. The procedure for determination of non-volatile matter. residue included oven storage of the tested bioactive compo 0242 Method 7: Method for Determination of Superoxide sition at 105° C. for 5 hours, cooling, and immediate deter Scavenging Activity. The enzymatic system, which uses Xan mination of the weight of the container with solid matter. thine oxidase (a purified enzyme preparation produced by 0236 Method 3: Method for Determination of L*a*b* “Sigma'), was used to generate Superoxide anions in high Values. The procedure for determination of L*a*b* values yield and in a controlled fashion. The conversion of xanthine utilized Hunter Labscan fixed geometry colorimeter with to hydroxanthine by this enzyme generates amounts of super measuring geometry of 0/45°. Standard illuminant Des with oxide anions and reduction of ferricytochrome c to ferrocy US 2009/O 185990 A1 Jul. 23, 2009
tochrome c was used as a sensitive measure of Superoxide (Ziegler-Heitbrock et al., “Establishement of a Human Cell levels. The measurements of ferrocytochrome c level (550 Line (Mono Mac 6) with Characteristics of Mature Mono nm), when tested bioactive compositions were added to the cytes.” International Journal of Cancer 41:456-461 (1988), reaction system, allow for determination of their Superoxide which is hereby incorporated by reference in its entirety). Scavenging activity. The final concentrations per well were Cells were maintained in RPMI 1640, supplemented with 2 for cytochrome c 75 uM, xanthine 425 um/L, and Xanthine mM L-glutamine, 100U/ml penicillin, 100 lug/ml streptomy oxidase 10 mU/ml. cin, 1 mM sodium pyruvate, 10% FCS, nonessential amino 0243 Method 8: Method for Determination of In Vitro acids, 9 g/ml insulin, and 1 mM oxalacetic acid. For assay Toxicity and Apoptosis. CellTiter 96 AQ, One Solution conditions, 0.2% glucose was also added. Cell Proliferation Assay and CytoTox 96 Non-radioactive Cytotoxicity Assay and Subsequent protocols were explored Example 22 (both assays produced by Promega Corporation, Madison, Wis.). Catechin Analyses of the Camelia Products 0244. The first assay is a colorimetric method for deter mining the number of viable cells which explores a tetrazo (0250. The cell walls fraction extract, the membrane frac lium compound (3-(4,5-dimethylthiaazol-2-yl)-5-(3-car tion extract, and the cell juice serum of the present invention boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, were analyzed for content of various catechins. A white tea inner salt; MTS and a electron coupling reagent (phenazine sample was used as a control. The following catechins were methosulfate; PMS). MTS is bioreduced by cells into a assayed: (-)-epigallocatechin; (+)-catechin, (-)-epicatechin; soluble in tissue culture medium formazan product that has an (-)-epigallocatechin gallate: (-)-gallocatechin gallate; and absorbance maximum at 490 nm. The conversion of MTS into (-)-epicatechin gallate. aqueous, soluble formazan is accomplished by dehydroge nase enzymes found in metabolically active cells and the 0251. The samples were extracted using 0.1% HPO and quantity of formazan product is directly proportional to the sonication for about 15 minutes. After centrifugation, the number of living cells in culture. extract was injected on HPLC. C-18 reverse phase column 0245. The second assay quantitatively measures lactate was used as the stationary phase. 0.1% phosphoric acid and dehydrogenase (LDH), a stable cytosolic enzyme that is acetonitrile were used as the mobile phases. The detection released upon cell lysis. Released LDH in cell culture super was at 280 nm. The calculation is based on comparing areas of natant is measured with a 30-minute coupled enzymatic each catechin listed with its pure standard. The results are assay, which results in the conversation of a tetrazolium salt shown in FIG.38 and Table 16 (below). (INT) into a red formazan product. The amount of color formed is proportional to the number of lysed cells. TABLE 16 0246 Method 9: Method for Determination of Level of Catechin Content of Bioactive Camelia Compositions Enzymes Secreted by Stimulated Cells. After incubation with Average PMA, Mono Mac 6 cells secrete two gelatinolytic matrix Average mg/g metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (ge mgig based based on latinase B). The levels of these enzymes in the presence of on 100% dry product tested bioactive compositions were