RU2394562C2 - Cocoa extract compound and methods of its obtaining and application - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine and pharmaceutics and deals with method of treating patients who need treating with NO modelling agent by introduction to patient composition, which contains polymer compound of formula A_n, where A represents monomer of formula

(I), and pharmacologically acceptable carrier.

EFFECT: invention ensures high treatment efficiency.

21 cl, 235 dwg, 17 tbl, 41 ex

Description

Reference is made to pending U.S. applications: No. 08/709406, filed September 6, 1996, 08/631661, filed April 2, 1996, and 08/317226, filed October 3, 1994 (U.S. Patent No. 5,554,545 ), and PCT / US 96/4497, each of which is incorporated herein by reference.

The present invention relates to cocoa extracts and compounds thereof, such as polyphenols, preferably procyanidin-enriched polyphenols. This invention also relates to methods for producing such extracts and compounds and their use, for example, as antitumor (anti-blastoma) agents, antioxidants, inhibitors of the enzymatic activity of DNA topoisomerase II, cyclooxygenase and / or lipoxygenase modulators, NO modulators (nitric oxide) or NO synthases as non-steroidal anti-inflammatory agents, apoptosis modulators, platelet aggregation modulators, blood glucose or in vitro modulators, antimicrobial agents and oxidative damage inhibitors ezhdeniya DNA.

A complete list of all documents cited in the present description is given at the end of the description before the claims in the section "Bibliography". These documents are within the scope of the present invention, with each cited document is incorporated into this description by reference.

Polyphenols are an incredibly diverse group of compounds (Ferreira et al., 1992), widespread in various plant species, some of which are part of the food chain. In some cases, this class of compounds is important in human nutrition. Although it is believed that some of the polyphenols have no nutritional value, interest in them is growing due to the fact that they are expected to have a beneficial effect on human health.

For example, studies in experimental animals have shown that quercetin (flavonoid) has anticancer activity (Deshner et al., 1991, and Kato et al., 1983). It has been shown that (+) - catechin and (-) - epicatechin (flavan-3-ol) inhibit leukemia virus reverse transcriptase activity (Chu et al., 1992). Nobotanin (oligomeric hydrolyzable tannin) has also been shown to have antitumor activity (Okuda et al., 1992). Statistical reports also report that mortality from stomach cancer is significantly lower in tea producing regions of Japan. There are reports that epigallocatechin gallate is a pharmacologically active substance present in green tea and inhibits the growth of skin tumors in mice (Okuda et al., 1992). It was also shown on the tumor model in various animals that ellagic acid has anticancer activity (Bukharta et al., 1992). Finally, proanthocyanidin oligomers are patented by Kikoman Corporation for use as antimutagens. Indeed, recently at the 202nd National Meeting of the American Chemical Society, reports were presented on the areas of distribution of phenolic compounds in foods and their modulation of tumor development in experimental animal models (But et al., 1992; Huang et al., 1992).

However, none of these reports proposed cocoa extracts or compounds from them, nor methods for producing such extracts or compounds, or the use of cocoa extracts or compounds from them as anti-blastoma agents, antioxidants, DNA topoisomerase II enzyme inhibitors, cyclooxygenase modulators and / or lipoxygenase, modulators of NO (nitric oxide) or NO synthase, as non-steroidal anti-inflammatory agents, modulators of apoptosis, modulators of platelet aggregation, modulators of blood glucose and in vitro, ant imicrobial agents or inhibitors of oxidative damage to DNA.

Since unfermented cocoa beans contain a significant amount of polyphenols, the authors of the present invention believe that similar activity and the ability to use cocoa extracts, for example compounds present in cocoa, can be detected by extracting such compounds from cocoa and then screening these extracts for the corresponding activity. The National Cancer Institute has screened various types of Theobroma and Herrania for anti-cancer activity as part of a wide selection of natural products. Extracts from cocoa bean tissue were found to have low levels of activity, and work was not continued. Thus, the researchers concluded that cocoa and its extracts could not be used as anti-blastoma or anti-cancer agents, and this led to the fact that experts in the field of antitumor or anti-cancer therapy refused to use cocoa and its extracts for the treatment of cancer.

Since a number of analytical methods have been developed to study the feasibility of using cocoa polyphenols for the manufacture of flavorings (Clapperton et al., 1992), the present applicants have decided to apply similar methods to obtain samples for screening for anticancer activity, despite the prevailing opinion regarding the uselessness of cocoa in antitumor or anticancer therapy. Unexpectedly and in contrast to the prior art, for example, screening results by the National Cancer Institute, the inventors of the present invention found that cocoa polyphenol extracts containing procyanidins have significant beneficial effects as anti-cancer or anti-blastoma agents.

In addition, applicants have demonstrated that cocoa extracts containing procyanidins and compounds from cocoa extracts can be used as anti-blastoma agents, antioxidants, inhibitors of the enzymatic activity of DNA topoisomerase II, cyclooxygenase and / or lipoxygenase modulators, NO (nitric oxide) modulators or NO synthases, non-steroidal anti-inflammatory agents, apoptosis modulators, platelet aggregation modulators, blood glucose and in vitro modulators, antimicrobial agents or oxidative inhibitors DNA damage. The subject of the present invention are methods for producing cocoa extract and / or compounds from them.

Another object of the present invention is an extract of cocoa and / or compounds from it.

Another object of the present invention is a polymeric compound of the formula A _n , where A is a monomer having the formula:

where n represents an integer from 2 to 18, so that

at least one terminal monomer unit A and a plurality of additional monomer units were present;

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

binding between adjacent monomers occurs at positions 4, 6 or 8,

the binding of additional monomer units at position 4 has α or β stereochemistry;

X, Y and Z are selected from the group consisting of the monomer unit A, hydrogen and sugar, provided that at least one terminal monomer unit associated with the additional monomer unit is in position 4 and that Y = Z = hydrogen;

the sugar is optionally substituted with a phenolic residue in any position, for example, via an ester linkage,

and pharmaceutically acceptable salts or derivatives thereof (including oxidized products).

Another object of the present invention is a polymeric compound of the formula A _n in which A is a monomer having the formula:

where n represents an integer from 2 to 18, for example 3-18;

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

adjacent monomers bind at positions 4 by (4 → 6) or (4 → 8);

each of X, Y and Z is hydrogen, sugar or an adjacent monomer, provided that if X and Y are adjacent monomers, Z is H or sugar, and if X and Z are adjacent monomers, Y is H or sugar, and so that at least one of the two terminal monomers associated with the adjacent monomer is in position 4, and optionally Y = Z = hydrogen;

the binding at position 4 has α or β stereochemistry;

the sugar is optionally substituted with a phenolic residue in any position, for example, via an ester linkage,

and pharmaceutically acceptable salts or derivatives thereof (including oxidized products).

Another object of the invention is an antioxidant composition.

Another object of the invention is a method of inhibiting the enzymatic activity of DNA topoisomerase II.

And another object of the present invention is a method of treating tumors or cancer.

Another object of the present invention are anticancer, antitumor or anti-blastoma compositions.

Another object of the present invention is an antimicrobial composition.

Another object of the present invention is a cyclooxygenase and / or lipoxygenase modulating composition.

Another object of the present invention is a composition modulating NO or NO synthase.

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

Another object of the present invention is a glucose-modeling composition in blood and in vitro.

Another object of the present invention is a method for the treatment of patients with anti-blastoma, antioxidant, antimicrobial, modulating cyclooxygenase and / or lipoxygenase, or modulating NO or NO synthase, modulating non-steroidal anti-inflammatory agents and / or modulating glucose in blood and in vitro compositions.

An additional object of the present invention is a composition and method of inhibiting oxidative damage to DNA.

Another additional object of the invention is a composition and methods for modulating platelet aggregation.

Another object of the present invention are compositions and methods for modulating apoptosis.

Another object of the present invention is a method for producing any of the above compositions.

Another object of the invention is a kit for use in the above methods or to obtain the above compositions.

It was unexpectedly discovered that cocoa extract and compositions therefrom have antitumor, anticancer or antineoplastic activity or are antioxidant, or have the ability to inhibit the enzymatic activity of DNA topoisomerase II, or are an antimicrobial agent, a cyclooxygenase modulator and / or lipoxygenase, an NO or NO modulator synthase, a non-steroidal anti-inflammatory agent, apoptosis modulator, platelet aggregation modulator or blood glucose modulator or in vitro, or an inhibitor hydrosoluble DNA damage.

Accordingly, the present invention provides a substantially pure cocoa extract and compounds therefrom. The extract or compounds preferably contain polyphenol (or polyphenols) such as polyphenol (s) enriched in cocoa polycyanidin (s), such as polyphenols containing at least one cocoa polycyanidin selected from (-) epicatechin, (+) catechin, procyanidin B-2, procyanidin oligomers from 2 to 18, for example 3-18, such as 2-12 or 3-12, preferably 2-5 or 4-12, more preferably 3-12 and more preferably 5-12, procyanidin B- 5, procyanidin A-2 and procyanidin C-1.

The present invention also provides an antitumor, anticancer or anti-blastoma or antioxidant composition, or a composition that inhibits the enzymatic activity of DNA topoisomerase II, or an antimicrobial, or modulating cyclooxygenase and / or lipoxygenase, or modulating NO or NO synthase, or a non-osteroidal anti-ospoalitis agent composition , an apoptosis modulator, a platelet aggregation modulator, or a glucose modulator in blood or in vitro, or an oxidative damage inhibitor composition DNA containing mainly pure cocoa extract or a compound from it, or synthetic cocoa polyphenols, such as procyanidin-enriched polyphenol (s), and a suitable carrier, for example, acceptable in the pharmaceutical, veterinary and food sciences. The extract or its compounds preferably contain cocoa procyanidin (s). The cocoa extract or compounds from it is preferably obtained by a method comprising grinding the cocoa beans into powder, degreasing the obtained powder and extracting the active compounds from this powder.

In addition, the present invention provides a method of treating patients in need of treatment with an antitumor, anti-cancer or anti-blastoma agent, or an antioxidant, or a DNA topoisomerase II inhibitor, or an antimicrobial agent, a cyclooxygenase and / or lipoxidase modulator, or an NO or NO synthase modulator, or a non-steroidal anti-inflammatory agent, apoptosis modulator, platelet aggregation modulator, or blood glucose modulator or in vitro, or an oxidative damage inhibitor of DNA containing an effective lichestvo substantially pure cocoa extract or compound therefrom or synthetic polyphenol (s) or cocoa procyanidin (s) and a carrier such as a carrier acceptable in pharmaceutical, veterinary and nutritional art. The cocoa extract or compound from it may be cocoa procyanidin (s) and is preferably obtained by grinding cocoa beans into a powder, degreasing the powder and extracting and purifying the active component (or active components) from the powder.

In addition, the present invention provides a kit for treating patients in need of treatment with an antitumor, anti-cancer or anti-blastoma agent, or an antioxidant, or a DNA topoisomerase II inhibitor, or an antimicrobial agent, a cyclooxygenase and / or lipoxidase modulator, or an NO or NO synthase modulator, or a non-steroidal anti-inflammatory agent, apoptosis modulator, platelet aggregation modulator or blood glucose modulator or in vitro, an inhibitor of oxidative damage to DNA containing an effective co a substantial amount of pure cocoa extract or a compound from it, or synthetic cocoa polyphenol (s) or procyanidin (s) and a suitable carrier, for example, a carrier acceptable in the pharmaceutical, veterinary and food field, for mixing with the extract or compound from it, or synthetic (them) polyphenol (s) or procyanidin (s).

The present invention provides the compounds illustrated in FIG. 38A-38B and 39A-39AA, with bonds at positions 4 → 6 and 4 → 8 are currently preferred.

The invention also includes compositions for preserving and preparing food products containing a compound of the invention, and methods for preparing or preserving food products by adding this composition to food.

And, in addition, the invention includes a DNA topoisomerase II inhibitor comprising a compound of the invention and a suitable carrier or diluent, and methods of treating a patient in need of such treatment by administering a composition.

If we consider in General the above run, including cocoa extracts, the invention also includes such embodiments in which the compound of the invention is used instead of or as cocoa extracts. Thus, the invention includes kits, methods and compositions similar to those described above for cocoa extracts and compounds of the invention.

These and other objects and accomplishments are disclosed or become apparent from the following detailed description.

The following detailed description is given with reference to the accompanying drawings.

Figure 1 shows a characteristic chromatogram obtained by fractionation of crude cocoa procyanidins by gel permeation chromatography;

figa shows a characteristic chromatogram obtained using reverse phase HPLC and showing the separation (elution profile) of cocoa procyanidins extracted from unfermented cocoa;

2B shows a characteristic separation of cocoa procyanidins extracted from unfermented cocoa using normal phase HPLC;

figure 3 shows several characteristic structures of procyanidin;

figa-4E show the characteristic HPLC chromatograms of the five fractions used in the screening for anticancer or antineoplastic activity;

5 and 6A-6D show the dose dependence between cocoa extracts and cancer cells ACHN (FIG. 5) and PC-3 (FIGS. 6A-6D) (proportion of surviving cells relative to dose, μg / ml); M & M2 F4 / 92, M & MA + E U12P1, M & MV + E Y192P1, M & MS + E U12P2, M & MD + E U12P2;

figa-7H show a typical dose dependence between the fractions of cocoa procyanidin A, B, C, D, E, A + B, A + E and A + D and the cell line PC-3 (the proportion of surviving cells relative to the dose, μg / ml ); ММ-1А 0212РЗ, ММ-1 В 0162Р1, ММ-1 С 0122РЗ, ММ-1 D 0122РЗ, ММ-1 Е 0292Р8, ММ-1 А / В 0292Р6, ММ-1 А / Е 0292Р6, ММ-1 A / D 0292P6;

figa-8H show a typical dose dependence between the fractions of cocoa procyanidin A, B, C, D, E, A + B, B + E and D + E and the nasopharyngeal cell line KB / HeLa (fraction of surviving cells / dose, μg / ml); ММ-1А 092К3, ММ-1 В 0212К5, ММ-1 С 0162К3, ММ-1 D 0212K5, ММ-1 Е 0292К5, ММ-1 А / В 0292К3, ММ-1 В / Е 0292К4, ММ-1 D / E 0292K5;

figa-9H show a typical dose dependence between the fractions of cocoa procyanidin A, B, C, D, E, B + D, A + E and D + E and the cell line HCT-116 (fraction of surviving cells / dose, μg / ml ); MM-1 С 0192Н5, D 019H5, Е 0192Н5, MM-1 B&D 0262H2, А / Е 0262Н3, MM-1 D&E 0262H1;

figa-10H show a typical dose dependence between the fractions of cocoa procyanidin A, B, C, D, E, B + D, C + D and A + E and the ACHN renal cell line (fraction of surviving cells / dose, μg / ml) ; ММ-1 А 092А5, ММ-1 В 092А5, ММ-1 С 0192А7, ММ-1 D 0192А7, М & М1 Е 0192А7, ММ-1 B&D 0302A6, ММ-1 C&D 0302A6, ММ-1 А & Е 0262А6;

11A-11H show a typical dose dependence between the cocoa procyanidin fractions A, B, C, D, E, A + E, B + E, and C + E by the A-549 lung cell line (fraction of surviving cells / dose, μg / ml); ММ-1 А 019258, ММ-1 В 09256, ММ-1 С 019259, ММ-1 D 019258, ММ-1 Е 019258, А / Е 026254, ММ-1 В & Е 030255, ММ-1 С & Е N6255;

figa-12H show a typical dose dependence between the fractions of cocoa procyanidin A, B, C, D, E, B + C, C + D and D + E melanoma cell line SK-5 (fraction of surviving cells / dose, μg / ml ); ММ-1 A 021S4, ММ-1 В 0212S4, ММ-1 С 0212S4, ММ-1 D 0212S4, ММ-1 Е N32S1, ММ-1 В&С N32S2, ММ-1 C&D N32S3, ММ-1 D&E N32S3;

figa-13H show a typical dose dependence between the fractions of cocoa procyanidin A, B, C, D, E, B + C, C + E and D + E breast cell line MCF-7 (fraction of surviving cells / dose, μg / ml); ММ-1 A N22M4, ММ-1 В N22M4, ММ-1 С N22M4, ММ-1 D N22M3, ММ-1 Е 0302М2, ММ-1 В / С 0302М4, ММ-1 С & Е N22M3, ММ-1 D&E N22M3;

Fig shows a typical dose dependence between cocoa procyanidin (in particular, fraction D) and the cell line CCRF-CEM of T-cell leukemia (cells / ml relative to the days of growth; open circle - control, filled circle - 125 μg - fraction D, open inverted triangle - 250 μg - fraction D, filled inverted triangle - 500 μg - fraction D).

Fig. 15A shows a comparison of analyzes using XTT and crystalline violet for cytotoxicity against MCF-7 p168 breast cancer cell lines treated with D + E fraction (an open circle indicates XTT and a filled circle indicates crystal violet);

Fig. 15B shows a typical dose-response curve plotted for the MDA MV231 breast cell line treated with various amounts of crude polyphenols obtained from the UIT-1 cocoa genotype (optical density (540 nm) - relative to days; an open circle indicates a control, filled the circle indicates the carrier, the unfilled inverted triangle indicates 250 μg / ml, the filled inverted triangle indicates 100 μg / ml, the unfilled square indicates 10 μg / ml; optical density 2.0 is max. Flax of plate reader and not necessarily correspond to the number of cells);

Fig. 15C shows a typical dose-response curve plotted for the PC-3 prostate cancer cell line treated with various amounts of crude polyphenols obtained from the cocoa genotype UIT-1 (optical density (540 nm) - relative to days; an open circle indicates control , a filled circle indicates a carrier, an open inverted triangle indicates 250 μg / ml, a filled inverted triangle indicates 100 μg / ml, an open square indicates 10 μg / ml);

Fig. 15D shows a typical dose-response curve plotted for the MCF-7 p168 breast cancer cell line treated with various amounts of crude polyphenols derived from the cocoa genotype UIT-1 (optical density (540 nm) - relative to days; an open circle indicates control, the filled circle indicates the carrier, the open inverted triangle indicates 250 μg / ml, the filled inverted triangle indicates 100 μg / ml, the open square indicates 10 μg / ml; optical density 2.0 is poppy ideal for a tablet reader and does not necessarily correspond to the number of cells);

Fig. 15E shows a typical dose-response curve constructed for a Hela cervical cancer cell line treated with various amounts of crude polyphenols obtained from the UIT-1 cocoa genotype (optical density (540 nm) - relative to days; an open circle indicates a control, a filled circle denotes a carrier, an open inverted triangle indicates 250 μg / ml, a filled inverted triangle indicates 100 μg / ml, an open square indicates 10 μg / ml; optical density 2.0 is maximum for pl anette reader and does not necessarily correspond to the number of cells);

Fig. 15F shows the effect against the Hela cervical cancer cell line treated with various fractions (optical density (540 nm) - relative to days; an open circle indicates 100 μg / ml of fraction AE, a filled circle indicates 100 μg / ml of fraction AC , an open, inverted triangle indicates 100 μg / ml of the D&E fraction; optical density 2.0 is maximum for a tablet reader and does not necessarily correspond to the number of cells);

15G shows a cytotoxic effect at a dose of 100 μg / ml against SKBR-3 breast cancer cell line treated with various cocoa polyphenol fractions (optical density (540 nm) - relative to days; an open circle indicates fractions AE, a filled circle indicates fractions AC, an unfilled inverted square denotes fractions D&E);

Fig. 15H shows a typical dose-response relationship between cocoa procyanidin D + E fraction and Hela cells (optical density (540 nm) versus days; an open circle indicates control, a filled circle indicates 100 μg / ml, an open inverted triangle indicates 75 μg / ml, the filled inverted triangle indicates 50 μg / ml, the empty square indicates 25 μg / ml, the filled square indicates 10 μg / ml; optical density 2.0 is maximum for the tablet reader and does not necessarily correspond to the amount cells);

Fig. 15I shows a typical dose-response relationship between cocoa procyanidin fraction D + E and SKBR-3 cells (optical density (540 nm) versus days; an open circle indicates control, a filled circle indicates 100 μg / ml, an open inverted triangle indicates 75 μg / ml, the filled inverted triangle indicates 50 μg / ml, the open square indicates 25 μg / ml, the filled square indicates 10 μg / ml);

Fig.15J shows a typical dose-response relationship between the D + E fraction of cocoa procyanidin and Hela cells in an assay performed by cloning in soft agar (bar graph; colony count relative to control, 1, 10, 50 and 100 μg / ml);

Fig. 15K shows growth inhibition of Hela cells treated with untreated polyphenol extracts obtained from eight different cocoa genotypes (% of control relative to concentration, μg / ml; an open circle represents C-1, a filled circle represents C-2, an open inverted triangle represents C-3, the filled inverted triangle represents C-4, the open square represents C-5, the filled square represents C-6, the filled triangle represents C-7, the filled triangle represents puts C-8;

C-1 = UF-12: Trinitario culture variety and description — crude extracts of cocoa polyphenols UF-12 (Brazil) (decaffeinated / de-brominated);

C-2 = NA-33: cultivar = Forastero and description — crude extracts of cocoa polyphenols NA-33 (Brazil) (decaffeinated / de-brominated);

C-3 = EEG-48: cultivar = Forastero and description — crude extracts of cocoa polyphenols EEG-48 (Brazil) (decaffeinated / de-brominated);

C-4 = unknown: cultivar = Forastero and description — crude extracts of unknown cocoa polyphenols (West Africa) (decaffeinated / de-brominated);

C-5 = UF-613: cultivar = Trinitario and description — crude extracts of cocoa polyphenols UF-613 (Brazil) (decaffeinated / de-brominated);

C-6 = ICS-100: cultivar = Trinitario (ancestor Nicaraguan Criollo) and description — crude cocoa polyphenol extracts ICS-100 (Brazil) (decaffeinated / de-brominated);

C-7 = ICS-139: cultivar = Trinitario (ancestor Nicaraguan Criollo) and description — crude cocoa polyphenol extracts ICS-139 (Brazil) (decaffeinated / de-brominated);

