KR100773647B1 - Cocoa extract compounds and methods for making and using the same - Google Patents

Abstract

The present invention relates to cocoa extracts, compounds, combinations thereof and compositions thereof, such as polyphenols or procyanidins, in particular polymeric compounds of the formula A _n and pharmaceutically acceptable salts or derivatives of A _n (including oxides), said extracts, Disclosed and claimed are methods of using the compounds and compositions as well as methods of their preparation, wherein A is a monomer having the formula:

Wherein n is an integer from 2 to 18 such that at least one terminal monomer unit A and a plurality of additional monomer units can be present;

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

Bonding between adjacent monomers at the 4, 6 or 8 positions;

Position 4 is an alpha or beta stereoisomer;

X, Y and Z are selected from the group consisting of monomers A, hydrogen and sugars, except that at least one terminal monomer further comprises monomer units bonded at 4 positions and Y = Z = hydrogen;

Sugars are optionally substituted at any site, eg with phenolic moieties, via ester bonds.

Description

Cocoa Extract Compounds, Method of Preparation and Use thereof {COCOA EXTRACT COMPOUNDS AND METHODS FOR MAKING AND USING THE SAME}

The present invention relates to a compound obtained from cocoa extracts such as cocoa extract and polyphenols rich in polyphenol specialty procyanidins. The present invention also provides methods for preparing and using the extracts and compounds and for example antitumor agents, antioxidants, DNA topoisomerase II inhibitors, cyclo-oxygenase and / or lipoxygenase modulators, NO (nitric oxide) or NO Synthase modulators, non-steroidal anti-inflammatory agents, apoptosis modulators, platelet aggregation modulators, blood or bioglucose modulators, antibacterial agents and inhibitors of oxidative DNA damage

References herein are then cited where necessary, or cited at the end of the detailed description. These documents relate to the field of the invention; Each document cited herein is incorporated herein by reference.

Polyphenols are a very diverse group of compounds present in a wide range of plants, some of which are linked to the food chain (Ferraira et al., 1992). In some cases they are an important class of compounds in food. Although some of the polyphenols are recognized as nonnutrative, there is a growing interest in these compounds because they can have a beneficial effect on health.

For example, quercetin (a type of flavonoid) has been shown to have antitumor activity in animal experiments (Dashner et al., 1991; Kato et al., 1983). (+)-Catechin and (-)-Epicatechin (Flaban-3-ol) have been shown to inhibit the reverse transcriptase action of leukemia virus (Chu et al., 1992). Novotannin (a type of hydrolyzable tannin oligomer) has antitumor activity (Okuda et al., 1992). According to statistical reports, deaths from gastric cancer are significantly lower in Japanese green tea production sites. Epigallocatechin gallate has been reported to be a pharmacological substance in green tea that inhibits skin tumors in mice (Okuda et al., 1992). Elazic acid has shown anticancer activity in several animal tumor models (Bukata et al., 1992). In addition, Kikoman's invention using a proanthocyanidin oligomer as an anti-mutant has recently been patented. The area of phenolic compounds in food and the regulation of tumor development of phenolic compounds in experimental animal models were recently published at the 20th American Chemical Society (Ho et al., 1992; Hwang et al., 1992).

However, any prior literature does not disclose cocoa extracts or compounds from cocoa extracts, methods for their preparation, such as antitumor agents, antioxidants, DNA topoisomerase II inhibitors, cyclo-oxygenase and / or lipoxygenase modulators, NO Look for any teaching or implications for use as (nitric oxide) or NO-synthase modulators, non-steroidal anti-inflammatory agents, apoptosis modulators, platelet aggregation modulators, blood or bioglucose modulators, antimicrobials and oxidative DNA damage inhibitors. Can't.

Summary and purpose of the invention

Since the non-fermented cocoa beans contain a significant amount of polyphenols, the inventors have found that, like the compounds contained in cocoa by extracting these compounds from cocoa and screening for activity, the cocoa extracts will exhibit similar activity and may be used as a cocoa extract. I thought. The National Cancer Institute (NCI ) screened several Theobroma and Hernia species that have anticancer activity as part of a large natural selection program. There were low levels of activity in some cocoa tissue extracts, and the study was no longer performed. Thus, cocoa and its extracts were not considered useful in the field of antitumor or anticancer; In other words, the prior art in the field of antitumor or anticancer teaches researchers in this field not to apply cocoa and its extracts in cancer therapy.

Since a number of analytical methods have been developed to study the contribution of cocoa polyphenols in the field of flavor development (Clapperton et al., 1992), the inventors have found that anti-cancer screening, unlike the NCI screening method, is known in the art of anti-tumor or anti-cancer. In order to apply the similar method in the field of flavoring. As a result, the present inventors found that cocoa polyphenol extracts containing procyanidins are useful as anticancer agents, antineoplastic agents or antitumor agents.

Furthermore, the present inventors have found that procyanidin-containing cocoa extracts and the compounds obtained therefrom are antitumor agents, antioxidants, DNA topoisomerase II inhibitors, cyclo-oxygenase and / or lipooxygenase modulators, NO (nitrogen oxides) or NO Synthetase modulators, non-steroidal anti-inflammatory agents, apoptosis modulators, platelet aggregation modulators, blood or bioglucose modulators, antibacterial agents and inhibitors of oxidative DNA damage.

It is an object of the present invention to provide a process for preparing cocoa extract and / or a compound therefrom.

Another object of the present invention is to provide cocoa extracts and / or compounds therefrom.

Of the present invention, another object is to provide a monomer the polymer compound of formula A _n A _n and a pharmaceutically acceptable salt or derivative capable of A (including the oxide) with the expression:

Wherein n is an integer from 2 to 18 such that at least one terminal monomer unit A and a plurality of additional monomer units can be present;

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

Bonding between adjacent monomers at the 4, 6 or 8 positions;

X, Y and Z are selected from the group consisting of monomers A, hydrogen and sugars, except that at least one terminal monomer further comprises monomer units bonded at 4 positions and Y = Z = hydrogen;

Sugars are optionally substituted at any site, for example with phenolic moieties, via ester bonds.

Of the present invention, another object is to provide a monomer the polymer compound of formula A _n A _n and a pharmaceutically acceptable salt or derivative capable of A (including the oxide) with the expression:

Wherein n is an integer from 2 to 18, for example from 3 to 18;

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

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

X, Y and Z are selected from the group consisting of monomers A, hydrogen and sugar, provided that Z is H or sugar if X and Y are adjacent monomers, Y is H or sugar if X and Z are adjacent monomers The monomer unit added to at least one of the monomers is bonded at the 4 position, optionally Y = Z = hydrogen;

Sugars are optionally substituted at any site, eg with phenolic moieties, via ester bonds.

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

Another object of the invention is to demonstrate the inhibition of DNA topoisomerase II activity.

Another object of the present invention is to provide a method for treating a tumor or cancer.

Another object of the present invention is to provide an anticancer, antitumor, antineoplastic composition.

Another object of the present invention is to provide an antimicrobial composition.

Another object of the present invention is to provide a cyclo-oxygenase and / or lipoxygenase modulator composition.

It is another object of the present invention to provide a NO (nitric oxide) or NO-synthase modulator composition.

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

Another object of the present invention is to provide a blood or bioglucose modulator composition.

Another object of the present invention is to provide an antitumor, antioxidant, antibacterial.

Treatment of patients with antitumor, antioxidant, cyclo-oxygenase and / or lipoxygenase modulators or NO (nitrous oxide) or NO-synthase modulators, non-steroidal anti-inflammatory agents and / or blood or bioglucose modulator compositions To provide a way.

It is another object of the present invention to provide compositions and methods for inhibiting oxidative DNA damage.

It is another object of the present invention to provide compositions and methods for controlling platelet aggregation.

It is another object of the present invention to provide compositions and methods for modulating apoptosis.

Another object of the present invention is to provide a method for preparing the above-mentioned compositions.

Another object of the present invention is to provide a kit for use in the methods or for the preparation of the composition.

Surprisingly, cocoa extracts and compounds therefrom may be antineoplastic, antitumor or anticancer, antioxidant composition, inhibit the action of DNA topoisomerase II, antibacterial agents, cyclo-oxygenase and / or lipoxygenase modulators, NO or NO-syntha It has been found to act as agent modulators, non-steroidal anti-inflammatory agents, apoptosis modulators, platelet aggregation modulators, blood or bioglucose modulators and oxidative DNA damage inhibitors.

Accordingly, the present invention provides substantially pure cocoa extracts and compounds therefrom. The extract or compound will consist of cocoa procyanidin-rich polyphenols such as polyphenols of at least one cocoa procyanidin selected from: (-) Epicatechin, (+) catechin, procyanidin B-2, procyanidin 2 to 18 oligomers, such as 3 to 18 oligomers, procyanidin 2 to 12 or 3 to 12 oligomers, preferably 3 to 12 oligomers, most preferably Are 2 to 5 or 4 or 12 oligomers, procyanidin B-5, procyanidin A-2 and procyanidin C-1.

The present invention also relates to synthetic cocoa polys such as those consisting of substantially pure cocoa extracts, compounds therefrom, or polyphenols rich in procyanidins and suitable carriers (e.g., pharmaceutically, veterinary or foodologically acceptable carriers). Anti-tumor, antioxidant, DNA topoisomerase II inhibitors containing phenols, cyclo-oxygenase and / or lipoxygenase modulators, NO (nitric oxide) or NO-synthase modulators, non-steroidal anti-inflammatory agents, apoptosis Modulators, platelet aggregation modulators, blood or bioglucose modulators, antibacterial agents and inhibitors of oxidative DNA damage. The extract or compound therefrom may comprise cocoa procyanidins. The extract or a compound therefrom may be prepared by powdering and degreasing cocoa beans and then extracting and purifying the active compound from the powder.

The invention also relates to antitumor agents, antioxidants, DNA topoisomerase II inhibitors, cyclo-oxygenase and / or lipoxygenase modulators, NO (nitric oxide) or NO-synthase modulators, non-steroidal anti-inflammatory agents, apoptosis Polyphenols rich in substantially pure cocoa extracts, compounds therefrom, or procyanidins, and suitable carriers for patients who need to be prescribed as modulators, platelet aggregation modulators, blood or bioglucose modulators, antibacterial agents and inhibitors of oxidative DNA damage. For example, there is provided a therapeutic method consisting of administering a composition containing an effective amount of synthetic cocoa polyphenols, such as consisting of pharmaceutically, veterinary or food acceptable carrier). The cocoa extract or a compound therefrom may be cocoa procyanidins; Cocoa beans can be prepared by powdering and degreasing followed by extracting and purifying the active compound from the powder.

The present invention also provides synthetic cocoa polys such as those consisting of substantially pure cocoa extracts, compounds therefrom, or polyphenols rich in procyanidins and suitable carriers (e.g., pharmaceutically, veterinary or foodologically acceptable carriers). Anti-tumor, antioxidant, DNA topoisomerase II inhibitors containing phenols, cyclo-oxygenase and / or lipoxygenase modulators, NO (nitric oxide) or NO-synthase modulators, non-steroidal anti-inflammatory agents, apoptosis Kits for the treatment of patients who need to be prescribed with modulators, platelet aggregation modulators, blood or bioglucose modulators, antibacterial agents and oxidative DNA damage inhibitors and the like are provided.

The present invention provides the compounds shown in FIGS. 38A-38P and 39A-39AA; 4 → 6 and 4 → 8 bonds are preferred.

The present invention includes a food preservation or manufacturing composition comprising the compound of the present invention and a method for producing or preserving food by adding the composition to food.

In addition, the present invention provides a DNA topoisomerase II inhibitor composed of the compound of the present invention and a suitable carrier or diluent, and a method for treating a patient administering the composition (inhibitor).

While widely contemplating embodiments comprising the aforementioned cocoa extract, the present invention also encompasses embodiments in which the compounds of the present invention are used in place of or as cocoa extracts. Accordingly, the present invention also encompasses kits, methods and compositions similar to those described above with regard to cocoa extract and compounds of the present invention.

These objects and embodiments of the invention are disclosed or will become apparent from the following detailed description.

The following detailed description will be understood in more detail with reference to the accompanying drawings.

1 is a representative gel permeation chromatogram of crude cocoa procyanidins fractions;

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

FIG. 2B is a representative normal HPLC chromatogram showing separation of cocoa procyanidins extracted from unfermented cocoa; FIG.

3 is a representative procyanidin structure;

4A-4E are representative HPLC chromatograms of five fractions used for the screening of anticancer or antineoplastic actions;

5 and 6A-6D are dose-response correlation charts (dose fraction versus viable fraction; μg / mL) of cocoa extract and cancer cells ACHN (FIG. 5) and PC-3 (FIGS. 6A-6D); M & M2 F4 / 92, M & MA + E U12P1, M & MB + E Y192P1, M & MC + E U12P2, M & MD + E U12P2;

7A-7H are typical dose-response correlation charts of cocoa procyanidin A, B, C, D, E, A + B, A + E and A + D fractions with PC-3 cell lines (administration versus fraction survival). Amount, μg / mL); MM-1 A 0212P3, MM-1 B 0162P1, MM-1 C 0122P3, MM-1 D 0122P3, MM-1 E 0292P8, MM-1 A / B 0292P6, MM-1 A / E 0292P6, MM-1 A / D 0292P6;

8A-8H are typical dose-response correlation charts of cocoa procyanidin A, B, C, D, E, A + B, B + E, and D + E fractions with KB nasoparingill / Hella cell lines (fraction survival Dose to water; μg / mL); MM-1 A 092K2, MM-1 B 0212K5, MM-1 C 0162K3, MM-1 D 0212K5, MM-1 E 0292K5, MM-1 A / B 0292K3, MM-1 B / E 0292K4, MM-1 D / E 0292K5;

9A-9H are typical dose-response correlation charts of cocoa procyanidin A, B, C, D, E, B + D, A + E and D + E fractions with HCT-116 cell line (fraction survival versus dose) Amount, μg / mL); MM-1 C 0192H5, MM-1 D 0192H5, MM-1 E 0192H5, MM-1 B & D 0262H2, MM-1 A / E 0262H3, MM-1 D & E 0262H1;

10A-10H are typical dose-response correlation charts of cocoa procyanidin A, B, C, D, E, C + D, D + E and A + E fractions with ACHN kidney cell lines (dose versus fraction viability). Μg / mL); MM-1 A 092 A5, MM-1 B 092 A5, MM-1 C 0192 A7, MM-1 D 0192 A7, MM-1 E 0192 A7, MM-1 B & D 0302 A6, MM-1 C & D 0302 A6, MM-1 A & E 0262 A6;

11A-11H are typical dose-response correlation charts of cocoa procyanidin A, B, C, D, E, A + E, B + E and C + E fractions with A549 lung cell lines (dose versus fraction viability). Μg / mL); MM-1 A 019258, MM-1 B 09256, MM-1 C 019259, MM-1 D 019258, MM-1 E 019258, MM-1 A / E 026254, MM-1 B & E 030255, MM-1 C & E N6255;

12A-12H are typical dose-response correlation charts of cocoa procyanidin A, B, C, D, E, B + C, C + D and D + E fractions with SK-5 melanoma cell lines (fraction survival numbers). Relative dose; μg / mL); MM-1 A 0212S4, MM-1 B 0212S4, MM-1 C 0212S4, MM-1 D 0212S4, MM-1 E N32S1, MM-1 B & C N32S2, MM-1 C & D N32S3, MM-1 D & E N32S3;

13A-13H are typical dose-response correlation charts of cocoa procyanidin A, B, C, D, E, B + C, C + E, and D + E fractions with MCF-7 breast cell lines (fraction viability versus Dosage; μg / mL); MM-1 A N22M4, MM-1 B N22M4, MM-1 C N22M4, MM-1 D N22M3, MM-1 E 0302M2, MM-1 B / C 0302M4, MM-1 C & E N22M3, MM-1 D & E N22M3;

FIG. 14 shows a typical dose-response correlation chart of cocoa procyanidins (particularly fraction C) with CCRF-CEM T-vertical leukemia cell line (proliferation days per cell / mL; empty circles are control, black circles are fraction D 125 μg, Empty inverted triangle of fraction D 250 μg, black inverted triangle of fraction D 500 μg);

FIG. 15A is a comparison of cytotoxicity experiments of XTT and crystal violet on fraction D + E treated MCF-7 p168 breast cancer cell line (empty circles are XYY, black circles are crystal violet);

FIG. 15B shows a typical dose-response curve obtained from MDA MB231 breast cell line treated with crude polyphenols extracted from UIT-1 cocoa genotype at various concentrations (days versus 540 nm absorbance; empty circles as control, black circles as excipients, empty inverted triangle) 250 μg / mL for silver, 100 μg / mL for black inverted triangle, 10 μg / mL for empty square; absorbance 2.0 is the highest limit of a plate reader, where the cell number may not be accurate);

Figure 15C shows a typical dose-response curve obtained from PC-3 prostate cancer cell lines treated with crude polyphenols extracted from the UIT-1 cocoa genotype at various concentrations (days versus 540 nm absorbance; empty circles are control, black circles are excipients, empty) An inverted triangle of 250 μg / mL, a black inverted triangle of 100 μg / mL, an empty rectangle of 10 μg / mL;

FIG. 15D shows a typical dose-response curve obtained from MCF-7 p168 breast cancer cell line treated with crude polyphenols extracted from UIT-1 cocoa genotype at various concentrations (days versus 540 nm absorbance; empty circles are control, black circles are excipients, empty) 250 μg / mL for the inverted triangle, 100 μg / mL for the black inverted triangle, 10 μg / mL for the empty rectangle; 1 μg / mL for the black rectangle; absorbance 2.0 is the upper limit of the plate reader, and the cell number may not be accurate. have);

FIG. 15E shows a typical dose-response curve obtained from HeLa cervical cancer cell lines treated with crude polyphenols extracted from the UIT-1 cocoa genotype at various concentrations (days versus 540 nm absorbance; the empty circle is the control, the black circle is the excipient, and the empty inverted triangle is 250 μg / mL, black inverted triangle is 100 μg / mL, empty rectangle is 10 μg / mL; absorbance 2.0 is the highest limit of the plate reader, where the cell count may not be accurate);

FIG. 15F is a cytotoxic effect chart for HeLa cervical cancer cell line treated with cocoa polyphenol fraction (days compared with 540 nm absorbance; empty circle fraction AE 100 µg / mL, black circle fraction AC 100 µg / mL, empty inverted triangle fraction D & E 100 μg / mL, absorbance 2.0 is the highest limit of the plate reader, where the cell count may not be accurate);

15G is a cytotoxic effect chart for SKBR-3 breast cancer cell line treated with 100 μg / mL of cocoa polyphenol fraction (days compared with 540 nm absorbance; empty circle is fraction AE, black circle is fraction AC, empty inverted triangle is fraction D &E);

FIG. 15H shows a typical dose-response relationship between cocoa procyanidin fraction D + E and HeLa cells (540 nm absorbance versus days; empty circles as control, black circles as 100 μg / mL, empty inverted triangle as 75 μg / mL, black inverse) Triangles 50 μg / mL, empty rectangles 25 μg / mL, black rectangles 10 μg / mL; absorbance 2.0 is the highest limit of the plate reader, where the cell count may not be accurate);

FIG. 15I shows a typical dose-response plot between cocoa procyanidin fraction D + E and SKBR-3 cells (days versus 540 nm absorbance; empty circles as control, black circles as 100 μg / mL, empty inverted triangle as 75 μg / mL, 50 μg / mL for the black inverted triangle, 25 μg / mL for the empty rectangle, 10 μg / mL for the black rectangle);

FIG. 15J shows a typical dose-response relationship plot between cocoa procyanidin fraction D + E and HeLa cells using a soft-agar-cloning assay (bar graph; control versus colony count, 1, 10, 50 and 100 μg / mL);

FIG. 15K shows inhibition of proliferation of HeLa cells when treated with crude polyphenol extracts obtained from eight cocoa genotypes (concentration compared to% control, μg / mL; empty circles are C-1, black circles are C-2, Empty inverted triangle is C-3, black inverted triangle is C-4, empty square is C-5, black square is C-6, empty triangle is C-7, black triangle is C-8; C-1 = UF- 12: hort species = Trintario and used crude extracts of UF-12 (Brazil) cocoa polyphenols (caffeine / theobromine removed); C-2 = NA-33: hort species = Forastero and used UF-12 (Brazil) crude extract of cocoa polyphenol (caffeine / theobromine removed); C-3 = EEG-48: hort species = Forastero and used is crude extract of EEG-48 (Brazil) cocoa polyphenol (caffeine) C4 = unknown: hort species = Forastero and used crude extracts of unknown (West African) cocoa polyphenols (caffeine / theobromine removed); C-5 = UF -613: hort species = Trintario and used are crude extracts of UF-613 (Brazil) cocoa polyphenols (caffeine / theobromin removed); C-6 = ICS-100: hort species = Trintario (prototype Nicaragua Criollo) Used is crude extract of ICS-100 (Brazil) cocoa polyphenol (with caffeine / theobromine removed); C-7 = ICS-139: hort species = Trintario (circa Nicaragua Criollo) and used ICS-139 (Brazil) crude extract of cocoa polyphenol (caffeine / theobromine removed); C-8 = UIT-1: hort species = Trintario and used was crude extract of UIT-1 (Malaysia) cocoa polyphenol (caffeine) / Theobromine removed);

FIG. 15L shows inhibition of proliferation of HeLa cells when treated with crude polyphenol extracts obtained from fermented cocoa beans and dried cocoa beans (each stage during fermentation and sun drying; concentration compared to% control, μg / mL; empty circle Silver fraction on the start of the experiment, black circle fraction on day 1, empty reverse triangle fraction on day 2, black reverse triangle fraction on day 3, empty square fraction on day 4, black square fraction on day 9);

FIG. 15M shows the effect of enzyme-oxidized cocoa procyanidins on HeLa cells (response to dose of crude cocoa polyphenols treated with polyphenol oxidase; concentration relative to% control, μg / mL; black square is caffeine And crude UIT-1 containing theobromine, empty circles for caffeine and theobromine removed crude UIT-1, black circles for crude UIT-1 treated with polyphenol oxidase);

FIG. 15N shows a representative semi-purified reverse phase HPLC separation for crude cocoa fraction D + E; FIG.

15O shows representative semi-purified normal HPLC separations for crude cocoa polyphenol extracts.

FIG. 16 shows a typical Rancimat oxidation curve (time relative to any unit; dashed and crossed (+) versus BHA and BHT; * is DE; FIG. 1) for cocoa procyanidin extracts and fractions compared to synthetic antioxidants BHA and BHT. x is crude extract, empty square is AC, empty rhombus is control);

Figure 17 shows a typical agarose gel showing the inhibition of cocoa procyanidin fractions against topoisomerase II catalyzing the decatenation of kinetoplast DNA chains (lane 1 is a label (M)). Containing 0.5 μg of monomer-length kinetoplasmic DNA ring; lanes 2 and 20 contain kinetoplasmic DNA treated with topoisomerase in 4% DMSO without cocoa procyanidins (Control-C); lanes 3 and 4 Contain kinetoplasmic DNA treated with topoisomerase in the presence of cocoa procyanidin fraction A 0.5 and 5.0 μg; lanes 5 and 6 contain kinetoplasmic DNA treated with topoisomerase in the presence of 0.5 and 5.0 μg of cocoa procyanidin fraction B Lanes 7, 8, 9, 13, 14 and 15 were kineto treated with topoisomerase in the presence of cocoa procyanidin fraction D 0.05, 0.5 and 5.0 μg; Replica of Last DNA; Lanes 10, 11, 12, 16, 17 and 18 are copies of Kinetoplasm DNA treated with topoisomerase in the presence of Cocoa Procyanidin Fraction E 0.05, 0.5 and 5.0 μg; Lane 19 is Cocoa Procyanidin Fraction E Copies of kinetoplasmic DNA treated with topoisomerase in the presence of 5.0 μg);

FIG. 18 is a dose-response correlation diagram of cocoa procyanidin fraction D for cell lines with and without DNA repair ability (dose versus fraction viability; μg / mL; left xrs-6 non-repairable cell line, MM-1 D). D282X1, right BR1 DNA repair cell line, MM-1 D D282B1);

EH 19 is the dose-response curve of adriamycin resistant MCF-7 cells compared to MCF-7 p168 parental cell line when treated with cocoa fraction D + E (concentration relative to% control, μg / mL; MCF-7 p168, black circle is MCF-7 ADR);

20A and B show dose-response effect plots for HeLa and SKBR-3 cells when 12 fractions obtained by semi-product normal HPLC were treated at 100 μg / mL and 25 μg / mL levels (bar graph,% control). Control and fractions 1-12);

21 shows normal HPLC separation of concentrated (enriched), purified crude pentamers from cocoa extracts;

22A, B and C show MALDI-TOF / MS of procanidine-rich pentamers, fractions A-C and fractions D-E, respectively;

FIG. 23A is the elution profile of procyanidin oligomer purified by modified semi-product HPLC;

FIG. 23B is the elution profile of procyanidin trimer purified by modified semi-product HPLC.

Figures 24A-D show an energy minimizing structure of pentacoids bound together (4-8), each based on epicatechin structure;

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

25B shows relative fluorescence of catechins upon thiol degradation with benzylmercapten;

25C shows the relative fluorescence of dimers (B2 and B5) upon thiol degradation with benzylmercapten;

26A shows the relative fluorescence of dimers upon thiol degradation;

FIG. 26B shows relative fluorescence of B5 dimer upon successive thiol degradation and desulfurization;

27A shows the relative tumor volume of the MDA MB 231 nude mouse model during treatment with pentamers;

FIG. 27B is a relative survival curve of MDA MB 231 nude mouse model treated with pentamers; FIG.

FIG. 28 is the elution profile of acetone extract of procyanidin from cocoa extract by halogen-free analytical separation;

FIG. 29 shows the effect of pore size of the stationary phase on separation of procyanine in normal HPLC;

30A shows the use of a substrate during fermentation of cocoa beans;

30B shows metabolic production during fermentation of cocoa beans;

30C shows the number of plates during fermentation of cocoa beans;

30D shows the relative concentrations of each component in the fermentation broth of cocoa beans;

FIG. 31 shows acetylcholine-induced dilatation of rat aorta pre-contracted with NO-related phenylephrine;

32 is a blood glucose tolerance profile from several experimental mixtures;

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

34A and B show the correlation between degree of polymerization and COX-1 / COX-2 (μM) versus IC ₅₀ ;

35 shows the correlation between the effect of compounds on COX-1 and COX-2 activity in μM;

36A-V show IC ₅₀ values (μM) of samples containing COX-1 / COX-2 and procyanidins;

FIG. 37 shows an overview of tablets for separating procyanine from cocoa; FIG.

