AU783239B2 - Cocoa extract compounds and methods for making and using the same - Google Patents

Description

P/001011 2815/91 Regulation 3.2(2)

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: COCOA EXTRACT COMPOUNDS AND METHODS FOR MAKING AND USING THE SAME The following statement is a full description of this invention, including the best method of performing it known to us COCOA EXTRACT COMPOUNDS AND METHODS FOR MAKING AND USING THE SAME REFERENCE TO RELATED APPLICATION Reference is made to copending U.S. application Nos.

08/709,406, filed September 6, 1996, 08/631,661, filed April 2, 1996, and 08/317,226, filed October 3, 1994 (now U.S. Patent No.

5,554,645) and PCT/US96/04497, each of which is 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 uses for such extracts and compounds, in particular as inhibitors of the i L oxidation of LDL (low density lipoprotein).

Documents are cited in this disclosure with a full citation for each appearing thereat or in a References section S at the end of the specification, preceding the claims. These documents pertain to the field of this invention; and, each -27 document cited herein is hereby incorporated herein by reference.

g 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 202 nd 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 S such extracts or compounds therefrom, or, any uses for cocoa extracts or compounds therefrom, as antineoplastic agents, antioxidants, DNA topoisomerase II enzyme inhibitors, cyclooxygenase and/or lipoxygenase modulators, NO (Nitric Oxide) or NO-synthase modulators, as non-steroidal antiinflammatory agents, apoptosis modulators, platelet aggregation modulators, blood or in vivo glucose modulators, antimicrobials,. or inhibitors of oxidative DNA damage.

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, 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 2 of activity were reported in some extracts of cocoa tissues, and the work was not pursued. Thus, in the antineoplastic or anticancer art, cocoa and its extracts were not deemed to be useful; 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 anticancer screening, contrary to the knowledge in the o* antineoplastic or anti-cancer art. Surprisingly, and contrary to the knowledge in the art, the National Cancer Institute screening, the present inventors discovered that cocoa :15 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 antineoplastic agents, antioxidants, DNA topoisomerase II enzyme inhibitors, cyclo-oxygenase and/or lipoxygenase modulators, NO (Nitric Oxide) or NO-synthase modulators, as non-steroidal antiinflammatory agents, apoptosis modulators, platelet aggregation modulators, blood or in vivo glucose modulators, antimicrobials, and inhibitors of oxidative DNA damage.

It has been surprisingly discovered that cocoa extract, and compounds therefrom, have activity as inhibitors of LDL oxidation. Accordingly, the present invention provides a method of inhibiting the oxidation of LDL in a mammal comprising administering to said mammal a composition comprising a compound which is epicatechin or a polymeric compound of the formula An, wherein A is a monomer of the formula: HO 0 4 3 Z R OH X wherein n is an integer from 2 to 18, such that there is at least one terminal monomer unit A and one or a plurality of additional monomeric units; R is 3-(a)-o-sugar or 3-(P)-O-sugar; and X has either a or P stereochemistry; bonding between 00 adjacent monomers takes place at positions 4, 6 or 8; of o T0 a bond of an additional monomeric unit in position 4 has a or p stereochemistry; X, Y, and Z are selected from a monomeric unit A, hydrogen and a sugar, with the proviso that as to the at least one terminal monomeric unit, bonding of the additional monomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; and 00 the sugar is optionally substituted with a phenolic moiety at any position; or a pharmaceutically acceptable salt, glycoside, ester or oxidation product of said compound; and a pharmaceutically, veterinarily or food science acceptable carrier.

The compound may be used in the form of a substantially pure cocoa extract, or compounds obtained therefrom. The extract or compounds preferably comprise 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 18, 3 through 18, such as 2 through 12 or 3 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-1.

The extract or compounds 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 provides the use, in the manufacture of a pharmaceutical composition, a veterinary composition or a food product, for use in inhibiting the oxidation of LDL, of a compound which is epicatechin or a polymeric compound of formula An wherein A is a monomer of the formula:

OH

OH

Y

8 HO 8 o Z R OH X wherein n is an integer from 2 to 18, such that there is at least one terminal monomer unit A and one or a plurality of additional monomeric units; R is 3-(a)-o-sugar or 3-(P)-O-sugar; and X has either a or 3 stereochemistry; bonding between adjacent monomers takes place at positions 4, 6 or 8; a bond of an additional monomeric unit in position 4 has a or P stereochemistry; X, Y, and Z are selected from a monomeric unit A, hydrogen and a sugar, with the proviso that as to the at least one terminal monomeric unit, bonding of the additional monomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; and the sugar is optionally substituted with a phenolic moiety at any position; or a pharmaceutically acceptable salt, glycoside, ester or oxidation product of said compound.

The compound may be presented as a kit for treating a patient in need of treatment with an inhibitor of LDL oxidation S comprising a substantially pure cocoa extract or compounds S therefrom or synthetic cocoa polyphenol(s) or procyanidin(s) and a suitable carrier, a pharmaceutically, veterinary or food science acceptable carrier, for admixture with the extract or compound therefrom or synthetic polyphenol(s) or procyanidin(s).

i 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 S 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 6 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; Fig 5. shows a representative normal phase semipreparative HPLC separation of a crude cocoa polyphenol extract; Fig 6. 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; SFig. 7 shows a normal phase HPLC separation of crude, enriched and purified pentamers from cocoa extract; Figs. 8A, B and C show MALDI-TOF/MS of pentamer enriched procyanidins, and of Fractions A-C and of Fractions D- E, respectively; Fig. 9A shows an elution profile of oligomeric 2.0 procyanidins purified by modified semi-preparative HPLC; Fig. 9B shows an elution profile of a trimer procyanidin by modified semi-preparative HPLC; Figs. 10A-D each show energy minimized structures of all linked pentamers based on the structure of epicatechin; Fig. 11A shows relative fluorescence of epicatechin upon thiolysis with benzylmercapten; Fig. 11B shows relative fluorescence of catechin upon thiolysis with benzylmercapten; Fig. 11C shows relative fluorescence of dimers (B2 and upon thiolysis with benzylmercapten; Fig. 12A shows relative fluorescence of dimer upon thiolysis; Fig. 12B shows relative fluorescence of B5 dimer upon 7 thiolysis of dimer and subsequent desulphurization; Fig. 13 shows the elution profile from halogen-free analytical separation of acetone extract of procyanidins from cocoa extract; Fig. 14 shows the effect of pore size of stationary phase for normal phase HPLC separation of procyanidins; Fig. 15A shows the substrate utilization during fermentation of cocoa beans; Fig. 15B shows the metabolite production during fermentation; Fig. 15C shows the plate counts during fermentation of cocoa beans; Fig. 15D shows the relative concentrations of each component in fermented solutions of cocoa beans; Fig. 16 shows the acetylcholine-induced relaxation of NO-related phenylephrine-precontracted rat aorta; Figs. 42A-B show the effects of indomethacin on COX-1 and COX-2 activities; Figs. 43A-B show the correlation between the degree of polymerization and IC50 vs. COX-1/COX-2 (pM); Fig. 44 shows the correlation between the effects of S compounds on COX-1 and COX-2 activities expressed as pM; Figs. 45A-V show the IC50 values (pM) of samples containing procyanidins with COX-1/COX-2; Fig. 17 shows the purification scheme for the isolation of procyanidins from cocoa; Fig. 18A to 18P shows the preferred structures of the pentamer; Figs. 19A-AA show a library of stereoisomers of pentamers; Figs. 20A-B show 70 minute gradients for normal phase HPLC separation of procyanidins, detected by UV and fluorescence, respectively; Figs. 21A-B show 30 minute gradients for normal phase 8 HPLC separation of procyanidins, detected by UV and fluorescence, respectively; Fig. 22 shows a preparation normal phase HPLC separation of procyanidins; Figs. 23A-G show CD (circular dichroism) spectra of procyanidin dimers, trimers, tetramers, pentamers, hexamers, heptamers and octamers, respectively; Fig. 24A shows the structure and 1 H/13C NMR data for epicatechin; Figs. 24B-F show the APT, COSY, XHCORR, 1H and 13C NMR spectra for epicatechin; Fig. 25A shows the structure and 1H/ 13 C NMR data for catechin; boo. o Figs. 25B-E show the 1 H, APT, XHCORR and COSY NMR spectra for catechin; Fig. 26A shows the structure and 1

H/

13 C NMR data for B2 dimer; 1 d e .Figs. 26B-G show the C, APT, H, HMQC, COSY and HOHAHA NMR spectra for the B2 dimer; Fig. 27A shows the structure and 1

H/

13 C NMR data for dimer; Figs. 27B-G show the 1 H, 1 3 C, APT, COSY, HMQC and HOHAHA NMR spectra for B5 dimer; Figs. 28A-D show the 1 H, COSY, HMQC and HOHAHA NMR spectra for epicatechin/catechin trimer; Figs. 29A-D show the 1 H, COSY, HMQC and HOHAHA NMR spectra for epicatechin trimer; Figs. 30A and B show the effects of cocoa procyanidin fraction A and C, respectively, on blood pressure; blood pressure levels decreased by 21.43% within 1 minute after administration of fraction A, and returned to normal after minutes, while blood pressure decreased by 50.5% within 1 minute after administration of fraction C, and returned to normal after minutes; Fig. 31 shows the effect of cocoa procyanidin fractions on arterial blood pressure in anesthetized guinea pigs; Fig 32 shows the effect of L-NMMA on the alterations of arterial blood pressure in anesthetized ginea pigs induced by cocoa procyanidin fraction c; Fig. 33 shows the effect of bradykinin on NO production by HUVEC; Fig. 34 shows the effect of cocoa procyanidin fractions on macrophage NO production by HUVEC; Fig. 35 shows the effect of cocoa procyanidin fractions on macrophage NO production; Fig. 36 shows the effect of cocoa procyanidin fraction on LPS induced and gamma-Interferon primed macrophages.

Fig. 37 shows a micellar electrokinetic capillary chromatographic separation of cocoa procyanidin oligomers; .L Fig. 38 A-F show MALDI-TOF mass spectra for Cu+2-, Zn 2 Fe2-, Fe 3 Ca 2 and Mg ions, respectively, complexed to a trimer; Fig. 39 shows a MALDI-TOF mass spectrum of cocoa S" procyanidin oligomers (tetramers to octadecamers); S* Fig. 40 shows time-temperature effects on hexamer hydrolysis; and Fig. 41 shows time-temperature effects on trimer formation.

DETAILED DESCRIPTION COMPOUNDS OF THE INVENTION As discussed above, it has now been surprisingly found that cocoa extracts or compounds derived therefrom exhibit activity as inhibitors of LDL oxidation.

The extracts, compounds or combination of compounds derived 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(s) can be by solvent extraction techniques. The extracts comprising the active compounds can be purified, to be substantially pure, for instance, by gel permeation chromatography or by preparative High Performance Liquid Chromatography (HPLC) techniques or by a combination of such techniques.

With reference to the isolation and purification of the compounds of the invention derived from cocoa, it will be understood that any species of Theobroma, Herrania or inter- and intra-species crosses thereof may be employed. In this regard, reference is made to Schultes, "Synopsis of Herrania," Journal of the Arnold Arboretum, Vol. XXXIX, pp. 217 to 278, plus plates :s I to XVII (1985), Cuatrecasas, "Cocoa and Its Allies, A Taxonomic Revision of the Genus Theobroma," Bulletin of the iUnited States National Museum, Vol. 35, part 6, pp. 379 to 613, plus plates 1 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 de Nortes, 25(3) (1951).

Additionally, Example 19 lists the heretofore never reported concentrations of the inventive compounds found in Theobroma and Herrania species and their inter- and intraspecies crosses; and Example 19 also describes methods of modulating the amounts of the inventive compounds which may be obtained from cocoa by manipulating cocoa fermentation conditions.

An outline of the purification protocol utilized in the isolation of substantially pure procyanidins is shown in Fig. 17. Steps 1 and 2 of the purification scheme are described in Examples 1 and 2; steps 3 and 4 are described in Examples 3, 19 and 17; step 5 is described in Examples 4 and 10; and step 6 is described in Examples 4, 10 and 12. The skilled artisan 11 would appreciate and envision modifications in the purification scheme outlined in Figure 17 to obtain the active compounds without departing from the spirit or scope thereof and without undue experimentation.

The extracts, compounds and combinations of compounds derived therefrom having activity, without wishing to necessarily 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; inhibit DNA topoisomerase II enzyme and oxidative damage to DNA; possess antimicrobial activity; have the ability to modulate cyclooxygenase and/or lipoxygenase, NO or NO-synthase, apoptosis, platelet aggregation and blood or in vivo glucose, and have efficacy as non-steroidal antiinflammatory agents.

In the compound of formula An as defined above, 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 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 S bond.

Thus, the compound can be of the formula:

OH

H

A= HO 8 O Z 6 4 3 R OH X wherein: n is an integer from 2 to 18, 3 to 12, advantageously 5 to 12, and preferably n is 5, such that there is a first monomeric unit A, .o oo ooo 2 5

H

Y

OH X and a plurality of other monomeric units of A; R is 3-(a)-O-sugar, or 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 and Y are hydrogen, or combinations thereof; and said sugar is optionally substituted with a phenolic moiety via an ester bond.

According to the present invention, in the polymeric of the formula An, A may be a monomer having the compound formula: HO 0 OH X wherein n is an integer from 2 to 18, such that there is at least one terminal monomeric unit A, and at least one or a plurality of additional monomeric units; R is 3-(a)-O-sugar, or o S* sugar; bonding between adjacent monomers takes place at positions 4, 6 or 8; a bond of an additional monomeric unit in position 4 has a or 1f stereochemistry; X, Y and Z are selected from the group consisting of monomeric unit A, hydrogen, and a sugar, with the provisos that as to the at least one terminal monomeric unit, bonding of the additional monomeric unit thereto the bonding of the monomeric unit adjacent the terminal monomeric unit) is at position 4 and optionally, Y Z hydrogen; the sugar is optionally substituted with a phenolic moiety at any position, for instance via an ester bond, and pharmaceutically acceptable salts or derivatives thereof (including oxidation products).

In preferred embodiments, n can be 3 to 18, 2 to 18, 3 to 12, 5 to 12; and, advantageously, n is The sugar is selected from the group consisting of 14 glucose, galactose, xylose, rhamnose and arabinose.

The sugar of any or all of R, X, Y and Z can optionally be substituted at any position with a phenolic moiety via an ester bond. The phenolic moiety is selected from the group consisting of caffeic, cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.

Additionally, in accordance with the present invention, in the polymeric compound of the formula An, A is a monomer having the formula:

H

HO

O

o

X

ooo wherein n is an integer from 2 to 18, 3 to 18, advantageously 3 to 12, 5 to 12, preferably n is R is 3-(a)-O-sugar, or sugar; adjacent monomers bind at position 4 by or each of X, Y and Z is H, a sugar or an adjacent monomer, with the provisos that if X and Y are adjacent monomers, Z is H or sugar and if X and Z are adjacent monomers, Y is H or sugar, and that as to at least one of the two terminal monomers, bonding of the adjacent monomer is at position 4 and optionally, Y Z hydrogen; a bond at position 4 has a or 8 stereochemistry; the sugar is optionally substituted with a phenolic moiety at any position, for instance, via an ester bond,and pharmaceutically acceptable salts or derivatives thereof (including oxidation products) With regard to the recitation of "at least one terminal monomeric unit it will be understood that the compounds have two terminal monomeric units, and that the two terminal monomeric unit A may be the same or different.

Additionally, it will be understood that the recitation of "at least one terminal monomeric unit A" includes embodiments wherein the terminal monomeric unit A is referred to as a "first monomeric unit", with the recitation of "first monomeric unit" relating to that monomer to which other monomeric units are 4e S added, resulting in a polymeric compound of the formula An.

Moreover, with regard to the at least one of the two terminal monomers, bonding of the adjacent monomer is at position 4 and :2 optionally, Y Z hydrogen.