determined by two-di Sample Chemical Analyzed latter as is mensional Sodium dodecyl Sulphate polyacrylamide gel elec White Tea (-)-epigallocatechin O.3 O.OO33 trophoresis. Extract (+)-catechin 1.33 O.O146 (-)-epicatechin O.15 O.OO17 Example 21 (-)-epigallocatechin gallate O.65 O.OO71 (-)-gallocatechin gallate O.OO3 OOOOO Cell Lines Used for Testing Certain Bioactive Char (-)-epicatechin gallate O.212 O.OO23 acteristics of the Camelia Products Cell Walls (-)-epigallocatechin O.OO OOOOO Fraction Extract (+)-catechin 2.39 O.O2O1 (-)-epicatechin O.O1 O.OOO1 0247. The cell line MDA-MB-435S which is considered a (-)-epigallocatechin gallate O.O1 O.OOO1 model of advanced breast cancer was obtained from Ameri (-)-gallocatechin gallate O.OO OOOOO can Type Culture Collection (ATCCNumber HTB-129). This (-)-epicatechin gallate O.OO6 O.OOO1 Membrane (-)-epigallocatechin 2.27 0.1552 cell line was cultivated at 37° C. in the following ATCC Fraction Extract (+)-catechin 8.96 O6121 medium: Leibovitz's L-15 medium with 2 mM L-glutamine (-)-epicatechin O.60 O.04.09 supplemented with 0.01 mg/ml insulin, 90%; fetal bovine (-)-epigallocatechin gallate 9.28 O.6340 serum, 10%. (-)-gallocatechin gallate O.O1 O.OOO6 (-)-epicatechin gallate 2.33 O.1589 0248. The cell line MCF-7 which is considered a model of Cell Juice (-)-epigallocatechin 3.01 O.1714 early or less de-differentiated breast cancer was obtained Serum (+)-catechin 6.09 O.3465 from ATCC (Number HTB-22) was cultivated at 37° C. in the (-)-epicatechin O.95 O.OS39 following ATCC medium: Minimum essential medium (-)-epigallocatechin gallate 1.97 O112O (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to (-)-gallocatechin gallate O.04 O.OO24 contain 1.5 g/L Sodium bicarbonate, 0.1 mM non-essential (-)-epicatechin gallate 0.57 O.O325 amino acids and 1 mM sodium pyruvate and Supplemented with 0.01 mg/ml bovine insulin, 90%; fetal bovine serum, 0252 Although preferred embodiments have been 10%. depicted and described in detail herein, it will be apparent to 0249. The cell line MonoMac6 (MM6, obtained from the those skilled in the relevant art that various modifications, German Collection of Microorganisms and Cell Cultures) additions, Substitutions, and the like can be made without which closely resembles a differentiated human monocyte departing from the spirit of the invention and these are there US 2009/O 185990 A1 Jul. 23, 2009 24 fore considered to be within the scope of the invention as 11. The bioactive composition according to claim 1, defined in the claims which follow. wherein said bioactive fraction is a cytoplasm fraction eXtract. 1. A bioactive composition comprising: 12. The bioactive composition according to claim 1, an isolated bioactive fraction derived from a Theacea plant, wherein said bioactive fraction is a cell juice serum. wherein said bioactive fraction is selected from the 13. The bioactive composition according to claim 12, group consisting of a cell walls fraction, a cell walls wherein said cell juice serum has a total catechin content of fraction extract, a membrane fraction, a membrane frac between about 8.0 and about 20.0 milligrams per gram of dry tion extract, a cytoplasm fraction, a cytoplasm fraction matter. extract, a cell juice serum, and combinations thereof. 14. The bioactive composition according to claim 12, 2. The bioactive composition according to claim 1, wherein wherein said cell juice serum has a catechin content profile said bioactive fraction is a cell walls fraction. comprising: 3. The bioactive composition according to claim 1, wherein between about 2.1 and about 4.4 milligrams of (-)-epigal said bioactive fraction is a cell walls fraction extract. locatechin per gram of dry matter of the celljuice serum, 4. The bioactive composition according to claim3, wherein between about 4.2 and about 8.6 milligrams of (+)-catechin said cell walls fraction extract has a total catechin content of per gram of dry matter of the cell juice serum, between about 2.1 and about 4.5 milligrams per gram of dry between about 0.2 and about 2.0 milligrams of (-)-epicat matter. echin per gram of dry matter of the cell juice serum, 5. The bioactive composition according to claim3, wherein between about 1.2 and about 3.2 milligrams of (-)-epigal said cell walls fraction extract has a catechin content profile locatechingallate per gram of dry matter of the cell juice comprising: Serum, between about 2.0 and