38A-P show a preferred structure of pentamers;

39A-AA depict stereoisomeric libraries of pentamers;

40A and B show concentration gradients for 70 minutes for normal HPLC separation of procyanidins detected by UV and fluorescence, respectively;

41A and B show 30 minute concentration gradients for normal HPLC separation of procyanidins detected by UV and fluorescence, respectively;

42 shows the preparation of normal HPLC to separate procyanidins;

43A-G show CD (circular dichroism) spectra of dimers, primers, tetramers, pentamers, hexamers, heptamers and octamers of procyanidins, respectively;

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

44B-F show APT, COZY, XHCORR, ¹ H and ¹³ C NMR spectra of epicatechin;

45A shows the structure of the catechin and ¹ H / ¹³ C NMR data;

45B-E show ¹ H, APT, XHCORR, and COSY NMR spectra of catechins;

46A shows the structure of the B2 dimer and ¹ H / ¹³ C NMR data;

46B-G show ¹³ C, APT, ¹ H, HMQC, COZY and HOHAHA NMR spectra of B2 dimers;

47A shows the structure of the B5 dimer and ¹ H / ¹³ C NMR data;

47B-G show ¹ H, ¹³ C, APT, COZY, HMQC and HOHAHA NMR spectra of B5 dimers;

48A-D show ¹ H, COZY, HMQC and HOHAHA NMR spectra of epicatechin / catechin trimer;

49A-D show ¹ H, COSY, HMQC and HOHAHA NMR spectra of epicatechin trimer;

50A and B show the effect of cocoa procyanidin fractions A and C on blood pressure; Fraction A decreased blood pressure level to 21.43% within 1 minute after administration and recovered to normal after 15 minutes, while fraction C decreased to 50.5% within 1 minute after administration and returned to normal after 5 minutes;

51 shows the effect of cocoa procyanidin fraction on arterial blood pressure of anesthetized guinea pigs;

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

Figure 53 shows the effect of bradykinin on NO production of HUVECs;

54 shows the effect of cocoa procyanidin fractions on macrophage NO production of HUVECs;

55 shows the effect of cocoa procyanidin fractions on macrophage NO production;

FIG. 56 shows the effect of cocoa procyanidin fractions on macrophage NO production induced by LPS and gamma-interferon;

FIG. 57 shows separation of cocoa procyanidin oligomers by micelle-micellar electrokinetic capillary chromatography;

58A-F show MALDI-TOF scalpel spectra of trimers bound to Cu ^{+ 2-} , Zn ^{+ 2-} , Fe ^{+ 2-} , Fe ^{+ 3-} , Ca ^{+ 2-} and Mg ^{+ 2-} ions, respectively;

FIG. 59 shows MALDI-TOF scalpel spectra of cocoa procyanidin oligomers (tetramers to octadecamers);

FIG. 60 shows a dose-response relationship between cocoa procyanidin oligomers and feline FeA lymphocytes producing leukemia virus;

FIG. 61 shows a dose-response relationship between cocoa procyanidin oligomer and feline CRFK normal kidney cell line producing leukemia virus;

FIG. 62 shows a dose-response relationship between cocoa procyanidin oligomer and canine MDCK normal kidney cell line producing leukemia virus;

FIG. 63 shows a dose-response relationship between cocoa procyanidin oligomer and canine GH normal kidney cell line producing leukemia virus;

64 shows the time-temperature effect on hexamer hydrolysis;

65 shows the time-temperature effect on trimer hydrolysis.

Compound of the Invention

Cocoa extracts or compounds derived therefrom, as described above, exhibit anticancer, antitumor, antineoplastic or antioxidant activity, prevent DNA topoisomerase II and oxidative DNA damage, antibacterial, cyclo-oxygenase and / or lipoxi It has been found that it has a syngenease regulation, NO (nitroxide) or NO-synthase regulation, apoptosis regulation, platelet aggregation regulation, blood or bioglucose control activity, and pharmacological activity as a non-steroidal anti-inflammatory agent.

Extracts, or compounds or combinations thereof, are generally obtained by grinding cocoa beans to powder, degrease the powder and extract and purify the active compound from the skim powder. The powder may be prepared by lyophilizing cocoa beans, pulping, depulping and dehulling the dried beans and grinding the dehaled beans. The active compound can be extracted by solvent extraction. Extracts containing the active substance can be purified substantially pure, for example by gel permeation chromatography or HPLC methods or combinations thereof.

Regarding the isolation and purification of the compounds of the invention derived from cocoa, any species of the genus Theobroma, Hernia , or heterologous and homologous thereof Any of the liver's hybrids can be applied. In this respect, Schultz's "Synopsis of Herrania " (J. Arnold Arboretum, Vol. 39, pp. 217-278, plus plates I to XVII, 1985), Cocoa and Its Allies, A Taxonomic Revision of the Genus Theobroma "(Bulletin fo the USNM, Vol. 35, part 6, pp. 379-613, plus plstes 1 to 11, Smithsonian Institution, 1964) and adipic Branson such as" Obsorvation on the Species of the Genus Theobroma Which Occurs in the Amazon "(Bol. Tech. Inst. Agronomico de Nortes, 25 (3), 1951).

In addition, Example 25 of the present invention was found in any species of Theobroma , Hernia genus, or hybrids of different and homologous species of the genus that have never been of interest in the prior art. Describes compounds; And Example 25 also describes a method of controlling the yield of a compound of the invention obtainable from cocoa by adjusting the cocoa fermentation conditions.

An overview of the purification method used for the separation of substantially pure procyanidins is shown in FIG. 37. Steps 1 and 2 of the purification method described in Examples 1 and 2; Steps 3, 4 are in Examples 3, 13, 23; Step 5 is in Examples 4, 14; Step 6 is described in Examples 4, 14 and 16, respectively. Those skilled in the art will be able to obtain the active compound by modifying the outline of the purification method shown in FIG. 37 without departing from the subject matter or scope of the present invention or from undue experimentation from the overview of the purification method shown in FIG.

Active extracts, compounds derived therefrom, and combinations of compounds have been identified as cocoa polyphenols, such as procyanidins, etc. without relying on any particular theory. These cocoa procyanidins have excellent anticancer, antitumor, antineoplastic activity; Antioxidant activity; Preventing DNA topoisomerase II and oxidative DNA damage; Antimicrobial activity; It has cyclo-oxygenase and / or lipoxygenase, NO or NO-synthase, apoptosis, platelet aggregation, blood or bio-glucose control ability, and also has pharmacological activity as a non-steroidal anti-inflammatory agent.

The present invention provides a compound of the formula:

Wherein n is an integer from 2 to 18, for example from 3 to 12, such that the first monomer unit A and a plurality of other monomer units can be present;

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

Position 4 is an alpha or beta stereoisomer;

In the first monomer unit, the other monomer units which bind to it are 4 positions and under the condition that Y = Z = hydrogen, X, Y and Z represent the bonding positions between the monomer units; X, Y and Z are hydrogen, Y, Z is sugar and X is hydrogen, or X is alpha or beta sugar and Y, Z is hydrogen, or when X, Y and Z are not the bonding positions between monomer units It is a combination of them. In the compound n may be 5 to 12, preferably 5 may be. The sugar may be selected from the group consisting of glucose, galactose, xylose, rhamnose and arabinose. The sugars of any one of R, X, Y and Z or both can be optionally substituted with phenolic moieties via ester bonds.

Thus, the present invention can provide a compound of the formula:

Wherein n is an integer from 2 to 18, for example from 3 to 12, advantageously from 5 to 12, preferably from 5, such that the following first monomer unit A and a plurality of further A monomer units are present;

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

Position 4 is an alpha or beta stereoisomer;

In the first monomer unit, the other monomer units which bind to it are 4 positions and under the condition that Y = Z = hydrogen, X, Y and Z represent the bonding positions between the monomer units; X, Y and Z are hydrogen, Y, Z is sugar and X is hydrogen, or X is alpha or beta sugar and Y, Z is hydrogen, or when X, Y and Z are not the bonding positions between monomer units Combinations thereof;

The sugars can be optionally substituted with phenolic moieties via ester bonds.

The present invention therefore provides a formula A _n polymer compound _wherein A is a monomer of the formula and a pharmaceutically acceptable salt or derivative of A _n , including oxides:

Wherein n is an integer from 2 to 18 such that at least one terminal monomer unit A and a plurality of additional monomer units can be present;

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

Bonding between adjacent monomers at the 4, 6 or 8 positions;

X, Y and Z are selected from the group consisting of monomers A, hydrogen and sugars, provided that at least one terminal monomer has an additional monomer unit (ie, a bond of monomer units adjacent to the terminal monomer unit) bonded at 4 positions, Optionally Y = Z = hydrogen;

Sugars are optionally substituted at any site, eg with phenolic moieties, via ester bonds.

In a preferred embodiment n can be 3 to 18, 2 to 12 eg 5 to 12; Advantageously n is 5. The sugar is selected from the group consisting of glucose, galactose, xylose, rhamnose and arabinose. The sugars of any one of R, X, Y and Z or both can be optionally substituted with phenolic moieties via ester bonds. The phenolic moiety is selected from the group consisting of caffeic acid, comarinic acid, ferulic acid, gallinic acid, hydrobenzoic acid and cinafinic acid.

The present invention also provides a formula A _n polymer compound _wherein A is a monomer of the formula and a pharmaceutically acceptable salt or derivative of A _n , including oxides:

Wherein n is an integer from 2 to 18, for example 3 to 18, advantageously 3 to 12 for example 5 to 12, preferably 5;

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

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

Z is H or a sugar when X and Y are adjacent monomers, and Y is H or a sugar when X and Z are adjacent monomers; In at least one of the two terminal monomers, adjacent monomers bind at the 4 position; under the conditions X, Y and Z are H, sugars or adjacent monomers, optionally Y = Z = hydrogen;

Binding of the 4 position is alpha or beta stereoisomer;

Sugars are optionally substituted with phenolic moieties, via ester bonds.

With respect to the expression “at least one terminal monomer unit A”, it will be appreciated that the present invention has two terminal monomer units, and that two terminal monomer units A may be the same or different. In addition, the expression "at least one terminal monomer unit A" includes an embodiment in which the terminal monomer unit A is referred to as "the first monomer unit", where the "first monomer unit" is further bonded to another monomer unit by the monomer. It will also be appreciated that the term refers to monomers that make up the polymer compound of formula A _n . Furthermore, in at least one of the two terminal monomers, adjacent monomers are bonded at the 4 position, optionally Y = Z = hydrogen.

With respect to the term "combination of them", it will be understood that more than one compound of the invention may be used simultaneously, such as for example to be administered to a subject that needs to be prescribed in a composition consisting of one or more compounds of the invention. .

The compounds of the present invention or combinations thereof show the utility of the cocoa extracts described above; It will be appreciated that the term "cocoa extract" throughout this specification may be expressed as a compound of the present invention or a combination thereof, so that the compound of the present invention or a combination thereof may have the same meaning as a cocoa extract.

The term "oligomer" as used herein terminates any compound of the above-mentioned formula or combinations thereof, wherein n is 2-18. when n is 2, a "dimer"; if n is 3, a "trimer"; if n is 4 "tetramer"; n is 5 is a "pentamer"; An "octadecamer" with n equal to 18 may also be called an oligomer.

The compounds of the present invention or combinations thereof may be isolated from natural sources, such as , for example , theobroma, any species of the genus Hernia or hybrids of different or homologous species thereof; The compounds of the present invention or combinations thereof can be purified, for example, to be substantially pure; For example, it may be purified to be apparently homogeneous. Purity is a relative concept, and since many embodiments describe the separation and purification of a compound of the present invention or combinations thereof, those skilled in the art can obtain substantially pure compounds of the present invention or combinations thereof according to the methods presented. They can be purified to be substantially homogeneous (eg, chromatographic peaks are separated, separated and purified). Considering an embodiment (eg Example 37), a substantially pure compound or combination thereof may be at least about 40% (eg at least about 50%) of purity, advantageously at least about 60% (eg at least about 70%) of Purity, more advantageously at least 75-80%, preferably at least about 90%, more preferably at least 90% (e.g. at least 90-95%), or at least 95% ( For example 95-98%).

Furthermore, monomer units constituting the oligomer used herein include (+)-catechin (abbreviated C) and (-)-epicatechin (abbreviated EC). Adjacent monomers are linked from 4 to 6 positions or from 4 to 8 positions; The combination of the 4 position of one monomer and the 6 or 8 position of an adjacent monomer unit is represented by (4 → 6) or (4 → 8). Four types of heterochemical linkages are possible between the 4 position of one monomer and the 6 and 8 positions of adjacent monomer units; Structural chemical bonds between monomer units are represented by (4α → 6), (4β → 6), (4α → 8) or (4β → 8), respectively. When C is bonded to another C or EC, the binding is represented by (4α → 6) or (4α → 8). When EC is bound to another C or EC, the binding is represented by (4β → 6) or (4β → 8).

As the compounds exhibiting the above-mentioned activity, dimers include EC- (4β → 8) -EC and EC- (4β → 6) -EC, with EC- (4β → 8) -EC being preferred; The trimers are [EC- (4β → 8)] ₂ -EC, [EC- (4β → 8)] ₂ -C and [EC- (4β → 6)] ₂ -EC, where [EC- (4β → 8)] ₂ -EC 8)] _2- EC is preferred; Tetramers are [EC- (4β → 8)] ₃ -EC, [EC- (4β → 8)] ₃ -C, [EC- (4β → 8)] ₃ -C, [EC- (4β → 8)] ₂ -EC- (4β → 6) -C, with [EC- (4β → 8)] ₃ -EC being preferred; Pentamers are [EC- (4β → 8)] ₄ -EC, [EC- (4β → 8)] ₃ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₃ -EC- ( 4β → 8) -C and [EC- (4β → 8)] ₃ -EC- (4β → 6) -C, wherein the 3-position of the terminal monomer unit of the pentamer is optionally gallate or substituted with β-D-glucose, with [EC- (4β → 8)] ₄ -EC being preferred.

In addition, the compounds exhibiting the above-mentioned activity include hexamerbunner dodecamer, examples of which are as follows:

As a hexamer, (4β → 8) or (4β → 6) bonds for ECs bound to other ECs or C, and (4α → 8) or (4α → 6) bonds for Cs bound to other C or ECs To one monomer (C or EC); The pentameric compounds described above connected by (4β → 8) bonds, eg [EC- (4β → 8)] ₅ -EC, [EC- (4β → 8)] ₄ -EC- (4β → 6) -EC , [EC- (4β → 8)] ₄ -EC- (4β → 8) -C and [EC- (4β → 8)] ₄ -EC- (4β → 6) -C, where the hexamer terminal monomo unit The 3-position of is optionally substituted with gallate or β-D-glucose, with [EC- (4β → 8)] ₅ -EC being preferred;

As heptamer, (4β → 8) or (4β → 6) bonds for ECs bound to other ECs or C, and (4α → 8) or (4α → 6) bonds for Cs bound to other C or ECs To any combination of two monomers with C and / or EC; The pentameric compounds described above connected by (4β → 8) bonds, eg [EC- (4β → 8)] ₆ -EC, [EC- (4β → 8)] ₅ -EC- (4β → 6) -EC , [EC- (4β → 8)] ₅ -EC- (4β → 8) -C and [EC- (4β → 8)] ₅ -EC- (4β → 6) -C, wherein the heptamer terminal monomo unit The 3-position of is optionally substituted with gallate or β-D-glucose, with [EC- (4β → 8)] ₆ -EC being preferred;

Octamer: (4β → 8) or (4β → 6) bonds for ECs linked to other ECs or C, and (4α → 8) or (4α → 6) bonds for Cs linked to other C or ECs To any combination of three monomers with C and / or EC; The pentameric compounds described above connected by (4β → 8) bonds, eg [EC- (4β → 8)] ₇ -EC, [EC- (4β → 8)] ₆ -EC- (4β → 6) -EC , [EC- (4β → 8)] ₆ -EC- (4β → 8) -C and [EC- (4β → 8)] ₆ -EC- (4β → 6) -C, where the octamer terminal monomo unit The 3-position of is optionally substituted with gallate or β-D-glucose, with [EC- (4β → 8)] ₇ -EC being preferred;

As a nonamer, (4β → 8) or (4β → 6) bonds for ECs bound to other ECs or Cs, and (4α → 8) or (4α → 6) bonds for Cs bound to other Cs or ECs To any combination of four monomers with C and / or EC; The pentameric compounds described above connected by (4β → 8) bonds, eg [EC- (4β → 8)] ₈ -EC, [EC- (4β → 8)] ₇ -EC- (4β → 6) -EC , [EC- (4β → 8)] ₇ -EC- (4β → 8) -C and [EC- (4β → 8)] ₇ -EC- (4β → 6) -C, wherein the octamer terminal monomo unit The 3-position of is optionally substituted with gallate or β-D-glucose, with [EC- (4β → 8)] ₈ -EC being preferred;

As a decamer, (4β → 8) or (4β → 6) bonds for ECs bound to other ECs or C, and (4α → 8) or (4α → 6) bonds for Cs linked to other C or ECs To any combination of five monomers (C and / or EC) with The pentameric compound described above connected by (4β → 8) bonds, eg [EC- (4β → 8)] ₉ -EC, [EC- (4β → 8)] ₈ -EC- (4β → 6) -EC , [EC- (4β → 8)] ₈ -EC- (4β → 8) -C and [EC- (4β → 8)] ₈ -EC- (4β → 6) -C, where the octamer terminal monomo unit The 3-position of is optionally substituted with gallate or β-D-glucose, with [EC- (4β → 8)] ₉ -EC being preferred;

As an undecamer, (4β → 8) or (4β → 6) bonds for ECs bound to other ECs or Cs, and (4α → 8) or (4α → 6) bonds for Cs bound to other Cs or ECs To any combination of six monomers (C and / or EC) with; The pentameric compounds described above connected by (4β → 8) bonds, eg [EC- (4β → 8)] ₁₀ -EC, [EC- (4β → 8)] ₉ -EC- (4β → 6) -EC , [EC- (4β → 8)] ₉ -EC- (4β → 8) -C and [EC- (4β → 8)] ₉ -EC- (4β → 6) -C, wherein the octamer terminal monomo unit The 3-position of is optionally substituted with gallate or β-D-glucose, with [EC- (4β → 8)] ₁₀ -EC being preferred;

As dodecamer, (4β → 8) or (4β → 6) bonds for ECs bound to other ECs or C, and (4α → 8) or (4α → 6) bonds for Cs bound to other C or ECs To any combination of seven monomers (C and / or EC) with The pentameric compounds described above connected by (4β → 8) bonds, for example [EC- (4β → 8)] ₁₁ -EC, [EC- (4β → 8)] ₁₀ -EC- (4β → 6) -EC , [EC- (4β → 8)] ₁₀ -EC- (4β → 8) -C and [EC- (4β → 8)] ₁₀ -EC- (4β → 6) -C, wherein the octamer terminal monomo unit The 3-position of is optionally substituted with gallate or β-D-glucose, with [EC- (4β → 8)] ₁₁ -EC being preferred.

From the detailed description of the invention, it is understood that the foregoing are exemplary and are provided for the purpose of limiting some of the compounds of the invention and do not limit the scope of the compounds of the invention which are possible by the invention. Will be done.

Examples 3A, 3B, 4, 14, 23, 24, 30 and 34 describe methods for separating compounds of the present invention. Examples 13, 14A-D and 16 describe methods for purifying compounds of the present invention. Examples 5, 15, 18, 19, 10 and 29 describe methods for identifying compounds of the present invention. 38A-P and 39A-AA show the structural chemical libraries of representative pentamers of the present invention. Example 17 describes a molecular model method of a compound of the invention. Example 36 provides evidence for the presence of high polymerization oligomers with n of 13-18 in cocoa.

Furthermore, although the present invention is preferably described with respect to cocoa extracts comprising cocoa procyanidins, organic chemists skilled in this disclosure will recognize and conceive that these active compounds can be obtained and / or prepared through synthetic methods. Could be. The present invention therefore encompasses synthetic cocoa polyphenols or procyanidins or derivatives thereof and / or synthetic precursors thereof including, but not limited to, glycosides, gallates, esters and the like. That is, the compounds of the present invention can be prepared not only from species of the cocoa or theobroma or hernia genus but also as synthetic methods; Derivatives and synthetic precursors of the compounds of the invention, such as glycosides, gallates, esters, correspond to the compounds of the invention. Derivatives include compounds wherein the sugar or gallate moiety is in the Y or Z position of the terminal monomer, or the substituted sugar or gallate moiety is in the Y or Z position of the terminal monomer.

For example, Method C of Example 8 describes the oxidation of the compounds of the present invention or combinations thereof with cocoa enzymes to improve cytotoxicity against certain cancer cell lines (see FIG. 15M). The present invention provides the ability to enzymatically modify (e.g., cleavage or addition of chemically important moieties) the compounds of the present invention, for example polyphenol oxidase, Enzymatic treatment with peroxidase, catalase combinations and / or hydrolases, esterases, reductases, transferases, and combinations thereof.

With regard to the synthesis of the compounds of the present invention, the skilled artisan will be able to design another method of synthesis based on the disclosure herein, without undue experimentation, if, for example, a careful reverse synthesis analysis of polymer compounds as well as monomers is made. For example, given the phenolic properties of the compounds of the present invention, the skilled person can utilize various selective protection / unprotection methods by combining organometallic addition, phenol coupling and photochemical reactions. The compounds of the present invention can be prepared synthetically without undue experimentation, for example, with convergent, linear or biomimetic approaches, or combinations thereof, with standard reactions well known in organic synthetic chemistry. In this respect, W. Carruthers' "Some Mordern Methods of Organic Synthesis" (3rd edition, Cambridge University Press, 1986), J. March's "Advanced Organic Chemistry" (3rd edition, John Willy & Sun, 1985), Van Lance See, eg, Zyma SA EP 0096 007 and the literature on 'quotation' below, which are hereby incorporated by reference.

Availability of Compounds of the Invention

With respect to the compounds of the present invention, it has been found that they have surprising characteristic activities, such as having broad applicability to the treatment of various conditions of the following compounds of the present invention.

COX / LOX-associated availability

Atherosclerosis, the most common cardiovascular disease, is the leading cause of heart attacks, heart attacks and circulatory problems. Atherosclerosis is a complex disease involving many cell types, biochemical reactions and partial factors. The disease, disease state, and disease progression depend on the interdependent consequences of low density lipoprotein (LDL) oxidation, cyclo-oxygenase (COX) / lipoxinase (LOX) biochemistry, and nitric oxide (NO) biochemistry. There are several aspects that are divided accordingly.

Clinical studies have shown that increasing the plasma concentration of LDL accelerates the development of atherosclerosis. Cholesterol deposited at the atherosclerotic site is mainly plasma lipoproteins, including LDL. Oxidation of LDL is a crystal at the onset of atherosclerosis and is associated with increased production of superoxide anion radicals (O ₂ ·-). Oxidation of LDL by O ₂ -or other reactive substances (e.g., OH, 0N00-, lipid peroxy radicals, copper ions, and metal-based proteins) results in cellular uptake of receptor-mediated cells. reduces the affinity of LDL for endocytosis. Oxidized LDL is rapidly inhaled by macrophages, which are transformed into cells that are very similar to the "foam cells" found in the atherosclerotic site at the onset of onset.

Lipoprotein oxide also damages blood vessels by the formation of lipid hydroperoxides in the LDL particles. Damage to blood vessels triggers a chain radical oxidation of unsaturated LDL lipids, thus further producing oxidized LDL for macrophage incorporation.

Intensive accumulation of oxidized LDL-filled foam cells results in early "fatty streak" damage, which is the result of atherosclerosis that eventually leads to heart disease (or coronary). Develops into complex damage;

As Jean Marx generalized and reviewed (Science, Vol. 265, p. 320, July 15, 1994), 330,000 patients a year in the United States occlude blood vessels obstructed by severe atherosclerotic plaque (atherosclerosis). Heart and / or peripheral vascular angioplasty surgery to dilate (eg, coronary arteries) is normalizing blood flow. For most patients, this surgery has the intended effect. However, in about 33% (possibly more) of patients, restenosis develops, in which the treated arteries rapidly occlude. These patients do not get better and even worse than before angioplasty surgery. Excessive proliferation of smooth muscle cells (SMC) in the vessel wall causes responsiveness. Increased accumulation of oxidized LDL in injured SMC may lead to atherosclerosis-like processes that are similar to restenosis. Encounter, et al., "Association Between Prior Cytomegalovirus Infection And The Risk Of Restenosis After Coronary Aterectomy" (New England Journal of Medicine, 335: 624-630, Aug. 29, 1996) and all references cited therein It is incorporated by reference. Thus, the usefulness of the present invention in connection with atherosclerosis can be applied to responentiation.

With regard to the inhibitory action of the compounds of the present invention on cyclo-oxygenase (COX; prostaglanding endoperoxide synthase), COX is a prostaglandin and other arachidonic acid metabolite (ie, eicosol) that is involved in many physiological processes. It is known that the enzyme is the central enzyme for the production of sanoids). While COX-1 is a constitutive enzyme expressed in many tissues, its isoform, COX-2, is induced by various cytokinin, hormone and tumor promoters. COX-1 is involved in platelet aggregation and thus produces thromboxane A2, which is involved in the progression of atherosclerosis. Preventing this is the basic principle of preventing cardiovascular disease.

The activity of COX-1 and COX-2 is inhibited by aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), and the side effects of NSAIDs are thought to be related to COX-1. Furthermore, patients who regularly take NSAIDs have a 40-50% lower risk of contractile colorectal cancer than patients who do not take these drugs; It is known that COX-2 mRNA levels are significantly increased to 86% of human colloidal adenocarcinoma.

One important feature of the taxonomy of COX-2 expression is the enhanced expression of genes involved in the regulation of apoptosis (ie, killing advanced cells). Several NSAIDs are known to be associated with increased necrosis of cells and induction of apoptosis in embryonic fibroblasts of chickens.

Cellular lipoxygenases are also involved in the oxidative modification of LDL through the peroxylation of unsaturated fats. The production of fatty peroxy radicals triggers the chain radical oxidation of unsaturated LDL lipids while further producing oxidized LDL for macrophage incorporation.

The compounds of the present invention have been found to be useful for the treatment of diseases involving COX / LOX. In Example 28, COX was inhibited by a compound of the present invention at a concentration similar to indomethacin, a known NSAID.