As to the recitation of the term "combinations o* o thereof", it will be understood that one or more of the inventive compounds may be used simultaneously, e.g., administered to a subject in need of treatment in a formulation comprising one or more inventive compounds.

The compounds or combinations thereof display the utilities noted above for cocoa extracts; and throughout the disclosure, the term "cocoa extract" may be substituted by compounds of the invention or combinations thereof, such that it will be understood that the inventive compounds or combinations thereof can be cocoa extracts.

The term "oligomer", as used herein, refers to any compounds or combinations thereof of the formula presented above, wherein n is 2 through 18. When n is 2, the oligomer is 16 termed a "dimer"; when n is 3, the oligomer is termed a "trimer"; when n is 4, the oligomer is termed a "tetramer"; when n is 5, the oligomer is termed a "pentamer"; and similar recitations may be designated for oligomers having n up to and including 18, such that when n is 18, the oligomer is termed an "octadecamer".

The compounds or combinations thereof can be isolated, from a natural source such as any species of Theobroma, Herrania or inter- or intra-species crosses thereof; or, the inventive compounds or combinations thereof can be purified, compounds or combinations thereof can be substantially pure; for instance, purified to apparent homogeneity. Purity is a relative concept, and the numerous Examples demonstrate S isolation of inventive compounds or combinations thereof, as well as purification thereof, such that by methods exemplified a S skilled artisan can obtain a substantially pure inventive compound or combination thereof, or purify them to apparent homogeneity purity by separate, distinct chromatographic peak). Considering the Examples, a substantially pure compound "0 or combination of compounds is at least about 40% pure, at least about 50% pure, advantageously at least about 60% pure, at least about 70% pure, more advantageously at least about 75-80% pure, preferably, at least about 90% pure, more preferably greater than 90% pure, at least 90-95% pure, or even purer, such as greater than 95% pure, 95-98% pure.

Further, examples of the monomeric units comprising the oligomers used herein are (+)-catechin and (-)-epicatechin, abbreviated C and EC, respectively. The linkages between adjacent monomers are from position 4 to position 6 or position 4 to position 8; and this linkage between position 4 of a monomer and position 6 and 8 of the adjacent monomeric units is designated herein as or There are four possible stereochemical linkages between position 4 of a monomer and position 6 and 8 of the adjacent monomer; and the stereochemical linkages between monomeric units is designated herein as (4a->6) or or or When C is linked to another C or EC, the linkages are designated herein as or (4ca-8) When EC is linked to another C or EC, the linkages are designated herein as or Examples of compounds eliciting the activities cited above include dimers, EC-(4f->8)-EC and EC-(48->6)-EC, wherein EC-(48-8)-EC is preferred; trimers [EC-(48-8)]2-EC, [EC- 2 -C and 2 -EC, wherein 2 -EC is preferred; tetramers 3 -EC, [EC-(415-8)] 3 -C and [EC- 2 -EC- wherein [EC-(48-8)]3-EC is preferred; and :pentamers 4 -EC, 3 -EC-(4f [EC- 3 and 3 wherein the 3-position of the pentamer terminal monomeric unit is optionally derivatized with a gallate or B-D-glucose; [EC-(48-8)] 4 -EC is S preferred.

Additionally, compounds which elicit the activities cited above also include hexamers to dodecamers, examples of Swhich are listed below: A hexamer, wherein one monomer (C or EC) having linkages to another monomer or for EC linked to another EC or C, and (4a-8) or for C linked to another C or EC; followed by a linkage to a pentamer compound listed above, 4 -EC-(4R-6)-EC, 4 and 4 wherein the 3-position of the hexamer terminal monomeric unit is optionally derivatized with a gallate or a B-D-glucose; in a preferred embodiment, the hexamer is [EC-(48->8)]5-EC; A heptamer, wherein any combination of two monomers (C and/or EC) having linkages to one another or for EC linked to another EC or C, and or for C linked to another C or EC; followed by a linkage to a pentamer compound listed above, [EC-(4-138)]5-ECand 5

-EC-

wherein the 3-position of the heptamer terminal monomeric unit is optionally derivatized with a gallate or a B- D-glucose; in a preferred embodiment, the heptamer is [EC- 6

-EC;

An octamer, wherein any combination of three monomers (C and/or EC) having linkages to one another or (48->6) for EC linked to another EC or C, and or for C o. linked to another C or EC; followed by a linkage to a pentamer compound listed above, 7 -EC, [ECand [EC- (4n8-8)] 6 wherein the 3-position of the octamer terminal monomeric unit is optionally derivatized with a gallate or a B-D-glucose; in a preferred embodiment, the octamer is [EC- (48->8)]7-EC; A nonamer, wherein any combination of four monomers (C and/or EC) having linkages to one another or for EC linked to another EC or C, and or for C linked to another C or EC; followed by a linkage to a pentamer compound listed above, [EC-(48-8) ]8-EC, ]7-ECand 7

-EC-

wherein the 3-position of the nonamer terminal monomeric unit is optionally derivatized with a gallate or a 8- D-glucose; in a preferred embodiment, the nonamer is [EC-

]-EC;

A decamer, wherein any combination of five monomers (C and/or EC) having linkages to one another or for EC linked to another EC or C, and or for C linked to another C or EC; followed by a linkage to a pentamer 19 compound listed above, 9 -EC, 8

-EC-

and 8

-EC-

wherein the 3-position of the decamer terminal monomeric unit is optionally derivatized with a gallate or a 3- D-glucose; in a preferred embodiment, the decamer is [EC- ]9-EC; An undecamer, wherein any combination of six monomers (C and/or EC) having linkages to one another or (4B->6) for EC linked to another EC or C, and (4a-8) or for C linked to another C or EC; followed by a linkage to a pentamer compound listed above, [EC-(4-8)]i 0 -EC, [ECand [EC- (4R8-8)] 9 wherein the 3-position of the undecamer terminal monomeric unit is optionally derivatized with a gallate .*15 or a f-D-glucose; in a preferred embodiment, the undecamer is ]o-EC; and A dodecamer, wherein any combination of seven monomers (C and/or EC) having linkages to one another or (4B->6) for EC linked to another EC or C, and or for C 20 linked to another C or EC; followed by a linkage to a pentamer compound listed above, 11 -EC, [EC- (48-8) 0 -EC-(48-6)-EC, and [EC- 10 wherein the 3-position of the dodecamer terminal monomeric unit is optionally derivatized with a gallate or a 8-D-glucose; in a preferred embodiment, the dodecamer is 1

-EC.

It will be understood from the detailed description that the aforementioned list is exemplary and provided as an illustrative source of several non-limiting examples of compounds of the invention, which is by no means an exhaustive list of the inventive compounds encompassed by the present invention.

Examples 3A, 3B, 4, 10, 17, 18 and 27 describe methods to separate the compounds of the invention. Examples 9, and 12 describe methods to purify the compounds of the invention. Examples 5, 11, 14, 15, 16 and 23 describe methods to identify compounds of the invention. Figures 18A-P and 19A-AA illustrate a stereochemical library for representative pentamers of the invention. Example 13 describes a method to molecularly model the compounds of the invention. Example 29 provides evidence for higher oligomers in cocoa, wherein n is 13 to 18.

Furthermore, while the invention is described with S. respect to cocoa extracts preferably comprising cocoa procyanidins, from this disclosure the skilled organic chemist will appreciate and envision synthetic routes to obtain and/or prepare the active compounds (see Example 7).

Accordingly, the invention comprehends synthetic cocoa polyphenols or procyanidins or their derivatives and/or their S synthetic precursors which include, but are not limited to Sglycosides, gallates, esters, etc. and the like. That is, the inventive compounds can be prepared from isolation from cocoa or from any species within the Theobroma or Herrania genera, as well as from synthetic routes; and derivatives and synthetic precursors of the inventive compounds such as glycosides, gallates, esters, etc. are included in the inventive compounds.

Derivatives can also include compounds of the above formulae wherein a sugar or gallate moiety is on the terminal monomer at positions Y or Z, or a substituted sugar or gallate moiety is on the terminal monomer at Y or Z.

The invention includes the ability to enzymatically modify cleavage or addition of a chemically significant moiety) the compounds of the invention, enzymatically with polyphenol oxidase, peroxidase, catalase combinations, and/or enzymes such as hydrolases, esterases, reductases, transferases, and the like and in any combination, taking into account kinetic and thermodynamic factors (see also Example 33 regarding 21 hydrolysis) With regard to the synthesis of the inventive compounds, the skilled artisan will be able to envision additional routes of synthesis, based on this disclosure and the knowledge in the art, without undue experimentation. For example, based upon a careful retrosynthetic analysis of the polymeric compounds, as well as the monomers. For instance, given the phenolic character of the inventive compounds, the skilled artisan can utilize various methods of selective 10 protection/deprotection, coupled with organometallic additions, oooe phenolic couplings and photochemical reactions, in a convergent, linear or biomimetic approach, or combinations thereof, together with standard reactions known to those well- S versed in the art of synthetic organic chemistry, as additional *-Oi synthetic methods for preparing the inventive compounds, without undue experimentation. In this regard, reference is made to W.

Carruthers, Some Modern Methods of Organic Synthesis, 3 rd ed., Cambridge University Press, 1986, and J. March, Advanced Organic Chemistry, 3 rd ed., John Wiley Sons, 1985, van Rensburg et al., Chem. Comm., 24: 2705-2706 (Dec. 21, 1996), Ballenegger et al., (Zyma SA) European Patent 0096 007 BI, and documents in the References section below, all of which are hereby incorporated herein by reference.

UTILITIES OF COMPOUNDS OF THE INVENTION With regard to the inventive compounds, it has been surprisingly found that the inventive compounds have discrete activities, and as such, the inventive compounds have broad applicability to the treatment of a variety of disease conditions, discussed hereinbelow.

Atherosclerosis, the most prevalent of cardiovascular diseases, is the principle cause of heart attack, stroke and vascular circulation problems. Atherosclerosis is a complex disease which involves many cell types, biochemical events and molecular factors. There are several aspects of this disease, 22 its disease states and disease progression which are distinguished by the interdependent consequences of Low Density Lipoprotein (LDL) oxidation, cyclo-oxygenase (COX)/lipoxygenase (LOX) biochemistry and Nitric Oxide (NO) biochemistry.

Clinical studies have firmly established that the elevated plasma concentrations of LDL are associated with accelerated atherogenesis. The cholesterol that accumulates in atherosclerotic lesions originate primarily in plasma lipoproteins, including LDL. The oxidation of LDL is a critical 1Q0 event in the initiation of atheroma formation and is associated with the enhanced production of superoxide anion radical (02 Oxidation of LDL by 02"- or other reactive species

.OH,

ONO'O-, lipid peroxy radical, copper ion, and iron based proteins) reduces the affinity of LDL for uptake in cells via receptor mediated endocytosis. Oxidatively modified LDLs are then rapidly taken up by macrophages which subsequently transform into cells closely resembling the "foam cells" observed in early atherosclerotic lesions.

Oxidized lipoproteins can also promote vascular injury through the formation of lipid hydroperoxides within the LDL particle. This event initiates radical chain oxidation reactions of unsaturated LDL lipids, thus producing more oxidized LDL for macrophage incorporation.

The collective accumulation of foam cells engorged with oxidized LDL from these processes results in early "fatty streak" lesions, which eventually progress to the more advanced complex lesions of atherosclerosis leading to coronary disease.

As discussed generally by Jean Marx at page 320 of Science, Vol. 265 (July 15, 1994), each year about 330,000 patients in the United States undergo coronary and/or peripheral angioplasty, a procedure designed to open up blood vessels, coronary arteries, clogged by dangerous atherosclerotic plaques (atherosclerosis) and thereby restore normal blood flow.

For a majority of these patients, the operation works as 23 intended. Nearly 33% of these patients (and maybe more by some accounts), however, develop restenosis, wherein the treated arteries become quickly clogged again. These patients are no better off, and sometimes worse off, than they were before angioplasty. Excessive proliferation of smooth muscle cells (SMCs) in blood vessel walls contributes to restenosis.

Increased accumulation of oxidized LDL within lesion SMCs might contribute to an atherogenic-related process like restenosis.

Zhou et al., "Association Between Prior Cytomegalovirus 10 Infection And The Risk Of Restenosis After Coronary Atherectomy," August 29, 1996, New England Journal of Medicine, Si.- 335:624-630, and documents cited therein, all incorporated herein by reference. Accordingly, utility of the present S invention with respect to atherosclerosis can apply to 1 restenosis.

With regard to the inhibition by the inventive compounds of cyclooxygenases (COX; prostaglandin endoperoxide synthase), it is known that cyclooxygenases are central enzymes S in the production of prostaglandins and other arachidonic acid metabolites eicosanoids) involved in many physiological Sprocesses. COX-1 is a constitutive enzyme expressed in many tissues, including platelets, whereas COX-2, a second isoform of the enzyme, is inducible by various cytokines, hormones and tumor promoters. COX 1 produces thromboxane A2, which is involved in platelet aggregation, which in turn is involved in the progression of atherosclerosis. Its inhibition is the basis for the prophylactic effects on cardiovascular disease.

The activity of COX-1 and COX-2 is inhibited by aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs), and the gastric side effects of NSAIDs are believed to be associated with the inhibiti'on of COX-1. Moreover, it has been found that patients taking NSAIDs on a regular basis have a to 50% lower risk of contracting colorectal cancer when compared to persons not being administered these type of medications; and COX-2 mRNA levels are markedly increased in 86% of human colorectal adenocarcinomas.

One significant property of COX-2 expressing cell lines is the enhanced expression of genes which participate in the modulation of apoptosis, programmed cell death.

Several NSAIDs have been implicated in increased cell death and the induction of apoptosis in chicken embryo fibroblasts.

Cellular lipoxygenases are also involved in the oxidative modification of LDL through the peroxidation of unsaturated lipids. The generation of lipid peroxy radicals contributes to the further radical chain oxidation of unsaturated LDL lipids, producing more oxidized LDL for macrophage incorporation.

The compounds have utility in the treatment of diseases associated with COX/LOX. In Example 22, COX was inhibited by individual inventive compounds at concentrations similar to a known NSAID, indomethacin.

For COX inhibition, the inventive compounds are S oligomers, where n is 2 to 18. In a preferred embodiment, the 2 inventive compounds are oligomers where n is 2 to 10, and more preferably, the inventive compounds are oligomers where n is 2 to Examples of compounds eliciting the inhibitory activity with respect to COX/LOX cited above include dimers, trimers, tetramers and pentamers, discussed above.

Hence, given the significant inhibitory potency of the inventive compounds on COX-2, coupled with the cytotoxic effects on a putative COX-2 expression colon cancer cell line, the inventive compounds possess apoptotic activity as inhibitors of the multistep progression leading to carcinomas, as well as activity as members of the NSAID family of medications possessing a broad spectrum of prophylactic activities.

Further, prostaglandins, the penultimate products of the COX catalyzed conversion of arachidonic acid to prostaglandin H 2 are involved in inflammation, pain, fever, fetal development, labor and platelet aggregation. Therefore, the inventive compounds are efficacious for the same conditions as NSAIDs, against cardiovascular disease, and stroke, etc. (indeed, the inhibition of platelet COX-1, which reduces thromboxane A 2 production, is the basis for the prophylactic effects of aspirin on cardiovascular disease).

Inflammation is the response of living tissues to injury. It involves a complex series of enzyme activation, mediator release, extravasation of fluid, cell migration, tissue breakdown and repair. Inflammation is activated by phospholipase A 2 which liberates arachidonic acid, the substrate for COX and LOX enzymes. COX converts arachidonic acid to the prostaglandin PGE 2 the major eicosanoid detected in inflammatory conditions ranging from acute edema to chronic arthritis. Its inhibition by NSAIDs is a mainstay for treatment.

Arthritis is one of the rheumatic diseases which encompass a wide range of diseases and pathological processes, most of which affect joint tissue. The basic structure affected 2C by these diseases is the connective tissue which includes synovial membranes, cartilage, bone, tendons, ligaments, and interstitial tissues. Temporary connective tissue syndromes include sprains and strains, tendonitis, and tendon sheath abnormalities. The most serious forms of arthritis are rheumatoid arthritis, osteoarthritis, gout and systemic lupus erythematosus.

In addition to the rheumatic diseases, other diseases are characterized by inflammation. Gingivitis and periodontitis follows a pathological picture resembling rheumatoid arthritis.

Inflammatory bowel disease refers to idiopathic chronic inflammatory conditions of the intestine, ulcerative colitis and Crohn's disease. Spondylitis refers to chronic inflammation of the joints of the spine. There is also a high incidence of osteoarthritis associated with obesity.

26 Thus, the inventive compounds have utility in the treatment of conditions involving inflammation, pain, fever, fetal development, labor and platelet aggregation.

The inhibition of COX by the inventive compounds would also inhibit the formation of postaglandins, PGD 2

PGE

2 Thus, the inventive compounds have utility in the treatment of conditions associated with prostaglandin PGD 2

PGE

2 Nitric oxide (NO) is known to inhibit platelet aggregation, monocyte adhesion and chemotaxis, and proliferation of vascular smooth muscle tissue which are critically involved in the process of atherogenesis. Evidence supports the view that NO is reduced in atherosclerotic tissues due to its reaction with oxygen free radicals. The loss of NO due to these reactions leads to increased platelet and inflammatory cell adhesion to vessel walls to further impair NO mechanisms of relaxation. In this manner, the loss of NO promotes atherogenic processes, leading to progressive disease states.

o Hypertension is a leading cause of cardiovascular diseases, including stroke, heart attack, heart failure, irregular heart beat and kidney failure. Hypertension is a condition where the pressure of blood within the blood vessels is higher than normal as it circulates through the body. When the systolic pressure exceeds 150 mm Hg or the diastolic pressure exceeds 90 mm Hg for a sustained period of time, damage is done to the body. For example, excessive systolic pressure can rupture blood vessels anywhere. When it occurs within the brain, a stroke results. It can also cause thickening and narrowing of the blood vessels which can lead to atherosclerosis. Elevated blood pressure can also force the heart muscle to enlarge as it works harder to overcome the elevated resting (diastolic) pressure when blood is expelled.

This enlargement can eventually produce irregular heart beats or heart failure. Hypertension is called the "silent killer" because it causes no symptoms and can only be detected when blood pressure is checked.

The regulation of blood pressure is a complex event where one mechanism involves the expression of constitutive Ca+2/calmodulin dependent form of nitric oxide synthase (NOS), abbreviated eNOS. NO produced by this enzyme produces muscle relaxation in the vessel (dilation), which lowers the blood pressure. When the normal level of NO produced by eNOS is not produced, either because production is blocked by an inhibitor or in pathological states, such as atherosclerosis, the vascular muscles do not relax to the appropriate degree. The resulting vasoconstriction increases blood pressure and may be responsible for some forms of hypertension.

Vascular endothelial cells contain eNOS. NO S synthesized by eNOS diffuses in diverse directions, and when it reaches the underlying vascular smooth muscle, NO binds to the heme group of guanylyl cyclase, causing an increase in cGMP.

Increased cGMP causes a decrease in intracellular free Ca Cyclic GMP may activate a protein kinase that phosphorylates Ca +2 transporters, causing Ca 2 to be sequestered in intracellular structures in the muscle cells. Since muscle contraction requires Ca 2 the force of the contraction is reduced as the Ca 2 S concentration declines. Muscle relaxation allows the vessel to dilate, which lowers the blood pressure. Inhibition of eNOS therefore causes blood pressure to increase.

When the normal level of NO is not produced, either because production is blocked by administration of an NOS inhibitor or possibly, in pathological states, such as atherosclerosis, the vascular muscles do not relax to the appropriate degree. The resulting vasoconstriction increases blood pressure and may be responsible for some forms of hypertension. There is considerable interest in finding therapeutic ways to increase the activity of eNOS in hypertensive patients, but practical therapies have not been reported. Pharmacological agents capable of releasing NO, such as nitroglycerin or isosorbide dinitrate, remain mainstays of vasorelaxant therapy.

Although the inventive compounds inhibit the oxidation of LDL, the more comprehensive effects of these compounds is their multidimensional effects on atherosclerosis via NO. NO modulation by the inventive compounds brings about a collage of beneficial effects, including the modulation of hypertension, lowering NO affected hypercholesterolemia, inhibiting platelet aggregation and monocyte adhesion, all of which are involved with the progression of atherosclerosis.

The role of NO in the immune system is different from its function in blood vessels. Macrophages contain a form of S. NOS that is inducible, rather than constitutive, referred to as iNOS. Transcription of the iNOS gene is controlled both positively and negatively by a number of biological response modifiers called cytokines. The most important inducers are gamma-interferon, tumor necrosis factor, interleukin-1, interleukin-2 and lipopolysaccharide (LPS), which is a component of the cell walls of gram negative bacteria. Stimulated macrophages produce enough NO to inhibit ribonuclease reductase, the enzyme that converts ribonucleotides to the deoxyribonucleotides necessary for DNA synthesis. Inhibition of DNA synthesis may be an important way in which macrophages and other tissues possessing iNOS can inhibit the growth of rapidly dividing tumor cells or infectious bacteria.

With regard to the effects of NO and infectious bacteria, microorganisms play a significant role in infectious processes which reflect body contact and injury, habits, profession, environment of the individual, as well as food borne diseases brought about by improper storage, handling and contamination.

The inventive compounds, combinations thereof and compositions containing the same are useful in the treatment of conditions associated with modulating NO concentrations.

Example 6 described the antioxidant activity (as inhibitors of free radicals) of the inventive compounds. Given that NO is a free radical and that the inventive compounds are strong antioxidants, it was suspected that the administration of the inventive compounds to experimental in vitro and in vivo models would have caused a reduction in NO levels. Any reduction in NO would have resulted in a hypertensive, rather than a hypotensive effect. Contrary to expectations, the inventive compounds elicited increases in NO from in vitro experiments and produced a hypotensive effect from in vivo studies (Examples 24 and 25). These results were unanticipated and completely unexpected.

Example 21 describes an erythmia (facial flush) shortly after drinking a solution containing the inventive compounds and glucose, thus implying a vasodilation effect.

Example 24 describes the hypotensive effects elicited by the inventive compounds in an in vivo animal model, demonstrating the efficacy of the inventive compounds in the treatment of hypertension. In this example, the inventive 0 compounds, combinations thereof and compositions comprising the same comprise oligomers wherein n is 2 to 18, and preferably, n is 2 to Example 25 describes the modulation of NO production by the inventive compounds in an in vitro model. In this example, the inventive compounds, combinations thereof and compositions comprising the same comprise oligomers wherein n is 2 to 18, and preferably n is 2 to Further, Example 28 provides evidence for the formation of Cu2_-, Fe+2 and Fe+ 3 -oligomer complexes detected by MALDI/TOF/MS. These results indicate that the inventive compounds can complex with copper and/or iron ions to minimize their effects on LDL oxidation.

Example 26 describes the effects of the inventive compounds on macrophage NO production. In this example, the results demonstrate that the inventive compounds induce monocyte/macrophage NO production, both independent and dependent of stimulation by lipopolysaccharide (LPS) or cytokines. Macrophages producing NO can inhibit the growth of infectious bacteria.

Example 6 describes the antioxidant activity by the compounds. The compounds of the invention inhibit DNA strand breaks, DNA-protein cross-links and free radical oxidation of nucleotides to reduce and/or prevent the occurrence of mutations.

Example 26 describes the effects of the inventive compounds on macrophage NO production. Macrophages which produce NO can inhibit the growth of rapidly dividing tumor cells.

0 FORMULATIONS AND METHODS Therefore, collectively, the inventive compounds, combinations thereof and compositions comprising the same have exhibited a wide array of activities against several aspects of goS.

atherosclerosis, cardiovascular disease, cancer, blood pressure modulation and/or hypertension, inflammatory disease, infectious agents and food spoilage.

Hence, the compounds of the invention, combinations thereof and compositions containing the same are COX inhibitors which affect platelet aggregation by inhibiting thromboxane A 2 formation, thus reducing the risk for thrombosis. Further, the inhibition of COX leads to decreased platelet and inflammatory cell adhesion to vessel walls to allow for improved NO mechanisms of relaxation. These results, coupled with the inhibition of COX at concentrations similar to a known NSAID, indomethacin, indicates antithrombotic efficacy.

Moreover, the compounds of the invention, combinations thereof and compositions containing the same are antioxidants which suppress the oxidation of LDL by reducing the levels of superoxide radical anion and lipoxygenase mediated lipid peroxy 31 radicals. The inhibition of LDL oxidation at this stage slows macrophage activation and retards foam cell formation to interrupt further progression of atherosclerosis. The inhibition of LDL oxidation can also slow the progression of restenosis. Thus, compounds of the invention or combinations thereof or compositions containing compounds of the invention or combinations thereof can be used for prevention and/or treatment of atherosclerosis and/or restenosis. And thus, the inventive compounds can be administered before or after angioplasty or similar procedures to prevent or treat restenosis in patients susceptible thereto.

For treatment or prevention of restenosis and/or atherosclerosis, an inventive compound or compounds or a composition comprising an inventive compound or compounds, alone T or with other treatment, may be administered as desired by the skilled medical practitioner, from this disclosure and knowledge in the art, at the first signs or symptoms of restenosis and/or atherosclerosis, immediately prior to, concomitant with or after angioplasty, or as soon thereafter as desired by the skilled medical practitioner, without any undue experimentation required; and the administration of the inventive compound or compounds or a composition thereof, alone or with other treatment, may be continued as a regimen, monthly, bimonthly, biannually, annually, or in some other regimen, by the skilled medical practitioner for such time as is necessary, without any undue experimentation required.

Further, the compounds of the invention, combinations thereof and compositions comprising the same have been shown to produce a hypotensive effect in vivo and induce NO in vitro.

These results have practical application in the treatment of hypertension and in clinical situations involving hypercholesterolemia, where NO levels are markedly reduced.

Formulations of the inventive compounds, combinations thereof and compositions comprising the same can be prepared 32 with standard techniques well known to those skilled in the pharmaceutical, food science, medical and veterinary arts, in the form of a liquid, suspension, tablet, capsule, injectable solution or suppository, for immediate or slow-release of the active compounds.

The carrier may also be a polymeric delayed release system. Synthetic polymers are particularly useful in the formulation of a composition having controlled release. An early example of this was the polymerization of methyl methacrylate into spheres having diameters less than one micron to form so-called nano particles, reported by Kreuter, J., Microcapsules and Nanoparticles in Medicine and Pharmacology, M.

Donbrow CRC Press, p. 125-148.

A frequent choice of a carrier for pharmaceuticals and more recently for antigens is poly (d,l-lactide-co-glycolide) (PLGA). This is a biodegradable polyester that has a long history of medical use in erodible sutures, bone plates and other temporary prostheses where it has not exhibited any toxicity. A wide variety of pharmaceuticals have been :"29 formulated into PLGA microcapsules. A body of data has accumulated on the adaption of PLGA for controlled, for example, as reviewed by Eldridge, et al. Current Topics in Microbiology and Immunology, 1989, 146:59-66. The entrapment in PLGA microspheres of 1 to 10 microns in diameter can have an effect when administered orally. The PLGA microencapsulation process uses a phase separation of a water-in-oil emulsion. The inventive compound or compounds is or are prepared as an aqueous solution and the PLGA is dissolved in a suitable organic solvents such as methylene chloride and ethyl acetate. These two immiscible solutions are co-emulsified by high-speed stirring. A non-solvent for the polymer is then added, causing precipitation of the polymer around the aqueous droplets to form embryonic microcapsules. The microcapsules are collected, and stabilized with one of an assortment of agents (polyvinyl alcohol (PVA), gelatin, alginates, methyl cellulose) and the solvent removed by either drying in vacuo or solvent extraction.

Additionally, with regard to the preparation of slowrelease formulations, reference is made to U.S. Patent Nos.

5,024,843, 5,091,190, 5,082,668, 4,612,008 and 4,327,725, hereby incorporated herein by reference.

Additionally, selective processing coupled with the identification of cocoa genotypes of interest could be used to prepare Standard-of-Identity (SOI) and non-SOI chocolate products as vehicles to deliver the active compounds to a patient in need of treatment for the disease conditions S described above, as well as a means for the delivery of conserved levels of the inventive compounds.

In this regard, reference is made to copending U.S.

Application Serial No. 08/709,406, filed September 6, 1996, hereby incorporated herein by reference. USSN 08/709,406 relates to a method of producing cocoa butter and/or cocoa S solids having conserved levels of polyphenols from cocoa beans using a unique combination of processing steps which does not :2c: require separate bean roasting or liquor milling equipment, S allowing for the option of processing cocoa beans without exposure to severe thermal treatment for extended periods of S time and/or the use of solvent extraction of fat. The benefit of this process lies in the enhanced conservation of polyphenols in contrast to that found in traditional cocoa processing, such that the ratio of the initial amount of polyphenol found in the unprocessed bean to that obtainable after processing is less than or equal to 2.

Compositions of the invention include one or more of the above noted compounds in a formulation having a pharmaceutically acceptable carrier or excipient, the inventive compounds having anti-cancer, anti-tumor or antineoplastic activities, antioxidant activity, inhibit DNA topoisomeriase II enzyme, inhibit oxidative damage to DNA, induce monocyte/macrophage NO production, have antimicrobial, cyclooxygenase and/or lipoxygenase, NO or NO-synthase, apoptosis, platelet aggregation and blood or in vivo glucose modulating activities, and have efficacy as non-steroidal antiinflammatory agents.

Another embodiment of the invention includes compositions comprising the inventive compounds or combinations thereof, as well as at least one additional antineoplastic, blood pressure reducing, antiinflammatory, antimicrobial, antioxidant and hematopoiesis agents, in addition to a pharmaceutically acceptable carrier or excipient.

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 the data herein, and such factors as the age, sex, weight, genetics and condition of the particular subject or patient, and the route of administration, relative concentration of particular oligomers, and toxicity LD 5 o).

The compositions can be co-administered or sequentially administered with other antineoplastic, anti-tumor or anti-cancer agents, antioxidants, DNA topoisomerase II enzyme S•inhibiting agents, inhibitors of oxidatively damaged DNA or cyclo-oxygenase and/or lipoxygenase, apoptosis, platelet aggregation, blood or in vivo glucose or NO or NO-synthase modulating agents, non-steroidal antiinflammatory 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, inhibitors of oxidatively damaged DNA, cyclo-oxygenase and/or lipoxygenase, apoptosis, platelet aggregation, blood or in vivo glucose or NO or NO-synthase modulating and/or non-steroidal antiinflammatory agents; again, taking into consideration such factors as the age, sex, weight, genetics and condition of the particular subject or patient, and, the route of administration.

Examples of compositions of the invention for human or veterinary use include edible compositions for oral administration, such solid or liquid formulations, for instance, capsules, 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 cocoa or chocolate flavored solid or liquid compositions); liquid preparations for orifice, 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 injectable administration) such as sterile suspensions or emulsions. However, the active ooo* X5 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, :.1Q 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, by a "salting out" procedure.

Example 30 describes the preparation of the inventive compounds in a tablet formulation for application in the pharmaceutical, supplement and food areas. Further, Example 31 describes the preparation of the inventive compounds in capsule formulations for similar applications. Still further, Example 32 describes the formulation of Standard of Identity (SOI) and non-SOI chocolates containing the compounds of the invention or 36 cocoa solids obtained from methods described in copending U.S.

Application Serial No. 08/709,406, hereby incorporated herein by reference.