about 3.0 milligrams of (+)-catechin between about 0.01 and about 0.1 milligrams of(-)-gallo per gram of dry matter of the cell walls fraction extract, catechin gallate per gram of dry matter of the cell juice between about 0.005 and about 0.02 milligrams of (-)- serum, and epicatechin per gram of dry matter of the cell walls between about 0.2 and about 1.3 milligrams of (-)-epicat fraction extract, echin gallate per gram of dry matter of the cell juice between about 0.005 and about 0.02 milligrams of (-)- S. epigallocatechin gallate per gram of dry matter of the 15. The bioactive composition according to claim 1, cell walls fraction extract, and wherein said Theacea plant is a Camelia plant or a Eurya between about 0.003 and about 0.01 milligrams of (-)- plant. epicatechin gallate per gram of dry matter of the cell 16. The bioactive composition according to claim 15, walls fraction extract. wherein said Camelia plant is selected from the group con 6. The bioactive composition according to claim 1, wherein sisting of Camelia sinensis, Camelia japonica, Camelia said bioactive fraction is a membrane fraction. reticulate, and Camellia Sasanqua. 7. The bioactive composition according to claim 1, wherein 17. The bioactive composition according to claim 15, said bioactive fraction is a membrane fraction extract. wherein said Eurya plant is Eurya Sandwicensis. 8. The bioactive composition according to claim 7, wherein 18. The bioactive composition according to claim 1 further said membrane fraction extract has a total catechin content of comprising a stabilizing agent. between about 15.0 and about 30.5 milligrams per gram of 19. The bioactive composition according to claim 18, dry matter. wherein said stabilizing agent is selected from the group 9. The bioactive composition according to claim 7, wherein consisting of an emulsifier, a preservative, an antioxidant, a said membrane fraction extract has a catechin content profile polymer matrix, and mixtures thereof. comprising: 20. A bioactive topical formulation suitable for topical between about 1.7 and about 3.3 milligrams of (-)-epigal application to a mammal, said bioactive topical formulation locatechin per gram of dry matter of the membrane comprising: fraction extract, a topically effective amount of the bioactive composition between about 6.1 and about 10.2 milligrams of (+)-cat according to claim 1 and echin per gram of dry matter of the membrane fraction a topically acceptable carrier. eXtract, 21. The bioactive topical formulation according to claim between about 0.3 and about 1.1 milligrams of (-)-epicat 20, wherein the topically acceptable carrier is selected from echin per gram of dry matter of the membrane fraction the group consisting of a hydrophilic cream base, a hydro eXtract, philic lotion base, a hydrophilic Surfactant base, a hydrophilic between about 6.2 and about 12.5 milligrams of (-)-epi gel base, a hydrophilic Solution base, a hydrophobic cream gallocatechingallate per gram of dry matter of the mem base, a hydrophobic lotion base, a hydrophobic Surfactant brane fraction extract, base, a hydrophobic gel base, and a hydrophobic solution between about 0.007 and about 0.03 milligrams of (-)- base. gallocatechingallate per gram of dry matter of the mem 22. The bioactive topical formulation according to claim brane fraction extract, and 20, wherein the bioactive composition is present in an amount between about 1.3 and about 3.3 milligrams of (-)-epicat ranging from between about 0.001 percent and about 90 echin gallate per gram of dry matter of the membrane percent of the total weight of the bioactive topical formula fraction extract. tion. 10. The bioactive composition according to claim 1, 23. A method for inhibiting inflammatory activity in skin wherein said bioactive fraction is a cytoplasm fraction. tissue of a mammal, said method comprising: US 2009/O 185990 A1 Jul. 23, 2009 25
