HK1020881B

HK1020881B - Cocoa extract compounds and methods for making and using the same

Info

Publication number: HK1020881B
Application number: HK99106158.7A
Authority: HK
Inventors: 小L‧J‧罗曼塞克; 小J‧F‧哈姆梅尔斯通; M‧M‧布克; L‧S‧波斯特; G‧G‧西波拉; C‧A‧麦克勒兰德; J‧A‧蒙德特; H‧H‧施米茨
Original assignee: 火星有限公司
Priority date: 1996-04-02
Filing date: 1997-04-02
Publication date: 2005-03-11

Description

Extractable compounds and methods of making and using the same

Reference to related applications

The references are co-pending U.S. application nos. 08/709, 406, 08/631, 661, and 08/317, 226(U.S. patent nos. 5,554, 645), filed on 6/1996, 4/2/1996, and 08/317, 226, filed on 3/10/1994, and PCT/US 96/04497, which are incorporated herein by reference.

Technical Field

The present invention relates to cocoa extracts and compounds such as polyphenols obtained therefrom, preferably polyphenols enriched in procyanidins. The invention also relates to methods for preparing such extracts and compounds and their use as anti-tumor agents, antioxidants, DNA topoisomerase II enzyme inhibitors, cyclooxygenase and/or lipoxygenase modulators, NO (nitric oxide) or NO synthase modulators, or as non-steroidal anti-inflammatory agents, apoptosis modulators, platelet aggregation modulators, blood or body fluid glucose modulators, antibacterial agents, and inhibitors of oxidative DNA damage.

Each of these documents is fully incorporated by reference herein where it appears at the end of the specification or in the portion of the reference preceding the claims. These documents are within the scope of the present invention; each of the documents cited herein is incorporated herein by reference.

Background

Polyphenols are a group of very different compounds that occur widely in a variety of different plants (Ferreira et al, 1992), some of which enter the food chain. In some cases, they represent an important class of compounds for use in the human diet. Although some phenols are considered to be non-nutritive, there is increasing interest in these compounds because they may have beneficial effects on health.

For example, quercetin (a flavonoid) has been shown to have anti-cancer activity in animal subjects (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). Nobotainin, an oligomeric hydrolyzable tannin, has also been shown to have antitumor activity (Okuda et al, 1992). Also, statistical reports show that stomach cancer mortality in tea production areas of Japan is significantly low. Epigallocatechin gallate has been reported to be a pharmacologically active substance in green tea that inhibits mouse skin cancer (Okua et al, 1992). Ellagic acid has been shown to have anti-cancer factor activity in various animal models (Bukharta et al, 1992). Recently, the use of proanthocyanidin oligomers as antimutagens has been patented by the Kikkoman company. Indeed, the field of phenolic compounds in food and their modulatory effects on tumor spread in animal models for testing have recently been proposed at the American chemical Association 202 national conference (Ho et al, 1992; Huang et al, 1992).

However, none of these reports teach or suggest cocoa extracts or compounds derived therefrom, methods of making such extracts and compounds derived therefrom, or the use of cocoa extracts or compounds derived therefrom as, for example, antineoplastic agents, antioxidants, DNA topoisomerase II enzyme inhibitors, cyclooxygenase and/or lipoxygenase modulators, NO (nitric oxide) or NO synthase modulators, or as non-steroidal anti-inflammatory agents, apoptosis modulators, platelet aggregation modulators, blood or body fluid glucose modulators, antimicrobial agents, and inhibitors of oxidative DNA damage.

Objects and summary of the invention

Since unfermented cocoa beans contain a large amount of polyphenols, the present inventors have considered that it is possible to make the extract exhibit similar activity and use by extracting a cocoa extract, i.e., a compound in cocoa, from cocoa and screening the extract for activity. The national cancer institute has screened various Theobroma (Theobroma) and Herrania genera for anti-cancer activity as part of their enormous natural product selection program. Low levels of activity were reported in some extracts of the composable tissues and the work was not continued. Thus, cocoa and its extracts are not considered useful in anti-tumor or anti-cancer means; that is, teachings in anti-tumor or anti-cancer means mislead one skilled in the art to not use cocoa and its extracts in cancer therapy.

Since a number of analytical steps have been provided to investigate the contribution of cocoa polyphenols to the development of the diet (Clapperton et al, 1992), the present inventors decided to use a similar approach to prepare samples for anti-cancer screening, in contrast to the knowledge in anti-tumor or anti-cancer approaches. Surprisingly, contrary to the knowledge in the prior art, such as the national cancer institute screen, the present inventors have found that cocoa polyphenol extracts containing procyanidins have significant utility as anti-cancer and anti-neoplastic agents.

Furthermore, the present inventors demonstrate that cocoa extracts containing procyanidins and compounds obtained from cocoa extracts can be used as antineoplastic agents, antioxidants, DNA topoisomerase II enzyme inhibitors, cyclooxygenase and/or lipoxygenase modulators, NO (nitric oxide) or NO synthase modulators, or as nonsteroidal anti-inflammatory agents, apoptosis modulators, platelet aggregation modulators, blood or body fluid glucose modulators, antibacterial agents, and inhibitors of oxidative DNA damage.

It is an object of the present invention to provide a process for the preparation of an extractable and/or a compound obtained thereby.

It is another object of the present invention to provide a cocoa extract and/or compounds derived therefrom.

It is a further object of the present invention to provide a compound of formula A_nAnd pharmaceutically acceptable salts or derivatives (including oxidation products) thereof, wherein A is of the formula

N is an integer of 2 to 18, such that at least one terminal monomer unit A and a plurality of additional monomer units are present;

r is 3- (. alpha. -OH, 3- (. beta. -OH), 3- (. alpha. -O-saccharide or 3- (. beta. -O-saccharide;

the bonding between adjacent monomers occurs at the 4, 6 or 8 position;

the bond to the additional monomeric unit in position 4 has alpha or beta stereochemistry;

x, Y and Z are selected from monomer units A, hydrogen and a sugar, provided that for at least one terminal monomer unit the additional monomer unit bonded thereto is at the 4-position and Y ═ Z ═ hydrogen;

the sugar is optionally substituted at any position with a phenolic moiety, for example via an ester linkage.

N is an integer of 2 to 18, for example 3 to 18;

adjacent monomers are bonded at the 4 position by (4 → 6) or (4 → 8);

the bond at position 4 has alpha or beta stereochemistry;

X, Y and Z are each hydrogen, a sugar or an adjacent monomer, with the proviso that if X and Y are adjacent monomers then Z is H or a sugar, if X and Z are adjacent monomers then Y is H and a sugar, and for at least one of the two terminal monomers the bond of the adjacent monomer is at the 4-position and optionally Y ═ Z ═ hydrogen;

the bond at position 4 has alpha or beta stereochemistry.

The sugar is optionally substituted on any material with a phenolic moiety, for example via an ester linkage.

It is another object of the present invention to provide an antioxidant composition.

It is a further object of the present invention to demonstrate inhibition of DNA topoisomerase II enzyme activity.

It is also an object of the present invention to provide a method of treating a tumor or cancer.

It is also an object of the present invention to provide an anti-cancer, anti-tumor or anti-tumor composition.

It is yet another object of the present invention to provide an antimicrobial composition.

It is a further object of the present invention to provide a cyclooxygenase and/or lipoxygenase modulating composition.

It is a further object of the present invention to provide a NO or NO synthase modulating composition.

It is a further object of the present invention to provide a non-steroidal anti-inflammatory composition.

It is still another object of the present invention to provide a blood or body fluid glucose-modulating composition.

It is also an object of the present invention to provide a method of treating a patient with an anti-tumor, anti-oxidant, anti-microbial, cyclooxygenase and/or lipoxygenase modulating or NO synthase modulating, non-steroidal anti-inflammatory modulating and/or blood or body fluid glucose modulating composition.

It is an additional object of the present invention to provide a composition and method for inhibiting oxidative DNA damage.

It is a further object of the present invention to provide a composition and method for the modulation of platelet aggregation.

It is a further object of the present invention to provide a composition and method for apoptosis modulation.

It is a further object of the present invention to provide a method of making any of the above compositions.

It is also an object of the present invention to provide a kit for use in the above method or for preparing the above composition

It has been surprisingly found that cocoa extracts and compounds obtained therefrom have anti-tumor, anti-cancer and anti-tumor activity, or are antioxidant compositions, or inhibit DNA topoisomerase II enzyme activity, or are antimicrobial agents, or are cyclooxygenase and/or lipoxygenase modulators, or are NO or NO synthase modulators, or are non-steroidal anti-inflammatory agents, are apoptosis modulators, are platelet aggregation modulators, or are blood or body fluid glucose modulators, or are inhibitors of oxidative DNA damage.

Accordingly, the present invention provides a substantially pure cocoa extract and compounds derived therefrom. The extract or compound preferably comprises polyphenol(s) such as polyphenol(s) rich in cocoa procyanidins, for example at least one cocoa procyanidin selected from (-) epicatechin, (+) catechin, procyanidin B-2, procyanidin oligomer 2-18, for example 3-18, such as 2-12 or 3-12, preferably 2-5 or 4-12, more preferably 3-12, most preferably 5-12, procyanidin B-5, procyanidin A-2, procyanidin C-1.

The present invention also provides a composition of an anti-neoplastic, anti-cancer and anti-neoplastic agent, or an antioxidant, or a DNA topoisomerase II enzyme inhibitor, or an antimicrobial agent, or a cyclooxygenase and/or lipoxygenase modulator, or a NO or NO synthase modulator, a non-steroidal anti-inflammatory agent, an apoptosis modulator, a platelet aggregation modulator, a blood or body fluid glucose modulator, or an inhibitor of oxidative DNA damage, consisting of a substantially pure cocoa extract or a compound therefrom or a synthetic cocoa polyphenol, such as a procyanidin-rich polyphenol, and a suitable carrier, such as a pharmaceutically, veterinarily or food scientifically acceptable carrier. The extract and compounds derived therefrom preferably include cocoa procyanidins. The cocoa extract or compounds obtained therefrom are preferably obtained by a process comprising grinding cocoa beans to a powder, defatting the powder, extracting the active compound from the powder and purifying.

The invention also includes a method of treating a subject in need of treatment with an anti-neoplastic, anti-cancer or antineoplastic agent, or an antioxidant, or a DNA topoisomerase II enzyme inhibitor, or an antimicrobial agent, or a cyclooxygenase and/or lipoxygenase modulator, or a NO or NO synthase modulator, a non-steroidal anti-inflammatory agent, an apoptosis modulator, a platelet aggregation modulator, a blood or body fluid glucose modulator, or an inhibitor of oxidative DNA damage, the method comprising administering to the subject an effective amount of a substantially pure cocoa extract or compound therefrom or a synthetic cocoa polyphenol or procyanidin and a carrier, e.g., a pharmaceutically, veterinary or food science acceptable carrier. The cocoa extract or compound derived therefrom may be a cocoa procyanidin; preferably obtained by grinding cocoa beans to a powder, defatting the powder, and extracting the active compound from the powder and purifying it.

In addition, the present invention provides a kit for treating a subject in need of treatment with an anti-neoplastic, anti-cancer or antineoplastic agent, or an antioxidant, or a DNA topoisomerase II enzyme inhibitor, or an antimicrobial agent, or a cyclooxygenase and/or lipoxygenase modulator, or a NO or NO synthase modulator, a non-steroidal anti-inflammatory agent, an apoptosis modulator, a platelet aggregation modulator, a blood or body fluid glucose modulator, or an inhibitor of oxidative DNA damage, comprising a substantially pure cocoa extract or a compound therefrom or a synthetic cocoa polyphenol or procyanidin and a carrier suitable for admixture with the extract or compound therefrom or synthetic polyphenol or procyanidin, e.g., a pharmaceutically, veterinarily or food science acceptable carrier.

The present invention provides a compound as shown in figures 38A-38P and 39A-39 AA; currently, the linkage of 4 → 6 and 4 → 6 is preferred.

The invention further comprises food preservation and preparation compositions comprising the compounds of the invention, and methods of preparing or preserving food by adding the compositions to food.

Also encompassed by the invention are DNA topoisomerase II inhibitors comprising a compound of the invention and a suitable carrier or diluent, and methods of treating a patient in need thereof by administering the composition.

In general, in view of the above-described embodiments comprising a cocoa extract, the present invention also includes embodiments wherein the compounds of the present invention are used in place of or as a cocoa extract. Thus, the invention includes kits, methods, and compositions similar to those described above in connection with cocoa extracts and compounds of the invention.

The following detailed description discloses or will make apparent these and other objects and embodiments.

Brief Description of Drawings

The following detailed description will be better understood with reference to the accompanying drawings, in which:

FIG. 1 shows a representative gel permeation chromatogram of a crude cocoa procyanidin fraction;

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

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

FIG. 3 shows several representative procyanidin structures;

FIGS. 4A-4E show representative HPLC chromatograms of 5 fractions employed in screening for anti-cancer or anti-tumor activity;

FIGS. 5 and 6A-6D show the dose-response relationship (residual fraction/dose, μ g/ml) between cocoa extract and cancer cells ACHN (FIG. 5) and PC-3 (FIGS. 6A-6D); m & M2F 4/92, M & MA + E U12P1, M & MB + E Y192P1, M & MC + E U12P2, M & MD + E U12P 2;

FIGS. 7A-7H show typical dose-response relationships between cocoa procyanidin fractions A, B, C, D, E, A + B, A + E and A + D and the PC-3 cell line (residual fraction/dose, μ g/ml); MM-1A 0212P3, MM-1B 0162P1, MM-1C 0122P3, MM-1D 0122P3, MM-1E 0292P8, MM-1A/B0292P 6, MM-1A/E0292P 6, MM-1A/D0292P 6;

FIGS. 8A-8H show typical dose-response relationships between cocoa procyanidin fractions A, B, C, D, E, A + B, B + E and D + E and the KB nasopharynx/Hela line (residual fraction/dose, μ g/ml); MM-1A 092K3, MM-1B 0212K5, MM-1C 0162K3, MM-1D 0212K5, MM-1E 0292K5, MM-1A/B0292K 3, MM-1B/E0292K 4, MM-1D/E0292K 5;

FIGS. 9A-9H show typical dose-response relationships between cocoa procyanidin fractions A, B, C, D, E, B + D, A + E and D + E and the HCT-116 cell line (residual fraction/dose, μ g/ml); MM-1C 0192H5, D0192H 5, E0192H 5, MM-1B & D0262H 2, A/E0262H 3, MM-1D & E0260H 1;

FIGS. 10A-10H show typical dose-response relationships between cocoa procyanidin fractions A, B, C, D, E, B + D, C + D and A + E and ACHN renal cell lines (residual fraction/dose, μ g/ml); MM-1A 092A5, MM-1B 092A5, MM-1C 0192A7, MM-1D 0912A7, M & M1E 0912A7, MM-1B & D0302A6, MM-1C & D0302A6, MM-1A & E0262A 6;

FIGS. 11A-11H show typical dose response relationships between cocoa procyanidin fractions A, B, C, D, E, A + E, B + E and C + E and the A-549 lung cell line (fraction survival/dose, μ g/ml); MM-1A 019258, MM-1B 09256, MM-1C 019259, MM-1D 091258, MM-1E 091258, A/E026254, MM-1B & E030255, MM-1C & E N6255;

FIGS. 12A-12H show typical dose-response relationships between cocoa procyanidin fractions A, B, C, D, E, B + C, C + D and D + E and the SK-5 melanoma cell line (residual fraction/dose, μ g/ml); MM-1A 0121S4, MM-1B 0121S4, MM-1C 0121S4, MM-1D 0121S4, MM-1E N32S1, MM-1B & C N32S2, MM-1C & D N32S3 and MM-1D & E N32S 3;

FIGS. 13A-13H show typical dose-response relationships between cocoa procyanidin fractions A, B, C, D, E, B + C, C + E and D + E and the MCF-7 mammary cell line (fraction survival/dose, μ g/ml); MM-1A N22M4, MM-1B N22M4, MM-1C N22M4, MM-1D N22M3, MM-1E 0302M2, MM-1B/C0302M 4, MM-1C & E N22M3, MM-1D & E N22M 3;

FIG. 14 shows a typical dose response relationship between cocoa procyanidins (especially fraction D) and CCRF-CEM T-cell leukemia cell line (cells/mL/days of growth; open circle is control, filled circle is 125 μ g fraction D, open inverted triangle is 250 μ g fraction D, filled inverted triangle is 500 μ g fraction D);

FIG. 15A shows a comparison of XTT and the identification of crystal violet cytotoxicity against MCF-7 p168 lung cancer cells treated with fraction D + E (open circles are XYY, filled circles are crystal violet);

FIG. 15B shows a typical dose response curve obtained from MDA MB231 lung cancer cell line treated with various levels of crude polyphenols from UIT-1 cocoa genotype (absorbance (540 nm) vs. days; open circle is control, filled circle is vehicle, open inverted triangle is 250 μ g/mL, filled inverted triangle is 100 μ g/mL, open square is 10 μ g/mL; absorbance of 2.0 is the maximum of the plate reader and does not necessarily represent cell number);

FIG. 15C shows typical dose response curves obtained from PC-3 prostate cancer cell line treated with various levels of crude polyphenols from UIT-1 cocoa genotype (absorbance (540 nm) vs. days; open circle is control, filled circle is vehicle, open inverted triangle is 250 μ g/mL, filled inverted triangle is 100 μ g/mL, open square is 10 μ g/mL);

FIG. 15D shows a typical dose-response curve obtained from MCF-7 p168 breast cancer cell line treated with various levels of crude polyphenols obtained from UIT-1 cocoa genotype (absorbance (540 nm) vs. days; open circle is control, filled circle is vehicle, open inverted triangle is 250 μ g/mL, filled inverted triangle is 100 μ g/mL, open square is 10 μ g/mL, filled square is 1 μ g/mL; absorbance of 2.0 is the maximum of the plate reader and does not necessarily represent cell number);

FIG. 15E shows typical dose response curves obtained from Hela uterine cancer cell lines treated with various levels of crude polyphenols obtained from UIT-1 cocoa genotypes (absorbance (540 nm) vs. days; open circle is control, filled circle is vehicle, open inverted triangle is 250 μ g/mL, filled inverted triangle is 100 μ g/mL, open square is 10 μ g/mL; absorbance of 2.0 is the maximum of the plate reader and does not necessarily represent cell number);

FIG. 15F shows cytotoxic effects against Hela uterine cancer cell line treated with different cocoa polyphenol fractions (absorbance (540 nm) vs. days; open circles are 100 μ g/mL fractions A-E, filled circles are 100 μ g/mL fractions A-C, open inverted triangles are 100 μ g/mL fractions D & E; absorbance of 2.0 is the maximum for the plate reader and does not necessarily represent cell number);

FIG. 15G shows cytotoxic effects (absorbance (540 nm) per day; open circles are fractions A-E, filled circles are fractions A-C, open inverted triangles are fractions D & E) against SKBR-3 breast cancer cell line treated with different cocoa polyphenol fractions;

FIG. 15H shows a typical dose response relationship between cocoa procyanidin fractions D + E and Hela (absorbance (540 nm) vs. days; open circle is control, closed circle is 100. mu.g/mL, open inverted triangle is 75. mu.g/mL, closed inverted triangle is 50. mu.g/mL, open square is 25. mu.g/mL, closed square is 10. mu.g/mL; absorbance of 2.0 is the maximum for the plate reader and does not indicate cell number);

FIG. 15I shows a typical dose response relationship between cocoa procyanidin fraction D + E and SKBR-3 cells (absorbance (540 nm) vs. days; open circle is control, closed circle is 100. mu.g/mL, open inverted triangle is 75. mu.g/mL, closed inverted triangle is 50. mu.g/mL, open square is 25. mu.g/mL, closed square is 10. mu.g/mL);

FIG. 15J shows a typical dose response relationship between cocoa procyanidin fraction D + E and Hela identified using soft agar clones (bar chart; number of colonies/control, 1, 10, 50 and 100. mu.g/mL);

figure 15K shows the inhibition of Hela growth when Hela was treated with crude polyphenol extracts obtained from 8 different cocoa genotypes (% control/concentration, μ g/mL; open circle is C-1, closed circle is C-2, open inverted triangle is C-3, closed inverted triangle is C-4, open square is C-5, closed square is C-6, open triangle is C-7, closed triangle is C-8; C-1. UF-12: crude extract of cocoa polyphenol of horticulture variety Trinitario, and UF-12 (brazil) (decaffeinated/decaffeinated), C-2. NA-33: horticulture variety Forastero, and crude extract of cocoa polyphenol of NA-33 (brazil) (decaffeinated/decaffeinated), C-3. EEG-48: horticulture variety forsterio, and is a crude extract (decaffeinated/decaffeinated) of EEG-48 (brazil) cocoa polyphenols; c-4 ═ unknown: horticulture cultivar ═ forsterio, and is an unknown crude extract of (texifil) cocoa polyphenols (decaffeinated/theobromine depleted); c-5 ═ UF-613: horticulture cultivar Trinitario, and is a crude extract of UF-613 (brazil) cocoa polyphenol (decaffeinated/theobromine depleted); c-6 ═ ICS-100: horticulture cultivar (Trinitario ancestor Criollo nigara) and is a crude extract (decaffeinated/theobromine-depleted) of ICS-100 (brazil) cocoa polyphenols; c-7 ═ ICS-139: horticulture cultivar (Trinitario ancestor Criollo of nigara melon) and is a crude extract (decaffeinated/decaffeinated) of ICS-139 (brazil) cocoa polyphenols; c-8 ═ UIT-1: horticulture grade ═ Trinitario, and is the crude extract (decaffeinated/decaffeinated) of cocoa polyphenols of UIT-1 (Malaysia); ) (ii) a

FIG. 15L shows the inhibition of Hela growth when treated with crude polyphenol extracts obtained from fermented and dried cocoa beans (complete fermentation step and sun drying;% control/concentration, μ g/mL; open circle is the zero day fraction, closed circle is the 1 day fraction, open inverted triangle is the 2 day fraction, closed inverted triangle is the 3 day fraction, open square is the 4 day fraction and closed square is the 9 day fraction);

FIG. 15M shows the effect of enzymatically oxidized cocoa procyanidins on Hela (dose response of polyphenol oxidase treated crude cocoa polyphenols;% control/concentration, μ g/mL; filled squares are crude UIT-1 (with caffeine and theobromine), open circles are crude UIT (without caffeine and theobromine) and filled circles are crude UIT-1 (catalyzed by polyphenol oxidase));

FIG. 15N shows a representative semi-preparative reverse phase HPLC separation of complex cocoa procyanidin fractions D and E;

FIG. 15O shows a representative normal phase semi-preparative HPLC separation of crude cocoa polyphenol extract;

FIG. 16 shows typical Rancimat oxidation curves for cocoa procyanidin extracts and fractions compared to the synthetic antioxidants BHA and BHT (arbitrary units/time; dotted and cross (+). BHA and BHT;. D-E;. times. bold; open squares: A-C; and open diamonds: control);

FIG. 17 shows a typical agarose gel showing inhibition of topoisomerase II catalyzed desmosoming of motile DNA by cocoa procyanidin fractions (lane 1 contains 0.5 μ g of labeled (M) monomer-long motile DNA loops; lanes 2 and 20 contain motile DNA incubated with topoisomerase II in the presence of 4% DMSO but no cocoa procyanidins. (control-C); lanes 3 and 4 contain motile DNA incubated with topoisomerase II in the presence of 0.5h and 5.0 μ g/mL cocoa procyanidin fraction A; lanes 5 and 6 contain motile DNA incubated with topoisomerase II in the presence of 0.5 and 5.0 μ g/mL cocoa procyanidin fraction B; lanes 7, 8, 9, 13, 14 and 15 are incubated with topoisomerase II in the presence of 0.05, 0.5 and 5.0 μ g/mL cocoa procyanidin fraction D; lanes 10, 11, 12, 16, 17 and 18 replicates of kinetoplast DNA incubated with topoisomerase II in the presence of cocoa procyanidin fraction D at 0.05, 0.5 and 5.0 μ g/mL; lane 19 is a replicate of kinetoplast DNA incubated with topoisomerase II in the presence of 5.0 μ g/mL cocoa procyanidin fraction E; )

FIG. 18 shows the dose-response relationship between cocoa procyanidin fraction D resistance to DNA repair competence and defective cell lines (residual fraction/. mu.g/mL; left xrs-6 DNA defect repair cell line, MM-1D D282X 1; right BRI competent DNA repair cell line, MM-1 DD282B 1);

FIG. 19 shows the dose-response curve (% control/concentration, μ g/mL; open circle is MCF-7 p 168; closed circle is MCF-7 ADR) for doxorubicin-resistant MCF-7 cells compared to the MCF-7 p168 parental cell line when treated with cocoa fraction D + E;

FIGS. 20A and B show the dose-response effect of Hela and SKBR-3 when treated with twelve fractions prepared by normal phase semi-preparative HPLC at 100. mu.g/mL and 25. mu.g/mL levels (bar chart,% control/control and fractions 1-12);

FIG. 21 shows normal phase HPLC separation of crude, enriched and purified pentamers from cocoa extract;

FIGS. 22A, B and C show MALDI-TOF/MS of procyanidin-rich pentamer, fractions A-C and fractions D-E, respectively;

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

FIG. 23B shows an elution profile of trimer procyanidins purified by modified semi-preparative HPLC;

FIGS. 24A-D each show the energy lowest structure of all (4-8) bonded pentamers based on the epicatechin structure;

FIG. 25A shows the relative fluorescence of epicatechin upon thiolysis with benzylthiol;

FIG. 25B shows the relative fluorescence of catechin upon thiolysis with benzylmercaptane;

FIG. 25C shows the relative fluorescence of dimers (B2 and B5) upon thiolysis with benzylmercaptan; (ii) a

FIG. 26A shows the relative fluorescence of dimers upon thiolysis;

FIG. 26B shows the relative fluorescence of B5 dimer upon thiolysis and subsequent desulfurization of the dimer;

FIG. 27A shows relative tumor volumes during treatment of MDA MB 231 nude mouse model with pentamer;

FIG. 27B shows the relative survival curves of the MDA MB 231 nude mouse model treated with pentamer;

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

FIG. 29 shows the effect of pore size of the stationary phase used for normal phase HPLC separation of procyanidins;

FIG. 30A shows the utilization of substrates during fermentation of cocoa beans;

FIG. 30B shows metabolite production during fermentation;

FIG. 30C shows plate readings during cocoa bean fermentation;

FIG. 30D shows the relative concentrations of components in a fermented solution of cocoa beans;

FIG. 31 shows acetylcholine-induced relaxation of NO-associated phenylephrine-precontracted rat aorta;

FIG. 32 shows a graph of blood glucose tolerance from various test mixtures;

FIGS. 33A-B show the effect of indomethacin on COX-1 and COX-2 activity;

FIGS. 34A-B show the correlation (μ M) between the degree of polymerization and IC 50/COX-1/COX-2;

FIG. 35 shows the correlation between the effect of compounds on COX-1 and COX-2 activity (μ M);

FIGS. 36A-V show the IC of samples containing procyanidins and COX-1/COX-2₅₀Value (μ M);

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

FIGS. 38A-38P show preferred structures for the pentamer;

FIG. 39A-AA show a stereoisomer library of pentamers;

FIGS. 40A-B show the 70 min gradient of normal phase HPLC separation of procyanidins, as determined by UV and fluorescence, respectively;

FIGS. 41A-B show 30 min gradients for normal phase HPLC separation of procyanidins, as determined by UV and fluorescence, respectively;

FIG. 42 shows a preparative normal phase HPLC separation of procyanidins;

FIGS. 43A-G show the CD (circular dichroism) patterns of procyanidin dimers, trimers, tetramers, pentamers, hexamers, heptamers and octamers, respectively

FIG. 44A shows the structure of epicatechin¹H/¹³C NMR data;

FIGS. 44B-F show the compounds of epicatechin, APT, COSY, XHCORR,¹H and¹³a CNMR map;

FIG. 45A shows the structure of catechin and¹H/¹³c NMR data;

FIGS. 45B-E show the preparation of catechin¹H. APT, XHCORR, and COSY NMR spectra;

FIG. 46A shows the structure of B2 dimer and¹H/¹³c NMR data;

FIGS. 46B-G show dimer of B2¹³C、APT、¹H. HMQC, COSY and HOHAHA NMR spectra;

FIG. 47A shows the structure of dimer B5 and¹H/¹³c NMR data;

FIGS. 47B-G show dimers of B5¹H、¹³C. APT, COSY, HMQC and HOHAHA NMR spectra;

FIGS. 48A-D show epicatechin/catechin trimers¹H. COSY, HMQC and HOHAHA NMR spectra;

FIGS. 49A-D show the preparation of epicatechin trimer¹H. COSY, HMQC and HOHAHA NMR spectra;

FIGS. 50A and B show the effect of cocoa procyanidin fractions A and C, respectively, on blood pressure; blood pressure levels decreased by 21.43% within 1 minute after fraction a administration and returned to normal after 15 minutes, while blood pressure levels decreased by 50.5% within 1 minute after fraction C administration and returned to normal after 5 minutes;

FIG. 51 shows the effect of cocoa procyanidins on arterial blood pressure in anesthetized guinea pigs;

FIG. 52 shows the effect of L-NMMA on changes in arterial blood pressure in anesthetized guinea pigs produced by cocoa procyanidin fraction C;

FIG. 53 shows the effect of bradykinin on the amount of NO produced by HUVEC;

FIG. 54 shows the effect of cocoa procyanidin fractions on the amount of macrophage NO production by HUVEC;

FIG. 55 shows the effect of cocoa procyanidin fraction on macrophage NO production;

FIG. 56 shows the effect of cocoa procyanidins on LPS-induced and gamma interferon-induced macrophages;

FIG. 57 shows micellar electrokinetic capillary chromatography of cocoa procyanidin oligomers;

FIGS. 58A-F show Cu complexed to trimer⁺²-、Zn⁺²-、Fe⁺²-、Fe⁺³-、Ca⁺²-and Mg⁺²-MALDI-TOF mass spectrometry of ions;

FIG. 59 shows MALDI-TOF mass spectrum (tetramer to decamer) of cocoa procyanidin oligomers;

FIG. 60 shows the dose response relationship between cocoa procyanidin oligomers and a leukemia virus producing feline FeA lymphoblastoid cell line;

FIG. 61 shows dose-response relationships between cocoa procyanidin oligomers and normal renal cell lines of feline CRFKs;

FIG. 62 shows dose-response relationships between cocoa procyanidin oligomers and canine MDCK normal renal cell lines;

FIG. 63 shows a dose-response relationship between cocoa procyanidin oligomers and canine GH normal renal cell lines;

FIG. 64 shows the time-temperature effect of hexamer hydrolysis; and

FIG. 65 shows the time-temperature effect in the trimer form;

Detailed Description

Compounds of the invention

As discussed above, it has now been surprisingly found that cocoa extracts or compounds derived therefrom exhibit anti-neoplastic, anti-cancer and anti-tumor activity, antioxidant activity, inhibition of DNA topoisomerase II enzyme and oxidative DNA damage, and possess antimicrobial, cyclooxygenase and/or lipoxygenase, NO or NO synthase, apoptosis, platelet aggregation and blood or body fluid glucose modulating activity, as well as efficacy as non-steroidal anti-inflammatory agents.