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

Examples of the compounds of the present invention which exhibit activity inhibition against COX / LOX described above are the dimers, trimers, tetramers and pentamers described above.

Thus, in addition to the significant inhibitory potential of the compounds of the present invention on COX-2 and the cytotoxic effects on COX-2, which are believed to express colorectal cancer cell lines, the compounds of the present invention are apopto like inhibitors for the stages of cancer progression. In addition to apoptotic activity, it possesses activities such as NASID species with a wide range of prophylactic actions (see Example 8, FIGS. 9D-H and the like).

Furthermore, prostaglandins, the second product in the conversion of arachidonic acid to prostaglandin H ₂ by the catalyst of COX, are involved in inflammation, pain, fever, lethal tremor, analgesic and platelet aggregation. Thus, the compounds of the present invention are effective in conditions such as, for example, NSAIDs, for cardiovascular diseases and seizures (substantially, inhibition of platelet COX-1, which inhibits the production of thromboxane A ₂ , is an effect of aspirin on cardiovascular diseases. Is the basis for preventive effects).

Inflammation is the response of living tissue to damage. It consists of a complex series of enzymatic reactions, secretion of regulatory substances, outflow of body fluids, cell migration, tissue breakdown and recovery. Inflammation is activated by prostaglandin H ₂ , which releases arachidonic acid, the substrate of COX and LOX enzymes. COX replaces arachidonic acid with prostaglandin PGE ₂ , a major eicosanoid found in inflammatory situations ranging from acute edema to chronic arthritis. Its inhibition by MSAID is at the heart of treatment.

Arthritis is a type of rheumatoid disease, a disease that occurs mostly in connective tissue. The basic areas affected by this disease are connective tissue, including the synovial membrane, cartilage, bone, tendons, ligaments and interstitial tissue. Temporary connective tissue symptoms include sprains, strains, tendon inflammation, and deformation of the tendon sheath. The most serious forms of arthritis are rheumatoid arthritis, osteoarthritis, gout and systemic lupus erythematosus.

In addition to rheumatic diseases, other diseases are also characterized by inflammation. Gingivitis and periodontitis have pathological symptoms similar to rheumatoid diseases. Enteritis diseases are called idiopathic chronic inflammatory conditions of the intestine, gastritis colitis and Crohn's disease. Spondylitis is a chronic inflammation of the spinal cord. The incidence of osteoarthritis associated with obesity is also high.

Thus, the compounds of the present invention have utility in the treatment of symptoms involving inflammation, pain, fever, lethal tremor, analgesic and platelet aggregation.

Inhibition of COX by the compounds of the invention may also inhibit the formation of prostaglandins (eg, PGD ₂ , PGE ₂ ). Therefore, the present invention is useful for the treatment of symptoms related to prostaglandin PGD ₂ , PGE ₂ .

Irrelevant usefulness

Nitric oxide (NO) is known to inhibit platelet aggregation, monocyte adhesion and chemotaxis, and proliferation of vascular smooth muscle tissue, which are critically involved in the development of atherosclerosis. Evidence is supported by the view that the amount of nitric oxide (NO) in atherosclerotic tissue is reduced due to reaction with oxygen free radicals. Loss of NO due to this reaction promotes adhesion of platelets and inflammatory cells to the vessel wall, further impairing the nitric oxide mechanism of relaxation. In this way, the loss of NO promotes the progression of atherosclerosis, leading to a progressive disease state.

Hypertension is a major cause of cardiovascular disease, including sleepiness, heart attacks, heart failure, irregular heartbeat, and kidney failure. Hypertension is a condition where blood pressure in blood vessels circulating in the body is higher than the standard value. When the systolic blood pressure exceeds 150 mm Hg or the diastolic blood pressure exceeds 90 mm Hg for a sustained period of time, the body is damaged. For example, too high systolic blood pressure can rupture blood vessels. When blood vessels rupture in the brain, stroke is caused. In addition, this may cause thickening and narrowing of blood vessels that may cause atherosclerosis. Elevated blood pressure can also enlarge myocardium, as it works more intensely to overcome elevated diastolic blood pressure when blood is released. Eventually, such myocardial dilatation can lead to irregular heart rate or heart failure. Hypertension is called a "silent killer" because it has no symptoms and can only be found when blood pressure is checked.

Regulation of blood pressure is a complex one involving the expression of the component Ca ⁺² / camodulin dependent form of nitric oxide synthase (NOS), the abbreviation eNOS. The NO synthesized by this enzyme causes the muscles in the blood vessels to relax (expansion) and lower blood pressure. If the standard level of NO synthesized by eNOS is not synthesized because it is inhibited by inhibitors or in pathological conditions such as atherosclerosis, the vascular muscle will not relax to an appropriate extent. As a result, blood vessel constriction increases blood pressure and may cause hypertension.

Vascular endothelial cells include eNOS. NO synthesized by eNOS diffuses in multiple directions, and when it reaches the underlying vascular smooth muscle, NO binds to the heme group of guanylyl cyclase and causes an increase in cGMP. Increasing cGMP causes increased free Ca ⁺² in the cell. Cyclic GMP can also activate protein kinases, which phosphorylate Ca ⁺² transporters, which cause Ca ⁺² sequestration in the intracellular structure of muscle cells. Because muscle contraction requires Ca ⁺² , muscle contractility decreases when the concentration of Ca ⁺² is reduced. Relaxation of the muscles causes blood vessels to dilate, which lowers blood pressure. Therefore, suppression of eNOS causes a rise in blood pressure.

When normal levels of NO are not synthesized, vascular muscles do not relax to an appropriate extent, either by administration of a NOS inhibitor or possibly because of the inhibition of synthesis in a pathological condition such as atherosclerosis. The resulting blood vessel contraction raises blood pressure, which can cause hypertension. There is considerable interest in finding treatments that increase the activity of eNOS in patients with hypertension, but no actual treatment has been reported. Pharmacological agents that can release NO, such as nitroglycerin or isosorbide dinitrate, serve as a girder for lowering blood pressure.

Although the compounds of the present invention inhibit the oxidation of LDL, the efficacy of these broader compounds is a multidimensional effect on NO by atherosclerosis. The regulation of NO by the compounds of the present invention results in beneficial effects, including the regulation of hypertension, the reduction of NO affecting hypercholesterolemia, the inhibition of platelet aggregation and monocyte adhesion, and all actions involved in the progression of atherosclerosis. . The role of NO in the immune system is different from its function in the blood vessels. Macrophages contain inducible NOS forms, called iNOS rather than constituents. Transcription of iNOS is positively and negatively regulated by many bioreactive modifiers called cytokinins. The most important inducers are lipopolysaccharide (LPS), the cell wall component of gamma-interferon, tumor necrosis factor, interleukin-1, interleukin-2 and gram negative bacteria. Stimulated macrophages produce enough NO to inhibit ribonuclease reductase, an enzyme that converts ribonucleotides to deoxyribonucleotides required for DNA synthesis. Inhibition of DNA synthesis may be an important way for macrophages and other tissues, including iNOS, to inhibit growth that rapidly divides tumor cells or infectious bacteria.

For the effects of NO and infectious bacteria, microorganisms play an important role in infectious processes that reflect physical contact and injury, personal constitution, occupation and environment, as well as nutritional diseases caused by improper storage, processing and contamination.

Compounds of the present invention, combinations thereof, and compositions comprising said components are useful for the treatment of diseases associated with the modulation of NO concentrations. Example 9 illustrates antioxidant activity (as an inhibitor of free radicals) of a compound of the invention. When NO is provided as free radicals and the compound of the present invention is provided as a strong antioxidant, it was doubtful whether administration of the compound of the present invention into the in vitro and in vivo experimental models would result in a reduction of the NO level. Reduction of NO results in hypertension rather than hypotension. Contrary to expectations, the compounds of the present invention induce an increase in NO from in vitro experimental models and suggest the effect of hypotension through in vivo irradiation (Examples 31 and 32). These results were unexpected and unexpected.

Example 27 exemplified erythema (facial scarlet) soon after drinking a solution comprising a compound of the present invention and glucose, accompanied by a vasodilating effect.

Example 31 exemplified the hypotensive effects induced by the compounds of the invention in an animal model in vivo, which illustrates the efficacy of the compounds of the invention in treating hypertension. In this embodiment, the compounds of the present invention, combinations thereof and compositions comprising said components comprise oligomers, where n is 2-18, preferably n is 2-10.

Example 32 illustrates the regulation of NO produced by the compounds of the present invention in an ex vivo model. In this embodiment, the compounds of the present invention, combinations thereof and compositions comprising said components comprise oligomers, where n is 2-18, preferably n is 2-10.

Example 35 also provided evidence regarding the formation of Cu ^+2- , Fe ⁺² -and Fe ⁺³ -oligomer complexes detected by MALDI / TOF / MS. These results can minimize the effects of the compounds of the present invention on LDL oxidation by complexing with copper and / or iron ions.

Moreover, the compounds of the present invention have antimicrobial activity useful for treating infections and preventing nutrient damage. Examples 22 and 30 illustrate the antimicrobial activity of the compounds of the present invention against various representative microbiota of clinical and nutritional importance as follows.

Example 33 illustrates the effect of a compound of the present invention on NO production of macrophages. In this example, the results obtained demonstrate that the compounds of the present invention induce NO production of monocytes / macrophages when irrelevant and dependent upon stimulation by lipopolysaccharide (LPS) or cytokinin. . Macrophages that produce NO can inhibit the growth of infectious bacteria.

Compounds of the invention which induce antimicrobial activity are oligomers, where n is 2-18, preferably n is 2, 4, 5, 6, 8 and 10.

Examples of compounds which induce antimicrobial activity against NO cited above are the dimers, tetramers, pentamers, hexamers, octamers and decamers described above.

Anti-cancer utility

Cancer is classified into three groups: carcinomas, sarcomas and lymphomas. Carcinomas are malignant tumors that occur in the skin, linings of various organs, glands, and tissues. Sarcoma is a malignant tumor that occurs in bone, muscle or connective tissue. The third group includes leukemias and lymphomas because they all occur in blood cells that form organs. The main types of cancer are prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, non-Hodgkin's lymphoma, uterine cancer, melanoma of the skin, kidney cancer, leukemia, ovarian cancer and pancreatic cancer.

Cancer occurs as a result of denaturation of the DNA of cells by several factors, such as genetic factors that damage DNA, ionizing radiation, contaminants, radon and free radicals. Cells with mutations become defective in ordered cell division. These cells do not experience apoptosis (expected cell necrosis) and continue to divide to mark the beginning of a malignant tumor or to allow more mutations to become malignant.

There are three main features that are common to very different cancers.

(1) the ability to multiply indefinitely; (2) infiltration of tumors as peripheral tissues; And (3) transition.

Certain types of cancer metastasize in a characteristic way. For example, thyroid cancer, lung cancer, breast cancer, kidney cancer and prostate cancer often metastasize to bone. Lung cancer usually spreads to the brain, and adrenal and colorectal cancers often metastasize to the liver. Leukemia was initially thought to be a generalized disease, which is found in the bone marrow of the body. Surprisingly, the compounds of the present invention have been found to be useful in the treatment of various cancers described above. Examples 6, 7, 8 and 15 illustrate that the compounds of the present invention induce anticancer activity against human HeLa (cervical), prostate cancer, breast cancer, kidney cancer, T-cell leukemia and colon cancer cell lines. It was. Example 12 (FIG. 20) illustrates the dose response effect on HeLa and SKBR-3 breast cancer cell lines treated with oligomeric (dimer-dodecamer) procanidine substantially purified by HPLC, and cytotoxins on these cancer cell lines. Was dependent on pentamer procyanidine to dodecarmer procyanidine, including lower oligomers that showed no efficacy at all.

While not wishing to be bound by theory, it is believed to be the least structural motif that describes the effects described above. Example 37 also exemplified the same cytotoxic effect of higher oligomers (pentamer-decamers) on feline lymphoblastic cell lines. Cytotoxins were also observed as cytotoxic effects of higher oligomers on standard canine and cat cell lines (FIGS. 58-61).

In Example 8 (FIGS. 9D-H), the compounds of the present invention have been shown to induce cytotoxins against putative COX-2 expressing human colon cancer cell line (HCT 116).

Example 9 illustrates antioxidant activity by the compounds of the present invention. Compounds of the present invention inhibit rupture of DNA strands, DNA-protein crosslinking and free radical oxidation of nucleotides to reduce and / or interfere with the occurrence of mutations.

Example 10 illustrates a compound of the present invention as a topoisomerase II inhibitor, which is the target of a chemotherapeutic agent such as doxorubicin.

Example 21 shows a substantially pure pentamer that induces anti-tumor activity against human breast cancer cell line (MDA-MB-231 / LCC6) in a nude mouse model (average weight of mouse is about 25 g). In vivo effects are illustrated. Because of unexpected animal toxicity, repeated in vivo experiments with high doses (5 mg) of pentamers were not entirely successful. Typically, it is believed that such toxicity may be related to the vasodilatory effect of the compounds of the present invention.

Example 33 illustrates the effect of a compound of the present invention on NO production of macrophages. NO producing macrophages can inhibit the growth of rapidly dividing tumor cells.

The invention also encompasses the use of the compounds of the invention to induce inhibition of cell proliferation by apoptosis.

For anticancer activity, the compounds of the present invention are oligomers, where n is 2 to 18, for example 3 to 18, such as 3 to 12, preferably n is 5 to 12, most preferably n Is five.

Compounds that induce inhibitory activity against cancer cited above include pentamers to dodecamers as described above.

Preparation and Method

Accordingly, the compounds of the present invention, combinations thereof, and compositions comprising the components of the present invention are directed to various aspects of atherosclerosis, cardiovascular disease, cancer, blood pressure control and / or hypertension, inflammatory diseases, infectious pathogens, and food poisoning. Extensive activity is shown.

Therefore, compounds of the present invention, combinations thereof, and compositions comprising these components are COX inhibitors that reduce the risk of thrombosis by inhibiting the formation of thromboxane A ₂ and thus affecting platelet aggregation. In addition, the inhibitory action of COX reduces the adhesion of platelets and inflammatory cells to the vessel wall, thereby improving the relaxation mechanism of NO. These results, combined with the inhibitory action of COX at concentrations similar to the known NSAID, indomethacin, show antithrombogenic efficacy.

Moreover, compounds of the present invention, combinations thereof and compositions comprising these components are antioxidants that inhibit the oxidation of LDL by reducing the levels of superoxide radical anions and lipoxygenase mediated lipid peroxyradicals. Inhibition of LDL oxidation at this stage slows macrophage activity and prevents the formation of foam cells to prevent further progression of atherosclerosis. Inhibition of LDL oxidation can also slow the progression of restenosis. Accordingly, the compounds of the present invention or combinations thereof or compositions comprising the compounds of the present invention or combinations thereof may be used for the prophylaxis and / or treatment of atherosclerosis and / or restenosis. Accordingly, the compounds of the present invention may be administered before or after angioplasty or similar procedures to prevent or treat responentiation in a patient.

For the treatment or prophylaxis of restenosis and / or atherosclerosis, the compounds of the present invention or compositions comprising the compounds or compounds of the present invention are skilled in the art, alone or in combination with other therapeutic agents, from known disclosure and knowledge. Can be administered as prescribed by a qualified practitioner, for example at the time of the initial symptoms or signs of restenosis and / or atherosclerosis, with or without angiogenesis or without angiogenesis at all. Administered immediately after angiogenesis as prescribed by an experienced practitioner; Administration of the compound or compounds of the present invention alone or in combination with other treatments is necessary, for example, once a month, bimonthly, twice a year, once a year or other diet without requiring excessive experimentation. It may last as long as your doctor's prescription says.

In addition, the compounds of the present invention, combinations thereof, and compositions comprising these components have been shown to exhibit hypotensive effects in vivo and to induce NO in vivo. These results apply practically to the treatment of hypertension and clinical conditions involving hypercholesterolemia, in which the NO level is significantly reduced.

Formulations of the compounds of the present invention, combinations thereof and compositions comprising these components are prepared according to standard techniques known in the pharmaceutical, food science, medicine and veterinary arts, in which liquids, suspensions, tablets, It may be prepared in the form of capsules, injectable solutions or suppositories.

The carrier may also be a polymeric delayed release system. Synthetic polymers are particularly useful for the preparation of compositions in which release is controlled. This initial example is to polymerize methyl methacrylate into spheres with diameters of less than 1 micron to form so-called nanoparticles (Kreuter, J. Microcapsules and Nanoparticles in Medicine and pharmacology, M. Donbrow (Ed)). CRC Press, P. 125-148).

A carrier often chosen for pharmaceuticals and more recently for antigens is poly (d, 1-lactide-co-glycolide) (PLGA). It has a long history of medical use in erodible sutures, bone plates and other temporary prostheses that show no toxicity. A wide range of drugs have been formulated with PLGA microcapsules. Data is accumulated according to the application of PLGA (Eldridge, J. H., et al. Current Topics in Microbiology and Immunology, 1989, 146: 59-66.). PLGA centroids with a diameter of 1 to 10 microns can be effective when administered orally. The PLGA microencapsulation process utilizes phase separation of a water-in-oil emulsion. The compound or compounds of the present invention are prepared in aqueous solution and PLGA is dissolved in suitable organic solvents such as methylene chloride and ethyl acetate. These two immiscible solutions are co-emulsified by rapid stirring. A non-solvent for the polymer is added to precipitate the polymer around the aqueous droplets to form undeveloped microcapsules. The microcapsules are collected, stabilized with one of several chemicals (polyvinyl alcohol (PVA), gelatin, alginate, methylcellulose) and then the solvent is removed by vacuum drying or solvent extraction.

Additionally, information on the preparation of slow-release preparations is disclosed in US Pat. Nos. 5,024,843, 5,091,190, 5,082,668 and 4,612,008 and 4,327,725, which are incorporated herein by reference.

Additionally, alternative processing methods associated with the identification of cocoa genotypes of interest are not only a means for delivering a compound of the present invention at a conservative level, but also for delivering the active compound to a patient in need of treatment for the aforementioned diseases. As an excipient can be used to prepare identity standards (SOI) and identity non-SOI chocolate products.

This is disclosed in United States Patent Application No. 08 / 709,406, filed September 6, 1996, which is incorporated herein by reference. USSN 08 / 709,406, which does not require roasting or liquid milling equipment of separate beans, allowing the selection of processing cocoa beans and / or the use of solvent extraction of fats without exposure to strong heat treatment for extended periods of time. A method of producing cocoa solids and / or cocoa butter having a preservation level of polyphenols from cocoa beans is disclosed using a unique combination of steps. The advantage of this method is the enhanced preservation of polyphenols in contrast to those found in traditional cocoa processing, where the ratio of the initial content of polyphenols found in raw beans to the content of polyphenols obtained after processing is less than two.

Compounds of the present invention comprise one or more of the aforementioned compounds in a pharmaceutical form with a pharmaceutically acceptable carrier or excipient, and include anti-cancer, anti-tumor or anti-neoplastic activity. Compounds of the invention having antioxidant activity inhibit DNA topoisomerase II enzymes, inhibit oxidative damage to DNA, induce NO production of monocytes / macrophages, and antibacterial cyclo-oxygenase and And / or have lipoxygenase, NO or NO-synthase, apoptosis, platelet aggregation and blood or in vivo glucose control activity, and have efficacy as a non-steroidal anti-infective factor.

Another embodiment of the invention, together with at least one additional antineoplastic agent, hypotensive agent, anti-infective agent, antimicrobial agent, antioxidant and hematopoietic agent, as well as a pharmaceutically acceptable carrier or excipient, a compound of the present invention or a Compositions comprising combinations.

Such compositions may be used in relation to the dosage required, relative to specific oligomers such as age, sex, weight, genetic conditions and the actual condition of the patient, route of administration, data considered by techniques well known in medicine, nutrition or veterinary medicine. It may be administered to the patient according to concentration toxicity (LD ₅₀ ).

Compositions of the present invention may contain other anti-neoplastic, anti-tumor or anti-cancer agents, antioxidants, DNA topoisomerase II enzyme inhibitors, oxidative damage inhibitors or cyclo-oxygenases and / or lipoxygenases, apoptosis, platelet aggregation of DNA. , With blood or in vivo glucose or NO or NO-synthase modulators, non-steroidal anti-infective agents and / or anti-neoplastic agents, antitumor agents, anticancer agents, antioxidants, DNA topoisomerase II enzyme inhibitors, DNA Reduce the adverse effects of oxidative damage inhibitors, cyclo-oxygenase and / or lipoxygenase, apoptosis, platelet aggregation regulators, blood or in vivo glucose or NO or NO-synthase modulators and / or non-steroidal anti-infective agents Or with the mitigating agent, co-administered or continuous administration taking into account age, sex, weight, genetic status, actual condition of the patient and route of administration.

Examples of compositions of the present invention applied to humans or livestock include not only chewable solid or beverage preparations, but also edible compositions such as, for example, solid or liquid preparations such as capsules, tablets, pills, and the like. The invention is very suitable because it is obtained from edible sources (e.g., cocoa or chocolate flavored solids or liquid compositions): the present invention is suitable for the mouth, nose, anus, vagina, for example, suspensions, syrups or elixirs. Includes liquid compositions administrable via, including compositions flavored with cocoa or chocolate; Parenteral administration, subcutaneous administration, intradermal administration, intramuscular administration or intravenous administration (eg injectable administration) such as sterile suspensions or emulsions. However, the active ingredient in the composition may form a complex with the protein, and when administered to the blood stream, coagulation may occur due to precipitation of blood proteins, which should be considered by those skilled in the art. The active cocoa extract in such compositions can be mixed with suitable carriers, diluents or excipients such as sterile water, physiological saline, glucose, DMSO, ethanol and the like. The active cocoa extract of the present invention may be provided in lyophilized recombination form, for example isotonic saline, glucose or DMSO buffer. Precipitation is observed in some isotonic salt solutions: and such precipitate formation can be used as a method of separating the compounds of the invention, for example as a "salting out" method.

Example 38 illustrates the preparation of a compound of the invention in the form of tablet formulations for application in the pharmaceutical and food sectors. Example 39 also illustrates the preparation of a compound of the invention in the form of a capsule formulation for similar applications. Example 40 is also an identity non-SOI chocolate and identity comprising a cocoa solid or a compound of the present invention obtained by a method disclosed in co-pending US patent 08 / 709,406, which is incorporated herein by reference. Formulations of standard (SOI) are illustrated.

The present invention also includes a kit in which the active cocoa extract is provided. The kit may comprise a separation vessel comprising a suitable carrier, diluent or excipient. In addition, the kit may be used for co-administration or continuous-dose additional anticancer, anti-tumor or anti-neoplastic agents, antioxidants, DNA topoisomerase II enzyme inhibitors or oxidative DNA damage inhibitors or antimicrobial agents or cyclo-oxygenases and And / or lipoxygenase, NO or NO-synthase non-steroidal anti-infective agents, apoptosis and platelet aggregation modulators or blood or in vivo glucose modulators and / or anti-neoplastic, anti-tumor or anticancer agents, antioxidants, DNA topo Isomerase II enzyme inhibitors or antibacterial or cyclo-oxygenase and / or lipoxygenase, NO or NO-synthase, apoptosis, platelet aggregation modulators and blood or in vivo glucose modulators and / or non-steroidal anti-infective agents May contain drugs that reduce or alleviate the adverse effects of The additive (s) may be provided in the form of separate container (s) or in admixture with active cocoa extract. In addition, the kit may comprise instructions for mixing or combining the ingredients and / or mode of administration.

Identification of genes

Another embodiment of the invention includes the regulation of genes expressed as a result of adjacent cell contact by a compound of the invention or a combination of these compounds. In such cases, the compounds of the present invention may be used by the compounds of the present invention or combinations of these compounds, including, but not limited to, various diseases including atherosclerosis, hypertension, cancer, cardiovascular disease and inflammation. Methods for identifying genes that are induced or inhibited. Specifically, genes that are differently expressed in the above-mentioned diseases states compared to those expressed in "normal" non-disease states are identified and described before or after contact with the compounds of the present invention or combinations of these compounds. do.

As mentioned above, the disease and disease state are based in part on free radical interaction with various biomolecules. The main theme of these diseases is that many free radical reactions carry reactive oxygen, which in turn causes the physiological conditions involved in the course of the disease. For example, reactive oxygen is involved in the regulation of transcription factors such as nuclear factor (NF) -KB. Target genes of NF-KB include genes linked to an integrated infectious response. These include genes encoding tumor necrosis factor (TNF) -α, interrokin (IL) -I, IL-6, IL-8, inducible NOS, major histocompatibility complex (MHC) class I antigens, and the like. In addition, genes that regulate transcription factor activity may be induced in turn by oxidative stress. Oxidative stress is an imbalance between the radical removal system and the radical generation system. Several known examples of these conditions (Winyard and Blake, 1997) include gadd153 (a gene induced by growth inhibition and DNA damage), a product that binds NF-IL6 and forms a heterodimer that cannot bind DNA. do. NF-IL6 regulates the expression of several genes, including genes encoding interrokin 6 and 8. Another example of oxidative stress inducible genes is gadd45, which modulates the effect of the transcription factor p53 in growth inhibition. p53 encodes a p53 protein that can stop cell division and cause abnormal cells (eg cancer) to progress apoptosis.

If a whole panoply is provided for the compounds of the invention useful for a variety of diseases based in part on free radical mechanisms or for unexpected and unknown new useful combinations of these compounds, the present invention is directed to a particular disease or condition. Methods for measuring transient effects on gene (s) or gene product (s) expressed by a compound of the invention in an in vitro and / or in vivo model of an animal in a state using a gene expression assay. . This assay is, but is not limited to, based on differential display, cDNA library sequencing, continuous analysis of gene expression (SAGE), expression monitoring by hybridization to high density oligonucleotide sequences and protocols or combinations thereof (Lockhard et al., 1996). One of various reverse transcriptase-polymerized chain reactions (RT-PCR).

The wide range of physiological effects of the compounds of the invention or combinations of these compounds exemplified in the present invention in conjunction with gene evaluation methods, whether known or novel, induced or inhibited, are found in genes and gene products. To be. For example, the present invention relates to in vivo and in vivo cytokine (eg, IL-1, IL-2, IL-6, IL-8, IL-12 and TNF-α) in lymphocytes using RT-PCR. Ex vivo induction and / or inhibition. Similarly, the present invention includes the application of parallax displays to confirm induction and / or inhibition of selection genes; Stimulated Conditions and / or Cardiovascular Fields (eg, TNF-α or H ₂ O ₂ ) under Stimulated Conditions (eg TNF-α or H ₂ O ₂ ) (eg, Superoxide Dismetases, Heme Oxidase, COX1 and 2 and other oxidants Defense genes). In the field of cancer, the present invention identifies the induction and / or inhibition of genes or gene products, such as CuZn-superoxide dismutase, Mn-superoxide dismutase, in cells stressed with regulated oxidants. Application of parallax displays.

The following non-limiting examples are merely illustrative and should not be considered as limiting the invention, and many variations are possible without departing from the spirit and scope of the invention.

Example 1 Cocoa Source and Preparation Method

Several theobroma cacao genotypes (Enriquez, 1967; Engels, 1981), representing three recognized horticultural varieties of cocoa, were obtained from three of the world's major cocoa producers. These genotypes used in this example are shown in Table 1. The harvested cocoa pods are opened and the pulp beans are removed for freeze drying. The pulp was manually removed from the lyophilized mass and then used to analyze the beans as follows. First, unfermented lyophilized cocoa beans were manually peeled and then crushed into fine powder masses using a TEKMAR mill. The resulting mass was degreased overnight by Soxhlet extraction using hexane re-distilled as solvent. The residual solvent was removed from the mass degreased under vacuum at ambient temperature.

Table 1. Description of Theobroma Cacao Material

Example 2 Procyanidin Extraction Process

A. Method 1

Prosanidine was extracted from the degreased and unfermented lyophilized cocoa beans of Example 1 using a method modified by Jaral and Choline (1977). Procyanidin was extracted from 50 grams of a batch of degreased cocoa lumps containing 2 × 400 mL of 70% acetone / deionized water and 400 mL of 70% methanol / deionized water. The extracts were collected and then the solvent was removed by evaporation at 45 ° C. using a rotary evaporator fixed under partial vacuum. The resulting aqueous phase was diluted to 1 L with deionized water and then extracted twice with 400 mL CHCl ₃ . The solvent phase was discarded. The aqueous phase was extracted four times with 500 mL ethyl acetate. The resulting emulsion was triturated with Sorval RC 285 centrifugation, which was operated at 2,000 x g for 30 minutes at 10 ° C. 100-200 mL of deionized water was added to the combined ethyl acetate extracts. The solvent was removed by evaporation at 45 ° C. using a rotary evaporator fixed under partial vacuum. The resulting aqueous phase was frozen with liquid N ₂ and then lyophilized according to the LABCONCO lyophilization system. The yield of crude procyanidins obtained from different cocoa citronoids is shown in Table 2.

Table 2: Yield of Zoprocyanidine

B. Method 2

Optionally, procyanidin was extracted from the degreased unfermented lyophilized cocoa beans of Example 1 using 70% aqueous acetone. Ten grams of degreased material was suspended for 5 minutes using 100 mL solvent. The slurry was centrifuged at 3000 x g for 15 minutes at 4 ° C, then the supernatant was passed through glass wool. The filtrate was distilled under partial vacuum, and the resulting aqueous phase was frozen in liquid N ₂ and then lyophilized according to the LABCONCO lyophilization system. The yield of zoprocyanidine was 15 to 20%.

Without being limited by a particular theory, it is believed that the difference in yield of zoprocyanidins varies according to different genotypes, geographic conditions, horticultural agricultural varieties and production methods.

Example 3 Partial Purification of Cocoa Procyanidins

A. Gel permeation chromatography

The procyanidins obtained in Example 2 were partially purified by liquid chromatography on Sephadex LH-20 (28 × 2.5 cm). The separation step was carried out by gradient from deionized water to methanol. The initial gradient composition, starting with 15% methanol in deionized water, consists of a staged composition every 25 minutes, 25% methanol in deionized water, 35% methanol in deionized water, 70% methanol in deionized water and finally 100% methanol. Was used. Eluates (caffeine and theobromine) following elution of xanthine alkaloids were collected as a single fraction. This fraction yielded a xanthine alkaloid free sub fraction, which was further subdistilled to yield five sub fractions designated MM2A to MM2E. The solvent was removed from each sub fraction by evaporation at 45 ° C. using a rotary evaporator fixed in partial vacuum. The resulting aqueous phase was frozen in liquid N ₂ and then lyophilized overnight according to the LABCONCO lyophilization system. A representative gel permeation chromatogram showing fractionation is shown in FIG. 1. Roughly, 100 mg of material was subfractionated in this manner.

Chromatography conditions: column; 28 × 2.5 cm Sephadex LH-20, Mobile phase: Methanol / water phase gradient, 15:85, 25:75, 35:65, 70:30, 100: 0, 1/2 hourly, flow rate ; 1.5 mL / min, detector; UV at λ ₁ = 254 nm and λ ₂ = 365 nm, chart speed: 0.5 mm / min, column load; 120mg

B. Semi-preparative high performance liquid chromatography (HPLC)

Method 1. Reverse phase separation

Procyanidins obtained from Example 2 and / or Example 3A were partially purified by semi-purified HPLC. A Leodine 7010 injection valve with a 1 mL injection loop, a Hewlett Packard 1050 HPLC system equipped with various wavelength detectors, was assembled with a Pharmacia FRAC-100 fraction collector. A separation step was performed on a Phenomenex Ultracarb 10μ ODS column (250 × 22.5mm) connected to a Phenomenex 10μ ODS Ultracarb (60 × 10 mm) guard column. The mobile phase compositions used under the following linear gradient conditions were A = water; B = methanol: [time,% A]; Compounds were detected by UV at (0, 85), (60, 50), (90, 0) and (110, 0) 254 nm at a flow rate of 5 mL / min.

Representative semi-purified HPLC results for the separation of procyanidins present in fraction D + E are shown in FIG. 15N. Individual peaks or selected chromatographic regions were collected at defined time intervals or manually according to collection of fractions for further purification and subsequent evaluation. The injection load of the material was 25-100 mg.

Method 2. Normal separation

The procyanidin extract obtained from Example 2 and / or Example 3A was partially purified by semi-purified HPLC. A Millipore-Waters Model 480LC detector set at 254 nm, a Hewlett Packard 1050 HPLC system, was assembled with a Pharmacia Frac-100 fraction collector set in peak mode. A separation step was performed on a Sufelco μm Sufelkosil LC-Si column (250 × 10 mm) connected to a Sufelco 5 μm Sufelguard LC-Si guard column (20 × 4.6 mm). Procyanidins were eluted by linear gradient under the following conditions: (time,% A,% B); (0, 82, 14), (30, 67.6, 28.4), (60, 46, 50), (65, 10, 86), (70, 10, 86). The equilibrium was then maintained for 10 minutes. 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 by UV at 245 nm and recorded with a Kipp & Zonan BD41 recorder. The injection volume was 100-250 μ of 10 mg of procyanidin extract dissolved in 0.25 mL of 70% aqueous acetic acid. Representative semi-purified HPLC results are shown in FIG. 15O. Individual peaks or selected chromatographic regions were collected at set time intervals or manually according to fraction collection for further purification and subsequent evaluation.

HPLC conditions: 250 × 10 mm Sufelco Sufelcosyl LC-Si

(5 μm) semi-preparation column

20 × 4.6mm Sufelco Sufelcosil LC-Si

(5 μm) guard column

Detector: Waters LC

Spectrophotometer model

480 @ 254nm

Flow rate: 3 mL / min

Column Temperature: Atmospheric Temperature

Injection amount: 250μL of 70% aqueous acetone extract

Fractions obtained are as follows:

Example 4 HPLC Analysis of Procyanidin Extract

Method 1. Reverse phase separation

The procyanidin extract obtained in Example 3 was filtered through a 0.45μ filter and analyzed by a Hewlett-Packard 1090 triple HPLC system equipped with a diode array detector and an HP model 1046A program fluorescence detector. Flavanol and procanidine were eluted by a column washed with B at a flow rate of 0.3 mL / min followed by a linear gradient from 60% B to A. The mobile phase composition was B = 0.5% acetic acid in methanol and A = 0.5% acetic acid in nano water pure water (hereinafter referred to as nano-pure water). The acetic acid level in the A and B mobile phases can be increased to 2%. Under the conditions of UV at 280 nm and λ _ex = 276 nm and λ _em = 316 nm, the components were detected by fluorometry. The concentrations of (+)-catechin and (-)-epicatechin were determined by comparison with standard solutions. Procyanidin levels were measured using response factors for (−)-epicatechin. Representative HPLC chromatograms showing the separation of various components for one cocoa genotype are shown in FIG. 2A. Similar HPLC profiles were obtained from different cocoa genotypes.

HPLC conditions: Column: 200 × 2.1 mm Hewlett Packard

Hypersil ODS (5μ)

Guard column: 20 × 2.1㎜ Hewlett Packard Hyper Seal ODS (5μ)

Detector: Diode Array @ 280nm

Fluorescence lambda _ex = 276 nm;

λ _em = 316 nm

Flow rate: mL / min

Column Temperature: 45 ℃

Method 2. Normal separation

Procyanidin extracts obtained from Examples 2 and / or 3 were filtered through a 0.45 μ filter and analyzed by a Hewlett Packard 1090 Series II HPLC system equipped with an HP Motel 1046A Program Fluorescence Detector and a Diode Array Detector. A separation step was performed on 100 μm (250 × 3.2 mm) 5 μ phenomenex licrosssphere silica connected to a Spellco Spellguard LC-Si 5 μ guard column (20 × 4.6 mm). Procyanidins were eluted by a linear gradient under the following conditions: (time,% A,% B); (0, 82, 14), (30, 67.6, 28.4), (60, 46, 50), (65, 10, 86), (70, 10, 86). Then re-equilibrium was maintained for 8 minutes. The mobile phase composition was A = dichloromethane, B = methanol and C = acetic acid: volume ratio with water = 1: 1, flow rate of 0.5 mL / min was used. The components were detected by fluorescence under λ _ex = 276 nm and λ _em = 316 nm or by UV at 280 nm. Representative HPLC chromatograms showing the separation of various procyanidins for one genotype are shown in FIG. 2B. Similar HPLC profiles were obtained from different cocoa genotypes.

HPLC conditions:

250 × 3.2 mm Phenomenex Licrosse Silica 100

Column (5μ) 20 x 4.6 mm

Soufelco Soufelgard

LC-Si (5μ) Guard Column

Detector: Photodiode Array @ 280nm

Fluorescence lambda _ex = 276 nm;

λ _em = 316 nm

Flow rate: 0.5 mL / min

Column Temperature: 37 ℃

Example 5 Identification of Procyanidins

Liquid chromatography using a Sephadex LH-20 (28 × 2.5 cm) column followed by a 10 μ Vanda semi-purified HPLC or 5 μ Sufelcosyl LC-Si (250 × 10 mm) using a Pack C18 (100 × 8 mm) column The procyanidins were purified by semi-purified HPLC using a column.

Isolates partially purified by Fast Atomic Impact-Mass Spectrometry (FAB-MS) using the VG ZAB-T high resolution MS system using liquid secondary ion mass spectrometry (LSIMS) in cationic and anionic mode. Analyzed. A cesium ion gun was used as the ionization source at 30 kV, and a "Magic Bullet Matrix" (1: 1 dithiothreitol / dithioerythritol) was used as proton donor.

Analysis by LSIMS for the three fractions revealed the presence of flavan-3-ol oligomers as shown in the table.

Table 3: LSIMS (Cation) Data Obtained from Cocoa Procyanidin Fractions

Major fragment ions were consistent with previously reported studies (Self et al., 1986 and Porter et al., 1991) through the cation and anion FAB-MS analysis of procyanidins. The ion corresponding to m / z 577 (M + H) ⁺ and its sodium adduct at m / z 599 (M + Na) ⁺ showed the presence of double-bonded procyanidin dimers in the isolate. It was noted that higher oligomers form sodium adducts (M + Na ⁺ ) rather than their protonated molecular ions (M + H) ⁺ . Procyanidin isomers B-2, B-5 and C-1 have been tested experimentally based on studies reported by Revila et al. (1991), Selfie et al. (1986) and Porter et al. Procyanidins, ranging from octamers and decamers, were identified by FAB-MS in partially purified fractions. In addition, procyanidins leading to dodecamer were observed by normal HPLC analysis (see FIG. 2B). Table 4 shows the relative concentrations of procyanidins measured in isolates without xanthine alkaloids based on reverse phase HPLC analysis. Table 5 shows the relative concentrations of procyanidins based on normal HPLC analysis.

Table 4: Relative Concentrations of Procyanidins in Isolates without Xanthine Alkaloids

Table 5: Relative Concentrations of Procyanidins in Aqueous Acetone Extracts

FIG. 3 shows various procyanidin structures, and FIGS. 4A-4E show representative HPLC chromatograms for the five fractions used to screen for the following anticancer activity or anti-neoplastic activity. HPLC conditions of Figures 4A-4E are as follows:

HPLC conditions: Hewlett Packard 1090 three

HPLC system with HP Model 1046A Programmable Fluorescence Detector

Column: Hewlett Packard 5μ Hyper Seal ODS (200 × 2.1mm)

Linear change from 60% B to A at a flow rate of 0.3 mL / min B = 0.5% acetic acid in methanol; A = 0.5% acetic acid in deionized water lambda _ex = 280 nm; λ _em = 316 nm.

FIG. 15 O shows a representative semi-purified HPLC chromatogram for 12 additional fractions used to screen anticancer activity or anti-neoplastic activity (HPLC conditions are as described above).

Example 6 Anticancer Activity, Antitumor Activity or Antineoplastic Activity of Cocoa Extract (Procyanidine)

MTT (3- [4,5-dimethylthiazol-2yl] -2,5-diphenyltetrazolium bromide) -microtiter plate tetrazolium toxicity assay, originally developed by Mosman (1983), It was used to screen test samples. Test samples, standard samples (cisplatin and chlorambucil) and MTT reagents were dissolved in 100% DMSO (dimethyl sulfoxide) at a concentration of 10 mg / mL. Serial dilutions were prepared from the stock solution. For test samples, 0.01-100 μg / mL dilutions were prepared in 0.5% DMSO.

All human tumor cell lines were obtained from the American type culture collection. Cells were grown in mono layers in alpha-MEM containing 10% fetal bovine serum, penicillin, 100 units / mL, 100 μg / mL streptomycin and 240 units / mL of nystatin. The cells were maintained in a humidified 5% CO ₂ atmosphere at 37 ° C.

After trypsinization, cells were counted and then adjusted to a concentration of 50 × 10 5 cells / mL (varies depending on cancer cell line). 200 μL of cell suspension was incubated in wells of four rows of 96-well microtiter plates. The cells were allowed to adhere for 4 hours and then 2 μL of DMSO containing the test sample solution was added to 4 wells. In early tour-response observations, test sample dilutions were used in order of size to determine dose ranges. Well absorbance at 540 nm was measured by a BIO RAD MP450 plate reader. The average absorbance of the four wells treated with the test sample was compared to the control and the results were expressed as a percentage of control absorbance +/- standard deviation. Reduction of MTT to the purple formazan product correlates with the linear method of the number of viable cells in the wells. Thus, by measuring the absorbance of the reduced product, one can quantify the probability of survival of cells at a given test sample dose. Control wells contained 1% DMSO at the final concentration.

Two samples were first tested according to this protocol. Sample MMI showed a crude isolate of cocoa procyanidins and contained a detectable amount of caffeine and theobromine. Sample MM2 showed partially separated cocoa procyanidins purified by gel permeation chromatography. Caffeine and theobromine were not present in MM2.

Using the method described above, samples were screened for activity against the following cancer cell lines:

HCT 116 Colon Cancer

ACHN kidney adenocarcinoma (adenocarcinoma)

SK-5 melanoma

A 498 kidney adenocarcinoma

MCF-7 Breast Cancer

PC-3 prostate cancer

CAPAN-2 pancreatic cancer

Little or no activity was observed in MMI for any cancer cell lines. MM2 was observed to have activity against HCT-116, PC-3 and ACHN cancer cell lines. However, MM1 and MM2 have been observed to interfere with MTT so that the decrease in absorbance, which affects the reduction of viable cell numbers, is unclear. In addition, this disturbance was a major error barrier as the chemical reaction proceeded faster in the wells along the perimeter of the plate. Typical examples of these effects are shown in FIG. 5. At high concentrations of test material, it was expected that a large decrease in viable cell numbers would be observed rather than high survival levels. Nevertheless, microscopic observations revealed that the cytotoxic effect appeared in spite of the MTT interference effect. For example, an IC ₅₀ value of 0.5 μg / mL was obtained for the effect of MM2 on ACHN cell lines.

In our opinion, these preliminary test results required supplementation of the analytical procedure to prevent MTT obstruction. This was done as follows. After incubating the plate at 37 ° C. for 18 hours under wet 5% CO ₂ atmosphere, the medium was carefully aspirated and replaced with fresh alpha-MEM medium. On day 3 of the assay the medium was aspirated out of the wells again and replaced with 100 μL of freshly prepared McCoy's medium. 11 μL of 5 mg / mL MTT stock solution in PBS (phosphate buffered saline) was added to the wells of each plate. After incubation at 37 ° C. for 4 hours under wet 5% CO ₂ atmosphere, 100 μL of 0.04N HCl in isopropanol was added to all wells of the plate, and the resulting formazan produced by viable cells. Mix to dissolve. In addition, procyanidins were subfractionated to determine specific components related to activity.

The subfractionation process described above was used to prepare samples for further screening. Five fractions representing the regions shown in FIG. 1 and the distribution component (s) shown in FIGS. 4A-4E were prepared. These samples were encoded with MM2A to MM2E to characterize the assay and to confirm the absence of caffeine and theobromine.

Each fraction was screened separately for HCT-11, PC-3 and ACHN cancer cell lines. The result was that the activity is not concentrated in one particular fraction. This type of result is not considered unusual because components in an "active" naturally occurring isolate can act interdependently. For cocoa procyanidin isolates (MM2), at least 20 detectable components included the isolates. The activity was believed to be related to the combination of components present in different fractions rather than to individual component (s).

Based on these results, it was determined to combine the fractions and to repeat the analysis for the same cancer cell lines. Several fraction combinations showed a cytotoxic effect against PC-3 cancer cell lines. Specifically, the IC ₅₀ values obtained were 40 μg / mL for the MM2A and MM2E combinations, and 20 μg / mL for the MM2C and MM2E combinations, respectively. In addition, activity on HCT-116 and ACHN cell lines was reported, but interference of the MTT indicator prevented accurate observation. In order to improve the data, replication experiments were repeated for HCT-116 and ACHN cell lines. However, these results did not reach conclusions due to bacterial contamination and consumption of test sample material. 6A-6D show dose-response correlations between a combination of cocoa extract and PC-3 cancer cells.

Nevertheless, from this data, cocoa extracts, especially cocoa polyphenols or procyanidins, have significant antitumor activity against PC-3 (prostate), HCT-116 (colon) and ACHN (kidney) cancer cell lines in humans; It has become clear that it has anticancer or anti-neoplastic activity. In addition, these results indicated that certain procyanidin fractions may be responsible for activity on PC-3 cell lines.

Example 7: Anticancer, Antitumor or Antineoplastic Activity of Cocoa Extract (Procyanidine)

In order to confirm the observations and further study the fraction combinations, another comprehensive screening was performed.

All prepared materials and methods are the same as described above, except that 4 standard replicates per test dose were increased to 8 or 12 standard replicates per test dose. For this experiment, five individual cocoa procyanidin fractions and combinations thereof were screened for the following cancer cell lines.

PC-3 prostate

KB Nasopharyngeal / HeLa

HCT-116 Colon

ACHN height

MCF-7 Breast

SK-5 melanoma

A-549 lung

CCRF-CEM T-Cell Leukemia

Individual screening consisted of analyzing different dose levels (0.01-100 μg / mL) of fractions A, B, C, D and E (supra) for each cell line. Combination screening yields fractions of equivalent dose levels for each cell line: A + B, A + C, A + D, A + E, B + C, B + D, B + E, C + D, and C + E. And adding to O + E. The results of these analyzes were discussed individually and summarized in their entirety.

A. PC-3 prostate cell line

7A-7H show typical dosing response relationships between cocoa procyanidin fractions and PC-3 cell lines. 7D and 7E showed that fractions D and E had activity at IC ₅₀ of 75 μg / mL. The IC ₅₀ obtained from the dose-response curves of the other procyanidin fraction combinations was in the range of 60-80 μg / mL when fraction D or E was present. Individual IC ₅₀ values are shown in Table 6.

B. KB nasopharynx / HeLa cell line

8A-8H show typical dose-response relationships between cocoa procyanidin fractions and KB nasopharynx / HeLa cell lines. 8D and 8E show that fractions D and E have activity at IC ₅₀ of 75 μg / mL. 8F-8H show representative results obtained from the study of fraction combinations. In this case, procyanidin fraction combination A + B showed no activity, while fraction combinations B + E and D + E showed activity at an IC ₅₀ of 60 μg / mL. When Fractions D or E were present, the IC ₅₀ obtained from the dose response curves of the other fraction combinations was in the range of 60-80 mg / mL. Individual IC ₅₀ is shown in Table 6. Basically these results were the same as those obtained for the PC-3 cell line.

C. HCT-116 Colon Cell Line

9A-9H show typical dose response relationships between cocoa procyanidin fractions and HCT-116 colon cell lines. 9D and 9E show that fraction E has activity at IC ₅₀ of about 400 μg / ml. This value was obtained by extrapolation of curves. It should also be noted that the slope of the dose response curve of fraction D indicates activity. However, since the slope of the curve was too shallow to obtain a reliable value, no IC ₅₀ value could be measured from this curve. 9F-9H show representative results obtained from the study of fraction combinations. In this case, the porosocyanidin fraction combination B + D exhibits no detectable activity, while the fraction combinations A + E and D + E exhibit an IC ₅₀ of 500 μg / mL and 85 μg / mL, respectively. It showed activity at. The IC ₅₀ value obtained from the dose response curves of the other fraction combinations when fraction E was present was on average about 250 μg / mL. The IC ₅₀ values measured by extrapolation are shown in Table 6.

D. ACHN kidney cell line

10A-10H show typical dose response relationships between cocoa procyanidin fractions and ACHN kidney cell lines. 10A-10E showed that the individual fractions showed no activity against this cell line. 10F-10H show representative results obtained from the study of fraction combinations. In this case, procyanidin fraction combination B + C showed inactivation, while fraction combination A + E showed an IC ₅₀ value of about 500 μg / mL, determined by extrapolation. Dose response curves similar to the C + D combinations were considered to be inactive because the slope was very shallow. IC ₅₀ values determined by extrapolation of the different fraction combinations are shown in Table 6.

E. A-549 Lung Cell Line

11A-11H show typical dose response relationships between cocoa procyanidin fraction and A-549 lung cell line. Individual fractions or combinations of these fractions at the doses used in the assay did not show activity. Nevertheless, procyanidin fractions may have utility for this cell line.

F. SK-5 melanoma cell line

12A-12H show typical dose response relationships between cocoa procyanidin fractions and SK-5 melanoma cell lines. Individual fractions or combinations of these fractions showed no activity at the doses used for analysis. Nevertheless, procyanidins may have utility for this cell line.

G. MCF-7 Breast Cell Line

13A-13H show typical dose response relationships between cocoa procyanidin fractions and MCF-7 breast cell lines. Individual fractions or combinations of these fractions showed no activity at the doses used for analysis. Nevertheless, procyanidin fractions may have utility for this cell line.

H. CORF-CEM T-Cell Lucemia Line

Typical dose response curves were originally obtained for the CCRF-CEM T-cell Lucemia line. However, a fine calculation of cell number versus time at different fraction concentrations indicates that 500 μg of fractions A, B, and D affect 80% growth reduction over 4 days. Representative dose response relationships are shown in FIG. 14.

I. summary

The IC ₅₀ values obtained in this assay are listed in Table 6 for all cell lines except CCRF-CEM T-cell Lucemia. T-cell Lucemia data were randomly omitted from the table because different assays were used. The general summary of the present results is that the most active are related to fractions D and E. These fractions are most active against PC-3 (prostate) and KB (nasoparingial / Hela) cell lines. These fractions were also demonstrated to be active against HCT-116 (colon) and ACHN (lenal) cell lines despite high doses. No activity was detected for MCF-7 (chest), SK-5 (melanoma) and A-549 (lung) cell lines. Nevertheless, however, procyanidin fractions have utility with respect to these cell lines. Activity was also shown for CCRF-CEM (T-cell Lucemia) cell line. It should also be mentioned that fractions D and E are the most complex in composition. Nevertheless, it is clear from this data that cocoa extracts, especially cocoa prasiadinin, have important anti-tumor, anti-cancer or anti-neoplastic activity.

Table 6. IC of Cocoa Procyanidin Fractions for Various Cell Lines ₅₀ value

(IC at μg / mL ₅₀ value)

Values above 100 μg / mL were estimated from dose response curves.

Example 8. Anti-cancer, anti-tumor or anti-neoplastic activity of cocoa extract (procyanidine)

Several additions to the ex vivo assay methods were used to supplement and expand the results shown in Examples 6 and 7.

Method A. Crystal Violet Staining Assay

All human tumor cell lines were obtained by American type culture collection.

The cells were grown as monolayers in IMEM with 10% young bovine serum without antibiotics. Cells were maintained in a humid, 5% CO ₂ environment at 37 ° C.

After trypsinization, cells were counted and adjusted to a concentration of 1,000-2,000 cells per 100 mL. Cell proliferation was determined by plating cells (1,000-2,000 cell / well) in 96 well microtiter plates. Cells were attached for 24 hours after addition of 100 μL cells per well. After 24 hours various cocoa fractions were added at different concentrations to obtain a dose response result. Cocoa fractions were dissolved in medium at 2x concentration and 100 μL of each solution was added to 3x wells. The next day, the plates were stained with 50 μL crystal violet (125 mL methanol, 2.5 g crystal violet dissolved in 375 mL water) for 15 minutes. The dye was removed and the plate was immersed in cold water to remove excess dye. The plates were dried after two more washes. The remaining dye was dissolved by adding 100 μL of 0.1 M sodium citrate / 50% ethanol to each well. After lysis, the number of cells was counted with an ELISA plate reader (reference filter at 410 nm) at 540 nm. The results from the ELISA reader are shown with the y-axis as absorbance and the x-axis as the daily growth rate.

Method B. Gentle Bovine Cloning Assay

The cells were cloned in soft daikon according to the method described by Nanata et al. (1981). Single cell suspensions were formed in medium with varying concentrations of cocoa fraction and 0.8% agar. Suspensions were divided into 35 mm dishes coated with medium containing 1.0% agar. After 10 days of incubation, the number of colonies with a diameter of 60 μm or more was determined on an Ominicron 3600 Image Analysis System. The results are plotted on the y-axis as the number of colonies and the x-axis as the concentration of cocoa fraction.

Method C. XTT-Microculture Tetrazolium Assay

The XTT assay method described by Scudiero et al. (1988) was used to screen various cocoa fractions. The XTT assay was completely identical to the assay described using the MTT method (Example 6) with the following modifications. XTT ((2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5-((phenylamino) carbonyl) -2H-tetrazolium hydroxide) was preheated to 37 ° C., Prepared in 1 mg / mL medium without serum PMS were prepared in 5 mM PBS XTT and PMS were mixed with each other; 100 μL of PMS and 50 μL of PMS-XX per mL of XTT were added to the wells, respectively. After incubation at 37 ° C. for 4 hours, the mixture was mixed for 30 minutes in a mechanical shaker, and the absorbance was measured at 450 to 600 nm. The results are shown with the y-axis as the absorbance and the x-axis as the daily growth rate.

For methods A and C, the results were also plotted as percentage control on the y-axis and daily growth rate or concentration on the x-axis.

A comparison of the XTT assay and the crystal violet assay consisted of cocoa fractions D & E on breast cancer cell line MCF-7 P168 (Example 3B) to determine which assay was the most sensitive. As shown in FIG. 15A, the two assays showed the same dose-response effect at concentrations above 75 μg / mL. At concentrations below 75 μg / mL, the crystal violet assay showed a higher standard deviation than the XTT assay. However, crystal violet analysis is easy to use, so all subsequent methods were performed this way unless otherwise specified.

Crystal violet assay results were shown to demonstrate the effect of crude polyphenol extract (Example 2) in breast cancer cell line MDA MB231, prostate cancer cell line PC-3, breast cancer cell line MCF-7 p163, and cervical cancer cell line HeLa, respectively (FIG. 15B). -15E). In all cases, the dose of 250 μg / mL completely inhibited the growth of all cancer cells over a period of 5-7 days or longer. The Hela cell line appeared to be more sensitive to extracts because the 100 μg / mL dose also inhibited growth. Cocoa fractions from Example 3B were also analyzed for Hela and other breast cancer cell lines SKBR-3. The results shown in fractions D & E (FIGS. 15F and 15G) have high activity. IC ₅₀ values of D & E of about 40 μg / mL were obtained from two cancer cell lines as shown in FIGS. 15H and 15I.

Cocoa fractions D & E were also tested in a gentle bovine cloning assay to determine the ability of the test compound (s) to inhibit anchorage independent growth. As shown in FIG. 15J, the concentration of 100 μg / mL completely inhibited clonal formation of Hela cells.

Crude polyphenol extracts from eight different cocoa genotypes representing three horticultural varieties of cocoa were also analyzed for Hela cell lines. As shown in FIG. 15K, all cocoa varieties showed similar dose-response effects. UIT-1 species showed the highest activity against Hela cell line. These results demonstrated that all cocoa genotypes have polyphenol fractions that induce activity against at least one human cancer cell line independent of geographic origin, horticultural variety and genotype.

Another series of analyzes was performed on crude polyphenol extracts, which were traditionally fermented on a one-day basis from Brazilian cocoa beans on a one-day basis for five days and after four days of sun drying. The results shown in FIG. 15L did not show the apparent effect of the previous process steps, suggesting little change in the polyphenol composition. However, it is known that polyphenol oxidase (PPO) will oxidize polyphenols during the fermentation step (Lehrian and Patterson, 1983). Other experiments will be conducted to determine how the polyphenols oxidized by the enzyme have an effect on activity. Crude PPO was prepared by extracting brazilian cocoa beans that were finely ground and not fermented with acetone at a ratio of 1 gm powder to 10 mL acetone. The slurry was centrifuged for 15 minutes at 3,000 rpm. Centrifugation was repeated three times with the fourth extract poured through a Buchner filtration funnel, discarding the supernatant every hour. Acetone powder was dried in air and then analyzed according to the method described by Mcrod and Calara (1983). To the solution of the crude polyphenol (100 mg / 10 mL citrate-phosphate buffer, 0.02 M, pH 5.5) was added 100 mg (4,000 units of active / mg protein) of acetone powder, followed by a stream of air through the slurry. The sample was centrifuged at 5,000 × g for 15 minutes and the supernatant was extracted 3 × with 20 mL ethyl acetate. Ethyl acetate extracts were mixed, dried by distillation in partial vacuum and 5 mL water was added and then lyophilized. Substances were then analyzed for Hela cells, and the dose-response was compared to crude polyphenol extracts that were not processed by enzymes. Results (FIG. 15M) show significant shifts in the dose-response curves for extracts oxidized by enzymes, indicating that the oxidized products are less inhibited than their original form.

Example 9 Antioxidant Activity of Cocoa Extracts Containing Procyanidins.

Evidence from the literature illustrates the relationship between the consumption of naturally occurring antioxidants (vitamins C, E and beta-carotene) and the low incidence of diseases, including cancer (Designing Foods, 1993; Karagai, 1992). It is believed that the agent will affect the process of oxidative free radicals, including the promotion of certain types of tumors. The polyphenolic compounds of some plants that exhibit anticarcinogenicity also have substantially antioxidant activity (Ho et al., 1992; Huang et al., 1992).

Standard Rancimat method was applied to determine whether the cocoa extract containing procyanidins had antioxidant properties. The methods described in Examples 1, 2 and 3 were used to produce more processed cocoa extracts to make two fractions from gel permeation chromatography. These two fractions are actually fractions A-C bound and D and E with antioxidant properties (see FIG. 1) are compared with synthetic antioxidants BHA and BHT.

Peanut oil was produced by peeling unbaked peanuts after peeling. Each test compound was spiked into oil at two levels, -100 ppm and -20 ppm, the actual levels given in Table 7.

50 μL of methanol in which antioxidant was dissolved was added to each sample to aid in the dispersion of the antioxidant. Control samples were prepared with 50 μL of methanol without antioxidants.

Samples were evaluated for replication for oxidative stability using the Rancimat stability test in air at 100 ° C., 20 cc / min. Experimental parameters were selected to match those used with the Active Oxygen Method (AOM) or the Swift Stability test (Ban Woosten et al., 1981). A typical lancet mat line is shown in FIG. 16. As a result, the time required to reach the peroxide level of 100 meq is shown in Table 8.

Table 7. Concentrations of Antioxidants

Table 8. Oxidative Stability of Peanut Oil with Various Antioxidants

These results demonstrate the increased oxidative stability of the peanut oil with all tested additives. The high rate of increase in oxidative stability is achieved by crude ethyl acetate extract of cocoa and spiked samples. These results demonstrated that cocoa extract containing procyanidins had the same or more antioxidant potential as synthetic BHA and BHT. Thus, the present invention can be applied in place of BHT or BHA, for example in the known use of BHA or BHT as an antioxidant and / or food additive. And in this connection it is also known that the present invention is derived from an edible source. Given the results, those skilled in the art can also readily determine the appropriate amount of the present invention for application to the use of "BHA or BHT", such as for example to add to food, without inappropriate experimentation.

Example 10: Topoisomerase II Inhibition Study

DNA topoisomerases I and II are enzymes that promote the breakdown and binding of DNA chains, thereby regulating the topological states of DNA (Wang, 1985). One of the most important findings besides studies of the intracellular function of topoisomerase is not only epipodophyllotoxins of interest, but also intercalating agents (m-AMSA, adriamcin). And ellipticin), demonstrating topoisomerase II as a primary cellular target for many clinically important anticancer compounds (Yamashita et al., 1990). Several lines of evidence are common to stabilize DNA-topoisomerase II complexes ("isolation complexes") exposed to denaturing reagents, which some anti-tumor drugs induce DNA isolation (Muller et al., 1989). It has a phosphorus property. The formation of isolation complexes by anti-tumor drugs suggests that they produce bulky DNA adducts that can kill cells.

According to this interesting model, specific new derivatives of the DNA topoisomerase II separation complex are useful as reagents for anti-cancer, anti-tumor, antineoplastic. In an attempt to prove that the cytotoxin compound with activity is the target DNA, cocoa procyanidins have been screened for enhanced cytotoxin activity against several DNA-damages sensitive to cell lines, and enzymatic analysis of human topoisomerase II Was obtained from lymphoma.

A. Isolation of Kinetoplasm DNA by Topoisomerase II.

In vitro inhibition of topoisomerase II isolation in kinetoplasmic DNA described by Muller et al. (1989) is performed as follows.

Nuclear extracts with topoisomerase II activity were prepared from human lymphomas by modifying the methods of Millet et al. (1981) and Gdansk et al. (1988). One unit of purified enzyme was sufficient to separate 0.25 μg of kinetoplasmic DNA at 34 ° C. for 30 minutes. Kinetoplasm DNA was obtained from trypanosome Crithidia fasciculata . Each reaction was carried out with 19.5 μL H ₂ O, 2.5 μL 10 × buffer (1 × buffer with Tris-HCl 50 mM, KCl 120 mM, MgCl _{2 10} mM, ATP 0.5 mM, 0.5 mM Dithiothreitol and 30 μg of PH 8.0). BSA / mL), 0.5 μg of microcentrifuge tube with 1 μL DMSO including Kineticplast DHA 1 μL (0.2 μg), and cocoa procanidine test fractions of varying concentrations. This combination was thoroughly mixed and left on ice. One unit of topoisomerase was added immediately and incubated in a 34 ° C. water bath for 30 minutes.

After incubation, the separation assay was stopped by the addition of 5 μL of stop buffer (5% sarcosyl, 0.0025% bromophenol blue, 25% glycerol) and placed on ice. DNA was electrophoresed on 1% agarose gel in TAE buffer containing ethidium bromide (0.5 μg / mL). Ultraviolet light at 310 nm wavelength predicted DNA. Gel was taken using a Polaroid Land Camera.

17 shows the results of these experiments. Fully bound Kinetoplasm DNA does not migrate to 1% agarose gel. Isolation of kinetoplasmic DNA by topoisomerase II produces a band of monomeric DNA (forming monomer circles, I and II) that migrate to the gel. Inhibition of the enzyme by the addition of cocoa procyanidins is evident as the monomer band gradually disappears with increasing concentration. Based on these results, it was known that cocoa procyanidin fractions A, B, D and E inhibited topoisomorase II at concentrations ranging from 0.5 to 5.0 μg / mL. These inhibitor concentrates were very similar to those obtained for mitoxantrone and m-AMSA (4 '-(9-acridinylamino) methanesulfon-m-anisidide).

B. Drug Sensitive Cell Lines

Cocoa procyanidins have been screened for cytotoxicity against several DNA-damaging sensitive cell lines. One of the cell lines was a repair mutant that cleaved the xrs-6 DNA double chain developed by Blood, Kego (Kemp et al., 1984).

DNA repair deletion of the xrs-6 cell line is particularly insensitive to X-rays, indirectly doubles as suggested by Walter et al. (1991) as it cleaves DNA double chains directly like bleomycin and inhibits topoisomerase II. May lead to cleavage of the chain. Cytotoxicity towards the repair deletion line was compared to cytotoxicity against the DNA repair proficient CHO line, BR1. Enhanced cytotoxicity towards the repair deletion (xrs-6) line was demonstrated by evidence of cleavage formation of the DNA isolated double chain.

The DNA repair competent CHO line, BR1, was developed by Barlows et al. (1987) and expressed as O ⁶ -alkylguanine-DNA-alkyltransferase by addition of normal CHO DNA repair enzymes. The repair deletion line (xrs-6), which cuts the CHO double chain, P. Uplifting and his collaborators (Uplifting et al., 1989) were generous gifts. These two lines grew monolayer in alpha-MEM containing serum and antibiotics as described in Example 6. Cells were maintained at 37 ° C. in a damp 5% CO ₂ environment. Prior to treatment with cocoa procyanidins, cell growth into monolayers was separated by trypsin treatment. It was analyzed using the MTT assay method described in Example 6.

The result of no enhanced cytotoxicity towards xrs-6 cells (FIG. 18) illustrates that cocoa procyanidins inhibit topoisomerase II in a different way than isolated double chain cleavage. That is, cocoa procyanidins interact with topoisomerase II before interacting with DNA to form a non-isolated complex.

Non-isolated complexes that form compounds are a relatively new discovery. Anthraclines, podophylin alkaloids, anthraceenediones, acridines and ellipticines are all clinical anti-cancer, anti-tumor or anti-neoplastic Has been demonstrated for their use, and they create a separation complex (Riu, 1989). Several new types of topoisomerase II inhibitors that have not been shown to form separation complexes have recently been demonstrated. These include amonafide (Hssien et al., 1989), tistamicin (Pessen et al., 1989), flavanoids (Yamasita et al., 1990), sintophins (Yamasita et al., 1991), Membraon (Drake) , 1989), terpenoids (Kawada et al., 1991), anthrapyrazole (Free et al., 1985), dioxopiperazine (Tanabe et al., 1991), and marine acridine-dercithin ( Bures et al., 1989).

Cocoa procyanidins inactivate topoisomerase II before the isolation complex is formed and therefore have chemotherapy values alone or in combination with other known and mechanically defined topoisomerase II inhibitors. In addition, cocoa procyanidin also appears to be a new class of topoisomerase II inhibitors (Kashiwada et al., 1993) and is less toxic to cells than other known inhibitors, thereby increasing utilization in chemotherapy.

Membrane-expressing human breast cancer cell line MCF-7 (ADR) was combined with glycoprotein (gp170) to confer multi-drug resistance (Leones et al., 1994), and its subordinate MCF-7 p168 was cocoa fraction D & It was used to analyze the effect of E. As shown in FIG. 19, paternity was suppressed at increasing dose levels of fractions D & E, where the adriamesin (ADR) resistance line was less effective at higher doses. Cocoa fractions D & E show results that are effective in multi-drug resistant cell lines.

Example 11 Synthesis of Procyanidins

Synthesis of procyanidins was performed by modifying the method developed by Delcoir et al. (1983). After concentrating (+)-catechin and dihydroquecetin under reducing conditions, (-)-epicatechin was also used to show high concentrations of (-)-epicatechin naturally occurring in unfermented cocoa beans. The synthesized product was isolated, purified, analyzed and demonstrated by the methods described in Examples 3, 4 and 5. In this way biflavanoids, triflavanoids and tetraflavanoids were prepared and used as the standard of analysis in the method described above in connection with cocoa extract.

Example 12; Analysis of normal semi-refined fractions

Since polyphenol extracts were structurally complex, the components were active against cancer cell lines for further purification, dose-response analysis and comprehensive structural confirmation. Normal semi-purified HPLC separation (Example 3B) was used to separate cocoa procyanidins based on oligomer size. In addition to the original extract, 12 fractions were prepared (FIGS. 2B and 15 0) and at 100 μg / mL and 25 μg / mL doses for HeLa and SKBR-3 cancer cell lines to determine which oligomers had maximum activity. Analyzed. As shown in FIGS. 20A and B, fractions 4-11 (pentamer-dodecamer) significantly inhibited HeLa and SKBr-3 cancer cell lines at the 100 μg / mL level. These results indicate that specific oligomers have maximum activity against Hela and SKBr-3 cells. In addition, normal HPLC analysis of cocoa fractions D & E showed that fractions used in known studies, for example, Example 7, were enriched with these oligomers.

Example 13 HPLC Purification Method

Method A. GPC Tablet

The procyanidins obtained in Example 2 were partially purified by solution chromatography on Sephadex LH 20 (72.5 × 2.5 cm) using 100% methanol as eluent at a flow rate of 3.5 mL / min. Fractions of the eluate were collected after the first 1.5 hours, and the fractions were concentrated on a rotary evaporator redissolved in water and then lyophilized. These fractions were referred to as pentamer enriched fractions. About 2.00 g of the extract obtained from Example 2 was subfractionated in this manner. The results are shown in Table 9.

Table 9. Composition of Acquired Fraction

Method B. Normal Separation

Procyanidins obtained in Example 2 can be selected from Sufelcosyl LC-Si, 100 μs, 5 μm (250 × 4.6 mm) at 1.0 mL / min, or alternatively 100, 100 μs, 5 μL of crosscross silica at 0.5 mL / min. Classification was carried out by normal chromatography at m (235 x 3.2 mm). Separation was facilitated by a phase 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). The mobile phase composition is A = diclotomentane; B = methanol; And C = acetic acid: water (1: 1). The components were detected by UV of 280 nm, by fluorescence with λ _ex = 276 nm and λ _em = 316 nm. The injection dose was 5.0 μL (20 mg / mL) of procyanidins obtained from Example 2. These results are shown in FIGS. 40A and 40B.

Optionally, separation was facilitated by a step gradient of the following conditions; (Time,% A,% B); (0, 76, 20): (25, 46, 50); (30, 10, 86). The mobile phase composition was 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

Procyanidins obtained in Example 2 were purified by Hewlett Packard Hypersil ODS (5 × m) reversed phase chromatography and Hewlett Packard Hypersil ODS 5μm guard columns (20 × 2.1mm). And separated. Procyanidin was eluted with a linear gradient of 20% B against A for 20 minutes and then the column was washed with 100% B at a flow rate of 0.3 mL / min. The mobile phase composition was a degassed mixture which was 1.0% acetic acid in B = methanol and 2.0% acetic acid in pure water (hereinafter referred to as nano-pure water) at the level of A = nano. The components were detected with UV of 280 nm and fluorescence with λ _ex = 276 nm and λ _em = 316; The injection dose was 2.0 μL (20 mg / mL).

Example 14 HPLC Separation of Pentamer Enhanced Fractions

Method A. Semi-purified Normal HPLC

Pentamer enriched fractions were further purified by semi-purified normal HPLC of a Hewlett Packard 1050 HPLC system equipped with a Millipo-water model 480LC detector set at 254 nm and assembled into a Palmacia Frax-100 fraction precipitator set in peak mode. It became. Separation was effective on a Sufelco 5μm Sufelcocel LC-Si, 100 mm3 column (250 × 10 mm) coupled with a Sufelco 5μ Sufelgard LC-Si guard column (20 × 4.6 mm). Procyanidins were eluted by a linear slope under the following conditions: (time,% A,% B); (0, 82, 14), (30, 67.6, 28.4), (60, 46, 50), (65, 10, 86), (70, 10, 86). Next was 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. Components were detected by UV of 254 nm; It was recorded on a Kipp & Zonan BD41 recorder. Injection doses range from 100-250 μl of 10 mg of procyanidin extract dissolved in 0.25 mL 70% aqueous acetone. Regions of each peak or selective chromatography were collected at regular intervals or manually by further collection of fractions for further purification and subsequent calculations.

HPLC conditions: 250 × 100 nm Sufelco Sufelcosyl LC-Si

(5μm) semi-preparation column

20 × 4.6mm Sufelco Sufelcosil LC-Si

(5μm) guard column

Detector: water LC

Spectrophotometer Model

480 @ 254nm

Flow rate: 3mL / min

Column Temperature: Ambient Temperature

Injection: Pentamer Fortified Extract 250μL

Acetic acid:

Method B. Reverse phase separation

The procyanidin extract obtained in Example 13 was filtered through a 0.45μ nylon filter and Hewlett Packard 1090 Ternary phase HPLC equipped with a diode array detector and an HP model 1046A programmable fluorescence detector. It was analyzed by the system. Separation was effective at 45 ° C., Hewlett Packard 5μ Hypersil ODS column (200 × 2.1 mm). Procyanidin was eluted with a linear gradient of 60% B relative to A, and then the column was washed with B at a flow rate of 0.3 mL / min. The mobile phase composition was a degassed mixture of B = 0.5% acetic acid in methanol and A = 0.5% acetic acid in nano-pure water. The acetic acid level in the A and B mobile phases can be increased by 2%. The components were detected by fluorescence with λ _ex = 276 nm and λ _em = 316 nm and UV at 280 nm. The concentrations of (+)-catechin and (-)-epicatechin were determined relative to the standard solution. Procyanidin levels were measured using the response coefficient of (-)-epicatechin.

Method C. Normal Separation

The pentamer enhanced procyanidin extract obtained in Example 13 was filtered through a 0.45μ nylon filter and analyzed by a Hewlett Packard 1090 Series II HPLC system equipped with an HP model 1046A programmable fluorescence detector and diode array detector. It was effective to separate at 100 ° C (250 × 3.2 mm) 5μ phenomenex recrosspair silica connected to a SUPELCO SUPPELGUARD LC-Si 5μ guard column (20 × 4.6 mm). Procyanidins were eluted by a linear slope 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 re-equilibrium . The composition of the mobile phase was A = dichloromethane, B = methanol, and C = acetic acid: water with a volume ratio of 1: 1. A flow rate of 0.5 mL / min was used. The components were detected by fluorescence with λ _ex = 276 nm, λ _em = 316 nm and UV at 280 nm. Representative HPLC chromatograms showing various procyanidin separations are shown in FIG. 2 for one genotype. Similar HPLC profiles were obtained from other Theobromas , Herrania and / or their crosses or internal specific crosses.

HPLC conditions:

250 × 3.2mm Phenomenex Recrosspair Siliga 100

Column (5μ) 20 × 4.6mm SUPELCO SUFELGUARD LC-Si (5μ) Guard Column

Detector: Photodiode Array @ 280nm

Fluorescence λ _ex = 276 nm; λ _em = 316 nm

Flow rate: 0.5mL / min

Column temperature: 37 ℃

Acetic acid:

Method 4. Manufactured normal separation

The pentamer enriched fractions obtained in Example 13 were further purified by normal chromatography prepared by modifying the method of Rigawood et al., (1993) J. chrome (654, 255-260).

Separation is influenced by ambient temperature in a 5μ Sufelcosyl LC-Si 100 mm column (50 × 2 cm) with a suitable guard column. Procyanidins were eluted by a linear slope 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). Mobile phase components were mixed with the following protocol before use.

Solvent A preparation (82% CH 2 Cl 2, 14% methanol, 2% acetic acid, 2% water);

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

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

3. Measure 560 mL of methanol and pour into the same 4 L bottle.

4. Measure 3280 mL of methylene chloride and pour into 4 L bottles.

5. Close the bottle cap and mix well.

6. Wash the mixture with high purity helium until gas is released for 5-10 minutes.

Repeat steps 1-6 twice until the volume of solvent A reaches 8.

Solvent B Preparation (96% Methanol, 2% Acetic Acid, 2% Water)

1. Measure 80 mL of water and place it in a 4L bottle.

2. Measure 80 mL of acetic acid and place it in the same 4L bottle.

3. Measure 3840 mL of methanol and place it in the same 4L bottle.

4. Close the bottle cap and mix well.

5. Wash the mixture with high purity helium until gas is released for 5-10 minutes.

Repeat steps 1-5 until Solution B has a volume of 4.

The mobile phase composition was A = 2% acetic acid and 2% water methylene chloride; B = methanol with 2% acetic acid and 2% water. Column load is 0.7 g at 7 mL. The components were detected by UV at 254 nm. Normal HPLC separation of typically prepared cocoa procyanidins is shown in FIG. 42.

HPLC conditions:

Column: 50 × 20cm 5μ Sufelcosyl LC-Si Run @ Ambient

Mobile phase: A = methylene chloride with 2% acetic acid and 2% water

B = methanol with 2% acetic acid and 2% water

Gradient / flow profile

Example 15 Identification of Procyanidins

Procyanidins obtained by Example 14, Method 4 were matrix assisted laser dissolution ionization-time of flight / mass spectrometry using an HP G2025A MALDI-TOF / MS system equipped with a LeCroy 9350 500 MHz oscilloscope. (Matrix Assisted Laser Desorption Ionization-Time of Flight / Mass Spectrometry; MALDI-TOF / MS). The device is a low molecular weight peptide standard (HP Part No. G2051A) or peptide standard (HP Part No. G2052A) of 2,5-dihydroxybenzoic acid (DHB) (HP Part No. G2056A) as a sample matrix according to the manufacturer's instructions. It was calibrated with. One (1.0) mg of sample was dissolved in 500 mL of 70/30 methanol / water, after which the sample was mixed with the DHB matrix at a ratio of 1: 1, 1:10 or 1:50 (sample: matrix) and mesa in vacuo. dried in mesa. The sample was analyzed in cation mode with a detector voltage set at 4.75 kV and the laser power was set between 1.5-8 μ J. The data was collected as the sum of many single shots and expressed in units of molecular weight and time flow. Representative MALDI-TOF / MS is shown in FIG. 22A.

The MALDI-TOF / MS spectra obtained from partially purified procyanine prepared as described in Example 3, Method A and used for in vitro evaluation as described in Examples 6 and 7 are shown in FIGS. 22 and C. And the results are summarized in Table 6. This data illustrates that the compounds of the invention described herein are mainly found in fraction D-E and not in fraction A-C.

The spectrum obtained is as follows:

With the exception of the fractions initially purified by the SEP-PACK C-18 cartridge, the purified D-E fractions are by MALDI-TOF / MS as described above. Five (5) mg fractions in 1 mL of nano-pure water were loaded into a pre-equilibrated SEP-PACK cartridge. The column was washed with 5 mL of nano-pure water to remove contaminants and procyanidins eluted with 1 mL of 20% methanol. No further purification, fractions A-C were used directly as isolated in Example 3, Method A.

These results were confirmed and extended the initial results (Example 5, Table 3, see FIGS. 20A and B), indicating that the compounds of the present invention are used as a cation collector. In particular, MALDI-TOF / MS results showed that n = 5 and higher procedanidine oligomers (FIGS. 20A and B; and the structural formulas in the present invention are summarized in HeLa and SKBR-3 cancer cell line models and anti-cancer activity). It is concluded that they are quite related. Oligomers with n = 4 or less have no effect with this model. Pentamer structures clearly have a structural motif that provides activity in and in higher order oligomers. It was also observed that MALDI-TOF / MS data showed strong M ⁺ ions of Na ⁺ , 2Na ⁺ , K ⁺ , 2K ⁺ , Ca ⁺⁺ , demonstrating its use as a cation collecting agent.

Example 16 Purification of Oligomeric Fractions

Method A. Purification by Semi-Purified Reverse Phase HPLC

Procyanidins obtained by Example 14, Methods A, B and D were further separated to obtain experimental amounts of the same oligomers for further structural identification and explanation (e.g. Examples 15, 18, 19 and 20). The Hewlett Packard 1050 HPLC system, equipped with various wavelength detectors and a Leodyne 7010 injection valve with 1 mL injection loop, was assembled with a Pharmacia FRAC-100 fraction collector. It was effective to separate on a Phenomenex Ultracarb 10μ ODS column (250 × 22.5mm) connected to a Phenomenex 10μ ODS Ultracarb (60 × 10 mm) guard column. The mobile phase composition was obtained with the following linear tilt conditions: (time,% A); A = water used at a flow rate of (0, 85), (60, 50), (90, 0) and (110, 0) and 5 mL / min; B = methanol. Each peak or selective chromatography region was collected manually at regular time intervals by fraction collection for further measurement by MALDI-TOF / MS and NMR. Injection loads ranged from 25 to 100 mg of material. Representative elution profiles are shown in FIG. 23B.

Method B. Modified semi-purified HPLC

Procyanidins obtained from Example 14, Methods A, B, and D were further separated to obtain experimental amounts of the same oligomer for further structural identification and explanation (eg, Examples 15, 18, 19, and 20). Sufelcosyl LC-Si 5μ (20 × 2 mm) Sufelcosyl LC-Si 5μ (250 × 10 mm) connected to a guard column. Separation was effective at ambient temperature, flow rate of 3.0 mL / min. The mobile phase composition is A = dichloromethane; B = methanol and C = acetic acid: water (1: 1); It was used under the following linear slope conditions: (time,% A,% B); (0, 82, 14); (22, 74, 21); (32, 74, 21); (60, 74, 50, 4); (61, 82, 14), then the column was re-equilibrated for 7 minutes. Injection amount was 60 μL with 12 mg of enhanced pentamer. The components were detected by UV at 280 nm. Representative eluent profiles are shown in FIG. 23A.

Example 17 Molecular Modeling of Pentamers

The energy to minimize the structure was determined by the 3.0 version of the Desktop Molecular Modeller (Oxford University Press, 1994). Four representative figures of [EC (4 → 8)] _4- EC (EC = epicatechin) based on the structure of epicatechin are shown in FIGS. 24A-D. A spiral structure is expected. In general, the beta sequence appears when epicatechin is the first monomer and the bond is 4 → 8, and the alpha sequence appears when the first monomer is catechin and the bond is 4 → 8; These results are obtained regardless of whether the second monomer is epicatechin or catechin (exception is ent-EC (4 → 8) ent-EC). Figures 38A-38P show preferred pentamers and Figures 39A-39P show libraries of stereoisomers up to and including pentamers. From this, other compounds within the scope of the present invention can be prepared without inappropriate experimentation.

Example 18 NMR Measurement of Pyocyanidins

¹³ C NMR spectroscopy is generally regarded as a useful technique for the study of procyanidins, particularly as a phenol that generally provides a good quality spectrum, where the proton NMR spectrum is quite broad. The ¹³ C NMR spectra of the oligomers provide useful information on the type of A or B ring substitution, in some cases the relative stereochemistry of the C ring, the location of the interflavanoid linkages.

HOHAHA also uses a pulse technique that shifts magnetization from the first hydrogen to the second hydrogen in sequence to obtain cross peaks corresponding to alpha, beta, gamma or delta protons. COSY is a 2D-fourure modified NMR technique, where the vertical and horizontal axes provide ¹ H chemical shift and 1D spectrum; The intersection provides the correlation between protons. This allows the spin-spin coupling to be determined. The HMQC spectrum increases the sensitivity of the NMR spectrum of the nucleus rather than the proton and can exhibit cross peaks from the secondary and tertiary carbons to each proton. APT is a ¹³ C technique used to determine the number of hydrogens in carbon. The even protons on the carbon will result from the plus signal, while the odd protons on the carbon will result from the negative signal.

Therefore, ¹³ C NMR, ¹ H NMR, HOHAHA (homonuclear Hartmann-Hahn), HMQC (heteronuclear multiple quantum coherence), COSY (homonuclear correlation spectro) Scopi Homonuclear correlation spectroscopy, APT (attach proton test) and XHCORR (a variation on HMQC) spectroscopy were used to describe the structure of the compounds of the present invention.

Method A. Monomer

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

44A-E show NMR spectra used to characterize the structure of epicatechin monomers. 44A shows the chemical shifts of ¹ H and ¹³ C in the plate form. 44 BE shows the spectra of ¹ H, APT, XHCORR and COSY of epicatechin.

Similarly, Figures 45A-F show the NMR spectra used to characterize the structure of the catechin monomer. 45A shows the chemical shifts of ¹ H and ¹³ C in the plate form. 44B-F show spectra of ¹ H, ¹³ C, APT, XHCORR and COSY of catechin.

Method B. Dimer

All spectra were obtained at 75% deuterated acetone of D ₂ O using acetone as internal standard, sample concentration of about 10 mg / mL.

46A-G show the spectra used to characterize the structure of the B2 dimer. FIG. 46A shows the chemical shifts of ¹ H and ¹³ C in the plate form. The terms T and B refer to the top half of the dimer and the bottom half of the dimer.

46B and C show ¹³ C and APT spectra obtained at room temperature, Bruker 500MHZ NMR, respectively.

46D-G show ¹ H, HMQC, COZY and HOHAHA, respectively, which were obtained at −7 ° C., AMZ-360 MHZ NMR. COSY spectra were obtained using gradient pulses.

47A-G show the spectra used to characterize the structure of the B5 dimer. 47A shows chemical shifts of ¹³ C and ¹ H in the platy form.

47B-D show ¹ H, ¹³ C and APT, respectively, obtained at room temperature, Bruker 500 MHZ NMR.

47E shows the COSY spectrum obtained at room temperature, AMX-360 using gradient pulses.

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

Method C. Trimmer-Epicatechin / Catechin

All spectra were obtained in 75% deuterated acetone of D ₂ O at −3 ° C. using acetone as internal standard with AMX-360 MHZ NMR at an appropriate sample concentration of 10 mg / mL.

48A-D show the spectra used to characterize the structure of epicatechin / catechin trimer. These figures show ¹ H, COZY, HMQC and HOHAHA, respectively. COSY spectra were obtained using gradient pulses.

Method D. Trimmer-All Epicatechin

All spectra were obtained in 70% deuterated acetone of D ₂ O at −1.8 ° C. using acetone as internal standard with AMX-360 MHZ NMR at an appropriate sample concentration of 10 mg / L.

49A-D show the spectra used to characterize the structure of all epicatechin trimers. These figures represent ¹ H, COZY, HMQC and HOHAHA, respectively. COSY spectra were obtained using gradient pulses.

Example 19 Gathiol Degradation of Procyanidins

In an effort to characterize the structure of procyanidins, benzyl mercaptan (BM) was reacted with catechin, epicatechin or dimers B2 and B5. Floroglucinol and thiophenol as well as benzyl mercaptan can be used for hydrolysis (gathiol degradation) of procyanidins in an alcohol / acetic acid environment.

Catechin, epicatechin or dimer (1: 1 mixture of B2 and B5 dimers) (2.5 mg) was dissolved in 1.5 mL of ethanol, 100 μM BM and 50 μL acetic acid, the vessel (Beckman amino acid assay vessel) emptied and the last removed The reaction vessel was washed with nitrogen until it disappeared and then the reaction vessel was sealed. The reaction vessel was left in a heat block at 95 ° C. and aliquots of the reaction were obtained at 30, 60, 120 and 240 minutes. Relative fluorescence of each aliquot is shown in Figures 25A-C, showing epicatechin, catechin and dimer, respectively. Higher oligomers are similarly hydrolyzed.

Example 20: Gatriol Decomposition and Sulfur Removal of Dimers

Dimers B2 and B5 were hydrolyzed to benzylmercaptan by dissolving dimers (B2 or B5; 1.0 mg) in 600 μl ethanol, 40 μL BM and 20 μL acetic acid. The mixture was heated at 95 ° C. for 4 hours under nitrogen in a Beckman Amino Acid Analysis vessel. Aliquots were removed for analysis by reverse-phase HPLC and 75 μL of ethanol Raney nickel and gallic acid (10 mg / mL) each were added to the remaining reaction medium with 2 mL hypovial. The vessel was washed with hydrogen and shaken frequently for 1 hour. The product was filtered through a 0.45μ filter and analyzed by reverse-phase HPLC. Representative elution profiles are shown in FIGS. 26A and B. Higher oligomers similarly desulfurize. This data anticipates the polymerization of epicatechin or catechin and represents a synthetic route for the preparation of the compounds of the present invention.

Example 21 In Vivo Activity of Pentamers in MDA MB 231 Nude Mouse Model

MDA-MB-231 / LCC6 cell line. Cell lines were grown in improved minimal essential medium (IMEM) containing 10% young bovine serum and maintained at 37 ° C. with wet and 5% CO ₂ .

mouse. Female 6 to 8-week-old NCr nu / nu (artificial) mice were purchased through NCI and housed in animal facilities for the American Association for the Recognition of Laboratory Animal Protection with the United States Department of Agriculture. It was raised in accordance with the regulations issued by the Accreditation of Laboratory Animal Care. Tumored mice were weighed daily as well as weekly to determine the appropriate drug dose.

Tumor transplantation. MDA-MD-231 prepared by tissue culture was diluted to 3.3 × 10 ⁶ cells / mL with IMEM, and 0.15 mL (ie 0.5 × 10 ⁶ cells) was subcutaneously between 2-3 nipples on the side of the mouse. Was injected into. Tumor volume was calculated by multiplication of length × width × height × 0.5. Tumor volumes over the treatment group were averaged and students' tests were used to calculate P values.

Sample manufacturing. Plasma samples were obtained by puncturing the heart and stored at −70 ° C. with 15-20 mM EDTA for blood chemistry determination. No difference was seen between the control and experimental groups.

Fifteen nude mice previously infected with 500,000 subcutaneous cells with tumor cell line MDA-MB-231 were randomly divided into three groups of five each and treated by intraperitoneal injection with one of the following: Plasbo with only DMSO); (Ii) 2 mg / mouse of purified pentamer procyanidin extract isolated from Example 14, Method D in excipients (DMSO); And (iv) 10 mg / mouse of purified pentamer procyanidin extract isolated from Example 14, Method D in excipients (DMSO).

The mice in group (iii) died within about 48-72 hours after administration of 10 mg, but the mice in group (ii) seemed to be normal. The cause of group mice death is unknown; And is unlikely to be essential for the administration of the compounds of the present invention. Nevertheless, 10 mg was considered to be the upper limit on toxicity.

Treatment of groups (iii) and (ii) was repeated once a week, and tumor growth was checked in each experimental and control group. Two weeks after treatment no signs of toxicity were observed in the mice in group (ii), and the doses to these groups were increased to 1/2 log rate every other week. The following table shows the dosages during the treatment schedule of mice in group (ii):

The processing results are shown in Figs. 27A, B and Table 10.

Table 10. In vivo anti-cancer consequences

These results demonstrate that the fractions of the present invention and the compounds of the present invention are indeed useful in anti-neoplastic composition and are less toxic when administered in medium that can be determined without inadequate experimentation, and that when administered in high doses.

Example 22 Antimicrobial Activity of Cocoa Extracts

Method A:

This study was conducted to determine the antimicrobial activity of crude procyanidin extracts from cocoa beans against the types of microorganisms important in food rot or onset. Cocoa extract from Example 2, Method A was used in this study. Dairy medium suitable for the growth of each test culture (99 mL) was propagated in 1 mL of each cell culture suspension in 0.45% saline (final state density 10 ² -10 ⁴ cfu / mL) and then poured into a Petri dish. The hardened radish was cut with a # 2 cork borer (5 mm diameter). The plate was frozen overnight at 4 ° C., the extract was spread with radish and then incubated at a growth temperature appropriate for text organisms. The result is as follows:

Sample area of suppression (mm)

The antimicrobial activity of the purified procyanidin extract from cocoa beans was demonstrated in another study using the diffusion assay (method A) described above with Staphalococcus aureus as text culture.

The result is as follows:

Cocoa Extract: 10 mg / 100 μL of decaffeinated / detheobrominated acetone extract in Example 13, Method A.

10 mg / 100 μL dimer (99% purity) in Example 14, Method D

10 mg / 100 μL tetramer (99% purity) in Example 14, Method D

10 mg / 100 μL hexamer (88% purity) in Example 14, Method D

10 mg / 100 μL octamer / nonamer (92% purity) in Example 14, Method D

10 mg / 100 μL nonamer and higher in Example 14, Method D (87% purity)

Sample area of inhibitor (mm)

Method B:

The crude procyanidin extract of Example 2, Method 2 was added to TSB (Trypticase Soy Broth) with phenol red (0.08 g / L) at various concentrations. TSB is Salmonella enteritidis or S. aureus . Inoculated with S. newport culture (10 ⁵ cfu / mL) and incubated for 18 hours at 35 ℃. The result is as follows:

The acid product shows + = natural growth,-= non-growth, as evidenced by the change from red to yellow in fermentation broth. Inhibition was confirmed by plates from TSB tubes on XLD plates.

This example demonstrates that the chemicals of the present invention are useful in food manufacturing and preservation.

This example also demonstrates gram negative and gram positive bacterial growth that can be inhibited by the compounds of the invention. From this, the compounds of the present invention can be used to inhibit Helicobacter pylori . Helicobacter pylori is associated with the cause of gastric ulcers and gastric cancer. Therefore, the compounds of the present invention can be used for the treatment or prevention of diseases caused by bacteria. Appropriate route of administration, dosage and labeling may be determined without inadequate experimentation, taking into account ingredients well known in the art, such as disease, and the age, weight, sex, and general health of the subject.

Example 23 Analytical Separation of Halogen-Free of Extracts

Procyanidins obtained from Example 2 were partially purified by analytical separation by halogen-glass standard phase chromatography on 100 μs Sufelcosyl LC-Si 5 μm (250 × 4.6 mm) at a column temperature of 37 ° C. and a flow rate of 1.0 mL / min. Separation was aided by the linear gradient under the following conditions: (time,% A,% B); (0, 82, 14); (30, 67.6, 28.4); (60, 46, 50).

The mobile phase composition is A = 30/70% diethyl ether / toluene; B = methanol; And C = acetic acid / water (1: 1). The components were detected by UV at 280 nm. Representative elution profiles are shown in FIG. 28.

Example 24: Pore Size Effect of Stationary Phase for Standard Phase HPLC Separation of Procyanidins

In order to enhance the separation of procadiines, the use of large pore sizes on silica stationary phases was investigated. Separation was carried out on silica-300, 5 μm, 300 mm (250 × 2.0 mm) or optionally silica-1000, 5 μm, 1000 mm (250 × 2.0 mm).

Linear gradients were used as mobile phase compositions, where A = dichloromethane; B = methanol; And C = acetic acid / water (1: 1). The components were detected by fluorometry, where λ _ex = 276 nm and λ _em = 316 nm, which was detected by a UV detector at 280 mm. The flow rate was 1.0 mL / min, and the oven temperature was 37 ° C. Representative chromatograms obtained from three different columns (100 mm 3 pore size, Example 13 Method D) are shown in FIG. This shows an effective pore size for the separation of procyanidins.

Example 25 Obtaining Desired Procyanidins Through Fermentation

Representative microbial strains of strains associated with cocoa fermentation were purchased from M & M / Mars cocoa culture collection:

The following isolates were used:

Acetobacteria Acety ATCC 15973

Lactobacillus sp. (BH 42)

Canda Cruise (BA 15)

Saccharomyces cerevisiae (ba 13)

Bacillus cereus (BE 35)

Bacillus spericus (ME 12)

Each strain was transferred from storage medium to fresh medium. Yeast and acetobacteria were incubated at 26 ° C. for 72 hours, and basil and Lactobacillus were incubated at 37 ° C. for 48 hours. The slants were harvested with 5 ml of phosphate buffer before use.

Cocoa beans are harvested from fresh pods and pulp, and the outer skin is removed. The cocoa beans were sterilized with hydrogen peroxide (35%) for 20 seconds and then treated with hydrogen peroxidase until the end of bubbling. The beans were washed twice with sterile water and then the process was repeated. The beans were divided into glass jars and processed according to the therapies detailed in the table below:

Bench scale fermentation was performed in duplicate.

All treatments were incubated as indicated below.

Day 1: 26 ℃

Day 2: 26 ℃ -50 ℃

Day 3: 50 ℃

Day 4: 45 ℃

Day 5: 40 ℃

Model fermentation was monitored by plate counts over the duration of the study to assess microbial populations and HPLC analysis of fermentation media for microbial metabolites.

After treatment, the beans were dried in a laminar flow hood and roasted at 66 ° C. for 15 minutes to ensure a water activity of 0.64. Samples were prepared for the analysis of procyanidins. Three beans per treatment were ground finely, ground to a powder, followed by fat removal with hexane, followed by extraction with acetone: water: acetic acid (70: 29.5: 0.5%) solution. The acetone solution extract was filtered through a vial and the polyphenol levels were quantified by standard phase HPLC of Example 13 Method B. The remaining beans are ground and ground to taste. Cultures and assay profiles of the model bench-top fermentation process are shown in FIGS. 30A-C. The procyanidin profile of various fermented cocoa beans is shown in FIG. 30D.

This example does not require that the invention be limited to any particular cocoa genotype; And, by fermentation manipulation, it exemplifies the fact that the level of procyanidins produced by specific theobroma or hernia species or their specific heterologous or homologous hybridization can be regulated, eg enhanced.

The following table shows procyanidin levels determined as a sample representing the specific hybrids of theobroma genus and heterologous and allogeneic. Samples were prepared according to Examples 1 and 2 (Methods 1 and 2) and analyzed according to Example 13 Method B. These data show that extracts comprising the compounds of the present invention are found in theobroma and hernia species and their specific hybrids.

Example 26 Effect of Procyanidins on Nitric Oxide (NO)

Method A.

The aim of this study was to establish a relationship between NO and procyanidins known to induce cerebral vasodilation (as in Example 14 Method D) and to nitrates (degradation metabolites of NO) from human umbilical vein endothelial cells (HUVEC). ), The effects of monomers and higher oligomers ranging in concentration from 100 μg / ml to 0.1 μg / ml were evaluated. HUVEC (chronetics) was assessed in the presence or absence of each procyanidin for 24 to 48 hours. At the end of the experiment, the supernatants were collected and collected to determine the nitrate content by thermal calorimetry. In each experiment, HUVECs were incubated with acetylcholine, known to induce NO production, in the presence or absence of procyanidins for 24 to 48 hours. At the end of the experiment, the supernatant was collected and the nitrate content was determined by thermal calorimetry. The role of NO is confirmed by the addition of nitroarginine or (1) -N-methylarginine, which are specific blockers of NO synthase.

Method B Vasorelaxation of Phenylephrine-Induced Contractile Rat Arteries

The effect of each of procyanidins in concentrations of 100 μg / ml to 0.1 μg / ml on rat arteries is a study target for vasorelaxation of phenylephrine-induced contracted rat arteries. The isolated rat arteries are cultured in the presence or absence of procyanidin (Example 14 Method D) and muscular degeneration is confirmed by visual inspection. Both relaxation or contraction of the rat arteries is determined. Then, using other organs, precontraction of the isolated rat artery is induced following the addition of epinephrine.

When contraction is stabilized, the addition of procyanidins determines the contraction or relaxation of rat arteries. The role of NO is confirmed by the addition of nitroarginine or (1) -N-methyl arginine. Acetylcholine-induced relaxation of NO is shown in FIG. 31 because it is caused by rat aorta pre-contracted with phenylephrine.

Method C Induction of hypotension in rats

This method relates to the effect of each procyanidin (as in Example 14, Method D) on blood pressure. An instrument is installed to monitor the peak and trough blood pressure of rats. Each of procyanidins is injected intravenously (dose range = 100-0.1 μg / μg) and the change in blood pressure is assessed. In addition, the effect of each procyanidin on the change in blood pressure induced by epinephrine is determined. The role of NO is confirmed by the addition of nitroarginine or (1) -N-methyl arginine.

Together with the following examples, these studies illustrate that the compounds of the present invention are useful for regulating vasodilation and even more useful with regard to blood pressure control or coronary artery and migraine addressing.

Example 27 Effect of Cocoa Polyphenols on Satin

Five to six 48-year-old healthy adult men aged 48 to determine if cocoa polyphenols regulate glucose levels, using blood glucose levels as indicators of in vivo signal events to control appetite and satiety Experiments were run on the subject.

Cocoa polyphenols of Brazilian cocoa beans were partially purified according to the method described by Glaperton et al. (1992). This substance did not contain caffeine or theobromine at all. Fasted blood glucose levels were tested for the Taxcorasa 75 (caffeine-free) glycos resistance test with and without 75 mg of cocoa polyphenols (Curtain Massison). 091-421) 10 FL. Analyze based on time after feeding oz. This level of polyphenols represents 0.1% of the total glucose of the test beverage and reflects the approximate amount present in the standard 100 g chocolate. Glucose levels of blood were determined using the Accu-Ju III blood glocos monitoring system (Berlinge Mannheim Company). Blood glucose levels were measured before feeding the test beverage and at the following time intervals after feeding the test beverage. : 15, 30, 45, 60, 75, 90, 120 and 180 minutes. Before beginning each glucose tolerance test, a high and low glucose level control was determined. Each glucose tolerance test was run in duplicate. Distilled water 10 fl. A control test solution comprising 75 mg of cocoa phenol dissolved in oz (no glucose) was also run.

Table 11 below shows the dates and controls obtained for each glucose tolerance experiment conducted in this study.

32 depicts the mean values along with the standard deviation of blood glucose levels obtained over a three hour course. It is very clear that blood glucose levels in blood obtained after feeding of a test mixture comprising cocoa polyphenols are significantly increased. The difference between the two major glucose tolerance profiles cannot be resolved by the profile obtained after feeding on a solution of cocoa polyphenol only. The addition of cocoa polyphenols to beverages for glucose testing significantly raises the glucose tolerance profile.

This increase in blood glucose levels is a range that is carefully considered to be diabetes, even though typical glucose tolerance profiles are considered normal (Davidson, I. et al., Eds. Todd-Sandford clinical Diagnosis by Lab oratory.Methods 14; WB Saunders). Co .; Philadelphia, PA 1969, 1025, 550-9). The additional glucose difference is a result of the compounds of the present invention, indicating that it is released from the glycogen reservoir into the bloodstream. Thus, the compounds of the present invention in the presence of sugars can be used to regulate glucose levels in the blood.

Table 11. Glucose Tolerance Test Data and Regulatory Results

Subjects also experienced facial flushing and redness of the head, followed by control of vasodilation following feeding on the compounds of the present invention.

The data in Tables 12 and 13 illustrate that extracts of the present invention belonging to cocoa raw materials and commercial chocolate, and the compounds of the present invention contained therein, can be used as excipients for pharmaceutical, veterinary and food science manufacturing and applications.

Table 13: Procyanidin levels (μg / g) in commercial chocolate

Table 14: Procyanidin Levels (μg / g) in Cocoa Raw Materials

Example 28 Effect of Procyanidins on Cyclooxygenases 1 and 2

The effect of procyanidins on cyclooxygenase 1 and 2 (COX1 / COX2) activity was that arachidonic acid (10 minutes at room temperature for 10 minutes in the presence of a solution of varying concentrations of procyanidins, including monomer-decamer and procanidine mixtures). 5 μm) and cultured the enzymes derived from the ram sperm vesicles and the placenta, respectively. Turnover was assessed using the PGE2EIA kit from Interkim (France). Indomethacin was used as a reference substance.

The results are shown in the table below, where IC ₅₀ values are expressed in units of μM. (Example 13 Except for S11, which represents a procyanidin mixture prepared from Method A, samples SI to S10 here represent procyanidin oligomers (monomers to decamers) according to Example 14 Method D and IC ₅₀ is mg / mL. In units of).

table

Results for inhibition studies are shown in Figures 33A and B, which show the effect of indomethacin on COX1 and COX2 activity. Figure 34A shows the correlation between the degree of polymerization of procyanidins and IC ₅₀ with BCOX1 and COX2: Figure 35 shows the correlation of IC ₅₀ values for COX1 and COX2; And, Fig. 36 through Y shows the IC ₅₀ value of each of the samples (S1 to S11) together with COX1 and COX2. These results indicate that the compounds of the present invention prevent analgesic blood clotting and have anti-inflammatory availability. Moreover, COX2 is also linked to colon cancer. Inhibition of COX2 activity by the compounds of the invention is exemplified by a plausible mechanism that the compounds of the invention have antitumor activity against colon cancer. COX1 and COX2 are also involved in the synthesis of prostaglandins. Thus, the results of this example indicate that the compounds of the present invention are renal function, immune response, fever, pain, mitogenesis, apoptosis, prostaglandin synthesis, ulceration (eg, stomach ) And the reproduction can be adjusted. It should be noted that modulation of renal function may affect blood pressure: compounds of the invention also affect blood pressure control, vasodilation and coronary artery disease (eg, regulation of angiotensyl, bradykinyl). Crazy

References are made to Cybert et al., PNAS USA 91: 12013-12017 (Dec. 1994), Mitchell et al., PNAS USA 90: 11693-11697 (Dec. 1994), Dewet et al. Cell 83: 345-348. (November 3, 1995), Langenbach et al., Cell 83: 483-92 (November 3, 1995), and Shuji et al. Cell 83: 493-501 (November 3, 1995), Morham et al. cell 83: 473-82 (Nov. 3, 1995).

Reference is made further to Examples 9, 26 and 27. Example 9 illustrates the antioxidant activity of the compounds of the present invention. Example 26 illustrates the effect on NO. And Example 27 provides evidence of facial vasodilation.

From the results of this example, in conjunction with Examples 9, 26 and 27, the compounds of the present invention can modulate the free radical mechanisms that induce physiological effects. Similarly, free radical type reactions of lipoxygenase mediated biochemically directed leucotriene synthesis can be modulated by the compounds of the present invention and thus subsequent physiological effects (eg inflammation, immune response). , Coronary artery disease, carcinogenic mechanisms, fever, pain, ulceration).

Thus, in addition to analgesic, the compound of the present invention may have a synergistic effect when administered with other analgesics. Likewise, in addition to having antitumor properties, synergistic effects may be exhibited by the compounds of the invention when administered in combination with other antitumor agents.

Example 29: Study of Circular Dichroism / Procyanidine

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

At a bandwidth of 1.50 nm, samples were scanned every 1.00 nm from 300 nm to 185 nm. Representative CD spectra are shown in FIGS. 43A-G, which show the CD spectrum of octamers from dimers.

These results show the helical properties of the compounds of the present invention.

Example 30 Inhibitory Effect of Cocoa Procyanidins on Helicobacter pyrroli and Staphylococcus Oreus

This study was carried out to evaluate the antimicrobial activity of procyanidin oligomers against H.e. bacteria pylori and Staphylococcus oreus. The pentamer-reinforced material was prepared according to Example 13, Method A and analyzed as described in Example 14 Method C with a pentamer of 89% and an oligo of 11% (n is 6-12). ). Purified pentamers (96.3%) were prepared as described in Example 14 Method D.

Helicobacter pyrroli and Staphylococcus Oreus were purchased from ATCC. In the case of Helicobacter pylori, the vial is hydrated again with 0.5 ml of trypticase soive rod medium and the suspension is transferred to a slant of fresh TSA containing 5% defiber sheep blood. Slants were incubated at 37 ° C. for 3 to 5 days under aerobic conditions in an anaerobic glass vessel (5-10% CO ₂ ; CampyPakplus, BBL) when good growth was achieved in the pool of broad at the bottom of the slant. Broad was used to inoculate additional slants of TSA with sheep blood. The viability harvested from the slant was collected and stored at −80 ° C. as viability decreased with subsequent umbilical culture. Cultures for analysis were used directly from frozen vials. s. Oreus medium was maintained on TSA slants and transferred to fresh slates 24 hours before use.

Cell suspensions of each culture were prepared (H. Pylori, 10 ^{8-10 9} cfu / mL; S. aureus 10 ^{6-10 7} cfu / mL) 0.5 mL was sprayed onto TSA plates with 5% sheep blood. Standard Standard assay discs (manufactured by Difco) were immersed in sterile filters and pentamers were serially diluted (23 mg / mL in sterile water). The test disc and the blank control disc (sterile water) are placed on the inoculated plate. A control disc containing 80 μg metronidazole ( H. pylori inhibitor) or 30 μg vancomycin ( S. aureus inhibitor) (BBL Sensidiscs) is placed on an appropriate plate set. Plates inoculated with H. pylori were cultured under aerobic conditions, and S. aureus sets were cultured aerobicly. Zones with inhibitory effects appeared to multiply well.

Table 14. Biological Analysis of H. pylori and S. aureus Using Pentamers

Example 31 NO-Dependent Hypotension in Guinea Pigs

The effect of five cocoa procyanidin fractions on the blood pressure of guinea pigs was investigated. In brief, guinea pigs (approximately 400 g body weight; male and female) were anesthetized by injection of 40 mg / μg sodium pentobarbital. Cannulating carotid arteries to monitor arterial blood pressure. Each of the five cocoa procyanidin fractions was injected intravenously through the jugular vein (dosage range 0.1 mg / kg-100 mg / kg). The change in blood pressure was recorded on a polygraph. In this experiment, the role of NO was confirmed by dosing L-N-methyl arginine (1 mg / kg) 10 minutes before the cocoa procyanidin fraction was administered.

Cocoa procyanidin fractions were prepared and analyzed according to the method disclosed in US Pat. No. 5,554,645.

Fraction A; Shows the prepared HPLC fractions composed of monomers to tetramers, where the HPLC analysis results are as follows:

Monomer 47.2%

Dimer 23.7%

Trimmer 18.7%

Tetramer 10.3%

Fraction B; The preparative HPLC fractions comprised of pentamers to decamers are shown, and the HPLC analysis results are as follows:

Pentamer 64.3%

Hexamer 21.4%

Heptamer 7.4%

Octamer 1.9%

Nonamer 0.9%

Decamer 0.2%

Fraction C; The nutritionally enriched cocoa procyanidin fraction used in the preparation of fractions A and B is shown, and the HPLC analysis results are as follows:

Monomer 34.3%

Dimer 17.6%

Trimmer 16.2%

Tetramer 12.4%

Pentamer 8.5%

Hexamer 5.2%

Heptamer 3.1%

Octamer 1.4%

Nonamer 0.7%

Decamer 0.3%

Fraction D; Procyanidin extract prepared from milk chocolate is shown. HPLC analysis revealed a composition similar to that described in Table 12 showing Brand 8. There was additionally 6.3% caffeine 10% theobroin.

Fraction E; Represents a procyanidin extract made from dark chocolate made with alkaline liquid. HPLC analysis revealed a composition similar to that described in Brand 12. Caffeine 16.0% Theobroin 5.8% was further present.

In three independent experiments, the effect of the administration of cocoa procyanidin fraction 10 mg / μg on synaptic blood pressure of anesthetized guinea pigs was investigated. At intravenous injection, procyanidin fractions A and E caused a decrease in blood pressure by about 20%. This reduction was not significantly different from that obtained from solvent (DMSO) control (15 ± 5%, n = 5). In contrast, the procyanidin fractions B, C, and D (10 mg / kg) resulted in significant blood pressure reduction, such as 50-60% in C. The order of the hypotensive effects in these experiments was C> B> D> A = E.

Typical records of blood pressure induced after injecting the procyanidin fraction are shown in FIG. 50A for fraction A and FIG. 50B for fraction C. 51 shows the relative effect on blood pressure by these fractions.

L-N-methylarginine (LNMMA) was used to analyze the contribution of NO induced by fraction C administration in hypotensive guinea pigs. Such pharmacological agents inhibit NO production by inhibiting NO synthase. 1 mg / kg of L-NMMA was administered 10 minutes prior to injection of the cocoa procyanidin fraction. As can be seen in Figure 52 administration of L-NMMA to the animal completely block the hypotension caused by procyanidin fraction C. For reference, when the inhibitor was administered as follows, the change in blood pressure caused by Fraction C was similar to that of using only solvent.

Example 32

Effect of Cocoa Procyanidin Fraction on NO Production in Endothelial Cells of Human Umbilical Vein

Human umbilical vein endothelial cells (hereinafter HUVEC) were obtained from Clonetics and cultured according to the manufacturer's instructions. HUVEC cells were seeded in 12 well plate (Falcon). Three cultures under the same conditions resulted in full growth of the cultured cells. Supernatants were replaced with fresh medium containing constant concentrations of bradykinin (25, 50 and 100 nM) or cocoa procyanidin fraction AE (100 u g / mL) as described in Example 31. The incubation lasted for 24 hours and the cell free supernatant was obtained and stored frozen before measuring the NO content described below. In certain experiments, NO synthase (NOS) antagonist N-nitro-L-arginine methyl ester (L-NAME, 10 uM ) was added to assess the association of NOS in NO production.

The nitrite concentration of the culture supernatant by the Griess reaction was measured to evaluate HUVEC NO production. Griess reagent contained 1% sulfonylamide and 0.1% N- (1-naphthyl) -ethylenediamine dihydrochloride. 50 u L aliquots were extracted 4 times from various supernatants and incubated with 150 u L of Griess reagent. Absorbency of 540 nm was measured on a multiscan device (Labsystems Multiskans MCC / 340). Standard curves were obtained using predetermined concentrations of sodium nitrate. The uptake capacity of the cell-free medium (base value) was subtracted from the value of the supernatant including the cells.

Figure 53 shows the effect of bradykinin on NO production of HUVECs when dose dependent NO release was observed. L-NAME antagonists completely inhibited the release of NO induced by bradykinin.

Figure 54 illustrates the effect of cocoa procyanidin fraction on NO production by HUVEC cells. Fractions B, C and D caused some significant NO production by HUVEC. Among them, the measurement by nitrite production showed that fraction C was the most efficient fraction causing NO formation and fraction F had little effect. In the presence of L-NAME, the effect of fraction C on NO production was significantly reduced. For reference, fractions B, C and D contained more procyanidin oligomers than fractions A and B. A notable difference between Fraction D and Fraction E is that Fraction E is made from dark chocolate using alkalized cocoa liquor in the area of making chocolate. Alkalization leads to polymerization of procyanidins by base catalysts and rapidly consumes levels of their compounds. Table 12 analyzes and compares the levels of procyanidins found in this type of chocolate, where brand 12 is dark chocolate with alkaline cocoa liquor and brand 11 is ordinary milk chocolate. Therefore, the extract obtained from milk chocolate contains a high proportion of procyanidin oligomer which can cause NO. NO was clearly inhibited by adding L-NAME, a inhibitor of NO, to fraction C sample. The results obtained in the procyanidin fractions were consistent with those observed in the NO experiments induced with bradykinin (see FIG. 53).

As with the HUVEC results, cocoa procyanidin fraction C induced a major hypotensive effect in guinea pigs, and fractions A and E were most ineffective. High molecular weight procyanidin oligomers have been involved in the regulation of NO production.

Example 33

Effect of Cocoa Procyanidin Fractions on Macrophage NO Production

Heparinized fresh human blood (70 mL) was added to phosphate buffer (PBS) in equal portions at room temperature. A piscol-Hippark solution layer was formed underneath the blood-PBS using a 3 mL Ficoll-Hypaque to 10 mL blood-PBS dilution ratio. The tubes were centrifuged at 2,000 rpm for 30 minutes at 18-20 ° C. The upper layer containing plasma and platelets was discarded. The monosite layer was transferred to another centrifuge tube and the cells were washed twice with Hanks equilibrium saline. The monosite layer was resuspended in complete RPMI 1640 supplemented with 10% fetal calf serum and aggregated and its validity was confirmed by trypan-blue-exclusion. Cell pellets were resuspended at a final concentration of 1 × 10 6 cells / mL in complete RPMI 1640 supplemented with 20% fetal calf serum. Aliquots of the cell suspension were washed three times with complete RPMI 1640 supplemented with 10% fetal calf serum after seeding in 96 well plates and the adherent cells (lymphocytes) were discarded.

The cells were incubated for 48 hours with or without the five procyanidin fractions described in Example 31. After incubation, the medium was obtained and centrifuged and the cell-free supernatant was stored frozen by nitrate assay measurement.

Macrophage NO production was confirmed by measuring the nitrite concentration by Greiss reaction. Griess reagent contained 1% sulfonylamide and 0.1% N- (1-naphthyl) -ethylenediamine dihydrochloride. 50 u L aliquots were extracted four times from various supernatants and incubated with 150 u L of Griess reagent. Absorbency of 540 nm was measured on a multiscan device (Labsystems Multiskans MCC / 340). A predetermined concentration of sodium nitrate was used to build a standard curve. The uptake capacity of the cell-free medium (base value) was subtracted from the value of the supernatant including the cells.

In a separate experiment macrophages were primed in the presence of 5 U / mL of gamma-interferon and then stimulated for 3 hours with 10 u g / mL of LPS in the presence or absence of 100 u g / mL of five procyanidin fractions.

FIG. 55 shows that procyanidin fraction C alone can induce NO production by monosite / macrophage at a concentration of 100 u g / mL. Basal NO production by these cells was not detectable and no nitrite was found in any cocoa procyanidin fraction used at a concentration of 100 u g / mL. 56 demonstrates that procyanidin fractions A and D enhanced LPS-induced NO production by first antigen-stimulated monosite / macrophage with γ-interferon. LPS-stimulated monosite / macrophage cultured without procyanidin fraction produced only 4μ mole / 10 ⁵ cell counts / 48 hours, so procyanidin fraction C appeared to have some negligible effect. γ-interferon alone was ineffective in inducing NO production.

Overall, these results demonstrate that certain concentrations of mixtures of compounds of the present invention can induce monosite / macrophage NO production independent or dependent on stimulation by LPS or cytokines.

As mentioned above, the extracts and cocoa polyphenols, in particular the compounds, compositions, methods and equipment of the present invention, are believed to be of major and versatile utility.

These in vivo and in vitro data clearly demonstrate the utility of the anti-neoplastic agents of the present invention and show that the compounds of the present invention can be used or replaced with existing anti-neoplastic agents.

The compounds of the present invention have antioxidant activity and oxidative stability such as BHT and BHA. Therefore, the present invention can utilize or substitute BHT and BHA in the practical field of BHT and BHA known as antioxidants and food additives.

The present invention can be utilized by replacing or integrating topoisomerase II-inhibitors for the above known practical use.

The compounds of the present invention can be used to preserve and prepare foods, and can also be used to treat or prevent bacteria-induced diseases. It is simply that the compounds of the present invention can be used as fungicides.

Compounds of the present invention may be used as cyclo-oxygenase and lipoxygenase or either, NO, NO-synthase, blood or in vivo glucose modulators, resulting in pain, fever, coronary inflammatory conditions, ulceration, carcinogen mechanisms It is also useful as an agent for the treatment, prevention or alteration of vasodilation, and can also be used as an analgesic, anticoagulant, anti-inflammatory and immune response modulator.

The present invention also encompasses the use of the compounds or extracts as excipients in pharmaceutical formulations. Accordingly, the present invention illustrates several compositions and methods. For example, the present invention includes uses such as antioxidant or preservative compositions, topoisomerase II-inhibitor compositions, methods of preserving food from oxidation, and the like, methods of inhibiting topoisomerase II, and the like. have. The compounds of the present invention may also consist of compositions. The method may consist of contacting food, item and topoisomerase II with the composition or the compound of the present invention. Other compositions, methods, and embodiments of the present invention can be deduced from the above description.

In this regard, the present invention is prepared from edible raw materials, suggesting that the activity of the test tube is at least predetermined in vivo. Based on the in vitro and in vivo experimental data, the dosage, route of administration and composition can be obtained without undue experimentation.

Example 34

Micellar electrokinetic capillary chromatography of cocoa procyanidins

A rapid method was developed using micelle-electrodynamic-capillary chromatography (MECC) to isolate procyanidin oligomers, a modification of the process reported by Delgado et al. In 1994. The same amount separated for 70 minutes by normal normal HPLC analysis can be treated with only 12 minutes by the MECC method. FIG. 57 shows the MECC separation of cocoa procyanidin obtained in Example 2. FIG.

MECC condition

Cocoa procyanidin extract was prepared by the method as described in Example 2. That is, 200 mM boric acid and 50 mM sodium dodecyl sulfate (purified by electrophoresis) consisted of sodium hydroxide to dissolve the cocoa procyanidin extract in a concentration of 1 mg / mL in a MECC buffer adjusted to pH 8.5.

Samples were filtered through a 0.45 um filter and electrophoresed using the Hewlett Packard HP-3D CZE system under the following conditions:

Inlet Buffer: Run the buffer as described above

Outlet buffer: run the buffer as described above

Capillary: 50cm x 75um, bare fused silica

Detection: 200nm, Diode Array Detector

Injection: 50 mBar in 3 seconds (150 mBar per second)

Voltage: 6 Watt

Amperage: system limit line (<300uA)

Temperature: 25 ℃

Capillary Condition: Permeate for 5 minutes with driving buffer before and after operation

The method can be altered by adjusting the temperature, barometric pressure, voltage parameters and organic and chiral selective agents of the operating buffer.

Example 35

MALDI-TOF / MS Analysis of Procyanidin Oligomers with Metal Salt Solutions

A series of MALDI-TOF / MS analyzes were performed on trimers mixed with various metal salt solutions to detect the cationic adducts of oligomers. Chelation or metastasis of metal cations important for physiological processes and diseases demonstrated that procyanidin oligomers play a physiological role in vitro and in vivo. The method described in Example 15 was used. A 2 uL 10 nM solution containing zinc sulfate dihydrate, calcium chloride, magnesium sulfate, ferric chloride hexahydrate, ferrous sulfate heptahydrate and cupric sulfate was prepared in Example 14. As described, the tablets were mixed (10 mg / mL) in each of 4 uL of trimmers purified to homogeneity and then 44 uL of DHB was added.

The results of Figs. 58A-F show ± 1 amu standard deviation values where the m / z values of copper and iron (ferrous and ferric) [metal-trimer + H] + ions correspond to the theoretical estimate of mass. It shows the same. The [metal-trimer + H] + mass of calcium and magnesium can be clearly determined from the [metal-trimer + H] + mass of sodium and potassium whose m / z values fall within the range of ± 1 amu standard deviation. There was no. NO [Zn + 2-trimer + H] + ions were detected. Since some of these cations are polyvalent, either the multimetal-oligomeric ligand species and the metal-oligomeric species or either are possible. However, attempts to scan their adducts at the predicted mass have failed.

The results of copper, iron, calcium, magnesium and zinc may be used in future analysis of the compound reactions of the present invention with other metal ions with reference to the relative positions of the periodic rates of the ions and the oxidation states.

Example 36

MALDI-TOF / MS Analysis of High Molecular Weight Procyanidin Oligomers

The GPC eluent associated with the high molecular weight procyanidin oligomer prepared in the method (A) of Example 3 was assayed. The purpose was to determine if there was a procyanidin oligomer having a value of n> 12. If present, these oligomers illustrate additional compounds of the invention. Anti-cancer, anti-tumor, anti-neoplastic activity, antioxidant activity, DNA topoisomerase II enzyme inhibition, breakdown of DNA by oxidation, antibacterial, NO or NO-synthase, apoptosis, platelet aggregation, The oligomers can be obtained by adjusting the existing sequestration, separation and purification methods implemented in the present invention for later in vitro and in vivo experimental evaluation with respect to blood or in vivo glucose control activity and activity of nonsteroidal anti-inflammatory agents. .

59 shows the MALDI-TOF mass spectrum of the GPC eluent described above. The [M + Na] +, [M + K] + and [M + 2Na] + ions, which can be characterized as tetramers to ten hexamers of procyanidin oligomers, or both ions are clearly shown. have.

It has been found that hydrolysis of procyanidin oligomers is accomplished by acid and heat treatment. Therefore, the controlled hydrolysis method of high molecular weight procyanidin oligomers includes the present invention as part of the process for the preparation of low molecular weight procyanidin oligomers (eg when n is 2 to 12).

Example 37

Dose Response Relationship between Procyanidin Oligomers and Dog and Cat Cell Lines

Dose response of procyanidin oligomers was evaluated in dog and cat cell lines. The cell line was obtained from the Waltham Center for Pet Nutrition, Waltham on-the-Wolds, Melton Mowbray, Leicestershire, UK. . More specifically the cell line is as follows:

Dog normal kidney GH cell line;

Dog normal kidney MDCK cell line;

Cat normal kidney CRFK cell line and

Feline lymphoblast FeA cell line cultured under the conditions described in the method (A) of Example 8 and producing leukemia virus.

In the method described in method (d) of Example 14, monomers of n to 2 to 10 and procyanidin oligomers were purified. As described in the method (C) of Example 14, the oligomer was examined due to analytical normal HPLC to give the following results.

If the purity of the oligomer is less than 90%, it has been refined by the method embodied in the present invention.

Monomers and each procyanidin oligomer were administered to each cell line at concentrations of 10 ug / mL, 50 ug / mL and 100 ug / mL and the results are shown in FIGS. 60-63. As shown in the figure, the high dose (100 ug / mL) of each oligomer resulted in a similar inhibitory effect on feline lymphoblast FeA cell line and feline normal kidney CRFK cell line. In this case, the cytotoxicity was shown in the case of pentamers, and as the number of oligomers increased, high cytotoxicity was induced. In contrast, each oligomer administered at high dose (100 ug / mL) to canine GH and MDCK normal kidney cell lines had higher constituent numbers but developed cytotoxicity. Cytotoxicity occurred in pentamers in the canine GH normal kidney cell line. Cytotoxicity was seen in heptamers in the canine MDCK normal kidney cell line. In both cases, elevated cytotoxicity occurred as the oligomers with high constituents were administered.

Example 38

Composition of tablets

Tablet compositions were prepared using cocoa solids by the method described in US application Ser. No. 08 / 709,406, filed Sep. 6, 1996. Compared to the compound level detected in conventional processed cocoa, the edible substance was prepared by a method for improving the naturally occurring compound level of the present invention. Due to this method, the content of the compound of the present invention before and after processing was treated to be 2 or less. For convenience, the cocoa solids are referred to as CP-cocoa solids. Compounds of the invention treated as isolated and purified or in either form as mentioned in this example can be used to replace or incorporate CP-cocoa solids into tablets.

The tablet composition consisted of the following (in percentages expressed in weight percent):

CP-Cocoa Solids 24.0%

Quadruple Natural Vanilla Extract

Bush Boake Allen 1.5%

Magnesium stearate

(Made by AerChem, Inc., as a dry lubricant) 0.5%

Dipac tabletting sugar 37.0%

Xylitol

American Xyrofin, Inc. 37.0%

100.0%

CP-Cocoa solids and vanilla extract were mixed in a food processor (mixer) for 2 minutes. Sugar and Magnesium stearate were mixed gently and then blended into a CP-cocoa solid / vanilla mixture. This material was treated with a Manesty Tablet Press (B3B) at maximum pressure and molding settings to produce round tablets weighing 1.5-1.8 g. Separate tablets according to this composition were made using commercially available low fat wild cocoa powder (11% fat) instead of CP-cocoa solids. The two tablet compositions provided a level of product that would tolerate seasoning properties and texture.

Analysis of the two tablet compositions was carried out by the method mentioned in the method (2) of Example 4. The assay was focused on the concentration of pentamers and the total levels of monomers and compounds of the invention with n to 2 to 12 and the results are as follows:

The data demonstrate that pentamers and compounds of the invention have higher levels in CP-Cocoa solid tablets than other tablet compositions. Therefore, in oral administration, tablet compositions formulated with CP-cocoa solids are considered excipients suitable for pharmaceutical, supplement and food applications.

Those of ordinary skill in the art will be able to formulate other tablet compositions with a wide range of properties such as: seasonings, colors, excipients, vitamins, minerals, OTC drugs, sugar additives, UV protective agents ( Eg, titanium dioxide, colorants, etc.), binders, aluminum hydroxide gels (hydrogels) and the remaining analogs thereof except polyvinyl pyrrolidone which irreversibly binds the compounds of the present invention or combinations thereof. The dosage of the compounds of the present invention or combinations thereof can be manipulated by adjusting the amount of sugar additives.

Superficial modifications of the present invention are considered to be apparent and can be made without departing from the scope and spirit of this embodiment.

Example 39

Capsule composition

In oral administration of the compounds of the present invention, a modification of Example 38 is a push-fit capsula consisting of gelatin and a sealed soft capsula consisting of plasticizers such as gelatin and glycerol. Push-fit capsulations contain a compound of the invention, combinations thereof, or CP-Cocoa solid powders illustrated in Examples 38 and 40. The dosage of the present invention may optionally be mixed with the powder an additive such as lactose or sucrose. In the case of soft capsules, the compounds of the invention, combinations thereof or CP-cocoa solids are suspended in a suitable liquid such as fatty oil, cocoa butter or mixtures thereof. Since the compound of the present invention may have photosensitivity to UV light, the capsule may contain a UV protective agent such as titanium dioxide or a color to protect from UV. The additives mentioned in the above examples can also be contained in capsules.

Superficial modifications of the present invention are considered to be apparent and can be made without departing from the scope and spirit of this embodiment.

Example 40

Standard of identity (SOI) and Non-standard of identity (non-SOI) dark and milk chocolate

Medical and veterinary SOI and non-SOI dark chocolate and milk chocolate thereof can be prepared from the composition of a compound or combination thereof prepared by the method of the present invention. Co-pending US patent application Ser. No. 08 / 709,406 (filed Sep. 6, 1996) is incorporated herein by reference to a cocoa butter containing a compound of the present invention from cocoa fruit using a unique combination of process steps. And a process for preparing a cocoa solid or a composition of either. The edible cocoa solid obtained by the above process is contrasted with the corresponding compound level of cocoa processed by conventional methods by preserving the naturally occurring compound of the present invention. . That is, the treatment was carried out so that the content ratio of the compound of the present invention before and after processing the cocoa fruit was 2 or less. For convenience, the cocoa solids are referred to as CP-cocoa solids. In making SOI chocolate, non-SOI chocolate, beverages, confectionery, confectionery and cooking ingredients, CP-cocoa solids can be used as powders or liquids.

As used herein, "SOI Chocolate" refers to the Standard of Identity, as amended by the US Food and Drug Administration (FDA) under the Federal Food, Drug and Cosmetic Act. By all means chocolate used in the United States. In the United States, a variety of chocolate definitions and standards are well established, where "non-SOI chocolate" refers to any non-standard chocolate that has a composition that falls outside of a certain range of standard chocolate.

Non-standard chocolate is formed by the following process: for example, when cocoa butter or milk fat is partially or completely replaced; Nutritional carbohydrate sweeteners have been partially or completely replaced; Flavoring flavoring milk, butter, cocoa powder or chocolate is added; And other additions or cuts outside the US FDA Standard of Identity for chocolate or combinations thereof.

As a kind of confectionery, chocolate can have a bar or a new shape of solid form. Chocolate can also be contained in more complex confections as a component. The chocolate may be optionally blended with the following substances: flavors and natural juices, spices and extracts by the Flavor & Extract Manufacturers Association (FEMA), which are classified as natural flavoring substances; The same substance as natural products; International Organization of the Flavor Industry (IOFI) as adopted by the FEMA GRAS Inventory, the FEMA and FDA Inventory, the Council of Europe (CoE) Inventory, and the FAO / WHO Food Standard Program ) Artificial antiseptics as defined in the list, Codex Alimentarius, Food Chemicals Codex list; Artificial flavors which usually coat other foods, such as caramels, nougat, nuts, wafers or the like, The foods are characterized by microbial storage stability at normal air pressure at a temperature of 65-85 ° F. Other composite confections are made by enclosing them in chocolate soft enclosures such as nectar cherries or peanut butter. Composite sweets can also be made by coating ice cream and other frozen or cooled desserts with chocolate. In general, chocolates used for coating or enveloping foods should be more fluid than solid bars or new shaped chocolates.

Incidentally, the chocolate may also be low fat chocolate, consisting of fat and fat free solids, nutritive carbohydrate sweeteners and edible emulsifiers. The following patents may be cited for low-fat chocolate: US Pat. Nos. 4,810,516, 4,701,337, 5,464,649, 5,474,795 and WO 96/19923.

Dark chocolate obtains their dark killer from the chocolate liquor or alkalized liquid or cocoa solid or alkalized cocoa solid used in any given formulation. However, the use of alkalized cocoa solids or liquids is not used in the dark chocolate formulations of the invention, as the contents of Table 13 in Example 27 indicate a loss of the compounds of the invention according to the alkalizing process.

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

The following describes the process steps used to make these chocolate preparations.

Process of non-standard dark chocolate

1. Cover all mixers and refiners throughout the process to avoid light rays.

2. Batch all components except 40% free fat (cocoa butter and anhydrous milk fat) while maintaining the temperature between 30 and 35 ° C.

3. Refine to 20 microns.

4. Dry the conche for 1 hour at 35 ° C.

5. Add a large amount of lecithin and 10% cocoa butter at the beginning of the wet conch cycle: wet the conch for 1 hour.

6. Add all remaining fat, standardize if necessary and mix for 1 hour at 35 ° C.

7. Knead and knead chocolate.

Treatment of Identity Standard Dark Chocolate

1. Batch all ingredients except milk fat at 60 ° C.

2. Refine to 20 microns.

3. Dry the conch at 60 ° C. for 3.5 hours.

4. Add fpcitin and milk fat and wet the conch at 60 ° C for 1 hour.

5. Standardize if necessary and mix for 1 hour at 35 ° C. Knead and knead the chocolate and wrap it.

Processing of Non-Identity Milk Chocolate

1. Cover all mixers and refiners throughout the process to avoid light rays.

2. Batch 66% of the sugar, whole milk powder, malted milk powder and cocoa butter at 75 ° C. for 2 hours.

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

4. Refine to 20 microns.

5. Add the remaining cocoa butter and dry the conch for 1.5 hours at 35 ° C.

6. Add dry milk fat and lecithin and wet the conch for 1 hour at 35 ° C.

7. Standardize, knead and mold chocolate.

Treatment of Identity Standard Milk Chocolate

1. Batch all ingredients except for cocoa butter 65% and milk fat at 60 ° C.

2. Refine to 20 microns.

3. Dry the conch at 60 ° C. for 3.5 hours.

4. Add lecithin, cocoa butter 10% and anhydrous milk fat; Wet the conch for 1 hour at 60 ° C.

5. Add the remaining cocoa butter, normalize if necessary and mix for 1 hour at 35 ° C.

6. Knead and knead chocolate.

CP-Cocoa solids and commercial chocolate liquors used in the formulations were analyzed for the overall levels of pentamers and monomers and for the compounds of the invention, and as described in Examples 4 and 2 before mixing into the formulations, where n is 2-12 These values were used to calculate the expected levels in each chocolate formulation, as shown in Tables 16 and 17. In the case of non-intense non-standard dark chocolate and non-intense non-standard milk chocolate, their products were analyzed similarly for the overall levels of pentamers and monomers and for the compounds of the present invention, where n is 2-12. Are shown in Tables 16 and 17.

The results from these formulation examples show that identity and non-standard dark and milk chocolates formulated with CP-cocoa solids are expected to be about 6.5 times more than expected pentamers, and 3.5 times more than expected identity and identity non-standard dark chocolates. Full level; And about 4.5-fold in identity standard and non-identity milk chocolate; It includes 7.0 times more expected pentamers and 2.5 times more than 3.5 times overall expected levels.

Analysis of some chocolate products is because the difference between the expected levels of the compounds of the present invention present in the final chocolates prepared with CP-cocoa solids is considerably larger than the formulations prepared with commercially available cocoa solids. , Was not performed. However, the effect of the processing was evaluated on identity non-standard dark chocolate products and milk chocolate products. As shown in the table, pentamers experienced 25-50% loss, while only slight differences were observed at the overall level. While not wishing to be bound by any theory, these losses are lower chains from milk components (eg, acetic acid, propionic acid and butyric acid) that can hydrolyze oligomers (eg, trimers can be hydrolyzed into monomers and dimers). It is thought to be due to fatty acids and / or heat. In addition, time consuming process steps may allow oxidation or may allow irreversible binding of the compounds of the invention to protein sources in the formulation. Accordingly, the present invention includes a modification of the methods of chocolate preparation and a process for exhibiting such effects in order to prevent or minimize such losses.

Skilled artisans will recognize numerous modifications in these examples covering a wide variety of formulations, ingredients, processes and mixtures, to reasonably control the naturally present rails of the compounds of the present invention for various chocolate uses. .

Example 41

Hydrolysis of Procyanidin Oligomers

Example 14, Method D, describes a normal HPLC method for purifying compounds of the present invention. Oligomers are obtained as aliquots dissolved in the mobile phase. The solvent is then removed by standard vacuum distillation (20-29 in Hg 40 ° C.) on a Rotovap apparatus.

It has been observed that when the distillation residence time is extended or when the temperature is used above 40 ° C, the loss of certain oligomers occurs as the smaller oligomers increase.

The loss of certain oligomers accompanied by an increase in smaller oligomers was due to time-temperature acid hydrolysis from residual acetic acid present in the mobile phase solvent mixture. This observation was confirmed by the following conformation, where 100 mg of hexamer was dissolved in 50 mL of the mobile phase containing ethylene chloride, acetic acid, water and methane (solvent ratio see Example 14, method D), Treatment with temperature dependent distillation At certain times, aliquots were removed for normal HPLC analysis as described in Example 4, Method 2 The results are illustrated in FIGS. 64 and 65, where hexamer levels were It is reduced in a manner that depends on time-temperature. 65 illustrates the appearance of one of the hydrolysis products (trimers) in a time-temperature dependent manner. Monomers and other oligomers (dimers to pentamers) also appeared in a time-dependent manner.

These results indicated that great care and care must be taken while handling the polymer compounds of the present invention.

In conjunction with Examples 5, 15, 18, 19, 20 and 29, the results provided above illustrate that the method described above can be used to complement other methods embodied in the present invention to identify any given oligomer of the present invention. have.

For example, the complete hydrolysis of any given oligomer that yields (+)-catechin or (-)-epicatechin exclusively eliminates the possibility of oligomer structures based on many "mixed" monomers and removes any given oligomer. It reduces the stereochemical linkability characteristic for each monomer comprising.

Moreover, the complete hydrolysis of any given oligomer yielding both (+)-catechin and (-)-epicatechin in a specific ratio provides the skilled technician with information about the monomer composition of any given oligomer and includes oligomers. It provides specific stereochemical linkage for each monomer.

The skilled artisan can expect that acid catalyzed epimerization reactions of individual monomers can occur, and that moderately controlled experiments and non-violent hydrolysis conditions must be considered (e.g. organic acids such as acetic acid instead of concentrated hydrochloric acid, nitric acid, etc.). Use) will recognize the fact.

Having been described in detail in the preferred embodiments of the present invention, the invention defined by the appended claims is capable of many apparent changes in the art, without departing from the spirit or scope of the invention. It should be understood that it is not limited by the specific details described.

Related Applications

US Application No. 08 / 709,406, filed Sep. 6, 1996, US Application No. 08 / 631,661, filed Apr. 2, 1996, US Application No. 08 / 317,226 filed Oct. 3, 1994 ( US Patent No. 5,554,645) and PCT / US96 / 04497 are still in related applications and are incorporated herein by reference.

Claims (28)

Formula A _n , wherein A is a monomer of the general formula wherein n is an integer from 2 to 18, or a polymer compound or a pharmaceutically acceptable salt, glycoside, ester or oxide thereof and pharmaceutically or veterinary Pharmaceutical or veterinary compositions for the treatment of cardiovascular disease, stroke, rheumatoid arthritis, osteoarthritis, gout, systemic lupus erythema, inflammatory enteritis or spondylitis, including acceptable carriers or excipients: From here, R and X each have an α or β stereochemical structure; R is OH or O-sugars; The substituents for C-4, C-6 and C-8 are X, Z and Y, respectively, and the bonding of the monomer units is at C-4, C-6 or C-8; X, Y and Z are hydrogen, Z and Y are sugars and X is hydrogen, or X is sugar and Z and Z are not bonded to any of the other monomeric units, either C-4, C-6 or C-8. Y is hydrogen or a combination thereof, provided that Y and Z of the first monomer unit are hydrogen when the second monomer unit is bonded to C-4 of the first monomer unit; The sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety by an ester bond. Represented by the general formula An, wherein A is a monomer of the general formula wherein n is an integer from 2 to 18, or a polymer compound or a pharmaceutically acceptable salt, glycoside, ester or oxide thereof and pharmaceutically or veterinarily acceptable Pharmaceutical or veterinary compositions for treating patients in need of nonsteroidal anti-inflammatory agents, including possible carriers or excipients: From here, R and X each have an α or β stereochemical structure; R is OH or O-sugars; The substituents for C-4, C-6 and C-8 are X, Z and Y, respectively, and the bonding of the monomer units is at C-4, C-6 or C-8; X, Y and Z are hydrogen, Z and Y are sugars and X is hydrogen, or X is sugar and Z and Y is hydrogen or a combination thereof, provided that Y and Z of the first monomer unit are hydrogen when the second monomer unit is bonded to C-4 of the first monomer unit; The sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety by an ester bond. Trimmer selected from [EC- (4β → 8)] _2- EC, [EC- (4β → 8)] _2- C and [EC- (4β → 6)] _2- EC, [EC- (4β → 8)] )] ₃ -EC, [EC- (4β → 8)] ₃ -C and [EC- (4β → 8)] ₂ -EC- (4β → 6) -C, a tetramer selected from [EC- (4β] → 8)] ₄ -EC, [EC- (4β → 8)] ₃ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₃ -EC- (4β → 8) -C and Pentamers selected from [EC- (4β → 8)] ₃ -EC- (4β → 6) -C, [EC- (4β → 8)] ₅ -EC, [EC- (4β → 8)] _4- EC- (4β → 6) -EC, [EC- (4β → 8)] ₄ -EC- (4β → 8) -C and [EC- (4β → 8)] ₄ -EC- (4β → 6)- Hexamer selected from C, [EC- (4β → 8)] ₆ -EC, [EC- (4β → 8)] ₅ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₅ -EC- (4β → 8) -C and [EC- (4β → 8)] ₅ -EC- (4β → 6) -C is a hepta selected from [EC- (4β → 8)] ₇ -EC , [EC- (4β → 8)] ₆ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₆ -EC- (4β → 8) -C and [EC- (4β → 8)] )] Octamer selected from _6- EC- (4β → 6) -C, [EC- (4β → 8)] _8- EC, [EC- (4β → 8)] _7- EC- (4β → 6) Nonamer selected from -EC, [EC- (4β → 8)],-EC- (4β → 8) -C and [EC- (4β → 8)] ₇ -EC- (4β → 6) -C, and [EC- (4β → 8)] ₉ -EC, [EC- (4β → 8)] ₈ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₈ -EC- (4β → 8)- A compound selected from decamers selected from C and [EC- (4β → 8)] ₈ -EC- (4β → 6) -C (wherein "EC" represents an epicatechin moiety and "C" represents a catechin moiety) Or a pharmaceutically acceptable salt, glycoside, ester or oxide thereof and a pharmaceutically or veterinary acceptable carrier or excipient. Trimmer selected from [EC- (4β → 8)] _2- EC, [EC- (4B → 8)] _2- C and [EC- (4β → 6)] _2- EC, [EC- (4β → 8)]-EC )] ₃ -EC, [EC- (4β → 8)] ₃ -C and [EC- (4β → 8)] ₂ -EC- (4β → 6) -C, a tetramer selected from [EC- (4β] → 8)] ₄ -EC, [EC- (4β → 8)] ₃ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₃ -EC- (4β → 8) -C and Pentamers selected from [EC- (4β → 8)] ₃ -EC- (4β → 6) -C, [EC- (4β → 8)] ₅ -EC, [EC- (4β → 8)] _4- EC- (4β → 16) -EC, [EC- (4β → 8)] ₄ -EC- (4β → 8) -C and [EC- (4β → 8)] ₄ -EC- (4β → 6)- Hexamer selected from C, [EC- (4β → 8)] ₆ -EC, [EC- (4β → 8)] ₅ -EC- (4β → 6) -EC, [EC- (4β → 8)] Heptamer selected from ₅ -EC- (4β → 8) -C and [EC- (4β → 8)] ₅ -EC- (4β → 6) -C, [EC- (4β → 8)] ₇ -EC , [EC- (4β → 8)] ₆ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₆ -EC- (4β → 8) -C and [EC- (4β → 8)] )] Octamer selected from _6- EC- (4β → 6) -C, [EC- (4β → 8)] _8- EC, [EC- (4β → 8)] _7- EC- (4β → 6) Nonamer selected from -EC, [EC- (4β → 8)] ₇ -EC- (4β → 8) -C and [EC- (4β → 8)] ₇ -EC- (4β → 6) -C, and [EC -(4β → 8)] ₉ -EC, [EC- (4β → 8)] ₈ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₈ -EC- (4β → 8) A compound selected from decamers selected from -C and [EC- (4β → 8)] ₈ -EC- (4β → 6) -C (wherein "EC" represents a (-)-epicatechin moiety and " C ″ represents a (+)-catechin moiety) or a pharmaceutically acceptable salt, glycoside, ester or oxide thereof and a pharmaceutically or veterinary acceptable carrier or excipient of an inflammatory periodontal disease in a mammal. Pharmaceutical or veterinary compositions for use in the treatment or prophylaxis. Formula A _n , wherein A is a monomer of the general formula wherein n is an integer from 2 to 18, or a polymer compound or a pharmaceutically acceptable salt, glycoside, ester or oxide thereof and pharmaceutically or veterinary A pharmaceutical or veterinary composition for treating a patient in need of a platelet aggregation inhibitor, comprising an acceptable carrier or excipient: From here, R and X each have an α or β stereochemical structure; R is OH or O-sugars; The substituents for C-4, C-6 and C-8 are X, Z and Y, respectively, and the bonding of the monomer units is at C-4, C-6 or C-8; X, Y and Z are hydrogen, Z and Y are sugars and X is hydrogen, or X is sugar and Z and Z are not bonded to any of the other monomeric units. Y is hydrogen or a combination thereof, provided that Y and Z of the first monomer unit are hydrogen when the second monomer unit is bonded to C-4 of the first monomer unit; The sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety by an ester bond. Formula A _n , wherein A is a monomer of the general formula wherein n is an integer from 2 to 18, or a polymer compound or a pharmaceutically acceptable salt, glycoside, ester or oxide thereof and pharmaceutically or veterinary Pharmaceutical or veterinary compositions for use in the treatment, prevention or reduction of atherosclerosis or restenosis in a mammal comprising an acceptable carrier or excipient: From here, R and X each have an α or β stereochemical structure; R is OH or O-sugars, The substituents of C-4, C-6 and C-8 are X, Z and Y, respectively, and the bonding of the monomer units is made in C-4, C-6 or C-8, X, Y and Z are hydrogen, Z and Y are sugars and X is hydrogen, or X is sugar and Z and Y is hydrogen or a combination thereof, provided that Y and Z of the first monomer unit are hydrogen when the second monomer unit is bonded to C-4 of the first monomer unit; The sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety by an ester bond. Represented by the general formula An, wherein A is a monomer of the general formula and n is an integer of 2 to 18, or a polymer compound or a pharmaceutically acceptable salt, glycoside, ester or oxide thereof and pharmaceutically or veterinarily acceptable. Pharmaceutical or veterinary compositions for use in inhibiting bacterial growth in mammals, including possible carriers or excipients: From here, R and X each have an α or β stereochemical structure; R is OH or O-sugars, The substituents of C-4, C-6 and C-8 are X, Z and Y, respectively, and the bonding of the monomer units is made in C-4, C-6 or C-8, X, Y and Z are hydrogen, Z and Y are sugars and X is hydrogen, or X is sugar and Z and Y is hydrogen or a combination thereof, provided that Y and Z of the first monomer unit are hydrogen when the second monomer unit is bonded to C-4 of the first monomer unit; The sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety by an ester bond. Formula A _n , wherein A is a monomer of the general formula wherein n is an integer from 2 to 18, or a polymer compound or a pharmaceutically acceptable salt, glycoside, ester or oxide thereof and pharmaceutically or veterinary Pharmaceutical or veterinary compositions for use in oral treatment of hypertension in a mammal, including an acceptable carrier or excipient: From here, R and X each have an α or β stereochemical structure; R is OH or O-sugars; The substituents for C-4, C-6 and C-8 are X, Z and Y, respectively, and the bonding of the monomer units is at C-4, C-6 or C-8; X, Y and Z are hydrogen, Z and Y are sugars and X is hydrogen, or X is sugar and Z and Z are not bonded to any of the other monomeric units, either C-4, C-6 or C-8. Y is hydrogen or a combination thereof, provided that Y and Z of the first monomer unit are hydrogen when the second monomer unit is bonded to C-4 of the first monomer unit; The sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety by an ester bond. Formula A _n , wherein A is a monomer of the general formula wherein n is an integer from 2 to 18, or a polymer compound or a pharmaceutically acceptable salt, glycoside, ester or oxide thereof and pharmaceutically or veterinary Pharmaceutical or veterinary compositions for use in regulating renal function in mammals, including acceptable carriers or excipients: From here, R and X each have an α or β stereochemical structure, R is OH or O-sugars; The substituents for C-4, C-6 and C-8 are X, Z and Y, respectively, and the bonding of the monomer units is at C-4, C-6 or C-8; X, Y and Z are hydrogen, Z and Y are sugars and X is hydrogen, or X is sugar and Z and Z are not bonded to any of the other monomeric units, either C-4, C-6 or C-8. Y is hydrogen or a combination thereof, provided that Y and Z of the first monomer unit are hydrogen when the second monomer unit is bonded to C-4 of the first monomer unit; The sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety by an ester bond. Represented by the general formula A _n , wherein A is a monomer of the general formula wherein n is an integer derived from 2 to 18 or derived from a composition comprising a pharmaceutically acceptable salt, glycoside, ester or oxide thereof A method of identifying at least one gene that is inhibited, the method comprising contacting at least one gene or gene product thereof with a composition containing the polymer compound using a gene expression assay: From here, R and X each have an α or β stereochemical structure, R is OH or O-sugars; The substituents for C-4, C-6 and C-8 are X, Z and Y, respectively, and the bonding of the monomer units is at C-4, C-6 or C-8; X, Y and Z are hydrogen, Z and Y are sugars and X is hydrogen, or X is sugar and Z and Z are not bonded to any of the other monomeric units, either C-4, C-6 or C-8. Y is hydrogen or a combination thereof, provided that Y and Z of the first monomer unit are hydrogen when the second monomer unit is bonded to C-4 of the first monomer unit; The sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety by an ester bond. 10. The composition or method of any one of claims 1, 2-6 and 7-9 or the method of claim 10, wherein n is 5-12. 10. The composition or method of any one of claims 1, 2-6 and 7-9 or the method of claim 10, wherein n is 5. 10. The composition of any one of claims 1, 2-6 and 7-9 or the method of claim 10, wherein in the polymer compound one (-)-epicatechin monomer unit is the other ( +)-Catechin or (-)-epicatechin unit with a composition or method which is bound by the combination of (4β → 6) or (4β → 8). The composition of any one of claims 1, 2-6 and 7-9 or the method of claim 10, wherein in the polymer compound one (+)-catechin monomer unit is the other ( +)-Catechin or (-)-epicatechin unit A composition or method which is bound by the combination of (4α → 6) or (4α → 8). The composition of any one of claims 1, 2-6 and 7-9 or the method of claim 10, wherein the sugar is selected from glucose, galactose, xylose, rhamnose and arabinose. Composition or method. The composition of any one of claims 1, 2-6 and 7-9 or the method of claim 10, wherein the phenolic moiety is selected from caffeic acid, cinnamic acid, coumarin acid, ferulic acid, gallinic acid, A composition or method selected from hydroxybenzoic acid and cinafinic acid. The composition of any one of claims 1, 2-6 and 7-9 or the method of claim 10, wherein in the compound of Formula An, between C-4 and C-6 of adjacent monomers Or a bond is made between C-4 and C-8 of adjacent monomers; Each of X, Y and Z is H, a sugar or an adjacent monomer, provided that Z is H or a sugar when X and Y are adjacent monomers, and Y is H or a sugar when X and Z are adjacent monomers. The bonding of adjacent monomers to at least one of the terminal monomers is at the C-4 position, optionally Y = Z = hydrogen. The composition of any one of claims 1, 2, 5, 6 and 7-9 or the method of claim 10, wherein the polymer compound is [EC- (4β → 8)] ₂ Trimmer selected from -EC, [EC- (4β → 8)] ₂ -C and [EC- (4β → 6)] ₂ -EC, [EC- (4β → 8)] ₃ -EC, [EC- ( 4β → 8)] ₃ -C and [EC- (4β → 8)] ₂ -EC- (4β → 6) -C, a tetramer selected from [EC- (4β → 8)] ₄ -EC, [EC -(4β → 8)] ₃ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₃ -EC- (4β → 8) -C and [EC- (4β → 8)] ₃ Pentamer selected from -EC- (4β → 6) -C, [EC- (4β → 8)] ₅ -EC, [EC- (4β → 8)] ₄ -EC- (4β → 6) -EC, Hexamers selected from [EC- (4β → 8)] ₄ -EC- (4β → 8) -C and [EC- (4β → 8)] ₄ -EC- (4β → 6) -C, [EC- (4β → 8)] ₆ -EC, [EC- (4β → 8)] ₅ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₅ -EC- (4β → 8)- Heptamer selected from C and [EC- (4β → 8)] _5- EC- (4β → 6) -C, [EC- (4β → 8)] _7- EC, [EC- (4β → 8)] 6-EC- (4β → 6) -EC, [EC- (4β → 8)] _6- EC- (4β → 8) -C and [EC- (4β → 8)] _6- EC- (4β → 6 Jade selected from) -C Tammer, [EC- (4β → 8)] ₈ -EC, [EC- (4β → 8)] ₇ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₇ -EC- ( 4β → 8) -C and [EC- (4β → 8)] ₇ -EC- (4β → 6) -C, a nonamer selected from [EC- (4β → 8)] ₉ -EC, [EC- ( 4β → 8)] ₈ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₈ -EC- (4β → 8) -C and [EC- (4β → 8)] ₈ -EC A composition or method selected from decamers selected from-(4β → 6) -C, wherein "EC" represents a (-)-epicatechin moiety and "C" represents a (+)-catechin moiety. The composition or method according to any one of claims 1, 2 to 6 and 7 to 9 or the method of claim 10, wherein the ester is a gallate. 10. A composition according to any one of claims 1, 2-6 and 7-9, which takes the form of capsules, tablets, pills, chewable solids or beverage preparations, Of claim 1, claim 2 to claim 6 and claim 7 to in claim 9 according to any one of the composition or method claim 10 of wherein the polymer compound is a Te of Rome (Theonroma) or spatula California (Herrania) ( A composition or method, which is a substantially pure compound derived from a hybrid of any species of i) or heterologous or homologous thereof. 10. The composition or method of any one of claims 1, 2-6 and 7-9 or the method of claim 10, wherein the polymer compound is present in a cocoa extract. The composition of claim 8 further comprising at least one additional antihypertensive agent. The composition of claim 1 further comprising at least one additional nonsteroidal anti-inflammatory agent. The method of claim 10, wherein said gene expression assay is selected from differential display, cDNA library sequencing, continuous analysis of gene expression, and expression monitoring by hybridization to high density oligonucleotide sequences. Pentamers selected from [EC- (4β → 8)] ₃ -EC- (4β → 6) -EC and [EC- (4β → 8)] ₃ -EC- (4β → 6) -C, [EC- (4β → 8)] hexamer selected from _4- EC- (4β → 6) -EC and [EC- (4β → 8)] _4- EC- (4β → 6) -C, [EC- (4β → 8)] heptamer selected from _5- EC- (4β → 6) -EC and [EC- (4β → 8)] _5- EC- (4β-) 6) -C, [EC- (4β → 8) ] ₇ -EC, [EC- (4β → 8)] ₆ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₆ -EC- (4β → 8) -C and [EC- (4β → 8)] octamer selected from _6- EC- (4β → 6) -C, [EC- (4β → 8)] _8- EC, [EC- (4β → 8)] ₇ -EC- ( 4β → 6) -EC, [EC- (4β → 8)] ₇ -EC- (4β → 8) -C and [EC- (4β → 8)] ₇ -EC- (4β → 6) -C Nonamer and [EC- (4β → 8)] ₉ -EC, [EC- (4β → 8)] ₈ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₈ -EC Decamer selected from-(4β → 8) -C and [EC- (4β → 8)] ₈ -EC- (4β → 6) -C, [EC- (4β → 8)] ₁₀ -EC, [EC -(4β → 8)] ₉ -EC- (4β → 6) -EC, [EC- (4β → 8)] ₉ -EC- (4β → 8) -C and [EC- (4β → 8)] ₉ Undecamer selected from -EC- (4β → 6) -C, [EC- (4β → 8)] ₁₁ -EC, [EC- (4β-8)] ₁₀ -EC- (4β → 6) -EC, [EC- (4 β → 8)] ₁₀ -EC- (4β → 8) -C and [EC- (4β → 8)] ₁₀ -EC- (4β → 6) -C. EC " represents a (-)-epicatechin moiety, " C " represents a (+)-catechin moiety, wherein the pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers and dodecamers The 3-position of the terminal monomer unit is derived from gallate or β-D-glucose. Formula A _n , wherein A is a monomer of the general formula wherein n is an integer from 2 to 18, or a polymer compound or a pharmaceutically acceptable salt, glycoside, ester or oxide thereof and pharmaceutically or veterinary A pharmaceutical or veterinary composition for oral treatment of a patient suffering from NO-affected hypercholesterolemia comprising an acceptable carrier or excipient: From here, R and X each have an α or β stereochemical structure, R is OH or O-sugars; The substituents for C-4, C-6 and C-8 are X, Z and Y, respectively, and the bonding of the monomer units is at C-4, C-6 or C-8; X, Y and Z are hydrogen, Z and Y are sugars and X is hydrogen, or X is sugar and Z and Z are not bonded to any of the other monomeric units, either C-4, C-6 or C-8. Y is hydrogen or a combination thereof, provided that Y and Z of the first monomer unit are hydrogen when the second monomer unit is bonded to C-4 of the first monomer unit; The sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety by an ester bond. Formula A _n , wherein A is a monomer of the general formula wherein n is an integer from 2 to 18, or a polymer compound or a pharmaceutically acceptable salt, glycoside, ester or oxide thereof and pharmaceutically or veterinary A pharmaceutical or veterinary composition for oral treatment of a patient suffering from atherosclerosis, thrombosis, heart attack, stroke or vascular circulatory system, including an acceptable carrier or excipient: From here, R and X each have an α or β stereochemical structure; R is OH or O-sugars; The substituents of C-4, C-6 and C-8 are X, Z and Y, respectively, and the bonding of the monomer units is made in C-4, C-6 or C-8, X, Y and Z are hydrogen, Z and Y are sugars and X is hydrogen, or X is sugar and Z and Z are not bonded to any of the other monomeric units, either C-4, C-6 or C-8. Y is hydrogen or a combination thereof, provided that Y and Z of the first monomer unit are hydrogen when the second monomer unit is bonded to C-4 of the first monomer unit; The sugar is an unsubstituted sugar or a sugar substituted with a phenol moiety by an ester bond.