C-6 = UIT-1: cultivar = Trinitario and description — crude cocoa polyphenol extracts UIT-1 (Malaysia) (decaffeinated / de-brominated);

Fig. 15L shows the growth inhibition of Hela cells treated with crude extracts of polyphenols obtained from fermented cocoa beans and dried cocoa beans (in all cases, fermentation and drying were performed in the sun;% of the control relative to concentration, μg / ml; open circles represent the fraction zero day, a filled circle represents a fraction of the first day, an unfilled inverted triangle represents a fraction of the second day, a filled inverted triangle represents a fraction of 3 days, not akrashenny square represents a fraction of the day 4, a filled square represents a fraction of the day 9);

Fig. 15M shows the effect of enzymatically oxidized cocoa procyanidins against Hela cells (dose-dependent effect of cocoa polyphenols treated with polyphenol oxidase); % of control relative to concentration; mcg / ml; the filled square represents the crude UIT-1 (with caffeine and theobromine), the empty circle represents the crude UIT-1 (caffeine and theobromine) and the filled circle represents the crude UIT-1 (catalyzed by polyphenol oxidase);

Fig. 15N shows a characteristic separation of the combined fractions D and E of cocoa procyanidins by semi-preparative reverse phase HPLC;

figo shows a characteristic separation of the crude cocoa polyphenol extract using semi-preparative normal phase HPLC;

Fig. 16 shows the characteristic racemate oxidation curve for the extract and cocoa polycyanidine fractions compared to the curves obtained for synthetic antioxidants BHA and BHT (conventional units relative to time; the dashed line and crosses (+) represent BHA and BHT; * represents DE; X represents crude extract; unfilled squares represent AC fractions; unfilled diamonds represent control);

FIG. 17 shows a typical agarose gel indicative of inhibition of topoisomerase II catalyzed decenation of kinetoplast DNA by cocoa procyanidin fractions (lane 1 contains 0.5 μg monomer rings of “marker” (M) kinetoplast DNA; lanes 2-20 contain kinetoplast DNA that was incubated with topoisomerase II in the presence of 4% DMSO, but in the absence of any cocoa procyanidins (control C); lanes 3 and 4 contain kinetoplast DNA that was incubated with topoisomerase II in the presence of 0.5 and 5.0 μg / ml of procyanidin fraction A ka ao; tracks 5 and 6 contain kinetoplast DNA, which was incubated with topoisomerase II in the presence of 0.5 and 5.0 μg / ml of cocoa procyanidin fraction B; tracks 7, 8, 9, 13, 14 and 15 represent copies of kinetoplast DNA incubated in the presence of 0.05, 0.5 and 5.0 μg / ml of fraction D of cocoa procyanidins; lanes 10, 11, 12, 16, 17 and 18 represent copies of kinetoplast DNA that were incubated with topoisomerase II in the presence of 0.05, 0.5 and 5.0 μg / ml fraction E of cocoa procyanidins; lane 19 represents a copy of kinetoplast DNA, which was incubated with topoisomerase II in the presence of 5.0 μg / ml fraction E of cocoa procyanidins);

Fig. 18 shows the dose-dependent activity of fraction D of cocoa procyanidins against cell lines capable of DNA repair and cell lines with insufficient DNA repair ability (proportion of surviving cells relative to μg / ml; left graph is xrs-6 cell line with insufficient repair ability DNA, MM-1 D D282X1; the right graph is the BR1 cell line capable of DNA repair, MM-1 D D282B1);

Fig. 19 shows dose-response curves for comparing MCF-7 adriamycin-resistant cells with the MCF-7 p168 parent cell line treated with D + E fraction (% of control relative to concentration, μg / ml; open circles represent MCF-7 p168 ; filled circles MCF-7 ADR);

figa and b show the dose-dependent effect on cells of the Hela and SKBR-3 lines when they are processed with concentrations of 100 μg / ml and 25 μg / ml of twenty fractions obtained using semi-preparative HPLC with a normal phase (histogram;% of control relative to control and fractions 1-12);

Figure 21 shows the separation of crude, enriched, and purified pentamers from cocoa extracts using normal phase HPLC;

figa, b and C show the MALDI-TOF / MS pentamer enriched with procyanidins, fractions aC and fractions DE, respectively;

figa shows the elution profile of oligomeric procyanidins purified using modified semi-preparative HPLC;

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

figa-B show the structure with minimum energy for all pentamers with a bond (4 → 8) based on the structure of epicatechin;

figa shows the relative fluorescence of epicatechin after thiolysis with benzyl mercaptene;

figv shows the relative fluorescence of catechin after thiolysis with benzyl mercaptene;

figs shows the relative fluorescence of dimers (B2 and B5) after thiolysis with benzyl mercaptene;

figa shows the relative fluorescence of dimers after thiolysis;

figv shows the relative fluorescence of B5 dimer after thiolysis of the dimer and subsequent desulfurization;

figa shows the relative volume of the tumor during treatment on a model of nude mice MDA MB 231 when they are treated with a pentamer;

27B shows the relative survival curve of pentamer-treated experimental nude mice of MDA 231;

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

Fig. 29 shows the effect of the pore size of the stationary phase on the separation of procyanidins by normal phase HPLC;

figa shows the use of the substrate during the fermentation of cocoa beans;

figv shows the production of metabolite during fermentation;

fig.30D shows the calculations of the tablets during the fermentation of cocoa beans;

figv shows the relative concentration of each component in a fermented solution of cocoa beans;

Fig. 31 shows acetylcholine-induced relaxation of a rat aorta, NO-dependent and pre-contracted by phenylephrine;

32 shows blood glucose tolerance profiles using various test mixtures;

figa-In show the effect of indomethacin on the activity of COX-1 and COX-2;

Figa-B show the correlation between the level of polymerization and IC ₅₀ relative to COX-1 / COX-2 (μm);

Fig. 35 shows the correlation between the effect of the compounds on the activity of COX-1 and COX-2, expressed in microns;

Figa-V show the values of the IC ₅₀ (μm) of samples containing procyanidins with COX-1 / COX-2;

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

figa-38B show the preferred structure of the pentamers;

figa-AA show a stereochemical library of pentamers;

FIGS. 40A-B show 70 minute gradients for separation of procyanidins by normal phase HPLC, as shown by UV irradiation and fluorescence, respectively;

FIGS. 41A-B show 30 minute gradients for the separation of procyanides by normal phase HPLC, as shown by UV irradiation and fluorescence, respectively;

Fig. 42 shows separation of procyanidins by normal phase HPLC;

figa-G show the CD (circular dichroism) of the spectrum of dimers, trimers, tetramers, pentamers, hexamers, heptamers and procyanidin octamers, respectively;

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

Figs. 44B-F show the spectrum of APT, COZY, XHCORR, ¹ H and ¹³ C NMR for epicatechin;

figa shows the structure and data ¹ H and ¹³ With NMR for catechin;

Figures 45B-E show a ¹ H, APT, XHCORR, and COZY NMR spectrum for catechin;

Fig. 46A shows the structure and ¹ H and ¹³ C NMR data for a B2 dimer;

Figures 46B-G show a spectrum of ¹³ C, APT, ¹ H, HMQC and COZY and NONAN NMR for a B2 dimer;

Fig. 47A shows the structure and ¹ H and ¹³ C NMR data for a B5 dimer;

Figv-C show the spectrum of ¹ H, ¹³ C, APT, COZY, HMQC, NONAN NMR for B5 dimer;

figa-D show the spectrum of ¹ H, COZY, HMQC and NONAN NMR for epicatechin / catechin trimer;

49A-B show a ¹ H, COZY, HMQC, and NONAN NMR spectrum for an epicatechin trimer;

50A and B show the effects of fractions A and C of cocoa procyanidin, respectively, on blood pressure; the blood pressure level decreases by 21.43% within a minute after applying fraction A, returns to normal after 15 minutes, whereas after applying fraction C, blood pressure decreases by 50.5% within 1 minute and returns to normal after 5 minutes;

Fig. 51 shows the effect of cocoa procyanidin fractions on blood pressure of guinea pigs under anesthesia;

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

Fig. 53 shows the effect of bradykinin on NO production in HUVEC (human umbilical vein endothelial cells);

Fig. 54 shows the effect of cocoa procyanidin fractions on macrophage production of NO in HUVEC.

Fig. 55 shows the effect of cocoa procyanidin fractions on NO production by macrophages;

Fig. 56 shows the effect of procyanidin fractions on LPS-induced and gamma-interferon-primed macrophages;

Fig. 57 shows a micellar electrokinetic capillary chromatographic separation of cocoa procyanidin oligomers;

58A-F show MALDI-TOF mass spectral analysis data for Cu ⁺² -, Zn ⁺² -, Fe ⁺² -, Fe ⁺³ -, Ca ⁺² - and Mg ⁺² - ions attached to the trimer, respectively;

Fig. 59 shows mass spectral analysis data of MALDI-TOF oligomers of cocoa procyanidin (from tetramers to octade chambers);

Fig. 60 shows the dose-dependent effect of cocoa procyanidin oligomers on feline lymphoblastic leukemia virus FeA cell line;

61 shows the dose-dependent effect of cocoa procyanidin oligomers on feline normal kidney CRFK cell line;

Fig.62 shows the dose-dependent effect of cocoa procyanidin oligomers on a normal kidney MDCK canine cell line;

Fig.63 shows a dose-dependent effect between the cocoa procyanidin oligomers and the canine GH cell line of a normal kidney;

Fig.64 shows the effect of the dependence of time - temperature on the hydrolysis of hexamer and

Fig.65 shows the effect of the dependence of time - temperature on the formation of the trimer.

Compounds of the Invention

As discussed above, it was unexpectedly found that cocoa extracts or compounds extracted from them exhibit anticancer, antitumor or antineoplastic activity, antioxidant activity, inhibit the DNA topoisomerase II enzyme and oxidative DNA damage, have antibacterial activity, modulate cyclooxygenase and / or lipoxygenase , NO (nitric oxide) or NO synthase, apoptosis, platelet aggregation, blood glucose and in vitro, are also effective as non-steroidal anti-inflammatory agents. These extracts, compounds, or a combination of the compounds isolated from them, are mainly obtained by grinding cocoa beans into a powder, degreasing the powder and extracting and purifying the active component of the compound (s) from defatted powder. The powder can be obtained by sublimating the cocoa beans and the pulp of the fruit, freeing the cocoa beans from the pulp, removing the peel from the sublimated cocoa beans and grinding the peeled beans. The extraction of the active component of the compound (s) can be carried out using the technique of extraction from solution. Extracts containing the active compounds can be purified, for example, to significant purity, for example, by gel permeation chromatography or preparative high performance liquid chromatography (HPLC), or a combination of such methods.

The references regarding the isolation and purification of the compounds of the invention extracted from cocoa make it clear that any species of Theobroma, Herrania or hybrids from their intraspecific or interspecific crosses can be used. From this point of view, reference is made to Schultes, "Synopsis of Herrania" Journal of the Arnold Arboretum, Vol. XXXIX, pp. 217-278, plus plates I-XVII (1985), Cuatrecasas, "Cocoa and Its Allies, A Taxonomic Revision of the Genus Teobroma", Bulletin of the United States National Museum, Vol. 35, part 6, pp .379-613, plus plates 1-11 (Smithsonian Institution, 1964), and Addison et al., "Observation 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 provides a list of previously undescribed species of Theobroma and Herrania and their hybrids from intraspecific and interspecific crosses in which certain concentrations of the compounds of the invention were found; Example 25 also describes methods for modulating the amount of compounds of the invention that can be obtained from cocoa by skillfully selecting cocoa fermentation conditions.

The basic principles of constructing a purification scheme used in the isolation of mainly pure polycyanide are shown in Fig. 37. Stage 1 and 2 of the purification scheme are described in examples 1 and 2; stages 3 and 4 are described in examples 3, 13 and 23; stage 5 is described in examples 4 and 14 and stage 6 is described in examples 4, 14 and 16. However, one skilled in the art will understand what modifications of the purification scheme shown in FIG. 37 to obtain the active component can be made without departing from the essence and scope , and without conducting optional experiments.

Extracts, compounds of them and a combination of compounds, possessing activity without the need to be attached to any particular theory, are identified as cocoa polyphenol (s), such as procyanidins. These cocoa procyanidins have significant anticancer, antitumor or antineoplastic activity, antioxidant activity; inhibit the enzyme DNA topoisomerase II and oxidative damage to DNA; possess antibacterial activity; capable of modulating cyclooxygenase and / or lipoxygenase, NO or NO synthase, apoptosis, platelet aggregation and blood glucose and in vitro, and are effective as non-steroidal anti-inflammatory agents.

The present invention provides a compound of the formula:

where n represents an integer from 2 to 18, for example 3-12, so that the first monomer unit A and many other monomer units are present;

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

position 4 represents alpha or beta stereochemistry;

X, Y, and Z represent positions for binding between monomer units, provided that the binding of the first monomer unit of the other monomer units occurs at position 4 and Y = Z = hydrogen, and that if you do not touch the binding of monomer units, X, Y and Z are hydrogen, or Z, Y represents sugar, and X represents hydrogen, or X represents alpha or beta sugar, and Z, Y represents hydrogen, or combinations thereof. In compound n, it can be 5-12, and in some preferred compounds n is 5. Sugar can be selected from the group consisting of glucose, galactose, xylose, ramnose and arabinose. Sugar of any or all of R, X, Y, and Z may optionally be substituted with a phenolic moiety through an ester linkage.

Thus, the invention provides a compound of the formula:

where n represents an integer from 2 to 18, for example 3-12, preferably 5-12, and preferably n is 5, so that the first monomer unit A has the following form:

and many other monomer units A;

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

position 4 represents alpha or beta stereochemistry;

X, Y, and Z represent positions for linking between monomer units, provided that the binding of the first monomer unit of another monomer unit occurs at position 4 and Y = Z = hydrogen, and that if you do not touch the binding of monomer units, X, Y and Z are hydrogen or Z, Y represents sugar, and X represents hydrogen, or X represents alpha or beta sugar, and Z, Y represents hydrogen, or combinations thereof;

said sugar is optionally substituted with a phenolic residue through an ester linkage.

Accordingly, the present invention provides a polymer compound of the formula A _n where A is a monomer having the formula:

where n represents an integer from 2 to 18, so that at least one terminal monomer unit and at least one or a plurality of additional monomer units are present;

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

the binding of adjacent monomers occurs at position 4, 6 or 8;

the addition of an additional monomer unit at position 4 has α and β stereochemistry;

X, Y and Z are selected from the group consisting of the monomer unit A, hydrogen and sugar, provided that the binding of at least one terminal monomer unit of the additional monomer unit (i.e., the binding of the monomer unit adjacent to the terminal monomer unit) occurs at the position 4, and that optionally Y = Z = hydrogen;

the sugar is optionally substituted with a phenolic residue at any position, for example via an ester linkage, and pharmaceutically acceptable salts or derivatives thereof (including oxidation products).

In a preferred embodiment, n may be 3-18, 2-18, 3-12, for example 5-12, and preferably n is 5. Sugar is selected from the group consisting of glucose, galactose, xylose, ramnose or arabinose. Sugar of any or all of R, X, Y, and Z may optionally be substituted at any position with a phenolic moiety through an ester linkage. The phenolic residue is selected from the group consisting of caffeic, cinnamon, coumaric, ferulic, gallic, hydroxybenzoic and synapinic acids.

In addition, the present invention provides a polymer compound of the formula A _n where A is a monomer having the formula:

where n represents an integer from 2 to 18, for example 3-18, mainly 3-12, for example 5-12, preferably n is 5;

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

the binding of adjacent monomers occurs at position 4, as (4 → 6) or (4 → 8);

each X, Y and Z represents H, sugar or an adjacent monomer: provided that if X and Y are adjacent monomers, Z represents H or sugar, and if X and Z are adjacent monomers, Y represents H or sugar and so that at least one of the two terminal monomers associated with the adjacent monomer was in position 4, and optionally Y = Z = hydrogen;

binding at position 4 has α or β stereochemistry;

the sugar is optionally substituted with a phenolic residue in any position, for example, via an ester linkage,

and pharmaceutically acceptable salts or derivatives thereof (including oxidation products).

Regarding the expression “at least one terminal monomer unit A”, it should be understood that the compounds of the invention have two terminal monomer units and that the two terminal monomer units A may be the same or different. In addition, it should be understood that the words about “at least one terminal monomeric unit A” include an execution in which they refer to the “first monomer unit”, the words about the “first monomer unit” refer to that monomer to which others are attached monomer units, resulting in a polymer compound of the formula A _n . Moreover, with regard to at least one of the two terminal monomers, the binding of adjacent monomers occurs at position 4 and, optionally, Y = Z = hydrogen.

Regarding the expression “combination thereof”, it should be understood that one or more compounds of the invention can be used simultaneously, for example, when administering to a patient in need of treatment a finished formulation containing one or more compounds of the invention.

The compounds of the invention or combinations thereof demonstrate the beneficial effects noted above for cocoa extracts, and as a result, the term "cocoa extract" can be replaced by the words - compounds of the invention or combinations thereof, so it is understood that the compounds of the invention or combinations thereof cocoa extracts.

The term “oligomer” as used herein refers to compounds (or a combination thereof) of the formula above, in which n represents a number 2-18. When n is 2, the oligomer is called a "dimer"; when n is 3, the oligomer is called a "trimer"; when n is 4, the oligomer is called a "tetramer"; when n is 5, the oligomer is called a "pentamer"; such a statement may apply to oligomers having a larger n value, including 18, so that when n is 18, the oligomer is called an “octadecamera”.

The compounds of the invention or their combinations can be isolated, for example, from a natural source of cocoa, such as any kind of Theobroma, Herrania or their hybrids from interspecific or intraspecific crosses; or the compounds of the invention or combinations thereof may be purified, for example, the compounds or combinations thereof may be substantially pure; for example, can be cleaned to manifest homogeneity. Purity is a relative concept, and many examples demonstrate the isolation of the compounds of the invention or their combinations, as well as their purification, so that using the methods given as examples, a qualified specialist can obtain fairly pure compounds of the invention or their combinations, or purify them to an obviously homogeneous state ( for example, purity from an isolated individual peak in chromatography). Based on examples (e.g., example 37), a compound or combination of compounds of significant purity has at least 40% purity, for example at least about 50%, mainly at least 60% purity, for example at least 70% purity, more preferably at least about 75-80% purity, preferably at least about 90% purity, more preferably more than 90% purity. For example, at least 90-95% purity, or even cleaner, more than 95% purity, for example 95-98%.

In addition, examples of monomer units containing the oligomers used herein are (+) - catechin and (-) - epicatechin, abbreviated as C and EC, respectively. The bonds between adjacent monomers are between positions 4 and 6 or between position 4 and position 8; and this relationship between position 4 of the monomer and position 6 and 8 of the adjacent monomer unit is denoted here as (4 → 6) or (4 → 8). There are 4 possible stereochemical bonds between position 4 of the monomer and position 6 and 8 of the adjacent monomer; and stereochemical bonds between monomer units are referred to herein as (4α → 6) or (4β → 6), or (4α → 8), or (4β → 8). If C binds to another C or EC, such a relationship is denoted here as (4α → 6) or (4α → 8). When the EU binds to another C or EU, such a relationship is referred to herein as (4β → 6) or (4β → 8).

Examples of compounds exhibiting the above activity include dimers, EC- (4β → 8) -EC and EC- (4β → 6) -EC, where EC- (4β → 8) is preferred; trimers [EC- (4β → 8)] _2-EU, [EC- (4β → 8)] ₂ -C and [EC- (4β → 6)] _2-EU, wherein [EC- (4β → 8)] ₂ -EC is preferred; tetramers of [EC- (4β → 8)] ₃ -EC, [EC- (4β → 8)] ₃ -C and [EC- (4β → 8)] ₂ -ES- (4β → 6) -C, where [ EC- (4β → 8)] ₃ -EC is preferred; and pentamers [EC- (4β → 8)] ₄ -ES, [EC- (4β → 8)] ₃ -ES- (4β → 6) -EC, [EC- (4β → 8)] ₃ -ES- ( 4β → 8) -C and [EC- (4β → 8)] ₃ -ES- (4β → 6) -C, where the position 3 of the terminal monomer unit of the pentamer is optionally derivatized with gallate or β-D-glucose; [EC- (4β → 8)] ₄ -EC is preferred.