The extract, compound or mixture of compounds derived therefrom is typically prepared by pulverizing cocoa beans to a powder, defatting the powder, and extracting and purifying the active compound from the powder. The powder can be prepared by freeze-drying the cocoa beans and pulp, removing the pulp and hull from the freeze-dried cocoa beans and milling the de-shelled cocoa beans. The extraction of the active compound can be carried out by solvent extraction techniques. Extracts including the active compounds may be purified, for example, by gel permeation chromatography or by preparative High Performance Liquid Chromatography (HPLC) techniques or by a combination of such techniques, e.g., to obtain a substantially pure extract.

With respect to the isolation and purification of compounds of the invention derived from cocoa, it will be understood that any species of Theobroma, Herrania or interspecies or intraspecies hybrids thereof will be employed. In this connection, the references are the summary of Herrania ("Synopsis of Herrania") by Schultes, the Journal of the Arnold plant park ("Journal of the Arnold arbor") at XXXIX, pages 217 to 278, and the pictures I to XVII (1985), Cuatrecasas Cocoa and Its homologues, the Classification Revision of the Genus Theobroma ("Cocoa and Its Allies, A Taxomic review of the Genus Theobroma") at 35, part 6, pages 379 to 613, and the pictures 1 to 11(Smithsonian institute of technology), and the observation of the Genus Theobroma of the Asian university of America ("Ochron of the United states National Musum") at 35, part 6, pages 379 to 1954, and the observation of the Genus Theobroma of America ("Ochron of America et al university of Japan, Inc. (Octozoa 1953).

Furthermore, example 25 lists concentrations of compounds of the invention found in theobroma and Herrania and interspecies and intraspecies mixtures thereof that have not been previously reported; also described in example 25 is a method of adjusting the amount of the compound of the invention that is obtainable by controlling the fermentation conditions of cocoa.

A schematic of the purification process used in the isolation of substantially pure procyanidins is shown in FIG. 37. Steps 1 and 2 of the purification scheme are described in examples 1 and 2; steps 3 and 4 are described in examples 3, 13 and 23; step 5 is described in examples 4 and 14; step 6 is described in examples 4, 14 and 16. Those skilled in the art will understand and envision modifications to the purification scheme outlined in FIG. 37 to obtain the active compounds without departing from the spirit and scope of the present invention and without undue experimentation.

The active extracts, compounds and mixtures of compounds derived therefrom, without wishing to be bound by any particular theory, have been considered cocoa polyphenols such as procyanidins. These cocoa procyanidins have significant anti-tumor, anti-cancer and anti-tumor activity; antioxidant activity; inhibition of DNA topoisomerase II enzyme and oxidative DNA damage; has antimicrobial activity; has the ability to modulate cyclooxygenase and/or lipoxygenase, NO or NO synthase, apoptosis, platelet aggregation, and blood or body fluid glucose, and efficacy as a non-steroidal anti-inflammatory agent.

The invention provides a formula

The compound of (1), wherein: n is an integer from 2 to 18, for example from 3 to 12, such that there is a first monomer unit A and a plurality of other monomer units;

alpha or beta stereochemistry at position 4;

x, Y and Z represent the bond between the monomer units and the position, but with the proviso that for the first monomer unit the other monomer units bonded thereto are at the 4 position and Y ═ Z ═ hydrogen, and with the proviso that, when not used for the bond and monomer units, X, Y and Z are hydrogen, or Z, Y is a saccharide and X is hydrogen, or X is an alpha or beta saccharide and Z, Y is hydrogen, or combinations thereof. In the compound, n may be 5 to 12, and a compound in which n is 5 is preferable. The sugar may be selected from glucose, galactose, xylose, rhamnose and arabinose. Any or all of the sugars of R, X, Y and Z may optionally be substituted with a phenol moiety through an ester linkage.

Accordingly, the present invention provides

Wherein n is an integer from 2 to 18, such as from 3 to 12, advantageously from 5 to 12, preferably n is 5, such that a first monomer unit A is present,

and a plurality of other monomer units of A.

Alpha or beta stereochemistry at position 4;

x, Y and Z represent the bond between the monomer units and the position, but with the proviso that for the first monomer unit the other monomer units bonded thereto are at the 4 position and Y ═ Z ═ hydrogen, and with the proviso that, when not used for the bond and monomer units, X, Y and Z are hydrogen, or Z, Y is a saccharide and X is hydrogen, or X is an alpha or beta saccharide and Z, Y is hydrogen, or combinations thereof.

The sugar may optionally be substituted with a phenolic moiety through an ester linkage.

Accordingly, the invention provides a compound of formula A_nAnd pharmaceutically acceptable salts or derivatives (including oxidation products) thereof, wherein A is of the formula

N is an integer from 2 to 18, such that at least one terminal monomer unit A and at least one or more additional monomer units are present;

the bonding between adjacent monomers occurs at the 4, 6 or 8 position;

x, Y and Z are selected from the group consisting of monomer units a, hydrogen and a saccharide, provided that for at least one terminal monomer unit, the additional monomer unit bonded thereto (that is to say the bond of the monomer unit adjacent to the terminal monomer unit) is at the 4-position and optionally Y ═ Z ═ hydrogen;

In preferred embodiments, n can be 3 to 18, 2 to 18, 3 to 12, such as 5 to 12; and advantageously n is 5. The sugar is selected from glucose, galactose, xylose, rhamnose and arabinose. Any or all of the sugars of R, X, Y and Z may optionally be substituted with a phenol moiety through an ester linkage. The phenolic moiety is selected from the group consisting of caffeic, cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.

Additionally, the present invention provides a compound of formula A_nAnd pharmaceutically acceptable salts or derivatives (including oxidation products) thereof, wherein A is of the formula

N is an integer from 2 to 18, such as from 3 to 18, advantageously from 3 to 12, such as from 5 to 12, preferably n is 5;

adjacent monomers are bonded at the 4-position by (4 → 6) or (4 → 8);

x, Y and Z are each hydrogen, a sugar or an adjacent monomer, with the proviso that if X and Y are adjacent monomers then Z is H or a sugar, if X and Z are adjacent monomers then Y is H and a sugar, and with the proviso that for at least one of the two terminal monomers the bond of the adjacent monomer is at the 4-position and optionally Y ═ Z ═ hydrogen;

The bond at position 4 has alpha or beta stereochemistry;

By the expression "at least one terminal monomeric unit A" it is of course understood that the compounds of the invention have two terminal monomeric units, and that these two terminal monomeric units may be identical or different. Furthermore, it is to be understood that the recitation of "at least one terminal monomeric unit A" includes embodiments in which the terminal monomeric unit is referred to as a "first monomeric unit", and the recitation of "first monomeric unit" refers to embodiments in which other monomeric units are appended thereto to obtain formula A_nMonomers of the polymeric compound. Furthermore, for at least one of the two terminal monomers, the bond of the adjacent monomer is in the 4-position and optionally Y ═ Z ═ hydrogen.

By the term "mixture thereof" it is understood that one or more compounds of the invention may be used simultaneously, that is to say a preparation comprising one or more compounds of the invention is administered to a patient in need of treatment.

The compounds of the invention or their mixtures show the use of the cocoa extracts mentioned above; and throughout this disclosure the term "cocoa extract" may be replaced by the compounds of the present invention or mixtures thereof, so it is understood that the compounds of the present invention and mixtures thereof are cocoa extracts.

The term "oligomer" as used herein refers to any compound of the structural formula presented above or mixtures thereof, wherein n is 2 to 18. When n is 2, the oligomer is referred to as a "dimer"; when n is 3, the oligomer is referred to as a "trimer"; when n is 4, the oligomer is referred to as a "tetramer"; when n is 5, the oligomer is referred to as a "pentamer"; and similar terminology may be used to denote oligomers with n up to and including 18, such that when n is 18, the oligomer is referred to as an "octadecomer".

The compounds of the invention or mixtures thereof may for example be isolated from natural sources such as any of the genus theobroma, Herrania or interspecies or intraspecies hybrids thereof; or the compounds of the invention or mixtures thereof may be purified, that is to say the compounds of the invention or mixtures thereof are substantially pure; for example, purification to apparent homogeneity. Purification is a relative concept and many examples demonstrate that isolating the compounds of the invention or mixtures thereof and purifying them, for example by methods enumerated by those skilled in the art, can result in substantially pure compounds of the invention or mixtures thereof, or purifying them to apparent homogeneity (i.e., by separation purification, characteristic chromatographic peaks). In view of the examples (e.g., example 37), a substantially pure compound or mixture of compounds is at least about 40% pure, e.g., at least about 50% pure, advantageously at least about 60% pure, e.g., at least about 70% pure, more advantageously at least about 75% to 80% pure, preferably at least about 90% pure, more preferably greater than 90% pure, e.g., at least 90 to 95% pure, or even more pure, e.g., greater than 95% pure, such as 95 to 98% pure.

Further, examples of monomer units comprising oligomers used herein are (+) -catechin and (-) -epicatechin, abbreviated as C and EC, respectively. The linkage between adjacent monomers is from 4 to 6 or from 4 to 8; such bonding between the 4-position of a monomer and the 6-and 8-positions of adjacent monomer units is referred to as (4 → 6) or (4 → 8) as such. There are 4 possible stereochemical bonds between the 4-position of a monomer and the 6-and 8-positions of its adjacent monomers; stereochemical bonding between monomers is referred to herein as (4 α → 6) or (4 β → 6) or (4 α → 8) or (4 β → 8). When C is bonded to another C or EC, the bond is referred to herein as (4 α → 6) or (4 α → 8). When EC is bonded to another C or EC, the bond is referred to herein as (4 β → 6) or (4 β → 8).

Examples of compounds that release the above-mentioned activity include dimers, EC- (4 β → 8) -EC and EC- (4 β → 6) -EC, with EC- (4 β → 8) -EC being preferred; trimer [ EC- (4. beta. → 8)]₂-EC、[EC-(4β→8)]₂-C and [ EC- (4. beta. → 6)]₂-EC, wherein [ EC- (4. beta → 8)]₂-EC; tetramer [ EC- (4. beta → 8)]₃-EC、[EC-(4β→8)]₃-C and [ EC- (4. beta. → 8)]₂-EC- (4 β → 6) -C, wherein [ EC- (4 β → 8)]₃-EC is preferred; and pentamer [ EC- (4. beta. → 8)]₄-EC、[EC-(4β→8)]₃-EC-(4β→6)-EC、[EC-(4β→8)]₃-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₃-EC- (4 β → 6) -C, wherein the 3-position of the pentamer terminal monomer unit is optionally derived from gallic acid or β -D-glucose; [ EC- (4. beta. → 8) ]₄-EC is preferred.

In addition, the compounds which release the above-mentioned activity also include hexamers to dodecamers, examples of which are as follows:

hexamers, wherein one monomer (C or EC) has a bond (4 β → 8) or (4 β → 6) to the other monomer for bonding EC to the other EC or C, and a bond (4 α → 8) or (4 α → 6) to the other monomer for bonding C to the other C or EC; and then bonded to the above pentamer compound by a (4. beta. → 8) bond, e.g. [ EC- (4. beta. → 8 ]]₅-EC、[EC-(4β→8)]₄-EC-(4β→6)-EC、[EC-(4β→8)]₄-EC- (4 β → → 8) -C and [ EC- (4 β → 8)]₄-EC- (4 β → 6) -C, wherein the 3-position of the hexamer terminal monomer unit is optionally derived from gallic acid or β -D-glucose; in a preferred embodiment the hexamer is [ EC- (4. beta. → 8)]₅-EC；

Heptamers in which any combination of two monomers (C and/or EC) has a bond (4 β → 8) or (4 β → 6) to another monomer for bonding EC to another EC or C, and has a bond to another monomerA bond (4 α → 8) or (4 α → 6) for bonding C to another C or EC; and then bonded to the above pentamer compound by a (4. beta. → 8) bond, e.g. [ EC- (4. beta. → 8 ]]₆-EC、[EC-(4β→8)]₅-EC-(4β→6)-EC、[EC-(4β→8)]₅-EC- (4 β → 8) -C and [ EC- (4 β → 8) ]₅-EC- (4 β → 6) -C, wherein the 3-position of the heptamer terminal monomer unit is optionally derived from gallic acid or β -D-glucose; in a preferred embodiment the heptamer is [ EC- (4. beta. → 8)]₆-EC；

An octamer in which any combination of three monomers (C and/or EC) has a bond (4 β → 8) or (4 β → 6) to another monomer for bonding EC to another EC or C, and a bond (4 α → 8) or (4 α → 6) to another monomer for bonding C to another C or EC; and then bonded to the above pentamer compound by a (4. beta. → 8) bond, e.g. [ EC- (4. beta. → 8 ]]₇-EC、[EC-(4β→8)]₆-EC-(4β→6)-EC、[EC-(4β→8)]₆-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₆-EC- (4 β → 6) -C, wherein the 3-position of the terminal monomer unit of the octamer is optionally derived from gallic acid or β -D-glucose; in a preferred embodiment the octamer is [ EC- (4. beta. → 8)]₇-EC；

Nonamer, where any combination of four monomers (C and/or EC) has a bond (4 β → 8) or (4 β → 6) to another monomer for bonding EC to another EC or C, and a bond (4 α → 8) or (4 α → 6) to another monomer for bonding C to another C or EC; and then bonded to the above pentamer compound by a (4. beta. → 8) bond, e.g. [ EC- (4. beta. → 8 ] ]₈-EC、[EC-(4β→8)]₇-EC-(4β→6)-EC、[EC-(4β→8)]₇-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₇-EC- (4 β → 6) -C, wherein the 3-position of the terminal monomer unit of the nonamer is optionally derived from gallic acid or β -D-glucose; in a preferred embodiment the nonamer is [ EC- (4. beta. → 8)]₈-EC；

Decamer in which any combination of five monomers (C and/or EC) has a bond to another monomerA bond (4 β → 8) or (4 β → 6) for bonding EC to another EC or C, and a bond (4 α → 8) or (4 α → 6) with another monomer for bonding C to another C or EC; and then bonded to the above pentamer compound by a (4. beta. → 8) bond, e.g. [ EC- (4. beta. → 8 ]]₉-EC、[EC-(4β→8)]₈-EC-(4β→6)-EC、[EC-(4β→8)]₈-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₈-EC- (4 β → 6) -C, wherein the 3-position of the terminal monomer unit of the decamer is optionally derived from gallic acid or β -D-glucose; in a preferred embodiment the decamer is [ EC- (4. beta. → 8)]₉-EC；

Undepolymers in which any combination of six monomers (C and/or EC) has a bond (4 β → 8) or (4 β → 6) to another monomer for bonding EC to another EC or C, and a bond (4 α → 8) or (4 α → 6) to another monomer for bonding C to another C or EC; and then bonded to the above pentamer compound by a (4. beta. → 8) bond, e.g. [ EC- (4. beta. → 8 ] ]₁₀-EC、[EC-(4β→8)]₉-EC-(4β→6)-EC、[EC-(4β→8)]₉-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₉-EC- (4 β → 6) -C, wherein the 3-position of the terminal monomer unit of the undecapmer is optionally derived from gallic acid or β -D-glucose; in a preferred embodiment the undecapolymer is [ EC- (4. beta. → 8)]₁₀-EC; and

a dodecamer in which any combination of seven monomers (C and/or EC) has a bond (4 β → 8) or (4 β → 6) to another monomer for bonding EC to another EC or C, and a bond (4 α → 8) or (4 α → 6) to another monomer for bonding C to another C or EC; and then bonded to the above pentamer compound by a (4. beta. → 8) bond, e.g. [ EC- (4. beta. → 8 ]]₁₁-EC、[EC-(4β→8)]₁₀-EC-(4β→6)-EC、[EC-(4β→8)]₁₀-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₁₀-EC- (4 β → 6) -C, wherein the 3-position of the terminal monomer unit of the dodecamer is optionally derived from gallic acid or β -D-glucose; in a preferred embodiment the dodecamer is [ EC- (4. beta. → 8)]₁₁-EC。

From this detailed description it will be clear that the above list is exemplary and serves as an explanation of a few non-limiting examples of the compounds of the invention, which by no means is an exhaustive list of the compounds of the invention encompassed by the present invention.

Examples 3A, 3B, 4, 14, 23, 24, 30 and 34 describe methods for isolating compounds of the invention. Examples 13, 14A-D and 16 describe methods for purifying compounds of the invention. Examples 5, 15, 18, 19, 20 and 29 describe methods for identifying compounds of the invention. FIGS. 38A-P and 39A-AA are schematic representations of a library of stereochemical configurations of the pentamers of the invention. Example 17 describes a method of making a molecular model of a compound of the invention. Example 36 provides evidence of the presence of higher oligomers (where n is 13-18) in cocoa.

Furthermore, because the present invention is described with respect to cocoa extracts that preferably include cocoa procyanidins, those skilled in organic chemistry will understand and envision synthetic routes to obtain and/or prepare the active compounds from this disclosure (see, e.g., example 11). Accordingly, the present invention includes synthetic cocoa polyphenols or procyanidins or derivatives thereof and/or synthetic precursors thereof and the like, wherein the precursors include, but are not limited to, glycosides, gallates, esters and the like. That is, the compounds of the present invention may be prepared by isolation from cocoa or from any of the genera theobroma or Herrania, as well as from synthetic routes; derivatives and synthetic precursors of the compounds of the invention, such as glycosides, gallates, esters and the like, are also included within the scope of the invention. Derivatives also include compounds of the above formula wherein the sugar or gallate moiety is on the terminal monomer at the Y or Z position or a substituted sugar or gallate moiety is on the terminal monomer at the Y or Z position.

For example, example 8, method C describes the use of cocoa enzymes to oxidatively modify a compound of the invention or a composition thereof to obtain improved cytotoxicity against a cancer cell line (see fig. 15M). The invention includes the ability to enzymatically modify the compounds of the invention, for example, using polyphenol oxidases, peroxidases, catalase mixtures, and/or enzymes such as hydrolases, esterases, reductases, transferases, and the like, and mixtures thereof, while taking into account kinetic and thermodynamic factors (see example 41, note hydrolysis).

With respect to the synthesis of the compounds of the present invention, based on this disclosure and the knowledge in the prior art, the skilled person will be able to foresee other synthetic routes without undue experimentation, e.g. based on careful retrosynthetic analysis of the polymeric compounds and on monomers. For example, given the nature of the compounds of the invention, the skilled worker may use a wide variety of selective protection/deprotection methods, methods for coupling with organometallic adducts, phenol coupling and photochemical methods, for example in convergent, linear or biomimetic methods, or combinations of these methods, together with standard reactions well known to the skilled worker in synthetic organic chemistry as additional synthetic methods for preparing the compounds of the invention, without undue experimentation. In this regard, references are W.Carruther, Modern Methods of Some Organic syntheses ("Some Modern Methods of Organic Synthesis") 3 rd edition, Cambridge University Press, 1986, and J.March, Advanced Organic Chemistry ("Advanced Organic Chemistry") 3 rd edition, John Wiley & Sons, 1985, Van Rensburg et al, chem.Comm., 24: 2705 to 2706(1996, 12, 21), Balllenegger et al, (Zyma SA) European patent 0096007B 1 and documents in the following reference section, all of which are incorporated herein by reference.

Use of the Compounds of the invention

With respect to the compounds of the present invention, it has been surprisingly found that the compounds of the present invention have different activities, and thus, the compounds of the present invention can be widely used for the treatment of various diseases, which will be discussed below.

COX/LOX-related uses

Atherosclerosis, the most prevalent of cardiovascular diseases, is the leading cause of heart disease, stroke, and vascular circulation problems. Atherosclerosis is a complex disease involving many cell types, biochemical phenomena, and molecular factors. Several aspects of the disease, its disease state and disease progression are identified by the dependent consequences of Low Density Lipoprotein (LDL) oxidation, Cyclooxygenase (COX)/Lipoxygenase (LOX) biochemistry and Nitric Oxide (NO) biochemistry.

Clinical studies have conclusively established that elevated plasma concentrations of LDL are associated with increased atherosclerosis. Cholesterol accumulated in atherosclerotic lesions is mainly produced from plasma lipoproteins, including LDL. The oxidation reaction of LDL is a critical event in the initiation of atherogenesis and is associated with superoxide anion radical (O)₂·^-) Is concerned with the increase in yield. By O₂·^-Or other active species (e.g.. OH, ONOO. cndot.) ^-Lipid peroxidation free radicals, copper ions and iron-based proteins) decreases the affinity of LDL for uptake into cells by receptor-mediated endocytosis. The oxidatively modified LDLs are then rapidly taken up by macrophages, which then transform into cells that closely resemble the "foam cells" found in early atherosclerotic lesions.

Oxidized lipoproteins can also promote vascular damage through the formation of lipid hydroperoxides in LDL particles. This event causes radical chain oxidation reactions of unsaturated LDL lipids, thus producing more oxidized LDL for macrophage incorporation.

The concentrated accumulation of foam cells laden with oxidized LDL by these methods leads to early "fatty streak" lesions that eventually progress to more advanced complex atherosclerotic lesions leading to coronary disease.

As discussed generally by Jean MarX in Science 265 (1994, 7, 15) page 320, approximately 330,000 patients undergo coronary and/or peripheral angioplasty, a procedure that opens a vessel blocked by a dangerous atherosclerotic plaque (atherosclerosis), such as a coronary vessel, and thus restores normal blood flow, annually in the united states. For most patients, the procedure is planned. However, almost 33% (and perhaps more by some reports) of these patients develop restenosis, in which the treated vessel is rapidly occluded again. These patients are not better, and sometimes worse, than they were prior to angioplasty. The restenosis is promoted by excessive proliferation of Smooth Muscle Cells (SMCs) in the vessel wall. The rapid accumulation of oxidized LDL in the lesion may contribute to processes associated with atherosclerotic formation mechanisms such as restenosis. Zhou et al relation Between The Risk Of primary cytomegalovirus Infection And Restenosis After coronary Athetomy ("Association Between PriorCytome Infection And The Risk Of Restenosis After coronary Athetomy") 1996, 8, 29, New England Journal Of Medicine, 335: 624-630, and the documents cited therein, all of which are incorporated herein by reference. Accordingly, the use of the invention in connection with atherosclerosis may be applicable to restenosis.

In inhibiting cyclooxygenase by the compounds of the present invention, cyclooxygenase is known to be a key enzyme in the production of prostaglandins and other arachidonic acid metabolites (e.g., eicosanoids) involved in many physiological processes. COX-1 is a constitutive enzyme expressed in many tissues, including platelets, while COX-2, the second isoform of the enzyme, is inducible by various cytokines, hormones, and tumor promoters. COX1 produces thromboxane a2, which is associated with platelet aggregation, which in turn is involved in the development of atherosclerosis. Its inhibition is the basis for the prophylactic effect of cardiovascular diseases.

COX-1 and COX-2 activity is inhibited by aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs), and the gastric side effects of NSAIDs are thought to be associated with COX-1 inhibition. Additionally, it has been found that patients who regularly take NSAIDs are at a 40-50% risk of contracting colorectal cancer compared to humans not administered such drugs; and COX-2 mRNA levels were significantly elevated in 86% of colorectal adenocarcinoma patients.

One significant property of COX-2 expressing cell lines is the greater expression of genes involved in apoptosis, i.e., modulation of apoptosis. Several NSAIDs have been implicated in increased cell death and induction of apoptosis in chicken embryo fibroblasts.

Cellular lipoxygenases are also involved in the oxidative modification of LDL by unsaturated lipids. Lipid peroxidation free radicals contribute to further oxidation of the free radical chain of unsaturated LDL lipids to produce more oxidized LDL for macrophage incorporation.

It has surprisingly been found that the compounds of the present invention are useful in the treatment of COX/LOX related diseases. In example 28, COX was inhibited by a separate compound of the invention at a concentration similar to that of the known NSAID, indomethacin.

For COX inhibition, the compounds of the invention are oligomers wherein n is 2-18. In a preferred embodiment, the compounds of the present invention are oligomers wherein n is 2 to 10, more preferably the compounds of the present invention are oligomers wherein n is 2 to 5.

Examples of compounds that release inhibitory activity with respect to COX/LOX as described above include dimers, trimers, tetramers and pentamers as discussed above.

Therefore, due to the significant inhibitory potency of the compounds of the present invention on COX-2 and cytotoxic effects on putative COX-2 expressing colon cancer cell lines, the compounds of the present invention have activity as inhibitors of apoptosis, which leads to the multistep progression of cancer, and as a member of the NSAID class of drugs with a broad spectrum of prophylactic activity (see, e.g., example 8, FIGS. 9D-9H).

In addition, prostaglandins, COX-catalyzed conversion of arachidonic acid to prostaglandin H₂The penultimate products of (a), are associated with inflammation, pain, fever, fetal development, labor, and platelet aggregation. Thus, the compounds of the invention are effective against the same conditions as NSAIDs, e.g., cardiovascular disease and stroke, etc. (in effect, reducing thromboxane A)₂Produced platelet COInhibition of X-1 is the basis for a prophylactic effect of aspirin on cardiovascular disease).

Inflammation is the response of living tissue to injury. It involves a complex series of enzyme activation, mediator release, extravasation of body fluids, cell migration, destruction of tissues and repair. Inflammation is caused by phospholipase A which releases arachidonic acid (substrates for COX and LOX)₂Is activated. COX converts arachidonic acid to prostaglandin PGE, the major eicosanoid found in inflammatory conditions ranging from acute edema to chronic arthritis₂. Inhibition thereof by NSAIDs is the primary means of treatment.

Arthritis is one of the wind-warm diseases, and rheumatism involves a wide variety of diseases and pathological processes, most of which damage joint tissues. The basic structure damaged by these diseases is connective tissue including synovium, cartilage, bone, tendons, ligaments, and interstitial tissue. Transient connective tissue syndromes include sprains and strains, tendonitis, and ganglioneural deformities. The most severe forms of arthritis are rheumatoid arthritis, osteoarthritis, gout, and systemic lupus erythematosus.

In addition to wind-warm diseases, other diseases are also characterized by inflammation. Gingivitis and periodontal periostitis follow pathological pictures similar to rheumatoid arthritis. Inflammatory bowel disease refers to idiopathic chronic inflammatory conditions of the bowel, ulcerative colitis and crohn's disease. Spondylitis involves chronic inflammation of the joints of the spine. Here again, the incidence of osteoarthritis associated with obesity is high.

Thus, the compounds of the present invention are useful for treating conditions involving inflammation, pain, fever, fetal development, labor, and platelet aggregation.

Inhibition of COX by the compounds of the invention will also inhibit prostaglandins, i.e., PGD₂、PGE₂Is performed. Thus, the compounds of the present invention are useful for the treatment of ulcers and prostaglandin PGD₂、PGE₂The associated condition.

Use in connection with NO

Nitric oxide is known to inhibit platelet aggregation, monocyte adhesion and chemotaxis, and proliferation of vascular smooth muscle tissue, primarily associated with the process of atherogenesis. Evidence supporting this view is the reduction of NO in atherosclerotic tissues due to its reaction with oxygen radicals. The reduction of NO due to these reactions results in a large number of platelets and inflammatory cells adhering to the vessel wall, further compromising the relaxation mechanism of NO. In this case, the reduction of NO promotes the atherogenic process, leading to an exacerbation of the condition.

Hypertension is the leading cause of cardiovascular diseases including stroke, heart attack, heart failure, irregular heart beat and renal failure. Hypertension is a condition in which the blood pressure in a blood vessel is higher than normal blood pressure when it flows through the body. When the systolic pressure exceeds 150 mmhg or the diastolic pressure exceeds 90 mmhg for a sustained period, it is harmful to the body. For example, excessive systolic blood pressure can destroy blood vessels anywhere. When it occurs in the brain, it causes a stroke. It can also cause thickening and narrowing of blood vessels, leading to atherosclerosis. The increase in blood pressure also forces the heart muscle to enlarge because it doubles to overcome the elevated resting (diastolic) pressure when blood is expelled. This enlargement eventually leads to irregular heart beats and heart failure. Hypertension is called "silent killer" because it does not produce any symptoms and is only found when blood pressure is measured.

Regulation of blood is a complex event, where one mechanism involves constitutive Ca⁺²Expression of calmodulin-dependent form of nitric oxide synthase (abbreviated eNOS). NO produced by this enzyme relaxes (relaxes) muscles in the blood vessels, which relaxes blood pressure. When normal levels of NO produced by eNOS are not available, either because production is prevented by inhibitors or in pathological situations, such as atherosclerosis, vascular muscles cannot relax to the appropriate degree. The resulting vasoconstriction increases blood pressure and may be responsible for some forms of hypertension.

Vascular endothelial cells include eNOS. NO synthesized by eNOS diffuses in the opposite direction, andand when it reaches the underlying vascular smooth muscle, NO adheres to the heme group of guanylate cyclase, causing cGMP to increase. The increase in cGMP results in intracellular free Ca⁺²Is reduced. Cyclic GMP activates protein kinases that mobilize Ca⁺²The transporter phosphorylates, thereby phosphorylating Ca⁺²Concealed within the intracellular structures in muscle cells. Since Ca is required for muscle contraction⁺²Therefore, due to Ca⁺²The concentration of (2) is reduced and the contractile force is reduced. Relaxation of muscles causes vasodilatation, which lowers blood pressure. Inhibition of eNOS therefore causes an increase in blood pressure.

When normal levels of NO are not produced, either because the inhibitor prevents production or in pathological conditions, such as atherosclerosis, the vascular muscle fails to relax to an appropriate degree. The resulting vasoconstriction increases blood pressure and may be responsible for some forms of hypertension. There is great interest in finding a treatment to increase eNOS activity in hypertensive patients, but no specific treatment is reported. Agents capable of releasing NO, such as nitroglycerin or isosorbide dinitrate, remain the primary means of vasodilation therapy.

Although the compounds of the present invention inhibit the oxidation of LDL, a more complex effect of these compounds is their diversification of the effects of atherosclerosis by NO. Modulation of NO by the compounds of the present invention produces a number of beneficial effects, including hypertension regulation, reduction of hypercholesterolemia affected by NO, inhibition of platelet aggregation and monocyte adhesion, all of which are associated with the development of atherosclerosis.

The role of NO in the immune system is different from its function in blood vessels. Macrophages contain a type of NOS, known as iNOS, which is inducible, rather than constitutive. Transcription of the iNOS gene is under positive or negative control by a number of biological response regulators called biological factors. The most important inducers are gamma interferon, tumor necrosis factor, interleukin 1, interleukin 2 and Lipopolysaccharide (LPS), wherein lipopolysaccharide is a component of the cell wall of gram-negative bacteria. Stimulated macrophages produce sufficient NO to inhibit the ribonuclease reductase enzyme that is used to convert nucleotides to deoxynucleotides that are required for DNA synthesis. Inhibition of DNA synthesis is an important approach in which macrophages and other tissues bearing iNOS can inhibit the growth of rapidly dividing tumor cells or infectious bacteria.

With respect to the role of NO and infectious bacteria, microorganisms play an important role in the infectious process reflecting physical contact and damage, habits, occupation, individual environment, and food-borne diseases through abnormal storage, handling, and contamination.

The compounds of the present invention, mixtures thereof and compositions containing them are useful in treating conditions associated with the modulation of NO concentrations. Example 9 describes the antioxidant activity (as inhibitors of free radicals) of the compounds of the present invention. Assuming that NO is a free radical and that the compounds of the invention are strong oxidants, it is speculated that administration of the compounds of the invention in vivo and in vitro experiments will result in reduced NO levels. Any reduction in NO will result in a hypertensive effect, not a hypotensive effect. Contrary to what was expected, the compounds of the invention increased NO in vitro experiments, while producing a hypotensive effect in vivo experiments (examples 31 and 32). These results are not expected and are completely unexpected.

Example 27 describes erythrocytosis (erythrocytosis) (flushing of the face) shortly after oral administration of a solution containing a compound of the invention and glucose, thus suggesting a vasodilating effect.

Example 31 describes the hypotensive effect obtained by the compounds of the present invention in an in vivo animal model, which demonstrates that the present invention is effective in treating hypertension. In this embodiment, the compounds of the present invention, mixtures thereof and compositions comprising them comprise oligomers wherein n is 2 to 18, preferably 2 to 10.

Example 32 describes the modulation of NO production by compounds of the invention in an in vitro model. In this embodiment, the compounds of the present invention, mixtures thereof and compositions containing them include oligomers wherein n is 2 to 18, preferably 2 to 10.

Furthermore, example 35 provides Cu formed as found by MALDI/TOF/MS⁺²-、Fe⁺²-、Fe⁺³Evidence of the complex of the oligomer. These results indicate that the compounds of the present invention can coordinate with copper and/or iron ions to reduce their effect on LDL oxidation.

In addition, the compounds of the present invention have potent antimicrobial activity in the treatment of infections and the prevention of food spoilage. Examples 22 and 30 describe the antimicrobial activity of the compounds of the present invention against several representative microorganisms of clinical and food significance listed below.

Clinical/food significance of microbial types

Helicobacter pylori gram-negative gastritis, ulcer and gastric cancer

Gram-positive food poisoning and wound sensation of bacillus

Infection, mastitis and septicemia of cattle

Disease and illness

Salmonella gram-negative food poisoning, diarrhea

Staphylococcus aureus gram-positive furuncle, carbuncle, wound infection,

Septicemia and breast abscess

Escherichia coli gram-negative infantile diarrhea and urinary tract infection

Pseudomonas gram-negative urinary tract infection and wound infection

Dyeing 'swimming ear'

Saccharomyces cerevisiae food rancidity

Acetobacter pasteurii gram-negative food rancidity

Example 33 describes the effect of compounds of the invention on macrophage NO formation. In this example, the results demonstrate that the compounds of the invention induce monocyte/macrophage NO formation, both independent and dependent on stimulation by Lipopolysaccharide (LPS) or cytokines. Macrophage ulceration of NO formation inhibits the growth of infected bacteria.

The compounds of the invention which release antimicrobial activity are oligomers wherein n is 2 to 18, preferably oligomers wherein n is 2, 4, 5, 6, 8 and 10.

Examples of compounds that release antimicrobial activity associated with NO as described above include dimers, tetramers, pentamers, hexamers, decamers and dodecamers as discussed above.

Anticancer use

Cancers are divided into three groups: carcinomas (carcinomas), sarcomas, and lymphomas. Cancer is a malignant tumor that develops in the skin, lining of various organs, glands and tissues. Sarcomas are malignant tumors that arise in bone, muscle or connective tissue. The third group includes leukemia and lymphoma, as both occur in blood-forming organs. The major types of cancer are prostate, breast, lung, colorectal, bladder, non-hodgkin's lymph, uterine, cutaneous melanoma, renal, leukemia, ovarian and pancreatic.

Cancer formation is caused by changes in DNA cells caused by many factors, such as genetic factors, ionizing radiation, contamination, radon, and free radicals that damage DNA. Cells carrying mutations are defective in the ordered course of cell division. These cells cannot undergo apoptosis (apoptosis) and continue to divide, either marking the onset of the malignancy or allowing more mutations to be made over a period of time, resulting in a malignancy.

There are three main common features for various cancers. They are (1) capable of unlimited proliferation; (2) tumor invasiveness to plant tissue; (3) and (5) transferring.

Certain types of cancer metastasize in a unique manner. For example thyroid, lung, breast and prostate cancers, often metastasize to bone. Lung cancer usually spreads to the brain and adrenal glands, and colorectal cancer often metastasizes to the liver. Leukemia is considered to be a systemic disease at onset, and is found in the bone marrow throughout the body.

It has been surprisingly found that the compounds of the present invention are effective in treating the various cancers discussed above. Examples 6, 7, 8 and 15 describe the effect of the compounds of the invention on human Hela (cervix), prostate, breast, kidney, T-cell leukemia, colon cancer cell lines, which release anticancer activity. Example 12 (FIG. 20) demonstrates the dose-response effect of Heal and SKBR-3 breast cancer cell lines treated with procyanidins of oligo (dimer to dodecamer) substantially purified by HPLC. Cytotoxicity against these cancer cell lines was dependent on pentamer to dodecamer procyanidins, whereas lower oligomers showed no effect.

Without wishing to be bound by any theory, the smallest structural primitive explaining the above-described effect occurs here. Example 37 also shows the same cytotoxic effect of higher oligomers (pentamer to dodecamer) against feline lymphoblast cell lines. Also in the higher oligomers (FIGS. 58-61) against cytotoxicity in normal canine and feline cell lines.

In example 8 (FIGS. 9D-H), it was shown that the compounds of the invention can release cytotoxicity against putative COX-2 expressing human colon cancer cell line (HCT 116).

Example 9 describes the antioxidant activity of the compounds of the present invention. The compounds of the present invention inhibit DNA strand breaks, DNA protein cross-linking and free radical oxidation of nucleotides to reduce and/or prevent the incidence of mutations.

Example 10 describes compounds of the present invention as topoisomerase II inhibitors, which are targets for chemotherapeutic agents such as doxorubicin.

Example 21 describes the in vivo effect of a substantially pure pentamer that releases anti-tumor activity against a human breast cancer cell line (MDA-MB-231/LCC6) in a nude mouse model (average weight of mouse is about 25 grams). Due to unexpected animal toxicity, in vivo experiments repeated with higher doses (5 mg) of pentamer were not completely successful. It is presently believed that this toxicity may be related to the vasodilating effect of the compounds of the present invention.

Example 33 describes the effect of compounds of the invention on macrophage NO formation. NO-producing macrophages can inhibit the growth of rapidly dividing tumor cells.

Furthermore, the invention includes the use of a compound of the invention to cause inhibition of cell proliferation by apoptosis.

Compounds of the invention for use in anti-cancer activity are oligomers wherein n is 2 to 18, for example 3 to 18, such as 3 to 12, preferably oligomers wherein n is 5 to 12, more preferably wherein n is 5.

Compounds that release inhibitory activity with respect to the above cancers include the pentamers through dodecamers discussed above.

Formulations and methods

Thus, in general, the compounds of the present invention, mixtures thereof and compositions containing them have shown a range of activities against atherosclerosis, cardiovascular diseases, cancer, blood pressure regulation and/or hypertension, inflammatory diseases, infectious agents and food spoilage.

Thus, the compounds of the present invention, mixtures thereof and compositions containing them are COX inhibitors through inhibition of thromboxane A₂Platelet aggregation is affected by the formation and thus by a reduced risk of thrombosis. In addition, inhibition of COX results in less platelets and inflammatory cells adhering to the vessel wall, thus allowing for improved relaxation mechanisms for NO. These results show anticoagulant potency in combination with inhibition of COX at concentrations similar to one of the known NSAIDs, indomethacin.

In addition, the compounds of the present invention, mixtures thereof and compositions containing them are antioxidants which inhibit the oxidation of LDL by lowering the levels of superoxide radical anions and lipoxygenase-mediated lipid peroxidation radicals. Inhibition of LDL oxidation at this stage reduces macrophage activity and delays foam cell formation, further interrupting the atherosclerotic process. Also inhibition of LDL oxidation may delay the process of restenosis. Thus, the compounds of the invention or mixtures thereof or compositions containing them may be used for the prevention and/or treatment of atherosclerosis and/or restenosis. Also, the compounds of the present invention can therefore be administered before or after angioplasty or similar surgery to prevent or treat restenosis in patients susceptible to restenosis.

For the treatment or prophylaxis of restenosis and/or atherosclerosis, one of the compounds of the invention or mixtures thereof or compositions containing one or more of the compounds of the invention, alone or together with other treatments as desired by the medical professional, is administered as soon as desired, for example at the time of the initial occurrence of the restenosis and/or atherosclerotic condition or symptom, immediately before, simultaneously with or after angioplasty, or as required by the medical professional after angioplasty, without any undue experimentation, in accordance with the disclosure and the general knowledge in the art; the compounds of the present invention or mixtures thereof or compositions containing them may be administered continuously without any other undue experimentation for a course of treatment, for example, a course of one month, two months, half a year, one year, or for a period of time deemed necessary by some other medical professional.

In addition, the compounds of the present invention, mixtures thereof and compositions containing them have been shown to produce hypotensive effects in vivo and to induce NO in vitro. Indeed, these results can be applied in the treatment of hypertension and in clinical situations where a significant reduction in NO levels is involved in hypercholesterolemia.

The formulations of the compounds of the present invention, mixtures thereof and compositions containing them may be prepared in the form of liquids, suspensions, tablets, capsules, injections or suppositories for immediate or sustained release of the active compound according to standard procedures well known to those skilled in the art of pharmaceutical, food science, medicine and veterinary medicine.

The carrier may be a polymeric delayed release system. Synthetic polymers are particularly useful in the formulation of controlled release compositions. An early example of this is the polymerization of methyl methacrylate into spheres with a diameter of less than 1 micron to form so-called nanoparticles, see Kreuter, j., "microcapsules and nanoparticles in medicine and pharmacy"Microcapsules and Nanoparticles in Medicine and Pharmacology"), M.Donbrow (ED). CRC Press, pages 125- & 148.

The carrier of choice for pharmaceutical agents and more recently for antigens is poly (d, 1-lactide-co-glycolide) (PLGA). This is a biodegradable polyester which has long been used in erodible pharmaceutical sutures, bone plates (boneplates) and other temporary prostheses, which do not exhibit any toxicity therein. A wide variety of agents have been formulated into PLGA microcapsules. As described by Eldridge, j.h. et al, "contemporary topics in Microbiology and Immunology" ("CurrentTopics in Microbiology and Immunology") 1989, 146: 59-66, a great deal of data has been accumulated regarding the suitability of PLGA for control. When the PLGA microspheres are orally taken, the capture substance (entrapm ent) in the PLGA microspheres with the diameter of 1-10 microns plays a role. The method of microencapsulation of PLGA employs a phase separation method of water-in-oil emulsion. The compounds of the invention or mixtures thereof are prepared as an aqueous solution with PLGA dissolved in a suitable organic solvent such as dichloromethane and ethyl acetate. The two immiscible solutions are co-emulsified by high speed stirring. A non-solvent for the polymer is then added, causing the polymer to precipitate around the aqueous droplets, forming the initial microcapsules. The microcapsules are collected, stabilized with a reagent (polyvinyl alcohol (PVA), gelatin, alginate, methyl cellulose) and the solvent is removed by vacuum drying or solvent extraction.

Further, with respect to the preparation of sustained release formulations, references are made to U.S. Pat. Nos. 5,024,843, 5,091,190, 5,082,668, 4,612,008, and 4,327,725, which are incorporated herein by reference.

In addition, the selection method and identification of cocoa genotypes of interest are used to prepare unit-normal (SOI) and non-SOI chocolate products that can be used as vehicles for delivering active ingredients to patients in need of treatment for the above conditions and as a means for delivering the compounds of the invention at a conserved level.

In this regard, the reference is co-pending U.S. application Ser. No. 08/709,406, filed on 9/6/1996, which is incorporated herein by reference. USSN08/709,406 relates to a process for producing cocoa butter and/or cocoa solids from cocoa beans with horizontally conserved polyphenols using a unique combination of processing steps that do not require separate cocoa bean roasting or refining equipment to process the cocoa beans optionally without exposure to intense heat treatment for a sustained period of time and/or using solvent extraction methods of fats. The method has the advantage that the conservation of polyphenols can be increased compared to polyphenols found in traditional cocoa processing methods, such that the ratio of the initial content of polyphenols found in the raw cocoa beans to the content of polyphenols obtained after processing is less than or equal to 2.

The compositions of the present invention include one or more of the above-mentioned compounds in a dosage form with a pharmaceutically acceptable carrier or excipient. The compounds of the invention have anti-tumor, anti-cancer and anti-tumor activity, antioxidant activity, inhibit DNA topoisomerase II enzyme, inhibit oxidative DNA damage, induce monocyte/macrophage NO formation, have antimicrobial, cyclooxygenase and/or lipoxygenase, NO or NO synthase, apoptosis, platelet aggregation and blood or in vivo glucose regulating activity, and efficacy as non-steroidal anti-inflammatory agents.

Other embodiments of the present invention include compositions comprised of the compounds of the present invention and mixtures thereof and at least one additional antineoplastic, antihypertensive, anti-inflammatory, antimicrobial, antioxidant and hematopoetic agent in addition to a pharmaceutically acceptable carrier or excipient.

Data set forth herein, route of administration, concentration of individual oligomers, and toxicity (e.g., LD) are considered₅₀) Such compositions may be administered to a patient in need of such regimen, either by dose or by techniques well known to those skilled in the medical, nutritional or veterinary arts, as data on such factors as age, sex, weight, genetics and condition of the individual subject or patient.

The composition may be co-administered or sequentially administered with other antineoplastic, anticancer, antineoplastic, antioxidant, DNA topoisomerase II inhibitor, inhibitor of oxidative DNA damage or cyclooxygenase and/or lipoxygenase inhibitor, apoptosis modulator, platelet aggregation modulator, blood or in vivo glucose modulator or NO synthase modulator, non-steroidal anti-inflammatory agent and/or with an agent capable of reducing or alleviating the adverse effects of antineoplastic, anticancer, antineoplastic, antioxidant, DNA topoisomerase II inhibitor, inhibitor of oxidative DNA damage or cyclooxygenase and/or lipoxygenase inhibitor, apoptosis modulator, platelet aggregation modulator, blood or in vivo glucose modulator or NO synthase modulator, non-steroidal anti-inflammatory agent, taking into account such factors as age, weight, and the like, Sex, weight, genetics and condition of the individual subject or patient, and route of administration.

Examples of compositions of the invention for human or veterinary use include edible compositions for oral administration, such as solid or liquid dosage forms, e.g. capsules, tablets, pills and the like, and chewable solid or drinkable dosage forms, to which the invention is particularly suitable, since the invention is derived from edible sources (e.g. cocoa or chocolate flavored solid or liquid compositions); liquid preparations suitable for needle injection, oral, nasal, anal, vaginal, etc., such as suspensions, syrups or elixirs (including cocoa or chocolate flavored compositions); formulations suitable for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g. injectable modes of administration) are for example sterile suspensions or emulsions. However, the active ingredient in the composition may be complexed with a protein, such that when administered in the blood, coagulation may occur due to precipitation of blood proteins; this should be taken into account by a person skilled in the art. In such compositions, the active cocoa extract may be mixed with suitable carriers, diluents or excipients such as sterile water, physiological saline, dextrose, DMSO, ethanol and the like. The active cocoa extracts of the invention may be provided in lyophilized form and then re-used, for example, in isotonic aqueous solution, saline, dextrose or DMSO buffer. In a certain saline solution, some precipitation was found; this finding can be employed as a means of isolating the compounds of the invention, for example by a "salting out" method.

Example 38 describes the formulation of a compound of the invention in tablet dosage form for use in the pharmaceutical, supplement and food fields. Additionally, example 39 describes the formulation of the compounds of the present invention for use in capsule dosage forms in the same field. In addition, example 40 describes dosage forms containing a compound of the present invention or cocoa solids obtained according to the methods described in co-pending U.S. application Ser. No. 08/709,406, which is incorporated herein by reference, standard identification (SOI) and non-SOI chocolate.

Reagent kit

In addition, the invention also includes a kit having an active cocoa extract therein. The kit may comprise a separate container containing a suitable carrier, diluent or excipient. The kit may further comprise an additional anti-neoplastic, anti-cancer, anti-neoplastic agent, antioxidant, DNA topoisomerase II enzyme inhibitor, inhibitor of oxidative DNA damage or cyclooxygenase and/or lipoxygenase inhibitor, apoptosis modulator, platelet aggregation modulator, blood or in vivo glucose modulator or NO synthase modulator, non-steroidal anti-inflammatory agent and/or agent capable of reducing or alleviating the adverse effects of the anti-neoplastic, anti-cancer, anti-neoplastic agent, antioxidant, DNA topoisomerase II enzyme inhibitor, inhibitor of oxidative DNA damage or cyclooxygenase and/or lipoxygenase inhibitor, apoptosis modulator, platelet aggregation modulator, blood or in vivo glucose modulator or NO synthase modulator, non-steroidal anti-inflammatory agent. The additional agent can be in a separate container or mixed with the active cocoa extract. In addition, the kit may include means for mixing or combining the components and/or a method of administration.

Identification of genes

Another embodiment of the invention comprises the modulation of genes expressed as a result of close cell contact by means of the compounds of the invention or mixtures of these compounds. Thus, the present invention includes the identification of genes associated with several diseases, including but not limited to atherosclerosis, hypertension, cancer, cardiovascular disease and inflammation, induced or inhibited by the compounds of the present invention or mixtures thereof. More precisely, genes that are differentially expressed in these disease states (relative to their expression in a "normal" disease-free state) are identified and described before and after contact by the mixture of the invention or mixtures thereof.

As mentioned in the discussion above, these diseases and disease states are based in part on free radical interactions and the diversity of biomolecules. The central topic associated with these diseases is that many free radical reactions involve reactive oxygen species, which in turn induce physiological states associated with disease progression. For example, reactive oxygen species have been implicated in the regulation of transcription factors such as Nuclear Factor (NF) - κ B. The target genes for (NF) - κ B include a series of genes associated with a synergistic inflammatory response. These genes include genes encoding Tumor Necrosis Factor (TNF) - α, Interleukin (IL) -I, (IL) -6 and (IL) -8, inducible NOS, Major Histocompatibility Complex (MHC) class I antigen, and others. Also, in turn, genes that regulate transcription factor activity can be induced by oxidative stress. The oxidation pressure between the radical scavenger and the radical generating system is unbalanced. Some known examples of these cases (Winyard and Blake, 1977) include gaddl 53 (a gene induced by growth inhibition and DNA damage), the product of which has been shown to bind NF-IL6 and form heterodimers that cannot bind DNA. NF-IL6 upregulates the expression of several genes, including those encoding interleukins 6 and 8. Another example of an oxidative stress inducible gene is gadd45, which regulates the role of the transcription factor p53 in growth inhibition. p53 encodes a p53 protein that can stop cell division and induce apoptosis in abnormal cells (e.g., cancer cells).

Giving an overview of the surprising, unobvious and novel uses of the compounds or mixtures of compounds of the present invention in the treatment of a variety of diseases based in part on free radical mechanisms. The invention further includes methods for determining the transient effects of compounds of the invention on the expression of genes or gene products in animal in vitro and in vivo models of specific diseases or disease states by using gene expression assays. These include, but are not limited to, differentiation, sequencing of cDNA libraries, Serial Analysis of Gene Expression (SAGE), expression monitoring by hybridization to high density oligonucleotide species and various reverse transcriptase-polymerase chain reactions (RT-PCR) based on this method or combinations thereof (Lockhart et al, 1996).

The combined physiological effects of the compounds or mixtures of compounds of the invention comprised in the present invention, combined with genetic evaluation methods, enable the discovery of genes and gene products, whether known or new, induced or inhibited. For example, the invention encompasses the use of RT-PCR to induce and/or inhibit cytokines (e.g., IL-1, IL-2, IL-6, IL-8, IL-12, and TNF- α) in lymphocytes in vitro and in vivo. Similarly, the invention includes Applying the differential to determine induction and/or inhibition of the selection gene; for use under conditions of stimulation and/or oxidant stimulation (e.g., TNF-alpha or H)₂O₂Conditions) of the cardiovascular field (e.g., superoxide dismutase, heme oxidase, COXI and 2, and other oxidant defense genes). For the cancer field, the present invention includes the use of differentiation displays to determine the induction and/or inhibition of genes or gene products, such as CuZn-superoxide 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 construed as limiting the present invention, as many apparent variations thereof are possible without departing from the spirit or scope thereof.

Examples

Example 1:cocoa sources and methods of preparation

Some generic species of cocoa (Theobroma cocao) representing three established horticultural varieties of cocoa were obtained from three major cocoa production places in the world. Typical species of those used in this study are shown in table 1. The harvested cocoa pods were opened and the beans with pulp were removed for freeze drying. The pulp was manually removed from the freeze-dried product, and the beans were analyzed as follows. Unfermented, freeze-dried cocoa beans were first peeled manually and ground to a very fine powder with a TEKMAR Mill. The resulting material was then defatted overnight by Soxhlet extraction using redistilled hexane as the solvent. Residual solvent was removed from the degreased material at room temperature using vacuum.

Table 1: description of cocoa (Theobroma cacao) derived materials

Generic species of genus	Producing area	Species of horticulture
Generic species of genus	Producing area	Species of horticulture	UIT-1	Malaysia	Trinitario
Is unknown	West Fei	Forastero	UIT-1	Malaysia	Trinitario
Is unknown	West Fei	Forastero	ICS-100	Brazil rubber	Trinitario(NicaraguanCriollo ancestor)
ICS-39	Brazil rubber	Trinitario(NicaraguanCriollo ancestor)	ICS-100	Brazil rubber	Trinitario(NicaraguanCriollo ancestor)
ICS-39	Brazil rubber	Trinitario(NicaraguanCriollo ancestor)	UF-613	Brazil rubber	Trinitario
EEG-48	Brazil rubber	Forastero	UF-613	Brazil rubber	Trinitario
EEG-48	Brazil rubber	Forastero	UF-12	Brazil rubber	Trinitario
NA-33	Brazil rubber	Forastero	UF-12	Brazil rubber	Trinitario

Example 2:method for extracting cyanidin

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 a 50g batch of defatted cocoa mass using 2X 400mL 70% acetone/deionized water followed by 400mL 70% methanol/deionized water. The extract was collected and the solvent was removed by evaporation at 45 ℃ using a rotary evaporator placed under partial vacuum. The resulting aqueous phase was diluted to 1L with deionized water and 400mL of CHCl₃The extraction was performed twice. The solvent phase was discarded. The aqueous phase is then extracted 4 times with 500mL of ethyl acetate. All resulting emulsions were broken by centrifugation at 2,000 Xg for 30 minutes at 10 ℃ on a Sorvall RC 28S centrifuge. To the combined ethyl acetate extracts was added 100-200mL of deionized water and the solvent was removed by evaporation at 45 ℃ using a rotary evaporator placed under partial vacuum. The resulting aqueous phase was frozen in liquid N2, followed by lyophilization in a laboco lyophilization system. The yields of crude procyanidins obtained from different typicals of the genus Theobroma are listed in Table 2.

Table 2: yield of crude procyanidins

Generic species of genus	Producing area	Yield (g)
Generic species of genus	Producing area	Yield (g)	UIT-1	Malaysia	3.81
Is unknown	West Fei	2.55	UIT-1	Malaysia	3.81
Is unknown	West Fei	2.55	ICS-100	Brazil rubber	3.42
ICS-39	Brazil rubber	3.45	ICS-100	Brazil rubber	3.42
ICS-39	Brazil rubber	3.45	UF-613	Brazil rubber	2.98
EEG-48	Brazil rubber	3.15	UF-613	Brazil rubber	2.98
EEG-48	Brazil rubber	3.15	UF-12	Brazil rubber	1.21
NA-33	Brazil rubber	2.23	UF-12	Brazil rubber	1.21

B. Method 2

In addition, procyanidins were extracted from defatted, unfermented, freeze-dried cocoa beans of example 1 with 70% aqueous acetone, and 10g of the defatted material was stirred with 100mL of solvent for 5-10 minutes. The slurry was centrifuged at 3000 Xg for 15 minutes at 4 ℃ and the supernatant was passed through glass wool. The filtrate was subjected to distillation under partial vacuum, and the resulting aqueous phase was frozen in liquid N₂Followed by lyophilization in a laboconco lyophilization system. The yield of crude procyanidins is 15-20%.

Without wishing to be bound by any particular theory, it is believed that the difference in crude yield reflects the differences between different generic species, geographical origin, horticultural cultivars and preparation methods.

Example 3:partial purification of cocoa procyanidins

A. Gel permeation chromatography

The procyanidins obtained from example 2 were partially purified by liquid chromatography on Sephadex LH-20 (28X 2.5 cm). A step gradient from deionized water to methanol was used to aid separation. The initial gradient composition started with a 15% methanol in deionized water followed by stepwise use of 25% methanol in deionized water, 35% methanol in deionized water, 70% methanol in deionized water and finally 100% methanol every 30 minutes. The effluent after elution of the xanthine alkaloids (caffeine and theobromine) was collected as a single fraction. This fraction yielded a sub-fraction of xanthine-free alkaloids which was further sub-fractionated to yield 5 sub-fractions (subfractions) designated MM2A-MM 2E. The solvent was removed from each subfraction by evaporation at 45 ℃ on a rotary evaporator placed under partial vacuum. Freezing the resultant water in liquid N ₂And lyophilized overnight in a laboconco freeze-drying system. A representative gel permeation chromatography showing fractionation is shown in fig. 1. About 100mg of material is fractionally separated in this way.

Chromatography conditions are as follows: a column; sephadex LH-20 of 28 x 2.5cm,mobile phase: a methanol/water step gradient, 15: 85, 25: 75, 35: 65, 70: 30, 100: 0, at 1/2 hour intervals, flow rate; 1.5 mL/min, detector; UV at λ₁254nm and λ₂365nm, recording speed: 0.5 mm/min, column load; 120 mg.

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

The method comprises the following steps: reverse phase separation

The 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 1mL injection port was equipped with a Pharmacia FRAC-100 fraction collector. In the field of Phenomenex 10 mu ODS Ultracarb^TMPhenomenex Ultracarb connected to a (60X 10mm) protective column^TMThe separation was carried out on a 10. mu. ODS column (250X 22.5 mm). Mobile phase conjugate a ═ water; b ═ methanol, used with the following linear gradient conditions: [ time,% A](ii) a (0, 85), (60, 50), (90, 0) and (110, 0) at a flow rate of 5 mL/min. The compounds were detected with UV light at 254 nm.

Representative semi-preparative HPLC amounts used to isolate procyanidins present in fraction D + E are shown in figure 15N. Individual peaks or selected chromatographic regions were collected at specified time intervals or by manual fraction collection for further purification and later evaluation. The injection loading is 25-100mg substance.

Method 2. normal phase separation

The procyanidin extract obtained from example 2 and/or 3A was partially purified by semi-preparative HPLC. A Hewlett Packard 1050 HPLC system with a Millipore-Waters model 480 LC detector set at 254nm was fitted with a pharmaciFrac-100 fraction collector set at peak shape. The separation was carried out on a Supelco 5 μm Supelcosil LC-Si column (250X 10mm) connected to a Supelco 5 μm Supelguard LC-Si column (20X 4.6 mm). Procyanidins were eluted with a linear gradient under the following conditions: (time: 0% A,% B); (0, 82, 14), (30, 67.6, 28.4), (60, 46, 50), (65, 10, 86), (70, 10, 86), followed by a 10 minute rebalance. The mobile phase composition is A ═ dichloromethane; b ═ methanol; and C ═ acetic acid: water (1: 1). A flow rate of 3 mL/min was used. The components were detected with uv at 254nm and recorded on a Kipp and Zonan BD41 recorder. Injection volume was 100-. The amounts of representative semi-preparative HPLC are shown in figure 15. The peaks or selected chromatographic zones are collected at specified time intervals or by manual collection of fractions for further purification and later evaluation. HPLC conditions: supelco Supelcosil LC-Si (5 μm) 250X 10mm

Semi-preparative column 20X 4.6mm Supelco Supelcosil LC-Si

(5 μm) guard column

A detector: waters LC

Spectrophotometer type 480@254nm

Flow rate: 3 mL/min of the reaction solution is added,

column temperature: at room temperature

And (3) injection: 250 μ L of 70% acetone extract

Gradient: time (minutes)	CH₂Cl₂	Methanol	Acetic acid H₂O(1∶1)
Gradient: time (minutes)	CH₂Cl₂	Methanol	Acetic acid H₂O(1∶1)	0	82	14	4
30	67.6	28.4	4	0	82	14	4
30	67.6	28.4	4	60	46	50	4
65	10	86	4	60	46	50	4
65	10	86	4	70	10	86	4

The fractions obtained were as follows:

fraction(s) of	Type (B)
fraction(s) of	Type (B)	1	Dimer
2	Trimer	1	Dimer
2	Trimer	3	Tetramer
4	Pentameric polymers	3	Tetramer
4	Pentameric polymers	5	Hexamer
6	Heptamer	5	Hexamer
6	Heptamer	7	Octamer
8	Nonomer	7	Octamer
8	Nonomer	9	Decamer
10	Undecamer	9	Decamer
10	Undecamer	11	Dodecamer
12	Higher oligomers	11	Dodecamer

Example 4:analytical HPLC analysis of procyanidins

The method comprises the following steps: reverse phase separation

Procyanidins obtained from example 3 were filtered through a 0.45 μ filter and analyzed using a hewlett packard 1090 three-way HPLC system equipped with a Diode Array detector and a HP type 1046A intensity controlled fluorescence detector. The separation was carried out at 45 ℃ on a Hewlett-Packard5 μ Hypersil ODS column (200X 2.1 mm). The flavanols (flavanols) and procyanidins were eluted with a linear gradient from 60% B to a followed by washing the column with B at a flow rate of 0.3 mL/min. The mobile phase composition was a methanol solution of 0.5% acetic acid B and a nano-pure water solution of 0.5% acetic acid a. The acetic acid level in the a and B mobile phases can be increased to 2%. With λ therein _ex276nm and λ_emFluorescence at 316nm and detection of each component with uv at 280 nm. The concentrations of (+) catechin and (-) epicatechin were determined from the reference standard solution. The levels of procyanidins were calculated by using the response factor of (-) epicatechin. A representative HPLC chromatogram showing the separation of various components for a typical species of the genus theobroma is shown in fig. 2A. A similar HPLC profile was obtained from another Cocospora species.

HPLC conditions: column: 200X 2.1mm Hewlett Packard Hypersil ODS

(5μ)

Protection of the column: 20X 2.1mm Hewlett packard Hypersil

ODS(5μ)

A detector: diade Array @280nm

Fluorescence lambda_ex＝276nm；λ_em＝316nm

Flow rate: 0.3 mL/min

Column temperature: 45 deg.C

Gradient: time (minutes)	0.5% acetic acid in nano-pure water	0.5% acetic acid in methanol
Gradient: time (minutes)	0.5% acetic acid in nano-pure water	0.5% acetic acid in methanol	0	100	0
50	40	60	0	100	0
50	40	60	60	0	100

The method 2 comprises the following steps: normal phase separation

The procyanidin extract obtained from example 2 and/or 3 was filtered through a 0.45 μ filter and analyzed using a Hewlett packard 1090 series II HPLC system equipped with an HP model 1046A programmable fluorescence detector and a diode system detector. At 37 ℃ in a 5. mu. Phenomenex Lichrosphere attached to a Supelco Supelguard LC-Si 5. mu. guard column (20X 4.6mm) ^_The separation was carried out on a column of silicon 100 (250X 3.2 mm). Procyanidins were eluted with a linear gradient under the following conditions: (time,% A,% B); (0, 82, 14), (30, 67.6, 28.4), (60, 46, 50) (65, 10, 86), (70, 10, 86), followed by 8 minutes of rebalancing. The mobile phase composition is A ═ dichloromethane, B ═ methanol, and C ═ acetic acid: water in a volume ratio of 1: 1. Using a flow of 0.5 mL/minAnd (4) speed. With λ therein_ex276nm and λ_emFluorescence at 316nm or detection of each component by ultraviolet light at 280 nm. A representative HPLC chromatogram showing the separation of various procyanidins for an atypical species is shown in figure 28. A similar HPLC profile was obtained from another Cocospora species.

HPLC conditions:

250×3.2mm Phenomenex Lichrosphere^_silica 100 column (5 μ)

20×4.6mm

Supelco Supelguard LC-Si (5 μ) guard column Detector: photodiode

Array @280nm fluorescence lambda_ex＝276nm；λ_em＝316nm

Flow rate: 0.5 mL/min

Column temperature: 37 deg.C

Gradient: time (minutes)	CH₂-Cl₂	Methanol	Acetic acid/water (1: 1)
Gradient: time (minutes)	CH₂-Cl₂	Methanol	Acetic acid/water (1: 1)	0	82	14	4
30	67.6	28.4	4	0	82	14	4
30	67.6	28.4	4	60	46	50	4
65	10	86	4	60	46	50	4
65	10	86	4	70	10	86	4

Example 5:identification of procyanidins

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

The partially purified isolate was analyzed by fast atom bombardment-mass spectrometry (FAB-MS) in a VG ZAB-T high resolution MS system using a liquid phase secondary ion mass spectrometry (LSIMS) method of positive and negative ion type. At 30KV, a cesium ion gun was used as the ionization source, and "MagicBullet Matrix" (1: 1 dithiothreitol/dithioerythritol) was used as the proton donor.

Analytical studies of these fractions with LSIMS revealed the presence of a variety of flavan-3-ol oligomers as shown in the following table.

Table 3: LSIMS (Positive ion) data from cocoa procyanidin fractions.

Oligomers	(M+1)⁺m/z	(M+Na)⁺m/z	Molecular weight
Oligomers	(M+1)⁺m/z	(M+Na)⁺m/z	Molecular weight	Monomer (Catechin)	291	313	290
Dimer (S)	577/579	599/601	576/578	Monomer (Catechin)	291	313	290
Dimer (S)	577/579	599/601	576/578	Trimer (S)	865/867	887/889	864/866
Tetramer (S)	1155	1177	1154	Trimer (S)	865/867	887/889	864/866
Tetramer (S)	1155	1177	1154	Pentamer (S)	1443	1465	1442
Hexamer (S)	1731	1753	1730	Pentamer (S)	1443	1465	1442
Hexamer (S)	1731	1753	1730	Heptamer (S)	---	2041	2018
Octamer (S)	---	2329	2306	Heptamer (S)	---	2041	2018
Octamer (S)	---	2329	2306	Nonmer (S)	---	2617	2594
Decamer (S)	---	2905	2882	Nonmer (S)	---	2617	2594
Decamer (S)	---	2905	2882	Undecamer (S)	---	---	3170
Dodecamer (S)	---	---	3458	Undecamer (S)	---	---	3170

The ions of the major mass fraction were consistent with the previously reported work on FAB-MS analysis of positive and negative ions of procyanidins (Self et al, 1986 and Porter et al, 1991). Corresponding to M/z 577(M + H)⁺And its ion at M/z 599(M + Na)⁺The sodium adduct of (a) indicates the presence of a doubly linked procyanidin dimer in the isolate. Of great interest is the higher oligomer with its protonated molecular ion (M + H) ⁺More likely to form sodium adduct (M + Na)⁺. Based on work reported by Revila et al (1991), Self et al (1986) and Porter et al (1991), procyanidin isomers B-2, B-5 and C-1 were speculatively identified. In the partially purified fraction, procyanidins up to octamers and decamers were confirmed by FAB-MS. In addition, evidence of procyanidins up to the dodecamer was observed from normal phase HPLC analysis (see fig. 2B). Table 4 lists the relative concentrations of procyanidins found in the isolate without xanthine alkaloids based on reverse phase HPLC analysis. Table 5 lists the relative concentrations of procyanidins based on normal phase HPLC analysis.

Table 4: relative concentrations of procyanidins in isolate free of xanthine alkaloids.

Composition (I)	Measurement of
Composition (I)	Measurement of	(+) -Catechin	1.6％
(-) epicatechin	38.2％	(+) -Catechin	1.6％
(-) epicatechin	38.2％	B-2 dimer	11.0％
B-5 dimer	5.3％	B-2 dimer	11.0％
B-5 dimer	5.3％	C-1 trimer	9.3％
Double-bonded dimers	3.0％	C-1 trimer	9.3％
Double-bonded dimers	3.0％	Tetramer (S)	4.5％
Pentamer-octamer	24.5％	Tetramer (S)	4.5％
Pentamer-octamer	24.5％	Unknown and higher oligomers	2.6％

Table 5: relative concentration of procyanidins in aqueous acetone extract

Composition (I)	Measurement of
Composition (I)	Measurement of	(+) -catechin and (-) -epicatechin	41.9％
B-2 and B-5 dimers	13.9％	(+) -catechin and (-) -epicatechin	41.9％
B-2 and B-5 dimers	13.9％	Trimer	11.3％
Tetramer	9.9％	Trimer	11.3％
Tetramer	9.9％	Pentameric polymers	7.8％
Hexamer	5.1％	Pentameric polymers	7.8％
Hexamer	5.1％	Heptamer	4.2％
Octamer	2.8％	Heptamer	4.2％
Octamer	2.8％	Nonomer	1.6％
Decamer	0.7％	Nonomer	1.6％
Decamer	0.7％	Undecamer	0.2％
Dodecamer	＜0.1％	Undecamer	0.2％

FIG. 3 shows the structure of some procyanidins, FIGS. 4A-4E show representative HPLC chromatograms of five fractions employed in the following screening for anti-cancer or anti-tumor activity. The conditions for the HPLC of FIGS. 4A-4E were as follows:

HPLC conditions: a hewlett packard 1090 ternary HPLC system equipped with an HP model 1046A programmable fluorescence detector.

Column: hewlett Packard 5. mu. Hypersil ODS (200X 2.1mm) with linear gradient from 60% B to A, flow rate 0.3 mL/min, B ═ 0.5% acetic acid in methanol; a 0.5% acetic acid in deionized water. Lambda [ alpha ]_ex＝280nm；λ_em＝316nm。

Figure 15O shows representative semi-preparative HPLC chromatography (HPLC conditions as described above) of another 12 fractions employed in screening for anti-tumor or anti-cancer activity.

Example 6:anticancer, antitumor or antitumor activity of cocoa extract (procyanidins)

MTT (3- [4, 5-dimethylthiazol-2-yl ] -2, 5-diphenyltetrazolium bromide), originally developed by Mosmann (1983), microtiter plate tetrazolium cytotoxicity assay was used to screen test samples from example 5. The test samples, standards (cisplatin and chlorambucil) and MTT reagent were dissolved in 100% DMSO (dimethyl sulfoxide) at a concentration of 10 mg/mL. Serial dilutions were prepared from stock solutions. In the case of the test samples, dilutions varying from 0.01 to 100. mu.g/mL were prepared in 0.5% DMSO.

All human tumor cell lines were obtained from the American type culture Collection. Cells were grown as monolayers in α -MEM containing 10% fetal bovine serum, 100 units/mL penicillin, 100 μ g/mL streptomycin, and 240 units/mL nystatin. Cells were stored in humidified 5% CO at 37 deg.C₂In an atmosphere.

After trypsinization, cells were counted and adjusted to 50X 10⁵Concentration of cells/mL (varied according to cancer cell lines). 200 μ l of the cell suspension was plated into 4 rows of wells of a 96 well microtiter plate. After allowing the cells to adhere for four hours, 2 μ Ι DMSO containing test sample solution was added to four rows of wells. Experiments looking for initial dose response using size order of test sample dilutions were used to determine the dose range to be examined. The absorbance at 540nm of each well was then measured in a BIO RAD MP 450 plate counter. The average absorbance of four rows of test sample treated wells was compared to the control and the results were expressed as the percent of control absorbance plus/minus the standard deviation. The reduction of MTT to purple formazan product correlates in a linear fashion with the number of viable cells in the well. Thus, by measuring the absorbance of the reduced product, the percentage of viable cells at a given dose of test sample can be obtained. Control wells contained a final concentration of 1% DMSO.

Two samples were first tested in this way. Sample MM1 represents a very coarse isolate of cocoa procyanidins and contains identifiable amounts of caffeine and theobromine. Sample MM2 represents a partially purified cocoa procyanidin isolate using gel permeation chromatography. Caffeine and theobromine were absent in MM 2. Two samples were screened for activity against the following cancer cell lines using the methods described previously:

HCT 116 Colon cancer

ACHN renal adenocarcinoma

SK-5 melanoma

A498 renal adenocarcinoma

MCF-7 Breast cancer

PC-3 prostate cancer

CAPAN-2 pancreatic cancer

In any of the tumor cell lines observed, little or no activity was observed with MM 1. MM2 was found to be active against HCT-116, PC-3 and ACHN tumor cell lines. However, MM1 and MM were found2 interferes with MTT so that it blurs the decrease in absorbance that reflects a decrease in the number of viable cells. This interference is also due to the large number of false obstacles, since the chemical reaction appears to occur more rapidly in the well along the periphery of the plate. A typical example of these effects is shown in figure 5. In the case of high concentrations of test material, it would be desirable to observe a substantial reduction in viable cells, rather than exhibiting high levels of survival. However, microscopic examination revealed that cytotoxic effects were produced despite MTT interference. For example, an IC of 0.5. mu.g/mL of MM2 effect on ACHN cells was obtained in this manner ₅₀The value is obtained.

In the inventors' view, these preliminary results require modification of the analysis method to exclude interference from MTT. This is done as follows. Plates were incubated at 37 ℃ in wet, 5% CO₂After incubation in the atmosphere for 18 hours, the medium was carefully aspirated and replaced with fresh α -MEM medium. On the third day of analysis, the medium was again aspirated from each well and replaced with 100. mu.l of fresh McCoy's medium. Subsequently, 11. mu.l of a 5mg/mL stock of MTT in PBS (phosphate buffered saline) was added to the wells of each plate. At 37 ℃ in moist, 5% CO₂After incubation in the atmosphere for 4 hours, 100 μ l of 0.04N isopropanol hydrochloride solution was added to each well of the plate, followed by thorough mixing to generate formazans from any living cells. In addition, it was decided to fractionate procyanidins to establish the specific components responsible for activity.

The aforementioned fractionation methods were used to prepare samples for further screening. Five fractions were prepared representing the area shown in figure 1 and the distribution of the ingredients shown in figures 4A-4E. Samples were numbered MM2A-MM2E to reflect these analytical characteristics and indicate the absence of caffeine and theobromine.

Each fraction was screened separately for HCT-116, PC-3 and ACHN tumor cell lines. The results show that the activity is not concentrated in any one particular fraction. This result is considered to be common because the components in the "active" natural product isolate may act synergistically. In the case of a cocoa procyanidin isolate (MM2), more than twenty detectable components make up the isolate. It is considered possible that the activity is related to the mixing of the ingredients present in the different fractions, rather than the activity being related to the individual ingredients.

Based on this result, pooled fractions were determined and the analysis against the same tumor cell line was repeated. Some fractions were mixed to produce cytotoxic effects against the PC-3 tumor cell line. Specifically, 40. mu.g/mL ICs were obtained each mixed for MM2A and MM2E₅₀Value, and IC of 20. mu.g/mL for MM2C and MM2E blends₅₀The value is obtained. Activity against HCT-16 and ACHN cell lines was also reported, but as before, interference with MTT indicators precluded clear observations. Duplicate experiments were performed in HCT-116 and ACHN lines to improve data. However, these results are not stable due to bacterial contamination and consumption of the test sample standards. FIGS. 6A-6D show dose-response relationships between mixtures of cocoa extracts and PC-3 tumor cells.

However, it is clear from this data that cocoa extracts, especially cocoa polyphenols or procyanidins, have significant anti-tumor properties; anti-cancer or anti-tumor activity, especially against human PC-3 (prostate), HCT-116 (colon) and ACHN (kidney) tumor cell lines. Furthermore, those results suggest that specific procyanidins may be the main cause of activity against the PC-3 cell line.

Example 7:anticancer, antitumor or antitumor activity of cocoa extract (procyanidins)

To confirm the above findings and to further study the fraction mixtures, additional comprehensive screens were performed.

All prepared materials and methods were identical to those described above, except that 4 replicates of each test dose standard were increased to 8 or 12 replicates of each dose. For this study, each of the five cocoa procyanidin fractions and mixtures thereof was screened for anti-following tumor cell line activity:

PC-3 prostate gland

KB nasopharynx/Hela

HCT-116 Colon

ACHN Kidney

MCF-7 mammary gland

SK-5 melanoma

A-549 Lung

CCRF-CEM T-cell leukemia

Each screen consisted of analyzing fractions A, B, C, D and E (see FIGS. 4A-4E and discussion thereof, supra) at different dose levels (0.01-100. mu.g/mL) for activity against various cell lines. The mixture was screened for resistance by mixing equal dose levels of fractions A + B, A + C, A + D, A + E, B + C, B + D, B + E, C + D, C + E and D + E

Active composition of various cell lines. The results of these analyses are discussed separately, followed by a comprehensive summary.

A. PC-3 prostate cell line

FIGS. 7A-7H show typical dose response relationships between cocoa procyanidin fractions and PC-3 cell lines. FIGS. 7D and 7E demonstrate the IC at 75. mu.g/mL for fractions D and E₅₀The value is active. IC obtained from dose response curves of mixtures of other procyanidin fractions when fractions D or E are present ₅₀Values varied between 60-80. mu.g/mL. Each IC₅₀The values are shown in Table 6.

B. KB nasopharyngeal/Hela cell line

FIGS. 8A-8H show typical dose response relationships between cocoa procyanidin fractions and the KB nasopharyngeal/HeLa cell line. FIGS. 8D and 8E demonstrate that fractions D and E are in IC₅₀The activity was found at a value of 75. mu.g/mL. Representative results obtained from the fraction mixture study are depicted in FIGS. 8F-8H. In this case, the procyanidin fraction mixture A + B had no effect, while the fraction mixtures B + E and D + E were in IC₅₀The activity was found to be 60. mu.g/ml. IC obtained from other dose response curves from other fraction mixtures when fractions D or E are present₅₀A value of 60-80. mu.g/mLAnd (4) change. Each IC₅₀The values are shown in Table 6. These results are essentially the same as those obtained for the PC-3 cell line.

C. HCT-116 Colon cell line

FIGS. 9A-9H show typical dose response relationships between cocoa procyanidin fractions and the HCT-116 colon cell line. FIGS. 9D and 9E demonstrate that fraction E is in IC₅₀A value of about 400. mu.g/mL was active. This value is obtained by extrapolating the existing curve. Note that the slope of the dose response curve for fraction D also indicates activity. However, no IC is present, since the slope of the curve is too low to obtain a reliable value ₅₀Values were obtained from this curve. Representative results obtained from the fraction mixture study are depicted in FIGS. 9F-9H. In this case, the procyanidin fraction mixture B + D showed no significant activity, whereas the fraction mixtures A + E and D + E were in IC respectively₅₀Values of 500. mu.g/mL and 85. mu.g/mL were active. IC obtained from dose response curves of other fraction mixtures when fraction E is present₅₀The values averaged about 250. mu.g/mL. Extrapolation IC₅₀The values are listed in table 6.

D. ACHN renal cell line

FIGS. 10A-10H show typical dose-response relationships between cocoa procyanidin fractions and ACHN kidney cell lines. FIGS. 10A-10E illustrate that the fractions are inactive against this cell line. FIGS. 10F-10H depict representative results obtained from a fraction mixture study. In this case, procyanidin fraction mixture B + C was inactive, while fraction mixture A + E gave an extrapolated IC₅₀The value was about 500. mu.g/mL. Dose response curves similar to the C + D mixtures were considered inactive because their slopes were too low. Extrapolated IC of other fraction mixtures₅₀The values are listed in table 6.

E. A-549 lung cell line

FIGS. 11A-11H show typical dose-response relationships between cocoa procyanidins and the A-549 lung cell line. No activity could be detected from any of the various fractions or mixtures of fractions at the dose used for the assay. However, procyanidins may have utility in this cell line.

F. SK-5 melanoma cell line

FIGS. 12A-12H show typical dose response relationships between cocoa procyanidin fractions and SK-5 melanoma cell lines. At the doses used in this assay, no activity could be detected from any of the various fractions or mixtures of fractions. However, procyanidins may have utility in this cell line.

G. MCF-7 mammary gland cell line

FIGS. 13A-13H show typical dose response relationships between cocoa procyanidin fractions and the MCF-7 mammary cell line. At the doses used in this assay, no activity could be detected from any of the various fractions or mixtures of fractions. However, procyanidins may have utility in this cell line.

H. CCRF-CEM T-cell leukemia cell line

A typical dose response curve for an anti-CCRF-CEM T-cell leukemia cell line was initially obtained. However, microscopic counting of cell number against time at different fraction concentrations indicated that 500 μ g of fractions a, B and D completed a 80% drop in growth over a 4 day period. Representative dose response relationships are shown in figure 14.

I. Summary of the invention

IC obtained from these assays for all cell lines, except CCRF-CEM T-cell leukemia ₅₀The values are collectively listed in table 6. T-cell leukemia data were purposely omitted from the table because of the different analytical methods used. A general summary of these results indicates that the maximum activity is associated with fractions D and E. These fractions were most active on PC-3 (prostate) and KB (nasopharyngeal/Hela) cell lines. Although only at such high doses, these fractions also showed resistance to HCT-116 (colon) and ACHN (kidney)) Activity of the cell line. No activity was detected against MCF-7 (mammary gland), SK-5 (melanoma) and A-549 (lung) cell lines. However, procyanidins may have utility for these cell lines. Also shown are activities against CCRF-CEM (T-cell leukemia) cell lines. It should also be noted that fractions D and E are the most complex mixtures in composition. However, it is clear from these data that cocoa extracts, and in particular cocoa procyanidins, have significant anti-tumor, anti-cancer or anti-tumor activity.

Table 6: IC of cocoa procyanidins on various cell lines₅₀Value of

(IC₅₀Value,. mu.g/mL)

Fraction(s) of	PC-3	KB	HCT-116	ACHN	MCF-7	SK-5	A-549
Fraction(s) of	PC-3	KB	HCT-116	ACHN	MCF-7	SK-5	A-549	A
B								A
B								C
D	90	80						C
D	90	80						E	75	75	400
A+B								E	75	75	400
A+B								A+C	125	100
A+D	75	75						A+C	125	100
A+D	75	75						A+E	80	75	500	500
B+C								A+E	80	75	500	500
B+C								B+D	75	80
B+E	60	65	200					B+D	75	80
B+E	60	65	200					C+D	80	75		1000
C+E	80	70	250					C+D	80	75		1000
C+E	80	70	250					D+E	80	60	85

Extrapolation of values greater than 100. mu.g/mL from dose-response curves

Example 8:anticancer, antitumor or antitumor activity of cocoa extract (procyanidins)

Several additional in vitro assays were used to complement and extend the results shown in tables 6 and 7.

Method A. Crystal Violet staining analysis

All human tumor cell lines were obtained from the American type culture Collection. Cells were grown to monolayer in IMEM containing 10% fetal bovine serum without antibiotics. Cells were stored in humidified, 5% CO at 37 deg.C₂In an atmosphere.

After trypsinization, cells were counted and adjusted to a concentration of 1,000-2,000 cells/100 mL. Cell proliferation was determined by plating cells (1,000-2,000 cells/well) in 96-well microtiter plates. After adding 100. mu.l of cells per well, the cells were allowed to grow adherently for 24 hours. At the end of 24 hours, various cocoa fractions were added at different concentrations to obtain dose response results. Cocoa fractions were dissolved in 2-fold concentration medium and 100. mu.l of each solution was added to three wells. Over the course of several consecutive days, the plates were stained with 50. mu.l of crystal violet (2.5g of crystal violet dissolved in 125mL of methanol, 375mL of water) for 15 minutes. The dye was removed and the plate gently dipped into cold water to remove excess dye. The washing was repeated two or more times and the plates were allowed to dry. The remaining dye was dissolved by adding 100. mu.l of 0.1M sodium citrate/50% ethanol to each well. After lysis, the cell number was quantified on an ELISA plate counter at 540nm (reference filter at 410 nm). Results from the ELISA counter were plotted using absorbance as the Y-axis and days of growth as the X-axis.

Method B.Soft agar cloning assay

Cells were cloned on soft agar according to the method described by Nawata et al (1981). Single cell suspensions were prepared in media containing 0.8% agar and various concentrations of cocoa fractions. The suspension was aliquoted into 35mm dishes covered with medium containing 1.0% agar. After 10 days incubation, the number of colonies with a diameter greater than 60 μm was determined in the Ominicron 3600 Image Analysis System. The number of colonies was plotted as the Y-axis and the concentration of cocoa fractions as the X-axis.

Method C.XTT microculture tetrazolium assay

The XTT assay described by Scudiero et al (1988) was used to screen various cocoa fractions. The XTT assay is essentially the same as described using the MTT method (example 6) except for the following modifications. XTT ((2, 3-bis (2-methoxy-4-nitro-5-sulfonylphenyl) -5- ((phenylamino) carbonyl) -2H-tetrazolium hydroxide) was prepared in 1mg/mL serum-free medium and preincubated to 37 ℃ PMS was prepared in 5mM PBS, XTT and PMS were mixed together, 10. mu.L PMS/mL XTT and 50. mu.L PMS-XTT.37 ℃ were added to each well, after 4 hours of incubation, the plates were mixed for 30 minutes on a mechanical shaker and the absorbance was measured at 450 and 600nm, and the results were plotted using absorbance as the Y-axis and growth days or concentrations as the X-axis.

For methods A and C, the results were also plotted with the percent control as the Y-axis and days of growth or concentration as the X-axis.

A comparison of the XTT and crystal violet assays of MCF-7p168 against breast cancer cell lines was performed with cocoa fractions D and E (example 3B) to determine which assay was the most sensitive. As shown in fig. 15A, both assays showed the same dose-response effect for concentrations > 75 μ g/mL. At concentrations below this value, the crystal violet analysis shows a higher standard deviation than the XTT analysis results. However, since the crystal violet assay is easy to use, all subsequent assays are performed in this manner unless otherwise indicated.

The results of the crystal violet assay (FIGS. 15B-15E) are provided to demonstrate the effect of the crude polyphenol extract (example 2) on the breast cancer cell line MDA MB231, the prostate cancer cell line PC-3, the breast cancer cell line MCF-7p 163 and the neck cancer cell line Hela, respectively. In all cases, a dose of 250 μ g/mL completely inhibited all cancer cell growth over a 5-7 day period. The Hela cell line appeared to be more sensitive to the extract, since the 100. mu.g/mL dose also inhibited growth. The cocoa fraction from example 3B was also analyzed against Hela and other breast cancer cell line SKBR-3. The results (fig. 15F and 15G) show that fractions D and E have the highest activity. As shown in FIGS. 15H and 15I, ICs of about 40. mu.g/mL D and E were obtained from two cancer cell lines ₅₀The value is obtained.

Cocoa fractions D and E were also tested in a soft agar clone assay to determine the ability of the test compound to inhibit anchorage-independent growth. As shown in FIG. 15J, colony formation of Hela cells was completely inhibited at a concentration of 100. mu.g/mL.

Crude polyphenol extracts obtained from generic species of 8 different species of cocoa representing three horticultural species of cocoa were also analyzed against the Hela cell line. As shown in fig. 15K, all cocoa variants showed similar dose-response effects. UIT-1 variant showed the greatest activity against Hela cell line. These results demonstrate that all cocoa generic species possess polyphenol fractions that produce activity against at least one human cancer cell line independent of geographic origin, horticultural cultivars and generic species.

Another series of analyses was performed on a daily basis from a crude polyphenol extract prepared from a traditional 5-day fermentation of brazilian cocoa beans on a one ton scale, followed by a 4-day solar drying phase. The results shown in figure 15L do not show significant effects of these early processing stages, indicating that there is little variation in the composition of polyphenols. However, it is known (Lehrian and Patterson, 1983) that polyphenol oxidase (PPO) will oxidize polyphenols during the fermentation stage. To determine what effect enzymatic oxidation of polyphenols will have on activity, additional experiments were performed. Crude PPO was prepared by extracting finely ground, unfermented, freeze-dried defatted Brazilian cocoa beans with acetone at a ratio of 1mg powder to 10mL acetone. The slurry was centrifuged at 3,000rpm for 15 minutes. This was repeated three times, the supernatant discarded each time, and the fourth extract was poured into a Buchner filter funnel. The acetone powder was allowed to air dry and then analyzed according to the method described by McLord and Kilara (1983). To the crude polyphenol solution (100mg/10mL citrate-phosphate buffer, 0.02M, ph5.5) was added 100mg of acetone powder (4,000 units activity/mg protein) and stirred for 30 minutes by passing a stream of bubbles through the slurry. The sample was centrifuged at 5,000 Xg for 15 minutes, and the supernatant was extracted 3 times with 20mL of ethyl acetate. The ethyl acetate extracts were combined, dried under partial vacuum by distillation and 5mL of water were added, followed by freeze drying. The material was then analyzed against Hela cells and the dose response was compared to crude polyphenol extract without enzymatic treatment. The results (fig. 15M) show a clear deviation in the dose-response curve of the enzymatically oxidized extract, indicating that the oxidation products are more inhibitory than their native form.

Example 9:antioxidant activity of cocoa extract containing procyanidins

Evidence in the literature suggests a relationship between the consumption of naturally occurring antioxidants (vitamins C, E and β -carotene) and the low incidence of disease including cancer (design foods, 1993; Caragay, 1992). These antioxidants are generally thought to affect certain oxidative and free radical processes involved in the initiation of some types of tumors. In addition, some plant polyphenol compounds that have been shown to be anticancer agents also have antioxidant activity per se (Ho et al, 1992; Huang et al, 1992).

To determine whether a procyanidin-containing cocoa extract has antioxidant properties, the standard Rancimat method was used. The methods described in examples 1, 2 and 3 were used to prepare cocoa extracts which were further processed to yield two fractions from gel permeation chromatography. These two fractions are in fact mixed fractions a-C, and D and E (see fig. 1), where their antioxidant properties are compared to the synthetic antioxidants BHA and BHT.

The peanut oil is extruded from the unbaked peanuts after peeling. Each test compound was blended into the oil at two levels, 100ppm and 20ppm, with the actual levels shown in Table 7. To each sample, 50 μ l of methanol-dissolved antioxidant was added to aid in the dispersion of the antioxidant. Control samples were prepared with 50 μ l of methanol without antioxidant.

The oxidative stability of the samples was evaluated twice using the Rancimat stability test at 100 ℃ and 20 cc/min air. The experimental parameters were chosen to match those using either the Active Oxygen Method (AOM) or the Swift stability test (Van Oosten et al, 1981). A typical Rancimat is shown in fig. 16. The results are reported in table 8 in terms of the number of hours required to reach a 100meq peroxide level.

Table 7: concentration of antioxidant

Sample level 1 level 2ppm
Sample level 1 level 2ppm			Butylated Hydroxytoluene (BHT)	24	120
Butylated Hydroxyanisole (BHA)	24	120	Butylated Hydroxytoluene (BHT)	24	120
Butylated Hydroxyanisole (BHA)	24	120	Crude ethyl acetate fraction of cocoa	22	110
Fractions A to C	20	100	Crude ethyl acetate fraction of cocoa	22	110
Fractions A to C	20	100	Fractions D to E	20	100

Table 8: oxidation stability of peanut oil and various antioxidants

Samples 20ppm 100ppm average
Samples 20ppm 100ppm average			Control	10.5±0.7
BHT	16.5±2.1	12.5±2.1	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	BHA	13.5±2.1	14.0±1.4
Crude cocoa fraction	18.0±0.0	19.0±1.4	Fractions A to C	16.0±6.4	17.5±0.0
Fractions D to E	14.0±1.4	12.5±0.7	Fractions A to C	16.0±6.4	17.5±0.0

These results demonstrate the increased oxidative stability of peanut oil containing all the tested additives. The greatest increase in oxidative stability was achieved by incorporating the crude ethyl acetate extract of cocoa into the sample. These results demonstrate that the procyanidin-containing cocoa extract has antioxidant capacity equal to or greater than the equivalent of synthetic BHA and BHT. Thus, the present invention may be used to replace BHT or BHA in known uses of BHA or BHT, for example as an antioxidant and/or food additive. And in this respect, it should also be noted that the present invention is derived from edible sources. Given these results, one skilled in the art can readily determine without undue experimentation the appropriate amount of the present invention, such as the amount to be added to the food, for use in such "BHA or BHT" applications.

Example 10:topoisomerase II inhibition study

DNA topoisomerases I and II are enzymes that catalyze DNA strand breaks and rejoins, thereby controlling the topological state of DNA (Wang, 1985). In addition to studying the intracellular function of topoisomerase, one of the most compelling findings was the identification of topoisomerase II as a primary cellular target for a number of clinically important anti-tumor compounds (Yamashita et al, 1990) including intercalators (m-AMSA, Adriamycin)^_And ellipticine) and non-intercalated epipodophyllotoxins. Some lines of evidence suggest that some antineoplastic drugs share a common property of stabilizing the DNA-topoisomerase II complex ("cleavable complex"), which is exposed to denaturing reagentsThe assay results in induction of DNA cleavage (Muller et al, 1989). It has been proposed that the formation of cleavable complexes by antineoplastic drugs results in the production of a number of DNA adducts that can lead to cell death.

According to this attractive model, specific novel inducers of DNA topoisomerase II cleavable complexes are useful as anti-cancer, anti-tumor or anti-neoplastic agents. In an attempt to identify cytotoxic compounds with targeted DNA activity, cocoa procyanidins were screened for increased cytotoxic activity against some DNA-damage sensitive cell lines and subjected to enzymatic analysis using human topoisomerase II obtained from lymphoma.

A. De-linkage of kinetoclast DNA of topoisomerase II

In vitro inhibition of topoisomerase II unlinked of kinetoplast DNA was performed as described by Muller et al (1989) as follows. Nuclear extracts containing topoisomerase II activity were prepared from human lymphoma carcinomas using modifications of the methods of Miller et al (1981) and Danks et al (1988). At 34 ℃ 1 unit of purified enzyme is sufficient to unlink 0.25. mu.g of kinetoplast DNA within 30 minutes. Kinetoplast was obtained from Trypanosoma brachypomum (Crithia fascicularia). In a solution containing 19.5. mu.l H₂O, 2.5. mu.l 10 Xbuffer (1 Xbuffer containing 50mM tris-HCl, pH8.0, 120mM KCl, 10mM MgCl)₂Each reaction was carried out in 0.5mL microcentrifuge tubes of 0.5mM ATP, 0.5mM dithiothreitol, and 30. mu.g BSA/mL), 1. mu.l kinetoclast DNA (0.2. mu.l), and 1. mu.l of each concentration of DMSO-containing cocoa procyanidin assay fraction. The mixture was mixed well and placed on ice. Immediately prior to incubation in the water bath for 30 minutes at 34 ℃ 1 unit of topoisomerase was added.

After incubation, the unlinked assay was terminated by adding 5 μ l of stop buffer (5% Sarkosyl, 0.0025% bromophenol blue, 25% glycerol) and placing on ice. The DNA was electrophoresed on a 1% agarose gel in TAE buffer containing ethidium bromide (0.5. mu.g/ml). The DNA was developed by ultraviolet irradiation at a wavelength of 310 nm. The gel was photographed with a Polaroid Land/camera.

Fig. 17 shows the results of these experiments. The fully linked kinetoplast DNA did not migrate into a 1% agarose gel. The unlinked kinetoclast DNA by topoisomerase II produces monomeric DNA bands (single loop, type I and type II) that do not migrate into the gel. The gradual disappearance of the monomer band as a function of increasing concentration made apparent the inhibition of the enzyme by the addition of cocoa procyanidins. Based on these results, cocoa procyanidin fractions A, B, D and E showed inhibition of topoisomerase II at varying concentrations from 0.5 to 5.0 μ g/mL. These inhibitor concentrations are very similar to those obtained for anthracyclines and m-AMSA (4' - (9-acridinylamino) methanesulfonyl-m-anisamide).

B. Drug sensitive cell lines

Cocoa procyanidins were screened for cytotoxicity against several DNA damage sensitive cell lines. One cell line is the xrs-6 DNA double strand break repair mutant developed by P.Jeggo (Kemp et al, 1984). The deficiency in DNA repair of the xrs-6 cell lines makes them particularly sensitive to X-rays, compounds that cause direct breaks in the DNA double strand, such as bleomycin, and to compounds that inhibit topoisomerase II, and therefore may indirectly induce double strand breaks as suggested by Warter et al (1991). Cytotoxicity against the repair deficient cell line was compared with that against the CHO cell line BR1 with good DNA repair. Enhanced cytotoxicity against repair-deficient (xrs-6) cell lines was explained as evidence of DNA-cleavable double strand break formation.

A CHO cell line with good DNA repair BR1 was developed by Barrows et al (1987) and expresses 0 in addition to the normal CHO DNA repair enzyme⁶-alkylguanine-DNA-alkyltransferase. The CHO double-strand break repair deficient cell line (xrs-6) is a gift from Dr.P.Jeggo and colleagues (Jeggo et al, 1989). Both cells were grown as monolayers in α -MEM containing serum and antibiotics, as described in example 6. Cells were stored in humidified, 5% CO at 37 deg.C₂In an atmosphere. Cells grown as monolayers were detached by treatment with trypsin prior to treatment with cocoa procyanidins. The analysis was performed using the MTT assay described in example 6.

The results (FIG. 18) indicate no increased cytotoxicity against the xrs-6 cell line, suggesting that cocoa procyanidins inhibit topoisomerase II in a manner different from cleavable double strand break formation. I.e., interacting with topoisomerase II before it interacts with DNA to form a non-cleavable complex.

Non-cleavable complex-forming compounds are a relatively new discovery. Members of the anthracyclines, podophyllin alkaloids, anthracenediones, acridines, and ellipticine are useful for clinical anticancer, antitumor or antitumor use, and they produce cleavable complexes (Liu, 1989). Some new classes of topoisomerase II inhibitors have recently been identified that appear not to produce cleavable complexes. This includes aminonapellin (Hsiang et al, 1989), selectamycin (Fesen et al, 1989), flavonoids (flavanoids) (Yamashita et al, 1990), saintopin (Yamashita et al, 1991), membrane ketones (Drake et al, 1989), terpenes (Kawada et al, 1991), anthrapyrazoles (Fry et al, 1985), dioxopiperazines (Tanabe et al, 1991), and marine acridine-dericitins (Burres et al, 1989).

Because cocoa procyanidins inactivate topoisomerase II prior to formation of the cleavable complex, they are of chemotherapeutic value alone or in admixture with other known and mechanistically defined topoisomerase II inhibitors. In addition, cocoa procyanidins appear to be a new class of topoisomerase II inhibitors (Kashiwada et al, 1993) and thus may be less cytotoxic than other known inhibitors, thereby increasing their utility in chemotherapy.

Cocoa fractions D and E were analyzed for their effects using the human breast cancer cell line MCF-7(ADR) (Leonesa et al, 1994) expressing membrane-bound glycoprotein (gp 170) to deliver multi-drug resistance and its parental cell line MCF-7. As shown in figure 19, the parental cell line was inhibited at increasing dose levels of fractions D and E, while the doxorubicin (ADR) -resistant cell line was less affected at higher doses. These results indicate that cocoa fractions D and E have an effect on multi-drug resistant cell lines.

Example 11:synthesis of procyanidins

The synthesis of procyanidins was carried out according to a modification of the method developed by Delcour et al (1983). In addition to the condensation of (+) -catechin with dihydroquercetin under reducing conditions, a high concentration of (-) -epicatechin, reflecting that naturally produced in unfermented cocoa beans, was employed. The synthesis products were isolated, purified, analyzed and characterized as described in examples 3, 4 and 5. In this method, the di-, tri-and tetra-flavonoids were prepared and used as analytical standards using the method described above for cocoa extracts.

Example 12:analysis of the normal phase semi-preparative fraction

Since polyphenol extracts are complex compositions, it is necessary to determine which components have anti-cancer cell line activity for further purification, dose response analysis and comprehensive structural identification. A normal phase semi-preparative HPLC separation (example 3B) was used to isolate cocoa procyanidins based on oligomer size. Twelve fractions (FIGS. 2B and 15O) were prepared except for the initial extract and analyzed for anti-Hela and SKBR-3 cancer cell line activity at doses of 100. mu.g/mL and 25. mu.g/mL to determine which oligomers possessed the greatest activity. As shown in FIGS. 20A and B, fractions 4-11 (pentamer-dodecamer) significantly inhibited Hela and SKBr-3 cancer cell line at a level of 100. mu.g/mL. These results indicate that these particular oligomers have the greatest activity against Hela and SKBR-3 cells, and in addition, normal phase HPLC analysis of cocoa fractions D and E indicated that this fraction, as employed in example 7, was enriched in these oligomers in previous studies.

Example 13:HPLC purification method

Method A.GPC purification

The procyanidins obtained as in example 2 were partially purified by liquid chromatography on Sephadex LH 20 (72.5X 2.5cm) using 100% methanol as eluting solvent at a flow rate of 3.5 mL/min. After the first 1.5 hours, the eluted fractions were collected and the fractions were concentrated on a rotary evaporator, redissolved in water and freeze dried. These fractions are referred to as pentamer enriched fractions. About 2.00g of the extract obtained from example 2 was fractionally separated in this way. The results are shown in Table 9.

Table 9: composition of the fractions obtained

Fraction (time)	Monomer (% area)	Dimer (% area)	Trimer (% area)	Tetramer (% area)	Pentamer (% area)	Hexamer (% area)	Heptamer (% area)	Octamer (% area)	Nonamer (% area)	Decamer (% area)	Undecamer (% area)	Others (% area)
Fraction (time)	Monomer (% area)	Dimer (% area)	Trimer (% area)	Tetramer (% area)	Pentamer (% area)	Hexamer (% area)	Heptamer (% area)	Octamer (% area)	Nonamer (% area)	Decamer (% area)	Undecamer (% area)	Others (% 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	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	16	31	28	15	6	2	tr	tr	tr	tr	tr	2:13	30	29	24	11	4	1	tr	tr	tr	tr	tr	tr
2:42	2	16	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	18	13	18	20	15	10	5	2	tr	tr	tr	3:11	1	12	17	25	22	13	7	2	1	tr	tr	tr
3:40	tr	18	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 is not detected

tr is a trace amount

Method B. normal phase separation

By normal phase chromatography on Supelcosil LC-Si, 100. mu.m, 5 μm (250X 4.6mm), at a flow rate of 1.0 mL/min or else on Lichrosphere^_The procyanidin obtained as in example 2 was isolated and purified using Silica 100, 100. mu.m, 5 μm (235X 3.2mm) at a flow rate of 0.5 mL/min. A fractionation gradient was used to aid separation under the following conditions: (time,% A,% B); (0, 82, 14),(30, 67.6, 28.4),(60, 46, 50),(65, 10, 86),(70, 10, 86). The mobile phase composition is A ═ dichloromethane; b ═ methanol; and C ═ acetic acid: water (1: 1). With λ therein _ex276nm and λ_emFluorescence at 316nm, and detection of each component by ultraviolet irradiation at 280 nm. The injection volume was 0.5. mu.l (20mg/mL) of the procyanidin obtained from example 2. These results are shown in fig. 40A and 40B.

Another method, a fractionation gradient under the following conditions was used to assist the separation: (time,% A,% B); (0, 76, 20); (25, 46, 50); (30, 10, 86). The mobile phase composition is A ═ dichloromethane; b ═ methanol; and C ═ acetic acid: water (1: 1). The results are shown in FIGS. 41A and 41B.

Method C. reverse phase separation

The procyanidins obtained as in example 2 were isolated and purified by reverse phase chromatography on a Hewlett Packard Hypersil ODS 5 μm, (200X 2.2mm), and a Hewlett Packard Hypersil ODS 5 μm guard column (20X 2.1 mm). The procyanidins were eluted over 20 min with a linear gradient from 20% B to A, followed by washing of the column with 100% B at a flow rate of 0.3 mL/min. The mobile phase composition was a degassed mixture of a solution of acetic acid, B ═ 1.0% in methanol, and a solution of acetic acid, a ═ 2.0% in nano-purified water. Ultraviolet light at 280nm, and lambda_ex276nm and λ_emFluorescence at 316nm to detect each component; the injection volume was 2.0. mu.l (20 mg/mL).

Example 14:HPLC separation of pentamer enriched fractions

Method A. semi-preparative normal phase HPLC

The pentamer enriched fraction was further purified by semi-preparative normal phase HPLC using a Hewlett Packard 1050 HPLC system equipped with a Millipore-water type 480LC detector set at 254nm, equipped with a Pharmacia Frac-100 fraction collector set at the peak. The separation was performed on a Supelco 5 μm Supelcousel LC-Si, 100-column (250X 10mm) connected to a Supelco 5 μ Supelguard LC-Si guard column (20X 4.6 mm). Procyanidins were eluted with a linear gradient under the following conditions: (time,% A,% B); (0, 82, 14), (30, 67.6, 28.4), (60, 46, 50), (65, 10, 86), (70, 10, 86) followed by 10 minutes of rebalancing, the mobile phase composition being a ═ dichloromethane; b ═ methanol; and C ═ acetic acid: water (1: 1). A flow rate of 3 mL/min was used. Detecting each component with ultraviolet ray at 254 nm; and recorded on a Kipp & Zonan BD41 recorder. Injection volume ranged from 100-. The peaks or selected chromatographic regions are collected at specified time intervals or by manual fraction collection for further purification and later evaluation.

HPLC conditions: 250X 100mm Supelco Supelcosil LC-Si (5 μm)

Semi-fabricated column

20×4.6mm Supelco Supelcosil LC-Si(5μm)

Protective column

A detector: waters LC

Spectrophotometer type 480@254nm

Flow rate: 3 mL/min

Column temperature: at room temperature

And (3) injection: 250 μ l pentamer enriched fraction

Gradient: CH (CH)₂Cl₂Methanol acetic acid water (1: 1)

0 82 14 4

30 67.6 28.4 4

60 46 50 4

65 10 86 4

70 10 86 4

Method B. reverse phase separation

The procyanidin extract obtained in example 13 was filtered through a 0.45 μ nylon membrane and analyzed by a Hewlett Packard1090 three-phase HPLC system with a diode array detector and a HP model 1046A degree fluorescence detector. The separation was carried out on a Hewlett Packard 5. mu. Hypersil ODS column (200X 2.1mm) at 45 ℃. Procyanidins were eluted with a 60% B-to-A linear gradient followed by column washing with B at a flow rate of 0.3 ml/min. The composition of the mobile phase is a degassed mixture: 0.5% acetic acid in methanol and 0.5% acetic acid in nano-purified water. The concentration of acetic acid in the mobile phases a and B can be increased to 2%. Detection of fluorescence of the fractions at UV 280m, where lambda_ex＝276nm，λ_em316 nm. The concentrations of (+) -catechin and (-) -epicatechin were determined with reference to standard solutions. The levels of procyanidins were assessed by the coefficient of response to (-) -epicatechin.

Method C. normal phase separation

The procyanidin extract obtained as in example 13 was filtered through a 0.45 μ nylon filter and analyzed using a Hewlett Packard 1090 series II HPLC system equipped with a diode system detector and a HP model 1046A programmable fluorescence detector. At 37 ℃ in a 5. mu. Phenomenex Lichrosphere attached to a Supelco Supelguard LC-Si 5. mu. guard column (20X 4.6mm)^_The separation was carried out on a Silica 100 column (250X 3.2 mm). Procyanidins were eluted because of a linear gradient of the following conditions: (time,% A,% B); (0, 82, 14), (30, 67.6, 28.4), (60, 46, 50), (65, 10, 86), (70, 10, 86), followed by 8 minutes of rebalancing, the mobile phase composition being a ═ dichloromethane; b ═ methanol; and C ═ acetic acid: water in a volume ratio of 1: 1. A flow rate of 0.5 mL/min was used. With λ therein_ex＝276nm，λ_emFluorescence at 316nm, or detection of each component by ultraviolet light at 280 nm. Representative HPLC chromatograms showing the separation of various procyanidins for an atypical species are shown in FIG. 2. From other genus Theobroma (Theobroma), Theobroma (Herrania) and/or their derivativesSimilar HPLC profiles were obtained for specific hybrids within interspecies.

HPLC conditions: 250X 3.2mm Phenomenex Lichrosphere^_Silica 100

Column (5. mu.) 20X 4.6mm Supelco Supelguard LC-Si

(5 mu) protective column

A detector: light diode system @280nm

Fluorescence lambda_ex＝276nm；λ_em＝316nm

Flow rate: 0.5 mL/min

Column temperature: 37 deg.C

Acetic acid:

gradient: CH (CH)₂Cl₂Methanol water (1: 1)

0 82 14 4

30 67.6 28.4 4

60 46 50 4

65 10 86 4

70 10 86 4

Method D. preparation of Normal phase separation

The pentamer enriched fraction obtained as in example 13 was further purified by preparative normal phase chromatography by modification of the method of Rigaud et al (1993) J.chromatographies 654, 255-260.

The separation was carried out on a 5. mu. Supelcosil LC-Si 100-column (50X 2cm) at room temperature using a suitable guard column. Cornflower ligands were eluted with a linear gradient under the following conditions: (time,% A,% B, flow rate); (0, 92.5, 7.5, 10), (10, 92.5, 7.5, 40); (30, 91.5, 18.5, 40); (145, 88, 22, 40); (150, 24, 86, 40); (155, 24, 86, 50); (180,0, 100, 50). Before use, the components of the mobile phase are mixed according to the following scheme:

preparation of solvent A (82% CH)₂Cl₂14% methanol, 2% acetic acid, 2% water):

1. 80mL of water was measured and poured into a 4L bottle.

2. 80mL of acetic acid was measured and poured into the same 4L bottle.

3. 560mL of methanol was measured and poured into the same 4L bottle.

4. 3280mL of methylene chloride was measured and poured into the 4L bottle.

5. The bottle was capped and mixed well.

6. The mixture was purged with high purity helium for 5-10 minutes to degas.

Steps 1-6 were repeated twice to yield 8 volumes of solvent a.

Preparation of solvent B (96% methanol, 2% acetic acid, 2% water).

1. 80mL of water was measured and poured into a 4L bottle.

2. 80mL of acetic acid was measured and poured into the same 4L bottle.

3. 3840mL of methanol was measured and 3840mL of methanol was poured into the same 4L bottle.

4. The bottle was capped and mixed well.

5. The mixture was purged with high purity helium for 5-10 minutes to degas.

Steps 1-5 were repeated to yield 4 volumes of solvent B. The mobile phase composition was a ═ dichloromethane containing 2% acetic acid and 2% water; b ═ methanol, 2% acetic acid and 2% water. The column load was 0.7g in 7 mL. Each component was detected by ultraviolet ray at 254 nm. A typical preparative normal phase HPLC separation of cocoa procyanidins is shown in FIG. 42.

HPLC conditions:

column: 50X 2cm 5. mu. Supelcosil LC-Si run at room temperature

Mobile phase: a-dichloromethane containing 2% acetic acid and 2% water

Methanol containing 2% acetic acid and 2% water

Gradient/flow velocity profile:

time (minutes)	％A	％B	Flow rate (mL/min)
time (minutes)	％A	％B	Flow rate (mL/min)	0	92.5	7.5	10
10	92.5	7.5	40	0	92.5	7.5	10
10	92.5	7.5	40	30	91.5	8.5	40
145	88.0	22.0	40	30	91.5	8.5	40
145	88.0	22.0	40	150	24.0	86.0	40
155	24.0	86.0	50	150	24.0	86.0	40
155	24.0	86.0	50	180	0.0	100.0	50

Example 15:identification of procyanidins

The procyanidins obtained as in example 14, method D were analysed using an HP G2025A MALDI-TOF/MS system equipped with a Lecroy 9350500 MHz oscilloscope using the ionization time of matrix assisted laser desorption (MALDI-TOF/MS) of a plate/mass spectrometer. The instrument was calibrated using either a low molecular weight peptide standard (HP part No. G2051A) or a peptide standard (HP part No. G2052A) with 2, 5-dihydroxybenzoic acid (DHB) (HP part No. G2056A) as the same matrix according to the manufacturer's instructions. 1.0mg of the sample was dissolved in 500. mu.l of 70/30 methanol/water and the sample was mixed with DHB matrix in a ratio of 1: 1, 1: 10 or 1: 50 (sample: matrix) and dried in a bench dryer under vacuum. The sample was analyzed cationically with the detector voltage set at 4.75KV and the laser energy set between 1.5 and 8 muj. Data were collected as the sum of multiple single trials and expressed in molecular weight units and time to flight. A representative MALDI-TOF/MS is shown in FIG. 22A.

FIGS. 22 and C show MALDI-TOF/MS spectra obtained from partially purified procyanidins prepared as described in example 3 and used for in vitro evaluation as described in examples 6 and 7, the results of which are summarized in Table 6. This data demonstrates that the compounds of the invention described herein are found predominantly in fractions D-E, but not A-C.

The spectra were obtained as follows:

the purified D-E fraction was subjected to MALDI-TOF/MS as described above, except that SEP-PACK was used initially^_C-18 column purification fractions. A solution of 5mg fraction D-E in 1mL of nano-purified water was loaded onto the pre-equilibrated SEP-PACK^_In the column. The column was washed with 5mL of milli-purified water to eliminate contaminants, and procyanidins were eluted with 1mL of 20% methanol. Since they have been purified in example 3 using method 3, fractions A-C were used without further purification.

These results confirm and extend the earlier results (see example 5, table 3, fig. 20A and B) and demonstrate that the compounds of the present invention have utility as cationic polyvalent chelates. In particular, the MALDI-TOF/MS results collectively indicate that n ═ 5 and higher procyanidin oligomers (see FIGS. 20A and B; and formulas in the objects and summary of the invention) are strongly associated with antitumor activity against Hela and SKBR-3 cancer cell line types. Oligomers with n-4 or less are not effective for these models. The pentamer structure apparently has structural units present therein and in higher oligomers that provide this activity. In addition, it was observed that MALDI-TOF/MS data showed strong Na⁺，2Na⁺，K⁺，2K⁺，Ca⁺⁺M of (A)⁺Ions, this proves Their use as cationic polyvalent chelates.

Example 16:purification of oligomer fractions

Method A. purification by semi-preparative reverse phase HPLC

Procyanidins obtained from example 14, methods a and B and D were further isolated to obtain similar oligomers in experimental amounts for further structural identification and elucidation (as in examples 15, 18, 19 and 20). A Hewlett Packard 1050 HPLC system equipped with a variable wavelength detector, a Rheodyne7010 injection valve with 1mL injection orifice was equipped with a pharmaciFRAC-100 fraction collector. In the field of Phenomenex 10 mu ODS Ultracarb^_Phenomenex Ultracarb connected to a (60X 10mm) protective column^_The separation was carried out on a 10. mu. ODS column (250X 22.5 mm). The mobile phase composition is A ═ water; b ═ methanol, used under the following linear gradient conditions: (time,% A); (0, 85), (60, 50), (90, 0) and (110, 0) at a flow rate of 5 mL/min. The individual peaks or selected chromatographic regions were collected at specified time intervals or by manual fraction collection for further evaluation by MALDI-TOF/MS and NMR. The injection load varied from 25 to 100mg of substance. A representative elution profile is shown in figure 23 b.

Method B improved semi-preparative HPLC

Procyanidins obtained from example 14, methods a and B and D were further isolated to obtain similar oligomers in experimental amounts for further structural identification and elucidation (as in examples 15, 18, 19 and 20). Supelcosil LC-Si 5. mu. column (250X 10mm) with Supelcosil LC-Si 5. mu. protective column (20X 2 mm). The separation was carried out at room temperature at a flow rate of 3.0 mL/min. The mobile phase composition is A ═ dichloromethane; b ═ methanol; and C ═ acetic acid: water (1: 1); used under the following linear gradient conditions: (time,% A,% B); (0, 82, 14); (22, 74, 21); (32, 74, 21); (60, 74, 50, 4); (61, 82, 14), and then the column was re-equilibrated for 7 minutes. The injection volume was 60. mu.l containing 12mg of the pentamer-rich fraction. The components were detected by ultraviolet light at 280 nm. A representative elution profile is shown in fig. 23A.

Example 17:building of pentamer molecular model

The energy-minimal structure was determined by the establishment of a Molecular model using a Desktop Molecular model, version 3.0, Oxford university press, 1994. Based on the structure of epicatechin [ EC (4 → 8)]₄Four representative plots of-EC (EC ═ epicatechin) pentamers are shown in fig. 24A-D. Presumably a helical structure. Typically, when epicatechin is the first monomer, the binding is a 4 → 8, β configuration result, when the first monomer is catechin, the binding is a 4 → 8, α configuration result, and these results are obtained regardless of whether the second monomer is epicatechin or catechin (one exception is ent-E (4 → 8) ent-EC). FIGS. 38A-38P show preferred pentamers, and FIGS. 39A-39P show a library of stereoisomers up to and including the pentamer, from which other compounds falling within the scope of the invention can be prepared without undue experimentation.

Example 18:NMR identification of procyanidins

¹³C NMR spectroscopy is considered a method commonly used for the study of procyanidins, especially since acids generally provide a well-defined mass spectrum, whereas proton NMR spectroscopy is quite broad. Of oligomers¹³C NMR spectroscopy yields information about the substitution pattern of the a or B ring, the relevant stereochemistry of the C ring and in some cases the position of the flavonoid internal linkage. In spite of this, it is possible to provide,¹h NMR spectroscopy still yields useful information.

Furthermore, HOHAHA transfers the magnetization of the first hydrogen sequentially to the second using a pulsed method to obtain cross peaks corresponding to alpha, beta, gamma or delta protons. COSY is a 2D-Fourier transfer NMR method in which the vertical and horizontal axes are provided¹H chemical shift and 1D spectroscopy; the transverse position provides the correlation between protons from which the spin-spin coupling can be determined. HMQC spectroscopy increases the sensitivity of nuclear NMR spectroscopy, but not protons and can reveal differences from the second and fourthCross peaks of carbon to corresponding protons. APT for determining the number of hydrogens in a carbon position¹³And (C) a method. An even number of protons located at a carbon site will produce a positive signal, while an odd number of protons located at a carbon site will produce a negative signal.

Thus, it is possible to provide¹³C NMR，¹H NMR, HOHAHA (homonuclear Hartmann-Hahn) HMQC (heteronuclear multiple quantum coherence), COSY (homonuclear correlation spectroscopy), APT (attached proton test) and XHCORR (modified HMQC) spectroscopy were used to illustrate the structures of the present invention.

Method A. monomer

All spectra were obtained in deuterated methanol at room temperature with a sample concentration of about 10 mg/mL. Spectra were obtained on Bruker 500MHZ NMR using methanol as internal standard.

FIGS. 44A-E represent NMR spectra used to characterize the structure of epicatechin monomers. FIG. 44A shows a flat sheet form¹H and¹³chemical change of C. FIGS. 44B-E show the preparation of epicatechin¹H, APT, XHCORR and COSY spectra.

Similarly, FIGS. 45A-F represent NMR spectra used to characterize the structure of catechin monomers. FIG. 45A shows a flat sheet form¹H and¹³chemical transformation of C. FIGS. 44B-F show the production of catechin¹H，¹³C, APT, XHCORR and COSY spectra.

Method B. dimerization

D in 75% deuterated methanol using acetone as an internal standard, samples at a concentration of about 10mg/mL₂All spectra were obtained in O solution.

FIGS. 46A-G represent spectra used to characterize the structure of B2 dimer. FIG. 46A shows a flat sheet form¹H and¹³chemical transformation of C. The terms T and B represent the upper half of the dimer and the lower half of the dimer.

FIGS. 46B and C show results obtained on Bruker 500MHZ NMR at room temperature¹³C and APT spectra.

FIGS. 46D-G show results obtained on AMZ-360MHZ NMR at-7 deg.C¹H, HMQC, COSY, and HOHAHA. COSY spectra were obtained using gradient pulses.

FIGS. 47A-G represent spectra used to characterize the structure of B5 dimer. FIG. 47A shows a flat sheet form¹³C and¹chemical transformation of H.

FIGS. 47B-D show results obtained on Bruker 500MHZ NMR at room temperature¹H，¹³C and APT.

FIG. 47E shows a COSY spectrum obtained on AMX-360 using gradient pulses at room temperature.

FIGS. 47F and G show HMQC and HOHAHA, respectively, obtained on AMX-360MHZ NMR at room temperature.

Method C. trimer-epicatechin/catechin

D at-3 ℃ on AMX-360MHZ NMR using acetone as an internal standard, and a suitable sample concentration of 10mg/mL in 75% deuterated acetone₂All spectra were obtained in O solution.

FIGS. 48A-D represent spectra used to characterize the structure of epicatechin/catechin trimers. These figures show respectively¹H, COSY, HMQC and HOHAHA. COSY spectra were obtained using gradient pulses.

Method D. trimer-all is epicatechin

D on AMX-360MHZ NMR using acetone as an internal standard and a suitable sample concentration of 10mg/mL in 70% deuterated acetone ₂All spectra were obtained in O solution.

FIGS. 49A-D represent spectra used to characterize the structure of all epicatechin trimers. These figures show respectively¹H, COSY, HMQC and HOHAHA. COSY spectra were obtained using gradient pulses.

Example 19: thiolysis of procyanidins

In an effort to characterize procyanidins, Benzylthiol (BM) was reacted with catechin, epicatechin, or dimers B2 and B5. Benzyl mercaptan and resorcinol and thiophenol are useful for the hydrolysis (thiolation) of procyanidins in an alcohol/acetic acid environment. The catechin, epicatechin, or dimer (1: 1 mixture of B2 and B5 dimer) (2.5mg) was dissolved in 1.5mL ethanol, 100 μ l BM, and 50 μ l acetic acid, and the vessel (Beckman amino acid analysis vessel) was evacuated and repeatedly purged with nitrogen until after the final purge with nitrogen was followed by a sealed reaction vessel. The reaction vessel was placed in a hot set at 95 ℃ and aliquots of the reaction were obtained at 30, 60, 120 and 240 minutes. The relative fluorescence of each aliquot, representing epicatechin, catechin, and dimer, respectively, is shown in FIGS. 25A-C. Higher oligomers are similarly thiolated.

Example 20:thiolysis and desulfurization of dimers

Dimers B2 and B5 were hydrolyzed with thiol by dissolving the dimer (B2 or B5; 1.0mg) in 600. mu.l ethanol, 40. mu.l BM and 20. mu.l acetic acid. The mixture was heated at 95 ℃ for 4 hours in a Beckman amino acid analysis vessel under nitrogen, aliquots were withdrawn for reverse phase HPLC analysis, and 75. mu.l of each ethanolic Raney (Raney) nickel and gallic acid (10mg/mL) were added to the remaining reaction in a 2mL vial. The vessel was purged under hydrogen atmosphere, occasionally shaking for 1 hour. The product was filtered through a 0.45 μ filter and analyzed by reverse phase HPLC. Representative elution profiles are shown in fig. 26A and B. Higher oligomers are similarly devulcanized. This data indicates the polymerization of epicatechin or catechin and thus represents a synthetic route to the compounds of the present invention.

Example 21:in vivo Activity of the pentamer in the MDA MB231 nude mouse model

MDA-MB-231/LCC6 cell line. The cell lines were grown on modified minimal essential Medium (IMEM) containing 10% fetal bovine serum and maintained at 37 ℃ in humidified, 5% CO₂In an atmosphere.

A mouse. Female six to eight week old NCr nu/nu (nude mice) mice were purchased from NCI and housed in animal laboratories and housed according to the regulations set forth by the american ministry of agriculture and the american society for laboratory animal husbandry and identification. Tumor bearing mice were weighed every two days and weekly to determine the appropriate drug dose.

And (5) tumor transplantation. Tissue cultured MDA-MD-231 was diluted to 3.3X 10 with IMEM⁶cells/mL, 0.15mL (i.e., 0.5X 10) were injected subcutaneously between papillae 2 and 3 on each side of the mouse⁶A cell). Tumor volume was calculated by multiplication: length x width x height x 0.5. Mean values of tumor volumes for treatment groups were calculated and student experiments were used to calculate p-values.

And (4) preparing a sample. Plasma samples were obtained by cardiac puncture and stored at-70 ℃ with 15-20mM EDTA for the purpose of blood chemistry assays. No difference was noted between the control group and the experimental group.

15 nude mice previously infected with 500,000 cells by subcutaneous injection of the tumor cell line MDA-MB-231 were randomly divided into three groups of 5 animals each and treated with peritoneal injections of one of the following: (i) placebo with vehicle (DMSO) only; (ii) vehicle (DMSO) contained 2mg of purified pentameric procyanidin extract isolated as in method D of example 14/mouse; and (iii) 10mg of purified pentameric procyanidin extract isolated as in example 14, method D/mouse in vehicle (DMSO).

After 10mg administration, the group (iii) mice died within about 48-72 hours, while the group (ii) mice showed normal. Group (iii) mice were of uncertain cause of death; and may not necessarily be due to administration of a compound of the invention. However, 10mg is considered as a higher limit of toxicity.

Treatments of groups (i) and (ii) were repeated weekly, and tumor growth was monitored for each experimental and control group. Two weeks after treatment, no toxicity signal was observed in the group (ii) mice, and the dose administered to this group was subsequently increased weekly in 1/2 log-sized increments. The following table shows the treatment process in group (ii) miceThe administration dose of (1):

sky	% survival group (i)	% active set (ii)	% active set (iii)
sky	% survival group (i)	% active set (ii)	% active set (iii)	18	100	100
19	100	100		18	100	100
19	100	100		20	100	100
21	100	100		20	100	100
21	100	100		22	75	100
23	75	100		22	75	100
23	75	100		24	75	100
25	75	100		24	75	100
25	75	100		26	75	100
27	75	100		26	75	100
27	75	100		28	75	100
29	50	100		28	75	100
29	50	100		30	50	100
31	50	100		30	50	100
31	50	100		32	50	100
33	50	100		32	50	100
33	50	100		34	50	100
35	50	100		34	50	100
35	50	100		36	25	100
37	25	100		36	25	100
37	25	100		38	25	100
39	25	100		38	25	100
39	25	100		40	25	100
41	25	100		40	25	100
41	25	100		42	25	100
43	25	80		42	25	100
43	25	80		44	25	80
45	25	80		44	25	80
45	25	80		46	25	80

Weekly dose (mg/mouse)

1 2

2 2

3 4

4 5

5 5

6 5

7 5

The processing results are shown in fig. 27A and B and table 10.

Table 10: in vivo anti-cancer effects

Sky	% survival group (i)	% active set (ii)	% active set (iii)
Sky	% survival group (i)	% active set (ii)	% active set (iii)	1	100	100	100
2	100	100	100	1	100	100	100
2	100	100	100	3	100	100	0
4	100	100		3	100	100	0
4	100	100		5	100	100
6	100	100		5	100	100
6	100	100		7	100	100
8	100	100		7	100	100
8	100	100		9	100	100
10	100	100		9	100	100
10	100	100		11	100	100
12	100	100		11	100	100
12	100	100		13	100	100
14	100	100		13	100	100
14	100	100		15	100	100
16	100	100		15	100	100
16	100	100		17	100	100

Sky	% survival group (i)	% active set (ii)	% active set (iii)
Sky	% survival group (i)	% active set (ii)	% active set (iii)	47	25	80
48	25	80		47	25	80
48	25	80		49	25	80
50	25	60		49	25	80
50	25	60		51	25	60
52	25	60		51	25	60
52	25	60		53	25	60
54	25	60		53	25	60
54	25	60		55	25	60
56	25	60		55	25	60
56	25	60		57	0	40
58		40		57	0	40
58		40		59		40
60		40		59		40
60		40		61		40
62		40		61		40
62		40		63		40
64		40		63		40

These results indicate that the fractions of the invention and the compounds of the invention do find utility in antitumor compositions and are not toxic at low and medium doses, and are toxic at higher doses as determined without undue experimentation.

Example 22:antimicrobial activity of cocoa extracts

Method A：

Studies were conducted to evaluate the antimicrobial activity of crude procyanidin extracts from cocoa beans against various microorganisms that are important in food spoilage or pathogenesis. The cocoa extract from method a of example 2 was used in this study. 1mL of 0.45% saline solution per cell culture suspension (final population number 10)²-10⁴cfu/mL) was inoculated with agar medium suitable for growth of the various test cultures and poured into petri dishes. The holes were cut into hardened agar using a #2 cork borer (5mm diameter). The plates were left in a refrigerator at 4 ℃ overnight to allow diffusion of the extract into the agar, and then incubated at a growth temperature appropriate for the microorganism of interest. The results are as follows:

sample area of inhibition (mm)

Concentration of extract (mg/mL)	Bacillus circulans	Bacillus cereus	Staphylococcus aureus	Pseudomonas aeruginosa	Bacillus subtilis
Concentration of extract (mg/mL)	Bacillus circulans	Bacillus cereus	Staphylococcus aureus	Pseudomonas aeruginosa	Bacillus subtilis	0	Nl	Nl	Nl	Nl	Nl
25	Nl	12	Nl	11	Nl	0	Nl	Nl	Nl	Nl	Nl
25	Nl	12	Nl	11	Nl	250	12	20	19	19	11
500	14	21	21	21	13	250	12	20	19	19	11

No inhibitory effect of Nl

Antimicrobial activity of purified procyanidins from cocoa beans was demonstrated in studies using the pore diffusion assay described above (in method a) with staphylococcus aureus as the culture of interest. The results are as follows:

Cocoa extract: decaffeinated/theobromine-depleted acetone extract 10mg/100 μ l as in method A of example 13

10 mg/100. mu.l dimer (99% purity) as in example 14, method D

10 mg/100. mu.l of tetramer (95% pure) as in example 14, method D

10 mg/100. mu.l hexamer (88% purity) as in example 14 method D

10 mg/100. mu.l octamer/nonamer as in example 14, method D (92% purity)

10 mg/100. mu.l nonamer and higher as in example 14, method D (87% purity)

Sample area of inhibition (mm)

0.45% salt solution 0

Dimer 33

Tetramer 27

Hexamer 24

0.45% salt solution 0

Octamer 22

Nonomer 20

decaffeination/Theobromine removal 26

Method B：

The crude procyanidin extract as in method 2 of example 2 was added at varying concentrations to TSB (tryptic Soy broth) with phenol red (0.08g/L) and the TSB was treated with Salmonella choleraesuis or Salmonella neobaumannii (10) ⁵cfu/mL) and incubated at 35 ℃ for 18 hours. The results are as follows:

salmonella choleraesuis, Salmonella newbornel

0mg/mL + +

50 + +

100 + +

250 + -

500 - -

750 - -

Where + growth-no growth, and acid production from broth medium was confirmed by a change from red to yellow. Inhibition was confirmed by plating from TSB tubes onto XLD plates.

This example demonstrates that the compounds of the invention are useful in food preparation and preservation.

This example further demonstrates that the compounds of the present invention inhibit the growth of gram negative and gram positive bacteria. From which the compounds of the invention can be used for the inhibition of helicobacter pylori. Helicobacter pylori has been implicated in causing gastric ulcers and gastric tumors. Thus, the compounds of the present invention may be used to treat or prevent these and other diseases of bacterial origin. Appropriate routes of administration, dosages and formulations can be determined without undue experimentation, taking into account factors well known in the art, such as the disease, age, weight, sex and general health of the individual.

Example 23:halogen-free analytical separation of extracts

The procyanidins obtained from example 2 were partially purified by halogen-free normal phase chromatography on 100_ Supelcosil LC-Si at a flow rate of 1.0 mL/min and a column temperature of 37 ℃. The separation was assisted with a linear gradient under the following conditions: (time,% A,% B); (0, 82, 14); (30, 67.6, 28.4); (60, 46, 50). The mobile phase composition was a-30/70% diethyl ether/toluene; b ═ methanol; and C ═ acetic acid/water (1: 1). Each component was detected by ultraviolet light at 280 nm. A representative elution profile is shown in figure 28.

Example 24:pore size of stationary phase for normal phase HPLC separation of procyanidins By using

To improve the separation of procyanidins, the use of larger pore sizes of the silicon stationary phase was investigated. The separation was performed on silicon-300, 5 μm, 300 a (250X 2.0mm) or another method, on silicon-1000, 5 μm, 1000 a (250X 2.0 mm). The linear gradient used as mobile phase composition was: a ═ dichloromethane; b ═ methanol; and C ═ acetic acid/water (1: 1). With λ therein_ex＝276nm，λ_emFluorescence at 316nm, and each component was detected at 280nm with an ultraviolet detector. Flow rate of 10 mL/min, oven temperature 37 ℃. Representative chromatograms from three different columns (100_ well size, from example 13, method D) are shown in fig. 29. This indicates an effective pore size for procyanidin separation.

Example 25:obtaining desired procyanidins by performing fermentation

Representative of a series of microbial strains associated with cocoa fermentation are selected from the M & M/Mars cocoa culture group. The following isolates were used:

acetobacter aceti ATCC 15973

Lactobacillus (BH 42)

Candida cruzii(BA 15)

Saccharomyces cerevisiae (BA 13)

Bacillus cereus (BE 35)

Spherical bacillus (ME 12)

Each strain was transferred from the stock culture to fresh medium. Yeast and Acetobacter aceti were incubated at 26 ℃ for 72 hours, and Bacillus and Lactobacillus were incubated at 37 ℃ for 48 hours. The slant cultures were collected with 5mL of phosphate buffer prior to use.

Cocoa beans were harvested from fresh pods, and the pulp and shell were removed. The beans were sterilized with hydrogen peroxide (35%) for 20 seconds and then treated with catalase until foaming ceased. The beans were rinsed twice with sterile water and the process was repeated. The beans were divided into two glass jars and treated according to the method detailed in the following table:

water (W)	Ethanol/acid	Fermentation soakingCrumbs	Fermentation of samples
water (W)	Ethanol/acid	Fermentation soakingCrumbs	Fermentation of samples	Daily transfer to fresh water	At various stages of the fermentation of the sample pulp, it was transferred daily to an alcohol and acid solution corresponding to the measured levels	Daily transfer to fermented pasteurized pulp during successive days of fermentation	Bench scale sample fermentation in sterile fruit pulp co-inoculated with test strains

The bench scale fermentation was performed twice. All treatments were incubated as follows:

the first day: 26 deg.C

The next day: 26-50 deg.C

And on the third day: 50 deg.C

The fourth day: 45 deg.C

The fifth day: 40 deg.C

During the study, sample fermentation was monitored by plate counting to assess microbial flora and HPLC analysis of the fermentation medium for microbial metabolite production. After treatment, the beans were dried to a water activity of 0.64 under laminar flow hood hyperstatic and baked at 66 ℃ for 15 minutes. Samples were prepared for procyanidin analysis. Three beans from each treatment were ground and defatted with hexane, followed by extraction with acetone, water, acetic acid (70: 29.5: 0.5%). The acetone solution extract was filtered into vials and the polyphenol levels quantified using n-butanol HPLC as in example 13, method B. The remaining beans were ground and tested. The culture and analysis profiles of the sample desktop fermentation are shown in FIGS. 30A-C. The procyanidin profiles of the various fermentation-treated cocoa beans are shown in FIG. 30D.

This example demonstrates that the invention need not be limited to any particular cocoa genus species and that by fermentation, the levels of procyanidins produced by a particular cocoa genus (Theobroma) or cocoa genus (Herrania) species or specific hybrids of interspecies or intraspecies thereof can be modulated, e.g. increased.

The following table shows the levels of procyanidins measured in species that are representative of the Theobroma genus (Theobroma) and specific hybrids of interspecies or intraspecies thereof. Samples were prepared as in examples 1 and 2 (methods 1 and 2) and analyzed as in example 13, method B. This data demonstrates that extracts containing the compounds of the invention are found in the species Theobroma cacao (Theobroma) and Theobroma cacao (Herrania), and in the specific hybridization of intraspecies and interspecies thereof.

Procyanidin levels ppm (μ g/g) of cocoa and Herrania species in defatted powder

Oligomer
Oligomer													Sample (I)	Monomer	Dimer	Trimer	Tetramer	Pentameric polymers	Hexamer	Heptamer	Octamer	Nonomer	Decamer	Undecamer	Total number of
T.grandillorum x T.obovatum 1¹	3822	3442	5384	4074	3146	2060	850	421	348	198	tr⁺	23,765	Sample (I)	Monomer	Dimer	Trimer	Tetramer	Pentameric polymers	Hexamer	Heptamer	Octamer	Nonomer	Decamer	Undecamer	Total number of
T.grandillorum x T.obovatum 1¹	3822	3442	5384	4074	3146	2060	850	421	348	198	tr⁺	23,765	T.grandillorum x T.obovatum 2¹	3003	4098	5411	3983	2931	1914	1090	577	356	198	tr	23,561
T.grandillorum x T.obovatum 3A¹	4990	4980	7556	5341	4008	2576	1075	598	301	144	tr	31,569	T.grandillorum x T.obovatum 2¹	3003	4098	5411	3983	2931	1914	1090	577	356	198	tr	23,561
T.grandillorum x T.obovatum 3A¹	4990	4980	7556	5341	4008	2576	1075	598	301	144	tr	31,569	T.grandillorum x T.obovatum 3B¹	3880	4498	6488	4930	3706	2560	1208	593	323	174	tr	28,360
T.grandillorum x T.obovatum 4¹	2647	3591	5328	4240	3304	2380	1506	815	506	249	tr	24,566	T.grandillorum x T.obovatum 3B¹	3880	4498	6488	4930	3706	2560	1208	593	323	174	tr	28,360
T.grandillorum x T.obovatum 4¹	2647	3591	5328	4240	3304	2380	1506	815	506	249	tr	24,566	T.grandillorum x T.obovatum 6¹	2754	3855	5299	3872	2994	1990	1158	629	359	196	88	23,194
T.grandillorum x T.obovatum SIN¹	3212	4134	7608	4736	3590	2274	936	446	278	126	ND^*	23,750	T.grandillorum x T.obovatum 6¹	2754	3855	5299	3872	2994	1990	1158	629	359	196	88	23,194
T.grandillorum x T.obovatum SIN¹	3212	4134	7608	4736	3590	2274	936	446	278	126	ND^*	23,750	T.obovatum 1¹	3662	5683	9512	5358	3858	2454	1207	640	302	144	ND	32,820
T.grandillorum TEFFE²	2608	2178	3090	2704	2241	1586	900	484	301	148	tr	16,240	T.obovatum 1¹	3662	5683	9512	5358	3858	2454	1207	640	302	144	ND	32,820
T.grandillorum TEFFE²	2608	2178	3090	2704	2241	1586	900	484	301	148	tr	16,240	T.grandillorum TEFFE x T.grandiflorum²	4773	4096	5289	4748	3804	2444	996	737	335	156	tr	27,380
T.grandillorum x T.subincanum¹	4752	3336	4916	3900	3064	2039	782	435	380	228	ND	23,832	T.grandillorum TEFFE x T.grandiflorum²	4773	4096	5289	4748	3804	2444	996	737	335	156	tr	27,380
T.grandillorum x T.subincanum¹	4752	3336	4916	3900	3064	2039	782	435	380	228	ND	23,832	T.obovatum x T.subincanum¹	3379	3802	5836	3940	2868	1807	814	427	271	136	tr	23,280
T.speciosum x T.sylvestris¹	902	346	1350	217	152	120	60	tr	tr	ND	ND	3,147	T.obovatum x T.subincanum¹	3379	3802	5836	3940	2868	1807	814	427	271	136	tr	23,280
T.speciosum x T.sylvestris¹	902	346	1350	217	152	120	60	tr	tr	ND	ND	3,147	T.microcarpum²	5694	3250	2766	1490	822	356	141	tr	ND	ND	ND	14,519
T.cacao，SIAL 659，t0	21,929	10,072	10,106	7788	5311	3242	1311	626	422	146	tr	60,753	T.microcarpum²	5694	3250	2766	1490	822	356	141	tr	ND	ND	ND	14,519
T.cacao，SIAL 659，t0	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	381	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，t24	21,088	9762	9119	7094	4774	2906	1364	608	381	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	N0	27,910
T.cacao，SIAL 659，1120	8581	4665	4070	2527	1628	888	326	166	123	tr	ND	22,974	T.cacao，SIAL 659，t96	9552	5780	5062	3360	2140	1160	464	254	138	tr	N0	27,910
T.cacao，SIAL 659，1120	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 recordings before 10/96 Herrania mariae	130	354	151	131	116	51	tr	ND	N0			933	Pod Rec.10/96，Herrania mariae	869	1295	545	347	175	97	tr	^*ND	ND			3329

ND-undetected sample designated CPATU tr-trace (< 50. mu.g/g)^*Sample designation ERJON

Example 26: action of procyanidin on NO

Method A

The objective of this study was to establish a relationship between procyanidins (as in example 14, method D) and NO, which is known to induce cerebral vasodilation. The effect of monomers and higher oligomers at concentrations ranging from 100. mu.g/mL to 0.1. mu.g/mL on the production of nitrate (catabolite of NO) from HUVEC (human umbilical vein endothelial cells) was evaluated. HUVECs (from Clnetics) were studied for 24 or 48 hours in the presence or absence of various procyanidins. At the end of the experiment, the supernatant was collected and calorimetrically determined for nitrate content. In each individual experiment, HUVECs were incubated with acetylcholine known to induce NO production for 24-48 hours in the presence or absence of procyanidins. At the end of the experiment, the supernatant was collected and calorimetrically determined for nitrate content. The effect of NO was confirmed by the addition of nitroarginine or (1) -N-methylarginine, which are NO synthase-specific blockers.

Method B vascular relaxation of the arteries of rats induced by phenylephrine contraction

The effect of various procyanidins at 100. mu.g/mL to 0.1. mu.g/mL on rat arteries was the goal of studying phenylephrine-induced systolic rat arterial vascular relaxation. Isolated rat arteries were incubated in the presence or absence of procyanidins (as in example 14, method D) and changes in muscle tone were assessed visually. The contraction or relaxation of the rat artery was determined. Next, using other organs, a pre-constriction of isolated rat arteries was induced upon addition of epinephrine. Once the contraction stabilized, procyanidins were added and the contraction or relaxation of the rat artery was determined. The effect of NO was confirmed by the addition of nitroarginine or (1) -N-methylarginine. Acetylcholine-induced release of NO as accomplished by phenylephrine preshrinking of rat aorta is shown in figure 31.

Method C. Induction of hypotension in rats

The present method is directed to the effect of various procyanidins (e.g., example 14, method D) on blood pressure. Rats were fitted with a sphygmomanometer to monitor systolic and diastolic blood pressure. Changes in blood pressure were determined by intravenous injection of various procyanidins (dose range 100-0.1 μ g/kg). In addition, the effect of various procyanidins on blood pressure changes caused by epinephrine was determined. The effect of NO was confirmed by the addition of nitroarginine or (1) -N-methylarginine.

These studies, together with the next example, are shown to be useful for modulating vasodilation, and may further be useful for modulating blood pressure or ameliorating coronary artery conditions, and migraine headaches.

Example 27:effect of cocoa polyphenols on satiety

Using blood glucose levels as an indicator of the occurrence of signaling events in the body for regulating appetite and satiety, healthy male adult volunteers aged 48 years were selected for a series of simple experiments to determine whether cocoa polyphenols regulated glucose levels. Cocoa polyphenols were partially purified from brazil cocoa beans according to the method described by Clapperton et al (1992). The substance does not contain caffeine or theobromine. Accelerated blood glucose levels were analyzed on a timed basis after drinking 10 fluid ounces of Dexicola 75 (caffeine-free) glucose tolerance test beverage (Curtin Matheson 091-421) with and without 75mg of cocoa polyphenols. This polyphenol level represents 0.1% of the total glucose of the test beverage and reflects the approximate amount that would be present in a standard 100g chocolate bar form. The blood glucose level was determined using the Accu-Chek III blood glucose monitoring system (Boehringer Mannheim). Blood glucose levels were measured after drinking the test beverages, and after drinking the test beverages at the following specified time intervals: 15, 30, 45, 60, 75, 90, 120 and 180 minutes. Controls of high and low glucose levels were determined prior to the initiation of each glucose tolerance test. Each glucose tolerance test was repeated twice. Control test solutions containing 75mg of cocoa polyphenols dissolved in 10 fluid ounces of distilled water (no glucose) were also tested.

Table 11 below lists the data and control values obtained from each glucose tolerance test performed in this study. Fig. 32 represents the mean and standard deviation with blood glucose levels obtained over a three hour time course. It is readily seen that there was a significant increase in blood glucose levels obtained after drinking the test mixture containing cocoa polyphenols. The difference between the two major glucose tolerance profiles cannot be addressed by the profile obtained after drinking a solution of cocoa polyphenols only. The addition of cocoa polyphenols to the glucose test beverages significantly improved the glucose tolerance profile. Although their typical glucose tolerance profile is considered normal (Davidson, I. et al, editor Todd-Sandford clinical diagnosis by laboratory methods 14 th edition; W.B.Saunders Co.; Philadelphia, PA 1969 Ch.10, pp.550-9), this increase in blood glucose levels is in a range considered to be moderate diabetes. This suggests that additional differences in glucose are released into the blood from glycogen storage sites due to the compounds of the present invention. Thus, the present invention can be used to regulate blood glucose levels in the presence of sugar.

TABLE 11 glucose tolerance test data and control results

Week (week)	Description of the invention	High control^a	Low control^b
Week (week)	Description of the invention	High control^a	Low control^b	0	Glucose tolerance	265mg/dL	53mg/dL
1	Glucose tolerance to 0.1% polyphenols	310	68	0	Glucose tolerance	265mg/dL	53mg/dL
1	Glucose tolerance to 0.1% polyphenols	310	68	2	Glucose tolerance	315	66
4	Glucose tolerance to 0.1% polyphenols	325	65	2	Glucose tolerance	315	66
4	Glucose tolerance to 0.1% polyphenols	325	65	5	0.1% polyphenol	321	66

a is the expected range: 253-373mg/dL

b-expected range: 50-80mg/dL

Individuals also experience flushing (erythema) and light headaches after ingestion of the compounds of the invention, indicating modulation of vasodilation.

The data presented in tables 12 and 13 illustrate the fact that the extract of the invention, which is dependent on cocoa raw material and commercial chocolate and in which the compounds of the invention are contained, can be used as an excipient for pharmaceutical, veterinary and food science products and applications.

Table 13: procyanidin levels in commercial chocolate

μg/g

Sample (I)	Monomer	Dimer	Trimer	Tetramer	Pentameric polymers	Hexamer	Heptamer and higher	Total of
Sample (I)	Monomer	Dimer	Trimer	Tetramer	Pentameric polymers	Hexamer	Heptamer and higher	Total of	Number plate 1	366	166	113	59	56	23	18	801
Number plate 2	344	163	111	45	48	ND^*	ND	711	Number plate 1	366	166	113	59	56	23	18	801
Number plate 2	344	163	111	45	48	ND^*	ND	711	Number plate 3	316	181	100	41	40	7	ND	685
Number 4	310	122	71	27	28	5	ND	563	Number plate 3	316	181	100	41	40	7	ND	685
Number 4	310	122	71	27	28	5	ND	563	Number 5	259	135	90	46	29	ND	ND	559
Number 6	308	139	91	57	47	14	ND	656	Number 5	259	135	90	46	29	ND	ND	559
Number 6	308	139	91	57	47	14	ND	656	Number 7	196	98	81	58	54	19	ND	506
Number plate 8	716	472	302	170	117	18	ND	1,795	Number 7	196	98	81	58	54	19	ND	506
Number plate 8	716	472	302	170	117	18	ND	1,795	Number 9	1,185	951	633	298	173	25	21	3,286
Number plate 10	1,798	1,081	590	342	307	93	ND	4,211	Number 9	1,185	951	633	298	173	25	21	3,286
Number plate 10	1,798	1,081	590	342	307	93	ND	4,211	Number plate 11	1,101	746	646	372	347	130	75	3,417
Number plate 12	787	335	160	20	10	8	ND	1,320	Number plate 11	1,101	746	646	372	347	130	75	3,417

ND^*Not detected out

Table 14: procyanidin levels in cocoa materials

μg/g

Sample (I)	Monomer	Dimer	Trimer	Tetramer	Pentameric polymers	Hexamer	Heptamer and higher	Total of
Sample (I)	Monomer	Dimer	Trimer	Tetramer	Pentameric polymers	Hexamer	Heptamer and higher	Total of	Not fermented	13,440	6,425	6,401	5,292	4,236	3,203	5,913	44,910
Fermentation of	2,695	1,538	1,362	740	470	301	277	7,383	Not fermented	13,440	6,425	6,401	5,292	4,236	3,203	5,913	44,910
Fermentation of	2,695	1,538	1,362	740	470	301	277	7,383	Baking	2,656	1,597	921	337	164	ND^*	ND	5,675
Chocolate solution	2,805	1,446	881	442	184	108	ND	5,866	Baking	2,656	1,597	921	337	164	ND^*	ND	5,675
Chocolate solution	2,805	1,446	881	442	184	108	ND	5,866	Cocoa shell	114	53	14	ND	ND	ND	ND	181
Cocoa powder 1% fat	506	287	112	ND	ND	ND	ND	915	Cocoa shell	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 Holland cocoa powder, pH7.4, 11% fat	1,222	483	103	ND	ND	ND	ND	1,808	Cocoa powder 11% fat	1,523	1,224	680	46	ND	ND	ND	3,473
Red Holland cocoa powder, pH7.4, 11% fat	1,222	483	103	ND	ND	ND	ND	1,808	Red Holland cocoa powder, pH8.2, 23% fat	168	144	60	ND	ND	ND	ND	372

ND^*Not detected out

Example 28:effect of procyanidin on cyclooxygenase 1 and 2

The effect of procyanidins on cyclooxygenase 1 and 2(COX1/COX2) activity was evaluated by incubating enzymes from ram semen vesicles and sheep placenta with arachidonic acid (5 μ M) for 10 minutes in the presence of various concentrations of procyanidin solutions containing monomers to decamers and a mixture of procyanidins, respectively, at room temperature. The conversion was evaluated using PGF2 EIA cassette (france) from Interchim. Indomethacin was used as reference compound. The results are provided in the table below, where IC is as in example 14, method D₅₀Values are expressed in units of μ M (except S11, which represents the procyanidin mixture prepared from example 13, method A, and where samples S1-S10 in turn represent procyanidin oligomers (monomers to decamers), and IC₅₀Expressed in mg/mL units).

Sample #	IC₅₀COX-1(*)	IC₅₀COX-2(*)	Proportional IC₅₀COX2/COX1
Sample #	IC₅₀COX-1(*)	IC₅₀COX-2(*)	Proportional IC₅₀COX2/COX1	1	0.074	0.197	2.66
2	0.115	0.444	3.86	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	3	0.258	0.763	2.96
4	0.154	3.73	24.22	5	0.787	3.16	4.02
6	1.14	1.99	1.75	5	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	7	1.89	4.06	2.15
8	2.25	7.2	3.20	9	2.58	2.08	0.81
10	3.65	3.16	0.87	9	2.58	2.08	0.81
10	3.65	3.16	0.87	11	0.0487	0.0741	1.52
Indometacin	0.599	13.5	22.54	11	0.0487	0.0741	1.52

(. about.) is expressed in. mu.M, except that sample 11 is mg/mL.

The results of the inhibition study are provided in fig. 33A and B, where it shows the effect of indomethacin on COX1 and COX2 activities. FIGS. 34A and B show the degree of polymerization of procyanidins with IC against COX1 and COX2₅₀The interrelationship between them; FIG. 35 shows IC's for COX1 and COX2₅₀Correlation between values. And FIGS. 36A-Y show the IC50 values for each sample (S1-S11) against COX1 and COX 2.

These results demonstrate that the compounds of the present invention have analgesic, anticoagulant, and anti-inflammatory uses. Furthermore, COX2 has been associated with colon cancer. The inhibition of COX2 activity by the compounds of the invention illustrates one possible mechanism by which the compounds of the invention have anti-tumor activity against colon cancer.

COX1 and COX2 are also involved in the synthesis of prostaglandins. Thus, the results of this example also demonstrate that renal function, immune response, fever, pain, mitosis, partial cell death, prostaglandin synthesis, ulcer formation (e.g., gastric ulcer), and reproduction can be modulated. It is noted that modulation of renal function can affect blood pressure; again, it is intended that the compounds of the present invention are involved in the regulation of blood pressure, vasodilation and coronary blood vessels (e.g., regulating angiotensin, bradykinin).

Reference to Seibert et al, PNAS USA 91: 12013-12017(12, 1994), Mitchell et al, PNAS USA 90: 11693-11697(12, 1994), Dewitt et al, cell 83: 345-: 483-92(113, 1995) and Sujii et al, cell 83: 493-501(113, 1995), Morham et al, cell 83: 473-82(113, 1995).

Reference is further made to examples 9, 26 and 27. In example 9, the antioxidant activity of the compounds of the present invention is shown. In example 26, the effect on NO was confirmed. And in example 27 evidence of facial vasodilation is provided. From the results of this example, in combination with examples 9, 26 and 27, the compounds of the present invention modulate the free radical mechanism that drives physiological action. Similarly, the present invention can modulate lipoxygenase-mediated free radical type reactions directed biochemically to leukotriene synthesis, thereby affecting later physiological activities (e.g., inflammation, immune response, coronary vascular conditions, carcinogenesis, fever, pain, ulceration).

Thus, in addition to having analgesic properties, the compounds of the present invention may also have a synergistic effect when taken together with other analgesics. Likewise, in addition to having anti-tumor properties, the compounds of the present invention may also have synergistic effects when taken with other anti-tumor agents.

Example 29:study of circular dichroism/procyanidins

CD studies were performed in an effort to explain the structure of purified procyanidins as in method D of example 14. Spectra were collected at 25 ℃ using CD Spectroscopy software AVIV 60DS V4.1f.

The samples were scanned at 1.00nm each time at a bandwidth of 1.50nm between 300nm and 185 nm. Representative CD spectra are shown in FIGS. 43A-G, which show the CD spectra of dimers through octamers.

These results illustrate the helical structure of the compounds of the present invention.

Example 30:inhibition of helicobacter pylori and staphylococcus aureus by cocoa procyanidins By using

Studies were conducted to evaluate the antimicrobial activity of procyanidin oligomers against helicobacter pylori and Staphylococcus aureus. An enrichment of pentamers was prepared as described in example 13, method a and analyzed as described in example 14, method C, with 89% pentamers and 11% higher oligomers (n is 6-12). Purified pentamer (96.3%) was prepared as described in example 14, method D.

Helicobacter pylori and staphylococcus aureus were obtained from the American Type Culture Collection (ATCC). For H.pylori, the vial was rehydrated with 0.5mL trypticase medium and the suspension was transferred to a fresh TSA slant containing 5% defibrinated sheep blood. The slant was incubated at 37 ℃ for 3-5 days in an anaerobic jar (5-10% carbon dioxide; CampyPakPlus, BBL) under microaerophilic conditions. When good growth was established in the culture broth layer at the bottom of the slant, the culture broth was used to inoculate additional TSA slants containing sheep blood. Since the survival rate decreased with the successive subcultures, the culture broth collected from the slant was pooled and stored at-80 ℃. Cultures for analysis from frozen tubular flasks were used directly. Staphylococcus aureus was cultured on TSA slants and transferred to fresh slants 24 hours prior to use.

Preparation of cell suspensions of various cultures (helicobacter pylori, 10)⁸-10⁹cfu/mL; staphylococcus aureus 10⁶-10⁷cfu/mL), and 0.5mL was plated on TSA plates containing 5% sheep blood. Standard assay trays (Difco) were immersed in a series of dilutions (23mg/mL to sterile water) of filter-sterilized pentamer. Test and blank control discs (sterile water) were placed on the inoculated plates. Control plates containing 80. mu.g metronidazole (inhibitory to H.pylori) or 30. mu.g vancomycin (inhibitory to Staphylococcus aureus) (BBL sensisics) were also placed on the appropriate sites of the plates. The H.pylori-inoculated plates were incubated under microaerobic conditions. Carrying out aerobic heat preservation on the staphylococcus aureus. After growth inhibition zones were determined.

TABLE 14 bioassay of pentamers against H.pylori and S.aureus

Pentamer enriched fraction (mg/ml)	Inhibition of Staphylococcus aureus (mm)	Inhibition of helicobacter pylori (mm)
Pentamer enriched fraction (mg/ml)	Inhibition of Staphylococcus aureus (mm)	Inhibition of helicobacter pylori (mm)	0	NI	NI
15	0	10	0	NI	NI
15	0	10	31	10	10
62	11	11	31	10	10
62	11	11	125	13	13
250	15	13	125	13	13
250	15	13	Vancomycin standard	15	--
Metronidazole standard	--	11	Vancomycin standard	15	--
Metronidazole standard	--	11	96% pure pentamer	15	11

NI-not inhibited

Example 31: NO-dependent hypotension in guinea pigs

The effect of five cocoa procyanidin fractions on guinea pig blood pressure was investigated. Briefly, guinea pigs (approximately 400g body weight; male and female) were anesthetized with an injection of 40mg/kg sodium pentobarbital. The carotid artery was cannulated to monitor arterial blood pressure. Five cocoa procyanidin fractions (dose range 0.1mg/kg-100mg/kg) were injected intravenously via jugular vein. The changes in blood pressure were recorded on a multi-channel recorder. In these experiments, the effect of NO was determined by administering L-N-methylarginine (1mg/kg) 10 minutes prior to administration of the cocoa procyanidin fraction.

The cocoa procyanidin fraction was prepared and analyzed according to the method described in U.S. patent 5,554,645, which is incorporated herein by reference.

And (2) component A: represents a preparative HPLC fraction consisting of monomer-tetramer. HPLC analysis revealed the following composition:

47.2 percent of monomer

Dimer 23.7

Trimer 18.7

Tetramer 10.3

Fraction B: represents a preparative HPLC fraction consisting of pentamer-decamer. HPLC analysis revealed the following composition:

pentamer 64.3%

Hexamer 21.4

Heptamer 7.4

Octameric 1.9

Nonamer 0.9

Decamer 0.2

Fraction C: represents the enriched cocoa procyanidin fraction used to prepare fractions a and B (above). HPLC analysis revealed the following composition:

34.3 percent of monomer

Dimer 17.6

Trimer 16.2

Tetramer 12.6

Pentameric 8.5

Hexamer 5.2

Heptamer 3.1

Octameric 1.4

Nonamer 0.7

Decamer 0.3

Fraction D: represents a procyanidin extract prepared from milk chocolate. HPLC analysis revealed a composition similar to that listed in table 12 for designation 8. In addition, 10% caffeine and 6.3% theobromine were present.

Fraction E: represents a procyanidin extract prepared from dark chocolate prepared with an aqueous alkaline solution. HPLC analysis revealed a composition similar to that listed in table 12 for designation 12. In addition, 16.0% caffeine and 5.8% theobromine were present.

In three separate experiments, the effect of administration of 10mg/kg cocoa procyanidin fraction on arterial blood pressure in anesthetized guinea pigs was studied. When injected intravenously, procyanidin fractions a and E resulted in a decrease in blood pressure of about 20%. This reduction differs only to some extent from that obtained from the solvent (DMSO) control (15 ± 5%, n ═ 5). In contrast, procyanidin fractions B, C and D (10mg/kg) induced a significant decrease in blood pressure, up to 50-60% for fraction C. In these experiments, the sequence of hypotensive effects was as follows: c > B > D > A ═ E.

Typical recordings of blood pressure generated after injection of the procyanidin fraction are provided in fig. 50A for fraction and fig. 50B for fraction C. FIG. 51 illustrates the comparative effect of these fractions on blood pressure.

The possible effect of NO on guinea pig hypotension induced by the administration of fraction C was analyzed using L-N-methylarginine (LNMMA). The agent inhibits NO formation by inhibiting NO synthase. L-NMMA was administered at a dose of 1mg/kg 10 minutes prior to injection of the cocoa procyanidin fraction. As shown in FIG. 52, treatment of animals with L-NMMA completely blocked

Hypotension induced by procyanidin fraction C. Indeed, the changes in blood pressure produced by fraction C after treatment with the inhibitor were similar to those observed with solvent alone.

Example 32: production of NO in human umbilical vein endothelial cells by cocoa procyanidin fractionInfluence of (2)

Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from Clonetics and cultures were performed according to the manufacturer's instructions. HUVEC cells were cultured at 5,000 cells/cm²Seeded in 12-well plates (Falcon). After a third generation under the same conditions, they were allowed to reach confluency. The supernatants were refreshed with fresh media containing either the specified concentrations of bradykinin (25, 50 and 100nM) or cocoa procyanidin fractions A-E (100. mu.g/mL) as described in example 31. The incubation was continued for 24 hours and cell-free supernatants were collected and stored frozen before assessment of NO content as described below. In selected experiments, the NO Synthase (NOs) antagonist N ω -nitro-L-arginine methyl ester (L-NAME, 10 μ M) was added to evaluate engagement of NOs in observed NO production.

HUVEC NO production was determined by measuring nitrite concentration in culture supernatants using the Griess reaction. Griess reagent is 1% sulfonamide, 0.1% N- (1-naphthyl) -ethylenediamine dihydrochloride. Briefly, 50. mu.l aliquots were taken from each supernatant in four replicates and incubated with 150. mu.l Griess reagent. The absorbance was measured at 540nm in a multiplex scanner (multiscan) (Labsystems Multiskans MCC/340). A standard curve was established using a defined concentration of sodium nitrite. The absorbance of the cell-free medium (blank) was subtracted from the value obtained with the cell-containing supernatant.

Figure 53 illustrates the effect of bradykinin on NO production by HUVEC, where dose-dependent NO release was observed. The inhibitor L-NAME completely inhibits bradykinin-induced NO release.

FIG. 54 illustrates the effect of cocoa procyanidin fractions on NO production by HUVEC cells. Fractions B, C and D induced moderate but significant amounts of NO production by HUVEC. Fraction C is the most effective fraction for inducing NO formation, while fraction E is almost ineffective, as assessed by nitrite production. The effect of fraction C on NO production was dramatically reduced in the presence of L-NAME. Interestingly, fractions B, C and D contained a greater amount of procyanidin oligomers than fractions a and E. The clear difference between fractions D and E is that E was prepared from dark chocolate in which an alkalized cocoa solution was used as the chocolate formulation. Basification results in the polymerization of base-catalyzed procyanidins that rapidly reduces the levels of these compounds. A comparison of the analysis of the procyanidin levels found in these types of chocolate is shown in table 12, where designation 12 is dark chocolate prepared with an alkalized cocoa solution and designation 11 is typical of milk chocolate. Thus, the extract obtained from milk chocolate contains a high proportion of NO-inducing procyanidin oligomers. The addition of the NO inhibitor L-NMMA to the fraction C samples clearly resulted in NO inhibition. The results obtained from the procyanidin fraction were consistent with those obtained from experiments with bradykinin-induced NO (see figure 53).

As in the case of HUVEC results, cocoa procyanidin fraction C produced the major hypotensive effect in guinea pigs, while fractions a and E were least effective. Furthermore, the presence of high molecular weight procyanidin oligomers is associated with the modulation of NO production.

Example 33: effect of cocoa procyanidins on macrophage NO production

To fresh heparin-added human blood (70mL) was added an equal volume of Phosphate Buffered Saline (PBS) at room temperature. The ratio of 3mL of sodium polysucrose-3, 5-diacetamido-2, 4, 6-triiodobenzoate to 10mL of blood-PBS solution was used to laminate the sodium polysucrose-3, 5-diacetamido-2, 4, 6-triiodobenzoate solution under the blood-PBS mixture, centrifuge the tubes at 2,000rpm for 30 minutes at 18-20 ℃. The upper layer containing plasma and platelets was discarded. The monocyte layer was transferred to another centrifuge tube and the cells were washed twice in Hanks balanced salt solution. Monocytes were resuspended in complete RPMI 1640 supplemented with 10% fetal bovine serum, counted and viability determined by trypan blue exclusion. Resuspend the cell pellet in complete RPMI 1640 supplemented with 20% fetal bovine serum to a final concentration of 1X 10⁶cells/mL. Aliquots of the cell suspension were plated into 96-well culture plates, rinsed 3 times with RPMI 1640 supplemented with 10% fetal bovine serum, and non-adherent cells (lymphocytes) were discarded.

These cells were incubated for 48 hours in the presence or absence of the five procyanidins described in example 31. At the end of the incubation period, the medium was collected, centrifuged and the cell-free supernatant was stored frozen for nitrite analysis.

Macrophage NO production was determined by measuring nitrite concentration using the Greiss reaction. Greiss reagent was 1% sulfonamide, 0.1% N- (1-naphthyl) -ethylenediamine dihydrochloride. Briefly, a 50. mu.l aliquot was removed from the supernatant in quadruplicate and incubated with 150. mu.l of Greiss reagent. The absorbance was measured at 540nm in a multiplex scanner (Labsystems Multiskans MCC/340). A standard curve was established using a defined concentration of sodium nitrite. The absorbance of the cell-free medium (blank) was subtracted from the value obtained with the cell-containing supernatant.

In a separate experiment, macrophages were exposed to antigen for 12 hours in the presence of 5U/mL interferon-gamma, followed by restimulation with 10. mu.g/mL LPS for 36 hours in the presence or absence of the five procyanidin fractions of 100. mu.g/mL.

FIG. 55 illustrates that only 100. mu.g/mL of procyanidin C induces NO production by monocytes/macrophages. No basal NO production by these cells could be detected, and NO nitrite could be detected in any cocoa procyanidin fraction taken at 100. mu.g/mL. FIG. 56 illustrates that procyanidin fractions A and D increase LPS-induced NO production by monocytes/macrophages exposed to the interferon-gamma antigen. Since LPS-stimulated monocytes/macrophages cultured in the absence of the procyanidin fraction produced only 4. mu. mol/10 ⁵Cells/48 hours, therefore procyanidin fraction C was effective to some extent. Only gamma interferon is ineffective in inducing NO.

Taken together, these results demonstrate that mixtures of the compounds of the invention used at specific concentrations induce the production of monocyte/macrophage NO independently and dependently on LPS or cytokine stimulation.

From the foregoing, it will be apparent that extracts and cocoa polyphenols, particularly the compounds of the invention and the compositions, methods and kits of the invention have significant and many uses.

The anti-tumor utility is clearly demonstrated by the in vivo and in vitro data herein and indicates that the compounds of the present invention can be used in place of or in combination with conventional anti-tumor agents.

The present invention has antioxidant activity, such as BHT and BHA, as well as oxidative stability. Thus, the present invention may be used instead of or in combination with BHT or BHA in known uses of BHA and BHT, e.g., antioxidants, such as antioxidants, food additives.

The present invention may also be employed in place of or in combination with topoisomerase II-inhibitors in their currently known uses.

The compounds of the invention can be used for the preservation or preparation of food products and for the prevention or treatment of diseases of bacterial origin. Briefly, the compounds of the present invention may be used as antimicrobial agents.

The compounds of the invention may also be used as cyclooxygenase and/or lipoxygenase, NO or NO-synthase, or blood or in vivo glucose modulator, and thus may be useful in the treatment or prevention or modulation of pain, fever, inflammatory coronary artery disease, ulcers, carcinogenesis, vasodilation, and analgesia, anti-coagulant anti-inflammatory agents, and immune response modifiers.

Furthermore, the invention includes the use of the compound or extract as an excipient for pharmaceutical formulations. Thus, there are many compositions and methods envisioned by this invention. For example, antioxidant or preservative compositions, topoisomerase II-inhibiting compositions, methods for preserving food or any desired substance, e.g., from oxidation, and methods for inhibiting topoisomerase II. The compositions may comprise a compound of the invention. The methods can comprise contacting a food, substance, or topoisomerase II with each composition or with a compound of the invention. Other compositions, methods and embodiments of the invention will be apparent from the foregoing.

In this respect, it has been mentioned that the invention is derived from edible sources and that in vitro activity may demonstrate at least some in vivo activity; based on the in vitro and in vivo data presented herein, dosages, routes of administration and formulations can be obtained without undue experimentation.

Example 34.Micellar electrokinetic capillary chromatography of cocoa procyanidins

A rapid method was developed for the isolation of procyanidin oligomers using capsule electrokinetic capillary chromatography (MECC). The method is an improvement of the method reported by Delgado et al, 1994. The MECC method requires only 12 minutes to achieve the same separation obtained with a 70 minute normal phase HPLC analysis. FIG. 57 represents the MECC isolation of cocoa procyanidins obtained from example 2.

MECC conditions:

a cocoa procyanidin extract was prepared as described in example 2 and dissolved at a concentration of 1mg/mL in MECC buffer consisting of 200mM boric acid, 50mM sodium dodecyl sulfate (electrophoresis) and NaOH to adjust the pH to 8.5.

The samples were run through a 0.45 μm filter and electrophoresed using a Hewlett packard HP-3D CZE system under the following conditions:

inlet buffer: run buffer as described above

Outlet buffer: run buffer as described above

Capillary tube: 50cm x 75 μm inner diameter uncoated molten silicon

And (3) detection: 200nm, detector with diode system

And (3) injection: 50m Bar for 3 seconds (150m bar second)

Voltage: 6 watt

Current: system limitations (< 300 μ A)

Temperature: 25 deg.C

Capillary conditions: wash with assay buffer for 5 minutes before and after each assay.

The method can be improved by mimicking varying temperature, pressure and voltage parameters, and including organic modifiers and chiral selectors in the assay buffer.

Example 35.MALDI-of procyanidin oligomers mixed with a metal salt solution TOF/MS analysis

A series of MALDI-TOF/MS analyses were performed on trimers mixed with various metal salt solutions to determine whether the cationic adducts of the oligomers could be detected. The importance of this experiment is to provide evidence that procyanidin oligomers exert physiological effects in vitro and in vivo by sequestering or transporting cations that are important to physiological processes and diseases.

The procedure used is as described in example 15. Briefly, 2. mu.l of 10mM zinc sulfate dihydrate, calcium chloride, magnesium sulfate, ferrous chloride hexahydrate, ferrous sulfate heptahydrate, and copper sulfate solution were mixed with 4. mu.l of trimer purified to apparent homogeneity as described in example 14 (10mg/mL), respectively, and 44. mu.l of DHB was added.

The results (FIGS. 58A-F) show [ metal-trimer + H ] for copper and iron (iron and ferrous iron)]⁺Ions with m/z values equal to ± 1amu standard deviation of the theoretically calculated mass. [ Metal-trimer + H of calcium and magnesium]⁺Cannot be unambiguously derived from sodium and potassium [ metal-trimer + H ]⁺The mass of (1) is eliminated, and the m/z value is within a standard deviation of + -1 amu. No detection of [ Zn ]⁺²-trimer + H]⁺Ions. Since some cations are multivalent, the possibility of a multimetal-oligomeric ligand species and/or a metal-multioligomeric species is possible. However, screening of these adducts for their expected mass proved unsuccessful.

The results shown above for copper, iron, calcium, magnesium and zinc can be used as a routine technique for later analysis of the reaction between other metal ions and the compounds of the invention, taking into account factors such as oxidation state and the relative position of the ions in question in the periodic table of the elements.

Example 36.MALDI-TOF/MS analysis of high molecular weight procyanidin oligomers

Analytical experiments were performed on GPC eluates associated with high molecular weight procyanidin oligomers prepared as in example 3, method a. The objective was to determine whether procyanidin oligomers with n > 12 were present. These oligomers, if present, replace additional compounds of the present invention. In line with the existing isolation procedures, the isolation and purification encompassed by the present invention can be used to obtain these oligomers for later evaluation in vitro and in vivo of anti-cancer, anti-tumor or antineoplastic activity, antioxidant activity, inhibition of DNA topoisomerase II, inhibition of oxidative damage of DNA, and having antimicrobial, NO or NO-synthetase, apoptosis, platelet aggregation, and blood or in vivo glucose regulating activity, as well as efficacy as non-steroidal anti-inflammatory agents.

FIG. 59 represents a MALDI-TOF mass spectrum of the GPC eluted sample described above. Characterization by tetramer to octamer[ M + Na ] of epicyandrin oligomers]⁺And/or [ M + K]⁺And/or [ M +2Na]⁺The ions are clearly visible.

It is recognized that acid and heat treatment will result in hydrolysis of procyanidin oligomers. Thus, the present invention encompasses the controlled hydrolysis of high molecular weight procyanidin oligomers as a means of preparing lower molecular weight procyanidin oligomers (e.g. when n is 2 to 12).

Example 37.Dose response relationships of procyanidin oligomers with canine and feline cell lines

The dose response effect of procyanidin oligomers on several canine and feline cell lines obtained from Waltham Center for Pet Nutrition, Walthamon-the-Wolds, Melton Mowbray, Leicestershire, U.K. was evaluated. These cell lines were for the production of leukemia virus cultured under the conditions described in example 8, method A

Dog normal kidney GH cell line;

dog normal kidney MDCK cell line;

feline normal renal CRFK cell line; and

feline lymphoblast FeA cell line.

Monomers and procyanidin oligomers where n is 2-10 were purified as described in example 14, method D. Oligomers were also detected by analytical normal phase HPLC as described in example 14, method C, where the following results were obtained.

Procyanidin HPLC purity%

Monomer 95.4

Dimer 98.0

Trimer 92.6

Tetramer 92.6

Pentameric 93.2

Hexamer 89.2 (containing 4.4% pentamer)

Heptamer 78.8 (containing 18.0% hexamer)

Octamer 76.3 (containing 16.4% heptamer)

Nonamer 60.3 (containing 27.6% octamer)

Decamer 39.8 (containing 22.2% nonamer, 16.5% octamer)

And 13.6% heptamer)

In those cases where the oligomer purity is less than 90%, the methods encompassed by the present invention can be used for their purification.

Monomers and various procyanidin oligomers were administered at 10. mu.g/mL, 50. mu.g/mL and 100. mu.g/mL to various cell lines. The results are shown in FIGS. 60-63. As shown in the figure, administration of high doses (100. mu.g/mL) of various oligomers produced similar inhibitory effects on feline FeA lymphoblasts and feline normal renal CRFK cell lines. In these cases, cytotoxicity occurred with the tetramer, and increasing higher oligomers produced a greater cytotoxic effect. In contrast, high dose (100. mu.g/mL) administration of each oligomer to dog GH and MDCK normal renal cell lines required a higher oligomer to initiate the appearance of cytotoxicity. For dog GH normal renal cell lines, cytotoxicity occurred with the pentamer. For canine MDCK normal renal cell lines, cytotoxicity occurred with hexamers. In both cases, administration of higher oligomers resulted in an increased degree of cytotoxicity.

Example 38.Tablet formulation

Tablets were prepared using cocoa solids obtained by the method described in U.S. application serial No. 08/709,406 filed on 6/9/1996, which is incorporated herein by reference. In short, the edible material is prepared by a process that increases the naturally occurring level of the compounds of the present invention to the level they are found in traditionally processed cocoa, such that the ratio of the initial amount of the compounds of the present invention found in the raw beans to the amount obtained after processing is less than or equal to 2. For simplicity, the cocoa solids material is referred to herein as CP-cocoa solids. The compound or compounds of the invention, e.g., in isolated and/or purified form, can be used in tablets as described in this example, instead of or in combination with CP-cocoa solids.

The tablet formulation comprised the following ingredients (percentages are expressed in weight percent):

CP-cocoa solids 24.0%

4 times of natural vanilla extract

(Bush Boake Allen) 1.5％

Magnesium stearate

(Dry Lubricant) (Aerchem, Inc.) 0.5%

Dipeck tabletting sugar

(Amstar Sugar Corp.) 37.0％

Xylitol (American XYrofin, Inc.)37.0％

100.0％

The CP-cocoa solids and vanilla extract were mixed together in a food processor for 2 minutes. The sugar and magnesium stearate were gently mixed together and then mixed in the CP-cocoa solids/vanilla extract mixture. The material was subjected to Manesty Tablet Press (B3B) at maximum pressure and pressed to produce round tablets (15 mm. times.5 m). The weight of the powder is 1.5-1.8 g. Another tablet of the above form was prepared with a commercially available low fat natural cocoa powder (11% fat) instead of CP-cocoa solids (11% fat). Both tablet formulations produced products with acceptable flavor profiles and structural properties.

The analysis of both tablet formulations was performed using the method described in example 4, method 2. In this case, the analysis focused on the pentamer concentration and the total level of monomers and compounds of the invention reported below where n is 2 to 12.

Tablet sample	Pentamer (μ g/g)	In total (μ g/g)	Pentamer (μ g/1.8g sample)	In total (μ g/1.8g of one sample)
Tablet sample	Pentamer (μ g/g)	In total (μ g/g)	Pentamer (μ g/1.8g sample)	In total (μ g/1.8g of one sample)	CP-cocoa solids containing tablets	239	8,277	430	14,989
Tablet containing commercial low-fat cocoa powder	ND	868	ND	1563	CP-cocoa solids containing tablets	239	8,277	430	14,989

ND is not detected

The data clearly show higher pentamer levels and overall levels of the compounds of the invention for CP-cocoa solids tablets than for other tablet formulations. Thus, tablet formulations prepared with CP-cocoa solids are ideal delivery vehicles for oral administration of the compounds of the present invention for pharmaceutical, additive and food applications.

Other tablet formulations containing a wide range of flavors, colors, excipients, vitamins, minerals, OTC drugs, carbohydrate fillers, uv protectants (e.g., titanium dioxide, colorants, etc.), binders, hydrogels, etc. other than polyvinylpyrrolidone which will irreversibly bind the compound or mixture of compounds of the present invention can be readily prepared by one of ordinary skill in the art. The amount of sugar filler can be adjusted to control the dosage of the compound or mixture of compounds of the invention.

Many apparent variations to the above are possible and will be apparent without departing from the spirit and scope of the embodiments.

Example 39.Capsule preparation

A modification of example 38 for oral delivery of the compounds of the present invention was 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 the compound or compound mixture of the invention or CP-cocoa solids in powder form, as described in examples 38 and 40, optionally mixed with a filler such as lactose or sucrose to control the dosage of the compound of the invention. In soft capsules, the compound or mixture of compounds of the invention or CP-cocoa solids are suspended in a suitable liquid, such as fatty oil or cocoa butter or mixtures thereof. Since the compound or mixture of compounds of the invention may be sensitive to light, such as ultraviolet light, the capsules may contain an ultraviolet light protectant, such as titanium dioxide or a suitable colorant, to protect against ultraviolet light. The capsule may also contain fillers such as those mentioned in the previous embodiments.

Many apparent variations to the above would be apparent to those of ordinary skill in the art that could be made without departing from the spirit and scope of the embodiments.

Example 40.Identification Standard (SOI) and non-identification standard (non-SOI) dark chocolate And milk chocolate preparation

The compound or compound mixture formulations of the invention produced by the processes encompassed by the invention can be prepared as dark and milk chocolates for human and veterinary use. Reference is made to pending U.S. application serial No. 08/709,406, filed on 6.9.1996, which is hereby incorporated by reference. USSN 08/709,406 relates to a process for making cocoa butter and/or cocoa solids having a constant level of a compound of the invention from cocoa beans using a unique combination of processing steps. In short, the edible cocoa solids obtained from this process retain the natural presence of the compounds of the present invention to the levels they are found in traditionally processed cocoa. The ratio of the starting amount of the compound of the invention thus found in the end-processed beans to the amount obtained after processing is less than or equal to 2. For simplicity, the cocoa solids material is referred to herein as CP-cocoa solids. CP-cocoa solids are used in powder or solution form to make SOI and non-SOI chocolates, beverages, snacks, baked goods and as an ingredient in nutritional applications.

The term "SOI chocolate" as used herein refers to any chocolate used in the united states for food products according to federal food, drug, cosmetic regulations, by the standards of accreditation by the U.S. committee for food and drug administration. The us definitions and standards for various types of chocolate are well established. The term "non-SOI chocolate" as used herein refers to any non-standardized chocolate having a composition that falls outside the specified range of standardized chocolates.

When cocoa butter or milk fat is partially or fully substituted; or when the nutritive carbohydrate sweetener is partially or fully substituted; or the addition of flavors to simulate milk, butter, cocoa or chocolate, or other additions or deletions made to the formulation are outside of the U.S. FDA quality standards for chocolate or mixtures thereof.

As a blended product, chocolate may take the form of a solid piece of chocolate, such as a stick or novel shape, and may be incorporated as an ingredient in other more complex blended pharmaceutical agents, optionally with any of the Flavor and Extract Manufacturers Association (FEMA) materials, natural juices, condiments, herbs and materials classified as natural flavors; a substance of natural nature; and those listed by FEMA GRAS, FEMA and FDA, by the European Association (CoE), by FAO/WHO Food Standard Programme, by Codexbearing artisius, by the International organization for the fragrance industry (IOFI) and by Food chemical codex, and generally contain other foods such as caramel, nougat, bits of small fruit, nuts, cake pieces, and the like. These foods are characterized as being microbiologically shelf stable at 65-85 ° F under normal air conditions. Other complex mix-ins result from being surrounded by a soft chocolate wrapper, such as a cordial cherry or peanut butter. Other complex mix products are made from coating ice cream or other frozen or refrigerated snacks with chocolate. Generally, the chocolate used to coat or surround the food product must be more fluid than chocolate used for ordinary chocolate solid bars or fresh shapes.

Additionally, the chocolate may also be a low fat chocolate comprising fat and non-fat solids, with nutritive carbohydrate sweeteners and edible emulsifiers. For low fat chocolate, reference is made to U.S. Pat. nos. 4,810,516, 4,701,337, 5,464,649, 5,474,795 and WO 96/19923.

Dark chocolate its dark color comes from the chocolate solution, or the alkalized solution or cocoa solids or the amount of alkalized cocoa solids in any given formulation. However, due to example 27, table 13 teaches that the loss of the compounds of the present invention is due to the alkalization process, therefore, the use of alkalized cocoa solids or solutions would not be used in the dark chocolate products of the present invention.

Examples of SOI and non-SOI dark chocolate and milk chocolate products are shown in tables 16 and 17. In these preparations, the amount of the compounds of the invention present in the CP-cocoa solids is similar to the compounds of the invention present in commercially available cocoa solids.

The processing steps for preparing these chocolate products are described below.

Method for non-SOI black chocolate

1. All mixers and homogenizers involved in the whole process were protected from light.

2. All ingredients except 40% of the flowing fat (cocoa butter and dehydrated milk fat) were kept at a temperature between 30-35 ℃ in batches.

3. Grind to 20 μm.

Dried in a chocolate machine for 2 hours at 4.35 ℃.

5. All lecithin and 10% cocoa butter were added at the beginning of the wet cycle in the chocolate machine and moistened for 1 hour in the machine.

6. All remaining fat was added, calibrated if necessary, and mixed at 35 ℃ for 1 hour.

7. Tempering, pressing and packaging the chocolate.

Method for SOI black chocolate

1. All ingredients except milk fat were kept at a temperature of 60 ℃ in batches.

2. Grind to 20 μm.

Drying in a chocolate machine for 3.5 hours at 3.60 ℃.

4. Lecithin and milk fat were added and moistened in a chocolate machine at 60 ℃ for 1 hour.

5. Standardisation was carried out if necessary and mixing was carried out at 35 ℃ for 1 hour. Tempering, pressing and packaging the chocolate.