KITS

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 an inhibitor of oxidative DNA damage or antimicrobial, or cyclo-oxygenase and/or S lipoxygenase, NO or NO-synthase non-steroidal antiinflammatory, apoptosis and platelet aggregation modulating or blood or in vivo glucose modulating agent and/or an agent which reduces or 5 alleviates ill effects of antineoplastic, anti-tumor or anticancer agents, antioxidant, DNA topoisomerase II enzyme inhibitor or antimicrobial, or cyclo-oxygenase and/or S lipoxygenase, NO or NO-synthase, apoptosis, platelet aggregation and blood or in vivo glucose modulating and/or non-steroidal antiinflammatory agents for co- or sequential-administration.

S The additional agent(s) can be provided in separate container(s) or in admixture with the active cocoa extract. Additionally, the kit can include instructions for mixing or combining ingredients and/or administration.

IDENTIFICATION OF GENES A further embodiment of the invention comprehends the modulation of genes expressed as a result of intimate cellular contact by the inventive compounds or a combination of compounds. As such, the present invention comprehends methods for the identification of genes induced or repressed by the inventive compounds or a combination of compounds which are associated with several diseases, including but not limited to atherosclerosis, hypertension, cancer, cardiovascular disease, and inflammation. Specifically, genes which are differentially expressed in these disease states, relative to their expression in "normal" nondisease states are identified and described before and after contact by the inventive compounds or a combination of compounds.

As mentioned in the previous discussion, these diseases and disease states are based in part on free radical interactions with a diversity of biomolecules. A central theme in these diseases is that many of the free radical reactions involve reactive oxygen species, which in turn induce physiological conditions involved in disease progression. For instance, reactive oxygen species have been implicated in the regulation of transcription factors such as nuclear factor (NF)- KB. The target genes for NF-KB comprise a list of genes linked to coordinated inflammatory response. These include genes X5 encoding tumor necrosis factor (TNF)-a, interleukin IL- 6, IL-8, inducible NOS, Major Histocompatabilty Complex (MHC) class I antigens, and others. Also, genes that modulate the activity of transcription factors may in turn be induced by oxidative stress. Oxidative stress is the imbalance between radical scavenging and radical generating systems. Several known examples (Winyard and Blake, 1997) of these conditions include gaddl53 (a gene induced by growth arrest and DNA damage), the product of which has been shown to bind NF-IL6 and form a heterodimer that cannot bind to DNA. NF-IL6 upregulates the expression of several genes, including those encoding interleukins 6 and 8. Another example of oxidative stress inducible genes are gadd45 which regulates the effects of the transcription factor p53 in growth arrest. p53 codes for the p53 protein which can halt cell division and induce abnormal cells cancer) to undergo apoptosis.

Given the full panoply of unexpected, nonobvious and novel utilities for the inventive compounds or combination of compounds for utility in a diverse array of diseases based in part by free radical mechanisms, the invention further 38 comprehends strategies to determine the temporal effects on gene(s) or gene product(s) expression by the inventive compounds in animal in vitro and/or in vivo models of specific disease or disease states using gene expression assays. These assays include, but are not limited to Differential Display, sequencing of cDNA libraries, Serial Analysis of Gene Expression (SAGE), expression monitoring by hybridization to high density oligonucleotide arrays and various reverse transcriptasepolymerization chain reaction (RT-PCR) based protocols or their combinations (Lockhart et al., 1996).

The comprehensive physiological effects of the inventive compounds or combination of compounds embodied in the invention, coupled to a genetic evaluation process permits the discovery of genes and gene products, whether known or novel, I induced or repressed. For instance, the invention comprehends S the in vitro and in vivo induction and/or repression of cytokines IL-1, IL-2, IL-6, IL-8, IL-12, and TNF-a) in Slymphocytes using RT-PCR. Similarly, the invention comprehends 0. the application of Differential Display to ascertain the 0 .:oQ induction and/or repression of select genes; for the cardiovascular area superoxide dismutase, heme oxidase, COX I and 2, and other oxidant defense genes) under stimulated and/or oxidant stimulated conditions TNF-a or H 2 0 2 conditions. For the cancer area, the invention comprehends the application of Differential Display to ascertain the induction and/or repression of genes or gene products such as CuZnsuperoxide dismutase, Mn-superoxide dismutase, catalase, etc., in control and oxidant stressed cells.

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 1: Cocoa Source and Method of Preparation Several Theobroma cacao genotypes which represent the three recognized horticultural races of cocoa (Enriquez, 1967; Engels, 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 f redistilled hexane as the solvent. Residual solvent was removed J5 from the defatted mass by vacuum at ambient temperature.

Table 1: Description of Theobroma cacao Source Material 0 0 00 0000 0990 0 GENOTYPE ORIGIN HORTICULTURAL RACE UIT-1 Malaysia Trinitario Unknown West Africa Forastero ICS-100 Brazil Trinitario (Nicaraguan Criollo ancestor) ICS-39 Brazil Trinitario (Nicaraguan Criollo ancestor) UF-613 Brazil Trinitario EEG-48 Brazil Forastero UF-12 Brazil Trinitario NA-33 Brazil Forastero 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 0 by 400mL 70% methanol/deionized water. The extracts were pooled and the solvents removed by evaporation at 45 0 C with a rotary oS* evaporator held under partial vacuum. The resultant aqueous phase was diluted to 1L with deionized water and extracted 2X with 400mL CHCl 3 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 S 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 N 2 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 GENOTYPE ORIGIN YIELDS (g) UIT-1 Malaysia 3.81 Unknown West Africa 2.55 ICS-100 Brazil 3.42 ICS-39 Brazil 3.45 UF-613 Brazil 2.98 EEG-48 Brazil 3.15 UF-12 Brazil 1.21 NA-33 Brazil 2.23 B. Method 2 Alternatively, procyanidins are extracted from defatted, unfermented, freeze dried cocoa beans of Example 1 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 0 C at 3000 xg and the supernatant passed through glass wool. The filtrate was subjected to distillation under partial vacuum and the resultant aqueous :i phase frozen in liquid N 2 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 *1'S variations encountered with different genotypes, geographical origin, horticultural race, and method of preparation.

Example 3: Partial Purification of Cocoa Procyanidins Procyanidins obtained from Example 2 were partially purified by liquid chromatography on Sephadex LH-20 (28 x cm). Separations were aided by a step gradient from deionized water into methanol. The initial gradient composition started with 15% methanol in deionized water which was followed step wise every 30 min. with 25% methanol in deionized water, 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 single fraction. The fraction yielded a xanthine alkaloid free subfraction which was submitted to further subfractionation to yield five subfractions designated MM2A through MM2E. The solvent was removed from each subfraction by evaporation at 4 0 5C with a rotary evaporator held under partial vacuum. The resultant aqueous phase was frozen in liquid N 2 and freeze dried overnight on a LABCONCO Freeze Dry System. A representative gel permeation chromatogram showing the fractionation is shown in Figure 1. Approximately, 100mg of material was subfractionated in this manner.

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 Hour Intervals, Flow Rate; 1.5mL/min, Detector; UV at X 1 254 nm and X 2 365 nm, Chart Speed: 0.5mm/min, Column Load; 120mg.

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 ImL injection loop was assembled with a Pharmacia FRAC-100 Fraction Collector.

Separations were effected on a Phenomenex Ultracarb M 10 p ODS column (250 x 22.5mm) connected with a Phenomenex 10 p ODS Ultracarb (60 x 10 mm) guard column. The mobile phase composition was A water; B methanol used under the following linear gradient conditions: [Time, (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 5.pm Supelcosil LC-Si column (250x10mm) connected with a Supelco 5 pm Supelguard LC-Si guard column (20x4.6mm).

Procyanidins were eluted by a linear gradient under the following conditions: (Time, (0,82,14), (30, 67.6, 28.4), (60, 46, 50), (65, 10, 86), (70, 10, 86) followed by a 5 min. re-equilibration. Mobile phase composition was A dichloromethane; B methanol; and C acetic acid: water 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 100-250 pL of 10mg of procyanidin extracts dissolved in 0.25mL 70% aqueous acetone. A representative semi-preparative HPLC trace is shown in O. 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 10mm Supelco Supelcosil LC-Si (5 pm) Semipreparative Column 20 x 4.6mm Supelco Supelcosil LC-Si (5 pm) Guard Column Detector: Waters LC Spectrophotometer Model 480 254nm Flow rate: 3mL/min, Column Temperature: ambient, Injection: 2500L of 70% aqueous acetone extract.

44 Gradient: CH 2 C1 2 Methanol Acetic Time (min) Acid: H 2 0 0 82 14 4 67. 6 28.4 4 46 50 4 10 86 4 10 86 4 The fractions obtained were as follows: FRACTION TYPE 1 dimers 2 trimers 3 tetramers 4 pentamers hexamers 6 heptamers 7 octamers 8 nonamers 9 decamers undecamers 11 dodecamers 12 higher oligomers

S.

Example 4: Analytical HPLC Analysis of Procyanidin Extracts Method 1. Reverse Phase Separation Procyanidin extracts obtained from Example 3 were filtered through a 0.45 p 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 0 C on a Hewlett- Packard 50 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 46 acid levels in A and B mobile phases can be increased to 2%.

Components were detected by fluorescence, where Aex 276nm and Aex 316nm 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 Guard column: 20 x 2.1mm Hewlett Packard Hypersil ODS 5 p) Detectors: Diode Array 280nm -i Fluorescence Xex 276nm; .o Aem 316nm.

Flow rate: 0.3mL/min.

Column Temperature: 45 0

C

p a Gradient: 0.5% Acetic Acid 0.5% Acetic acid Time (min) in nanopure water in methanol 0 100 0 40 0 100 Method 2. Normal Phase Separation Procyanidin extracts obtained from Examples 2 and/or 3 were filtered through a 0.45p 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 0 C on a 5p Phenomenex Lichrosphere® Silica 100 column (250x3.2mm) connected to a Supelco Supelguard LC-Si 5p guard column (20x4.6mm).

Procyanidins were eluted by linear gradient under the following conditions: (Time, 82, 14), (30, 67.6, 28.4), 46, 50), (65, 10, 86), (70, 10, 86) followed by an 8 min. reequilibration. 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 Xex 276nm and Xem 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 (5p) 20 x 4.6mm Supelco Supelguard LCf Si 5 p) guard column 6 64 6 *6.

6 a 6 a

S

S.r Detectors: Photodiode Array 280nm Fluorescence Xex 276nm; Aem 316nm.

Flow rate: 0.5 mL/min.

Column Temperature: 37 0

C

Gradient: CH 2 -C1 2 Methanol Acetic Time (min.) Acid/Water (1:1) 0 82 14 4 67.6 28.4 4 46 50 4 10 86 4 10 86 4 Example 5: Identification of Procyanidins Procyanidins were purified by liquid chromatography on 48 Sephadex LH-20 (28x2.5cm) columns followed by semi-preparative HPLC using a 10p Bondapak C18 (100x8mm) column or by semipreparative HPLC using a 5p Supelcosil LC-Si (250x10mm) 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 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.

i* Table 3: LSIMS (Positive Ion) Data from Cocoa Procyanidin 1 5 Fractions Oligomer (M 1) (M Na) Mol. Wt.

m/z m/z Monomers 291 313 290 (catechins) Dimer(s) 577/579 599/601 576/578 Trimer(s) 865/867 887/889 864/866 Tetramer(s) 1155 1177 1154 Pentamer(s) 1443 1465 1442 Hexamer(s) 1731 1753 1730 Heptamer(s) 2041 2018 Octamer(s) 2329 2306 Nonamer(s) 2617 2594 Decamer(s) 2905 2882 Undecamer(s) 3170 Dodecamer(s) 3458 The major mass fragment ions were consistent with work previously reported for both positive and negative ion FAB-MS analysis of procyanidins (Self et al., 1986 and Porter et al., 1991). The ion corresponding to m/z 577 and its sodium adduct at m/z 599 suggested the presence of doubly linked procyanidin dimers in the isolates. It was interesting to note that the higher oligomers were more likely to form sodium adducts than their protonated molecular ions The procyanidin isomers B-2, B-5 and C-1 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). 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 I concentrations of the procyanidins based on normal phase HPLC 20- analysis.

Table 4: Relative Concentrations of Procyanidins in the Xanthine Alkaloid Free Isolates Component Amount (+)-catechin 1.6% (-)-epicatechin 38.2% B-2 Dimer 11.0% Dimer 5.3% C-i Trimer 9.3% Doubly linked dimers Tetramer(s) Pentamer-Octamer 24. Unknowns and higher 2.6% oligomers a a.

a a a Table 5: Relative Concentrations of Procyanidins in Aqueous Acetone Extracts 5 Component Amount (+)-catechin and 41.9% -epicatechin B-2 and B-5 Dimers 13.9% Trimers 11.3% Tetramers 9.9% Pentamers 7.8% Hexamers 5.1% Heptamers 4.2% Octamers 2.8% Nonamers 1.6% Decamers 0.7% Undecamers 0.2% Dodecamers <0.1% Figure 3 shows several procyanidin structures and Figures 4A-4E show the representative HPLC chromatograms of the five fractions employed in the following screening for anticancer or antineoplastic 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.

Hewlett Packard 5 p Hypersil ODS (200 x Column: 2.1mm) Linear Gradient of 60% B into A at a flow rate of 0.3mL/min. B acetic acid in methanol; A acetic acid in deionized water. Xex 280nm; hem 316nm.

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: Antioxidant Activity of Cocoa Extracts Containing Procyanidins Evidence in the literature suggests a relationship between the consumption of naturally occurring antioxidants (Vitamins C, E and Beta-carotene) and a lowered incidence of :1 disease, including cancer (Designing Foods, 1993; Caragay, 1992). It is generally thought that these antioxidants affect i .i certain oxidative and free radical processes involved with some Stypes of tumor promotion. Additionally, some plant polyphenolic compounds that have been shown to be anticarcinogenic, also 2 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 extracts 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. 50uL of methanol solubilized antioxidant was added to each sample to aid in dispersion of the antioxidant. A control sample was prepared with 50 pL of methanol containing no antioxidant.

The samples were evaluated in duplicate, for oxidative stability using the Rancimat stability test at 100 0 C and 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). A typical Rancimat trace is shown in Figure 6. Results are reported in Table 8 as S hours required to reach a peroxide level of 100 meq.

Table 7: Concentrations of Antioxidants *0

S.

0 0 0 00*0 0 SAMPLE LEVEL 1 LEVEL 2 ppm Butylated Hydroxytoluene (BHT) 24 120 Butylated Hydroxyanisole (BHA) 24 120 Crude Ethyl Acetate Fraction of 22 110 Cocoa Fraction A-C 20 100 Fraction D-E 20 100 Table 8: Oxidative Stability of Peanut Oil with Antioxidants Various SAMPLE 20 ppm 100 ppm average Control 10.5 0.7 BHT 16.5 2.1 12.5 2.1 BHA 13.5 2.1 14.0 1.4 Crude Cocoa Fraction 18.0 0.0 19.0 1.4 Fraction A-C 16.0 6.4 17.5 0.0 Fraction D-E 14.0 1.4 12.5 0.7 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 as an antioxidant and/or food additive. And, in this regard, it is noted too that the invention is from an edible source. Given these results, 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.

5e A c eeo555 Example 7: 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 standards and, in the manner Sdescribed above with respect to cocoa extracts.

Example 8: Assay of Normal Phase Semi-Preparative Fractions Since the polyphenol extracts are compositionally complex, it was necessary to determine which components were active against cancer cell lines for further purification, doseresponse assays and comprehensive structural identification. A :normal phase semi preparative HPLC separation (Example3B) 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 5) and assayed at 100.pg/mL and doses against Hela and SKBR-3 cancer cell lines to determine which oligomer possessed the greatest activity.

Example 9: 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 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.OOg of the extract obtained from Example 2 was subfractionated in this manner. Results are shown in Table 9.

e

-S

5 S S .5-S

S.-

5 0

S

S

S 55

S

I S

S

S S

S

S.

6 S S

S.

4555

I

*5S5

S.

0 4SS

S

Table 9: Composition of Fractions Obtained: tjndecame Fraction Mmonomer DDimer TTrimer Tetramer Pentamer Hexamer Heptamer Octamer Nonamer Decamer r Others (Time) Area) Area) Area) (%Area) (%Area) Area) Area) Area) Area) Area) Area) Area) 1:15 73 8 16 3 ND ND ND ND ND ND ND ND 1:44 67 19 10 3 1 tr tr tr tr tr tr tr 2:13 30 29 24 11 4 1 tr tr tr tr tr tr 2 :42 2 161 31 28 15 6 2 tr tr tr tr tr 3:11 1 12 17 25 22 13 7 2 1 tr tr tr 3:40 tr 1 8 13 18 20 15 10 5 2 tr tr tr 4 :09 tr 6 8 17 21 19 14 8 4 2 tr tr ND tr not detected trace amount Method B. Normal Phase Separation Procyanidins obtained as Example 2 were separated purified by normal phase chromatography on Supelcosil LC-Si, 100A, 5.pm (250 x 4.6mm), at a flow rate of l.0mL/min, or, in the alternative, Lichrosphere® Silica 100, 100A, 5.pm (235 x 3.2mm), at a flow rate of 0.5mL/min. Separations were aided by a step gradient under the following conditions: (Time, 82, 14), (30, 67.6, 28.4), (60, 46, 50), (65, 10, 86), 86). Mobile phase composition was A dichloromethane; B methanol; and C acetic acid:water Components were detected by fluorescence where Aex 276nm and \em 316nm, and by UV at 280nm. The injection volume was 5.OpL (20mg/mL) of the procyanidins obtained from Example 2. These results are shown in Fig. 40A and In the alternative, separations were aided by a step gradient under the following conditions: (Time, (0, 76, 20); (25, 46, 50); (30, 10, 86). Mobile phase composition was A dichloromethane; B methanol; and C acetic acid water The results are shown in Fig. 41A and 41B.

Method C. Reverse Phase Separation Procyanidins obtained as in Example 2 were separated purified by reverse phase chromatography on Hewlett Packard Hypersil ODS 5.pm. (200 x 2.1mm), and a Hewlett Packard Hypersil S ODS 5.pm guard column (20 x 2.1mm). The procyanidins were eluted with a linear gradient of 20% B into A in 20 minutes, followed by a column wash 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 Aex 276nm and Aem 316nm; and the injection volume was 2.0.pL Example 10: 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 59 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 5 pm Supelcosel LC-Si, 100A column (250 x 10mm) connected with a Supelco 5p Supelguard LC-Si guard column x 4.6mm). Procyanidins were eluted by a linear gradient under the following conditions: (Time, 82, 14), 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 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- 2 of 10mg of procyanidin extracts dissolved in 0.25mL 70% aqueous acetone. Individual peaks or select chromatographic regions were collected on timed intervals or manually by fraction Scollection for further purification and subsequent evaluation.

HPLC conditions: 250 x 100mm Supelco Supelcosil LC- Si (5.pm) Semipreparative Column x 4.6mm Supelco Supelcosil LC-Si (5 Guard Column Detector: Waters LC Spectrophotometer Model 480 254nm Flow rate: 3mL/min., Column Temperature: ambient, Injection: 250.pL of pentamer enriched extract acetic acid: Gradient: CH 2 C12 methanol water (1:1) 0 82 14 4 67.6 28.4 4 46 50 4 10 86 4 10 86 4 Method B. Reverse Phase Separation Procyanidin extracts obtained as in Example 13 were filtered through a 0.

45 p nylon filter and analyzed by a Hewlett Packard 1090 ternary phase HPLC system equipped with a Diode Array detector and a HP model 1046A Programmable Fluorescence Detector. Separations were effected at 45 0 C on a Hewlett Packard 5p Hypersil 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-gassed 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 Components were detected by fluorescence, where Aex 276nm and Aem 316nm, 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.

Method C. Normal Phase Separation Pentamer enriched procyanidin extracts obtained as in Example 13 were filtered through a 0.45p 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 0 C on a 5p Phenomenex Lichrosphere® Silica 100 column (250 x 3.2mm) connected to a Supelco Supelguard LC-Si 5 p guard column x 4.6mm). Procyanidins were eluted by linear gradient under the following conditions: (time, 82, 14), 67.6, 28.4), (60, 46, 50), (65, 10, 86), (70, 10, 86), followed by an 8 minute 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 0.5mL/min was used.

Components were detected by fluorescence, where Xex 276nm and Xem 316nm or by UV at 280nm. A representative HPLC chromatogram 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: 250 x 3.2mm Phenomenex Lichrosphere® Silica 100 column (5p) 20 x 4.6mm Supelco Supelguard LC-Si guard column Detectors: Photodiode Array 280nm Fluorescence Xex 276nm; Xem 316nm Flow rate: 0.5 mL/min.

Column temperature: 37 0

C

acetic acid: Gradient: CH 2 C12 methanol water (1:1) :0 82 14 4 30 67.6 28.4 4 46 50 4 65 10 86 4 10 86 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 p Supelcosil LC-Si 100A column (50 x 2cm), with an appropriate guard column. Procyanidins were eluted by a linear gradient under the following conditions: (time, 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, Prior to use, the mobile phase components were mixed by the following protocol: Solvent A preparation (82% CH 2 C12, 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 dispense 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 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): 2 w 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 3840mL of methanol and dispense 3840mL of methanol and dispense into the same 4L bottle.

4. Cap the bottle and mix well.

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

Repeat steps 1-5 to yield 4 volumes 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 was 0.7g in 7mL. components were detected 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 5 p Supelcosil LC-Si run ambient temperature.

Mobile Phase: A Methylene Chloride with 2% Acetic Acid and 2% Water.

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

Gradient/Flow Profile: TIME %A %B FLOW RATE (MIN) (mL/min) 0 92.5 7.5 92.5 7.5 91.5 8.5 145 88.0 22.0 150 24.0 86.0 155 24.0 86.0 180 0.0 100.0

S.

S

4r

S

Sr

S.

'S

4 0

S

Example 11: Identification of Procyanidins Go Procyanidins obtained as in Example 10, method D were .1b analyzed by Matrix Assisted Laser Desorption Ionization-Time of SFlight/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 .4.

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 mg of sample was dissolved in 500.pL 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 8.pJ. 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 8A.

Figures 8B and C show MALDI-TOF/MS spectra obtained from partially purified procyanidins prepared as described in Example 3, Method A.

The spectra were obtained as follows: The purified D-E fraction was subjected to MALDI- TOF/MS as described above, with the exception that the fraction was initially purified by SEP-PACK® C-18 cartridge. Five mg of fraction D-E in 1 mL nanopure water was loaded onto a preequilibrated SEP-PACK® cartridge. The column was washed with nanopure water to eliminate contaminants, and procyanidins were eluted with ImL 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 5 (see Example 5, Table 3) and indicate that the inventive .o compounds have utility as sequestrants of cations. In particular, MALDI-TOF/MS results 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 less were S 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 Ca demonstrating the utility as cation sequestrants.

Example 12: Purification of Oligomeric Fractions Method A. Purification by Semi-Preparative Reverse Phase HPLC Procyanidins obtained from Example 10, Method A and B and D were further separated to obtain experimental quantities of like oligomers for further structural identification and elucidation Example 11, 14, 15, and 16). A Hewlett Packard 1050 HPLC system equipped with a variable wavelength detector, Rheodyne 7010 injection valve with ImL injection loop was assembled with a Pharmacia FRAC-100 Fraction Collector.

Separations were effected on a Phenomenex Ultracarb® 10p ODS column (250 x 22.5mm) connected with a Phenomenex 10p ODS Ultracarb® (60 x 10mm) guard column. The mobile phase composition was A water; B methanol used under the following linear gradient conditions: (time, (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 shown in Fig. 9B.

Method B. Modified Semi-Preparative HPLC S: Procyanidins obtained from Example 10, Method A and B and D were further separated to obtain experimental quantities of like oligomers for further structural identification and elucidation Example 11, 14, 15, and 16). Supelcosil LC- S Si 5p column (250 x 10mm) with a Supelcosil LC-Si 5p (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 used under the following linear gradient conditions: (time, 82, 14); (22, 74, 21); (32, 74, 21); 74, 50, (61, 82, 14), followed by column re-equilibration for 7 minutes. Injection volumes were 60.pL containing 12mg of enriched pentamer. Components were detected by UV at 280nm. A representative elution profile is shown in Figure 9A.

Example 13: Molecular Modeling of Pentamers Energy minimized structures were determined by molecular modeling using Desktop Molecular Modeller, version Oxford University Press, 1994. Four representative views of [EC(4 8)] 4 -EC (EC epicatechin) pentamers based on the structure of epicatechin are shown in Figures 10 A-D. A helical structure is suggested. In general when epicatechin is the first monomer and the bonding is a beta configuration results, when the first monomer is catechin and the bonding is an alpha configuration results; and, these results are obtained regardless of whether the second monomer is epicatechin or catechin (an exception is ent-EC(4-+8)ent-EC). Figures 18A 18P show preferred pentamers, and, Figures 19A to 19P show a library of stereoisomers up to and including the pentamer, from which other compounds within the scope of the invention can be prepared, without undue experimentation.

Example 14: NMR Evaluation of Pyrocyanidins 1C NMR spectroscopy was deemed a generally useful technique for the study of procyanidins, especially as the S: phenols usually provide good quality spectra, whereas proton NMR spectra are considerably broadened. The 3C NMR spectra of :5 oligomers yielded useful information for A or B ring substitution patterns, the relative stereochemistry of the C ring and in certain cases, the position of the interflavanoid linkages. Nonetheless, H 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 sequence to obtain cross peaks corresponding to alpha, beta, gamma or delta protons. COSY is a 2D-Fourier transform NMR technique wherein vertical and horizontal axes provide 'H chemical shift and 1D 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 peaks from secondary and tertiary carbons to the respective protons. APT is a 3C 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, IH NMR, HOHAHA (homonuclear Hartmann- Hahn), HMQC (heteronuclear multiple quantum coherence), COSY (Homonuclear correlation spectroscopy), APT (attached proton test), and XHCORR (a variation on HMQC) spectroscopy were used 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 Spectra were taken on a Bruker 500 MHZ NMR, using methanol as an internal standard.

Figures 24A-E represent the NMR spectra which were used to characterize the structure of the epicatechin monomer.

Figure 24A shows the 1H and 13C chemical shifts, in tabular form.

Figures 24 B-E show H, APT, XHCORR and COSY spectra for 5 epicatechin.

Similarly, Figures 25A-F represent the NMR spectra which were used to characterize the structure of the catechin monomer. Figure 25A shows the 'H and 1C chemical shifts, in tabular form. Figures 24 B-F show 1 H, 1C, APT, XHCORR and COSY spectra for catechin.

Method B. Dimers All spectra were taken in 75% deuterated acetone in S D 2 0, using acetone as an internal standard, and an approximate sample concentration of Figures 26A-G represent the spectra which were used to characterize the structure of the B2 dimer. Fig. 26A shows 1H and 13C chemical shifts, in tabular form. The terms T and B indicate the top half of the dimer and the bottom half of the dimer.

Figures 26B and C show the 13C and APT spectra, respectively, taken on a Bruker 500 MHZ NMR, at room temperature.

Figures 26D-G show the 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 27A-G represent the spectra which were used to characterize the structure of the B5 dimer. Figure 47A shows the 13C and 1 H chemical shifts, in tabular form.

Figures 27B-D show the 13C and APT, respectively, which were taken on a Bruker 500 MHZ NMR, at room temperature.

Figure 27E shows the COSY spectrum, taken on an AMX- 360, at room temperature, using a gradient pulse.

Figures 27F and G show 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

D

2 0, at -30C using acetone as an internal standard, on an AMX-360 MHZ NMR, and an appropriate sample concentration of Figures 28A-D represent the spectra which were used to :5 characterize the structure of the epicatechin/catechin trimer.

These figures show 1H, COSY, HMQC and HOHAHA, respectively. The COSY spectrum was taken using a gradient pulse.

Method D. Trimer -All Epicatechin All spectra were taken in 70% deuterated acetone in

D

2 0, at using acetone as an internal standard, on an AMX- 360 MHZ NMR, and an appropriate sample concentration of Figures 29A-D represent the spectra which were used to characterize the structure of all epicatechin trimer. These figures show 1H, COSY, HMQC and HOHAHA, respectively. The COSY spectrum was taken using a gradient pulse.

Example 15: Thiolysis of Procyanidins In an effort to characterize the structure of procyanidins, benzyl mercaptan (BM) was 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 dissolved in 1.5mL ethanol, 100 pL BM and 50 pL 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 0 C, and aliquots of the reaction were taken at 60, 120 and 240 minutes. The relative fluorescence of each aliquot is shown in Figures 25A-C, representing epicatechin, catechin and dimers, respectively. Higher oligomers are similarly thiolyzed.

Example 16: Thiolysis and Desulfurization of Dimers Dimers B2 and B5 were hydrolyzed with benzylmercaptan by dissolving dimer (B2 or B5; 1.0 mg) in 600.pl ethanol, 40 pL BM and 20 pL acetic acid. The mixture was heated at 95 0 C for 4 hours under nitrogen in a Beckman Amino Acid Analysis vessel.

Aliquots were removed for analysis by reverse-phase HPLC, and 75.pL of each of ethanol Raney Nickel and gallic acid e l~ were added to the remaining reaction medium in a 2mL hypovial.

The vessel was purged under hydrogen, and occasionally shaken for 1 hour. The product was filtered through a 0.45p filter and analyzed by reverse-phase HPLC. Representative elution profiles are shown in Figures 12 A and B. Higher oligomers are similarly desulfurized. This data suggests polymerization of epicatechan or catechin and therefore represents a synthetic route for preparation of inventive compounds.

Example 17: Halogen-free Analytical Separation of Extract Procyanidins obtained from Example 2 were partially purified by Analytical Separation by Halogen-free Normal Phase Chromatography on 100A Supelcosil LC-Si 5pm (250 x 4.6mm), at a flow rate of l.0mL/min, and a column temperature of 37 0

C.

Separations were aided by a linear gradient under the following conditions: (time, 82, 14); (30, 67.6, 28.4); 46, 50). Mobile phase composition was A 30/70 diethyl ether/Toluene; B= Methanol; and C acetic acid/water Components were detected by UV at 280nm. A representative elution profile is shown in Figure 13.

Example 18: 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 investigated. Separations were effected on Silica-300, 300A (250 x 2.0mm), or, in the alternative, on Silica-1000, 1000A (250 x 2.0mm). A linear gradient was employed as mobile phase composition was: A= Dichloromethane; B Methanol; and C acetic acid/water Components were detected by fluorescence, wherein hex 276nm and \em 316nm, by UV detector at 280nm. The flow rate was l.0mL/min, and the oven temperature was 37 0 C. A representative chromatogram from three different columns (100A pore size, from Example 10, Method D) is shown in Figure 14. This shows effective pore size for separation of procyanidins.

Example 19: Obtaining Desired Procyanidins Via Manipulating Fermentation Microbial strains representative of the succession Sassociated with cocoa fermentation were selected from the M&M/Mars cocoa culture collection. The following isolates were used: Acetobacter aceti ATCC 15973 Lactobacillus sp. (BH 42) Candida cruzii (BA Saccharomyces cerevisiae (BA 13) Bacillus cereus (BE Bacillus 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 0

C.

The slants were 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 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: a.

a a Fermentation Model Water Ethanol/acid infusate Fermentation daily daily transfer daily transfer bench scale transfer to solutions of to fermented model to fresh alcohol and acid pulp pasteurized fermentation in water corresponding to on each sterile pulp levels successive day coinoculated determined at of fermentation with test each stage of a strains model pulp fermentation a.

0* a The bench scale fermentation was performed in S duplicate. All treatments were incubated as indicated below: SDay 1: 260C Day 2: 260C to 500C Day 3: 50 0

C

Day 4: 450C Day 5: 400C 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 66C 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 9, 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 15A C. The procyanidin profiles of cocoa beans subjected to various fermentation treatments is shown in Figure 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 S1.3 Theobroma genus and their inter and intra species specific crosses. Samples were prepared as in Examples 1 and 2 (methods 1 and and analyzed as in Examples 13, method B. This data illustrates that the extracts containing the inventive compounds 0 are found in Theobroma and Herrania species, and their intra and inter species specific crosses.

*00 *9 *9 9 5 5* 9 9 5 S 5t *5 99 9* *5*9 S 9 5 5* 5 *5 S S 69 *5 9. 9 S. S. *S *S t Theobroma and Herrania Species Procyanidin Levels ppm (pg/g) in defatted powder Oligomer SAMPLE Monomer Dimer Trimer Tetramer Pentamer Hexamer Heptamer Octamer Nonamer Decamer Undecamer Total T. grandiflorum x T. obovatum 1 3822 3442 5384 4074 3146 2080 850 421 348 198 tr* 23,765 T. grandiflorum x T. obovatum 2 3003 4098 5411 3983 2931 1914 1090 577 356 198 tr 23,561 T. grandiflorum x T. obovatum 3A 4990 4980 7556 5341 4008 2576 1075 598 301 144 tr 31,569 T. grandiflorum x T. obovatum 3B 3880 4498 6488 4930 3706 2560 1208 593 323 174 tr 28,360 T. grandiflorum x T. obovatum 4 2647 3591 5328 4240 3304 2380 1506 815 506 249 tr 24,566 T. grandiflorum x T. obovatum 6 2754 3855 5299 3872 2994 1990 1158 629 359 196 88 23,194 T. grandiflorum x T. obovatum SIN 3212 4134 7608 4736 3590 2274 936 446 278 126 ND 23,750 T. obovatumn 1 3662 5683 9512 5358 3858 2454 1207 640 302 144 ND 32,820 T. grandiflorum TEFFE 2608 2178 3090 2704 2241 1586 900 484 301 148 tr 16,240 T. grandiflorum TEFFE x T. 4773 4096 5289 4748 3804 2444 998 737 335 156 tr 27,380 grandiflorum' T. grandiflorum x T. subincanum 4752 3336 4916 3900 3064 2039 782 435 380 228 ND 23,832 T. obovatum x T. subincanum 3379 3802 5836 3940 2868 1807 814 427 271 136 tr 23,280 T. speciosun x T. sylvestris 902 346 1350 217 152 120 60 tr tr ND ND 3,147 T. microcarpum 5694 3250 2766 1490 822 356 141 tr ND ND ND 14,519 T. cacao, SIAL 659, to 21,929 10,072 10,106 7788 5311 3242 1311 626 422 146 tr 60,753 T. cacao, SIAL 659, t24 21,088 9762 9119 7094 4774 2906 1364 608 361 176 tr 57,252 T. cacao, SIAL 659, t48 20,887 9892 9474 7337 4906 2929 1334 692 412 302 tr 58,165 T. cacao, SIAL 659, t96 9552 5780 5062 3360 2140 1160 464 254 138 tr ND 27,910 T. cacao, SIAL 659, t120 8581 4665 4070 2527 1628 888 326 166 123 tr ND 22,974 Pod Rec. 10/96, Herrania mariae 869 1295 545 347 175 97 tr *ND ND 3329 Sample Rec. prior to 10/96, 130 354 151 131 116 51 tr ND ND 933 Herrania mariae I_ IL_ r I Nu none aetected 2 sample designated

ERJON

sample aesignate CtPATU tr trace Example 20: Effect of Procyanidins on NO Method A.

The purpose of this study is to establish the relationship between procyanidins (as in Example 10, method D) and NO, which is known to induce cerebral vascular dilation.

The effects of monomers and higher oligomers, in concentrations ranging from 100 pg/mL to 0.1 pg/mL, on the production of nitrates (the catabolites of NO), from HUVEC (human umbilical vein endothelial cells) is evaluated. HUVEC (from Clonetics) is investigated in the presence or absence of each procyanidin for S24 to 48 hours. At the end of the experiments, the supernatants are collected and the nitrate content determined by calorimetric assay. In separate experiments, HUVEC is incubated with a acetylcholine, which is known to induce NO production, in the presence or absence of procyanidins for 24 to 48 hours. At the li end of the experiments, the supernatants are collected and Snitrate content is determined by calorimetric assay. The role of NO is ascertained by the addition of nitroarginine or methyl arginine, which are specific blockers of NO synthase.

Method B. Vasorelaxation of Phenylephrine-Induced Contracted Rat Artery The effects of each of the procyanidins (100pg/mL to 0.lg/mL on the rat artery is the target for study of vasorelaxation of phenylephrine-induced contracted rat artery.

Isolated 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 is 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 16.

Method C. Induction of Hypotension in the Rat This method is directed to the effect of each procyanidin (as in Example 10, method D) on blood pressure.

Rats are instrumented in order to monitor systolic and diastolic blood pressure. Each of the procyanidins are injected intravenously (dosage range 100 0.1 pg/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-methyl arginine.

These studies, together with next Example, illustrate that the inventive compounds are useful in modulating vasodilation, and are further useful with respect to modulating S blood pressure or addressing coronary conditions, and migraine headache conditions.

Example 21: Effects of Cocoa Polyphenols on Satiety

*S.

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 100g 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, 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 S study. 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 eeoc 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 14 th edition; W.B. Saunders Co.; Philadelphia, PA 1969 Ch. 10, pp. 550-9).

This suggests that the difference in additional glucose 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 HIGH LOW WEEK DESCRIPTION CONTROLa CONTROLb 0 Glucose Tolerance 265 mg/dL 53 mg/dL 1 Glucose Tolerance 310 68 with 0.1% polyphenols 2 Glucose Tolerance 315 66 4 Glucose Tolerance 325 with 0.1% polyphenols 5 0.1% polyphenols 321 66 a Expected range: 253 373mg/dL b Expected range: 50-80mg/dL The subject also experienced a facial flush (erythema) 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.

Table 12: Procyanidin Levels in Commercial Chocolates Pg/g Heptamers and Sample Monomers Dimers Trimers Tetramers Pentamers Hexamers Higher Total Brand 1 366 166 113 59 56 23 18 801 Brand 2 344 163 il1 45 48 ND* ND 711 Brand 3 316 181 100 41 40 7 ND 685 Brand 4 310 122 71 27 28 5 ND 563 Brand 5 259 135 90 46 29 ND ND 559 Brand 6 308 139 91 57 47 14 ND 656 Brand 7 196 98 81 58 54 19 ND 506 Brand 8 716 472 302 170 117 18 ND 1,795 Brand 9 1,185 951 633 298 173 25 21 3,286 Brand 10 1,798 1,081 590 342 307 93 ND 4,211 Brand 11 1,101 746 646 372 347 130 75 3, 417 Brand 12 787 335 160 20 10 8 ND 1,320 ND* None detected.

9* 6 6* *6 6 6 66 6 6 6 6 6 6* *666* **6 *6 66 6 *6 6 66 Table 13: Procyaniclin Levels in Cocoa Raw Materials pg/ g Heptamers Sample Monomers Dimers Trimers Tetramers Pentamers Hexamers and Higher Total Unfermented 13,440 6,425 6,401 5,292 4,236 3,203 5,913 44,910 Fermented 2,695 1,538 1,362 740 470 301 277 7,383 Roasted 2,656 1,597 921 337 164 ND* ND 5,675 Choc. Liquor 2,805 1, 446 881 442 184 108 ND 5,866 Cocoa Hulls 114 53 14 ND ND ND ND 181 Cocoa Powder 1% Fat 506 287 112 ND ND ND ND 915 Cocoa Powder 11% Fat 1,523 1,224 680 46 ND ND ND 3,473 Red Dutch Cocoa 1,222 483 103 ND ND ND ND 1,808 Powder, pH 7.4, 11% fat Red Dutch Cocoa 168 144 60 ND ND ND ND 372 Powder, pH 8.2, 23% fat ND* None detected.

Example 22: The Effect of Procyanidins on Cyclooxygenase 1 2 1 e The effect of procyanidins on cyclooxygenase 1 2 (COX1/COX2) activities was assessed by incubating the enzymes, derived from ram seminal vesicle and sheep placenta, respectively, with arachidonic acid (5 pM) for 10 minutes at room temperature, in the presence of varying concentrations of procyanidin solutions containing monomer to decamer and procyanidin mixture. Turnover was assessed by using PGE2 EIA .0 kits from Interchim (France). Indomethacin was used as a Sreference compound. The results are presented in the following Table, wherein the IC50 values are expressed in units of pM (except for Sl, which represents a procyanidin mixture prepared from Example 9, Method A and where the samples S1 to Eg represent sequentially procyanidin oligomers (monomer through decamer) as in Example 10, Method D, and IC50 is expressed in units of mg/mL).

r ICso COX-1 IC50 COX-2 RATIO SAMPLE COX2/COXl 1 0.074 0.197 2.66 2 0.115 0.444 3.86 3 0.258 0.763 2.96 4 0.154 3.73 24.22 0.787 3.16 4.02 6 1.14 1.99 1.75 7 1.89 4.06 2.15 8 2.25 7.2 3.20 9 2.58 2.08 0.81 3.65 3.16 0.87 11 0.0487 0.0741 1.52 Indomethacin 0.599 13.5 22.54 expressed as uM with the exception of sample 11, which is mg/mL.

The results of the inhibition studies are presented in Figures 42 A and B, which shows the effects of Indomethacin on COX1 and COX2 activities. Figures 43 A and B shows the correlation between the degree of polymerization of the procyanidin and IC50 with COX1 and COX2; Figure 44 shows the correlation between IC50 values on COX1 and COX2. And, Figures 45 A through Y show the IC50 values of each sample (S1 Sil) with COX1 and COX2.

These results indicate that the inventive compounds have analgesic, anti-coagulant, and anti-inflammatory utilities.

Further, COX2 has been linked to colon cancer. Inhibition of 5 COX2 activity by the inventive compounds illustrates a plausible mechanism by which the inventive compounds have antineoplastic activity against colon cancer.

COX1 and COX2 are also implicated in the synthesis of "o prostaglandins. Thus, the results in this Example also indicate that the inventive compounds can modulate renal functions, immune responses, fever, pain, mitogenesis, apoptosis, prostaglandin synthesis, ulceration 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 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 6, 20, and 21.

In Example 6, the anti-oxidant activity of inventive compounds is shown. In Example 20, the effect on NO is demonstrated.

And, Example 21 provides evidence of a facial vasodilation.

From the results in this Example, in combination with Examples 6, 20 and 21, the inventive compounds can modulate free radical mechanisms driving physiological effects. Similarly, lipoxygenase mediated free radical type reactions biochemically directed toward leukotriene synthesis can be modulated by the inventive compounds, thus affecting subsequent physiological effects inflammation, immune response, coronary conditions, carcinogenic mechanisms, fever, pain, ulceration).

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 .5 addition to having antineoplastic properties, there may also be a synergistic effect by the inventive compounds when S administered with other antineoplastic agents.

Example 23: Circular Dichroism/Study of Procyanidins CD studies were undertaken in an effort to elucidate the structure of purified procyanidins as in Example 14, Method S D. The spectra were collected at 25°C using CD spectrum software AVIV 60DS V4.1f.

Samples were scanned from 300nm to 185nm, every 1.00nm, at 1.50nm bandwidth. Representative CD spectra are shown in Figures 23A through G, which show the CD spectra of dimer through octamer.

These results are indicative of the helical nature of the inventive compounds.

Example 24: NO DEPENDENT HYPOTENSION IN THE GUINEA PIG The effect of five cocoa procyanidin fractions on guinea pig blood pressure were investigated. Briefly, guinea pigs (approximately 400g body weight; male and female) were anesthetized upon injection of 40 mg/kg sodium pentobarbital.

The carotid artery was cannulated for monitoring of the arterial blood pressure. Each of the five cocoa procyanidin fractions was injected intravenously (dose range 0.1 mg/kg 100 mg/kg) through the jugular vein. Alterations of blood pressure were recorded on a polygraph. In these experiments, the role of NO was ascertained by the administration of L-N-methylarginine (1 mg/kg) ten minutes prior to the administration of cocoa procyanidin fractions.

Cocoa procyanidin fractions were prepared and analyzed according to the procedures described in U.S. Patent 5,554,645, S hereby incorporated herein by reference.

Fraction A: Represents a preparative HPLC fraction comprised of monomers-tetramers. HPLC analysis revealed the following composition: Monomers 47.2% :Dimers 23.7 Trimers 18.7 Tetramers 10.3 Fraction B: Represents a preparative HPLC fraction comprised of pentamers-decamers. HPLC analysis revealed the following composition: Pentamers 64.3% Hexamers 21.4 Heptamers 7.4 Octamers 1.9 Nonamers 0.9 Decamers 0.2 Fraction C: Represents an enriched cocoa procyanidin fraction used in the preparation of Fractions A and B (above). HPLC analysis revealed the following composition: Monomers 34.3% Dimers 17.6 Trimers 16.2 Tetramers 12.6 Pentamers Hexamers 5.2 Heptamers 3.1 Octamers 1.4 Nonamers 0.7 Decamers 0.3 S S Fraction D: Represents a procyanidin extract prepared from a S. milk chocolate. HPLC analysis revealed a composition similar to that listed in the Table 12 for Brand 8. Additionally, caffeine and theobromine 6.3% were present.

S Fraction E: Represents a procyanidin extract prepared from a dark chocolate prepared with alkalized liquor. HPLC analysis revealed a composition similar to that listed in the Table 12 for Brand 12. Additionally, caffeine 16.0% and theobromine 5.8% were present.

In three separate experiments, the effects of administering 10mg/kg cocoa procyanidin fractions on arterial blood pressure of anesthetized guinea pigs was investigated.

Upon intravenous injection, procyanidin fractions A and E evoked a decrease in blood pressure of about 20%. This decrease was only marginally different from that obtained from a solvent (DMSO) control (15 In contrast, procyanidin fractions B, C and D (10mg/kg) induced marked decreases in blood pressure, up to 50-60% for C. In these experiments the order of hypotensive effect was as follows: C>B>D>>A=E.

Typical recordings of blood pressure elicited after injection of procyanidin fractions appear in Figure 30A for fraction A and Figure 30B for fraction C. Figure 31 illustrates the comparative effects on blood pressure by these fractions.

The possible contribution of NO in the hypotension in the guinea pig induced by administration of fraction C was analyzed using L-N-methyl arginine (LNMMA). This pharmacological agent inhibits the formation of NO by inhibiting NO synthase. L-NMMA was administered at the dose of 1 mg/kg, ten minutes prior to injection of the cocoa procyanidin 0 fractions. As shown in Figure 32, treatment of the animals with L-NMMA completely blocked the hypotension evoked by the procyanidin fraction C. Indeed, following treatment with this inhibitor, the alterations of blood pressure produced by fraction C were similar to those noted with solvent alone.

Example 25: Effect of Cocoa Procyanidin Fractions on NO Production in Human Umbilical Vein o* :2 Endothelial Cells Human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics and cultures were carried out according to the manufacturer's specifications. HUVEC cells were seeded at 5,000 cells/cm 2 in 12-well plates (Falcon). After the third passage under the same conditions, they were allowed to reach confluence. The supernatant was renewed with fresh medium containing defined concentrations of bradykinin (25,50 and 100nM) or cocoa procyanidin fractions A-E (100 pg/mL) as described in example 31. The culture was continued for 24 hr.

and the cell free supernatants were collected and stored frozen prior to assessment of NO content as described below. In selected experiments, the NO synthase (NOS) antagonist, Nonitro-L-arginine methyl ester (L-NAME, 10 pM) was added to assess the involvement of NOS in the observed NO production.