In addition, compounds exhibiting the above activity include compounds from hexamers to dodecamers, examples of which are given below:

hexamer in which one monomer (C or EC) having bonds with another monomer (4β → 8) or (4β → 6) when the EU binds to another EC or C, and (4α → 8) or (4α → 6) when binding of C to another C or EU; followed by binding (4β → 8) to the pentameric compound, from the above, for example, [EC- (4β → 8)] ₅ -ES, [EC- (4β → 8)] ₄ -ES- (4β → 6) - EU, [EU- (4β → 8)] ₄ -ES- (4β → 8) -C and [EU- (4β → 8)] ₄ -ES- (4β → 6) -C, where position 3 of the terminal monomer unit hexamer optionally derivatized with gallate or β-D-glucose; In a preferred embodiment, the hexamer is [EC- (4β → 8)] _5-EU;

heptamer in which a combination of two monomers (C and / or EC) having bonds with one another (4β → 8) or (4β → 6) when the EU binds to another EU or C, and (4α → 8) or (4α → 6) when binding With another C or the EU; followed by binding (4α → 8) to the pentameric compound, from the above, for example, [EC- (4β → 8)] ₆ -ES, [EC- (4β → 8)] ₅ -ES- (4β → 6) - EU, [EU- (4β → 8)] ₅ -ES- (4β → 8) -C and [EU- (4β → 8)] ₅ -ES- (4β → 6) -C, where position 3 of the terminal monomer unit heptamer is optionally derivatized with gallate or β-D-glucose; in a preferred embodiment, the heptamer is [EC- (4β → 8)] ₆ -EC;

an octamer in which any combination of three monomers (C and / or EC) having bonds with one other monomer (4β → 8) or (4β → 6) when the EU binds to another EU or C, and (4α → 8) or ( 4α → 6) upon binding of C to another C or EC; followed by binding (4β → 8) to the pentameric compound of the above, for example, [EC- (4β → 8)] ₇ -ES, [EC- (4β → 8)] ₆ -ES- (4β → 6) - EU, [EU- (4β → 8)] ₆ -ES- (4β → 8) -C and [EC- (4β → 8)] ₆ -ES- (4β → 6) -C, where position 3 of the terminal monomer unit the octamera is optionally derivatized with gallate or β-D-glucose; in a preferred embodiment, the octamer is [EC- (4β → 8)] ₇ -EC;

nonamer in which any combination of four monomers (C and / or EC) having bonds with one other monomer (4β → 8) or (4β → 6) when the EU binds to another EC or C, and (4α → 8) or ( 4α → 6) upon binding of C to another C or EC; followed by binding (4β → 8) to the pentameric compound of the above, for example, [EC- (4β → 8)] ₈ -ES, [EC- (4β → 8)] ₇ -ES- (4β → 6) - EC, [EC- (4β → 8)] ₇ -ES- (4β → 8) -C and [EC- (4β → 8)] ₇ -ES- (4β → 6) -C, wherein the 3-position terminal monomeric unit nonamera is optionally derivatized with gallate or β-D-glucose; in a preferred embodiment, the nonamer is [EC- (4β → 8)] ₈ -EC;

a decamer in which any combination of five monomers (C and / or EC) having bonds with one other monomer (4β → 8) or (4β → 6) when the EU binds to another EU or C, and (4α → 8) or ( 4α → 6) upon binding of C to another C or EC; followed by binding (4β → 8) to the pentameric compound, from the above, for example, [EC- (4β → 8)] ₉ -ES, [EC- (4β → 8)] ₈ -ES- (4β → 6) - EU, [EU- (4β → 8)] ₈ -ES- (4β → 8) -C and [EU- (4β → 8)] ₈ -ES- (4β → 6) -C, where position 3 of the terminal monomer unit the decamera is optionally derivatized with gallate or β-D-glucose; in a preferred embodiment, the decamer is [EC- (4β → 8)] ₉ -EC;

undecamera, in which any combination of six monomers (C and / or EC) having bonds with one other monomer (4β → 8) or (4β → 6) when binding the EU to another EU or C, and (4α → 8) or ( 4α → 6) upon binding of C to another C or EC; followed by binding (4β → 8) to the pentameric compound, from the above, for example, [EC- (4β → 8)] ₁₀ -ES, [EC- (4β → 8)] ₉ -ES- (4β → 6) - EU, [EU- (4β → 8)] ₉ -ES- (4β → 8) -C and [EU- (4β → 8)] ₉ -ES- (4β → 6) -C, where position 3 of the terminal monomer unit undecamera is optionally derivatized with gallate or β-D-glucose; in a preferred embodiment, the undecamera is [EC- (4β → 8)] ₁₀ -EC;

dodecamer, in which any combination of seven monomers (C and / or EC) having bonds with one other monomer (4β → 8) or (4β → 6) when the EU binds to another EU or C, and (4α → 8) or ( 4α → 6) upon binding of C to another C or EC; followed by binding (4β → 8) to the pentameric compound, from the above, for example, [EC- (4β → 8)] ₁₁ -ES, [EC- (4β → 8)] ₁₀ -ES- (4β → 6) - EU, [EU- (4β → 8)] ₁₀ -ES- (4β → 8) -C and [EU- (4β → 8)] ₁₀ -ES- (4β → 6) -C, where position 3 of the terminal monomer unit dodecamera is optionally derivatized with gallate or β-D-glucose; in a preferred embodiment, the dodecamer is [EC- (4β → 8)] ₁₁ -EC.

From the detailed description it becomes clear that the above listing is only an example and is offered as an illustrative source of many, not limited to these examples, compounds of the invention, which, of course, are not an exhaustive list of compounds of the present invention.

Examples 3A, 3B, 4, 14, 23, 24, 30, and 34 describe methods for separating the compounds of the invention. Examples 13, 14A-D and 16 describe methods for purifying such compounds. Examples 5, 15, 18, 19, 20 and 29 describe methods for determining the compounds of the invention. 38A-B and 39A-AA illustrate a stereochemical library of representative pentamers of the invention. Example 17 describes a method for producing the molecular model of the invention. Example 36 provides features for determining higher oligomers contained in cocoa, where n is 13-18.

In addition, despite the fact that the invention primarily describes cocoa extracts, preferably containing cocoa procyanidins, from this description a specialist will no doubt determine a way to artificially produce and / or prepare the active compound (see, for example, example 11). Accordingly, the invention includes synthetic cocoa polyphenols or procyanidins or their derivatives and / or their synthetic precursors, which include, but are not limited to, glycosides, galates, esters, etc. and the like. The compounds of the invention can be obtained by isolation from cocoa or from any other species of Theobroma and Herrania, as well as artificially; and derivatives and artificial precursors of the compounds of the invention, such as glucosides, gallates, esters, etc. included in the compounds of the invention. Derivatives may also include compounds of the above formula, wherein the sugar or gallate residue is on the terminal monomer in the Y or Z position, or substituted sugar or gallate residues are on the terminal monomer in the Y or Z position.

For example, method C of example 8 describes the use of cocoa enzymes for the oxidative modification of compounds of the invention or a combination thereof in order to improve cytotoxicity (see FIG. 15M) directed against several cancer cell lines. The invention includes the possibility of enzymatic modification (e.g., cleavage or addition of a chemically important fragment) of a compound of the invention, for example, enzymatically using a combination of polyphenol oxidase, peroxidase, catalase and / or enzymes such as hydrolases, esterases, reductases, transferases and the like, and any combination, taking into account kinetic and thermodynamic factors (see also example 41 concerning hydrolysis).

As for the synthesis of compounds according to the invention, qualified specialists without undue experimentation will be able to find additional synthesis routes based on this description and prior art, for example, on a thorough retrosynthetic analysis of both polymer and monomeric compounds. For example, having a phenolic characterization of the compounds of the invention, a qualified person can use various methods for the selective introduction / removal of protective groups combined with organometallic additives, phenolic binding and photochemical reactions, for example, in a convergent, linear or biomimetric approach, or combinations thereof, together with standard reactions , known to specialists in the field of chemistry of organic synthesis, and additional methods of synthesis to obtain the compounds of the invention Without undue experimentation. In this regard, references are made to W. Carruthers, Some Modern Methods of Organic Syntesis, 3 ^rd ed., Cambridge University Press, 1986, and J. March, Advanced Organic Chemistry, 3 ^rd ed., John Willey & Sons, 1985, van Rensburg et al., Chem. Comm., 24: 2705-2706 (Dec. 21, 1996), Ballenegger et al., (Zyma SA) Europian Patent 0096 007 Bl, and the documentation below in the Bibliography section, the contents of which are incorporated herein by reference.

The usefulness of the compounds of the invention

As for the compounds of the invention, it was surprisingly discovered that the compounds of the invention have different types of activity and, as such, have a wide range of applications in the treatment of various pathological conditions discussed above.

COX / LOX Utility

Atherosclerosis, the most common of cardiovascular diseases, is the main cause of heart attacks, strokes and other problems associated with blood circulation. Atherosclerosis is a complex disease in which many types of cells, biochemical processes and molecular factors are involved. There are several aspects of this disease, its disease states and progression, which are characterized by interdependent sequential processes of oxidation of low density lipoproteins (LDL), biochemistry of cyclooxygenase (COX) / lipogenase (LOX) biochemistry of nitric oxide (NO).

Clinical studies have made it possible to accurately establish that an increase in plasma LDL concentrations is associated with an accelerated development of atherosclerosis. Cholesterol, which accumulates in atherosclerotic foci, originates primarily from plasma lipoproteins, including LDL. Oxidation of LDL is a critical event in the triggering mechanism of atheroma formation and is associated with increased production of superoxide anion radical (O ₂ • -). Oxidation of LDL O ₂ • - or other reactive groups (for example, • OH, ONOO • -, lipid peroxy radical, copper ion and protein base ion) reduces the affinity of LDL for uptake by cells through receptor-mediated endocytosis. Oxidation-modified LDLs are then rapidly captured by macrophages, which subsequently transform into cells very similar to the “foam cells” observed in atherosclerotic foci at an early stage of formation.

Oxidized lipoproteins can also contribute to vascular damage by the formation of hydroxy oxides inside LDL particles. This triggers a radical chain of oxidation reactions of unsaturated LDL, thereby producing more oxidized LDL captured by macrophages.

A massive accumulation of foam cells saturated with oxidized LDL resulting from these processes leads to early damage in the form of "fat bands", which eventually progress to form a more developed complex of atherosclerotic lesions leading to coronary diseases.

As discussed in a general sense by Jean Marx on page 320 Science, Vol.265 (July 15, 1994), every year about 330,000 patients in the United States undergo plastic surgery of coronary and / or peripheral vessels, a process leading to the release of the lumen of blood vessels, such as coronary vessels arteries clogged with dangerous atherosclerotic plaques (atherosclerosis), and in this way to restore normal blood flow. For most of these patients, surgery is as intended. However, about 33% of these patients (and according to some estimates perhaps more) again develop stenosis, when the operated arteries again quickly become clogged. These patients have no improvement, and sometimes they get worse than before angioplasty. Excessive smooth muscle cell proliferation (SMC) in the walls of a blood vessel promotes restenosis. Increased accumulation of oxidized LDL inside the damaged (SNC) can contribute to atherogenic-dependent processes like restenosis. Zhou et al., "Association Between Prior Cytomegalovirus Infection And The Risk Of Restenosis After Coronary Atherectomy", August 29, 1996, New England Journal of Medicine, 335: 624-630, and the documentation therein is incorporated by reference. Accordingly, the usefulness of the present invention from the point of view of atherosclerosis may consist in its use in restenosis.

From the point of view of the inhibition of cyclooxygenase (COX; prostaglandin endoperoxide synthase) by the compounds of the invention, cyclooxygenase is known to be a central enzyme in the production of prostaglandins and other arachidonic acid metabolites (i.e., eicosanoids) involved in many physiological processes. COX-1 is a constitutive enzyme expressed in many tissues, including platelets, while COX-2, the second isoform of the enzyme, is stimulated by various cytokines, hormones, and tumor activators. COX 1 produces thromboxane A2, which is included in platelet aggregation, which in turn is involved in the development of atherosclerosis. The prophylactic effect against cardiovascular diseases is based on the suppression of these processes.

The activity of COX-1 and COX-2 is suppressed by aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs), and it is believed that the gastric side effects of NSAIDs are associated with the suppression of COX-1. Moreover, it was found that in patients who regularly received NSAIDs, the risk of developing bowel cancer was 40-50% lower than those who did not receive such medications, and mRNA values for COX-2 significantly increased in 86% of cases of intestinal adenosarcoma .

One important property of cell lines expressing COX-2 is the increased expression of genes involved in the modulation of apoptosis, that is, programmed cell death. Several NSAIDs are related to increased cell mortality and induction of apoptosis in chick embryonic fibroblasts.

Cellular lipoxygenase is also involved in the oxidative modification of LDL via peroxidation of unsaturated lipids. The formation of lipid peroxy radicals leads to a further radical chain of oxidation of unsaturated LDL lipids, producing more oxidized LDL for macrophage uptake.

Surprisingly, the compounds of the invention have been found to be useful in the treatment of diseases associated with COX / LOX. In Example 28, COX was inhibited by individual compounds of the invention at concentrations similar to the known NSAID, indomethacin.

Compounds of the invention designed to suppress COX are oligomers where n is 2-18. In a preferred embodiment, the compounds of the invention are oligomers in which n is 2-10, and more preferably the compounds of the invention are oligomers in which n is 2-5.

Examples of compounds having inhibitory activity with respect to the above COX / LOX include dimers, trimers, tetramers and pentamers discussed above.

Therefore, given the significant ability of the compounds of the invention to inhibit COX-2, together with the cytotoxic effect on the putative expression of COX-2 in the colon cancer cell line, the components of the invention have apoptotic activity as inhibitors of the multi-stage process leading to carcinomas, and, in addition, possess activity as drugs of the NSAID drug group, which has a wide range of different types of prophylactic activity (see, for example, example 8, Fig. 9D-9H).

Furthermore, prostaglandins - penultimate products of the COX catalyzed conversion of arachidonic acid to prostaglandin _H2, are involved in processes such as inflammation, pain, fever, fetal development, labor and platelet aggregation. Therefore, the compounds of the invention are effective under the same conditions as the NSAIDs, for example, against cardiovascular disease, stroke, etc. (indeed, on the inhibition of COX-1 platelets, which reduces the production of thromboxane A ₂ , the prophylactic effects of aspirin in cardiovascular diseases are based).

Inflammation is the reaction of living tissues to their damage. It includes a complex set of enzyme activation, mediator release, fluid transudation, cell migration, tissue damage and restoration. Inflammation is activated by phospholipase A ₂ , which releases arachidonic acid, a substrate for COX and LOX enzymes. MOR converts arachidonic acid into prostaglandin PGE ₂ , the main eicosanoid found in various inflammatory processes from acute edema to chronic arthritis. Inhibition by NSAIDs underlies treatment.

Arthritis is one of the rheumatic diseases that covers a wide range of diseases and pathological processes, most of which involve joint tissues. The main structure that suffers from these diseases is the connective tissue, which includes the synovial membranes, cartilage, bones, tendons, ligaments, and interstitial tissues. The intermittent syndromes associated with connective tissue include sprains and strains, tendonitis and abnormalities of the tendon sheath. The most serious forms of arthritis are rheumatoid arthritis, osteoarthritis, gout, and systemic erythematous ("red") lupus.

In addition to rheumatic, other diseases are accompanied by inflammation. With gingivitis and periodontitis, a pathological picture develops similar to the symptoms of rheumatoid arthritis. Inflammatory bowel diseases include idiopathic chronic inflammatory bowel conditions, ulcerative colitis and Crohn's disease. Spondylosis refers to chronic inflammation of the joints of the vertebrae. There are also many cases of osteoarthritis associated with obesity.

Thus, the compounds of the invention are useful in the treatment of conditions including inflammation, pain, fever, abnormalities of the fetus and the birth process, and platelet aggregation.

Inhibition of COX and with the compounds of the invention would also slow down the formation of prostaglandins, for example PGD ₂ and PGE ₂ . Thus, the compounds of the invention are useful in the treatment of conditions associated with prostaglandins PGD ₂ and PGE ₂ .

Usefulness associated with NO

Nitric oxide (NO) is known to slow down platelet aggregation, monocyte adhesion and chemotaxis, and the proliferation of smooth muscle tissue in the vascular wall, that is, processes involved in atherogenesis. This clearly confirms the point that the amount of NO is reduced in atherosclerotic tissues due to the reaction with free oxygen radicals. The loss of NO due to these reactions leads to an increase in the number of platelets and the adhesion of inflammatory cells to the walls of the vessel, which leads to a further weakening of the relaxation mechanisms associated with NO. Thus, the loss of NO supports atherogenic processes, leading to the progression of a disease state.

Arterial hypertension is a leading cause of cardiovascular disease, including stroke, heart attack, heart disease, arrhythmias, and kidney damage. Arterial hypertension is a condition in which the blood pressure inside the blood vessels during its circulation through the vessels of the body is higher than normal. When for a continuous period of time, systolic pressure exceeds 150 mmHg. or diastolic pressure exceeds 90 mmHg, then the whole body suffers. For example, excessive systolic pressure can lead to rupture of blood vessels in some places. When this happens inside the brain, the result is a stroke (stroke). It can also cause thickening and narrowing of blood vessels, which can lead to atherosclerosis. Elevated blood pressure can also cause hypertrophy of the heart muscle, since it is forced to work harder to overcome elevated (diastolic) pressure when the blood is expelled. This hypertrophy can ultimately lead to arrhythmias or heart disease. Arterial hypertension is called the “silent killer” because it can be asymptomatic and can only be detected by checking blood pressure.

Blood pressure regulation is a complex of phenomena in which one mechanism involves the expression of a constitutive Ca ^{+ 2} / calmodulin-dependent form of nitric oxide synthase (NOS), abbreviated eNOS. The NO produced by this enzyme causes muscle relaxation of the vessel (dilatation), which lowers blood pressure. The muscles of the vascular wall do not relax properly when there is no production of a normal level of nitric oxide produced by eNOS, or as a result of the fact that its production is blocked by an inhibitor, or in pathological conditions such as atherosclerosis. The resulting vasoconstriction leads to an increase in blood pressure and may be responsible for some forms of arterial hypertension.

Vascular endothelial cells contain eNOS. NO synthesized by eNOS diffuses in different directions and, when it reaches the main smooth muscle layer of the vascular wall, binds to the hemanyl group of guanylyl cyclase, which causes an increase in cGMP. An increase in cGMP causes a decrease in intracellular free Ca ⁺² . Cyclic GMP can activate a protein kinase that phosphorylates Ca ^{+ 2} transporters, causing Ca ^{+ 2 to} sequester in the intercellular structures of muscle cells. Since Ca ^{+ 2 is} required for muscle contraction, the force of contraction decreases with decreasing Ca ²⁺ concentration. Muscle relaxation leads to vasodilation, which lowers blood pressure. The suppression of eNOS therefore causes an increase in blood pressure.

When normal amounts of NO are not produced, or due to blockages in production when using an NOS inhibitor or, possibly, in pathological conditions, the vascular muscle does not relax properly. The resulting vasoconstriction raises blood pressure and may be responsible for some forms of arterial hypertension. Of considerable interest is the discovery of therapeutic methods for increasing eNOS activity in patients with hypertension, but there are no reports of the practice of such treatment. Pharmacological agents that promote the release of NO, such as nitroglycerin or isosorbite dinitrate, remain the main vasodilators.

Although the compounds of the invention inhibit the oxidation of LDL, a more general effect of these compounds is their multidimensional effect on atherosclerosis by NO. The modulation of NO by the compounds of the invention causes a complex of beneficial effects, including modulation of arterial hypertension, reduction of NO-induced hypercholesterolemia, slow platelet aggregation and monocyte adhesion, which are involved in the development of atherosclerosis.

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

Regarding the effects of NO and infectious bacteria, microorganisms play a significant role in infectious processes, which reflect the influence on the body of bad habits, occupational hazards, the human environment, as well as foodborne diseases caused by improper storage, handling and infection of products.

The compounds of the invention, their combinations, and compositions containing them are useful in treating conditions associated with modulating NO concentrations. Example 9 describes the antioxidant activity (as free radical inhibitors) of the compounds of the invention. Provided that NO is a free radical and that the compounds of the invention are strong antioxidants, it has been suggested that the use of the compounds of the invention in experiments in vitro and in vivo will lower NO levels. Any decrease in NO would lead to a hypertensive rather than a hypotensive effect. Contrary to expectations, the compounds of the invention showed an increase in NO in in vitro experiments and produced a hypotensive effect in in vivo studies (Examples 31 and 32). These results were unexpected and completely unforeseen.

Example 27 describes erythema (redness of the face) that appears shortly after drinking a solution containing the compounds of the invention and glucose, thereby demonstrating the effect of vasodilation.

Example 31 describes the antihypertensive effect caused by the compounds of the invention in an experimental animal in vivo, demonstrating the effectiveness of the compounds of the invention in the treatment of arterial hypertension. In this example, the compounds of the invention, their combinations and compositions comprising them contain oligomers in which n is 2-18 and preferably n is 2-10.

Example 32 describes the modulation of NO production by the compounds of the invention in an in vitro model. In this example, the compounds of the invention, their combinations and compositions containing them contain oligomers in which n is 2-18 and preferably n is 2-10.

In addition, Example 35 provides evidence of the formation of Cu ⁺² -, Fe ⁺² - and Fe ⁺³ - oligomeric complexes detected using MALDI / TOF / MS. These results indicate that the compounds of the invention can combine with copper and / or iron ions in order to minimize their effect on LDL oxidation.

In addition, the compounds of the invention have useful antimicrobial activity in the treatment of contagious diseases and for the prevention of food spoilage. Examples 22 and 30 describe the antimicrobial activity of the compounds of the invention against individual representatives of microorganisms having clinical and nutritional significance, as shown below.

Microorganism Type of Clinical / nutritional value Helicobacter pylori Gram negative Gastritis, ulcers, stomach cancer Species of Bacillus Gram positive Food poisoning, infected wounds, bovine mastitis, sepsis Species of Salmonella Gram negative Food poisoning, diarrhea Staphyloccocus aureus Gram positive Boiling, carbuncles, infected wounds, sepsis, breast abscesses Escherichia coli Gram negative Diarrhea of the babies, urinary tract infection Views of Pseudomonas Gram negative Urinary tract infections, infected wounds, swimmer's ear Saccharomyces cervisea Yeast Food spoilage Acetobacter pasteurianus Gram negative Food spoilage

Example 33 describes the effect of the compounds of the invention on the production of NO by macrophages. In this example, the results demonstrate that the compounds of the invention induce the production of NO by monocytes / macrophages, both independently and depending on stimulation with polysaccharides (LPS) or cytokines. Macrophage production of NO can inhibit the growth of infectious microorganisms.

The compounds of the invention having antimicrobial activity are oligomers in which n is 2-18, and preferably are oligomers in which n is 2, 4, 5, 6, 8, and 10.

Examples of formulations exhibiting antimicrobial activity associated with the above NO effect include dimers, tetramers, hexamers, octamers, and decamers discussed above.

Anti-cancer utility

Cancers are classified into three groups: carcinomas, sarcomas, and lymphomas. Carcinoma is a malignant neoplasm that occurs in the skin, epithelium, lining the internal surfaces of various organs, glands and tissues. Sarcoma is a malignant neoplasm that occurs in bones, muscles, or in connective tissue. The third group includes leukemia and lymphoma, as both develop in the organs that form blood cells. The main types of cancer are cancer of the prostate, breast, lung, colon, bladder, non-Hodgkin lymphoma, cancer of the uterus, skin melanoma, kidney cancer, leukemia, cancer of the ovary and pancreas.

Cancer develops as a result of a change in the DNA of cells, which is caused by many factors, such as a hereditary genetic factor, the effects of radiation, pollutants, radon, and DNA damage by free radicals. In cells carrying mutations, a violation of the normal process of cell division occurs. These cells are not adequately apoptotic and continue to divide, which marks the beginning of the development of a malignant tumor or allow the appearance of more mutations over time, which leads to the development of malignant neoplasms.

There are three main features common to various cancers. This is (1) the ability to unpredictably spread; (2) tumor growth into the surrounding tissue; and (3) metastasis.

Different types of cancer metastasize in a characteristic way. For example, cancers of the thyroid gland, lung, breast, kidney, and prostate often produce bone metastases. Lung cancer usually spreads to the brain and adrenal glands, colon cancer often metastasizes to the liver. Leukemia from the very beginning is considered as a generalized disease that is found in the bone marrow of the body.

Surprisingly, it has been found that the compounds of the invention are useful in the treatment of the cancers discussed above. Examples 6, 7, 8, and 15 describe compounds of the invention that exhibit anticancer activity against human cancer HeLa (cervical), cancer of the prostate, breast, kidney, against T-cell leukemia and colon cancer cell line. Example 12 (FIG. 20) illustrates the dose dependence of HeLa SKBR-3 breast cancer cell lines treated with oligomeric (dodecamer dimers) procyanidins that have been substantially purified by HPLC. Cytotoxicity against these cancer cell lines depended on the presence of procyanidins from a pentamer to a dodecamer, while lower oligomers did not show a similar effect.

Without associating this with any theory, it turned out that there is a minimal structural fragment responsible for the effects described above. Example 37 also shows the same cytotoxic effect of higher oligomers (decamer pentamers) against a feline lymphoblastoma cell line. Cytotoxicity of higher oligomers was also observed (FIGS. 58-61) against normal canine and feline cell lines.

Example 8 (Fig. 9D-H) shows that the compounds of the invention exhibit cytotoxicity against the intended expression of COX-2 human colon cancer cell line (HCT 116).

Example 9 describes the antioxidant activity of the compounds of the invention. The compounds of the invention inhibit the occurrence of breaks in the DNA chain, the formation of cross-linking protein-DNA and the oxidation of free radicals of nucleotides to reduce and / or prevent the occurrence of mutations.

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

Example 21 describes the in vivo effect of a substantially pure pentamer that showed antitumor activity against a human cancer cell line (MDA-MB-231 / LCC6) in a nude mouse model (average mouse weight of about 25 g). Repeated in vivo experiments with pentamers at higher dosages (5 mg) were not completely successful due to unforeseen animal toxicity. It is currently believed that this toxicity may be associated with the vasodilating effect of the compounds of the invention.

Example 33 describes the effect of a compound of the invention on macrophage production of NO. Macrophages producing NO can slow the growth of rapidly dividing tumor cells.

In addition, the invention includes the use of compounds of the invention to stimulate inhibition of cell proliferation by apoptosis.

In order to exhibit anticancer activity, the compounds of the invention should be oligomers in which n is 2-18, for example 3-18, type 3-12, and preferably n is 5-12, most preferably n is 5.

Compounds that exhibit inhibitory activity against the above types of cancer include the dodecameric pentamers discussed above.

Finished formulations and methods of use

Thus, the compounds of the invention, their combinations and compositions containing them exhibit a wide range of activities against individual manifestations of atherosclerosis, cardiovascular disease, cancer, modulation of blood pressure and / or arterial hypertension, inflammatory diseases, infectious agents and food spoilage.

Therefore, the compounds of the invention, their combinations and the content of their composition are COX inhibitors that affect platelet aggregation by inhibiting the formation of thromboxane A ₂ , thereby reducing the risk of thrombosis. In addition, inhibition of COX leads to a decrease in the adhesion of platelets and inflammatory cells to the walls of blood vessels, and the NO-mediated relaxation mechanism improves. These results, together with inhibition of COX at concentrations similar to those of the known NSAID, indomethacin, indicate the effectiveness of the compounds of the invention as antimicrobial agents.

In addition, the compounds of the invention, their combinations and compositions containing them are antioxidants that inhibit the oxidation of LDL by lowering the level of superoxide radical anion and lipoxygenase-mediated lipid peroxy radicals. Inhibition of LDL oxidation at this stage slows down the activation of macrophages and reduces the formation of "foam cells" (spongy tissue), which interrupts the further progression of atherosclerosis. Inhibition of LDL oxidation can also slow the progression of restenosis. Thus, the compounds of the invention, or combinations thereof, or compositions containing them, can be used to prevent and / or treat atherosclerosis and / or restenosis. And thus, the compounds of the invention can be used before or after plastic surgery on vessels or similar procedures to prevent or treat restenosis in patients prone to it.

For the treatment or prevention of restenosis and / or atherosclerosis, the compound of the invention or compound or composition containing the compound or compounds of the invention, alone or in combination with other therapeutic agents, can be used as directed by a qualified healthcare practitioner based on this description and prior art, for example, at the first signs or symptoms of restenosis and / or atherosclerosis, immediately before, simultaneously with or after angioplasty, or later in accordance with the instructions of a qualified doctor general doctor, without any undue experimentation; the use of a compound or compounds of the invention, or their compositions alone or together with other methods of treatment can be continued in the form of a course, for example, monthly, two-month, twice a year, annual or in any other mode determined by a qualified attending physician necessary time without any undue experimentation.

In addition, the compounds of the invention, their combinations, and compositions containing them demonstrated their in vivo antihypertensive effect and in vitro stimulation of NO. These results can be of practical use in the treatment of hypertension and in various clinical cases, including hypercholesterolemia, in which the level of NO is markedly reduced.

Formulations of the compounds of the invention, their combinations and compositions containing them can be prepared by standard methods known in the pharmaceutical industry of the food industry, in the field of medicine and veterinary medicine, in the form of a liquid, suspension, tablet, capsule, injection solution or suppository, with immediate or delayed release of active compounds.

The carrier may also be a delayed release polymer system. Synthetic polymers, in particular, find application in a controlled formulation of a controlled release composition. The first example of this form was the polymerization of methyl methacrylate into spheres with a diameter of less than one micron to form so-called nanoparticles, as reported by Kreuter, J., Microcapsules and Nanoparticles in Medicine and Pharmacology, M. Donbrow (Ed). CRC Press, p. 125-148. Often poly (d, l-lactide-glycolide) (PLGA) is chosen as the carrier of the pharmaceutical preparation and less often for antigens. It is a biodegradable polyester that has a long history of use in medicine as a substance for the preparation of absorbable suture material, metal plates for fastening bone fragments and for other temporary prostheses, and at the same time it did not show any toxicity. A variety of pharmaceuticals are formulated in PLGA microcapsules. Enough data has been collected on the adaptation of PLGA to provide a controlled yield, as discussed, for example, Eldridge, J.H., et al., Current Topics in Microbiology and Immunology, 1989, 146: 59-66. When administered orally, the content in PLGA microspheres of 1-10 microns in diameter is effective. The PLGA microencapsulation process uses water-in-oil phase emulsion separation. The compound or compounds of the invention are prepared as an aqueous solution and PLGA is dissolved in suitable organic solvents such as methylene chloride and ethyl acetate. These two immiscible solutions are jointly emulsified with high speed mixing. Then add a substance that is not a solvent for the polymer, causing it to precipitate around water droplets with the formation of germinal microcapsules. The microcapsules are collected and stabilized with one of the agents selected from polyvinyl alcohol (PVA), gelatin, alginates and methyl cellulose, and then the solvent is removed either by drying in vacuo or by extraction of the solvent.

Additionally, regarding the preparation of sustained release formulations, reference is made to US Pat. Nos. 5,024,843, 5,091,190, 5,026,668, 4,612,008 and 4,327,725, incorporated herein by reference.

In addition, selective procedures, together with the determination of cocoa genotypes of interest, can be used to create chocolate products, both those included in the Identity Standard (SOI), and not included, such as excipient, designed to deliver active ingredients to a patient in need of treatment suffering from the described higher diseases, as well as means to provide a certain level of compounds of the invention.

In this regard, reference is made to the simultaneously registered US application Serial No. 08/709406, filed September 6, 1966, incorporated herein by reference. USSN 08/709406 relates to a method for producing cocoa butter and / or cocoa solids, which maintains the level of cocoa bean polyphenols, and uses a unique combination of cooking steps that does not require separate equipment for roasting or wet grinding the beans, making it possible to process cocoa beans without being subjected to excessive heat treatment for a long period of time and / or without the use of solvent extraction of the fat component. The advantage of this process is the increased concentration of polyphenols compared with the concentration obtained with traditional methods for processing cocoa beans, so the ratio of the initial amount of polyphenols found in unprocessed beans to those determined after the processing process is less than or equal to 2.

Compositions of the invention include one or more of the above compounds in the form of formulations containing a pharmaceutically acceptable carrier or excipient, and compounds of the invention having anticancer, antitumor or antineoplastic activity, antioxidant activity, inhibit the enzymatic activity of DNA topoisomerase II, inhibit oxidative damage to DNA induce monocyte / macrophage production of NO, have antimicrobial activity, as well as activity as modulators of c iclooxygenase and / or lipoxygenase, NO or NO synthase, apoptosis, platelet and glucose aggregation in blood and in vivo, and are effective as non-steroidal anti-inflammatory agents.

Another embodiment of the invention includes compositions containing the compounds of the invention or combinations thereof, as well as at least one additional antitumor agent, a blood pressure lowering agent, anti-inflammatory, antimicrobial, antioxidant and hematopoietic agents, in addition to a pharmaceutically acceptable carrier or excipient.

Such compositions can be administered to a patient in need of them in such dosages and in such ways as are known to those skilled in the field of medicine, nutrition or veterinary medicine, taking into account the data discussed and such factors as age, gender, weight, heredity and living conditions of a particular subject or patient and also considering the route of administration, the relative concentration of certain oligomers and toxicity (e.g. LD ₅₀ ).

Compositions can be administered simultaneously or sequentially with other anti-blastoma and anti-cancer agents, antioxidants, agents that inhibit the enzymatic activity of DNA topoisomerase II, with agents that model inhibitors of oxidative damage to DNA or cyclooxygenase and / or lipoxygenase, platelet aggregation and glucose, glucose, vivo or NO or NO synthase, with non-steroidal anti-inflammatory agents and / or with agents that reduce or alleviate the painful effects of anti-blastoma, anti-cancer antitumor agents, antioxidants, agents that inhibit the enzymatic activity of DNA topoisomerase II, an inhibitor of oxidative DNA damage, agents that modulate cyclooxygenase and / or lipoxygenase, apoptosis, platelet aggregation, blood glucose and in vivo, NO or NO synthase, and / or non-steroidal anti-inflammatory agents; and this time taking into account factors such as age, gender, weight, heredity and living conditions of a particular subject or patient and the route of administration.

Examples of compositions of the invention intended for human or veterinary use include edible compositions for oral administration, such as solid or liquid formulations, for example capsules, tablets, pills, and the like, as well as solid preparations in the form of chewing gum, to which the present invention is fully applicable, since it used a source suitable for human consumption (for example, solid compositions flavored with cocoa or chocolate); liquid compositions for administration to various body cavities, for example, for oral, intranasal, anal, vaginal, etc. administration, such as suspensions, syrups or elixirs (including cocoa or chocolate flavored compositions); preparations for parenteral, subcutaneous, transdermal, intramuscular or intravenous administration (for example, by injection), such as suspensions or emulsions. However, each specialist should keep in mind that the active ingredient present in the compositions can form a complex with blood flow proteins, as a result of which blood coagulation can occur due to precipitation of blood proteins. In such compositions, the active cocoa extract may be mixed with a suitable carrier, diluent or excipient, such as sterile water, saline, glucose, DMSO, ethyl alcohol or the like. The active cocoa extract of the present invention can be obtained in lyophilized form, followed by reduction, for example, in isotonic aqueous, saline, glucose or DMSO buffer. A slight precipitation was observed in some physiological solutions, and this observation can be used as a method for isolating the compounds of the invention, for example, salting out.

Example 38 describes the preparation of the compounds of the invention in the form of tablets in the form of tablets for use in the pharmaceutical, nutritional and food fields. Example 39 further describes the preparation of the compounds of the invention in capsule formulations for the same use. Example 40 describes a formulation of chocolate included in the Identity Standard (SOI) and non-SOI chocolate containing the compounds of the invention or solid cocoa compositions prepared by the methods described in US Patent Application Serial Number 08/709406 included in this description by reference.

Sets

Further, the invention also relates to a kit comprising an active cocoa extract. This kit may be a separate container containing a suitable carrier, diluent or excipient. The kit may also contain an additional anticancer, antitumor or anti-blastoma agent, an antioxidant, an inhibitor of the enzymatic activity of DNA topoisomerase II, or an inhibitor of oxidative damage to DNA, or an antibacterial agent, or an agent that modulates cyclooxygenase and / or lipoxygenase, NO or NO synthase, non-steroidal anti agents, apoptosis and platelet aggregation, an agent modulating glucose in the blood or in vivo, and / or an agent that reduces or alleviates the effects of anti-blastoma, antitumor, or prot ivoracic agents, an inhibitor of the enzymatic activity of DNA topoisomerase II, antimicrobial agents, or cyclooxygenase and / or lipoxygenase, NO or NO synthase, apoptosis, platelet and glucose aggregation in blood or in vivo and / or non-steroidal anti-inflammatory agents intended for joint or application. The additional agent (or agents) can be placed in a separate container (s) or it can be mixed with an active cocoa extract. In addition, the kit may include instructions for mixing or combining the ingredients and / or their use.

Gene identification

Another embodiment of the invention includes the modulation of genes expressed as a result of close contact of cells with the compounds of the invention or a combination of compounds. From this point of view, the present invention contains methods for identifying genes induced or suppressed by the compounds of the invention or a combination of compounds that are associated with individual diseases, including, but not limited to, atherosclerosis, arterial hypertension, cancer, cardiovascular disease and inflammation. In particular, genes that are differentially expressed in these disease states compared to their expression in “normal” non-disease states are identified and described before and after contact with the compounds of the invention or a combination of compounds.

As indicated in the previous discussion, these diseases and disease states are partly based on free radical interactions with a variety of biomolecules. A key point in the development of these diseases is that many of the free radical reactions involve reactive oxygen particles, which in turn lead to physiological conditions involved in the progression of the disease. For example, reactive oxygen particles are involved in the regulation of transcription factors, such as nuclear factor (NF) -kB. Target genes for NF-kB include a number of genes associated with a coordinated inflammatory response. It includes genes encoding tumor necrosis factor (TNF) -α, interleukin (IL) -l, IL-6, IL-8, inducible NOS, major histocompatibility complex (MHC) class I antigens, and others. Also, genes that modulate the activity of transcription factors can, in turn, be induced by oxidative stress. Oxidative stress is the lack of balance between radical purification and radical generating systems. Several well-known examples (Winyard and Blake, 1997) of these conditions include gaddl53 (a gene induced by stunting and DNA damage), the product of which is shown to bind NF-IL-6 and form a heterodimer that cannot bind to DNA. NF-IL-6 regulates the expression of individual genes, including coding interleukins 6 and 8. Another example of inducible oxidative stress genes is qadd45, which regulates the effects of p53 transcription factor upon growth retardation. p53 encodes a p53 protein that can stop cell division and stimulate apoptosis of abnormal cells (such as cancer cells).

To give a complete picture of unforeseen, non-obvious and novel uses of the compounds of the invention or a combination of compounds useful in another group of diseases based in part on free radical mechanisms, the invention further includes a strategy for determining the temporal effect on the gene (s) or gene expression product (s) by the compounds of the invention in animal models of certain diseases or disease states in vitro or in vivo, using gene expression analysis. These assays include, but are not limited to, differential demonstration, sequencing of cDNA sequence libraries, serial gene expression analysis (SAGE), expression registration by hybridization with high density oligonucleotide sequences, and a variety of reverse transcriptase polymerization chain reactions (RT-PCR) based on protocols or a combination of the above analysis methods (Lockhart et al, 1996).

Comprehensive physiological effects of the compounds of the invention or combinations of compounds embodied in the invention, together with the genetic assessment process, allow the discovery of genes and gene products, both known and new, induced or suppressed. For example, the invention includes in vitro and in vivo induction and / or repression of cytokines (e.g., IL-I, IL-2, IL-6, IL-8, IL-12 and TNF-α) in lymphocytes using RT-PCR.

Similarly, the invention includes the use of differential demonstration to establish the induction and / or repression of selected genes for the cardiovascular region (e.g., superoxide dismutase, hemoxidase, COX 1 and 2 and other oxidation protection genes) under conditions of stimulation and / or oxidative stimulation (e.g., TNF-α or H ₂ O ₂ ). For the cancer field, the invention includes the use of a differential demonstration to confirm the induction and / or repression of genes or gene products such as CuZn superoxide dismutase, Mn superoxide dismutase, catalase, etc., in control cells and in cells subjected to oxidative stress. The following, non-limiting examples are for illustration only and should not be construed as limiting the invention, and many obvious changes can be made to these examples without departing from the scope and spirit of the invention.

EXAMPLES

EXAMPLE 1

Cocoa sources and methods for producing

Some genotypes of Teobroma cacao, which are three well-known cultivated cocoa varieties (Enriques, 1967; Engels, 1981), were obtained from the three main regions of the world producing cocoa. The genotypes used in this study are listed in Table 1. The collected cocoa fruits were opened and beans with pulp were extracted for freeze-drying. Then the pulp was manually separated from the dried mass, and the beans were subjected to the analysis described below. Unfermented and freeze-dried cocoa beans were first manually separated from the peel, and then ground in a TEKMAR mill to obtain a fine powder mass. Then the resulting mass was degreased until morning by extraction (Soxhlet) using re-distillation hexane as a solvent. Residual solvent was removed from the defatted mass by vacuum distillation at ambient temperature.

Table 1
Description of Teobroma cacao as a source of material Genotype Region of origin Cultivar UIT-1 Malaysia Trinitario Unknown West Africa Forastero ICS-100 Brazil Trinitario (ancestor of Nicaraguan Criollo) ICS-39 Brazil Trinitario (ancestor of Nicaraguan Criollo) UF-613 Brazil Trinitario EEG-48 Brazil Forastero UF-12 Brazil Trinitario NA-33 Brazil Forastero

EXAMPLE 2

Procyanidin Extraction Methods

A. Method 1

Procyanidins were extracted from fat-free, unfermented, and freeze-dried cocoa beans of Example 1 using a modification of the method described by Jalan & Collin (1977). Procyanidins were extracted twice from 50-gram batches of non-fat cocoa mass 400 ml of a mixture of 70% acetone / deionized water, and then 400 ml of 70% methanol / deionized water. The extracts were combined, and the solvents were removed by evaporation at a temperature of 45 ° C on a rotary evaporator in partial vacuum. The resulting aqueous phase was diluted with deionized water to a volume of 1 liter, and then was extracted twice with 400 ml of CHCl ₃ . The solvent phase was discarded. Then, the aqueous phase was extracted four times with 500 ml of ethyl acetate. Each of the emulsions obtained was destroyed by centrifugation on a Sorvall RC 208 centrifuge at 2000xg for 30 minutes at a temperature of 10 ° C. To the combined ethyl acetate extracts were added 100-200 ml of deionized water. Then, the solvent was removed at a temperature of 45 ° C on a rotary evaporator in a partial vacuum. The resultant aqueous phase was frozen in liquid N ₂ and then freeze-dried by using a freeze-drying system LABCONCO. The yields of crude procyanidins obtained from various cocoa genotypes are presented in table 2.

table 2
The yield of crude procyanidins Genotype Region of origin Yield (g) UIT-1 Malaysia 3, 81 Unknown West Africa 2,55 ICS-100 Brazil 3.42 ICS-39 Brazil 3.45 UF-613 Brazil 2.98 EEG-48 Brazil 3.15 UF-12 Brazil 1.21 NA-33 Brazil 2.23

B. Method 2

Alternatively, procyanidins were extracted from fat-free, non-fermented, freeze-dried cocoa beans, prepared as described in Example 1, using a 70% aqueous acetone solution. Ten grams of defatted material was suspended for 5-10 minutes with 100 ml of solvent. The resulting suspension was centrifuged at 3000 xg for 15 minutes at 4 ° C and the supernatant was passed through glass wool. The filtrate was distilled in a partial vacuum, and the resulting aqueous phase was frozen in liquid nitrogen, followed by freeze drying on a LABCONCO Freeze Dry System. The yield of crude procyanidins was 15-20%.

Without claiming to be a specific theory, it can be assumed that the differences in the yield of crude procyanidins are due to differences in genotypes, geographical origin, and production methods.

EXAMPLE 3

Partial purification of cocoa procyanidins

A. Gel Permeation Chromatography

The procyanidins obtained as described in Example 2 were partially purified by liquid chromatography on Sephadex (Sephadex LH-20) (28x2.5 cm). Separation was carried out using a stepwise gradient of deionized water → methanol. As the initial gradient composition, a 15% solution of methanol in deionized water was used, and then by incrementally increasing every 30 minutes, a 25% solution of methanol in deionized water, a 35% solution of methanol in deionized water, a 70% solution of methanol in deionized water, and finally 100% methanol. The liquid flowing after elution of xanthine alkaloids (caffeine and theobromine) was collected as one fraction. This fraction gave a xanthine alkaloid-free subfraction, which was then further fractionated to give five subfractions designated as MM2A-MM2E. The solvent was removed from each subfraction by evaporation at 45 ° C on a rotary evaporator operating in a partial vacuum. The resulting aqueous phase was frozen in liquid nitrogen and freeze dried overnight on the LABCONGO Freeze Dry System. A representative chromatogram obtained by gel permeation chromatography and illustrating fractionation is shown in FIG. In this way, approximately 100 mg of material was fractionated.

Chromatography conditions:

column: 28x2.5 cm, Sephadex LH-20;

mobile phase: stepwise gradient methanol / water 15:85, 25:75, 35:65, 70:30, 100: 0 at 30-minute intervals;

flow rate: 1.5 ml / min;

UV detector at λ ₁ = 254 nm and λ ₂ = 365 nm;

chart paper speed: 0.5 mm / min;

column loading: 120 mg.

B. Semi preparative high performance liquid chromatography (HPLC)

Method 1: reverse phase separation

The procyanidins obtained as described in Example 2 and / or 3A were partially purified using semi-preparative HPLC (a Hewlett Packard 1050 HPLC system equipped with a variable wavelength detector and a Rheodyne 7010 injection valve with a 1 mm injection loop) was connected to a collector Fraction Farmacia Frac-100, separation was carried out on a column (250 x 22.5 mm) of a Phenomenex Ultracard connected to a pre-column (60 x 10 mm) of Phenomenex 10 μm ODS Uitracarb. The mobile phase of composition A = water; B = methanol used in a linear gradient under the following conditions (time;% A): (0, 85), (60, 50), (90, 0) and (110, 0) at a flow rate of 5 ml / min. Components were detected by UV at 254 nm. The characteristic curve obtained by semi-preparative HPLC for the separation of procyanidins present in the D + E fractions is shown in FIG. 15N. Individual peaks or selected chromatographic zones were collected at regular intervals or manually, by collecting fractions for further purification and subsequent evaluation. The sample applied by syringe was 25-100 mg of material.

Method 2: Normal Phase Separation

The procyanidin extracts obtained as described in Example 2 and / or 3A were partially purified using semi-preparative HPLC, a Hewlett Packard 1050 HPLC system with a Millipore-Waters Model 480 LC detector set at 254 nm, coupled to a Farmacia Frac-100 fraction collector . Separation was performed on a Supelco 5 μm Supeicosil LC-Si column (250x10 mm) coupled to a Supelco 5 μm Supelguard LC-Si column (20x4.6 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), followed by a 10-minute rebalancing . The mobile phase of the composition: A = dichloromethane, B = methanol and C = acetic acid / water (1: 1). The flow rate was 3 ml / min. The components were detected using a UV detector at a wavelength of 254 nm and recorded using a Kipp & Zonan BD41 recorder. The volume of injected samples ranged from 100 to 250 μl of extracts of 10 mg of procyanidin diluted in 0.25 ml of 70% aqueous acetone. A representative chromatogram obtained by semi-preparative HPLC is shown in FIG. Individual peaks or selected chromatographic zones were collected at regular intervals or manually by collecting fractions for further purification and subsequent evaluation.

HPLC conditions:

250x10 mm semi-preparative column Supelco Supelcosil LC-Si (5 μm);

20x4.6 mm Supeico Supelcosil LC-Si pre-column (5 microns);

detector: Waters LC;

Model 480 spectrophotometer at 254 nm;

flow rate: 3 ml / min;

column temperature: ambient temperature;

application: 250 μl of extract in 70% aqueous acetone.

Gradient:
time (min) CH ₂ Cl ₂ Methanol Acetic acid: H ₂ O (1: 1) 0 82 fourteen four thirty 67.6 28,4 four 60 46 fifty four 65 10 86 four 70 10 86 four

The following fractions were obtained:

Fractions View one Dimers 2 Trimers 3 Tetramers four Pentamers 5 Hexamers 6 Heptamers 7 Octamers 8 Nonamers 9 Decamers 10 Undecamera eleven Dodecamers 12 Higher oligomers

EXAMPLE 4

Analysis of procyanidin extracts using analytical HPLC

Method 1: reverse phase separation

The procyanidin extracts obtained as described in Example 3 are filtered through a 0.45 micron filter and analyzed on a three-part Hewlett Packard 1090 HPLC system equipped with a HP Model 1046A Photodiode and Programmable Fluorescence Detector. Separation was carried out at 45 ° C on a Hewlett-Packard column (200 x 2.1 mm) with Hypersil ODS (5 μm). Flavanols and procyanidins were suirable linear gradient of 60% B to A, followed by washing the column with solvent B at a flow rate of 0.3 ml / min. The composition of the mobile phase: B = 0.5% acetic acid in methanol and A = 0.5% acetic acid in deionized water. The amount of acetic acid in the mobile phases A and B can be increased up to 25%. Components identified by measuring fluorescence radiation, where λ _exc. = 276 nm and λ _ex. = 316 nm. Concentrations of (+) - catechin and (-) - epicatechin were determined in relation to standard standard solutions. Procyanidin levels were evaluated using a reactivity factor for (-) - epicatechin. On figa shows a typical HPLC chromatogram illustrating the separation of the various components for a single cocoa genotype. Similar HPLC profiles were obtained for other cocoa genotypes.

HPLC conditions:

Column 200x2.1 mm, Hewlett-Packard Hypersil ODS (5 μm).

Pre-column: 20x2.1 mm, Hewlett-Packard Hypersil ODS (5 μm).

Detectors: diode array detector, at 280 nm, fluorescence detector, at λ _exc. = 276 nm and λ _ex. = 316 nm.

Flow rate: 3 ml / min.

Column temperature: 45 ° C.

Gradient:
time (min) 0.5% acetic acid in deionized water 0.5% acetic acid in methanol 0 one hundred 0 fifty 40 60 60 0 one hundred

Method 2: normal phase separation

The procyanidin extracts obtained as described in Examples 2 and / or 3 were filtered through a 0.45 micron filter and analyzed using a Hewlett-Packard 1090 HPLC system equipped with an HP Model 1046A Programmable Fluorescent Detector and a Photodiode Array Detector. Separation was carried out at 37 ° C on a column (250x3.2 mm) with a Phenomenex Lichroshere Silica 100 (5 μm) connected to a pre-column (20x4.6) with a Supelco Superguard LC-Si (5 μm). And procyanidins were eluted by a linear gradient under the following conditions: time,% A,% B): (0, 82, 14), (30, 67.6, 26.4), (60, 46, 50), (65, 10 , 86), (70, 10, 86) followed by an 8-minute rebalancing. The composition of the mobile phase is% A = dichloromethane, B = methanol and C = acetic acid / water in a volume ratio of 1: 1. The flow rate was 0.5 ml / min. The components were detected by measuring fluorescence radiation, where λ _exc. = 276 nm and λ _ex. = 316 nm, and UV radiation at 280 nm. 2B shows a representative HPLC chromatogram illustrating the separation of various procyanidins for a single genotype. Similar HPLC profiles were obtained for other cocoa genotypes.

HPLC conditions:

250 x 2.2 mm column with Phenoinenex Lichrosphere Silica 100 (5 μm) and a 20 x 4.6 mm pre-column with Superguad LC-Si (5 μm).

Detectors: photodiode array detector at 2380 nm, fluorescent detector; λ _exc. = 276 nm and λ _ex. = 316 nm.

Flow rate: 0.5 ml / min.

Column temperature: 37 ° C.

Gradient:
time (min) CH ₂ Cl ₂
methyl chloride Methanol Acetic acid / water (1: 1) 0 82 fourteen four thirty 67.6 28,4 four 60 46 fifty four 65 10 86 four 70 10 86 four

EXAMPLE 5

Procyanidin Identification

Procyanidins were purified by liquid chromatography on a column (28x2.5 cm) with Sufadex LH-20 followed by semi-preparative HPLC on a column (100x8 mm) with Bondapak C18 (10 μm) or by semi-preparative HPLC on a column (250x10 mm) with Sepelcosil LC-Si (5 μm).

Partially purified isolates were analyzed by fast atom bombardment mass spectrometry (FAB-MC = Fast Atom Bombardment - Mass Spectrometry) performed on a high resolution XG ZAD-T MS system using the secondary ion liquid spectrophotometry (LSIMS) technique in recording modes positive and negative ions. A cesium ion gun was used as an ionization source at 30 kV, and the Magic Bullet Matrix (1: 1 dithiothreitol / dithioerythritol) was used as a proton donor.

As a result of analytical studies of these fractions using LSIMS, the presence of a number of Flavan-3-ol oligomers was detected, as shown in Table 3.

Table 3
Data for cocoa procyanidin fractions obtained using LSIMS (positive ion) Oligomer (M + 1) ⁺ ,
m / z (M + Na) ⁺ ,
m / z Molecular mass Monomers (catechins) 291 313 290 Dimer (s) 577/579 599/601 576/578 Trimer (s) 865/867 887/889 864/866 Tetramer (s) 1155 1177 1154 Pentamer (s) 1443 1465 1442 Hexamer (s) 1731 1753 1730 Heptamer (s) - 2041 2018 Octamer (s) - 2329 2306 Nonamer (s) - 2617 2594 Decamer (s) - 2905 2882 Undecamera (s) - - 3170 Dodecamera (s) - - 3458

The bulk of the ionic fragments corresponded to the data obtained previously using the FAB-MC analysis of procyanidins by positive and negative ionization (Self et al., 1986, and Porter et al., 1991). It was assumed that the ion corresponding to m / z = 577 (M + H) ⁺ and its sodium adduct with m / z = 599 (M + Na) ⁺ indicate the presence of double bond procyanidin dimers in the isolates. It is interesting to note that higher oligomers are more likely to form sodium adducts (M + Na) ⁺ than their protonated molecular ions (M + H) ⁺ . The isomers of procyanidins B-2, B-5 and C-1 were tentatively identified based on data obtained by Revilla et al. (1991), Self et al. (1986) and Porter et al. (1991). Procyanidins up to octamers and decamers were confirmed using Fab-MC in partially purified fractions. In addition, the presence of procyanidins with a structure up to the decamer was confirmed by normal phase HPLC analysis (see FIG. 2B). Table 4 shows the relative concentrations of procyanidins found in the isolates not containing xanthine alkaloid by reverse phase HPLC analysis. Table 5 shows the relative concentrations of procyanidins detected by normal phase HPLC analysis.

Table 4
Relative concentrations of procyanidins in xanthine alkaloid-free isolates Component amount (+) - Catechin 1.6% (-) - Epicatechin 38.2% B-2 dimer 11.0% B-5 dimer 5.3% C-1 Trimer 9.3% Double bond dimers 3.0% Tetramer (s) 4,5% Pentameter Octamer 24.5% Unknown and higher oligomers 2.6%

Table 5
Relative concentrations of procyanidins in aqueous acetone extracts Component amount (+) - Catechin and (-) - epicatechin 41.9% B-2 dimer and B-5 dimer 13.9% Trimers 11.3% Tetramers 9.9% Pentamers 7.8% Hexamers 5.1% Heptamers 4.2% Octamers 2.8% Nonamers 1.6% Decamers 0.7% Undecamera 0.2% Dodecamers <0.1%

Figure 3 shows several structures of procyanidins, and Figures 4A-4E show typical HPLC chromatograms of five fractions used in the screening for anticancer or antitumor activity described below. For the HPLC illustrated in FIGS. 4A-4E, the following conditions were used:

HPLC conditions: a three-part Hewlett Packard 1090 HPLC system equipped with an HP Model 1046A Programmable Fluorescence Detector.

Column: Hewlett-Packard 5 μm Hypersil ODS (200 x 2.1 mm);

A linear gradient of 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. λ _exc. = 276 nm and λ _ex. = 316 nm.

On figo shows a characteristic chromatogram obtained by conducting semi-preparative HPLC for an additional 12 fractions used in screening for anticancer or antineoplastic activity (the conditions for the HPLC indicated above.

EXAMPLE 6

Anticancer, antitumor, or antineoplastic activity of cocoa extracts (procyanidins)

To screen test samples obtained as described in Example 5, a cytotoxicity assay was performed with tetrazole using microtiter plates and MTT reagent (3- [4,5-dimethyltriazol-2-yl] -2,5-diphenyltetrazolium bromide). Test samples, standards (ciplatin and chlorambucil) and MTT reagent were dissolved in 100% DMSO (dimethyl sulfoxide) at a concentration of 10 mg / ml. Serial dilutions were obtained from stock solutions. In the case of test samples, dilutions were obtained in the range from 0.01 to 100 μg / ml in 0.5% DMSO.

All human tumor cell lines were obtained from the American Type Culture Collection. The cultures were cultured as monolayers in alpha-MEM medium containing 10% fetal bovine serum, 100 units / ml penicillin, 100 μg / ml streptomycin and 240 units / ml nystatin. Cells were kept in a humid atmosphere of 5% CO ₂ at 37 ° C.

After trypsinization, the cells were counted and brought to a concentration of 50x10 ⁵ cells / ml, which varies depending on the cancer cell line. 200 ml of cell suspension were plated in the wells of four rows of a 96-well microtiter plate. Then the cells were left for 4 hours to bind, after which 2 μl of DMSO containing solutions of the test samples was added to the wells (in four duplicates). Initial experiments to identify the dose-response curve using a series of multiple dilutions of the test sample were performed to determine dose ranges that need to be evaluated. Then, using the BIO RAD MP450 plate reader, the optical densities of the wells were determined at 540 nm. The average optical density of the wells treated with the test sample in four duplicates was compared with the control value and the results were expressed as a percentage of the control optical density +/- standard deviation. The reduction of MTT to the purple formazan product is linearly correlated with the number of surviving cells in the well. Thus, by measuring the optical density, the percentage of cells surviving at a given dose of the test sample can be determined. Control wells contained a final concentration of 1% DMSO.

According to this scheme, two samples were first tested. Sample MM1 was a very coarse cocoa procyanidin isolate and contained amounts of caffeine and theobromine. Sample MM2 — Cocoa procyanidin isolate, partially purified by gel permeation chromatography. In the MM2 sample, caffeine and theobromine were absent. Using the method described above, both samples were screened for activity against the following cancer cell lines:

HCT - 116 - colon cancer cell line

ACHN - kidney adenocarcinoma cell line

SK-5 - melanoma cell line

A-498 - kidney adenocarcinoma cell line

MCF-7 - Breast Cancer Cell Line

PC-3 - prostate cancer cell line

CAPAN - Pancreatic Cancer Cell Line

Sample MM1 showed little activity or no activity at all with respect to the studied cancer cell lines. Sample MM2 was active against cancer cell lines HCT-116, PC-3 and ACHN. However, it was found that MM1 and MM2 interfere with MTT in such a way that this prevents a decrease in optical density, which correlates with a decrease in the number of surviving cells. Such interference also leads to an increase in the magnitude of the error, since the chemical reaction obviously takes place more quickly in the wells located along the perimeter of the tablet. A typical example of such an effect is shown in FIG. It should be expected that a significant decrease in the number of surviving cells instead of the indicated levels of surviving cells will be observed at high concentrations of the test material. Nevertheless, microscopic studies have shown that a cytotoxic effect takes place, despite the phenomenon of interference with MMT. So, for example, in this experiment, the IC ₅₀ value obtained for MM2 using the ACHN cell line was 0.5 μg / ml.

These preliminary results from the point of view of the authors of the present invention require a change in the methodology for the analysis in order to eliminate interference with MMT. These changes were carried out as follows. After the plates were incubated for 18 hours at 37 ° C in a humid 5% CO ₂ atmosphere, the medium was carefully aspirated and replaced with fresh alpha-MEM medium. Then on the third day of analysis, the medium was again aspirated from the wells and replaced with 100 milliliters of freshly prepared McCoy medium. Then, 11 μl of a 5 mg / ml stock solution of MTT in PBS (buffered phosphate saline) was added to the wells of each plate. After 4 hours of incubation in a humid 5% CO ₂ atmosphere at 37 ° C, 0.4 N was added to all wells of the plates. HCl in isopropanol and then thoroughly mixed to solubilize formazan produced by any viable cells. In addition, to determine the specific components responsible for the activity, it was decided to subfractionate procyanidins.

The subfractionation procedure described above was carried out in order to obtain samples for subsequent screening. As a result, five fractions were obtained representing the regions shown in FIG. 1 and the distribution of components shown in FIGS. 4A-4E. For analytical characterization and to indicate the absence of caffeine and theobromine, the samples were designated MM2A-MM2E.

Each fraction was individually screened for activity against cancer cell lines HCT-116, PC-3 and ACHN. The results showed that this activity is not concentrated in any one particular fraction. Such results were not considered unusual, since the components in the isolates of “active” natural products may have a synergistic effect. So in the case of the cocoa procyanidin isolate (MM2), it was found that it contains over twenty detectable components. It is considered possible that the activity of the isolate is most likely associated not with any individual component (s), but with a combination of components present in different fractions.

Based on these results, it was decided to combine the fractions and repeat the assays for activity against the same cancer cells. These analyzes showed that combinations of different fractions produced cytotoxic effects against cancer cell lines PC-3. In particular, the IC ₅₀ values obtained for the combination of MM2A and MM2E were 40 μg / ml, and the IC ₅₀ values obtained for the combination of MM2C and MM2E were 20 μg / ml. Activity against HCT-116 and ACHN cell lines was also observed, but as mentioned above, interference with the MMT indicator prevented accurate measurements. For specified data, repeated experiments were performed in duplicates for cell lines HCT-116 and ACHN. However, these results were unreliable due to bacterial contamination and depletion of the test material. 6A-6D show a dose response curve obtained as a result of the interaction between the fractions and the PC-3 cancer cell line.

Nevertheless, the data obtained clearly showed that cocoa extracts, especially cocoa polyphenols or procyanidins, have significant antitumor, anticancer or antineoplastic activity, especially against cell lines such as PC-3 (prostate cancer), HCT-116 (cancer colon) and ACHN (kidney cancer). In addition, the results suggest that specific procyanidin fractions may be responsible for activity against the PC-3 cell line.

EXAMPLE 7

Anticancer, antitumor and antineoplastic activity of cocoa extracts (procyanidins)

To confirm the results and further study of the combinations of fractions, another more detailed screening was performed.

All the materials obtained and the procedures performed are similar to those described above, except that instead of the standard 4 duplicates per dose, 8 and 12 duplicates per test dose were made. For the experiment, individual and combinations of five fractions of cocoa procyanidins were screened against the following cancer cell lines:

PC-3 - Prostate

KB - nasopharyngeal area / Hela

HCT - 116 - colon

ACHN - Kidney

MCF-7 - Breast

SK-5 - Melanomas

A-549 - lungs

CCRF-CEM - T Cell Leukemia

Screening of individual fractions consists in analyzing different doses (0.01-100 μg / ml) of fractions A, B, C, D and E (see FIGS. 4A-4E and their discussion above) against each of the cell lines. Screening for a combination of fractions included combining equal doses of these fractions A + B, A + C, A + D, A + E, B + C, B + D, B + E, C + D, C + E and D + E and analysis their effects on each of the cell lines. The effects on cell lines were discussed separately and then summarized.

A. PC-3 Prostate Cancer Cell Line

On figa-7H shows the curves of the dose-response, obtained by the interaction between the fractions of cocoa procyanidins and the cell line PC-3. On fig.7D-7E shows that fractions D and E have activity at IC ₅₀ = 75 μg / ml The IC ₅₀ values obtained from the dose-response curves for other combinations of procyanidin fractions including D or E fractions were 60-80 μg / ml. The remaining IC ₅₀ values are shown in table 6.

B. Nasopharyngeal Cancer Cell Line KB / HeLa

On figa-8H shows the curves of the dose-response, obtained as a result of the interaction between the fractions of cocoa procyanidins and the nasopharyngeal cell line KB / HeLa. On fig.8D and 8E shows that the fractions D and E have activity at IC ₅₀ = 75 μg / ml On Fig.8E-8H presents typical results obtained on the basis of the study of the combination of fractions. In these studies, it was found that the combination A + B does not have activity, while the combinations B + E and D + E have activity at an IC ₅₀ = 60 μg / ml. The IC ₅₀ values obtained from the dose-response curves for other combinations of procyanidin fractions, including fractions D or E, were 60-80 μg / ml. The individual IC ₅₀ values are shown in Table 6. These data are basically similar to the data obtained for the PC-3 cell line.

C. Colon cancer cell line HCT-116

On figa-9H shows a typical dose dependence between the fractions of procyanidin and the cell line of colon cancer HCT-116. Figures 9D and 9E demonstrate that fraction E has activity at an IC ₅₀ value of approximately 400 μg / ml. This value was obtained by extrapolating the existing curve. It should be noted that the slope of the dose-response curve for fraction D also indicates the activity of fraction D. However, the IC ₅₀ value was not determined from this curve, since this curve is too gentle to obtain a reliable result. On fig.9F-9H presents the characteristic results obtained on the basis of studies of combinations of fractions. In this study, it was found that the combination of procyanidin B + D fractions did not have significant activity, while combinations of fractions A + E and D + E showed activity at IC ₅₀ = 500 μg / ml and 85 μg / ml, respectively. IC ₅₀ values obtained from dose-response curves for other combinations containing fraction E averaged about 250 μg / ml. The following table 6 shows the IC ₅₀ values extrapolated.

D. ACHN Renal Adenocarcinoma Cell Line

On figa-10H shows a typical dose dependence between the fractions of cocoa procyanidin and the renal cell line ACHN. Figa-10E show that individual fractions do not have activity against this cell line. On fig.10F-10H presents the characteristic results obtained on the basis of studies of combinations of fractions. In these studies, the combination of the procyanidin fraction B + C did not show activity, and the extrapolated IC ₅₀ value obtained for the combination A + E was approximately 500 μg / ml. It was concluded that combinations giving dose-response curves similar to the curve for the C + D combination do not have activity, since this curve is too gentle. The extrapolated IC ₅₀ values for other combinations of fractions are given in table 6.

E. A-549 Lung Cancer Cell Line

On figa-11H shows a typical dose dependence between the fractions of cocoa procyanidin and the lung cancer cell line A-549. At the doses used in this experiment, no activity of either individual fractions or their combinations was detected. However, procyanidin fractions can nevertheless be used in relation to this cell line.

F. Cell line melanoma SK-5

On figa-12H presents a typical dose dependence between the fractions of cocoa procyanidin and the melanoma cell line SK-5. At the doses used in this experiment, no activity of either individual fractions or their combinations was detected. However, procyanidin fractions can nevertheless be used in relation to this cell line.

G. Breast Cancer Cell Line MCF-7

On figa-13H presents a typical dose dependence between the fractions of cocoa procyanidin and the breast cancer cell line MCF-7. At the doses used in this experiment, no activity of either individual fractions or their combinations was detected. However, procyanidin fractions can nevertheless be used in relation to this cell line.

H. T cell leukemia cell line CCRF-CEM

Typically, typical dose-response curves were obtained for the CCRF-CEM T-cell leukemia line. However, microscopic calculation of the number of cells versus time at various concentrations showed that 500 μg of fractions A, B and D caused 80% inhibition of cell growth over a period of 5 days. Typical dose-response curves are shown in FIG.

I. Brief Summary

All IC ₅₀ values obtained from the described experiments for all cell lines except the CCRF-CEM T-cell leukemia line are listed in Table 6. The data for T-cell leukemia were intentionally excluded from the table, because a different analysis procedure was used to obtain them. . A generalization of the results showed that the highest activity is associated with fractions D and E. These fractions are most active against cell lines PC-3 (prostate gland) and KB (nasopharyngeal carcinoma / HeLa). These fractions also showed activity against cell lines HCT-116 (colon) and ACHN (kidney), but only at significantly higher doses. No activity against MCF-7 (mammary gland), SK-5 (melanoma), and A-549 (lung) cell lines was observed. However, procyanidin fractions can nevertheless be used in relation to these cell lines. In addition, activity against the CCRF-CEM (T cell leukemia) cell line was observed. It should also be noted that the composition of fractions D and E are the most complex. Nevertheless, from the data obtained, it is obvious that cocoa extracts, and especially cocoa procyanidins, have significant antitumor, anticancer, or antineoplastic activity.

Table 6
IC ₅₀ values for cocoa procyanidin fractions having activity against various cell lines (IC ₅₀ values are expressed in μg / ml) Fraction RS-3 KB NST-116 ACHN Mcf-7 SK-5 A -549 A B C D 90 80 E 75 75 400 A + B A + c 125 one hundred A + d 75 75 A + e 80 75 500 500 B + c B + d 75 80 B + e 60 65 200 C + d 80 75 1000 C + e 80 70 250 D + E 80 60

The above values of 100 μg / ml were obtained by extrapolating the dose-response curves

EXAMPLE 8

Anticancer, antitumor and antineoplastic activity of cocoa extracts (procyanidins)

To complement and expand the results presented in examples 6 and 7, several additional in vitro assays were performed.

Method A. Analysis by Crystal Violet Staining

All human tumor cell lines were obtained from the American Type Culture Collection. Cells were cultured as monolayers in IMEM medium containing 10% fetal serum and not containing antibiotics. Cells were maintained in a humidified 5% CO ₂ atmosphere at 37 ° C.

After trypsinization, the cells were counted and adjusted to a concentration of 1000-2000 cells per 100 ml. Cell proliferation was determined by plating cells in a 96-well microtiter plate (1000-2000 cells / well). After adding 100 μl of cells to the well, the cells were left for 24 hours to bind. After the completion of the 24-hour period, various cocoa fractions were added at various concentrations, resulting in dose-response data. Then the cocoa fractions were dissolved in a 2-fold concentration medium and 100 μl of each solution was added to the wells in triplicate. On the following days, the plates were stained with 50 μl of crystalline violet for 15 minutes (2.5 g of crystalline violet dissolved in 125 ml of methanol, 375 ml of water). Then the dye was removed, the tablet was carefully immersed in cold water to remove excess staining. After this, the tablets were washed two more times and left to dry. The residual dye was solubilized by adding to each well 100 μl of a mixture of 0.1 M sodium citrate / 50% ethanol. After solubilization, the number of cells was counted using an ELISA plate reader at 540 nm (reference filter at 410 nm). Based on the results obtained, dependency graphs were constructed where the growth time (days) is plotted on the abscissa (x) axis and the optical density is plotted on the ordinate (y) axis.

Method B. Analysis of soft agar cloning

Cells were cloned in soft agar according to the method described by Nawata et al. (1981). Single cell suspensions were prepared in media containing 0.8% agar with various concentrations of cocoa fractions. Aliquots of suspensions were added to 35 mm plates coated with medium containing 1.0% agar. After 10-day incubation, the number of colonies with a diameter of more than 60 μm was determined using the Ominicron 3600 Image analytical system. Based on the results obtained, graphs of the dependence of the number of colonies (y axis) on the concentration of cocoa fractions (x axis) were constructed.

Method C. Microculture analysis using tetrazolium (XTT)

XTT analysis method described by Scudiero et al. (1988), used for screening various fractions of cocoa. The analysis using XTT was performed by the same method as the analysis using MTT (see Example 6), with the exception of some of the following modifications. XTT ((2,3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -5 - [(phenylamino) carbonyl] -2H-tetrazolium hydroxide) was prepared at a concentration of 1 mg / ml in serum-free medium preheated to 37 ° C. PMS was prepared in 5 mM PBS, then XTT and PMS were mixed together and 10 μl of PMS per 1 ml of XTT and 50 μl of PMS-XTT were added to each well. 30 minutes on a mechanical shaker and the optical density was determined at 450-600 nm. Based on the results obtained, graphs of the dependence of optical density (y axis) on time change (days) or concentration (x-axis).

When conducting analyzes using methods A and C, the results were expressed as% of control and graphs of the% control (y axis) versus time (days) or concentration (x axis) were plotted.

In order to determine which of the assays is the most sensitive, a comparison of XTT and crystal violet analysis methods was performed to evaluate the activity of fractions D and E (Example 3B) against the p168 MCF-7 breast cancer cell line. As shown in FIG. 15A, both analyzes gave similar dose-response relationships for concentrations> 75 μg / ml. At lower concentrations, the results of the analysis using crystalline violet showed higher standard deviations than the results of the analysis using XTT. However, since the method of conducting the analysis using crystalline violet is simpler, all subsequent analyzes, unless otherwise noted, were carried out by this method.

On figv-15E presents the results of analyzes performed using crystalline violet to illustrate the effect of the crude polyphenol extract (example 2) on the MDA breast cancer cell line MDA MB231, the prostate cancer cell line PC-3, the breast cancer cell line MCF- 7 p163 and Hela uterine cancer cell line, respectively. In all cases, a dose of 250 μg / ml completely inhibited the growth of all cancer cells over a period of 5-7 days. It is obvious that the Hela cell line is more susceptible to the action of the extract, since its dose of 100 μg / ml also inhibited cell growth. The cocoa fractions obtained in Example 3B were also analyzed for activity against the Hela cell line and against the SKBR-3 breast cancer cell line. The results of these analyzes (FIGS. 15F and 15G) showed that fractions D and E have the highest activity. As seen in fig.15N and 15I, IC ₅₀ values obtained for the two cell lines were about 40 ug / ml D and E.

The cocoa fractions D and E were also analyzed by soft agar cloning to determine the ability of the test compound (or compounds) to inhibit free ("armless") cell growth. As shown in FIG. 15J, a concentration of the compound of 100 μg / ml completely inhibited the formation of Hela cell colonies.

The crude polyphenol extracts obtained from eight different cocoa genotypes, representing three cultivated cocoa varieties, were also analyzed for activity against the Hela cell line. As can be seen in FIG. 15K, extracts of all cocoa varieties showed similar dose-response relationships. The highest activity against the Hela cell line was detected by the UIT-1 variety. The results obtained indicate that all cocoa genotypes have polyphenol fractions that are active against at least one cell line of a human cancer tumor, and this activity does not depend on the geographical origin of the culture variety and genotype.

Another series of analyzes was carried out using crude polyphenol extracts obtained by traditional 5-day fermentation of Brazilian cocoa beans with a production scale of 1 ton per day, followed by 4-day drying. The results presented in FIG. 15L did not show a noticeable effect from such early stages of processing, which suggests an insignificant change in the composition of the polyphenols. However, it is known (Lehrian and Patterson, 1983) that polyphenol oxidase (PPO) can oxidize polyphenols in a fermentation step. In order to determine what activity the enzymatically oxidized polyphenols will have, another experiment was carried out. Crude polyphenol oxidase (PPO) was obtained by extracting finely ground unfermented, freeze-dried non-fat Brazilian cocoa beans with acetone in a ratio of 1 g of powder per 10 ml of acetone. The resulting suspension was centrifuged at 3000 rpm for 15 minutes. This procedure was repeated three times, each time removing the supernatant, and after the fourth extraction it was poured through a Buchner filter funnel. The acetone-extracted powder was allowed to air dry and then analyzed in accordance with the procedure described by McLord and Kilara (1983). To a solution of crude polyphenols (100 mg / 10 ml citrate-phosphate buffer, 0.02 M, pH 5.5) was added 100 mg of acetone powder (4000 active units / mg of protein) and left to mix for 30 minutes at the same time bubbling air through the suspension. The sample was centrifuged at 5000xg for 15 minutes and the supernatant was extracted three times with 20 ml of ethyl acetate. The ethyl acetate extracts were combined, dried to dryness by partial vacuum distillation, 5 ml of water was added and then lyophilized. After that, the obtained material was analyzed for activity against Hela cells, the dose-response relationship obtained for this material was compared with the corresponding dependences obtained for crude polyphenol extracts that were not subjected to enzymatic treatment. As a result of this comparison (Fig. 15M), a significant shift in the dose-response curve was found for the enzymatic oxidation of the extract, which indicates that the oxidized products have a greater inhibitory ability than their native forms.

EXAMPLE 9

Antioxidant activity of cocoa extracts containing procyanidins

Data reported in the literature suggest that there is a correlation between the intake of natural antioxidants (vitamins C, E and B-carotene) and a low incidence, including cancer incidence (Designing Foods, 1993; Caragay, 1992). It is generally known that these antioxidants affect some oxidative processes and free radical formation processes that stimulate the growth of certain types of tumors. In addition, some plant polyphenol compounds that have been shown to have anticancer activity also have significant antioxidant activity (Ho et al., 1992; Huang et al., 1992).

In order to determine whether cocoa extracts containing procyanidins possess antioxidant properties, the standard Rancimat method was used. To obtain cocoa extracts, the methods described in examples 1, 2 and 3 were used, after which the obtained extracts were subjected to gel permeation chromatography to obtain two fractions. In fact, these two fractions were the combined fractions AC and D and E (see figure 1), the antioxidant properties of which were compared with the properties of the synthetic antioxidants BHA and BHT.

Peanut butter was squeezed from unroasted peanuts, which had previously been peeled. Each test compound was added to the oil in two concentrations of ~ 100 and ~ 20 ppm. (specific concentrations are shown in table 7). Then, 50 μl of an antioxidant solubilized in methanol was added to each sample to disperse the antioxidant. A control sample was prepared with 50 μl of antioxidant-free methanol.

The resulting samples were evaluated in duplicates for oxidation stability using the Rancimat test at a temperature of 100 ° C and air flow rate

20 cm ³ / min. The experimental parameters were chosen so that they corresponded to the parameters used using active oxygen (AOM) or a quick stability test (Van Oosten et al., 1981). A typical Rancimat curve is shown in FIG. The test results, expressed as the time (hours) required to reach the peroxide level of 100 meq, are shown in table 8.

Table 7
Antioxidant concentrations Sample Level 1 (ppm) Level 2 (ppm) Bottled Hydroxytoluene (BHT) 24 120 Bottled Hydroxyanisole (BHA) 24 120 Crude Cocoa Ethyl Acetate 22 110 Fraction A-C twenty one hundred Fraction D-E twenty one hundred

Table 8
Oxidation resistance of peanut butter with various antioxidants Sample 20 (ppm) 100 (ppm) average The control 10.5 ± 0.7 VNT 16.5 + 2.1 12.5 + 2.1 VNA 13.5 + 2.1 14.0 + 1.4 Unrefined Cocoa Fraction 18.0 + 0.0 19.0 + 1.4 Fraction A-C 16.0 + 6.4 17.5 + 0.0 Fraction D-E 14.0 + 1.4 12.5 + 0.7

The obtained results demonstrated the increased oxidation stability of peanut butter with all tested additives. The highest oxidation stability was observed in the sample treated with crude cocoa extract. These results showed that cocoa extracts containing procyanidins have antioxidant activity equal to or greater than the antioxidant activity of the synthetic antioxidants BHA and BHT at the same doses. Accordingly, the extracts of the present invention can be used, for example, as an antioxidant and / or food supplement instead of BHT and BHA. In this regard, it should also be noted that the extract according to the invention comes from a source suitable for human consumption. Based on the results obtained, each specialist can easily determine, without undue experimentation, the amount of the compound of the invention, for example, the amount of food additives that can be used instead of BHA and BHT.

EXAMPLE 10

The study of the inhibition of topoisomerase II

DNA topoisomerases I and II are enzymes that catalyze the breakdown and repair of DNA nucleotide chains, thereby controlling the topological state of DNA (Wang, 1985). In addition to studying the intracellular function of topoisomerase, the most valuable information was obtained by identifying topoisomerase II as the main cellular target for a number of clinically important antitumor compounds (Yamashita et al., 1990), including intercalating agents (m-AMSA, adriamycin and ellipticin), as well as intercalating epipodophyllotoxins. A number of available data suggest that some anticancer drugs have the common property of stabilizing the DNA topoisomerase II (cleavable complex) complex, which can be cleaved by denaturing agents that induce DNA cleavage (Muller et al., 1989). It has been suggested that the formation of a cleavable complex by antitumor drugs leads to the production of bulk DNA adducts, which, in turn, lead to cell death.

In accordance with this promising model, a new specific inducer that stimulates the formation of a cleavable complex of DNA topoisomerase II can be used as an anti-cancer, anti-tumor, or anti-blastoma agent. In an attempt to identify cytotoxic compounds with activity directed at the target DNA, cocoa procyanidins were scanned for increased cytotoxic activity against several cell lines susceptible to DNA damage, and an enzymatic analysis was performed using human topoisomerase II derived from lymphoma.

A. Decenation of kinetoplast DNA by topoisomerase II

In vitro inhibition of the decenation of kinetoplast DNA by topoisomerase II, as described by Muller et al., 1989, was carried out as follows. Nuclear extracts containing topoisomerase (II) activity were obtained from human lymphoma by modifying the methods of Miller et al. (1981) and Banks et al. (1988). One unit of purified enzyme was enough to decenate 0.25 μg kinetoplast DNA for 30 minutes at 34 ° C. Kinetoplast DNA was obtained from trypanosome Crithidia fasciculata. Each reaction was performed in 0.5 ml centrifuge tube containing 19.5 .mu.l of water, 2.5 l of 10X buffer (1X buffer contains 50 mM Tris-HCl, pH 8.0, 120 mM KCl, 10 mM MgCl _2, 0.5 mM ATP, 0.5 mM dithiotriol and 30 μg BSA / ml), 1 μl kinetoplast DNA (0.2 μg) and 1 μl DMSO containing the tested cocoa procyanidin fractions in various concentrations. This reaction mixture was thoroughly mixed and kept on ice. Then, immediately before incubation, one topoisomerase unit was added and the mixture was incubated in a water bath for 30 minutes at 34 ° C.

After incubation, the decenation analysis was stopped by adding 5 μl of a stop buffer (5% sarcosyl, 0.0025% bromphenol blue, 25% glycerol) and the mixture was placed on ice. DNA was subjected to electrophoresis on 1% agarose gel in a TAE buffer containing ethidium bromide (0.5 μg / ml). DNA was visualized by UV irradiation at a wavelength of 310 nm. Then the gel was photographed with a Polaroid Land camera.

The results of these experiments are presented in Fig.17. Fully catenated kinetoplast DNA does not migrate to a 1% agarose gel. As a result of the decenation of kinetoplast DNA by topoisomerase II, bands of monomeric DNA are formed (monomeric ring, forms I and II), which migrates into the gel. When cocoa procyanidins are added, the enzyme is inhibited, as evidenced by the progressive disappearance of bands as the concentration of procyanidins increases. Based on the results obtained, it was shown that the fractions of procyanidins A, B, D and E inhibit topoisomerase II at concentrations from 0.5 to 5.0 μg / ml. These inhibitory concentrations are very close to those obtained for mitoxantrone and m-AMSA [4 '- (9-acridylamino) methanesulfon-m-anisidide].

B. Cell lines susceptible to the drug

Cocoa procyanidins were screened for cytotoxicity against several cell lines sensitive to DNA damage. One of the cell lines was a mutant (xrs-6) for the repair of a double-stranded break in DNA obtained by Jeggo (Kemp et al., 1984). The inability of the xrs-6 cell line to repair DNA makes it susceptible to x-ray radiation, as well as to the effects of compounds that directly induce double-stranded breakage in DNA, such as bleomycin, and compounds that inhibit topoisomerase II, resulting in such a ability of the cell line to repair DNA can, as suggested by Warters et al., (1991), indirectly induce double-stranded breaks. Cytotoxicity towards a cell line incapable of DNA repair was compared with cytotoxicity against a CHO cell line (BR1) capable of DNA repair. Increased cytotoxicity against a cell line (xrs-6) incapable of repair was interpreted as evidence of the formation of a double-stranded break in cleavable DNA.

DNA cell line, BR1, capable of DNA repair, was obtained by Barrows et al. (1987), and in addition to the DNA repair enzymes of normal CHO, this cell line is able to express O ⁶ -alkylguanine-DNA-alkyl transferase. The line (xrs-6), which is not capable of repairing double-stranded DNA breaks of CHO DNA, is a valuable gift from Dr. P. Jeggo and his collaborators (Jeggo et al., 1989). Both of these cell lines were cultured as monolayers in alpha-MEM medium containing serum and antibiotics, as described in Example 6. The cells were kept at 37 ° C in a humid 5% CO ₂ atmosphere. Before treatment with cocoa procyanidins, cells cultured as monolayers were isolated by trypsin treatment. The analysis was performed using the MTT method as described in example 6.

The results obtained (Fig. 18) did not show increased cytotoxicity with respect to xrs-6 cells, which suggests that cocoa procyanidins inhibit topoisomerase II in accordance with a mechanism different from the formation of cleavable double-stranded rupture. That is, cocoa procyanidins interact with topoisomerase II before it interacts with DNA, thereby forming an indissoluble complex.

Compounds forming a non-cleavable complex are relatively new. All members of groups of compounds such as anthracyclines, podophylline alkaloids, anthracenedion, acridine, and elliptisins have been tested for clinical anticancer, antitumor, or antineoplastic uses and are capable of producing cleavable complexes (Liu, 1989). Recently, several new classes of topoisomerase II inhibitors have been identified that do not appear to produce cleavable complexes. Such compounds are amonafid (Hsiang et al., 1989), distamycin (Fesen et al., 1989), flavanoids (Yamashita et al., 1990), sintopin (Yanashita et al., 1991), membrane (Drake et al., 1989), terpenoids (Kawada et al., 1991), anthrapyrazoles (Fry et al., 1985), dioxopiperazines (Tanabe et al., 1991) and marine acridindercitin (Burres et al., 1989).

Since cocoa procyanidins inactivate topoisomerase II before the formation of cleavable complexes, they can be used in chemotherapy either individually or in combination with other known and mechanically determined topoisomerase II inhibitors. In addition, it is also obvious that cocoa procyanidins are a new class of topoisomerase II inhibitors (Kashiwada et al., 1993) and therefore they may be less toxic to cells than other known inhibitors, which will make them more effective in chemotherapy.

For analysis of the activity of the D + E fraction, the MCF-7 human breast cancer cell line (ADR) expressing membrane glycoprotein (gp170) was used as a multidrug-resistant cell line and its parent line MCF-7 p168. As shown in FIG. 19, the parent cell line was inhibited at increasing doses of the D and E fractions, while the andramycin-resistant line (ADR) was less affected at higher doses. These results showed that the cocoa fraction D and E was active against multidrug resistant cell lines.

EXAMPLE 11

Synthesis of Procyanidins

The synthesis of procyanidins was carried out according to the method developed by Delcour et al., (1983), but with some modification. In addition to condensation of (+) - catechin with dihydroquercetin under reduction conditions, (-) - epicatechin was also used to reproduce the high concentrations of (-) - epicatechin present in natural unfermented cocoa beans. The synthesized products were isolated, purified, analyzed and identified in accordance with the procedure described in examples 3, 4 and 5. According to this method, biflavonoids, triflavonoids and tetraflavonoids were obtained and used as analytical standards (see above) with respect to cocoa extracts.

EXAMPLE 12

Analysis of fractions obtained using semi-preparative chromatography with normal phase

Since polyphenol extracts are complex in composition, for subsequent purification, dose-response analysis and complete structural identification, it is necessary to determine which components are active against cancer cell lines. Based on the size of the oligomers, semi-preparative normal-phase HPLC was used to isolate cocoa procyanidins (Example 3B). To determine the oligomer with the highest activity, in addition to the initial extract, twelve fractions were obtained (Figs. 2B and 15O) and these twelve fractions were analyzed at doses of 100 μg / ml and 25 μg / ml for their activity against the Hela and SKBR-3 cell lines. As shown in FIG. 20A and 20B, fractions 4-11 (pentamer-dodecamera) significantly inhibit the Hela and SKBR-3 cancer cell lines at a value of 100 μg / ml. The results obtained indicate that these specific oligomers have the highest activity against Hela and SKBR-3 cells. In addition, analysis of cocoa fractions D and E using normal phase HPLC showed that this fraction used in previous studies, for example in Example 7, contains an increased amount of these oligomers.

EXAMPLE 13

Purification Methods Using HPLC

Method A. GPC Cleaning

The procyadins obtained as described in Example 2 were partially purified by liquid chromatography on Sephadex LH 20 (72.5 x 2.5 cm), using 100% methanol as an eluting solvent, at a flow rate of 3.5 ml / min . Eluent fractions were collected after the first 1.5 hours, concentrated on a rotary evaporator, redissolved in water and freeze dried. These fractions were enriched pentamer fractions. Similarly, approximately 2.00 g of the extract obtained in Example 2 was subfractionated. The results are shown in table 9.

Method B. Normal Phase Separation

The procyanidins obtained as described in Example 2 were purified by separation using normal phase chromatography on a Supelcosil LC-Si column, 100 Å, 5 μm (250 x 4.6 mm), at a flow rate of 1.0 ml / min. or alternatively, on a Lichrosphere® Silica 100 Å, 5 μm (235 x 3.2 mm) column at a flow rate of 0.5 ml / min. The separation was carried out by a stepwise 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 composition of the mobile phase: A = dichloromethane; B = methanol and C = acetic acid / water (1: 1). The components were determined by fluorescence at λ _exc. = 276 nm and λ _ex. = 316 nm and ultraviolet radiation at 280 nm. The applied sample volume was 5.0 μl (20 mg / ml) of procyanidins obtained as in Example 2. These results are shown in FIGS. 40A and 40B.

In another case, the separation was carried out by a stepwise gradient under the following conditions: (time,% A,% B); (0, 76, 20); (25, 46, 50); (30, 10, 86). The composition of the mobile phase: A = dichloromethane; B = methanol and C = acetic acid: water (1: 1). The results are shown in FIGS. 41A and 41B.

Method C. Reverse Phase Separation

The procyanidins obtained as described in Example 2 were purified by reverse phase chromatography on a Hewlett Packard Hypersil ODS 5 μm (200x2.1 mm) column, with a Hewlett Packard Hypersil ODS 5 μm pre-column (20x2.1 mm). Procyanidins were eluted with a linear gradient of 20% B to A for 20 minutes, followed by washing the column with 100% B at a flow rate of 0.3 ml / min. The composition of the mobile phase: a degassed mixture of B = 1.0% acetic acid in methanol and A = 2.0% acetic acid in deionized water. The components were determined by UV irradiation at 280 nm, and fluorescence at λ _exc. = 276 nm and λ _ex. = 316 nm; the volume of the applied sample was 2.0 μl (20 mg / ml).

EXAMPLE 14

Separation of enriched pentamer fractions by HPLC

Method A. Semi-preparative normal phase HPLC

The enriched pentamer fractions were further purified by normal phase semi-preparative HPLC using a Hewlett Packard 1050 HPLC system equipped with a Millipore-Waters Model 480 LC detector set to 254 nm and coupled to a Pharmacia Frac-100 fraction collector set to peak mode. Separations were performed on a Supelco 5 μm Supelcosel LC-Si column, 100Å (250 x 10 mm) connected to a Supelco 5 μm Supelguard LC-Si pre-column (20x4.6 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) followed by a 10-minute rebalancing. The composition of the mobile phase: A = dichloromethane; B = methanol and C = acetic acid: water (1: 1). The flow rate was 3 ml / min. The components were determined by UV irradiation and recorded using a Kipp & Zonan BD41 recorder. The volume of injected samples ranged from 100-250 μl of 10 mg of procyanidin extracts dissolved in 0.25 ml of 70% aqueous acetone. Individual peaks or selected chromatographic zones were collected at specific time intervals or by manually collecting fractions for further purification and subsequent evaluation.

HPLC conditions:

Semi-preparatory column: 250x100 mm Supelco Supelcosil LC-Si (5 microns), pre-column 20x4.6 mm Supelco Supelcosil LC-Si (5 microns).

Detector: Waters LC spectrophotometer model 480 with a wavelength of 254 nm.

Flow rate 3 ml / min.

Column temperature: ambient temperature.

Sample application: 250 μl of enriched pentamer extract.

Gradient CH ₂ Cl ₂ Methanol Acetic acid: water (1: 1) 0 82 fourteen four thirty 67.6 28,4 four 60 46 fifty four 65 10 86 four 70 10 86 four

Method B. Reverse Phase Separation

The procyanidin extracts obtained as described in Example 13 were filtered through a 0.45 micron nylon filter and analyzed on a three-part Hewlett Packard 1090 HPLC system equipped with an HP model 1046A programmable fluorescence array detector. Separation was carried out at 45 ° C on a Hewlett Packard 5 μm Hypersil ODS column (200 x 2.1 mm). Procyanidins were eluted with a linear gradient of 60% B to A, followed by washing the column with solvent B at a flow rate of 0.3 ml / min. The composition of the mobile phase: a degassed mixture of B = 0.5% acetic acid in methanol and A = 0.5% acetic acid in deionized water. The level of acetic acid in components A and B of the mobile phase can be increased to 2%. The components were determined by fluorescence at λ _exc. = 276 nm and λ _ex. = 316 nm and UV irradiation with a wavelength of 280 nm. Concentrations of (+) - catechin and (-) - epicatechin were determined relative to standard solutions. Procyanidin levels were evaluated using a response factor for (-) - epicatechin.

Method C. Normal Phase Separation

Pentameric enriched procyanidin extracts prepared as described in Example 13 were filtered through a 0.45 micron nylon filter and analyzed on a Hewlett Packard 1090 Series II HPLC system equipped with an HP Model 1046A programmable fluorescence detector and a photodiode array detector. Separation was carried out at 37 ° C on a 5 μm Phenomenex Lichrosphere® Silica 100 column (250x3.2 mm) connected to a Supelco Supelguard LC-Si 5 μ column (20x4.6 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), followed by an 8-minute rebalancing . The composition of the mobile phase: A = dichloromethane, B = methanol and C = acetic acid: water in a volume ratio of 1: 1. The flow rate was 0.5 ml / min. The components were determined by fluorescence at λ _exc. = 276 nm and λ _rad. = 316 nm or UV irradiation with a wavelength of 280 nm. Figure 2 shows a typical HPLC chromatogram for one genotype, showing the separation of different procyanidins. A similar HPLC profile was obtained by separating other species of Theobroma, Herrania and / or their hybrids from interspecific and intraspecific specific crosses.

HPLC conditions:

Column (250 x 3.2 mm) Phenomenex Lichrosphere® Silica 100 (5 microns).

Pre-column (20x4.6 mm) Supelco Supelguard LC-Si (5 microns).

Detectors: a photodiode array detector operating at 280 nm and a fluorescence detector at λ _exc. = 276 nm and λ _ex. = 316 nm.

Flow rate: 0.5 ml / min.

Column temperature: 37 ° C.

Gradient CH ₂ Cl ₂ Methanol Acetic acid: water (1: 1) 0 82 fourteen four thirty 67.6 28,4 four 60 46 fifty four 65 10 86 four 70 10 86 four

Method D. Normal Phase Preparative Separation

The enriched pentamer fractions obtained as described in Example 13 were further purified by normal phase preparative chromatography using a modified method of Rigaud et al. (1993), J. Chrom. 654, 255-260.

Separation was carried out at ambient temperature on a column of 5 μm Supelcosil LC-Si 100Å (50x2 cm) with the corresponding pre-column. Procyanidins were eluted by a linear gradient under the following conditions: (time,% A,% B, flow rate); (0, 92.5, 7.5, 10); (10, 92.5, 7.5, 40); (30, 91.5, 18.5, 40); (145, 88, 22, 40); (150, 24, 86, 40); (155, 24, 86, 50); (180, 0, 100, 50). Prior to use, the components of the mobile phase were mixed in accordance with the following scheme.

Preparation of solvent A (82% CH ₂ Cl ₂ , 14% methanol, 2% acetic acid, 2% water)

1. Measure 80 ml of water and pour into a 4-liter bottle.

2. Measure 80 ml of acetic acid and pour into the same 4-liter bottle.

3. Measure 560 ml of methanol and pour into the same 4-liter bottle.

4. Measure 3280 ml of methylene chloride and pour into a 4-liter bottle.

5. Close the bottle and mix well.

6. Clean the mixture with high purity helium for 5-10 minutes for degassing.

Repeat steps 1-6 twice to get 8 volumes of solvent A.

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

1. Measure 80 ml of water and pour into a 4-liter bottle.

2. Measure 80 ml of acetic acid and pour into the same 4-liter bottle.

3. Measure 3840 ml of methanol and pour into the same bottle.

4. Close the bottle and mix thoroughly.

5. Clean the mixture with high purity helium for 5-10 minutes for degassing.

Repeat steps 1-5 to obtain a yield of 4 volumes of solvent B. Composition of the mobile phase: A = methylene chloride with 2% acetic acid and 2% water; B = methanol with 2% acetic acid and 2% water. Column loading was 0.7 g in 7 ml. The components were determined by UV irradiation at a wavelength of 254 nm. A typical separation of cocoa procyanidins by preparative normal phase HPLC is shown in FIG. 42.

HPLC conditions:

Column: (50x2 cm) 5 microns Supelcosil LC-Si at ambient temperature.

Mobile phase: A = methylene chloride with 2% acetic acid and 2% water, B = methanol with 2% acetic acid and 2% water.

Gradient / Flow Profile:

Time
(min) %BUT %AT Flow rate (ml / min) 0 92.5 7.5 10 10 92.5 7.5 40 thirty 91.5 8.5 40 145 88.0 22.0 40 150 24.0 86.0 40 155 24.0 86.0 fifty 180 0,0 100.0 fifty

EXAMPLE 15

Procyanidin Identification

The procyanidins obtained as described in Example 14, Method D, were analyzed using a matrix forced laser desorption ionization-time-of-flight / mass spectrometry (MALDI-TOF / MS) method using an HP G2025A MALDI-TOF / MS system equipped with an oscilloscope Lecroy 9350 500 MHz. The instrumentation is calibrated according to the manufacturer's instructions for the standard low molecular weight protein (HP Part No G2051A) or the protein standard (HP No G2052A) with 2,5-dihydroxybenzoic acid (DHB) (HP No G2056A) as a standard matrix. One (1.0) ml of the sample was dissolved in 500 ml of 70/30 methanol / water, and then the sample was mixed with a DHB matrix in the ratio 1: 1, 1:10 or 1:50 (sample: matrix) and dried on a tray in vacuum. Samples were analyzed in the mode of registration of positive ions on a detector with a voltage of 4.75 kV and a laser with a power of 1.5 and 8 μJ. Data was collected as the sum of a set of individual frames and presented in units of molecular weight and flight time. Representative MALDI-TOF / MS spectra are shown in FIG. 22A.

Figv and 22C show the MALDI-TOF / MS spectra obtained in the study of partially purified procyanidins prepared as described in example 3, method A, and used for in vitro evaluation, as described in examples 6 and 7; the results are summarized in table 6. These data illustrate that the compounds of the invention described herein were primarily found in fractions D-E, but not in fractions AC.

Spectra were obtained as follows: the purified D-E fraction was analyzed by the MALDI-TOF / MS method as described above, except that the fraction was previously purified using a SEP-PACK® C-18 cartridge. Five milligrams of the D-E fraction in 1 ml of deionized water was loaded into a pre-equilibrated SEP-PACK® cartridge. The column was washed with 5 ml of deionized water to eliminate impurities, and procyanidins were eluted with 1 ml of 20% methanol. Fractions A-C were used directly since they were isolated in Example 3, Method A, without further purification.

These results confirm and extend the previously obtained results (see example 5, table 3, figa and 20B), and indicate that the compounds of the invention are suitable as sequestrants of cations. In particular, the results of the MALDI-TOF / MS analysis indicate conclusively that oligomers of procyanidins with n = 5 and higher (see FIGS. 20A and 20B and the formula in the section “Aspects and Summary of the Invention”) are directly related to anticancer activity in research on models of cancer cell lines HeLa and SKBR-3. Oligomers with n = 4 or less were ineffective on these models. The structure of the pentamer obviously includes a structural fragment, which is presented both in it and in higher oligomers and ensures their activity. In addition, it was observed that the MALDI-TOF / MS data showed strong M ⁺ ions Na ⁺ , 2 Na ⁺ , K ⁺ , 2 K ⁺ , Ca ⁺⁺ , demonstrating usefulness as cation sequestrants.

EXAMPLE 16

Purification of fractions of oligomers

Method A. Purification using semi-preparative reverse phase HPLC

The procyanidins obtained in Example 14, Methods A, B and D, were further separated to obtain experimental amounts of such oligomers for further structural identification and disclosure (for example, Examples 15, 18, 19 and 20). An Hawlett Packard 1050 HPLC system equipped with a variable wavelength detector and a Rheodyne 7010 injection valve with a 1 mm injection loop was attached to a Pharmacia FRAC-100 fraction collector. Separation was performed on a Phenomenex Ultracarb® 10 µ ODS column (250x22.5) connected to a Phenomenex 10 µ ODS Ultracarb® pre-column (60x10 mm). The composition of the mobile phase: A = water; B = methanol, used with a linear gradient under the following conditions: (time,% A); (0, 85), (60, 50), (90, 0) and (110, 0) at a flow rate of 5 ml / min. Individual peaks and selected chromatographic zones were collected at specific time intervals or by manually collecting fractions for further evaluation using MALDI-TOF / MS and NMR. Downloadable samples ranged from 25 to 100 mg of material. A representative elution profile is shown in FIG. 23B.

Method B. Modified Semi-Preparative HPLC

The procyanidins obtained in Example 14, Method A, B and D, were further separated to obtain experimental amounts of such oligomers for further structural identification and disclosure (for example, Examples 15, 18, 19 and 20). Supelcosil LC-Si column 5 microns (250x10 mm) with Supelcosil LC-Si column 5 microns (20x2 mm). Separation was carried out at a flow rate of 3.0 ml / min, at ambient temperature. The composition of the mobile phase: A = dichloromethane; B = methanol and C = acetic acid: water (1: 1), used with a linear gradient under the following conditions: (time,% A,% B); (0, 82, 14); (22, 74, 21); (32, 74, 21); (60, 74, 50,4), (61, 82, 14), followed by a 7-minute column re-balancing. The volume of the applied sample was 60 μl containing 12 mg of enriched pentamer. The components were determined by UV irradiation at a wavelength of 280 nm. A characteristic elution profile is shown in FIG.

EXAMPLE 17

Molecular modeling of pentamers

Minimum energy structures were determined by molecular modeling using the Desktop Molecular Modeller, version 3.0, Oxford University Press, 1994. Four characteristic patterns of [EC (4 → 8)] ₄ -EC (EC = epicatechin) pentamers based on the epicatechin structure shown in figa-24D. The spiral structure is assumed. In general, when epicatechin is the first monomer and the binding occurs at 4 → 8 positions, the beta configuration is formed as a result, and when the first monomer is catechin and binding occurs at 4 → 8 positions, the alpha configuration is formed as a result; these results are obtained regardless of whether the second monomer is catechin or epicatechin (the exception is ent-EC (4 → 8) ent-EC). FIGS. 38A-38B show preferred pentamers, and FIGS. 39A-39B show a stereoisomer library, up to and including pentamers, from which other compounds within the scope of this invention can be prepared without undue experimentation.

EXAMPLE 18

NMR assessment of procyanidins

¹³ C NMR spectroscopy is generally considered a useful technique for studying procyanidins, especially because phenols usually give good quality spectra, while proton NMR spectra are significantly blurred. ¹³ C NMR spectra of oligomers provided useful information on the substitution pattern in rings A and B, the comparative stereochemistry of ring C, and in some cases, the position of bonds between flavanoids. In any case, the ¹³ C NMR spectra were useful information.

In addition, NONANA uses a pulse technique to transfer magnetization sequentially from the first hydrogen to the second to obtain cross peaks corresponding to alpha, beta, gamma, or delta protons. COZY is a modification of the 2D-Fourier NMR technique, where the vertical and horizontal axes represent a chemical shift of ¹ N and a spectrum of 1D; the intersection point denotes the correlation between the protons, whereby the spin-spin interaction can be determined. The HMQC spectrum enhances the sensitivity of the NMR spectrum of nuclei other than protons, and can detect cross peaks from secondary and tertiary carbon with corresponding proton peaks. APT is a ¹³ C technique used to determine the number of hydrogen atoms present on carbon. An even number of protons in carbon will lead to a positive signal, while an odd number of protons in carbon will lead to a negative signal.

Thus, ¹³ C NMR, ¹ H NMR, NONANA (Hartman-Hahn homonuclear), HMQC (heteronuclear multiple quantum coherence), COSY (homonuclear spectroscopy correlation), APT (proton addition test) and XHCORR (variation of HMQC) spectroscopy methods to clarify the structure of the compounds of the invention.

Method A. Monomers

All spectra were determined in a deuterated methanol solution at room temperature, with an approximate sample concentration of 10 mg / ml. Spectra were determined on a Bruker 500 MHz NMR using methanol as an internal standard.

Figa-44E represent NMR spectra that were used to characterize the structure of the epicatechin monomer. On figa shows chemical shifts ¹ N and ¹³ C in tabular form. On figv-44E shows ¹ H, APT, XHCORR and COZY spectra for epicatechin.

Similarly, FIGS. 45A-45F represent NMR spectra that were used to characterize the structure of the catechin monomer. On figa shows a chemical shift of ¹ N and ¹³ C in tabular form. On figv-44F shows the spectra of ¹ H, ¹³ C, APT, XHCORR and COZY for catechin.

Method B. Dimers

All spectra were determined in 75% deuterated acetone in D ₂ O, using acetone as an internal standard, and at an approximate sample concentration of 10 mg / ml.

Figa-G represent the spectra that were used to characterize the structure of the dimer B2. On figa shows chemical shifts ¹ H and ¹³ C in tabular form. The terms T and B indicate the upper half of the dimer and the lower half of the dimer.

On figv and C shows the spectra of ¹³ C and APT, respectively, obtained on a Bruker 500 MHz NMR at room temperature.

FIG. 46D-G shows ¹ H, HMQC, COZY, and NONANA, respectively, which were obtained on an AMZ-360 MHz NMR at −7 ° C. The COZY spectrum was determined using a pulse gradient.

Figa-G represent the spectra used to characterize the structure of the dimer B5. Figa shows chemical shifts ¹ H and ¹³ C in tabular form.

Figv-D show ¹ H, ¹³ C and APT, respectively, obtained on a Bruker 500 MHz NMR, at room temperature.

Fig. 47E shows the COZY spectra obtained on AMX-360 at room temperature using a pulse gradient.

Figures 47F and G show HMQC and NONANA, respectively, obtained on an AMX-360 MHz NMR at room temperature.

Method C. Trimer - Epicatechin / Catechin

All spectra were determined in 70% deuterated acetone in D ₂ O at -3 ° C, using acetone as an internal standard, on an AMX-360 MHz NMR and at an appropriate typical concentration of 10 mg / ml.

Figa-D represent the spectra that were used to characterize the structure of the trimer epicatechin / catechin. These figures show ¹ H, COZY, HMQC and NONANA respectively. The COZY spectrum was obtained using a pulsating gradient.

Method D. Trimer - Fully Epicatechin

All spectra were determined in 70% deuterated acetone in D ₂ O at -1.8 ° C, using acetone as an internal standard, on an AMX-360 MHz NMR and at an appropriate sample concentration of 10 mg / ml.

Figa-49D represent the spectrum that was used to characterize the structure of the trimer, consisting entirely of epicatechin. These figures show ¹ H, COZY, HMQC and NONANA respectively. The COZY spectrum was obtained using a pulsating gradient.

EXAMPLE 19

Procyanidin Thiolysis

To characterize the structure of procyanidins, benzyl mercaptan (BM) was reacted with catechin, epicatechin, or B2 and B5 dimers. Benzyl mercaptan, as well as fluoroglucinol and thiophenol, can be used in the hydrolysis (thiolysis) of procyanidins in an alcohol / acetic acid medium. Catechin, epicatechin or dimer (1: 1 mixture of B2 and B5 dimers) (2.5 mg) was dissolved in 1.5 ml of ethanol, 100 μl of BM and 50 μl of acetic acid, and air was removed from the vessel (Beckman's vessel for amino acid analysis) , repeatedly purged with nitrogen and after the last purge, the vessel was tightly clogged. The reaction vessel was placed in a high temperature block at 95 ° C and reaction aliquots were sampled for 30, 60, 120, and 240 minutes. The relative fluorescence of each aliquot shown in FIGS. 25A-C represents epicatechin, catechin and dimer, respectively. Higher oligomers were subjected to thiolysis in a similar manner.

EXAMPLE 20

Thiolysis and desulfurization of dimers

Dimers B2 and B5 were hydrolyzed with benzyl mercaptan by dissolving the dimer B2 or B5; 1/0 ml) in 600 μl of ethanol, 40 μl of BM and 20 μl of acetic acid. The mixture was heated at 95 ° C. for 4 hours under nitrogen in a Beckman vessel for amino acid analysis. Separate portions were isolated for analysis by reverse phase HPLC, and 75 μl of each portion in Nickel Raney ethanol and gallic acid (10 mg / ml) was added to the remaining reaction medium in 2 ml of hypovial. The vessel was purged with a stream of hydrogen and shaken periodically for an hour. The reaction product was filtered through a 0.45 micron filter and analyzed by reverse phase HPLC. A characteristic elution profile is shown in FIGS. 26A and B. Higher oligomers were subjected to desulfurization in a similar manner. These data show the polymerization of epicatechin or catechin and thus represent a way to artificially produce the compounds of the invention.

EXAMPLE 21

In vivo pentamer activity in nude mouse model MDA MB 231

Cell line MDA-MB-231 / LCC6. The cell line was grown in improved minimal essential medium (IMEM), containing 10% fetal bovine serum and kept in a humidified atmosphere of 5% CO ₂ at 37 ° C.

The mice. Six-eight-week-old female nude / nude NCr (without thymus) were acquired through NCI, housed in a vivarium, and kept according to standards set forth by the United States Department of Agriculture and the American Laboratory Animal Protection Association. Tumor mice were weighed every other day and weekly to determine the appropriate dosage of the drug.

Tumor implantation. MDA-MD-231, prepared by diluting an IMEM tissue culture with 3.3x10 ⁶ cells / ml and 0.15 ml (i.e. 0.5x10 ⁶ cells), was injected subcutaneously between 2 and 3 nipples on each side of the mouse. Tumor volume was calculated with the following multiplication: length x width x height x 0.5. Tumor volume outside the treated group was averaged and Student's test was used to calculate the p-value.

Sample preparation. Plasma samples were obtained by cardiac puncture and stored at -70 ° C with 15-20 mm EDTA in order to determine the chemical composition of the blood. There were no differences between the control group and the experimental group.

Fifteen nude mice pre-infected with subcutaneous injection of 500,000 cells with MDA-MB-231 tumor cells were randomly divided into three groups of 5 animals each and were injected intraperitoneally with one of: (I) a placebo containing only solvent (DMSO); (II) 2 mg / mouse of purified pentameric procyanidin extract isolated as described in Example 14, Method D, in a solvent (DMSO); (III) 10 mg / mouse of the extract of purified pentameric procyanidin isolated as described in Example 14, Method D, in a solvent (DMSO).

Group (III) mice died over a period of approximately 48 to 72 hours after administration of 10 mg, while group (II) mice seemed to feel normal. The cause of death of group (III) mice has not been established; it may not necessarily be attributed to the use of a compound of the invention. However, 10 mg are considered an upper limit of toxicity.