Method for non-SOI milk chocolate

At 2.75 ℃, the sugar, whole milk powder, maltose-added as flour and 66% cocoa butter were processed in portions for 2 hours in a chocolate machine.

3. Cooling to 35 deg.C, adding cocoa powder, ethyl vanillin, cocoa solution and 21% cocoa butter, and mixing at 35 deg.C for 20 min.

4. Grind to 20 μm.

5. The remaining cocoa butter was added and dried in a chocolate machine at 35 ℃ for 1.5 hours.

6. Adding dehydrated milk fat and lecithin, and moistening in chocolate machine at 35 deg.C for 1 hr.

7. Standardizing, tempering, pressing and packaging the chocolate.

Method for SOI milk chocolate

1. All ingredients except 65% cocoa butter and milk fat were kept at a temperature of 60 ℃ in batches.

2. Grind to 20 μm.

Dried in a chocolate machine for 3.5 hours at 3.60 ℃.

4. Adding lecithin, 10% cocoa butter and dehydrated milk fat; at 60 ℃ for 1 hour in a chocolate machine.

5. The remaining cocoa butter was added, standardized if necessary and mixed at 35 ℃ for 1 hour.

6. Tempering, pressing and packaging the chocolate.

Before incorporation into the product, the total level of pentamers as well as monomers and compounds of the invention wherein n is 2-12 in CP-cocoa solids and commercial chocolate solutions used in the product was analyzed as described in example 4, method 4. These values were then used to evaluate the expected levels of the various chocolate formulations as shown in tables 16 and 17. In the case of non-SOI dark chocolate and non-SOI milk chocolate, the products were similarly analyzed for the total level of pentamers as well as monomers and compounds of the invention wherein n is 2-12. The results are shown in tables 16 and 17.

The results from these preparation examples show that the SOI and non-SOI dark chocolate and milk chocolate formulated with CP-cocoa solids contained approximately more than 6.5 times the expected pentamer and more than 3.5 times the expected total level in the SOI and non-SOI dark chocolate, respectively; and about more than 4.5 in SOI and non-SOI milk chocolates; 7.0 times the expected pentamer and more than 2.5; 3.5 times the expected total level.

Since the expected levels of the compounds of the invention present in the final chocolate made with CP-cocoa solids were significantly higher than those formulations made with commercially available cocoa solids, some chocolate products were not analyzed. However, the processing effect was evaluated in non-SOI dark chocolate and milk chocolate products. As shown in the table, a loss of pentamer of 25-50% was produced, while only a small difference in total level was observed. Without wishing to be bound by any theory, it is believed that these losses are due to heating and/or short chain fatty acids from hydrolysable oligomers (e.g., trimers hydrolysable to monomers and dimers) of milk constituents (e.g., acetic acid, propionic acid, and butyric acid). In addition, time consuming processing steps may allow for oxidation of the compounds of the invention or irreversible binding to the protein source in the formulation. Thus, the present invention includes improved methods of chocolate products and processes for achieving these effects to prevent or minimize these losses.

The skilled artisan will appreciate that the examples cover a wide range of formulations, ingredients, processing and mixtures to rationally adjust the naturally occurring levels of the compounds of the invention for various chocolate applications.

TABLE 16 dark chocolate products prepared with non-alkalized cocoa ingredients

non-SOI dark chocolate using CP-cocoa solids	Soi dark chocolate using CP-cocoa solids	Soi dark chocolate using commercial cocoa solids
non-SOI dark chocolate using CP-cocoa solids	Soi dark chocolate using CP-cocoa solids	Soi dark chocolate using commercial cocoa solids	Preparation:	preparation:	preparation:
41.49% sugar 3% whole milk powder 26% CP-cocoa solids 4.5% Normal solution 21.75% cocoa butter 2.75% Anhydrous milk fat 0.01% vanillin 0.5% lecithin	41.49% sugar 3% whole milk powder 52.65% CP-solution 2.35% Anhydrous milk fat 0.01% Vanillin 0.5% lecithin	41.49% sugar 3% whole milk powder 52.65% normal solution 2.35% anhydrous milk fat 0.01% vanillin 0.5% lecithin	Preparation:	preparation:	preparation:
			Total fat: 31 percent of	Total fat: 31 percent of	Total fat: 31 percent of
Particle size: 20 micron	Particle size: 20 micron	Particle size: 20 micron	Total fat: 31 percent of	Total fat: 31 percent of	Total fat: 31 percent of

Expected levels of pentameric and total oligomeric procyanidins (monomer and n ═ 2-12 in μ g/g)

Pentamer: 1205	Pentamer: 1300	Pentamer: 185
Pentamer: 1205	Pentamer: 1300	Pentamer: 185	All of the following: 13748	All of the following: 14646	All of the following: 3948

Actual levels of pentameric and total oligomeric procyanidins (monomer and n ═ 2-12 in μ g/g)

Pentamer: 561	Is not carried out	Is not carried out
Pentamer: 561	Is not carried out	Is not carried out	All of the following: 14097

TABLE 17 milk chocolate products prepared with non-alkalized cocoa ingredients

non-SOI milk chocolate using CP-cocoa solids	SOI milk chocolate using CP-cocoa solids	SOI milk chocolate using commercial cocoa solids
non-SOI milk chocolate using CP-cocoa solids	SOI milk chocolate using CP-cocoa solids	SOI milk chocolate using commercial cocoa solids	Preparation:	preparation:	preparation:
46.9965% sugar 15.5% whole milk powder 4.5% CP-cocoa solids 5.5% plain solution 21.4% cocoa butter 1.6% anhydrous milk fat 0.035% vanillin 0.5% lecithin 4.0% maltose added milk powder	46.9965% sugar 15.5% whole milk powder 13.9% CP-solution 1.6% anhydrous milk fat 0.0035% vanillin 0.5% lecithin 17.5% cocoa butter 4.0% maltose added milk powder	46.9965% sugar 15.5% totalMilk powder 13.9% common solution 1.60% anhydrous milk fat 0.0035% vanillin 0.5% lecithin 17.5% cocoa butter 4.0% maltose added milk powder	Preparation:	preparation:	preparation:
			Total fat: 31.75 percent	Total fat: 31.75 percent	Total fat: 31.75 percent
Particle size: 20 micron	Particle size: 20 micron	Particle size: 20 micron	Total fat: 31.75 percent	Total fat: 31.75 percent	Total fat: 31.75 percent

Pentamer: 225	Pentamer: 343	Pentamer: 49
Pentamer: 225	Pentamer: 343	Pentamer: 49	All of the following: 2734	All of the following: 3867	All of the following: 1042

Actual levels of pentameric and total oligomeric procyanidins (monomeric and n ═ 2-12 in units of μ g/g)

Pentamer: 163	Is not carried out	Is not carried out
Pentamer: 163	Is not carried out	Is not carried out	All of the following: 2399

Example 41.Hydrolysis of procyanidin oligomers

Example 14 method D describes a preparative normal phase HPLC method for purifying a compound of the invention. The oligomers were obtained as fractions dissolved in the mobile phase. The solvent was then removed by standard vacuum distillation (20-29 inch high: 40 ℃) on a Rotovap setup. When the vacuum distillation residence time is extended or temperatures > 40 ℃ are used, loss of specific oligomers is observed to occur with an increase in smaller oligomers.

The loss of specific oligomers with the concomitant increase in smaller oligomers is due to time-temperature acid hydrolysis from the remaining acetic acid present in the mobile phase solvent mixture. This observation was confirmed by the following experiment in which 100mg of the hexamer was dissolved in 50mL of a mobile phase containing dichloromethane, acetic acid, water and methanol (see example 14, solvent part of method D) and subjected to time-temperature dependent distillation. At a specific time, an aliquot was removed for analytical normal phase HPLC analysis as described in example 4, method 2. The results are illustrated in figures 64 and 65, where hexamer levels are reduced in a time-temperature dependent manner. FIG. 65 illustrates a hydrolysis product (trimer) occurring in a time-temperature dependent manner. Monomers and other oligomers (dimers through pentamers) also occur in a time-temperature dependent manner.

These results indicate that great care and caution must be taken in handling the polymeric compounds of the present invention.

Together with the findings in examples 5, 15, 18, 19, 20 and 29, the above results demonstrate that the above methods can be used to demonstrate any of the oligomers given in the present invention in combination with other methods included in the present invention.

For example, complete hydrolysis of any given (+) -catechin-or (-) -epicatechin-producing oligomer eliminates the possibility of many "mixed" monomer-based oligomer structures and reduces the possibility of the characteristic stereochemical bonding of each of the monomers comprising any given oligomer.

In addition, the complete hydrolysis of any given oligomer that produces (+) -catechin and (-) -epicatechin in a particular ratio provides the skilled artisan with information regarding the monomer composition of any given oligomer and, thus, the likelihood of a characteristic stereochemical bond of each of the monomers comprising the oligomer.

It will be appreciated by those skilled in the art that acid catalyzed epimerization of the individual monomers may occur and that suitable control measures and hydrolysis without activation should be considered (e.g. the use of organic acids such as acetic acid instead of concentrated hydrochloric acid, nitric acid, etc.).

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

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Claims

1. Formula A_nFor the manufacture of a medicament, veterinary composition or food product for the treatment of a subject in need of a cyclooxygenase and/or lipoxygenase modulator, wherein a is a monomer having the following structural formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

the substituents for C-4, C-6 and C-8 are X, Z and Y, respectively, and the bonding of the monomer units occurs at the C-4, C-6 and C-8 positions;

x, Y and Z are hydrogen, or Z and Y are a saccharide and X is hydrogen, or X is a saccharide and Z and Y is H, or a combination thereof, when none of C-4, C-6 and C-8 are bonded to another monomer unit, with the proviso that Y and Z of a first monomer unit are hydrogen when a second monomer unit is bonded to C-4 of said first monomer unit;

wherein the sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety through an ester bond.

2. Formula A_nFor use in the manufacture of a medicament, veterinary composition or food product for treating a subject in need of a NO modulator, wherein a is a monomer having the following structural formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

R is OH or an O-sugar;

3. Formula A_nOr a derivative or oxidation product thereof for the manufacture of a medicament, a veterinary for the treatment of a subject in need of a NO synthase modulatorUse of a pharmaceutical composition or a foodstuff wherein a is a monomer of the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

when none of C-4, C-6 and C-8 are bonded to another monomer unit, X, Y and Z are hydrogen, or Z and Y are a sugar and X is hydrogen, or X is a sugar and Z and Y is H, or a combination thereof,

provided however that when the second monomer unit is bonded to C-4 of said first monomer unit, Y and Z of the first monomer unit are hydrogen;

4. Use according to claim 2 or 3, wherein nitric oxide synthesis is increased.

5. The use of claim 4, wherein nitric oxide synthesis is increased by induction of Inducible Nitric Oxide Synthase (iNOS) in a mammal.

6. The use according to any one of claims 2 to 5, wherein the subject suffers from NO-affected hypercholesterolemia.

7. The use of any one of claims 2-5, wherein the subject has arteriosclerosis, thrombosis, a heart attack, stroke, or a vascular circulation disorder.

8. Formula A_nThe polymeric compound or the derivative or the oxidation product thereof is used for manufacturing the polymer for use in the nursingUse of a medicament, veterinary composition or food for inducing iNOS in monocytes and/or macrophages of a dairy animal, wherein a is a monomer having the following structural formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

9. Formula A_nOr a derivative or oxidation product thereof for the manufacture of a medicament, veterinary composition or foodstuff for stimulating the production of NO by mammalian monocytes and/or macrophages, wherein a is a monomer of the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

10. Formula A_nFor the manufacture of a medicament, veterinary composition or food product for the treatment of a subject in need of treatment with a glucose modulator, wherein a is a monomer having the following structural formula:

Wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

11. Formula A_nFor use in the manufacture of a medicament, veterinary composition or food product for treating a subject in need of a nonsteroidal anti-inflammatory agent, wherein a is a monomer having the following structural formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

12. Use of a compound selected from the group consisting of a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer and decamer, wherein the trimer is selected from [ EC- (4 β → 8), or a derivative or oxidation product thereof, for the manufacture of a medicament, veterinary composition or food product for the treatment of a mammal in need of gingivitis treatment]₂-EC、[EC-(4β→8)]₂-C and [ EC- (4. beta. → 6)]₂-EC, tetramer selected from [ EC- (4. beta. → 8)]₃-EC、[EC-(4β→8)]₃-C and [ EC- (4. beta. → 8)]₂-EC- (4 β → 6) -C, pentamer selected from [ EC- (4 β → 8)]₄-EC、[EC-(4β→8)]₃-EC-(4β→6)-EC、[EC-(4β→8)]₃-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₃-EC- (4 β → 6) -C, hexamer selected from [ EC- (4 β → 8)]₅-EC、[EC-(4β→8)]₄-EC-(4β→6)-EC、[EC-(4β→8)]₄-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₄-EC- (4 β → 6) -C, heptamer selected from [ EC- (4 β → 8)]₆-EC、[EC-(4β→8)]₅-EC-(4β→6)-EC、[EC-(4β→8)]₅-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₅-EC- (4 β → 6) -C, octamer selected from [ EC- (4 β → 8)]₇-EC、[EC-(4β→8)]₆-EC-(4β→6)-EC、[EC-(4β→8)]₆-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₆-EC- (4 β → 6) -C, nonamer selected from [ EC- (4 β → 8)]₈-EC、[EC-(4β→8)]₇-EC-(4β→6)-EC、[EC-(4β→8)]₇-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₇-EC- (4 β → 6) -C, the decamer being selected from [ EC- (4 β → 8)]₉-EC、[EC-(4β→8)]₈-EC-(4β→6)-EC、[EC-(4β→8)]₈-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₈-EC- (4 β → 6) -C; wherein "EC" represents an epicatechin moiety and "C" represents a catechin moiety.

13. Use of a compound selected from the group consisting of a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer and decamer, wherein the trimer is selected from [ EC- (4 β → 8), or a derivative or oxidation product thereof, for the manufacture of a medicament, veterinary composition or foodstuff for reducing the development of inflammatory periodontal disease in a mammal ]₂-EC、[EC-(4β→8)]₂-C and [ EC- (4. beta. → 6)]₂-EC, tetramer selected from [ EC- (4. beta. → 8)]₃-EC、[EC-(4β→8)]₃-C and [ EC- (4. beta. → 8)]₂-EC- (4 β → 6) -C, pentamer selected from [ EC- (4 β → 8)]₄-EC、[EC-(4β→8)]₃-EC-(4β→6)-EC、[EC-(4β→8)]₃-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₃-EC- (4 β → 6) -C, hexamer selected from [ EC- (4 β → 8)]₅-EC、[EC-(4β→8)]₄-EC-(4β→6)-EC、[EC-(4β→8)]₄-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₄-EC- (4 β → 6) -C, heptamer selected from [ EC- (4 β → 8)]₆-EC、[EC-(4β→8)]₅-EC-(4β→6)-EC、[EC-(4β→8)]₅-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₅-EC- (4 β → 6) -C, octamer selected from [ EC- (4 β → 8)]₇-EC、[EC-(4β→8)]₆-EC-(4β→6)-EC、[EC-(4β→8)]₆-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₆-EC- (4 β → 6) -C, nonamer selected from [ EC- (4 β → 8)]₈-EC、[EC-(4β→8)]₇-EC-(4β→6)-EC、[EC-(4β→8)]₇-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₇-EC- (4 β → 6) -C, the decamer being selected from [ EC- (4 β → 8)]₉-EC、[EC-(4β→8)]₈-EC-(4β→6)-EC、[EC-(4β→8)]₈-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₈-EC- (4 β → 6) -C; wherein "EC" represents a tableCatechin moiety, "C" represents a catechin moiety.

14. Use of a compound selected from the group consisting of a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer and decamer, wherein the trimer is selected from [ EC- (4 β → 8), or a derivative or oxidation product thereof, for the manufacture of a medicament, veterinary composition or food product for the treatment of inflammatory periodontal disease]₂-EC、[EC-(4β→8)]₂-C and [ EC- (4. beta. → 6)]₂-EC, tetramer selected from [ EC- (4. beta. → 8)]₃-EC、[EC-(4β→8)]₃-C and [ EC- (4. beta. → 8)]₂-EC- (4 β → 6) -C, pentamer selected from [ EC- (4 β → 8) ]₄-EC、[EC-(4β→8)]₃-EC-(4β→6)-EC、[EC-(4β→8)]₃-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₃-EC- (4 β → 6) -C, hexamer selected from [ EC- (4 β → 8)]₅-EC、[EC-(4β→8)]₄-EC-(4β→6)-EC、[EC-(4β→8)]₄-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₄-EC- (4 β → 6) -C, heptamer selected from [ EC- (4 β → 8)]₆-EC、[EC-(4β→8)]₅-EC-(4β→6)-EC、[EC-(4β→8)]₅-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₅-EC- (4 β → 6) -C, octamer selected from [ EC- (4 β → 8)]₇-EC、[EC-(4β→8)]₆-EC-(4β→6)-EC、[EC-(4β→8)]₆-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₆-EC- (4 β → 6) -C, nonamer selected from [ EC- (4 β → 8)]₈-EC、[EC-(4β→8)]₇-EC-(4β→6)-EC、[EC-(4β→8)]₇-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₇-EC- (4 β → 6) -C, the decamer being selected from [ EC- (4 β → 8)]₉-EC、[EC-(4β→8)]₈-EC-(4β→6)-EC、[EC-(4β→8)]₈-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₈-EC- (4 β → 6) -C; wherein "EC" represents an epicatechin moiety and "C" represents a catechin moiety.

15. Formula A_nOr a derivative or oxidation product thereof for the manufacture of a medicament, veterinary composition or food for modulating platelet aggregation in a mammal, wherein A is a compound having the formulaA monomer of the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

16. Formula A_nFor the manufacture of a medicament, veterinary composition or food product for modulating apoptosis, wherein a is a monomer having the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

17. Formula A_nThe use of a polymeric compound of (a) or a derivative or oxidation product thereof in the manufacture of a medicament, veterinary composition or foodstuff for inhibiting oxidative damage to DNA, wherein a is a monomer of the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

R and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

18. Formula A_nFor the manufacture of a medicament, veterinary composition or foodstuff for the treatment, prevention or reduction of atherosclerosis or restenosis in a mammal, wherein a is a monomer of the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

19. Formula A_nFor use in the manufacture of a medicament, veterinary composition or food product for inhibiting the oxidation of LDL in a mammal, wherein a is a monomer having the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

20. Formula A_nOr a derivative or oxidation product thereof for the manufacture of a medicament, veterinary composition or foodstuff for modulating thrombosis in a mammal, wherein a is a monomer of the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

R and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

21. Formula A_nFor the manufacture of a medicament, veterinary composition or food for the modulation of cyclooxygenase-1 (COX-1) for the treatment of inflammatory bowel disease,

wherein A is a monomer having the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

22. Formula A_nFor use in the manufacture of a medicament, veterinary composition or foodstuff for inhibiting bacterial growth in a mammal, wherein a is a monomer having the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

23. Formula A_nFor use in the manufacture of a medicament, veterinary composition or food product for reducing hypertension in a mammal, wherein a is a monomer having the formula:

wherein the content of the first and second substances,

n is a positive integer of 2-12;

R and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

24. Formula A_nOr a derivative or oxidation product thereof for the manufacture of a medicament, veterinary composition or foodstuff for modulating kidney function in a mammal, wherein a is a monomer of the formula:

wherein the content of the first and second substances,

n is a positive integer of 2-12;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

25. Identification pass-type A_nWherein a is a monomer having the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

wherein the sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety through an ester bond;

the method comprises contacting the at least one gene or gene product with a polymeric compound to perform a gene expression assay.

26. The use according to any one of claims 1 to 24 or the method according to claim 25, wherein n is 5 to 12.

27. The use according to any one of claims 1 to 24 or the method according to claim 25, wherein n is 5.

28. The use according to any one of claims 1 to 24 or the method according to claim 25, wherein the sugar is selected from the group consisting of glucose, galactose, xylose, rhamnose and arabinose.

29. The use according to any one of claims 1 to 24 or the method according to claim 25, wherein the phenolic moiety is selected from the group consisting of caffeic, cinnamic, coumaric, succinic, hydroxybenzoic and sinapic acids.

30. The use according to any one of claims 1 to 24 or the method according to claim 25, wherein in formula a_nIn which bonding occurs between C-4 and C-6 of adjacent monomers or between C-4 and C-8 of adjacent monomers; and

x, Y and Z are each hydrogen, a sugar or an adjacent monomer, with the proviso that if X and Y are adjacent monomers then Z is H or a sugar, if X and Z are adjacent monomers then Y is H and a sugar, and with the proviso that for at least one of the two terminal monomers the bond of the adjacent monomer is at C-4 and optionally Y ═ Z ═ hydrogen.

31. The use according to any one of claims 1-11 or 15-24 or the method according to claim 25, wherein the compound is selected from the group consisting of a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer and decamer, wherein the trimer is selected from [ EC- (4 β → 8) ]₂-EC、[EC-(4β→8)]₂-C and [ EC- (4. beta. → 6)]₂-EC, tetramer selected from [ EC- (4. beta. → 8)]₃-EC、[EC-(4β→8)]₃-C and [ EC- (4. beta. → 8)]₂-EC- (4 β → 6) -C, pentamer selected from [ EC- (4 β → 8)]₄-EC、[EC-(4β→8)]₃-EC-(4β→6)-EC、[EC-(4β→8)]₃-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₃-EC- (4 β → 6) -C, hexamer selected from [ EC- (4 β → 8)]₅-EC、[EC-(4β→8)]₄-EC-(4β→6)-EC、[EC-(4β→8)]₄-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₄-EC- (4 β → 6) -C, heptamer selected from [ EC- (4 β → 8)]₆-EC、[EC-(4β→8)]₅-EC-(4β→6)-EC、[EC-(4β→8)]₅-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₅-EC- (4 β → 6) -C, octamer selected from [ EC- (4 β → 8)]₇-EC、[EC-(4β→8)]₆-EC-(4β→6)-EC、[EC-(4β→8)]₆-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₆-EC- (4 β → 6) -C, nonamer selected from [ EC- (4 β → 8)]₈-EC、[EC-(4β→8)]₇-EC-(4β→6)-EC、[EC-(4β→8)]₇-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₇-EC- (4 β → 6) -C, the decamer being selected from [ EC- (4 β → 8)]₉-EC、[EC-(4β→8)]₈-EC-(4β→6)-EC、[EC-(4β→8)]₈-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₈-EC- (4 β → 6) -C; wherein "EC" represents an epicatechin moiety and "C" represents a catechin moiety.

32. The use according to any one of claims 1 to 24, wherein the pharmaceutical, veterinary composition or foodstuff is selected from a liquid, a suspension, a polymeric sustained release system, a tablet, a capsule, a unit standard chocolate or a non-unit standard chocolate.

33. The use according to any one of claims 1 to 24 or the method according to claim 25, wherein said polymeric compound is substantially a pure compound from the genus theobroma or Herrania or an interspecies or intraspecies hybrid thereof.

34. The use according to any one of claims 1 to 24 or the method according to claim 25, wherein R of the terminal monomer unit of the polymeric compound is O- β -D-glucose or O-R ', wherein R' is a succinic moiety.

35. The use of claim 1, wherein the medicament, veterinary composition or food further comprises at least one additional cyclooxygenase and/or lipoxygenase modulator.

36. The use according to any one of claims 2 to 7, wherein the medicament, veterinary composition or food product further comprises at least one additional NO or NO synthase modulator.

37. The use of claim 23, wherein the medicament, veterinary composition or food further comprises at least one hypertension-lowering agent.

38. The use of claim 10, wherein the medicament, veterinary composition or food further comprises at least one additional glucose modulator.

39. The use of claim 11, wherein the medicament, veterinary composition or food further comprises at least one additional non-steroidal anti-inflammatory agent.

40. The method of claim 25, wherein said gene expression assay is selected from the group consisting of differential display, sequencing of cDNA libraries, serial analysis of gene expression, and expression monitoring by hybridization to high density oligonucleotide arrays.

41. Chocolate-containing chocolate composition comprising an effective antioxidant amount of formula A_nWherein a is a monomer having the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

42. The chocolate composition of claim 41, wherein n is 5 to 12.

43. The chocolate composition of claim 41, wherein n is 5.

44. The chocolate composition of any one of claims 41-43, wherein the sugar is selected from the group consisting of glucose, galactose, xylose, rhamnose and arabinose.

45. The chocolate composition of any one of claims 41-44, wherein the phenolic moiety is selected from the group consisting of caffeic, cinnamic, coumaric, ferulic, hydroxybenzoic and sinapic acids.

46. The chocolate composition of any of claims 41-45, wherein in formula A_nIn which bonding occurs between C-4 and C-6 of adjacent monomers or between C-4 and C-8 of adjacent monomers; and X, Y and Z are each hydrogen, a sugar or an adjacent monomer, with the proviso that if X and Y are adjacent monomers then Z is H or a sugar, if X and Z are adjacent monomers then Y is H and a sugar, and with the proviso that for at least one of the two terminal monomers the bond of the adjacent monomer is to C-4 and optionally Y ═ Z ═ hydrogen.

47. The chocolate composition of any of claims 41-46, wherein the polymeric compound is selected from the group consisting of a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, and decamer, wherein the trimer is selected from [ EC- (4 β → 8)]₂-EC、[EC-(4β→8)]₂-C and [ EC- (4. beta. → 6)]₂-EC, tetramer selected from [ EC- (4. beta. → 8)]₃-EC、[EC-(4β→8)]₃-C and [ EC- (4. beta. → 8)]₂-EC- (4 β → 6) -C, pentamer selected from [ EC- (4 β → 8)]₄-EC、[EC-(4β→8)]₃-EC-(4β→6)-EC、[EC-(4β→8)]₃-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₃-EC- (4 β → 6) -C, hexamer selected from [ EC- (4 β → 8)]₅-EC、[EC-(4β→8)]₄-EC-(4β→6)-EC、[EC-(4β→8)]₄-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₄-EC- (4 β → 6) -C, heptamer selected from [ EC- (4 β → 8)]₆-EC、[EC-(4β→8)]₅-EC-(4β→6)-EC、[EC-(4β→8)]₅-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₅-EC- (4 β → 6) -C, octamer selected from [ EC- (4 β → 8) ]₇-EC、[EC-(4β→8)]₆-EC-(4β→6)-EC、[EC-(4β→8)]₆-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₆-EC- (4 β → 6) -C, nonamer selected from [ EC- (4 β → 8)]₈-EC、[EC-(4β→8)]₇-EC-(4β→6)-EC、[EC-(4β→8)]₇-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₇-EC- (4 β → 6) -C, the decamer being selected from [ EC- (4 β → 8)]₉-EC、[EC-(4β→8)]₈-EC-(4β→6)-EC、[EC-(4β→8)]₈-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₈-EC- (4 β → 6) -C; wherein "EC" represents an epicatechin moiety and "C" represents a catechin moiety.

48. The chocolate composition of any one of claims 41-47, having a form selected from the group consisting of a liquid, a suspension, a polymer sustained release system, a tablet, a capsule, a unit standard chocolate, or a non-unit standard chocolate.

49. The chocolate composition of any one of claims 41 to 48, wherein said polymeric compound is substantially a pure compound from the genus Theobroma or Herrania or an interspecies or intraspecies hybrid thereof.

50. The chocolate composition of any one of claims 41-48, wherein R of the terminal monomer unit of the polymeric compound is O- β -D-glucose or O-R ', wherein R' is a succinic moiety.

51. A kit comprising, in admixture or in separate containers:

(a) formula A_nWherein a is a monomer having the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

and a pharmaceutically, veterinarily or food science acceptable carrier; and

(b) an additional agent selected from the group consisting of: NO or NO synthase modulators, cyclooxygenase and/or lipoxygenase modulators, non-steroidal anti-inflammatory agents, platelet aggregation modulators, and blood or in vivo glucose modulators.

52. A kit comprising, in admixture or in separate containers:

(a) formula A_nWherein a is a monomer having the formula:

wherein the content of the first and second substances,

n is a positive integer of 2 to 18;

r and X each have an α or β stereochemical configuration;

r is OH or an O-sugar;

and a pharmaceutically, veterinarily or food science acceptable carrier; and

(b) an additional agent that reduces or alleviates the adverse effects of the agent selected from the group consisting of: NO or NO synthase modulators, cyclooxygenase and/or lipoxygenase modulators, non-steroidal anti-inflammatory agents, platelet aggregation modulators, and blood or in vivo glucose modulators.

53. A compound selected from the group consisting of pentamer, hexamer, heptamer, octamer, nonamer, decamer, undecammer, and dodecamer, wherein the pentamer is selected from [ EC- (4 β → 8)]₄-EC、[EC-(4β→8)]₃-EC-(4β→6)-EC、[EC-(4β→8)]₃-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₃-EC- (4 β → 6) -C, hexamer selected from [ EC- (4 β → 8)]₅-EC、[EC-(4β→8)]₄-EC-(4β→6)-EC、[EC-(4β→8)]₄-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₄-EC- (4 β → 6) -C, heptamer selected from [ EC- (4 β → 8)]₆-EC、[EC-(4β→8)]₅-EC-(4β→6)-EC、[EC-(4β→8)]₅-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₅-EC- (4 β → 6) -C, octamer selected from [ EC- (4 β → 8)]₇-EC、[EC-(4β→8)]₆-EC-(4β→6)-EC、[EC-(4β→8)]₆-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₆-EC- (4 β → 6) -C, nonamer selected from [ EC- (4 β → 8) ]₈-EC、[EC-(4β→8)]₇-EC-(4β→6)-EC、[EC-(4β→8)]₇-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₇-EC- (4 β → 6) -C, the decamer being selected from [ EC- (4 β → 8)]₉-EC、[EC-(4β→8)]₈-EC-(4β→6)-EC、[EC-(4β→8)]₈-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₈-EC- (4 β → 6) -C, the undecapolymer being selected from [ EC- (4 β → 8)]₁₀-EC、[EC-(4β→8)]₉-EC-(4β→6)-EC、[EC-(4β→8)]₉-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₉-EC- (4 β → 6) -C, the dodecamer being selected from [ EC- (4 β → 8)]₁₁-EC、[EC-(4β→8)]₁₀-EC-(4β→6)-EC、[EC-(4β→8)]₁₀-EC- (4 β → 8) -C and [ EC- (4 β → 8)]₁₀-EC- (4 β → 6) -C; wherein "EC" represents an epicatechin moiety and "C" represents a catechin moiety; wherein the 3-position terminal monomeric units of the pentamer, hexamer, heptamer, octamer, nonamer, decamer, undecamer and dodecamer are derived from a trimer acid ester or beta-D-glucose.