providing the bioactive composition according to claim 1 providing the bioactive composition according to claim 1 and and applying the bioactive composition to the skin tissue in an applying the bioactive composition to the skin tissue in an amount effective to inhibit inflammatory activity in the amount effective to normalize a cell disorder in the skin skin tissue. tissue. 24. The method according to claim 23, wherein said Thea cea plant is a Camelia plant or a Eurya plant. 37. The method according to claim 36, wherein said Thea 25. The method according to claim 23, wherein said bio cea plant is a Camelia plant or a Eurya plant. active composition further comprises a stabilizing agent. 38. The method according to claim 36, wherein said bio 26. The method according to claim 25, wherein said stabi active composition further comprises a stabilizing agent. lizing agent is selected from the group consisting of an emul 39. The method according to claim 38, wherein said stabi sifier, a preservative, an antioxidant, a polymer matrix, and lizing agent is selected from the group consisting of an emul mixtures thereof. sifier, a preservative, an antioxidant, a polymer matrix, and 27. The method according to claim 23, wherein said bio mixtures thereof. active composition further comprises a topically acceptable carrier. 40. The method according to claim 36, wherein said bio 28. The method according to claim 27, wherein the topi active composition further comprises a topically acceptable cally acceptable carrier is selected from the group consisting carrier. of a hydrophilic cream base, a hydrophilic lotion base, a 41. The method according to claim 40, wherein the topi hydrophilic Surfactant base, a hydrophilic gel base, a hydro cally acceptable carrier is selected from the group consisting philic solution base, a hydrophobic cream base, a hydropho of a hydrophilic cream base, a hydrophilic lotion base, a bic lotion base, a hydrophobic surfactant base, a hydrophobic hydrophilic Surfactant base, a hydrophilic gel base, a hydro gel base, and a hydrophobic solution base. philic solution base, a hydrophobic cream base, a hydropho 29. A method of protecting skin tissue of a mammal from bic lotion base, a hydrophobic surfactant base, a hydrophobic ultraviolet light-induced damage, said method comprising: gel base, and a hydrophobic solution base. providing the bioactive composition according to claim 1 42-50. (canceled) and 51. An isolated bioactive composition comprising a bioac applying the bioactive composition to the skin tissue in an tive fraction derived from cell juice of a Theacea plant amount effective to reduce ultraviolet light-induced wherein said bioactive fraction is produced according to a damage of the skin tissue and to prevent oxidative dam method comprising the steps of age of the skin tissue. providing a Theacea plant; 30. The method according to claim 29, wherein said Thea cea plant is a Camelia plant or a Eurya plant. separating the Theacea plant into cell juice and a cell walls 31. The method according to claim 29, wherein said bio component; active composition further comprises a stabilizing agent. treating the cell juice under conditions effective to yield a 32. The method according to claim 31, wherein said stabi bioactive fraction wherein said bioactive fraction is lizing agent is selected from the group consisting of an emul Selected from the group consisting of a membrane frac sifier, a preservative, an antioxidant, a polymer matrix, and tion a membrane fraction extract a cytoplasm fraction a mixtures thereof. cytoplasm fraction extract and a cell juice serum; and 33. The method according to claim 29, wherein said bio isolating said bioactive fraction from the treated cell juice. active composition further comprises a topically acceptable 52-57. (canceled) carrier. 58. An isolated bioactive fraction derived from a cell walls 34. The method according to claim 33, wherein the topi component of a Theacea plant wherein said bioactive fraction cally acceptable carrier is selected from the group consisting is produced according to a method comprising the steps of of a hydrophilic cream base, a hydrophilic lotion base, a hydrophilic Surfactant base, a hydrophilic gel base, a hydro providing a Theacea plant; philic solution base, a hydrophobic cream base, a hydropho separating the Theacea plant into cell juice and a cell walls bic lotion base, a hydrophobic surfactant base, a hydrophobic component; gel base, and a hydrophobic solution base. treating the cell walls component under conditions effec 35. The method according to claim 29, wherein said ultra tive to yield a bioactive fraction; and violet light-induced damage is caused by ultraviolet light in a isolating the bioactive fraction from the treated cell walls range of between about 320 and about 400 nanometers. component. 36. A method for normalizing skin disorders in skin tissue of a mammal, said method comprising: