WO1997036597A1 - Cocoa extract compounds and methods for making and using the same - Google Patents

Abstract

Disclosed and claimed are cocoa extracts such as polyphenols or procyanidins, methods for preparing such extracts, as well as uses for them, especially a compound of formula (I), wherein: n is an integer from 3 to 12, such that there is a first monomeric unit A, and a plurality of other monomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or 3-(β)-O-sugar; position 4 is alpha or beta stereochemistry; X, Y and Z represent positions for bonding between monomeric units, with the provisos that as to the first monomeric unit, bonding of another monomeric unit thereto is at position 4 and Y = Z = hydrogen, and, that when not for bonding monomeric units, X, Y and Z are hydrogen or alpha or beta sugar; and the sugar can be optionally substituted with a phenolic moiety via an ester bond.

Description

COCOA EXTRACT COMPOUNDS AND METHODS FOR MAKING AND USING THE SAME

REFERENCE TO RELATED APPLICATION Reference is made to copending U.S. application

Serial No. 08/317,226, filed October 3, 1994, incorporated herein by reference. FIELD OF THE INVENTION

This invention relates to cocoa extracts and compounds therefrom such as polyphenols preferably polyphenols enriched with procyanidins. This invention also relates to methods for preparing such extracts and compounds, as well as to uses for them; for instance, as antineoplastic agents, antioxidants, DNA topoisomerase II enzyme inhibitors, cyclo-oxygenase and/or lipoxygenase modulators, NO (Nitric Oxide) or NO-synthase modulators, blood or in vivo glucose modulators, and antimicrobials.

Documents are cited in this disclosure with a full citation for each appearing thereat or in a References section at the end of the specification, preceding the claims. These documents pertain to the field of this invention; and, each document cited herein is hereby incorporated herein by reference. BACKGROUND OF THE INVENTION Polyphenols are an incredibly diverse group of compounds (Ferreira et al., 1992) which widely occur in a variety of plants, some of which enter into the food chain. In some cases they represent an important class of compounds for the human diet. Although some of the polyphenols are considered to be nonnutrative, interest in these compounds has arisen because of their possible beneficial effects on health. For instance, quercetin (a flavonoid) has been _shown to possess anticarcinogenic activity in experimental animal studies (Deshner et al., 1991 and Kato et al., 1983). (+)-Catechin and (-) -epicatechin (flavan-3-ols) have been shown to inhibit Leukemia virus reverse transcriptase activity (Chu et al., 1992) . Nobotanin (an oligomeric hydrolyzable tannin) has also been shown to possess anti- tumor activity (Okuda et al., 1992). Statistical reports have also shown that stomach cancer mortality is significantly lower in the tea producing districts of Japan. Epigallocatechin gallate has been reported to be the pharmacologically active material in green tea that inhibits mouse skin tumors (Okuda et al., 1992) . Ellagic acid has also been shown to possess anticarcinogen activity in various animal tumor models (Bukharta et al., 1992) . Lastly, proanthocyanidin oligomers have been patented by the Kikkoman Corporation for use as antimutagens. Indeed, the area of phenolic compounds in foods and their modulation of tumor development in experimental animal models has been recently presented at the 202nd National Meeting of The American Chemical Society (Ho et al., 1992; Huang et al., 1992) .

However, none of these reports teaches or suggests cocoa extracts or compounds therefrom, any methods for preparing such extracts or compounds therefrom, or, any uses for cocoa extracts or compounds therefrom, as antineoplastic agents, anti-oxidants, anti-microbials, cyclo-oxygenase and/or lipoxygenase modulators, NO or NO-synthase modulators, and blood or in vivo glucose modulators. OBJECTS AND SUMMARY OF THE INVENTION

Since unfermented cocoa beans contain substantial levels of polyphenols, the present inventors considered it possible that similar activities of and uses for cocoa extracts, e.g., compounds within cocoa, could be revealed by extracting such compounds from cocoa and screening the extracts for activity. The National Cancer Institute has screened various Theobroma and Herrania species for anti- cancer activity as part of their massive natural product selection program. Low levels of activity were reported in some extracts of cocoa tissues, and the work was not pursued. Thus, in the antineoplastic or anti-cancer art, cocoa and its extracts were not deemed to be useful; i.e., the teachings in the antineoplastic or anti-cancer art lead the skilled artisan away from employing cocoa and its extracts as cancer therapy.

Since a number of analytical procedures were developed to study the contributions of cocoa polyphenols to flavor development (Clapperton et al., 1992), the present inventors decided to apply analogous methods to prepare samples for anti-cancer screening, contrary to the knowledge in the antineoplastic or anti-cancer art. Surprisingly, and contrary to the knowledge in the art, e.g., the National Cancer Institute screening, the present inventors discovered that cocoa polyphenol extracts which contain procyanidins, have significant utility as anti-cancer or antineoplastic agents. Additionally, the inventors demonstrate that cocoa extracts containing procyanidins and compounds from cocoa extracts have utility as antioxidants, antineoplastics, antimicrobials, cyclo-oxygenase and/or lipoxygenase modulators, NO or NO-synthase modulators, and blood or in vivo glucose modulators.

It is an object of the present invention to provide a method for producing cocoa extract and/or compounds therefrom.

It is another object of the invention to provide a cocoa extract and/or compounds therefrom. It is still another object of the present invention to provide compounds of the formula and methods for obtaining a compound of the formula:

wherein: n is an integer from 3 to 12, such that there is a first monomeric unit A, and a plurality of other monomeric units;

R is 3-(α)-0H, 3-(jS)-OH, 3-( )-0-sugar, or 3-(/3)- 0-sugar; position 4 is alpha or beta stereochemistry;

X, Y and Z represent positions for bonding between monomeric units, with the provisos that as to the first monomeric unit, bonding of another monomeric unit thereto is at position 4 and Y = Z = hydrogen, and, that when not for bonding monomeric units X, Y and Z are hydrogen, or Z, Y are sugar and X is hydrogen, or X is alpha or beta sugar and Z, Y are hydrogen, or combinations thereof. The compound can have n as 5 to 12, and certain preferred compounds have n as 5. The sugar can be selected from the group consisting essentially of glucose, galactose, xylose, rhamnose, and arabinose. The sugar of any or all of R, X, Y and Z can optionally be substituted with a phenolic moiety via an ester bond.

It is another object of the invention to provide an antioxidant composition. It is another object of the invention to demonstrate inhibition of DNA topoiso erase II enzyme activity.

It is yet another object of the present invention to provide a method for treating tumors or cancer. It is still another object of the invention to provide an anti-cancer, anti-tumor or antineoplastic composition.

It is still a further object of the invention to provide an antimicrobial composition. It is yet another object of the invention to provide a cyclo-oxygenase and/or lipoxygenase modulating composition.

It is still another object of the invention to provide an NO or NO-synthase-modulating composition. It is another object of the invention to provide a blood or in vivo glucose-modulating composition.

It is yet a further object of the invention to provide a method for treating a patient with an antineoplastic, antioxidant, antimicrobial, cyclo-oxygenase and/or lipoxygenase modulating or NO or

NO-synthase modulating and/or blood or in vivo glucose- modulating composition.

It is a further object of the invention to provide a method for making any of the aforementioned compositions. And, it is an object of the invention to provide a kit for use in the aforementioned methods or for preparing the aforementioned compositions. It has been surprisingly discovered that cocoa extract, and compounds therefrom, have anti-tumor, anti- cancer or antineoplastic activity or, is an antioxidant composition or, inhibits DNA topoisomerase II enzyme activity or, is an antimicrobial or, is a cyclo-oxygenase and/or lipoxygenase modulator or, is a NO or NO-synthase modulator or, is a blood or in vivo glucose modulator.

Accordingly, the present invention provides a substantially pure cocoa extract and compounds therefrom. The extract or compounds preferably comprises polyphenol(s) such as polyphenol (s) enriched with cocoa procyanidin(s) , such as polyphenols of at least one cocoa procyanidin selected from (-) epicatechin, (+) catechin, procyanidin B- 2, procyanidin oligomers 2 through 12, preferably 2 through 5 or 4 through 12, more preferably 3 through 12, and most preferably 5 through 12, procyanidin B-5, procyanidin A-2 and procyanidin C-l.

The present invention also provides an anti-tumor, anti-cancer or antineoplastic or antioxidant or DNA topoisomerase II inhibitor, or antimicrobial, or cyclo- oxygenase and/or lipoxygenase modulator, or an NO or NO- synthase modulator, or blood or in vivo glucose modulator composition comprising a substantially pure cocoa extract or compound therefrom or synthetic cocoa polyphenol (s) such as polyphenol(s) enriched with procyanidin(s) and a suitable carrier, e.g., a pharmaceutically, veterinary or food science acceptable carrier. The extract or compound therefrom preferably comprises cocoa procyanidin(s) . The cocoa extract or compounds therefrom is preferably obtained by a process comprising reducing cocoa beans to powder, defatting the powder and, extracting and purifying active compound(s) from the powder. The present invention further comprehends a method for treating a patient in need of treatment with an anti- tumor, anti-cancer, or antineoplastic agent or an antioxidant, or a DNA topoisomerase II inhibitor, or antimicrobial, or cyclo-oxygenase and/or lipoxygenase modulator, or an NO or NO-synthase modulator, or blood or in vivo glucose modulator comprising administering to the patient a composition comprising an effective quantity of a substantially pure cocoa extract or compound therefrom or synthetic cocoa polyphenol (s) or procyanidin(s) and a carrier, e.g., a pharmaceutically, veterinary or food science acceptable carrier. The cocoa extract or compound therefrom can be cocoa procyanidin(ε) ; and, is preferably obtained by reducing cocoa beans to powder, defatting the powder and, extracting and purifying active compound(s) from the powder.

Additionally, the present invention provides a kit for treating a patient in need of treatment with an anti- tumor, anti-cancer, or antineoplastic agent or antioxidant or DNA topoisomerase II inhibitor, or antimicrobial, or cyclo-oxygenase and/or lipoxygenase modulator, or an NO or NO-synthase modulator, or blood or in vivo glucose modulator comprising a substantially pure cocoa extract or compounds therefrom or synthetic cocoa polyphenol(s) or procyanidin(s) and a suitable carrier, e.g., a pharmaceutically, veterinary or food science acceptable carrier, for admixture with the extract or compound therefrom or synthetic polyphenol(s) or procyanidin(s) .

Further, the present invention provides a compound of the formula:

wherein: n is an integer from 3 to 12, such that there is a first monomeric unit A, and a plurality of other monomeric units;

R is 3-(α)-0H, 3-(3)-OH, 3-(α)-0-sugar, or 3-(/3)- O-εugar; position 4 is alpha or beta stereochemistry; X, Y and Z represent positions for bonding between monomeric units, with the provisos that as to the first monomeric unit, bonding of another monomeric unit thereto is at position 4 and Y = Z = hydrogen, and, that when not for bonding monomeric units, X, Y and Z are hydrogen, or Z, Y are sugra and X is hydrogen, or X is alpha or beta sugar and Z, Y are hydrogen, or combinations thereof. The compound can have n as 5 to 12, and certain preferred compounds have n as 5. The sugar can be selected from the group consisting essentially of glucose, galactose, xylose, rhamnose, and arabinose. The sugar of any or all of R, X, Y and Z can optionally be substituted with a phenolic moiety via an ester bond. Preferred compounds are illustrated in Figs. 38A to 38P and 39A to 39AA. Linkages of 4-6 and 4-8 are presently preferred. The present invention in another embodiment provides an antineoplastic composition comprising an inventive compound and a pharmaceutically, veterinary or food science acceptable carrier. In a further embodiment the invention provides an antimicrobial composition comprising an inventive compound and a suitable carrier or diluent.

The invention also provides a cyclo-oxygenase and/or lipoxygenase modulator composition comprising an inventive compound and a suitable carrier or diluent. The invention additionally provides a NO or NO-synthase-modulating composition comprising an inventive compound and a suitable carrier or diluent.

The invention comprehends a blood or in vivo glucose-modulating composition comprising an inventive compound and a suitable carrier or diluent.

Still further, the invention comprehends a method for treating a patient in need of treatment with an antineoplastic agent, or antioxidant agent/composition, or a DNA topoisomerase II inhibitor or composition, or antimicrobial agent/composition, or cyclo-oxygenase and/or lipoxygenase modulating agent/composition, or NO or NO- synthase modulating agent/composition, or blood or in vivo glucose-modulating agent/composition comprising administering to the patient a composition comprising an effective quantity of an inventive compound and a suitable carrier.

The invention even further encompasses food preservation or preparation compositions comprising an inventive compound, and methods for preparing or preserving food by adding the composition to food. And, the invention still further encompasses a DNA topoisomerase II inhibitor comprising an inventive compound and a suitable carrier or diluent, and methods for treating a patient in need of such treatment by administration of the composition.

Considering broadly the aforementioned embodiments involving cocoa extracts, the invention also includes such embodiments wherein an inventive compound is used instead of or as the cocoa extracts. Thus, the invention comprehends kits, methods, and compositions analogous to those above- stated with regard to cocoa extracts and with an inventive compound.

These and other objects and embodiments are disclosed or will be obvious from the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description will be better understood by reference to the accompanying drawings wherein: Fig. 1 shows a representative gel permeation chromatogram from the fractionation of crude cocoa procyanidins;

Fig. 2A shows a representative reverse-phase HPLC chromatogram showing the separation (elution profile) of cocoa procyanidins extracted from unfermented cocoa;

Fig. 2B shows a representative normal phase HPLC separation of cocoa procyanidins extracted from unfermented cocoa;

Fig. 3 shows several representative procyanidin structures; Figs. 4A-4E show representative HPLC chromatograms of five fractions employed in screening for anti-cancer or antineoplastic activity;

Figs. 5 and 6A-6D show the dose-response relationship between cocoa extracts and cancer cells ACHN (Fig. 5) and PC-3 (Figs. 6A-6D) (fractional survival vs. dose, μg/mL) ; M&M2 F4/92, M&MA+E U12P1, M&MB+E Y192P1, M&MC+E U12P2, M&MD+E U12P2;

Figs. 7A to 7H show the typical dose response relationships between cocoa procyanidin fractions A, B, C, D, E, A+B, A+E, and A+D, and the PC-3 cell line (fractional survival vs. dose, μg/mL) ; MM-1A 0212P3, MM-1 B 0162P1, MM-1 C 0122P3, MM-1 D 0122P3, MM-1 E 0292P8, M -1 A/B 0292P6, MM- 1 A/E 0292P6, M-1 A/D 0292P6; Figs. 8A to 8H show the typical dose response relationships between cocoa procyanidin fractions A, B, C,

D, E, A+B, B+E, and D+E and the KB Nasopharyngeal/HeLa cell line (fractional survival vs. dose, μg/mL) ;

MM-1A092K3, MM-1 B 0212K5, MM-1 C 0162K3, MM-1 D 0212K5, MM-1 E 0292K5, MM-1 A/B 0292K3, MM-1 B/E 0292K4, MM-1 D/E 0292K5;

Figs. 9A to 9H show the typical dose response relationship between cocoa procyanidin fractions A, B, C, D,

E, B+D, A+E and D+E and the HCT-116 cell line (fractional survival vs. dose, μg/mL); MM-1 C 0192H5, D 0192H5, E

0192H5, MM-1 B&D 0262H2, A/E 0262H3, MM-1 D&E 0262H1; Figs. 10A to 10H show typical dose response relationships between cocoa procyanidin fractions A, B, c, D, E, B+D, C+D and A+E and the ACHN renal cell line (fractional survival vs. dose, μg/mL); MM-1 A 092A5, MM-1 B 092A5, MM-1 C 0192A7, MM-1 D 0192A7, M&M1 E 0192A7, MM-1 B&D 0302A6, MM-1 C&D 0302A6, MM-1 A&E 0262A6; Figs. 11A to 11H show typical dose response relationships between cocoa procyanidin fractions A, B, C, D, E, A+E, B+E and C+E and the A-549 lung cell line (fractional survival vs. dose, μg/mL); MM-l A 019258, MM-l B 09256, MM-l C 019259, MM-l D 019258, MM-l E 019258, A/E 026254, MM-l B&E 030255, MM-l C&E N6255;

Figs. 12A to 12H show typical dose response relationships between cocoa procyanidin fractions A, B, C, D, E, B+C, C+D and D+E and the SK-5 melanoma cell line (fractional survival vs. dose μg/mL) ; MM-l A 0212S4, MM-l B 0212S4, MM-l C 0212S4, MM-l D 0212S4, MM-l E N32S1, MM-l B&C N32S2, MM-l C&D N32S3 , MM-l D&E N32S3;

Figs. 13A to 13H show typical dose response relationships between cocoa procyanidin fractions A, B, C, D, E, B+C, C+E, and D+E and the MCF-7 breast cell line

(fractional survival vs. dose, μg/mL); MM-l A N22M4, MM-l B N22M4, MM-l C N22M4, MM-l D N22M3, MM-l E 0302M2, MM-l B/C 0302M4, MM-l C&E N22M3 , MM-l D&E N22M3;

Fig. 14 shows typical dose response relationships for cocoa procyanidin (particularly fraction D) and the CCRF-CEM T-cell leukemia cell line (cells/mL vs. days of growth; open circle is control, darkened circle is I25μg fraction D, open inverted triangle is 250μg fraction D, darkened inverted triangle is 500μg fraction D) ; Fig. 15A shows a comparison of the XTT and Crystal

Violet cytotoxicity assays against MCF-7 pl68 breast cancer cells treated with fraction D+E (open circle is XTT and darkened circle is Crystal Violet) ;

Fig. 15B shows a typical dose response curve obtained from MDA MB231 breast cell line treated with varying levels of crude polyphenols obtained from UIT-1 cocoa genotype (absorbance (540nm) vs. Days; open circle is control, darkened circle is vehicle, open inverted triangle is 250μg/mL, darkened inverted triangle is lOOμg/ L, open square is lOμg/ L; absorbance of 2.0 is maximum of plate reader and may not be necessarily representative of cell number) ;

Fig. 15C shows a typical dose response curve obtained from PC-3 prostate cancer cell line treated with varying levels of crude polyphenols obtained from U T-1 cocoa genotype (absorbance (540nm) vs. Days; open circle is control, darkened circle is vehicle, open inverted triangle is 250μg/mL, darkened inverted triangle is lOOμg/mL and open square is lOμg/mL) ;

Fig. 15D shows a typical dose-response curve obtained from MCF-7 pl68 breast cancer cell line treated with varying levels of crude polyphenols obtained from UIT-1 cocoa genotype (absorbance (540nm) vs. Days; open circle is control, darkened circle is vehicle, open inverted triangle is 250μg/mL, darkened inverted triangle is lOOμg/mL, open square is lOμg/mL, darkened square is lμg/mL; absorbance of 2.0 is maximum of plate reader and may not be necessarily representative of cell number) ;

Fig. 15E shows a typical dose response curve obtained from Hela cervical cancer cell line treated with varying levels of crude polyphenols obtained from UIT-I cocoa genotype (absorbance (540nm) vs. Days; open circle is control, darkened circle is vehicle, open inverted triangle is 250μg/mL, darkened inverted triangle is lOOμg/mL, open square is lOμg/mL; absorbance of 2.0 is maximum of plate reader and may not be necessarily representative of cell number) ;

Fig. 15F shows cytotoxic effects against Hela cervical cancer cell line treated with different cocoa polyphenol fractions (absorbance (540nm) vs. Days; open circle is lOOμg/mL fractions A-E, darkened circle is lOOμg/mL fractions A-C, open inverted triangle is lOOμg/mL fractions D&E; absorbance of 2.0 is maximum of plate reader and not representative of cell number) ;

Fig. 15G shows cytotoxic effects at lOOul/mL against SKBR-3 breast cancer cell line treated with different cocoa polyphenol fractions (absorbance (540nm) vs. Days; open circle is fractions A-E, darkened circle is fractions A-C, open inverted triangle is fractions D&E) ;

Fig. 15H shows typical dose-response relationships between cocoa procyanidin fraction D+E on Hela cells (absorbance (540nm) vs. Days; open circle is control, darkened circle is lOOμg/mL, open inverted triangle is 75μg/mL, darkened inverted triangle is

50μg/mL, open square is 25μg/mL, darkened square is lOμg/mL; absorbance of 2.0 is maximum of plate reader and is not representative of cell number) ;

Fig. 151 shows typical dose-response relationship between cocoa procyanidin fraction D+E on SKBR-3 cells (absorbance (540nm) vs. Days; open circle is control, darkened circle is lOOμg/mL, open inverted triangle is 75μg/mL, darkened inverted triangle is 50μg/mL, open square is 25μg/mL, darkened square is lOμg/mL) ;

Fig. 15J shows typical dose-response relationships between cocoa procyanidin fraction D+E on Hela cells using the Soft Agar Cloning assay (bar chart; number of colonies vs. control, 1, 10, 50, and lOOμg/mL) ;

Fig. 15K shows the growth inhibition of Hela cells when treated with crude polyphenol extracts obtained from eight different cocoa genotypes (% control vs. concentration, μg/mL; open circle is C-l, darkened circle is C-2, open inverted triangle is C-3, darkened inverted triangle is C-4, open square is C-5, darkened square is C-6, open triangle is C-7, darkened triangle is C-8; C-l = UF-12: horti race = Trinitario and description is crude extracts of UF-12 (Brazil) cocoa polyphenols

(decaffeinated/detheobrominated) ; C-2 = NA-33: horti race = Forastero and description is crude extracts of NA-33 (Brazil) cocoa polyphenols (decaffeinated/ detheobrominated) ; C-3 = EEG-48: horti race = Forastero and description is crude extracts of EEG-48 (Brazil) cocoa polyphenols (decaffeinated/detheobro inated) ; C-4 = unknown: horti race = Forastero and description is crude extracts of unknown ( . African) cocoa polyphenols

(decaffeinated/detheobrominated) ; C-5 = UF-613: horti race = Trinitario and description is crude extracts of UF-613 (Brazil) cocoa polyphenols (decaffeinated/ detheobrominated); C-6 = ICS-100: horti race = Trinitario (to Nicaraguan Criollo ancestor) and description is crude extracts of ICS-100 (Brazil) cocoa polyphenols (decaffeinated/detheobrominated) ; C-7 = ICS-139: horti race = Trinitario (Nicaraguan Criollo ancestor) and description is crude extracts of ICS-139 (Brazil) cocoa polyphenols (decaffeinated/detheobrominated) ; C-8 = UIT-1: horti race = Trinitario and description is crude extracts of UIT-1 (Malaysia) cocoa polyphenols (decaffeinated/detheobrominated) ;

Fig. 15L shows the growth inhibition of Hela cells when treated with crude polyphenol extracts obtained from fermented cocoa beans and dried cocoa beans (stages throughout fermentation and sun drying; % control vs. concentration, μg/mL; open circle is day zero fraction, darkened circle is day 1 fraction, open inverted triangle is day 2 fraction, darkened inverted triangle is day 3 fraction, open square is day 4 fraction and darkened square is day 9 fraction) ;

Fig. 15M shows the effect of enzymatically oxidized cocoa procyanidins against Hela cells (dose response for polyphenol oxidase treated crude cocoa polyphenol; % control vs. concentration, μg/mL; darkened square is crude UIT-1 (with caffeine and theobromine) , open circle crude UIT-1 (without caffeine and theobromine) and darkened circle is crude UIT-l (polyphenol oxidase catalyzed) ;

Fig. 15N shows a representative semi- preparative reverse phase HPLC separation for combined cocoa procyanidin fractions D and E;

Fig. 150 shows a representative normal phase semi- preparative HPLC separation of a crude cocoa polyphenol extract; Fig. 16 shows typical Rancimat Oxidation curves for cocoa procyanidin extract and fractions in comparison to the synthetic antioxidants BHA and BHT (arbitrary units vs. time; dotted line and cross (+) is BHA and BHT; * is D-E; x is crude; open square is A-C; and open diamond is control) ; Fig. 17 shows a typical Agarose Gel indicating inhibition of topoisomerase II catalyzed decatenation of kinetoplast DNA by cocoa procyanidin fractions (Lane 1 contains 0.5μg of marker (M) monomer-length kinetoplast DNA circles; Lanes 2 and 20 contain kinetoplast DNA that was incubated with Topoisomerase II in the presence of 4% DMSO, but in the absence of any cocoa procyanidins. (Control -C) ; Lanes 3 and 4 contain kinetoplast DNA that was incubated with Topoisomerase II in the presence of 0.5 and 5.0μg/mL cocoa procyanidin fraction A; Lanes 5 and 6 contain kinetoplast DNA that was incubated with Topoisomerase II in the presence of 0.5 and 5.0μg/mL cocoa procyanidin fraction B; Lanes 7, 8, 9, 13, 14 and 15 are replicates of kinetoplast DNA that was incubated with Topoisomerase II in the presence of 0.05, 0.5 and 5.0μg/mL cocoa procyanidin fraction D; Lanes 10, 11, 12, 16, 17 and 18 are replicates of kinetoplast DNA that was incubated with Topoisomerase II in the presence of 0.05, 0.5, and 5.0μg/mL cocoa procyanidin fraction E; Lane 19 is a replicate of kinetoplast DNA that was incubated with Topoisomerase II in the presence of 5.0μg/mL cocoa procyanidin fraction E) ;

Fig. 18 shows dose response relationships of cocoa procyanidin fraction D against DNA repair competent and deficient cell lines (fractional survival vs. μg/mL; left side xrs-6 DNA Deficient Repair Cell Line, MM-l D D282X1; right side BR1 Competent DNA Repair Cell Line, MM-l D D282B1) ; Fig, 19 shows the dose-response curves for

Adriamycin resistant MCF-7 cells in comparison to a MCF-7 pl68 parental cell line when treated with cocoa fraction D+E (% control vs. concentration, μg/mL; open circle is MCF-7 pl68; darkened circle is MCF-7 ADR); Figs. 20A and B show the dose-response effects on

Hela and SKBR-3 cells when treated at 100 μg/mL and 25 μg/mL levels of twelve fractions prepared by Normal phase semi- preparative HPLC (bar chart, % control vs. control and fractions 1-12) ; Fig. 21 shows a normal phase HPLC separation of crude, enriched and purified pentamers from cocoa extract; Figs. 22A, B and C show MALDI-TOF/MS of penta er enriched procyanidins, and of Fractions A-C and of Fractions D-E, respectively;

Fig. 23A shows an elution profile of oligomeric procyanidins purified by modified semi-preparative HPLC;

Fig. 23B shows an elution profile of a trimer procyanidin by modified semi-preparative HPLC;

Figs. 24A-D each show energy minimized structures of all (4-8) linked pentamers based on the structure of epicatechin;

Fig. 25A shows relative fluorescence of epicatechin upon thiolysis with benzylmercapten;

Fig. 25B shows relative fluorescence of catechin upon thiolysis with benzylmercapten; Fig. 25C shows relative fluorescence of dimers (B2 and B5) upon thiolysis with benzylmercapten;

Fig. 26A shows relative fluorescence of dimer upon thiolysis;

Fig. 26B shows relative fluorescence of B5 dimer upon thiolysis of dimer and subsequent desulphurization;

Fig. 27A shows the relative tumor volume during treatment of MDA MB 231 nude mouse model treated with pentamer;

Fig. 27B shows the relative survival curve of pentamer treated MDA 231 nude mouse model;

Fig. 28 shows the elution profile from halogen- free analytical separation of acetone extract of procyanidins from cocoa extract;

Fig. 29 shows the effect of pore size of stationary phase for normal phase HPLC separation of procyanidins; Fig. 30A shows the substrate utilization during fermentation of cocoa beans;

Fig. 3OB shows the metabolite production during fermentation; Fig. 30C shows the plate counts during fermentation of cocoa beans;

Fig. 30D shows the relative concentrations of each component in fermented solutions of cocoa beans;

Fig. 31 shows the acetylcholine-induced relaxation of NO-related phenylephrine-precontracted rat aorta;

Fig. 32 shows the blood glucose tolerance profiles from various test mixtures;

Figs. 33A-B show the effects of indo ethacin on COX-l and COX-2 activities; Figs. 34A-B show the correlation between the degree of polymerization and IC₅₀ vs. COX-l/COX-2 (μM) ;

Fig. 35 shows the correlation between the effects of compounds on COX-l and COX-2 activities expressed as μM;

Figs. 36A-V show the IC₅₀ values (μM) of samples containing procyanidins with C0X-1/COX-2;

Fig. 37 shows the purification scheme for the isolation of procyanidins from cocoa;

Fig. 38A to 38P shows the preferred structures of the pentamer; Figs. 39A-AA show a library of stereoiso ers of pentamers;

Figs. 40A-B show 70 minute gradients for normal phase HPLC separation of procyanidins, detected by UV and fluorescence, respectively; Figs. 41A-B show 30 minute gradients for normal phase HPLC separation of procyanidins, detected by UV and fluorescence, respectively; Fig. 42 shows a preparation normal phase HPLC separation of procyanidins;

Figs. 43A-G show CD (circular dichrois ) spectra of procyanidin dimers, trimers, tetramerε, pentamers, hexa erε, heptamers and octamerε, respectively;

Fig. 44A shows the structure and ¹H/¹³C NMR data for epicatechin;

Figs. 44B-F show the APT, COSY, XHCORR, ^ and ¹³C NMR spectra for epicatechin; Fig. 45A shows the structure and ¹H/¹³C NMR data for catechin;

Figs. 45B-E show the ¹H, APT, XHCORR and COSY NMR spectra for catechin;

Fig. 46A shows the structure and H/¹³C NMR data for B2 dimer;

Figs. 46B-G show the ¹³C, APT, ¹H, HMQC, COSY and HOHAHA NMR spectra for the B2 dimer;

Fig. 47A shows the structure and ¹H/¹³C NMR data for B5 dimer; Figs. 47B-G show the ¹H, ¹³C, APT, COSY, HMQC and

HOHAHA NMR spectra for B5 dimer;

Figε. 48A-D show the ¹H, COSY, HMQC and HOHAHA NMR spectra for epicatechin/catechin trimer; and

Figs. 49A-D show the ¹H, COSY, HMQC and HOHAHA NMR spectra for epicatechin trimer. DETAILED DESCRIPTION

As discussed above, it has now been surprisingly found that cocoa extracts or a compound therefrom exhibit anti-cancer, anti-tumor or antineoplastic activity, antioxidant activity, inhibit DNA topoisomerase II enzyme and have antimicrobial, cyclo-oxygenase and/or lipoxygenase, NO or NO-synthase and blood or in vivo glucose modulating activities. The extracts or compound therefrom are generally prepared by reducing cocoa beans to a powder, defatting the powder, and extracting and purifying the active compound(s) from the defatted powder. The powder can be prepared by freeze-drying the cocoa beans and pulp, depulping and dehulling the freeze-dried cocoa beans and grinding the dehulled beans. The extraction of active compound(ε) can be by solvent extraction techniques. The extracts can be purified; for instance, by gel permeation chromatography or by preparative High Performance Liquid

Chromatography (HPLC) techniques or by a combination of such techniques. An outline of the purification protocol utilized in the isolation of substantially pure procyanidins is shown in Fig. 37. Steps l and 2 of the purification scheme are described in Examples 1 and 2; steps 3 and 4 are described in Examples 3, 13 and 23; step 5 is described in Examples 4 and 14; and step 6 is described in Examples 4, 14 and 16. The skilled artisan would appreciate and envision modifications in the purification scheme outlined in Figure 37 to obtain the active compounds without departing from the spirit or scope thereof and without undue experimentation. The extracts or compound therefor having activity, without wishing to neceεεarily be bound by any particular theory, have been identified as cocoa polyphenol (s) such as procyanidins. These cocoa procyanidins have significant anti-cancer, anti-tumor or antineoplastic activity; antioxidant activity; and inhibit DNA topoisomerase II enzyme; possess antimicrobial activity; and can modulate cyclo-oxygenase and/or lipoxygenase, NO or NO-synthase, and blood or in vivo glucose.

As recited above, the invention involves certain inventive compounds displaying the utilities noted above for cocoa extracts; and, throughout this disclosure, the term "cocoa extract" may be substituted by an inventive compound disclosed above, such that it is to be understood that an inventive compound can be the cocoa extract. Preferred inventive compounds are shown in Fig. 38A to 38P, and Fig. 39A to 39V show a library of εtereoisomers of the pentamer from which other compounds within the scope of the invention may be obtained without undue experimentation.

Anti-cancer, anti-tumor or antineoplastic or, antioxidant, DNA topoisomerase II enzyme inhibiting, antimicrobial, cyclo-oxygenase and/or lipoxygenase modulator NO- or NO-synthase and blood or in vivo glucose modulating activities, or compositions containing the inventive cocoa polyphenols or procyanidins can be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical or food science or veterinary art(s) .

Such compositions can be administered to a subject or patient in need of such administration in dosages and by techniques well known to those skilled in the medical, nutritional or veterinary arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject or patient, and the route of administration. The compositions can be co-administered or sequentially administered with other antineoplastic, anti- tumor or anti-cancer agents, antioxidants, DNA topoiεomerase II enzyme inhibiting agents, or cyclo-oxygenase and/or lipoxygenase, blood or in vivo glucose or NO or NO-synthase modulating agents and/or with agents which reduce or alleviate ill effects of antineoplastic, anti-tumor, anti- cancer agents, antioxidants, DNA topoisomerase II enzyme inhibiting agents, cyclo-oxygenase and/or lipoxygenase, _^ blood or in vivo glucose or NO or NO-synthase modulating agents; again, taking into consideration such factors as the age, sex, weight, and condition of the particular subject or patient, and, the route of administration.

Examples of co positionε of the invention include edible compositions for oral administration such solid or liquid formulations, for instance, capsuleε, tablets, pills and the like, as well as chewable solid or beverage formulations, to which the present invention may be well- suited since it is from an edible source (e.g., cocoa or chocolate flavored solid or liquid compositions) ; liquid preparations for orifice, e.g., oral, nasal, anal, vaginal etc., administration such as suspensions, syrups or elixirs (including cocoa or chocolate flavored compositions) ; and, preparations for parental, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions. However, the active ingredient in the compositions may complex with proteins such that when administered into the bloodstream, clotting may occur due to precipitation of blood proteins; and, the skilled artisan should take this into account. In such compositions the active cocoa extract may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, DMSO, ethanol, or the like. The active cocoa extract of the invention can be provided in lyophilized form for reconstituting, for instance, in isotonic aqueous, saline, glucose or DMSO buffer. In certain saline solutions, some precipitation has been observed; and, this observation may be employed as a means to isolate inventive compounds, e.g., by a "salting out" procedure. Further, the invention also comprehends a kit wherein the active cocoa extract is provided. The kit can include a separate container containing a suitable carrier, diluent or excipient. The kit can also include an additional anti-cancer, anti-tumor or antineoplastic agent, antioxidant, DNA topoisomerase II enzyme inhibitor or antimicrobial, or cyclo-oxygenase and/or lipoxygenase, NO or NO-synthase or blood or in vivo glucose modulating agent and/or an agent which reduces or alleviates ill effects of antineoplastic, anti-tumor or anti-cancer agents, antioxidant, DNA topoisomeraεe II enzyme inhibitor or antimicrobial, or cyclo-oxygenase and/or lipoxygenase, NO or NO-synthase and blood or in vivo glucose modulating agents for co- or sequential-administration. The additional agent(s) can be provided in separate container(s) or in admixture with the active cocoa extract. Additionally, the kit can include inεtructions for mixing or combining ingredients and/or administration.

Furthermore, while the invention is described with respect to cocoa extracts preferably comprising cocoa procyanidins, from this disclosure the skilled organic chemist will appreciate and envision synthetic routes to obtain the active compounds. Accordingly, the invention comprehends synthetic cocoa polyphenols or procyanidins or their derivatives which include, but are not limited to glycosideε, gallates, esters, etc. and the like. That is, the inventive compounds can be prepared from isolation from cocoa or from any species within the Theobro a or Herrania genera, as well as from synthetic routes; and derivatives of the inventive compounds such as glycosides, gallates, esterε, etc. are included in the inventive co poundε. Alεo, with reference to isolation from cocoa, any species of Theobroma or Herrania or their inter- and intra- specific crosεes thereof may be employed therefor, and reference in this regard is made to Schultes, "Synopsis of Herrania," Journal of the Arnold Arboretum, Vol. XXXIX, pp. 217 to 278 plus plates I to XVII (1958) , Cuatrecasas, "Cacao and its Allies A Taxonomic Revision of the Genus Theobroma," Bulletin of the United States National Museum, Vol. 35, part 6 , pp. 379 to 613, plus plates l to 11 (Smithsonian Institution 1964), and Addison et al., "Observations on the Species of the Genus Theobroma Which Occurs in the Amazon," Bol. Tech. Inst. Agronomico do Nortes, 25, 3 (1951) .

The following non-limiting Examples are given by way of illustration only and are not to be considered a limitation of this invention, many apparent variations of which are possible without departing from the spirit or scope thereof.

EXAMPLES Example l: Cocoa Source and Method of Preparation

Several Theobroma cacao genotypes which represent the three recognized horticultural races of cocoa (Enriquez, 1967; Engelε, 1981) were obtained from the three major cocoa producing origins of the world. A list of those genotypes used in this study are shown in Table 1. Harvested cocoa pods were opened and the beans with pulp were removed for freeze drying. The pulp was manually removed from the freeze dried mass and the beans were subjected to analysis as follows. The unfermented, freeze dried cocoa beans were first manually dehulled, and ground to a fine powdery mass with a TEKMAR Mill. The resultant mass was then defatted overnight by Soxhlet extraction using redistilled hexane as the solvent. Residual solvent was removed from the defatted ass by vacuum at ambient temperature. Table l: Description of Theobroma cacao Source Material

Example 2: Procyanidin Extraction Procedures A. Method 1 Procyanidins were extracted from the defatted, unfermented, freeze dried cocoa beans of Example 1 using a modification of the method described by Jalal and Collin (1977) . Procyanidins were extracted from 50 gram batches of the defatted cocoa mass with 2X 400 mL 70% acetone/deionized water followed by 400mL 70% methanol/deionized water. The extracts were pooled and the εolventε removed by evaporation at 45°C with a rotary evaporator held under partial vacuum. The resultant aqueous phase was diluted to IL with deionized water and extracted 2X with 400mL CHC1₃. The solvent phase was discarded. The aqueous phase was then extracted 4X with 500mL ethyl acetate. Any resultant emulsions were broken by centrifugation on a Sorvall RC 28S centrifuge operated at 2,000 xg for 30 min. at 10°C. To the combined ethyl acetate extracts, 100-200mL deionized water was added. The solvent was removed by evaporation at 45°C with a rotary evaporator held under partial vacuum. The resultant aqueous phase was frozen in liquid N₂ followed by freeze drying on a LABCONCO Freeze Dry System. The yields of crude procyanidins that were obtained from the different cocoa genotypes are listed in Table 2.

Table 2: Crude Procyanidin Yields

B. Method 2

Alternatively, procyanidins are extracted from defatted, unfermented, freeze dried cocoa beans of Example with 70% aqueous acetone. Ten grams of defatted material was slurried with 100 mL solvent for 5-10 min. The slurry was centrifuged for 15 min. at 4°C at 3000 xg and the supernatant passed through glass wool. The filtrate was subjected to distillation under partial vacuum and the resultant aqueous phase frozen in liquid N₂, followed by freeze drying on a LABCONCO Freeze Dry System. The yields of crude procyanidins ranged from 15-20%.

Without wishing to be bound by any particular theory, it is believed that the differences in crude yields reflected variations encountered with different genotypes, geographical origin, horticultural race, and method of preparation.

Example 3: Partial Purification of Cocoa Procyanidins A. Gel Permeation Chromatography Procyanidins obtained from Example 2 were partially purified by liquid chromatography on Sephadex LH- 20 (28 x 2.5 cm). Separations were aided by a step gradient from deionized water into methanol. The initial gradient composition εtarted with 15% methanol in deionized water which was followed step wise every 30 min. with 25% methanol in deionized water, 35% methanol in deionized water, 70% methanol in deionized water, and finally 100% methanol. The effluent following the elution of the xanthine alkaloids (caffeine and theobromine) was collected as a εingle fraction. The fraction yielded a xanthine alkaloid free subfraction which was submitted to further subfractionation to yield five subtractions designated MM2A through MM2E. The solvent was removed from each εubfraction by evaporation at 45°C with a rotary evaporator held under partial vacuum. The reεultant aqueous phase was frozen in liquid N₂ and freeze dried overnight on a LABCONCO Freeze Dry System. A representative gel permeation chromatogram showing the fractionation is shown in Figure 1. Approximately, lOOmg of material was subfractionated in this manner. Figure 1: Gel Permeation Chromatogram of Crude

Procyanidins on Sephadex LH-20 Chromatographic Conditions: Column; 28 x 2.5 cm Sephadex

LH-20, Mobile Phase: Methanol/Water Step Gradient, 15:85,

25:75, 35:65, 70:30, 100:0 Stepped at 1/2 Hour Intervals,

Flow Rate; 1.5mL/min, Detector; UV at λ, = 254 nm and λ₂ =

365 nm, Chart Speed: 0.5ιran/min, Column Load; I20mg.

B. Semi-preparative High Performance Liquid Chromatography (HPLC)

Method 1. Reverse Phase Separation

Procyanidins obtained from Example 2 and/or 3A were partially purified by semi-preparative HPLC. A Hewlett Packard 1050 HPLC System equipped with a variable wavelength detector, Rheodyne 7010 injection valve with lmL injection loop was assembled with a Pharmacia FRAC-100 Fraction Collector. Separations were effected on a Phenomenex Ultracarb™ 10 μ ODS column (250 x 22.5mm) connected with a Phenomenex 10 μ ODS Ultracarb"¹ (60 x 10 mm) guard column. The mobile phase composition was A = water; B = methanol used under the following linear gradient conditions: [Time, %A]; (0,85), (60,50), (90,0), and (110,0) at a flow rate of 5mL/min. Compounds were detected by UV at 254nm

A representative Semi-preparative HPLC trace is shown in Figure 15N for the separation of procyanidins present in fraction D + E. Individual peaks or select chromatographic regions were collected on timed intervals or manually by fraction collection for further purification and subsequent evaluation. Injection loads ranged from 25-100mg of material.

Method 2. Normal Phase Separation Procyanidin extracts obtained from Examples 2 and/or 3A were partially purified by semi-preparative HPLC. A Hewlett Packard 1050 HPLC system, Millipore-Waters Model 480 LC detector set at 254nm was assembled with a Pharmacia Frac-100 Fraction Collector set in peak mode. Separations were effected on a Supelco 5μm Supelcosil LC-Si column (250 x 10mm) connected with a Supelco 5μm Supelguard LC-Si guard column (20 x 4.6mm). Procyanidins were eluted by a linear gradient under the following conditions: (Time, %A, %B) ; (0,82,14), (30, 67.6, 28.4), (60, 46, 50) , (65, 10, 86), (70, 10, 86) followed by a 10 min. re-equilibration. Mobile phase composition was A = dichloromethane; B = methanol; and C = acetic acid: water (1:1). A flow rate of 3mL/min was used. Components were detected by UV at 254nm, and recorded on a Kipp & Zonan BD41 recorder. Injection volumes ranged from 100-250μl of lOmg of procyanidin extracts dissolved in 0.25mL 70% aqueous acetone. A representative semi-preparative HPLC trace is shown in

Figure 15 O. Individual peaks or select chromatographic regions were collected on timed intervals or manually by fraction collection for further purification and subεequent evaluation.

HPLC conditions: 250 x 10mm Supelco Supelcosil LC-Si (5μm) Semipreparative Column 20 x 4.6mm Supelco Supelcosil LC-Si (5μm) Guard Column

Detector: Waters LC Spectrophotometer Model

480 @ 254nm Flow rate: 3mL/min, Column Temperature: ambient, Injection: 250μL of 70% aqueous acetone extract.

The fractions obtained were aε followε:

Example : Analytical HPLC Analysis of Procyanidin Extracts

Method 1. Reverse Phase Separation

Procyanidin extracts obtained from Example 3 were filtered through a 0.45μ filter and analyzed by a Hewlett Packard 1090 ternary HPLC system equipped with a Diode Array detector and a HP model 1046A Programmable Fluorescence Detector. Separations were effected at 45°C on a Hewlett- Packard 5μ Hypersil ODS column (200 x 2.1mm). The flavanols and procyanidins were eluted with a linear gradient of 60% B into A followed by a column wash with B at a flow rate of 0.3mL/min. The mobile phase composition was B = 0.5% acetic acid in methanol and A = 0.5% acetic acid in nanopure water. Acetic acid levels in A and B mobile phases can be increased to 2%. Components were detected by fluorescence, where

λex - 276nm and λ„^β™, = 3l6nm and by UV at 280nm.

Concentrations of (+)-catechin and (-) -epicatechin were determined relative to reference standard solutions. Procyanidin levels were estimated by using the response factor for (-) -epicatechin. A representative HPLC chromatogram showing the separation of the various components is shown in Figure 2A for one cocoa genotype. Similar HPLC profiles were obtained from the other cocoa genotypes.

HPLC Conditions: Column: 200 x 2.1mm Hewlett Packard Hypersil ODS (5μ)

Guard column: 20 x 2.1mm Hewlett

Packard Hypersil ODS (5μ)

Detectorε: Diode Array @ 280nm

Fluorescence λ_eχ = 276nm; λ_Λem_m = 3l6nm.

Flow rate: 0.3mL/min. Column Temperature: 45°C

Method 2. Normal Phase Separation Procyanidin extracts obtained from Examples 2 and/or 3 were filtered through a 0.45μ filter and analyzed by a Hewlett Packard 1090 Series II HPLC syεtem equipped with a HP model 1046A Programmable Fluorescence detector and Diode Array detector. Separations were effected at 37°C on a 5μ Phenomenex Lichrosphere" Silica 100 column (250 x 3.2mm) connected to a Supelco Supelguard LC-Si 5μ guard column (20 x 4.6mm). Procyanidins were eluted by linear gradient under the following conditions: (Time, %A, %B) ; (0, 82, 14), (30, 67.6, 28.4) , (60, 46, 50) , (65, 10, 86) , (70, 10, 86) followed by an 8 min. re-equilibration. Mobile phase composition was A=dichloromethane, B=methanol, and C=acetic acid: water at a volume ratio of 1:1. A flow rate of 0.5 mL/min. was used. Components were detected by fluorescence, where λ_eχ = 276nm and λ_em = 316nm or by UV at 280 nm. A representative HPLC chromatogram showing the separation of the various procyanidins is shown in Figure 2B for one genotype. Similar HPLC profiles were obtained from other cocoa genotypes.

HPLC Conditions:

250 x 3.2mm Phenomenex Lichrosphere^® Silica 100 column (5μ) 20 x 4.6mm Supelco Supelguard LC-Si (5μ) guard column

Detectors: Photodiode Array @ 280nm Fluorescence λ_eχ = 276nm; λe_mm = 316nm.

Flow rate: 0.5 mL/min. Column Temperature: 37°C

Example 5:Identification of Procyanidins

Procyanidins were purified by liquid chromatography on Sephadex LH-20 (28 x 2.5cm) columns followed by semi-preparative HPLC using a lOμ Bondapak C18 (100 x 8mm) column or by semi-preparative HPLC using a 5μ Supelcosil LC-Si (250 x 10mm) column.

Partially purified isolates were analyzed by Fast Atom Bombardment - Mass Spectrometry (FAB-MS) on a VG ZAB-T high resolution MS system using a Liquid Secondary Ion Mass Spectrometry (LSIMS) technique in positive and negative ion modes. A cesium ion gun was used as the ionizing source at 30kV and a "Magic Bullet Matrix" (1:1 dithiothreitol/dithioerythritol) was used as the proton donor. Analytical investigations of these fractions by

LSIMS revealed the presence of a number of flavan-3-ol oligomers as shown in Table 3.

Table 3: LSIMS (Positive Ion) Data from Cocoa Procyanidin Fractions

The major mass fragment ions were consistent with work previously reported for both positive and negative ion FAB-MS analysiε of procyanidins (Self et al., 1986 and Porter et al., 1991). The ion corresponding to m/z 577 (M+H)⁺ and its εodium adduct at m/z 599 (M+Na)^* suggeεted the preεence of doubly linked procyanidin di ers in the isolates. It was interesting to note that the higher oligomers were more likely to form sodium adducts (M+Na)^* than their protonated molecular ions (M+H)^*. The procyanidin isomerε B-2, B-5 and C-l were tentatively identified based on the work reported by Revilla et al. (1991), Self et al. (1986) and Porter et al. (1991). _^Procyanidins up to both the octamer and decamer were verified by FAB-MS in the partially purified fractions.

Additionally, evidence for procyanidins up to the dodecamer were observed from normal phase HPLC analysis (see

Figure 2B) . Without wishing to be bound by any particular theory, it is believed that the dodecamer is the limit of solubility in the solvents used in the extraction and purification schemes. Table 4 lists the relative concentrations of the procyanidins found in xanthine alkaloid free isolates based on reverse phase HPLC analysis.

Table 5 lists the relative concentrations of the procyanidinε baεed on normal phase HPLC analysis.

Table 4: Relative Concentrations of Procyanidins in the Xanthine Alkaloid Free Isolates

Table 5: Relative Concentrations of Procyanidins in Aqueous Acetone Extracts

Figure 3 shows several procyanidin structures and Figures 4A-4E show the repreεentative HPLC chromatogramε of the five fractionε employed in the following εcreening for anti-cancer or antineoplaεtic activity. The HPLC conditions for Figs. 4A-4E were as follows:

HPLC Conditions: Hewlett Packard 1090 ternary

HPLC System equipped with HP Model 1046A

Programmable Fluorescence Detector.

Column: Hewlett Packard 5μ Hypersil ODS (200 x 2.1mm) Linear Gradient of 60% B into A at a flow rate of 0.3mL/min. B = 0.5% acetic acid in methanol; A = 0.5% acetic acid in deionized water. λ„ex_v = 280nm;' λ_Λe_mm = 316nm. Figure 15 0 shows a representative semi-prep HPLC chromatogram of an additional 12 fractions employed in the screening for anticancer or antineoplastic activity (HPLC conditions stated above) . Example 6: Anti-Cancer, Anti-Tumor or Antineoplastic

Activity of Cocoa Extracts (Procyanidins)

The MTT (3-[4,5-dimethyl thiazol-2yl]-2,5- diphenyltetrazoliu bromide) - icrotiter plate tetrazolium cytotoxicity assay originally developed by Mosmann (1983) was used to screen test sampleε from Example 5. Test samples, standards (cisplatin and chlorambucil) and MTT reagent were disεolved in 100% DMSO (dimethyl εulfoxide) at a lOmg/ L concentration. Serial dilutionε were prepared from the εtock solutions. In the case of the test samples, dilutions ranging from 0.01 through lOOμg/mL were prepared in 0.5% DMSO.

All human tumor cell lines were obtained from the American Type Culture Collection. Cells were grown as mono layers in alpha-MEM containing 10% fetal bovine serum, 100 units/ L penicillin, lOOμg/mL streptomycin and 240 units/mL nystatin. The cells were maintained in a humidified, 5% C0₂ atmosphere at 37°C.

After trypsinization, the cells are counted and adjusted to a concentration of 50 x 10⁵ cells/mL (varied according to cancer cell line) . 200μL of the cell suspension was plated into wells of 4 rows of a 96-well microtiter plate. After the cells were allowed to attach for four hours, 2μL of DMSO containing test sample solutions were added to quadruplicate wells. Initial dose-response finding experiments, using order of magnitude test sample dilutions were used to determine the range of doses to be examined. Well absorbancies at 540nm were then measured on a BIO RAD MP450 plate reader. The mean absorbance of quadruplicate test sample treated wells was compared to the control, and the results expressed as the percentage of control absorbance plus/minus the standard deviation. The reduction of MTT to a purple formazan product correlates in a linear manner with the number of living cells in the well. Thus, by measuring the absorbance of the reduction product, a quantitation of the percent of cell survival at a given dose of test sample can be obtained. Control wells contained a final concentration of 1% DMSO.

Two of the samples were first tested by this protocol. Sample MM1 represented a very crude isolate of cocoa procyanidins and contained appreciable quantities of caffeine and theobromine. Sample MM2 represented a cocoa procyanidin isolate partially purified by gel permeation chromatography. Caffeine and theobromine were absent in MM2. Both samples were screened for activity against the following cancer cell lines using the procedures previously described: HCT 116 colon cancer

ACHN renal adenocarcinoma SK-5 melanoma A498 renal adenocarcinoma MCF-7 breast cancer PC-3 proεtate cancer

CAPAN-2 pancreatic cancer Little or no activity was observed with MM1 on any of the cancer cell lines investigated. MM2 was found to have activity against HCT-116, PC-3 and ACHN cancer cell lines. However, both MM1 and MM2 were found to interfere with MTT such that it obscured the decrease in absorbance that would have reflected a decrease in viable cell number. This interference also contributed to large error bars, because the chemical reaction appeared to go more quickly in the wells along the perimeter of the plate. A typical example of these effects is shown in Figure 5. At the high concentrations of test material, one would have expected to observe a large decrease in survivors rather than the high survivor levels shown. Nevertheless, microscopic examinations revealed that cytσtoxic effects occurred, despite the MTT interference effects. For instance, an IC₅₀ value of 0.5μg/mL for the effect of MM2 on the ACHN cell line was obtained in this manner.

These preliminary results, in the inventors' view, required amendment of the asεay procedureε to preclude the interference with MTT. Thiε was accompliεhed as follows. After incubation of the plates at 37°C in a humidified, 5% C0₂ atmosphere for 18 hours, the medium was carefully aspirated and replaced with fresh alpha-MEM media. This media was again aspirated from the wells on the third day of the assay and replaced with lOOμL of freshly prepared McCoy's medium. llμL of a 5mg/mL stock solution of MTT in

PBS (Phosphate Buffered Saline) were then added to the wells of each plate. After incubation for 4 hours in a humidified, 5% C0₂ atmosphere at 37°C, lOOμL of 0.04 N HCl in isopropanol was added to all wells of the plate, followed by thorough mixing to solubilize the formazan produced by any viable cells. Additionally, it was decided to subfractionate the procyanidins to determine the specific components responsible for activity.

The subfractionation procedures previously described were used to prepare samples for further screening. Five fractions representing the areas shown in Figure 1 and component(s) distribution shown in Figures 4A - 4E were prepared. The samples were coded MM2A through MM2E to reflect these analytical characterizations and to designate the absence of caffeine and theobromine.

Each fraction was individually screened against the HCT-116, PC-3 and ACHN cancer cell lines. The results indicated that the activity did not concentrate to any one specific fraction. This type of result was not considered unusual, since the components in "active" natural product isolates can behave synergistically. In the case of the cocoa procyanidin isolate (MM2) , over twenty detectable components comprised the isolate. It was considered possible that the activity was related to a combination of components present in the different fractions, rather than the activity being related to an individual component(s) . On the basis of these results, it was decided to combine the fractions and repeat the assays against the same cancer cell lines. Several fraction combinations produced cytotoxic effects against the PC-3 cancer cell lines. Specifically, IC₅₀ values of 40μg/mL each for MM2A and MM2E combination, and of 20μg/mL each for MM2C and MM2E combination, were obtained. Activity was also reported against the HCT-116 and ACHN cell lines, but as before, interference with the MTT indicator precluded precise observations. Replicate experiments were repeatedly performed on the HCT-116 and ACHN lines to improve the data. However, these results were inconclusive due to bacterial contamination and exhaustion of the test εample material. Figures 6A-6D εhow the dose-response relationship between combinations of the cocoa extracts and PC-3 cancer cells. Nonetheless, from this data, it is clear that cocoa extracts, especially cocoa polyphenols or procyanidins, have significant anti-tumor; anti-cancer or antineoplastic activity, especially with respect to human

PC-3 (prostate) , HCT-116 (colon) and ACHN (renal) cancer cell lines. In addition, those results suggest that specific procyanidin fractions may be responsible for the activity against the PC-3 cell line.

Example 7: Anti-Cancer, Anti-Tumor or Antineoplastic Activity of cocoa Extracts (Procyanidins)

To confirm the above findings and further study fraction combinations, another comprehensive screening was performed.

All prepared materials and procedures were identical to those reported above, except that the standard 4-replicates per test dose was increased to 8 or 12- replicates per test dose. For this study, individual and combinations of five cocoa procyanidin fractions were screened against the following cancer cell lines.

PC-3 Prostate KB Nasopharyngeal/HeLa HCT-116 Colon

ACHN Renal MCF-7 Breast SK-5 Melanoma A-549 Lung CCRF-CEM T-cell leukemia

Individual screenings consisted of assaying different dose levels (0.0l-l00μg/mL) of fractions A, B, C, D, and E (See Figs. 4A-4E and discussion thereof, supra) against each cell line. Combination screenings consisted of combining equal dose levels of fractions A+B, A+C, A+D, A+E, B+C, B+D, B+E, C+D, C+E, and D+E against each cell line. The results from these assays are individually discussed, _^followed by an overall summary. A. PC-3 Prostate Cell Line

Figures 7A - 7H show the typical dose response relationship between cocoa procyanidin fractions and the PC- 3 cell line. Figures 7D and 7E demonstrate that fractions D and E were active at an IC₅₀ value of

75μg/mL. The IC_S0 values that were obtained from dose- response curves of the other procyanidin fraction combinations ranged between 60 - 80μg/mL when fractions D or E were present. The individual IC₅₀ values are listed in Table 6.

B. KB Nasopharynqeal/HeLa Cell Line

Figures 8A - 8H εhow the typical doεe response relationship between cocoa procyanidin fractions and the KB Nasopharyngeal/HeLa cell line. Figures 8D and 8E demonstrate that fractions D and E were active at an IC₅₀ value of 75μg/mL. Figures 8F - 8H depict representative results obtained from the fraction combination study. In this case, procyanidin fraction combination A+B had no effect, whereas fraction combinations B+E and D+E were active at an IC₅₀ value of 60μg/mL. The IC₅₀ values that were obtained from other dose reεponεe curves from other fraction combinations ranged from 60 - 80μg/mL when fractions D or E were present. The individual IC_$0 values are listed in Table 6. These results were essentially the same as those obtained against the PC-3 cell line.

C. HCT-116 Colon Cell Line

Figure 9A - 9H show the typical dose response relationships between cocoa procyanidin fractions and the HCT-116 colon cell line. Figures 9D and 9E demonstrate that fraction E was active at an IC₅₀ value of approximately

400μg/mL. This value was obtained by extrapolation of the existing curve. Note that the slope of the dose response curve for fraction D also indicated activity. However, no IC₅₀ value was determined from this plot, since the slope of the curve was too shallow to obtain a reliable value. Figures 9F - 9H depict representative results obtained from the fraction combination study. In this case, procyanidin fraction combination B+D did not show appreciable activity, whereas fraction combinations A+E and D+E were active at IC₅₀ values of 500μg/mL and 85μg/mL, respectively. The IC₅₀ values that were obtained from dose response curves of other fraction combinations averaged about 250μg/mL when fraction E was present. The extrapolated IC₅₀ values are listed in Table 6.

D. ACHN Renal Cell Line

Figure 10A - 10H show the typical dose response relationships between cocoa procyanidin fractions and the ACHN renal cell line. Figures 10A - 10E indicated that no individual fraction was active against this cell line. Figures 10F - 10H depict representative results obtained from the fraction combination study. In this case, procyanidin fraction combination B+C was inactive, whereas the fraction combination A+E resulted in an extrapolated IC₅₀ value of approximately 500μg/mL. Dose response curves similar to the C+D combination were considered inactive, since their εlopes were too shallow. Extrapolated IC₅₀ values for other fraction combinations are listed in Table 6.

E. A-549 Luncr Cell Line

Figures 11A - 11H show the typical dose response relationships between cocoa procyanidin fractions and the A- 549 lung cell line. No activity could be detected from any individual fraction or combination of fractions at the doses used in the assay. However, procyanidin fractions may nonetheless have utility with respect to this cell line.

T . SK-5 Melanoma Cell Line

Figure 12A - 12H show the typical dose response relationships between cocoa procyanidin fractions and the

SK-5 melanoma cell line. No activity could be detected from any individual fraction or combination of fractions at the doses used in the assay. However, procyanidin fractions may nonetheless have utility with respect to this cell line. G. MCF-7 Breast cell Line

Figures 13A - 13H show the typical dose response relationships between cocoa procyanidin fractions and the MCF-7 breast cell line. No activity could be detected from any individual fraction or combination of fractions at the doses used in the assay. However, procyanidin fractions may nonetheless have utility with respect to this cell line.

H. CCRF-CEM T-Cell Leukemia Line

A typical dose response curves were originally obtained against the CCRF-CEM T-cell leukemia line. However, microscopic counts of cell number versus time at different fraction concentrations indicated that 500 μg of fractions A, B and D effected an 80% growth reduction over a four day period. A representative dose response relationship is shown in Figure 14. I. Summary

The IC₅₀ values obtained from these assays are collectively listed in Table 6 for all the cell lines except for CCRF-CEM T-cell leukemia. The T-cell leukemia data was intentionally omitted from the Table, since a different assay procedure was used. A general summary of these results indicated that the most activity was associated with fractions D and E. These fractions were most active against the PC-3 (prostate) and KB (nasopharyngeal/HeLa) cell lines. These fractions also evidenced activity against the HCT-116 (colon) and ACHN (renal) cell lines, albeit but only at much higher doses. No activity was detected against the MCF-7 (breast) , SK-5 (melanoma) and A-549 (lung) cell lines.

However, procyanidin fractions may nonetheless have utility with respect to these cell lines. Activity was also shown against the CCRF-CEM (T-cell leukemia) cell line. It should also be noted that fractions D and E are the most complex compoεitionally. Nonetheleεε, from this data it is clear that cocoa extracts, especially cocoa procyanidins, have significant anti-tumor, anti-cancer or antineoplastic activity.

Table 6: IC₅₀ Values for Cocoa Procyanidin Fractions

Against Various Cell Lines

(IC₅₀ values in μg/mL)

Values above lOOμg/mL were extrapo ate rom dose response curves Example 8. Anti-cancer, Anti-Tumor or Antineoplastic Activity of Cocoa Extracts (Procyanidins)

Several additional in vitro assay procedures were used to complement and extend the resultε presented in Examples 6 and 7. Method A. Crystal Violet Staining Assay

All human tumor cell lines were obtained from the American Type Culture Collection. Cells were grown as monolayers in IMEM containing 10% fetal bovine serum without antibiotics. The cells were maintained in a humidified, 5% C0₂ atmosphere at 37°C.

After trypsinization, the cells were counted and adjusted to a concentration of 1,000-2,000 cells per 100 mL. Cell proliferation was determined by plating the cells (1,000-2,000 cells/well) in a 96 well microtiter plate. After addition of 100μL cellε per well, the cells were allowed to attach for 24 hours. At the end of the 24 hour period, various cocoa fractionε were added at different concentrations to obtain dose response resultε. The cocoa fractions were diεsolved in media at a 2 fold concentration and lOOμL of each solution was added in triplicate wells. On consecutive days, the plates were stained with 50μL crystal violet (2.5g crystal violet dissolved in 125mL methanol, 375mL water), for 15 min. The stain was removed and the plate was gently immersed into cold water to remove excess stain. The washingε were repeated two more times, and the plates allowed to dry. The remaining stain was solubilized by adding lOOμL of 0.1M εodium citrate/50% ethanol to each well. After solubilization, the number of cellε were quantitated on an ELISA plate reader at 540nm (reference filter at 410nm) . The results from the ELISA reader were graphed with absorbance on the y-axis and days growth on the x-axis. Method B. Soft Aσar Cloning Assay Cells were cloned in soft agar according to the method described by Nawata et al. (1981) . Single cell _^suspensions were made in media containing 0.8% agar with various concentrations of cocoa fractions. The suspensions were aliquoted into 35mm dishes coated with media containing 1.0% agar. After 10 days incubation, the number of colonies greater than 60μm in diameter were determined on an Ominicron 3600 Image Analysis System. The results were plotted with number of colonies on the y-axis and the concentrations of a cocoa fraction on the x-axis.

Method C. XTT-Microculture Tetrazolium Assay The XTT assay procedure described by Scudiero et al. (1988) was used to screen various cocoa fractions. The XTT assay was essentially the same as that described using the MTT procedure (Example 6) except for the following modifications. XTT ( (2,3-biε (2-methoxy-4-nitro-5- sulfophenyl) -5-( (phenylamino) carbonyl) -2H-tetrazolium hydroxide) was prepared at lmg/mL medium without serum, prewarmed to 37°C. PMS was prepared at 5mM PBS. XTT and PMS were mixed together; 10μL of PMS per L XTT and 50μL PMS-XTT were added to each well. After an incubation at 37°C for 4 hr, the plates were mixed 30 min. on a mechanical shaker and the absorbance measured at 450-600nm. The results were plotted with the absorbance on the y-axis and days growth or concentration on the x-axis.

For methodε A and C, the resultε were also plotted as the percent control as the y-axiε and dayε growth or concentration on the x-axis.

A comparison of the XTT and Crystal Violet Assay procedures was made with cocoa fraction D & E (Example 3B) against the breast cancer cell line MCF-7 pl68 to determine which assay was most εenεitive. As shown in Figure 15A, both asεayε εhowed the εame doεe-response effects for concentrations >75μg/mL. At concentrations below this value, the crystal violet assay showed higher standard deviations than the XTT assay results. However, since the crystal violet assay was easier to use, all subsequent assays, unless otherwise specified, were performed by this procedure. Crystal violet assay results are presented

(Figures 15B-15E) to demonstrate the effect of a crude polyphenol extract (Example 2) on the breast cancer cell line MDA MB231, prostate cancer cell line PC-3, breast cancer cell line MCF-7 pl63, and cervical cancer cell line Hela, respectively. In all cases a dose of 250μg/mL completely inhibited all cancer cell growth over a period of 5-7 days. The Hela cell line appeared to be more sensitive to the extract, since a lOOμg/mL dose also inhibited growth. Cocoa fractions from Example 3B were also assayed against Hela and another breaεt cancer cell line SKBR-3. The results (Figures 15F and 15G) showed that fraction D & E has the highest activity. As shown in Figures 15H and 151, IC₅₀ values of about 40μg/mL D & E were obtained from both cancer cell lines. The cocoa fraction D & E was also tested in the soft agar cloning assay which determines the ability of a test compound(s) to inhibit anchorage independent growth. As shown in Figure 15J, a concentration of lOOμg/mL completely inhibited colony formation of Hela cells. Crude polyphenol extracts obtained from eight different cocoa genotypes representing the three horticultural races of cocoa were also assayed against the Hela cell line. As shown in Figure 15K all cocoa varieties showed similar dose-response effects. The UIT-l variety exhibited the most activity against the Hela cell line. These results demonstrated that all cocoa genotypes possess a polyphenol fraction that elicits activity against at least one human cancer cell line that is independent of geographical origin, horticultural race, and genotype.

Another series of assays were performed on crude polyphenol extracts prepared on a daily basis from a one ton scale traditional 5-day fermentation of Brazilian cocoa beans, followed by a 4-day sun drying stage. The results shown in Figure 15L showed no obvious effect of these early processing stages, suggesting little change in the composition of the polyphenols. However, it is known

(Lehrian and Patterson, 1983) that polyphenol oxidase (PPO) will oxidize polyphenols during the fermentation stage. To determine what effect enzymatically oxidized polyphenols would have on activity, another experiment was performed. Crude PPO was prepared by extracting finely ground, unfermented, freeze dried, defatted Brazilian cocoa beans with acetone at a ratio of lgm powder to lOmL acetone. The slurry was centrifuged at 3,000 rpm for 15 min. This was repeated three times, discarding the supernatant each time with the fourth extraction being poured through a Buchner filtering funnel. The acetone powder was allowed to air dry, followed by assay according to the procedureε described by McLord and Kilara, (1983). To a solution of crude polyphenols (lOOmg/lOmL Citrate-Phosphate buffer, 0.02M, pH 5.5) lOOmg of acetone powder (4,000 units activity/mg protein) was added and allowed to stir for 30 min. with a stream of air bubbled through the slurry. The sample was centrifuged at 5,000xg for 15 min. and the supernatant extracted 3X with 20mL ethyl acetate. The ethyl acetate extracts were combined, taken to dryness by distillation under partial vacuum and 5mL water added, followed by , lyophilization. The material was then assayed against Hela cells and the dose-response compared to crude polyphenol extracts that were not enzymatically treated. The results

(Figure 15M) showed a significant shift in the dose-response curve for the enzymatically oxidized extract, showing that the oxidized products were more inhibitory than their native forms.

Example 9: Antioxidant Activity of Cocoa Extracts Containing Procyanidins _____ Evidence in the literature suggests a relationship between the consumption of naturally occurring antioxidantε (Vitaminε C, E and Beta-carotene) and a lowered incidence of disease, including cancer (Designing Foodε, 1993; Caragay, 1992) . It iε generally thought that theεe antioxidantε affect certain oxidative and free radical processes involved with some types of tumor promotion. Additionally, some plant polyphenolic compounds that have been shown to be anticarcinogenic, also possess substantial antioxidant activity (Ho et al., 1992; Huang et al., 1992). To determine whether cocoa extracts containing procyanidins possessed antioxidant properties, a standard Rancimat method was employed. The procedures described in Examples 1, 2 and 3 were used to prepare cocoa extractε which were manipulated further to produce two fractions from gel permeation chromatography. These two fractions are actually combined fractions A through C, and D and E (See Figure 1) whose antioxidant properties were compared against the synthetic antioxidants BHA and BHT.

Peanut Oil was pressed from unroasted peanuts after the skins were removed. Each test compound was spiked into the oil at two levels, - 100 ppm and - 20 ppm, with the actual levels given in Table 7. 50μL of methanol _^solubilized antioxidant was added to each sample to aid in dispersion of the antioxidant. A control sample was prepared with 50μL of methanol containing no antioxidant.

The samples were evaluated in duplicate, for oxidative stability using the Rancimat stability test at 100°C and 20 cc/min of air. Experimental parameters were chosen to match those used with the Active Oxygen Method (AOM) or Swift Stability Test (Van Oosten et al., 1981). 1 typical Rancimat trace iε shown in Figure 16. Results are reported in Table 8 as hours required to reach a peroxide level of 100 meq. Table 7: Concentrations of Antioxidants

Table 8: Oxidative Stability of Peanut Oil with Various Antioxidants

These results demonstrated increased oxidative stability of peanut oil with all of the additives tested. The highest increase in oxidative stability was realized by the sample spiked with the crude ethyl acetate extract of cocoa. These results demonstrated that cocoa extracts containing procyanidins have antioxidant potential equal to or greater than equal amounts of synthetic BHA and BHT.

Accordingly, the invention may be employed in place of BHT or BHA in known utilities of BHA or BHT, for instance aε an antioxidant and/or food additive. And, in thiε regard, it is noted too that the invention is from an edible source. Given these resultε, the skilled artisan can also readily determine a suitable amount of the invention to employ in such "BHA or BHT" utilities, e.g., the quantity to add to food, without undue experimentation. Example 10: Topoisomerase II Inhibition Study DNA topoisomerase I and II are enzymes that catalyze the breaking and rejoining of DNA strandε, thereby controlling the topological states of DNA (Wang, 1985) . In addition to the study of the intracellular function of topoisomerase, one of the most significant findings has been the identification of topoisomerase II as the primary cellular target for a number of clinically important antitumor compounds (Yamashita et al., 1990) which include intercalating agents ( -AMSA, Adriamycin® and ellipticine) as well as nonintercalating epipodophyllotoxins. Several lines of evidence indicate that some antitumor drugs have the common property of stabilizing the DNA - topoisomerase II complex ("cleavable complex") which upon exposure to denaturing agents results in the induction of DNA cleavage

(Muller et al., 1989). It has been suggested that the cleavable complex formation by antitumor drugs produces bulky DNA adducts that can lead to cell death. According to this attractive model, a specific new inducer of DNA topoisomerase II cleavable complex is useful as an anti-cancer, anti-tumor or antineoplastic agent. In an attempt to identify cytotoxic compounds with activities that target DNA, the cocoa procyanidins were screened for enhanced cytotoxic activity against several DNA - damage sensitive cell lines and enzyme asεay with human topoiεomeraεe II obtained from lymphoma.

A. Decatenation of Kinetoplast DNA by Topoisomerase II The in vitro inhibition of topoisomerase II decatenation of kinetoplast DNA, aε deεcribed by Muller et al. (1989), waε performed as follows. Nuclear extracts containing topoisomerase II activity were prepared from human lymphoma by modifications of the methods of Miller et al. (1981) and Dankε et al. (1988) . One unit of purified enzyme waε enough to decatenate 0.25 μg of kinetoplaεt DNA in 30 min. at 34°C. Kinetoplaεt DNA was obtained from the trypanosome Crithidia fasciculata . Each reaction was carried out in a 0.5mL icrocentrifuge tube containing 19.5μL H₂0, 2.5μL 10X buffer (IX buffer contains 50mM tris- HC1, pH 8.0, 12OmM KCl, lOmM MgCl₂, 0.5mM ATP, 0.5mM dithiothreitol and 30μg BSA/mL) , lμL kinetoplast DNA (0.2μg), and lμL DMSO-containing cocoa procyanidin test fractions at various concentrations. This combination was mixed thoroughly and kept on ice. One unit of topoisomerase was added immediately before incubation in a waterbath at 34°C for 30 min. Following incubation, the decatenation assay was stopped by the addition of 5μL stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol) and placed on ice. DNA was electrophoresed on a 1% agarose gel in TAE buffer containing ethidiu bromide (0.5μg/mL). Ultraviolet illumination at 310nm wavelength allowed the visualization of DNA. The gels were photographed uεing a Polaroid Land camera.

Figure 17 showε the results of these experiments. Fully catenated kinetoplaεt DNA does not migrate into a 1% agarose gel. Decatenation of kinetoplast DNA by topoisomerase II generates bands of monomeric DNA (monomer circle, forms I and II) which do migrate into the gel. Inhibition of the enzyme by addition of cocoa procyanidins is apparent by the progressive disappearance of the monomer bands as a function of increasing concentration. Based on these results, cocoa procyanidin fractions A, B, D, and E were shown to inhibit topoiεo eraεe II at concentrationε ranging from 0.5 to 5.0μg/mL. Theεe inhibitor concentrationε were very similar to those obtained for mitoxanthrone and m-AMSA (4'-(9- acridinyla ino) ethanesulfon-m-aniεidide) . B. Drug Sensitive Cell Lines

Cocoa procyanidins were screened for cytotoxicity against several DNA-damage sensitive cell lines. One of the cell lines was the xrs-6 DNA double strand break repair mutant developed by P. Jeggo (Kemp et al., 1984). The DNA repair deficiency of the xrs-6 cell line renders them particularly sensitive to x-irradiation, to compounds that produce DNA double strand breaks directly, such as bleomycin, and to compounds that inhibit topoisomerase II, and thus may indirectly induce double strand breaks as suggested by Warters et al. (1991) . The cytotoxicity toward the repair deficient line was compared to the cytotoxicity against a DNA repair proficient CHO line, BR1. Enhanced cytotoxicity towards the repair deficient (xrs-6) line was interpreted as evidence for DNA cleavable double strand break formation.

The DNA repair competent CHO line, BR1, was developed by Barrows et al. (1987) and expresses 0⁶- alkylguanine - DNA - alkyltransferase in addition to normal CHO DNA repair enzymes. The CHO double strand break repair deficient line (xrs-6) was a generous gift from Dr. P. Jeggo and co-workers (Jeggo et al., 1989) . Both of these lines were grown as monolayers in alpha-MEM containing serum and antibiotics as described in Example 6. Cells were maintained at 37°C in a humidified 5% C0₂ atmosphere. Before treatment with cocoa procyanidins, cells grown as monolayers were detached with trypsin treatment. Assays were performed using the MTT asεay procedure deεcribed in Example 6. The reεultε (Figure 18) indicated no enhanced cytotoxicity towards the xrs-6 cells suggesting that the cocoa procyanidins inhibited topoisomerase II in a manner different from cleavable double strand break formation. That is, the cocoa procyanidins interact with topoisomerase II before it has interacted with the DNA to form a noncleavable complex.

Noncleavable complex forming compounds are relatively new discoveries. Members of the anthracyclines, podophyllin alkaloids, anthracenediones, acridines, and ellipticineε are all approved for clinical anti-cancer, anti-tumor or antineoplastic use, and they produce cleavable complexes (Liu, 1989) . Several new clasεeε of topoisomerase II inhibitors have recently been identified which do not appear to produce cleavable complexes. These include a onafide (Hsiang et al., 1989), distamycin (Fesen et al., 1989), flavanoids (Yamashita et al., 1990), saintopin (Yamashita et al., 1991), membranone (Drake et al., 1989), terpenoids (Kawada et al., 1991), anthrapyrazoles (Fry et al., 1985), dioxopiperazines (Tanabe et al., 1991), and the marine acridine - dercitin (Burres et al., 1989) . Since the cocoa procyanidins inactivate topoisomerase II before cleavable complexes are formed, they have chemotherapy value either alone or in combination with other known and mechanistically defined topoisomerase II inhibitors. Additionally, cocoa procyanidins also appear to be a novel class of topoisomerase II inhibitors, (Kaεhiwada et al., 1993) and may thus be less toxic to cells than other known inhibitors, thereby enhancing their utility in chemotherapy.

The human breast cancer cell line MCF-7 (ADR) which expresεes a membrane bound glycoprotein (gpl70) to confer multi-drug resiεtance (Leonesεa et al., 1994) and its parental line MCF-7 pl68 were used to assay the effects of cocoa fraction D & E. As shown in Figure 19, the parental line was inhibited at increasing dose levels of fraction D & E, whereas the Adria ycin (ADR) resistant line was less effected at the higher doses. These results show that cocoa fraction D & E has an effect on multi-drug resistant cell lines. Example 11: Synthesis of Procyanidins

The synthesis of procyanidins was performed according to the procedures developed by Delcour et al. (1983) , with modification. In addition to condensing (+)-catechin with dihydroquercetin under reducing conditions, (-)-epicatechin was also used to reflect the high concentrations of (-)-epicatechin that naturally occur in unfermented cocoa beans. The synthesis products were isolated, purified, analyzed, and identified by the procedures described in Examples 3, 4 and 5. In this manner, the biflavanoids, triflavanoids and tetraflavanoids are prepared and used as analytical standardε and, in the manner described above with respect to cocoa extracts. Example 12: Assay of Normal Phase Semi-Preparative

Fractions

Since the polyphenol extracts are compoεitionally complex, it waε necessary to determine which componentε were active againεt cancer cell lineε for further purification, dose-response assayε and comprehenεive εtructural identification. A normal phase semi preparative HPLC separation (Example 3B) was used to separate cocoa procyanidins on the basis of oligomeric size. In addition to the original extract, twelve fractions were prepared

(Figures 2B and 15 0) and assayed at lOOμg/mL and 25μg/mL doses againεt Hela and SKBR-3 cancer cell lines to determine which oligomer possessed the greatest activity. As shown in Figures 20A and B, fractions 4-11 (pentamer-dodecamer) significantly inhibited HeLa and SKBr-3 cancer cell lines at the lOOμg/mL level. These resultε indicated that these specific oligomerε had the greatest activity against Hela and SKBR-3 cellε. Additionally, normal phase HPLC analysis of cocoa fraction D & E indicated that this fraction, used in previous investigations, e.g., Example 7, was enriched with these oligomers. Example 13: HPLC Purification Methods

Method A. GPC Purification

Procyanidins obtained as in Example 2 were partially purified by liquid chromatography on Sephadex LH 20 (72.5 x 2.5cm), using 100% methanol as the eluting solvent, at a flow rate of 3.5mL/min. Fractions of the eluent were collected after the first 1.5 hours, and the fractions were concentrated by a rotary evaporator, redissolved in water and freeze dried. These fractions were referred to as pentamer enriched fractions. Approximately

2.00g of the extract obtained from Example 2 was subfractionated in this manner. Reεults are εhown in Table

9.

Table 9: Composition of Fractions Obtained:

co z m ND = not detected m tr = trace amount

30

C m ro

Method B. Normal Phase Separation

Procyanidins obtained as Example 2 were εeparated purified by normal phase chromatography on Supelcosil LC-Si, lOoA, 5μm (250 x 4.6mm), at a flow rate of l.OmL/min, or, in the alternative, Lichrosphere* Silica 100, lOOA, 5μm (235 x

3.2mm), at a flow rate of 0.5mL/min. Separations were aided by a step gradient under the following conditions: (Time,

%A, %B) ; (0, 82, 14), (30, 67.6, 28.4), (60, 46, 50), (65,

10, 86), (70, 10, 86). Mobile phase composition was A = dichloromethane; B = methanol; and C = acetic acid:water

(1:1). Components were detected by fluorescence where λ_Λex_V = 276nm and λ„em_ = 316nm,' and by■* UV at 280nm. The injection volume was 5.OμL (20mg/mL) of the procyanidins obtained from Example 2. These results are shown in Fig. 40A and 40B.

In the alternative, separations were aided by a step gradient under the following conditions: (Time, %A, %B) ; (0, 76, 20); (25, 46, 50); (30, 10, 86). Mobile phase composition was A = dichloromethane; B = methanol; and C = acetic acid : water (1:1). The results are shown in Fig. 41A and 4IB.

Method C. Reverse - Phase Separation Procyanidins obtained as in Example 2 were separated purified by reverse phase chromatography on Hewlett Packard Hypersil ODS 5μm. (200 x 2.1mm), and a

Hewlett Packard Hypersil ODS 5μm guard column (20 x 2.1mm). The procyanidinε were eluted with a linear gradient of 20% B into A in 20 minuteε, followed by a column waεh with 100% B at a flow rate of 0.3mL/min. The mobile phase composition was a degassed mixture of B = 1.0% acetic acid in methanol and A = 2.0% acetic acid in nanopure water. Components were detected by UV at 280nm, and fluorescence where λ_eχ = 276nm and λem = 316nm; and the injection volume was 2.0μ' l

(20mg/mL) .

Example 14: HPLC Separation of Pentamer Enriched

Fractions

Method A. Semi-Preparative Normal Phase HPLC

The pentamer enriched fractions were further purified by semi-preparative normal phase HPLC by a Hewlett

Packard 1050 HPLC system equipped with a Millipore - Waters model 480 LC detector set at 254nm, which was assembled with a Pharmacia Frac-100 Fraction Collector set to peak mode. Separations were effected on a Supelco 5μm Supelcosel LC-Si, lOOA column (250 x 10mm) connected with a Supelco 5μ Supelguard LC-Si guard column (20 x 4.6mm) . Procyanidins were eluted by a linear gradient under the following conditions: (Time, %A, %B) ; (0, 82, 14), (30, 67.6, 28.4) , (60, 46, 50), (65, 10, 86), (70, 10, 86) followed by a 10 minute re-equilibration. Mobile phase composition was A = dichloromethane; B = methanol; and C = acetic acid:water (1:1) . A flow rate of 3mL/min was used. Components were detected by UV at 254nm; and recorded on a Kipp & Zonan BD41 recorder. Injection volumes ranged from 100-250μl of lOmg of procyanidin extractε diεsolved in 0.25mL 70% aqueous acetone. Individual peaks or select chromatographic regions were collected on timed intervals or manually by fraction collection for further purification and subsequent evaluation.

HPLC conditions: 250 x 100mm Supelco Supelcosil LC-Si (5μm) Semipreparative Column 20 x 4.6mm Supelco Supelcosil LC-Si (5μm) Guard Column

Detector: Waters LC

Spectrophotometer Model 480 <a 254nm Flow rate: 3mL/min. , Column Temperature: ambient, Injection: 250μl of pentamer enriched extract acetic acid:

CH,C1, water (1:1) 82 4

67.6 4 46 4 10 4

10 4

Method B. Reverse Phase Separation

Procyanidin extracts obtained as in Example 13 were filtered through a 0.45μ nylon filter and analyzed by a Hewlett Packard 1090 ternary phase HPLC εystem equipped with a Diode Array detector and a HP model 1046A Programmable Fluorescence Detector. Separations were effected at 45°C on a Hewlett Packard 5μ Hyperεil ODS column (200 x 2.1mm) . The procyanidins were eluted with a linear gradient of 60% B into A followed by a column wash with B at a flow rate of 0.3mL/min. The mobile phase composition was a de-gasεed mixture of B = 0.5% acetic acid in methanol and A = 0.5% acetic acid in nanopure water. Acetic acid levels in A and B mobile phases can be increased to 2%. Components were detected by fluorescence, where λ_eχ = 276nm and λ^ = 316nm, and by UV at 280nm. Concentrations of (+)-catechin and (-)- epicatechin were determined relative to reference standard solutions. Procyanidin levels were estimated by uεing the response factor for (-) -epicatechin.

Method C. Normal Phase Separation

Pentamer enriched procyanidin extracts obtained as 5 in Example 13 were filtered through a 0.45μ nylon filter and analyzed by a Hewlett Packard 1090 Series II HPLC system equipped with a HP Model 1046A Programmable Fluorescence detector and Diode Array detector. Separations were effected at 37°C on a 5μ Phenomenex Lichrosphere* Silica 100

10 column (250 x 3.2mm) connected to a Supelco Supelguard LC-Si 5μ guard column (20 x 4.6mm) . Procyanidins were eluted by linear gradient under the following conditions: (time, %A, %B) ; (0, 82, 14) , (30, 67.6, 28.4) , (60, 46, 50), (65, 10, 86), (70, 10, 86), followed by an 8 minute re-equilibration.

15 Mobile phase composition was A - dichloromethane, B = methanol, and C = acetic acid:water at a volume ratio of 1:1. A flow rate of 0.5mL/min was used. Components were detected by fluorescence, where λ_eχ = 276nm and λ_em = 3l6nm or by UV at 280nm. A representative HPLC chromatogram

20 showing the separation of the various procyanidins is shown in Figure 2 for one genotype. Similar HPLC profiles were obtained from other Theobroma , Herrania and/or their inter or intra specific crosses.

HPLC conditions:

25 250 x 3.2mm Phenomenex Lichrosphere* Silica 100 column (5μ) 20 x 4.6mm Supelco Supelguard LC-Si (5μ) guard column Detectors: Photodiode Array @ 280nm

_ Fluorescence λ.e.x = 27,6nm;' λ_mem = 316nm

30 Flow rate: 0.5 mL/min.

Column temperature: 37°C acetic acid: water d : l)

4 4 4 4

4

Method D. Preparative Normal Phase Separation The pentamer enriched fractions obtained as in

Example 13 were further purified by preparative normal phase chromatography by modifying the method of Rigaud et al., (1993) J. Chrom. 654, 255-260.

Separations were affected at ambient temperature on a 5μ Supelcosil LC-Si lOOA column (50 x 2cm) , with an appropriate guard column. Procyanidins were eluted by a linear gradient under the following conditions: (time, %A, %B, flow rate); (0, 92.5, 7.5, 10); (10, 92.5, 7.5, 40); (30, 91.5, 18.5, 40); (145, 88, 22, 40); (150, 24, 86, 40); (155, 24, 86, 50); (180, 0, 100, 50). Prior to use, the mobile phase components were mixed by the followng protocol:

Solvent A preparation (82% CH₂Cl₂, 14% methanol, 2% acetic acid, 2% water) :

1. Measure 80mL of water and dispense into a 4L bottle.

2. Measure 80mL of acetic acid and dispense into the same 4L bottle.

3. Measure 560mL of methanol and dispenεe into the same 4L bottle. 4. Measure 3280mL of methylene chloride and dispense into the 4L bottle.

5. Cap the bottle and mix well.

6. Purge the mixture with high purity Helium for 5-10 minutes to degas. Repeat steps 1-6 two times to yield 8 volumes of solvent A.

Solvent B preparation (96% methanol, 2% acetic acid, 2% water) : 1. Measure 80mL of water and dispense into a 4L bottle.

2. Measure 80mL of acetic acid and diεpenεe into the εame 4L bottle.

3. Meaεure 3840mL of methanol and diεpense 3840mL of methanol and diεpense into the same 4L bottle.

4. Cap the bottle and mix well.

5. Purge the mixture with high purity Helium for 5-10 minuteε to degas.

Repeat stepε 1-5 to yield 4 volumeε of solvent B. Mobile phase composition was A = methylene chloride with 2% acetic acid and 2% water; B = methanol with 2% acetic acid and 2% water. The column load waε 0.7g in 7mL. components were dected by UV at 254nm. A typical preparative normal phase HPLC separation of cocoa procyanidins is shown in Figure 42.

HPLC Conditions:

Column: 50 x 2cm 5μ Supelcosil LC-Si run @ ambient temperature. Mobile Phase: A = Methylene Choride with 2%

Acetic Acid and 2% Water.

B = Methanol with 2% Acetic Acid and 2% Water.

Gradient/Flow Profile:

Example 15: Identification of Procyanidins

Procyanidins obtained as in Example 14, method D were analyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight/Mass Spectrometry (MALDI-TOF/MS) using a HP G2025A MALDI-TOF/MS system equipped with a Lecroy 9350 500 MHz Oscilloscope. The instrument was calibrated in accordance with the manufacturer's instructions with a low molecular weight peptide standard (HP Part No. G2051A) or peptide standard (HP Part No. G2052A) with 2,5- dihydroxybenzoic acid (DHB) (HP Part No. G2056A) as the sample matrix. One (1.0) mg of sample was dissolved in 500μl of 70/30 methanol/water, and the sample was then mixed with DHB matrix, at a ratio of 1:1, 1:10 or 1:50 (sample.matrix) and dried on a mesa under vacuum. The samples were analyzed in the positive ion mode with the detector voltage set at 4.75kV and the laser power set between 1.5 and 8μJ. Data was collected as the sum of a number of single shots and displayed as units of molecular weight and time of flight. A representative MALDI-TOF/MS is shown in Figure 22A. Figures 22 and C show MALDI-TOF/MS spectra obtained from partially purified procyanidins prepared as described in Example 3, Method A and used for in vitro assessment as described in Examples 6 and 7, and whose results are summarized in Table 6. This data illustrates that the inventive compounds described herein were predominantly found in fractions D-E, but not A-C. The spectra were obtained as follows: The purified D-E fraction was subjected to MALDI- TOF/MS as deεcribed above, with the exception that the fraction was initially purified by SEP-PACK* C-18 cartridge. Five (5) mg of fraction D-E in 1 L nanopure water was loaded onto a pre-equilibrated SEP-PACK* cartridge. The column waε waεhed with 5mL nanopure water to eliminate contaminantε, and procyanidins were eluted with lmL 20% methanol. Fractions A-C were used directly, as they were isolated in Example 3, Method A, without further purification.

These results confirmed and extended earlier results (see Example 5, Table 3, Figs. 20A and B) and indicate that the inventive compounds have utility as εequestrants of cationε. In particular, MALDI-TOF/MS reεultε conclusively indicated that procyanidin oligomers of n = 5 and higher (see Figures 20A and B; and formula under Objects and Summary of the Invention) were strongly associated with anti-cancer activity with the HeLa and SKBR- 3 cancer cell line model. Oligomers of n = 4 or lesε were ineffective with these models. The pentamer structure apparently has a structural motif which is present in it and in higher oligomers which provides the activity.

Additionally, it was observed that the MALDI-TOF/MS data showed strong M⁺ ions of Na⁺, 2 Na⁺, K\ 2 K⁺, Ca⁺⁺, demonstrating the utility as cation sequestrants. Example 16: Purification of Oligomeric Fractions

Method A. Purification by semi-Preparative Reverse Phase HPLC

Procyanidins obtained from Example 14, Method A and B and D were further separated to obtain experimental quantities of like oligomers for further structural identifiction and elucidation (e.g., Example 15, 18, 19, and 20) . A Hewlett Packard 1050 HPLC system equipped with a variable wavelength detector, Rheodyne 7010 injection valve with lmL injection loop was assembled with a Pharmacia FRAC- 100 Fraction Collector. Separations were effected on a Phenomenex Ultracarb* 10μ ODS column (250 x 22.5mm) connected with a Phenomenex 10μ ODS Ultracarb* (60 x lOm ) guard column. The mobile phase composition was A = water; B = methanol used under the following linear gradient conditions: (time, %A) ; (0,85), (60,50), (90,0 and (110,0) at a flow rate of 5 mL/min. Individual peaks or select chromatographic regions were collected on timed intervals or manually by fraction collection for further evaluation by MALDI-TOF/MS and NMR. Injection loads ranged from 25-100mg of material. A representative elution profile is εhown in Fig. 23b. Method B. Modified Semi-Preparative HPLC

Procyanidinε obtained from Example 14, Method A and B and D were further εeparated to obtain experimental quantities of like oligomers for further structural identification and elucidation (e.g., Example 15, 18, 19, and 20) . Supelcosil LC-Si 5μ column (250 x 10mm) with a Supelcosil LC-Si 5μ (20 x 2mm) guard column. The separations were effected at a flow rate of 3.0mL/min, at ambient temperature. The mobile phase composition was A = dichloromethane; B = methanol; and C = acetic acid:water (1:1); used under the following linear gradient conditions: (time, %A, %B) ; (0, 82, 14); (22, 74, 21); (32, 74, 21); (60, 74, 50, 4) ; (61, 82, 14), followed by column re- equilibration for 7 minutes. Injection volumes were 60μl containing 12mg of enriched pentamer. Components were detected by UV at 280nm. A repreεentative elution profile is shown in Figure 23A.

Example 17: Molecular Modeling of Pentamers

Energy minimized structures were determined by molecular modeling using Desktop Molecular Modeller, version 3.0, Oxford University Presε, 1994. Four repreεentative viewε of (EC(4 → 8))₄EC (EC = epicatechin) pentamerε based on the structure of epicatechin are shown in Figures 24 A-D. A helical structure is suggested. In general when epicatechin is the firεt monomer and the bonding iε 4-8, a beta configuration results, when the first monomer is catechin and the bonding is 4-8, an alpha configuration results; and, these reεultε are obtained regardleεε of whether the second monomer is epicatechin or catechin (an exception iε ent-EC(4 — 8)ent-EC) . Figureε 38A - 38P show preferred pentamers, and, Figures 39A to 39P show a library of stereoiεomerε up to and including the pentamer, from which other compounds within the scope of the invention can be prepared, without undue experimentation. Example 18: NMR Evaluation of Pyrocyanidins

¹³C NMR spectroscopy was deemed a generally useful technique for the study of procyanidins, especially as the phenolε uεually provide good quality spectra, whereas proton _^ NMR spectra are considerably broadened. The ¹³C NMR spectra of oligomers yielded useful information for A or B ring substitution patterns, the relative sterochemistry of the C ring and in certain caseε, the poεition of the interflavanoid linkages. Nonetheless, ¹H NMR spectra yielded useful information.

Further, HOHAHA, makes use of the pulse technique to transfer magnetization of a first hydrogen to a second in a εequence to obtain croεε peaks corresponding to alpha, beta, gamma or delta protons. COSY is a 2D-Fourier transform NMR technique wherein vertical and horizontal axes provide ¹H chemical shift and ID spectra; and a point of intersection provides a correlation between protons, whereby spin-spin couplings can be determined. HMQC spectra enhances the sensitivity of NMR spectra of nuclei; other than protons and can reveal cross peakε from secondary and tertiary carbons to the respective protons. APT is a ¹³C technique used in determining the number of hydrogens present at a carbon. An even number of protons at a carbon will result in a positive signal, while an odd number of protons at a carbon will result in a negative signal.

Thus ¹³C NMR, ^!H NMR, HOHAHA (homonuclear Hartmann-

Hahn) , HMQC (heteronuclear multiple quantum coherence) , COSY

(Homonuclear correlation spectroscopy) , APT (attached proton test) , and XHCORR (a variation on HMQC) εpectroεcopy v/ere uεed to elucidate the structures of the inventive compounds.

Method A. Monomer

All spectra were taken in deuterated methanol, at room temperature, at an approximate sample concentration of lOmg/ L. Spectra were taken on a Bruker 500 MHZ NMR, using methanol as an internal standard.

Figures 44A-E represent the NMR spectra which were used to characterize the structure of the epicatechin monomer. Figure 44A shows the ¹H and ¹³C chemical shifts, in tabular form. Figures 44 B-E show ¹H, APT, XHCORR and COSY spectra for epicatechin.

Similarly, Figures 45A-F represent the NMR spectra which were used to characterize the structure of the catechin monomer. Figure 45A showε the *H and ¹³C chemical shiftε, in tabular form. Figureε 44 B-F εhow 'H, ¹³C, APT, XHCORR and COSY εpectra for catechin. Method B. Dimers All spectra were taken in 75% deuterated acetone in D₂0, using acetone as an internal standard, and an approximate sample concentration of lO g/mL.

Figures 46A-G represent the spectra which were used to characterize the structure of the B2 dimer. Fig. 46A shows ¹H and ¹³C chemical shiftε, in tabular form. The terms T and B indicate the top half of the dimer and the bottom half of the dimer.

Figureε 46B and C show the ¹³C and APT spectra, respectively, taken on a Bruker 500 MHZ NMR, at room temperature.

Figures 46D-G show the ¹H, HMQC, COSY and HOHAHA, respectively, which were taken on AMZ-360 MHZ NMR at a -7°C. The COSY spectrum was taken using a gradient pulse.

Figures 47A-G represent the εpectra which were used to characterize the structure of the B5 dimer. Figure 47A shows the ¹³C and ¹H chemical shifts, in tabular form.

Figures 47B-D show the ¹H, ¹³C and APT, respectively, which were taken on a Bruker 500 MHZ NMR, at room temperature. Figure 47E shows the COSY spectrum, taken on an

AMX-360, at room temperature, using a gradient pulse. Figureε 47F and G εhow the HMQC and HOHAHA, respectively, taken on an AMX-360 MHZ NMR, at room temperature.

Method C. Trimer - Epicatechin/Catechin

All spectra were taken in 75% deuterated acetone in D₂0, at -3°C using acetone as an internal εtandard, on an AMX-360 MHZ NMR, and an appropriate εample concentration of lOmg/ L. Figureε 48A-D represent the spectra which were used to characterize the structure of the epicatechin/catechin trimer. These figures show ¹H, COSY, HMQC and HOHAHA, respectively. The COSY spectrum waε taken uεing a gradient pulεe. Method D. Trimer -All Epicatechin

All spectra were taken in 70% deuterated acetone in D₂0, at -1.8°C, using acetone as an internal standard, on an AMX-360 MHZ NMR, and an appropriate sample concentration of lOmg/mL. Figures 49A-D represent the εpectra which were uεed to characterize the εtructure of all epicatechin trimer. These figures show ¹H, COSY, HMQC and HOHAHA, respectively. The COSY spectrum was taken using a gradient pulse. Example 19: Thiolysis of Procyanidins

In an effort to characterize the structure of procyanidinε, benzyl mercaptan (BM) waε reacted with catechin, epicatechin or dimers B2 and B5. Benzyl mercaptan, as well as phloroglucinol and thiophenol, can be utilized in the hydrolysis (thiolysis) of procyanidins in an alcohol/acetic acid environment. Catechin, epicatechin or _^dimer (1:1 mixture of B2 and B5 dimers) (2.5mg) was disεolved in 1.5mL ethanol, lOOμl BM and 50μl acetic acid, and the vessel (Beckman amino acid analysis vessel) was evacuated and purged with nitrogen repeatedly until a final purge with nitrogen was followed by sealing the reaction vessel. The reaction vessel was placed in a heat block at 95°C, and aliquots of the reaction were taken at 30, 60, 120 and 240 minutes. The relative fluorescence of each aliquot iε εhown in Figureε 25A-C, repreεenting epicatechin, catechin and di erε, reεpectively. Higher oligo erε are εi ilarly thiolyzed.

Example 20: Thiolysis and Desulfurization of Dimers

Dimerε B2 and B5 were hydrolyzed with benzyl ercaptan by diεεolving dimer (B2 or B5; 1.0 mg) in 600μl ethanol, 40μl BM and 20μl acetic acid. The mixture waε heated at 95°C for 4 hourε under nitrogen in a Beckman Amino Acid Analyεis vesεel. Aliquotε were removed for analyεiε by reverεe-phaεe HPLC, and 75μl of each of ethanol Raney Nickel and gallic acid (lOmg/mL) were added to the remaining reaction medium in a 2mL hypovial. The veεεel waε purged under hydrogen, and occaεionally εhaken for 1 hour.

The product waε filtered through a 0.45μ filter and analyzed by reverεe-phase HPLC. Representative elution profiles are shown in Figures 26 A and B. Higher oligomers are similarly deεulfurized. This data suggests polymerization of epicatechan or catechin and therefore represents a synthetic route for preparation of inventive compounds.

Example 21: In vivo Activity of Pentamer in MDA MB 231 Nude Mouse Model MDA-MB-231/LCC6 cell line. The cell line was grown in improved minimal essential medium (IMEM) containing 10% fetal bovine serum and maintained in a humidified, 5% CO- at oεphere at 37°C. Mice. Female six to eight week old NCr nu/nu (athymic) mice were purchased through NCI and housed in an animal facility and maintained according to the regulations set forth by the United States Department of Agriculture, and the American Association for the Accreditation of

Laboratory Animal Care. Mice with tumors were weighed every other day, as well as weekly to determine appropriate drug dosing.

Tumor implantation. MDA-MD-231 prepared by tissue culture waε diluted with IMEM to 3.3 x 10° cells/mL and

0.15mL (i.e. 0.5 x 10° cells) were injected subcutaneously between nipples 2 and 3 on each side of the mouse. Tumor volume was calculated by multiplying: length x width x height x 0.5. Tumor volumes over a treatment group were averaged and Student's t test waε used to calculate p values.

Sample preparation. Plasma samples were obtained by cardiac puncture and stored at -70°C with 15-20 mM EDTA for the purposes of blood chemistry determinations. No differences were noted between the control group and experimental groups.

Fifteen nude mice previously infected with 500,000 cells subcutaneously with tumor cell line MDA-MB- 231, were rando Ly separated into three groups of 5 animals each and treated by intraperitoneal injection with one of: (i) placebo containing vehicle alone (DMSO) ; (ii) 2mg/mouse of purified pentameric procyanidin extract as isolated in Example 14 method D in vehicle (DMSO) ; and (iii) lOmg/mouse purified pentameric procyanidin extract as isolated in Example 14, method D in vehicle (DMSO).

The group (iii) mice died within approximately 48 to 72 hours after administration of the lOmg, whereas the group (ii) mice appeared normal. The cause of death of the group (iii) mice was undetermined; and, cannot necessarily be attributed to the administration of inventive compounds.

Nonetheless, lOmg was considered an upper limit with respect to toxicity.

Treatment of groupε (i) and (ii) waε repeated once a week, and tumor growth was monitored for each experimental and control group. After two weekε of treatment, no signs of toxicity were observed in the mice of group (ii) and, the dose administered to this group waε incrementally increaεed by 1/2 log scale each subsequent week. The following Table represents the dosages administered during the treatment schedule for mice of group (ii) :

Week Dose f α/mouse)

1 2

2 2

3 4

4 5 5 5

6 5

7 5

The results of treatment are shown in Figures 27A and B and Table 10.

TABLE 10: IN VIVO ANTI-CANCER RESULTS

These results demonstrate that the inventive fractions and the inventive compounds indeed have utility in antineoplastic compositions, and are not toxic in low to medium dosages, with toxicity in higher dosages able to be determined without undue experimentation. Example 22: Antimicrobial Activity of Cocoa Extracts

Method A:

A study waε conducted to evaluate the antimicrobial activity of crude procyanidin extracts from cocoa beans against a variety of microorganisms important in food spoilage or pathogenesis. The cocoa extracts from Example 2, method A were used in the study. An agar medium appropriate for the growth of each test culture (99mL) was seeded with 1 mL of each cell culture suspenεion in 0.45% εaline (final population 10² - 10⁴ cfu/mL) , and poured into petri dishes. Wells were cut into hardened agar with a #2 cork borer (5mm diameter). The plates were refrigerated ^"at 4°C overnight, to allow for diffusion of the extract into the agar, and subsequently incubated at an appropriate growth temperature for the text organism. The results were as follows:

Sample Zone of Inhibition fmm)

Nl » no inhibition

Antimicrobial activity of purified procyanidin extracts from cocoa beans was demonstrated in another study using the well diffusion assay described above (in Method A) with Staphylococcuε aureus as the text culture. The results were as follows: cocoa extracts: lOmg/100 μL decaffeinated/ detheobrominated acetone extract as in Example 13, method A lOmg/lOO μL dimer (99% pure) as in Example 14, method D lOmg/100 μL tetra er (95% pure) as in Example 14, method D lOmg/100 μL hexamer (88% pure) as in Example 14, method D lOmg/100 μL octamer/nanomer (92% pure) as in Example 14, method D lOmg/100 μL nanomer & higher (87% pure) as in Example 14, method D

Sample Zone of Inhibition (mm)

0.45% saline 0 Dimer 33

Tetramer 27 Hexamer 24 0.45% saline 0

Octamer 22 Nanomer 20 Decaff. /detheo. 26 Method B:

Crude procyanidin extract as in Example 2 , method 2 was added in varying concentrations to TSB (Trypticase Soy Broth) with phenol red (0.08g/L), The TSB were inoculated with cultures of Salmonella enteritidis or S . newport (10⁵ cfu/ L) , and were incubated for 18 hours at 35°C. The results were as follows:

S . enteritidis 5. Newport +

+

+

where + = outgrowth, and - = no growth, as evidenced by the change in broth culture from red to yellow with acid production. Confirmation of inhibition was made by plating from TSB tubes onto XLD plates.

This Example demonstrates that the inventive compounds are useful in food preparation and preservation. This Example further demonstrates that gram negative and gram positive bacterial growth can be inhibited by the inventive compounds. From this, the inventive compounds can be used to inhibit Helicobacter pylori . Heliobacter pylori has been implicated in causing gastric ulcers and stomach cancer. Accordingly, the inventive compounds can be used to treat or prevent these and other maladies of bacteial origin. Suitable routes of administration, dosages, and formulations can be determined without undue experimentation considering factors well known in the art such as the malady, and the age, weight, sex, general health of the subject. Example 23: Halogen-free Analytical Separation of Extract

Procyanidinε obtained from Example 2 were partially purified by Analytical Separation by Halogen-free Normal Phase Chromatography on lOOA Supelcosil LC-Si 5μm (250 x 4.6mm), at a flow rate of l.OmL/min, and a column temperature of 37°C. Separations were aided by a linear gradient under the following conditions: (time, %A, %B) ; (0, 82, 14); (30, 67.6, 28.4) ; (60, 46, 50). Mobile phase composition was A = 30/70 % diethyl ether/Toluene; B = Methanol; and C = acetic acid/water (1:1) . Components were detected by UV at 280nm. A representative elution profile is εhown in Figure 28.

Example 24: Effect of Pore Size of Stationary Phase for

Normal Phase HPLC Separation of Procyanidins

To improve the separation of procyanidins, the use of a larger pore size of the silica stationary phase was inveεtigated. Separations were effected on Silica-300, 5μm, 3θθA (250 x 2.0mm), or, in the alternative, on Silica-1000, 5μm, lOOoA (250 x 2.0mm) . A linear gradient was employed as mobile phase composition was: A = Dichloromethane; B = Methanol; and C = acetic acid/water (1:1). Components were detected by fluorescence, wherein λ_eχ = 276nm and λ^, = 316nm, by UV detector at 280nm. The flow rate waε l._OmL/min, and the oven temperature was 37°C. A representative chromatogram from three different columns

(lOOA pore size, from Example 13, Method D) is shown in

Figure 29. This shows effective pore size for separation of procyanidins.

Example 25: obtaining Desired Procyanidins Via Manipulating Fermentation

Microbial strains representative of the succession associated with cocoa fermentation were selected from the

M&M/Mars cocoa culture collection. The following isolates were used:

Acetobacter aceti ATCC 15973 Lactobacillus εp . (BH 42) Candida krusei (BA 15)

Saccharomyces cerevisiae (BA 13) Bacillus cereus (BE 35) Bacill us sphaericus (ME 12) Each strain was transferred from stock culture to fresh media. The yeasts and Acetobacter were incubated 72 hours at 26°C and the bacilli and Lactobacillus were incubated 48 hours at 37^βC. The slants v/ere harvested with 5mL phosphate buffer prior to use. Cocoa beans were harvested from fresh pods and the pulp and testa removed. The beans were sterilized with hydrogen peroxide (35%) for 20 seconds, followed by treatment with catalase until cessation of bubbling. The beans were rinsed twice with sterile water and the process repeated. The beans were divided into glass jars and processed according to the regimens detailed in the following Table:

97/36597

The bench scale fermentation was performed in duplicate. All treatments were incubated as indicated below:

Day l: 26°C Day 2: 26°C to 50°C

Day 3: 50°C

Day 4: 45°C

Day 5: 40°C The model fermentation was monitored over the duration of the study by plate counts to assess the microbial population and HPLC analysis of the fermentation medium for the production of microbial metabolites. After treatment, the beans were dried under a laminar flow hood to a water activity of 0.64 and were roasted at 66°C for 15 min. Samples were prepared for procyanidin analysis. Three beans per treatment were ground and defatted with hexane, followed by extraction with an acetone:water:acetic acid (70:29.5:0.5%) solution. The acetone solution extract was filtered into vials and polyphenol levels were quantified by normal phase HPLC as in Example 13, method B. The remaining beans were ground and tasted. The cultural and analytical profiles of the model bench-top fermentation process is shown in Figures 30A - C. The procyanidin profiles of cocoa beans subjected to various fermentation treatments is shown in Figure 30D. This Example demonstrates that the invention need not be limited to any particular cocoa genotype; and, that by manipulating fermentation, the levels of procyanidins produced by a particular Theobroma or Herrania species or their inter or intra species specific crosses thereof can be modulated, e.g., enhanced.

The following Table shows procyanidin levels determined in specimens which are representative of the Theobroma genus and their inter and intra species specific crosses. Samples were prepared as in Examples 1 and 2

(methods 1 and 2), and analyzed as in Examples 13, method B. This data illustrates that the extracts containing the inventive compounds are found in Theobroma and Herrania species, and their intra and inter species specific crosses.

Theobroma Species Procyanidin Levels ppm (μg/g) In defatted powder

0) c

00 ω

H

H C H m cn

I m m

H C ι- m to CD

'ND = none detected' sample designated CPATU ^*tr - trace (< 50/vg/gf sample designated ERJON

Example 26: Effect of Procyanidins on NO

Method A.

The purpose of this study is to establish the relationship between procyanidins (as in Example 14, method D) and NO, which is known to induce cerebral vascular dilation. The effects of monomers and higher oligomers, in concentrationε ranging from lOOμg/mL to O.lμg/mL, on the production of nitrateε (the catabolites of NO) , from HUVEC (human umbilical vein endothelial cells) is evaluated.

HUVEC (from Cloneticε) iε investigated in the presence or absence of each procyanidin for 24 to 48 hours. At the end of the experiments, the supernatantε are collected and the nitrate content determined by colorimetric assay. In separate experiments, HUVEC is incubated with acetylcholine, which iε known to induce NO production, in the presence or absence of procyanidins for 24 to 48 hours. At the end of the experiments, the supernatantε are collected and nitrate content is determined by colorimetric assay. The role of NO is ascertained by the addition of nitroarginine or (1)-N- methyl arginine, which are εpecific blockerε of NO εynthase.

Method B. Vasorelaxation of Phenylephrine-Induced Contracted Rat Artery

The effects of each of the procyanidins (lOOμg/mL to O.lμg/mL on the rat artery iε the target for εtudy of vaεorelaxation of phenylephrine-induced contracted rat artery. Iεolated rat artery is incubated in the presence or absence of procyanidins (as in Example 14, method D) and alteration of the muscular tone is assessed by visual inspection. Both contraction or relaxation of the ray artery is determined. Then, using other organs, precontraction of the isolated rat artery is induced upon addition of epinephrine. Once the contraction is stabilized, procyanidins are added and contraction or relaxation of the rat artery is determined. The role of NO iε ascertained by the addition of nitroarginine or (1)-N- methyl arginine. The acetylcholine-induced relaxation of NO, as it is effected by phenylephrine-precontracted rat aorta is shown in Figure 31.

Method C. Induction of Hypotension in the Rat

This method is directed to the effect of each procyanidin (as in Example 14, method D) on blood pressure. Rats are instrumented in order to monitor systolic and diaεtolic blood preεεure. Each of the procyanidins are injected intravenously (dosage range = 100 - O.lμg/kg) , and alteration of blood pressure is assessed. In addition, the effect of each procyanidin on the alteration of blood pressure evoked by epinephrine is determined. The role of NO is ascertained by the addition of nitroarginine or (1)-N- ethyl arginine.

These studieε, together with next Example, illuεtrate that the inventive compounds are uεeful in modulating vasodilation, and are further useful with respect to modulating blood preεεure or addressing coronary conditions, and migraine headache conditions.

Example 27: Effects of Cocoa Polyphenols on Satiety Using blood glucose levels as an indicator for the signal events which occur in vivo for the regulation of appetite and satiety, a series of simple experiments were conducted using a healthy male adult volunteer age 48 to determine whether cocoa polyphenols would modulate glucose levels. Cocoa polyphenols were partially purified from

Brazilian cocoa beans according to the methods described by Clapperton et al. (1992). This material contained no caffeine or theobromine. Fasting blood glucose levels were analyzed on a timed basis after ingestion of 10 fl. oz of Dexicola 75 (caffeine free) Glucose tolerance test beverage (Curtin Matheson 091-421) with and without 75mg cocoa polyphenols. This level of polyphenols represented 0.1% of the total glucose of the test beverage and reflected the approximate amount that would be present in a standard lOOg chocolate bar. Blood glucose levels were determined by using the Accu-Chek III blood glucose monitoring system (Boehringer Mannheim Corporation) . Blood glucose levels were measured before ingestion of test beverage, and after ingestion of the test beverage at the following timed intervals: 15, 30, 45, 60, 75, 90, 120 and 180 minutes. Before the start of each glucose tolerance test, high and low glucose level controls were determined. Each glucose tolerance test was performed in duplicate. A control test solution containing 75mg cocoa polyphenols dissolved in 10 fl. oz. distilled water (no glucose) was also performed. Table 11 below lists the dates and control values obtained for each glucose tolerance experiment performed in this study. Figure 32 repreεentε plotε of the average values with εtandard deviations of blood glucose levels obtained throughout a three hour time course. It is readily apparent that there is a substantial increase in blood sugar levels was obtained after ingestion of a test mixture containing cocoa polyphenols. The difference between the two principal glucose tolerance profiles could not be resolved by the profile obtained after ingestion of a solution of cocoa polyphenols alone. The addition of cocoa polyphenols to the glucose test beverage raised the glucose tolerance profile significantly. This elevation in blood glucose levels is within the range considered to be mildly diabetic, even though the typical glucose tolerance profile was considered to be normal (Davidson, I. et al., Eds. Todd - Sandford Clinical Diagnosis by Laboratory Methods 14th edition; W.B. Saunders Co. ; Philadelphia, PA 1969 Ch. 10, pp. 550-9) . This suggestε that the difference in additional glucoεe was released to the bloodstream, from the glycogen stores, as a result of the inventive compounds. Thus, the inventive compounds can be used to modulate blood glucose levels when in the presence of sugars.

Table 11. Glucose Tolerance Test Dates and Control Results

a = Expected range: 253 - 373mg/dL b = Expected range: 50-80mg/dL

The subject also experienced a facial flush (erythemia) and lightheadedness following ingestion of the inventive compounds, indicating modulation of vasodilation.

The data presented in Tables 12 and 13 illustrates the fact that extracts of the invention pertaining to cocoa raw materials and commercial chocolates, and inventive compounds contained therein can be used as a vehicle for pharmaceutical, veterinary and food science preparations and applications.

Procyanidin Levels in Commercial Chocolates μg/g

CO c to

CO

3 V0 m co x m m

H

33 cr- m

ιo n

ND* None detected.

Procyanidin Levels in Cocoa Raw Materials μg/g

CO c

CD O

H

H

C 10 H m o x m

3 c r m ιo cn

ND* = None etecte .

Example 28: The Effect of Procyanidins on

Cyclooxyqenase 1 & 2

The effect of procyanidins on cyclooxygenase 1 & 2 (COX1/COX2) activitieε was assessed by incubating the enzymes, derived from ram seminal vesicle and εheep placenta, respectively, with arachidonic acid (5μM) for 10 minutes at room temperature, in the presence of varying concentrations of procyanidin solutionε containing monomer to decamer and procyanidin mixture. Turnover waε aεsesεed by using PGE2 EIA kits from Interchim (France) . Indomethacin was uεed as a reference compound. The resultε are preεented in the following Table, wherein the IC₅₀ valueε are expressed in units of μM (except for Sll, which represents a procyanidin mixture prepared from Example 13, Method A and where the the samples SI to S10 represent sequentially procyanidin oligomers (monomer through decamer) as in Example 14, Method D, and IC₅₀ is expresεed in units of mg/mL) .

(^*) expressed as mM with the exception of sample 11, which is mg/mL.

The results of the inhibition studies are presented in Figures 33 A and B, which shows the effects of Indomethacin on COXl and C0X2 activitieε. Figureε 34 A and B shows the correlation between the degree of polymerization of the procyanidin and IC₅₀ with COXl and COX2; Figure 35 shows the correlation between IC₅₀ values on COXl and COX2. And, Figures 36 A through Y show the IC₅₀ values of each εample (SI - Sll) with COXl and COX2.

These results indicate that the inventive compounds have analgesic, anti-coagulant, and anti- inflammatory utilities. Further, COX2 has been linked to Ξolon cancer. Inhibition of C0X2 activity by the inventive compounds illustrates a plausible mechanism by which the inventive compounds have antineoplastic activity against colon cancer.

COXl and C0X2 are also implicated in the synthesis of prostaglandins. Thus, the resultε in this Example also indicate that the inventive compounds can modulate renal functions, immune responεeε, fever, pain, itogeneεis, apoptosis, prostaglandin εynthesis, ulceration (e.g., gastric) , and reproduction. Note that modulation of renal function can affect blood pressure; again implicating the inventive compounds in modulating blood pressure, vasodilation, and coronary conditions (e.g., modulation of angiotensin, bradykinin) .

Reference is made to Seibert et al., PNAS USA 91:12013- 12017 (December, 1994), Mitchell et al. , PNAS USA 90:11693- 11697 (December 1994), Dewitt et al., Cell 83:345-348 (November 3, 1995), Langenbach et al., Cell 83:483-92 (November 3, 1995) and Sujii et al., Cell 83:493-501 (November 3, 1995), Morham et al., Cell 83:473-82 (November 3, 1995) .

Reference is further made to Examples 9, 26, and 27. In Example 9, the anti-oxidant activity of inventive compounds iε shown. In Example 26, the effect on NO is demonstrated. And, Example 27 provides evidence of a facial vasodilation. From the results in this Example, in combination with Exampleε 9, 26 and 27, the inventive compounds can modulate free radical mechanismε driving phyεiological effects. Similarly, lipoxygenase mediated free radical type reactions biochemically directed toward leukotriene εynthesiε can be modulated by the inventive compounds, thus affecting subsequent physiological effects (e.g., inflamation, immune response, coronary conditions, carciongenic mechanisms, fever, pain, ulcertation) .

Thus, in addition to having analgesic properties, there may also be a synergistic effect by the inventive compounds when administered with other analgesics. Likewise, in addition to having antineoplastic properties, there may also be a synergistiic effect by the inventive compounds when administered with other antineoplastic agents.

Example 29:circular Dichroism/Study of Procyanidius

CD studies were undertaken in an effort to elucidate the structure of purified procyanidins as in Example 14, Method D. The εpectra were collected at 25°C using CD spectrum software AVIV 60DS V4.1f.

Samples were scanned from 300nm to 185nm, every l.OOn , at 1.50nm bandwidth. Representative CD spectra are shown in Figures 43A through G, which show the CD spectra of dimer through octamer.

These resultε are indicative of the helical nature of the inventive compoundε.

From the foregoing, it iε clear that the extract and cocoa polyphenolε, particularly the inventive compoundε, as well as the compositions, methods, and kits, of the invention have significant and numerous utilities.

The antineoplastic utility is clearly demonstrated by the in vivo and in vitro data herein and shows that inventive compounds can be used instead of or in conjunction with conventional antineoplastic agents.

The inventive compounds have antioxidant activity like that of BHT and BHA, as well as oxidative stability. Thus, the invention can be employed in place of or in conjuction with BHT or BHA in known utilities of BHA and BHT, such aε an antioxidant, for instance, an antioxidant; food additive.

The invention can also be employed in place of or in conjuction with topoisomerase II-inhibitors in the presently known utilities therefor.

The inventive compounds can be used in food preservation or preparation, as well as in preventing or treating maladies of bacterial origin. Simply the inventive compoundss can be used as an antimicrobial.

The inventive compoundε can also be used aε a cyclo-oxygenaεe and/or lipoxygenase, NO or NO-synthase, or blood or in vivo glucose modulator, and are thus uεeful for treatment or prevention or modulation of pain, fever, inflammation coronary conditionε, ulceration, carcinogenic mechanisms, vasodilation, as well as an analgesic, anti¬ coagulant anti-inflammatory and an immune response modulator.

Further, the invention comprehends the use of the compounds or extracts as a vehicle for pharmaceutical preparations. Accordingly, there are many compositions and methods envisioned by the invention. For instance, antioxidant or preservative compositionε, topoisomerase II- inhibiting compositions, methods for preserving food or any desired item such as from oxidation, and methods for inhibiting topoisomeraεe II. The compoεitionε can comprise the inventive compounds. The methods can comprise contacting the food, item or topoisomerase II with the respective composition or with the inventive compounds.

Other compositions, methods and embodiments of the invention are apparent from the foregoing.

In this regard, it is mentioned that the invention is from an edible source and, that the activity in vitro can demonstrate at least some activity in vivo ; and from the in vi tro and in vivo data herein, doses, routes of administration, and formulations can be obtained without undue experimentation.

Having thus described in detail the preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited by particular details set forth in the above descriptions as many apparent variationε thereof are possible without departing from the spirit or scope of the present invention.

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Claims

WHAT IS CLAIMED IS: 1. A compound of the formula:

wherein: n is an integer from 3 to 12, such that there is a first monomeric unit A, and a plurality of other monomeric units;

R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or 3-(β) -

O-sugar; position 4 is alpha or beta stereochemistry;

X, Y and Z represent positions for bonding between monomeric units, with the provisos that as to the first monomeric unit, bonding of another monomeric unit thereto is at position 4 and Y = Z = hydrogen, and, that when not for bonding monomeric units, X, Y and Z are hydrogen or Z, Y are sugar and X is hydrogen, or X is alpha or beta sugar and Z + Y are hydrogen, or combinations thereof; and the sugar can be optionally ssubstituted with a phenolic moiety via an ester bond.

2. The compound of claim 1 wherein n is 5.

3. The compound of claim 1 wherein the sugar is selected from the group consisting essentially of glucose, galactose, xylose, rhamnose and arabinose.

4. An antineoplastic composition comprising a compound of claim 1 and a carrier or diluent.

5. An antineoplastic composition comprising a compound of claim 2 and a carrier or diluent.

6. An antioxidant composition comprising a compound of claim 1 and a carrier or diluent.

7. An antioxidant composition comprising a compound of claim 2 and a carrier or diluent.

8. An antimicrobial composition comprising a compound of claim 1 and a carrier or diluent.

9. An antimicrobial composition comprising a compound of claim 2 and a carrier or diluent.

10. A cyclo-oxygenase and/or lipoxygenase

modulator composition comprising a compound of claim 1 and a carrier or diluent.

11. A cyclo-oxygenase and/or lipoxygenase

modulator composition comprising a compound of claim 2 and a carrier or diluent.

12. An NO or NO-synthase modulating composition comprising a compound of claim 1 and a carrier or diluent.

13. An NO or NO-synthase modulating composition comprising a compound of claim 2 and a carrier or diluent.

14. An in vivo glucose-modulating composition comprising a compound of claim 1 and a carrier or diluent.

15. An in vivo glucose-modulating composition comprising a compound of claim 2 and a carrier diluent.

16. A method for treating a subject in need of treatment with an antineoplastic agent comprising

administering to the subject an antineoplastic composition of claim 4.

17. A method for treating a subject in need of treatment with an antineoplastic agent comprising

administering to the subject an antineoplastic composition of claim 5.

18. A method for treating a subject in need of treatment with an antioxidant agent comprising administering to the subject an antioxidant composition of a compound of claim 6.

19. A method for treating a subject in need of treatment with an antioxidant agent comprising administering to the subject an antioxidant composition of a compound of claim 7.

20. A method for treating a subject in need of treatment with an antimicrobial agent comprising administering to the subject an antimicrobial composition of a compound of claim 8.

21. A method for treating a subject in need of treatment with an antimicrobial agent comprising

administering to the subject an antimicrobial composition of claim 9.

22. A method for treating a subject in need of treatment with a cyclo-oxygenase or lipoxygenase modulating agent comprising administering to the subject a composition of claim 10.

23. A method for treating a subject in need of treatment with a cyclo-oxygenase or lipoxygenase modulating agent comprising administering to the patient a composition of claim 11.

24. A method for treating a subject in need of treatment with a NO or NO-synthase modulating agent

comprising administering to the subject a composition of claim 12.

25. A method for treating a subject in need of treatment with a NO-modulating agent comprising

administering to the subject a NO or NO-synthase modulating composition of claim 12.

26. A method for treating a subject in need of treatment with a glucose-modulating agent comprising

administering to the subject a composition of claim 14.

27. A method for treating a subject in need of treatment with a glucose-modulating agent comprising

administering to the subject a composition of claim 15.

28. An antioxidant or preservative composition comprising a compound as claimed in claim 1 and a diluent.

29. An antioxidant or preservative composition comprising a compound as claimed in claim 2 and a diluent.

30. A topoisomerase II-inhibiting composition comprising a compound as claimed in claim 1 and a carrier or diluent.

31. A topoisomerase II-inhibiting composition comprising a compound as claimed in claim 2 and a carrier or diluent.

32. A method for preserving or protecting a desired item from oxidation comprising contacting the item with a composition as claimed in claim 28.

33. A method for inhibiting topoisomerase II which comprises contacting topoisomerase II with a

composition as claimed in claim 30.

34. A carrier or vehicle for a pharmaceutical comprising a compound as claimed in claim 1.

35. A carrier or vehicle for a pharmaceutical comprising a cocoa extract.

36. A kit for a composition of claim 4 comprising the compound and the carrier or diluent separately packaged, and optionally instructions for admixture or administration.

37. A kit for a composition of claim 6 comprising the compound and the carrier or diluent separately packaged, and optionally instructions for admixture or administration.

38. A kit for a composition of claim 8 comprising the compound and the carrier or diluent separately packaged, and optionally instructions for admixture or administration.

39. A kit for a composition of claim 10 comprising the compound and the carrier or diluent

separately packaged, and optionally instructions for

admixture or administration.

40. A kit for a composition of claim 12 comprising the compound and the carrier or diluent

separately packaged, and optionally instructions for

admixture or administration.

41. A kit for a composition of claim 14 comprising the compound and the carrier or diluent

separately packaged, and optionally instructions for

admixture or administration.

42. A kit for a composition of claim 30 comprising the compound and the carrier or diluent

separately packaged, and optionally instructions for

admixture or administration.

43. A kit for a composition of claim 28 comprising the compound and the diluent separately packaged, and optionally instructions for admixture or use.

44. A substantially pure Theobroma or Herrania species or inter or their intra species specific crosses thereof extract comprising polyphenols comprising oligomers 3 through 12.

45. A compound of claim 1 having a structure giving an NMR spectra as set forth in Figure 48A-D.

46. A compound of claim 1 having a structure giving an NMR spectra as set forth in Figure 49A-D.

47. A method for enhancing the concentration levels and distribution of cocoa procyanidins in cocoa beans by manipulating fermentation conditions.