HUVEC NO production was estimated by measuring nitrite concentration in the culture supernatant by the Griess reaction.

Griess reagent was 1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride. Briefly, 50 pL aliquots were removed from the various supernatants in quadruplicate and incubated with 150 pL of the Griess reagent. The absorbency at 540 nm was determined in a multiscan (Labsystems Multiskans MCC/340) apparatus. Sodium nitrite was used at defined concentrations to establish standard curves. The absorbency of the medium without cells (blank) was subtracted from the value obtained with the cell containing supernatants.

Figure 33 illustrates the effect of bradykinin on NO production by HUVEC where a dose dependent release of NO was 1 S observed. The inhibitor L-NAME completely inhibited the bradykinin induced NO release.

Figure 34 illustrates the effect of the cocoa S procyanidin fractions on NO production by HUVEC cells.

Fractions B, C and D induced a moderate but significant amount of NO production by HUVEC. By far, Fraction C was the most 0 S efficient fraction to induce NO formation as assessed by the production of nitrites, while Fraction E was nearly ineffective.

S**

The effect of Fraction C on NO production was dramatically reduced in the presence of L-NAME. Interestingly, Fractions B, C and D contained higher amounts of procyanidin oligomers than Fractions A and E. A distinguishing difference between Fractions D and E was that E was prepared from a dark chocolate which used alkalized cocoa liquor as part of the chocolate recipe. Alkalization leads to a base catalyzed polymerization of procyanidins which rapidly depletes the levels of these compounds. An analytical comparison of procyanidin levels found in these types of chocolate appear in the Table 12, where Brand 12 is a dark chocolate prepared with alkalized cocoa liquor and Brand 11 is a typical milk chocolate. Thus, extracts obtained from milk chocolates contain high proportions of procyanidin oligomers which are capable of inducing NO. The addition of the NO inhibitor L-NMMA to the Fraction C sample clearly led to the inhibition of NO. The results obtained from the procyanidin fractions were consistent to those observed with the bradykinin induced NO experiment (see Figure 33).

As in the case of the HUVEC results, cocoa procyanidin fraction C elicited a major hypotensive effect in guinea pigs, whereas fractions A and E were the least effective. Again, the presence of high molecular weight procyanidin oligomers were implicated in the modulation of NO production.

Example 26: Effect of Cocoa Procyanidin Fractions on Macrophage NO Production '*IS Fresh, human heparinized blood (70 mL) was added with an equal volume of phosphate buffer saline (PBS) at room temperature. A Ficoll-Hypaque solution was layered underneath the blood-PBS mixture using a 3mL Ficoll-Hypaque to lOmL blood- PBS dilution ratio. The tubes were centrifuged for 30 minutes at 2,000 rpm at 18-200C. The upper layer containing plasma and platelets was discarded. The mononuclear cell layer was transferred to another centrifuge tube and the cells were washed 2X in Hanks balanced saline solution. The mononuclear cells were resuspended in complete RPMI 1640 supplemented with fetal calf serum, counted and the viability determined by the trypan blue exclusion method. The cell pellet was resuspended in complete RPMI 1640 supplemented with 20% fetal calf serum to a final concentration of 1 x 106 cells/mL. Aliquots of the cell suspension were plated into a 96 well culture plate and rinsed 3X with RPMI 1640 supplemented with 10% fetal calf serum and the nonadherent cells (lymphocytes) were discarded.

These cells were incubated for 48 hours in the presence or absence of five procyanidin fractions described in 88 Example 24. At the end of the incubation period, the culture media were collected, centrifuged and cell free supernatants were stored frozen for nitrate assay determinations.

Macrophage NO production was determined by measuring nitrite concentrations by the Greiss reaction. Greiss reagent was 1% sulfanilamide, 0.1% N-(l-naphthyl)-ethylenediamine dihydrochloride. Briefly, 50 pL aliquots were removed from the supernatants in quadruplicate and incubated with 150 pL of the Greiss reagent. The absorbency at 540 nm was determined in a multiscan (Labsystems Multiskans MCC/340) apparatus. Sodium nitrite was used at defined concentrations to establish standard curves. The absorbency of the medium without cells (blank) was subtracted from the value obtained with the cell containing supernatants.

In a separate experiment, macrophages were primed for 12 hours in the presence of 5U/mL gamma-interferon and then stimulated with 10 pg/mL LPS for the next 36 hours in the presence or absence of 100 pg/mL of the five procyanidin fractions.

Figure 35 indicates that only procyanidin fraction C, at 100 pg/mL, could induce NO production by monocytes/macrophages. Basal NO production by these cells was undetectable and no nitrite could be detected in any of the cocoa procyanidin fractions used at 100 pg/mL. Figure 36 indicates that procyanidin fractions A and D enhanced LPSinduced NO production by y-interferon primed monocytes/macrophages. Procyanidin fraction C was marginally effective, since LPS-stimulated monocytes/macrophages cultured in the absence of procyanidin fractions produced only 4p mole/10 cells/48 hours. y-Interferon alone was ineffective in inducing

NO.

Collectively, these results demonstrate that mixtures of the inventive compounds used at specific concentrations are capable of inducing monocyte/macrophage NO production both independent and dependent of stimulation by LPS or cytokines.

From the foregoing, it is clear that the extract and cocoa polyphenols, particularly the inventive compounds, 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 conjunction with BHT or BHA in known utilities of BHA and BHT, such as an antioxidant, for instance, an antioxidant; food additive.

The invention can also be employed in place of or in conjunction 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 compounds can be used as an antimicrobial.

S, The inventive compounds can also be used as a cyclooxygenase and/or lipoxygenase, NO or NO-synthase, or blood or in vivo glucose modulator, and are thus useful for treatment or prevention or modulation of pain, fever, inflammation coronary conditions, 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 compositions, topoisomerase II-inhibiting compositions, methods for preserving food or any desired item such as from oxidation, and methods for inhibiting topoisomerase II. The compositions 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 vitro and in vivo data herein, doses, routes of administration, and formulations can be obtained without undue experimentation.

Example 27. Micellar Electrokinetic Capillary Chromatography of Cocoa Procyanidins *5 A rapid method was developed using micellar electrokinetic capillary chromatography (MECC) to separate procyanidin oligomers. The method is a modification of that reported by Delgado et al., 1994. The MECC method requires only 12 minutes to achieve the same separation as that obtained by a S 70 minute normal phase HPLC analysis. Figure 37 represents a MECC separation of cocoa procyanidins obtained by Example 2.

•MECC Conditions: The cocoa procyanidin extract was prepared by the method described in Example 2 and dissolved at a concentration of 1 mg/mL in MECC buffer consisting of 200mM boric acid, sodium dodecyl sulfate (electrophoresis pure) and NaOH to adjust to pH The sample was passed through a 0.45um filter and electrophoresed using a Hewlett Packard HP-3D CZE System operated at the following conditions: Inlet buffer: Run buffer as described above Outlet buffer: Run buffer as described above 91 Capillary: 50cm x 75um i.d. uncoated fused silica Detection: 200nm, with Diode Array Detector Injection: 50 mBar for 3 seconds (150 mBar sec) Voltage: 6 watts Amperage: System limit (<300uA) Temperature: 25 0

C

Capillary Condition: 5 min flush with run buffer before and after each run.

This method can be modified by profiling temperature, pressure, and voltage parameters, as well as including organic modifiers and chiral selective agents in the run buffer.

Example 28. MALDI TOF/MS Analysis of Procyanidin Oligomers with Metal Salt Solutions S: A series of MALDI -TOF/MS analyses were performed on trimers combined with various metal salt solutions to determine whether cation adducts of the oligomer could be detected. The significance of the experiment was to provide evidence that the procyanidin oligomers play a physiological role in vitro and in vivo by sesquestering or delivering metal cations important to physiological processes and disease.

The method used was as described in Example S Briefly, 2uL of 10mM solutions of zinc sulfate dihydrate, S calcium chloride, magnesium sulfate, ferric chloride hexahydrate, ferrous sulfate heptahydrate, and cupric sulfate were individually combined with 4uL of a trimer purified to apparent homogeneity as described in Example 10, and 44uL of DHB added.

The results (Figures 38A-F) showed [Metal-Trimer H] ions for copper and iron (ferrous and ferric) whose m/z values matched 1 amu standard deviation value for the theoretical calculated masses. The [Metal-Trimer H] masses for calcium and magnesium could not be unequivocally resolved from the [Metal-Trimer masses for sodium and potassium, whose m/z values were within the 1 amu standard deviation values. No [Zn2 Trimer ion could be detected. Since some of these cations are multi-valent, the possibility for multimetaloligomer(s) ligand species and /or metal-multioligomer species were possible. However, scanning for these adducts at their predicted masses proved unsuccessful.

The results shown above for copper, iron, calcium, magnesium and zinc may be used as general teachings for subsequent analysis of the reaction between other metal ions and the inventive compounds, taking into account such factors as oxidation state and the relative position in the periodic table of the ion in question.

Example 29. MALDI TOF/MS Analysis of High Molecular Weight Procyanidin Oligomers An analytical examination was made on GPC eluants associated with high molecular weight procyanidin oligomers as prepared in Example 3, Method A. The objective was to determine whether procyanidin oligomers with n 12 were present. If present, these oligomers represent additional compounds of the invention. Adjustments to existing methods of isolation, separation and purification embodied in the invention can be made to obtain these oligomers for subsequent in vitro and in vivo evaluation for anti-cancer, anti-tumor or antineoplastic activity, antioxidant activity, inhibit DNA topoisomerase II enzyme, inhibit oxidative damage to DNA, and have antimicrobial, NO or NO-synthase, apoptosis, platelet aggregation, and blood or in vivo glucose-modulating activities, as well as efficacy as non-steroidal antiinflammatory agents.

Figure 39 represents a MALDI-TOF mass spectrum of the GPC eluant sample described above. The [M Na]+ and/or [M K] and/or [M 2Na]+ ions characterizing procyanidin oligomers representative of tetramers through octadecamers are clearly evident.

It was learned that an acid and heat treatment will cause the hydrolysis of procyanidin oligomers. Therefore, the invention comprehends the controlled hydrolysis of high molecular weight procyanidin oligomers where n is 13 to 18) as a method to prepare lower molecular weight procyanidin oligomers where n is 2 to 12).

Example 30. Tablet Formulations A tablet formulation was prepared using cocoa solids obtained by methods described in U.S. Application Serial No.

08/709,406 filed 6 September 1996, hereby incorporated herein by reference. Briefly, this edible material is prepared by a process which enhances the natural occurrence of the compounds of the invention in contrast to their levels found in traditionally processed cocoa, such that the ratio of the 15 initial amount of the compounds of the invention found in the unprocessed bean to that obtained after processing is less than or equal to 2. For simplicity, this cocoa solids material is designated herein as CP-cocoa solids. The inventive compound or S compounds, in isolated and/or purified form may be used in tablets as described in this Example, instead of or in S combination with CP-cocoa solids.

A tablet formula comprises the following (percentages expressed as weight percent): CP-cocoa solids 24.0% 4-Fold Natural vanilla extract (Bush Boake Allen) Magnesium stearate (dry lubricant)(AerChem, Inc.) Dipac tabletting sugar (Amstar 37.0% Sugar Corp.) Xylitol (American Xyrofin, Inc.) 37.0% 100.0% The CP-cocoa solids and vanilla extract are blended together in a food processor for 2 minutes. The sugars and magnesium stearate are gently mixed together, followed by blending in the CP-cocoa solids/vanilla mix. This material is run through a Manesty Tablet Press (B3B) at maximum pressure and compaction to produce round tablets (15mm x 5mm) weighing 1.8 gram. Another tablet of the above mentioned formula was prepared with a commercially available low fat natural cocoa powder (11% fat) instead of the CP-cocoa solids (11% fat). Both tablet formulas produced products having acceptable flavor characteristics and texture attributes.

An analysis of the two tablet formulas was performed using the procedures described in Example 4, Method 2. In this case, the analysis focused on the concentration of the pentamer and the total level of monomers and compounds of the invention where n is 2 to 12 which are reported below in Tab S Table pentamer total pentamer total (ug/l.8g (ug/l.8g Tablet sample (ug/g) (ug/g) serving) serving) tablet with 239 8,277 430 14,989 CP-cocoa solids tablet with ND 868 ND 1563 commercial low fat cocoa powder ND not detected The data clearly showed a higher level of pentamer and total level of compounds of the invention in the CP-cocoa solids tablet than in the other tablet formula. Thus, tablet formulas prepared with CP-cocoa solids are an ideal delivery vehicle for the oral administration of compounds of the invention, for pharmaceutical, supplement and food applications.

The skilled artisan in this area can readily prepare other tablet formulas covering a wide range of flavors, colors, excipients, vitamins, minerals, OTC medicaments, sugar fillers, UV protectants titanium dioxide, colorants, etc.), binders, hydrogels, and the like except for polyvinyl pyrrolidone which would irreversibly bind the compounds of the invention or combination of compounds. The amount of sugar fillers may be adjusted to manipulate the dosages of the 0.:0 compounds of the invention or combination of compounds.

Many apparent variations of the above are self-evident and possible without departing from the spirit and scope of the example.

Example 31 Capsule Formulations A variation of Example 30 for the oral delivery of the compounds of the invention is made with push-fit capsules made of gelatin, as well as soft sealed capsules made of gelatin and a plasticizer such as glycerol. The push-fit capsules contain S the compound of the invention or combination of compounds or CPcocoa solids as described in Examples 30 and 32 in the form of a powder which can be optionally mixed with fillers such as lactose or sucrose to manipulate the dosages of the compounds of the invention. In soft capsules, the compound of the invention or combination of compounds or CP-cocoa solids are suspended in a suitable liquid such as fatty oils or cocoa butter or combinations therein. Since an inventive compound or compounds may be light-sensitive, sensitive to UV, a capsule can contain UV protectants such as titanium dioxide or suitable colors to protect against UV. The capsules can also contain fillers such as those mentioned in the previous Example.

Many apparent variations of the above are self-evident 96 and possible to one skilled in the art without departing from the spirit and scope of the example.

Example 32 Standard of Identity (SOI) and Non-Standard of Identity (non-SOI) Dark and Milk Chocolate Formulations Formulations of the compounds of the invention or combination of compounds derived by methods embodied in the invention can be prepared into SOI and non-SOI dark and milk chocolates as a delivery vehicle for human and veterinary applications. Reference is made to copending U.S. Application Serial No. 08/709,406, filed September 6, 1996, hereby incorporated herein by reference. USSN 08/709,406 relates to a method of producing cocoa butter and/or cocoa solids having conserved levels of the compounds of the invention from cocoa beans using a unique combination of processing steps. Briefly, the edible cocoa solids obtained by this process conserves the natural occurrence of the compounds of the invention in contrast to their levels found in traditionally processed cocoa, such that the ratio of the initial amount of the compounds of the 2O*: invention found in the unprocessed bean to that obtained after processing is less than or equal to 2. For simplicity, this cocoa solids material is designated herein as CP-cocoa solids.

CP-cocoa solids are used as a powder or liquor to prepare SOT and non-SOI chocolates, beverages, snacks, baked goods, and as an ingredient for culinary applications.

The term "SOI chocolate" as used herein shall mean any chocolate used in food in the United States that is subject to a Standard of Identity established by the U.S. Food and Drug Administration under the Federal Food, Drug and Cosmetic Act.

The U.S. definitions and standards for various types of chocolate are well established. The term "non-SOI chocolate" as used herein shall mean any nonstandardized chocolates which have compositions which fall outside the specified ranges of the standardized chocolates.

Examples of nonstandardized chocolates result when the cocoa butter or milk fat are replaced partially or completely; or when the nutrative carbohydrate sweetener is replaced partially or completely; or flavors imitating milk, butter, cocoa powder, or chocolate are added or other additions or deletions in the formula are made outside the U.S. FDA Standards of Identity for chocolate or combinations thereof.

As a confection, chocolate can take the form of solid pieces of chocolate, such as bars or novelty shapes, and can also be incorporated as a component of other, more complex confections where chocolate is optionally combined with any Flavor Extract Manufacturers Association (FEMA) material, S natural juices, spices, herbs and extracts categorized as r natural-flavoring substances; nature-identical substances; and .0 artificial flavoring substances as defined by FEMA GRAS lists, .e FEMA and FDA lists, Council of Europe (CoE) lists, International Organization of the Flavor Industry (IOFI) adopted by the FA/W-O Food Standard Programme, Codex Alimentarius, and Food '0 Chemicals Codex and generally coats other foods such as caramel, go ~nougat, fruit pieces, nuts, wafers or the like. These foods are characterized as microbiologically shelf-stable at 65-85 0 F under 0:0-6: normal atmospheric conditions. Other complex confections result from surrounding with chocolate soft inclusions such as cordial cherries or peanut butter. Other complex confections result from coating ice cream or other frozen or refrigerated desserts with chocolate. Generally, chocolate used to coat or surround foods must be more fluid than chocolates used for plain chocolate solid bars or novelty shapes.

Additionally, chocolate can also be a low fat chocolate comprising a fat and nonfat solids, having nutrative carbohydrate sweetener(s), and an edible emulsifier. As to low fat chocolate, reference is made to U.S. Patent Nos. 4,810,516, 4,701,337, 5,464,649, 5,474,795, and WO 96/19923.

Dark chocolates derive their dark color from the amount of chocolate liquor, or alkalized liquor or cocoa solids or alkalized cocoa solids used in any given formulation.

However, the use of alkalized cocoa solids or liquor would not be used in the dark chocolate formulations in the invention, since Example 27, Table 13 teaches the loss of the compounds of the invention due to the alkalization process.

Examples of formulations of SOI and non-SOI dark and milk chocolates are listed in Tables 16 and 17. In these formulations, the amount of the compounds of the invention present in CP-cocoa solids was compared to the compounds of the invention present in commercially available cocoa solids.

The following describes the processing steps used in preparing these chocolate formulations.

Process for non-SOI Dark Chocolate 1. Keep all mixers and refiners covered throughout process to avoid light.

2. Batch all the ingredients excluding 40% of the free fat 2) (cocoa butter and anhy. milk fat) maintaining temperature between 30-35°C.

3. Refine to 20 microns.

4. Dry conche for 1 hour at 35 0

C.

Add full lechithin and 10% cocoa butter at the beginning of the wet conche cycle; wet conche for 1 hour.

6. Add all remaining fat, standardize if necessary and mix for 1 hour at 7. Temper, mould and package chocolate.

Process for SOI Dark Chocolate 1. Batch all ingredients excluding milk fat at a temperature 99 of 600C.

2. Refine to 20 microns.

3. Dry conche for 3.5 hours at 600C.

4. Add lecithin and milk fat and wet conche for 1 hour at 600C.

5. Standardize if necessary and mix for 1 hour at 350C.

Temper, mould and package chocolate.

Process for non-SOI Milk Chocolate 1. Keep all mixers and refiners covered throughout process to avoid light.

2. Batch sugar, whole milk powder, malted milk powder, and 66% of the cocoa butter, conche for 2 hours at 750C.

3. Cool batch to 35C and add cocoa powder, ethyl vanillin, chocolate liquor and 21% of cocoa butter, mix 20 minutes at 350C.

.1 4. Refine to 20 microns.

5. Add remainder of cocoa butter, dry conche for 1.5 hour at S: 350C.

6. Add anhy. milk fat and lecithin, wet conche for 1 hour at 350C.

7. Standardize, temper, mould and package the chocolate.

Process for SOI Milk Chocolate 1. Batch all ingredients excluding 65% of cocoa butter and milk fat at a temperature of 600C.

2. Refine to 20 microns.

3. Dry conche for 3.5 hours at 600C.

4. Add lecithin, 10% of cocoa butter and anhy. milk fat; wet conche for 1 hour at 600C.

Add remaining cocoa butter, standardize if necessary and mix for 1 hour at 350C.

6. Temper, mould and package the chocolate.

The CP-cocoa solids and commercial chocolate liquors used in the formulations were analyzed for the pentamer and total level of monomers and compounds of the invention where n is 2 to 12 as described in Method 2, Example 4 prior to incorporation in the formulations. These values were then used to calculate the expected levels in each chocolate formula as shown in Tables 16 and 17. In the cases for the non-SOI dark chocolate and non-SOI milk chocolate, their products were similarly analyzed for the pentamer, and the total level of monomers and the compounds of the invention where n is 2 to 12.

The results appear in Tables 16 and 17.

The results from these formulation examples indicated that SOI and non-SOI dark and milk chocolates formulated with CP-cocoa solids contained approximately 6.5 times more expected pentamer, and 3.5 times more expected total levels in the SOI and non-SOI dark chocolates; and approximately 4.5; 7.0 times *ooe more expected pentamer and 2.5; 3.5 times more expected total levels in the SOI and non-SOI milk chocolates, respectively.

Analyses of some of the chocolate products were not performed since the difference between the expected levels of the compounds of the invention present in finished chocolates prepared with CP-cocoa solids were dramatically higher than those formulas prepared with commercially available cocoa solids. However, the effects of processing was evaluated in the non-SOI dark and milk chocolate products. As shown in the tables, a 25-50% loss of the pentamer occurred, while slight differences in total levels were observed. Without wishing to be bound by any theory, it is believed that these losses are due to heat and/or low chain fatty acids from the milk ingredient (e.g.

101 acetic acid, propionic acid and butyric acid) which can hydrolyze the oligomers a trimer can hydrolyze to a monomer and dimer). Alternatively, time consuming processing steps can allow for oxidation or irreversible binding of the compounds of the invention to protein sources within the formula. Thus, the invention comprehends altering methods of chocolate formulation and processing to address these effects to prevent or minimize these losses.

The skilled artisan will recognize many variations in these examples to cover a wide range of formulas, ingredients, processing, and mixtures to rationally adjust the naturally occurring levels of the compounds of the invention for a variety of chocolate applications.

Table 16. Dark Chocolate Formulas PrAn~r~A 1.2,4-h LyL~~.k.L~I11~.S Non-SOI Dark Chocolate Using SOI Dark Chocolate Using CP- SOI Dark Chocolate Using CP-cocoa solids Cocoa Solids Commercial Cocoa Solids Formulation: Formulation: Formulation: 41.49 Sugar 41.49% sugar 41.49% sugar 3% whole milk powder 3% whole milk powder 3% whole milk powder 26% CP-cocoa solids 52.65% CP-liquor 52.65% com. liquor com. liquor 2.35% anhy. milk fat 2.35% anhy. milk fat 21.75% cocoa butter 0.01% vanillin 0.01% vanillin 2.75% anhy. milk fat 0.5% lecithin 0.5% lecithin 0.01% vanillin lecithin Total fat: 31% Total fat: 31% Total fat: 31% .S mL.L. cron Przicie size: 20 microns Particle size: 20 microns

I

Expected Levels of pentamer and total oligomeric procyanidins (monomers and n 2-12; units of ugig) Pentamer: 1205 Pentamer: 1300 Pentamer: 185 Total: 13748 Total: 14646 Total: 3948 Actual Levels of pentamer and total oligomeric procyandins (monomers and n 2-12; units of ugig) Pentamer: 561 [Not performed Not performed Total: 14097 .e Table 17. Milk Chocolate Formulas Prepared with non-Alkalized Cocoa Ingredients Non-SOI Milk Chocolate Using SOI Milk Chocolate Using CP- SOI Milk Chocolate Using CP-cocoa solids Cocoa Solids Commercial Cocoa Solids Formulation: Formulation: Formulation: 46.9965 Sugar 46.9965% sugar 46.9965% sugar 15.5% whole milk powder 15.5% whole milk powder 15.5% whole milk powder CP-cocoa solids 13.9% CP-liquor 13.9% cor. liquor corn. liquor 1.6% anhy. milk fat 1.60% anhy. milk fat 21.4% cocoa butter 0.0035% vanillin 0.0035% vanillin 1.6% anhy. milk fat 0.5% lecithin 0.5% lecithin 0.035% vanillin 17.5% cocoa butter 17.5% cocoa butter lecithin 4.0% malted milk powder 4.0% malted milk powder malted milk powder Total fat: 31.75% Total fat: 31.75% Total fat: 31.75% Particle size: 20 microns Particle size: 20 microns Particle size: 20 microns Expected Levels of pentamer and total oligomeric procyanidins (monomers and n 2-12; units of ug/g) Pentamer: 225 Pentamer: 343 Pentamer: 49 Total: 2734 Total: 3867 Total: 1042 Actual Levels of pentamer and total oligomeric procyandins (monomers and n 2-12; units of ug/g) Pentamer: 163 Not performed Not performed Total: 2399 Example 33. Hydrolysis of Procyanidin Oligomers Example 10, Method D describes the preparation normal phase HPLC procedure to purify the compounds of the invention.

The oligomers are obtained as fractions dissolved in mobile phase. Solvent is then removed by standard vacuum distillation (20-29 in. Hg: 400C) on a Rotovap apparatus. It was observed that losses of a particular oligomer occurred with increases in smaller oligomers when the vacuum distillation residence time was prolonged or temperatures >40°C were used.

The losses of a particular oligomer with accompanying increases in smaller oligomers was attributed to a timetemperature acid hydrolysis from residual acetic acid present in the mobile phase solvent mixture. This observation was confirmed by the following experiment where 100mg of hexamer was dissolved in 50mL of the mobile phase containing methylene chloride, acetic acid, water, and methanol (see Example 14, Method D for solvent proportions) and subjected to a timetemperature dependent distillation. At specific times, an aliquot was removed for analytical normal phase HPLC analysis as described in Example 4, Method 2. The results are illustrated in Figures 40 and 41, where hexamer levels decreased in a timetemperature dependent fashion. Figure 41 illustrates the appearance of one of the hydrolysis products (Trimer) in a timetemperature dependent fashion. Monomer and other oligomers (dimer to pentamer) also appeared in a time-temperature dependent fashion.

These results indicated that extreme care and caution must be taken during the handling of the inventive polymeric compounds.

The results provided above, together with that found in Examples 5, 11, 14, 15, 16 and 23, demonstrate that the method described above can be used to complement other methods embodied in the invention to identify any given oligomer of the invention.

For instance, the complete hydrolysis of any given oligomer which yields exclusively (+)-catechin or epicatechin eliminates many "mixed" monomer-based oligomer structure possibilities and reduces the stereochemical linkage possibilities characteristic for each monomer comprising any given oligomer.

Further, the complete hydrolysis of any given oligomer which yields both (+)-catechin and (-)-epicatechin in specific proportions provides the skilled artisan with information on the monomer composition of any given oligomer, and hence, the stereochemical linkage possibilities characteristic for each S monomer comprising the oligomer.

The skilled artisan would recognize the fact that acid S1.$ catalyzed epimerization of individual monomers can occur and S suitable control experiments and nonvigorous hydrolysis conditions should be taken into account the use of an organic acids, such as acetic acid, in lieu of concentrated HC1,

HNO

3 etc) S2 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 S limited by particular details set forth in the above descriptions as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

10 0 0 0 *0 *0 0

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Claims (19)

1. A method of inhibiting the oxidation of LDL in a mammal, comprising administering to said mammal a composition comprising a compound which is epicatechin or a polymeric compound of the formula An, wherein A is a monomer of the formula: OH OH HO 1 OH X 1 S wherein n is an integer from 2 to 18, such that there is at least one terminal monomer unit A and one or a plurality of additional monomeric units; 15 R is 3-(a)-o-sugar or sugar; and X has either a or p stereochemistry; bonding between adjacent monomers takes place at positions 4, 6 or 8; a bond of an additional monomeric unit in position 4 has a or P stereochemistry; X, Y, and Z are selected from a monomeric unit A, hydrogen and a sugar, with the proviso that as to the at least one terminal monomeric unit, bonding of the additional monomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; and the sugar is optionally substituted with a phenolic moiety at any position; or a pharmaceutically acceptable salt, glycoside, ester or oxidation product of said compound; and a pharmaceutically, veterinarily or food science acceptable carrier.

2. A method according claim 1 wherein n is 5 to 12.

3. A method according to claim 2 wherein n is

4. A method according to any one of the preceding claims wherein the ester of said polymeric compound is a 15 gallate.

A method according to any of the preceding claims wherein the sugar is selected from glucose, galactose,

6. A method according to any one of the preceding claims wherein the phenolic moiety is selected from caffeic, cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.

7. A method according to any of the preceding claims wherein, in the compound of formula An, bonding occurs between C-4 and C-6 of adjacent monomers or between C-4 and C-3 of adjacent monomers; and each of X, Z and Y is H, a sugar or an adjacent monomer, with the provisos that if X and Y are adjacent monomers, Z is H or sugar and if 123 X and Z are adjacent monomers, Y is H or sugar, and that as to at least one of the two terminal monomers, bonding of the adjacent monomer is at C-4 and optionally, Y Z hydrogen.

8. A method according to any one of the preceding claims wherein the polymeric compound is a substantially pure compound from a Theobroma or Herrania species or inter or intra-species specific crosses thereof.

9. A method according to any one of the preceding claims wherein R, of a terminal monomeric unit of said polymeric compound, is O-p-D-glucose or wherein R' Sis a gallic acid moiety.

Use,in the manufacture of a pharmaceutical composition, a veterinary composition or a food product, for use in inhibiting the oxidation of LDL, of a compound which is epicatechin or a polymeric compound of formula eo' 20 An wherein A is a monomer of the formula: OH OH Y HO O 4 3 Z R OH X wherein n is an integer from 2 to 18, such that there is at least one terminal monomer unit A and one or a 124 plurality of additional monomeric units; R is 3-(a)-o-sugar or sugar; and X has either a or P stereochemistry; bonding between adjacent monomers takes place at positions 4, 6 or 8; a bond of an additional monomeric unit in position 4 has a or P stereochemistry; X, Y, and Z are selected from a monomeric unit A, hydrogen and a sugar, with the proviso that as to the at 10 least one terminal monomeric unit, bonding of the additional monomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; and the sugar is optionally substituted with a phenolic moiety at any position; or a pharmaceutically acceptable salt, glycoside, ester or oxidation product of said compound.

11. Use according to claim 10 wherein n is 5 to 12. 20

12. Use according to claim 11 wherein n is 00

13. Use according to any one of claims 10 to 12 wherein the ester of said compound is a gallate.

14. Use according to any one of claims 10 to 13, wherein the sugar is selected from glucose, galactose, xylose, rhamnose and arabinose.

Use according to any one of claims 10 to 14, wherein the phenolic moiety is selected from caffeic, cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic 125 acids.

16. Use according to any one of claims 10 to wherein, in the compound of formula An, bonding occurs between C-4 and C-6 of adjacent monomers or between C-4 and C-3 of adjacent monomers; and each of X, Z and Y is H, a sugar or an adjacent monomer, with the provisos that if X and Y are adjacent monomers, Z is H or sugar and if X and Z are adjacent monomers, Y is H or sugar, and that 0 as to at least one of the two terminal monomers, bonding of the adjacent monomer is at C-4 and optionally, Y Z hydrogen. 4e 4 9* *0 1 *4 a 0* 0

17. Use according to any one of claims 10 to 16 wherein the polymeric compound is a substantially pure compound from a Theobroma or Herrania species or inter or intra- species specific crosses thereof.

18. Use according to any one of claims 10 to 17 wherein 20 said composition or food product takes the form of capsules, tablets, pills, a chewable solid or a beverage formulation.

19. A method according to any one of claims 1 to 9 or a use according to any one of claims 10 to 18 and substantially as hereinbefore described