The treatment of groups (I) and (II) was repeated once a week and tumor growth was monitored in each experimental and control group. After two weeks of treatment, no signs of toxicity were observed in the mice of group (II), and the dose used in this group was introduced with a incremental increase of 1/2 on a logarithmic scale in each subsequent week. The following table presents the dosages used during the treatment period of the mice of group (II):

A week Dose (mg / ml) one 2 2 2 3 four four 5 5 5 6 5 7 5

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

Table 10
In vivo anti-cancer treatment results Day % survival group (I) % survival group (II) % survival group (III) one one hundred one hundred one hundred 2 one hundred one hundred one hundred 3 one hundred one hundred 0 four one hundred one hundred 5 one hundred one hundred 6 one hundred one hundred 7 one hundred one hundred 8 one hundred one hundred 9 one hundred one hundred 10 one hundred one hundred eleven one hundred one hundred 12 one hundred one hundred 13 one hundred one hundred fourteen one hundred one hundred fifteen one hundred one hundred 16 one hundred one hundred 17 one hundred one hundred eighteen one hundred one hundred 19 one hundred one hundred twenty one hundred one hundred 21 one hundred one hundred 22 75 one hundred 23 75 one hundred 24 75 one hundred 25 75 one hundred 26 75 one hundred 27 75 one hundred 28 75 one hundred 29th fifty one hundred thirty fifty one hundred 31 fifty one hundred 32 fifty one hundred 33 fifty one hundred 34 fifty one hundred 35 fifty one hundred 36 25 one hundred 37 25 one hundred 38 25 one hundred 39 25 one hundred 40 25 one hundred 41 25 one hundred 42 25 one hundred 43 25 80 44 25 80 45 25 80 46 25 80 47 25 80 48 25 80 49 25 80 fifty 25 60 51 25 60 52 25 60 53 25 60 54 25 60 55 25 60 56 25 60 57 0 40 58 40 59 40 60 40 61 40 62 40 63 40 64 40

These results demonstrate that the fractions of the invention and compounds indeed have utility as antitumor (anti-blastoma) compositions and are not toxic at low or medium doses, and toxicity at higher dosages can be determined without undue experimentation.

EXAMPLE 22

Antimicrobial activity of cocoa extracts

Method a

The study was conducted with the aim of emitting the antimicrobial activity of extracts of crude cocoa bean procyanidins against various microorganisms involved in food spoilage or in the pathogenesis of diseases. The cocoa extracts from Example 2, Method A, were used in this study. Agar medium suitable for the growth of each test culture (99 ml) was seeded with 1 ml of a suspension of each cell culture in 0.45% saline (final content 10 ² -10 ⁴ cfu / ml ) and poured into Petri dishes. In solidified agar, holes # 2 were made with a cork drill (5 mm in diameter). The cups were cooled at 4 ° C. overnight, allowing the extract to diffuse into agar, and then incubated at a temperature suitable for the growth of the studied microorganism. Received the following results:

Sample Inhibition Area (mm) The concentration of the extract (mg / ml) B. sphericus B. cereus S.Aureus P. aeruginosa B. Subtilis 0 Ni Ni Ni Ni Ni 25 Ni 12 Ni eleven Ni 250 12 twenty 19 19 eleven 500 fourteen 21 21 21 13 NI = no suppression

The antimicrobial activity of extracts of purified procyanidins from cocoa beans was demonstrated in another study using the diffusion analysis from the wells described above (in method A) with a Staphyloccocus aureus test culture.

Received the following results:

Cocoa extracts: 10 mg / 100 ml of decaffeinated / de-brominated acetone extract, as in example 13, method A.

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

10 mg / 100 ml tetramer (95% purity), as in example 14, method D.

10 mg / 100 ml hexamer (88% pure), as in example 14, method D.

10 mg / 100 ml octamer / nonamer (92% pure), as in example 14, method D.

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

Sample Suppression Area (mm) 0.45% saline 0 Dimer 33 Tetramer 27 Hexamer 24 0.45% saline 0 Octamer 22 Nonamer twenty Decofein / Deteobrom 26

Method B

The crude procyanidins of Example 2, Method 2, were added at various concentrations to TSB (trypticase soy broth) with phenol red (0.08 g / L). Salmonella enteritidis or S.newport cultures (10 ⁵ cfu / ml) were plated on TSB and incubated for 18 hours at 35 ° C. Received the following results:

S. Enteritidis S.Newpor 0 mg / ml + + fifty + + one hundred + + 250 + - 500 - - 750 - -

where + = growth, - = lack of growth, as becomes apparent from the color change in the culture of the broth from red to yellow, associated with the production of acid. Confirmation of inhibition was obtained by transferring from TSB tubes to XLD plates.

These examples demonstrate that the compounds of the invention are useful in the preparation and storage of food.

This example also demonstrates that the growth of gram-negative and gram-positive bacteria can be inhibited by the compounds of the invention. Based on this, it can be said that the compounds of the invention can be used to inhibit Helicobacter pylori. Helicobacter pylori is involved in the development of stomach ulcers and gastric cancer. Accordingly, the compounds of the invention can be used to treat or prevent these and other diseases of bacterial origin. Given the known parameters, such as the type of disease, as well as the age, weight, gender and general health of the patient, suitable routes of administration, dosages and formulations can be determined without undue experimentation.

EXAMPLE 23

Halogen-free analytical separation of the extract

The procyanidins obtained in example 2 were partially purified by analytical separation using halogen-free chromatography with normal phase on 100Å Supelcosil LC-Si 5 μm (250x4.6 mm) at a flow rate of 1.0 ml / min and at a column temperature of 37 ° C . The separation was carried out by a linear gradient under the following conditions: (time,% A,% B); (0, 82, 14), (30, 67.6, 28.4); (60, 46, 50).

The composition of the mobile phase: A = 30/70% diethyl ether / toluene; B = methanol and C = acetic acid / water (1: 1). The components were determined by UV radiation with a wavelength of 280 nm. A typical elution profile is shown in FIG.

EXAMPLE 24

The value of the pore size of the stationary phase in the separation of procyanidins using HPLC with a normal phase

To improve the separation of procyanidins, the use of a silicone stationary phase with a large pore size was investigated. Separation was carried out on Silica-300, 5 μm, 300Å (250x2.0 mm), or alternatively, on Silica-1000, 5 μm, 1000Å (250x2.0 mm). A linear gradient was used since the mobile phase was: A = dichloromethane; B = methanol and C = acetic acid / water (1: 1). The components were determined by fluorescence at λ _exc. = 276 nm and λ _ex. = 316 nm and a UV detector at 280 nm. The flow rate was 1.0 ml / min and the thermostat temperature was 37 ° C. A representative chromatogram obtained on three different columns (pore size 100Å, from Example 13, Method D) is shown in FIG. 29. It shows the effective pore size for the separation of procyanidins.

EXAMPLE 25

Obtaining the desired procyanidins through controlled fermentation

Representatives of various types of microbes from the group associated with cocoa fermentation were selected from the M & M / Mars collection for cocoa culture. The following isolates were used:

Acetobacter aceti ATCC 15973

Lactobacillus sp. (BH 42)

Candida cruzii (BA 15)

Saccharomyces cerevisiae (BA 13)

Bacillus cereus (BE 35)

Bacillus sphaericus (ME 12).

Each species was transferred from the original culture to fresh medium. Yeast and Acetobacter were incubated for 72 hours at 26 ° C and bacilli and Lactobacillus were incubated for 48 hours at 37 ° C. Before use, beveled agar was treated with 5 ml of phosphate buffer.

Cocoa beans were collected, freeing from the peel, pulp and shell. The beans were sterilized with hydrogen peroxide (35%) for 20 seconds, followed by catalase treatment until the bubbling ceased. Then the beans were washed twice with sterilized water and the process was repeated. Beans were distributed in glass jars and processed in accordance with the regimen detailed in the following table:

Water Ethanol / acid Fermentation infusate Fermentation model Move to fresh water daily Transfer to a solution of alcohol and acid daily, corresponding to the level determined at each stage of the pulp fermentation model Transfer daily to fermented pulp pasteurized on each consecutive day of fermentation A model of bench fermentation of sterile pulp co-inoculated with test strains

Bench fermentation was duplicated. All samples were incubated as shown below:

Day 1 26 ° C Day 2 from 26 ° С to 50 ° С Day 3 50 ° C Day 4 45 ° C Day 5 40 ° C

The fermentation model was monitored throughout the radiation by counting the plates to evaluate the microbial population and an HPLC analysis of the fermentation medium in terms of the presence of microbial metabolites. After processing, the beans were dried in a laminar heat flow to a water activity of 0.64 and fried at 66 ° C for 15 minutes. Samples were prepared for analysis of procyanidins. Three beans for one treatment were ground and degreased with hexane, followed by extraction with a solution of acetone: water: acetic acid (70: 29.5: 0.5%). The acetone solution of the extract was filtered into bottles and the polyphenol levels were quantified using normal phase HPLC, as in Example 13, Method B. The remaining beans were ground and tasted. The cultural and analytical profile of the bench fermentation process is shown in FIGS. 30A-C. The procyanidin profile of cocoa beans subjected to various enzymatic treatments is shown in FIG. 30D.

This example demonstrates that the invention should not be limited to any particular cocoa genotype and that, thanks to controlled fermentation, the levels of procyanidins produced by certain Theobroma and Herrania varieties or their hybrids from interspecific or intraspecific crosses can be modulated, for example expanded.

The following table shows the levels of procyanidins determined in the samples that are characteristic of Theobroma species and their hybrids from interspecific and intraspecific specific crosses. Samples were prepared as in examples 1 and 2 (methods 1 and 2) and analyzed as described in example 13, method B. These data explain that extracts containing the compounds of the invention were found in Theobroma and Herrania species and their hybrids from inter- and intraspecific specific crossbreeding.

EXAMPLE 26

The effect of procyanidins on NO

Method a

The purpose of this study is to establish a relationship between procyanidins (as in Example 14, method D) and NO, which is known to stimulate vasodilation of the brain. The effects of monomers and higher oligomers, in concentrations ranging from 100 μg / ml to 0.1 μg / ml, on the production of nitrates (NO catabolites) from HUVEC (human umbilical vein endothelial cells) were evaluated. HUVEC (from Clonetics) was examined in the presence or absence of each procyanidin for 24 to 48 hours. At the end of the experiments, supernatants were collected and the nitrate content was determined by calorimetric studies. In separate experiments, HUVECs were incubated with acetylchlorine, which is known to stimulate NO production in the presence or absence of procyanidins for 24 to 48 hours. At the end of the experiments, supernatants were collected and the nitrate content was determined by calorimetric studies. The role of NO is established by the addition of nitroarginine or (1) -N-methylarginine, which are specific blockers of NO synthase.

Method B. Vasorelaxation of rat arteries contracted by phenylephrine

The effect of each of the procyanidins (100 μg / ml - 0.1 μg / ml) on the rat artery is the goal of studying the relaxation of a rat artery in a state of phenylephrine-induced contraction. The isolated rat artery is incubated in the presence or absence of procyanidins (as in Example 14, method D) and the change in muscle tone is evaluated by visual inspection. Both the contraction and relaxation of the rat artery are determined. Then, using other organs, a preliminary contraction of the isolated rat artery is caused by the addition of epinephrine. Having achieved a stable contraction, procyanidins were added and rat arterial contraction or relaxation was determined. The action of NO was accelerated by the addition of nitroarginine or (1) -N-methylarginine. Acetylchlorine-induced relaxation of NO, as manifested on a rat aorta previously contracted with phenylephrine, is shown in FIG.

Method C. Induction of Rat Hypertension

This method aims at the effect of each procyanidin (as in example 14, method D), exerted on blood pressure. Equipment for monitoring systolic and diastolic blood pressure was connected to rats. Each of the procyanidins was administered intravenously (dosage range = 100-0.1 μg / kg) and the change in blood pressure was evaluated. In addition, the effect of each procyanidin on the change in pressure caused by adrenaline was determined. The role of NO was determined by the addition of nitroarginine or (1) -N-methylarginine.

These studies, together with the following example, demonstrate that the compounds of the invention are useful for modulating vascular relaxation, as well as modulating blood pressure, or are aimed at altering the state of coronary circulation and the state of migraine.

EXAMPLE 27

Effect of Cocoa Polyphenols on Saturation

Using blood glucose as an indicator of the appearance of results that occur in vivo in regulating appetite and satiety, a series of simple experiments were carried out in which healthy adult male volunteers, 48 years old, participated, and which were carried out to determine whether cocoa polyphenols would be modulate glucose levels. Cocoa polyphenols were partially purified from Brazilian cocoa beans according to the methods described by Clapperton et al. (1992). This material did not contain any caffeine or theobromine. Fast changes in blood glucose during fasting were analyzed as a function of time after drinking 10 fl oz Dexicol 75 (caffeine-free) using a glucose tolerance test (Curtin Matheson 091-421) without and with 75 mg of cocoa polyphenols. This polyphenol level represented 0.1% of the total glucose of the test drink and reflected the approximate amount that would be present in a standard 100 gram chocolate bar. Blood glucose levels were determined using an Accu-Chek III blood glucose control system (Boehringer Mannheim Corporation). Blood glucose levels were measured before drinking the test drink and after drinking the test drink at the following time intervals: 15, 30, 45, 60, 75, 90, 120 and 180 minutes. Before the start of each glucose tolerance test, controls for high and low glucose were determined. Each glucose tolerance test was performed twice. A control test solution was also prepared containing 75 mg of cocoa polyphenols dissolved in 10 fluid ounces of distilled water (without glucose).

Table 11 below presents the test and control data obtained for each glucose tolerance experiment conducted in this study. Fig. 32 is a graph of mean values with standard deviations of blood glucose levels obtained over the entire three-hour course. It is clear that there is a real increase in blood sugar obtained after drinking the test mixture containing cocoa polyphenols. The difference between the two main profiles of glucose tolerance could not be determined only by the profile obtained after drinking a solution of cocoa polyphenols. The addition of cocoa polyphenols to the glucose test solution significantly increased the glucose tolerance profile. A similar increase in blood glucose does not go beyond what is considered moderately diabetic, although a typical glucose tolerance profile was considered normal (Davidson, I. et al., Eds. Todd - Sandford Clinical Diagnosis by Laboratory Methods 14th edition ; WDSaunders Co; Philadelfia, PA 1969 Ch. 10, pp. 550-9). Supposedly, the extra glucose that determined this difference was released into the bloodstream from glycogen stores as a result of exposure to the compounds of the invention. Thus, the compounds of the invention can be used to modulate blood glucose in the presence of sugars.

Table 11
Glucose tolerance test results
(experience and control) A week Description High value control Low value control 0 Glucose tolerance 265 mg / dl 53 mg / dl one Glucose tolerance with 0.1% polyphenols 310 68 2 Glucose tolerance 315 66 four Glucose tolerance with 0.1% polyphenols 325 65 5 0.1% polyphenols 321 66

a = expected range: 253-373 mg / dl,

b = expected range: 50-80 mg / dl.

A participant in the experiment also observed facial redness (erythema) and dizziness (lightheadedness), appearing after consuming the compounds of the invention, which indicates modulation of vasodilation with the compounds of the invention.

The data presented in tables 12 and 13 illustrate the fact that the extracts of the invention related to cocoa powder and commercial types of chocolate and the compounds of the invention contained therein can be used as carriers in pharmaceutical, veterinary and food preparations and their use .

Table 13
Commercial Chocolate Procyanidins Levels (mg / g) Sample Monomers Dimers Trimers Tetramers Pentamers Hexamers Heptamers and above General Brand 1 366 166 113 59 56 23 eighteen 801 Brand 2 344 163 111 45 48 ND * Nd 711 Brand 3 316 181 one hundred 41 40 7 Nd 685 Brand 4 310 122 71 27 28 5 Nd 563 Brand 5 259 135 90 46 29th Nd Nd 559 Brand 6 308 139 91 57 47 fourteen Nd 656 Brand 7 196 98 81 58 54 19 Nd 506 Brand 8 716 472 302 170 117 eighteen Nd 1795 Brand 9 1185 951 633 298 173 25 21 3286 Brand 10 1798 1081 590 342 307 93 Nd 4211 Brand 11 1101 746 646 372 347 130 75 3417 Brand 12 787 335 160 twenty 10 8 Nd 1320 ND = not defined

Table 14
Procyanidin Levels in Cocoa Raw Material (mg / g) Sample Monomers Dimers Trimers Tetramers Pentamers Hexamers Heptamers and above General Unfermented 13440 6425 6401 5292 4236 3203 5913 44910 Fermented 2695 1538 1362 740 470 301 277 7383 Fried 2656 1597 921 337 164 Nd Nd 5675 Liquid chocolate 2805 1446 881 442 184 108 Nd 5866 Cocoa shell 114 53 fourteen Nd Nd Nd Nd 181 Cocoa Powder, 1% Fat 506 287 112 Nd Nd Nd Nd 915 Cocoa Powder, 11% Fat 1523 1224 680 46 Nd Nd Nd 3473 Dutch Red Cocoa Powder, pH 7.4, 11% fat 1222 483 103 Nd Nd Nd Nd 1808 Dutch Red Cocoa Powder, pH 8.2, 23% Fat 168 144 60 Nd Nd Nd Nd 372 ND = not defined

EXAMPLE 28

The effect of procyanidins on cyclooxygenase 1 and 2

The effect of procyanidins on the activity of cyclooxygenase 1 and 2 (COX1 / COX2) was evaluated by incubating the enzymes obtained from the seminal vesicles of the sheep and placenta of the sheep, respectively, with arachidonic acid (5 μM) for 10 minutes at room temperature in the presence of various concentrations of procyanidin solutions, containing oligomers of the monomer-decamer and a mixture of procyanidins. The change in activity was also evaluated using PGE2 EIA kits from Interchim (France). Indomethacin was used as a compound for comparison. The results are presented in the following table, where the IC ₅₀ values are expressed in units of μM (except for S11, which is a mixture of procyanidins prepared as in example 13, method A, and where samples S1-S10 are consecutive procyanidin oligomers (decamer monomers) as in example 14, method D, and IC ₅₀ are expressed in mg / ml).

Sample # IC ₅₀ SOX-1 (*) IC ₅₀ COX-2 (*) IC ₅₀ ratio COX2 / COX1 one 0,074 0.197 2.66 2 0.115 0.444 3.86 3 0.258 0.763 2.96 four 0.154 3.73 24.22 5 0.787 3.16 4.02 6 1.14 1.99 1.75 7 1.89 4.06 2.15 8 2.25 7.2 3.20 9 2,58 2.08 0.81 10 3.65 3.16 0.87 eleven 0,0487 0,0741 1,52 Indomethacin 0.599 13.5 22.54 (*) expressed as μM with the exception of sample 11, which is expressed in mg / ml.

The results of the inhibition study are presented in FIGS. 33A and B, which show the effect of indomethacin on the activity of COX1 and COX2. Figa and b show the correlation between the degree of polymerization of procyanidin and IC ₅₀ with COX1 and COX2; Fig. 35 shows the correlation between the IC ₅₀ value for COX1 and COX2. and FIGS. 36A-Y show IC ₅₀ values for each sample (S1-S11) with COX1 and COX2.

These results indicate that the compounds of the invention may be useful as an analgesic, anticoagulant and anti-inflammatory agent. In addition, COX2 is associated with colon cancer. The suppression of COX2 activity by the compounds of the invention illustrates the likely mechanism of the antitumor activity of the compounds of the invention against colon cancer.

COX1 and COX2 are also involved in the synthesis of prostaglandins. Thus, the results of this example also indicate that the compounds of the invention can modulate the functioning of the kidneys, immune responses, fever, pain, mitogenesis, apoptosis, prostaglandin synthesis, ulcer formation (e.g., stomach) and reproduction mechanisms. Note that modulation of kidney function can affect blood pressure, again including compounds of the invention in modulation of blood pressure, vasodilation, and coronary circulation (e.g., modulation of angiotensin, bradykin).

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

In addition, reference is made to examples 9, 26 and 27. Example 9 shows the antioxidant activity of the compounds of the invention. Example 26 demonstrates the effect on NO. And example 27 clearly demonstrates the expansion of the vessels of the face. The results of these examples in combination with examples 9, 26 and 27 show that the compounds of the invention can modulate free radical mechanisms for controlling physiological effects. Similarly, lipoxygenase indirectly involved in typical free radical reactions biochemically directed to the synthesis of leukotrein can be modulated by the compounds of the invention, thereby affecting subsequent physiological effects (e.g., inflammation, immune response, coronary circulation, carcinogenic mechanisms, fever, pain, ulceration).

Thus, in addition to the presence of analgesic properties, there is a synergistic effect of the compounds of the invention when they are administered together with analgesics. Similarly, in addition to having antitumor properties, there may also be a synergistic effect of the compound of the invention when used with other anti-blastoma agents.

EXAMPLE 29

Study of circular dichroism (CD) of procyanidins

CD studies were undertaken to determine the structure of the purified procyanidins obtained as in Example 14, Method D. Spectra were collected at 25 ° C. using a spectral soft carrier AVIV 60DS V4.1f.

Samples were scanned at 300-185 nm, each 1.0 nm, with a bandwidth of 1.50 nm. Representative spectra are shown in FIGS. 43A-G, which show CD spectra of dimer to octamer oligomers.

These results are indicative of the spiral nature of the compounds of the invention.

EXAMPLE 30

The effect of inhibition of cocoa procyanidins of cocoa Helicobacter pylori and Staphylococcus aureus

The study was conducted to evaluate the antimicrobial activity of procyanidin oligomers against Helicobacter pylori and Staphylococcus aureus. Enriched pentamer material was prepared as described in Example 13, Method A, and analyzed as described in Example 14, Method C with 89% pentamers and 11% higher oligomers (n is 6-12). Purified pentamer (96.3%) was prepared as described in example 14, method D.

Helicobacter pylori and Staphylococcus aureus were obtained from the American Type Culture Collection (ATCC). For H. pylori, the vesicle was rehydrated with 0.5 ml trypticase soy broth and the suspension was transferred to fresh TSA containing 5 μg of defibrinated sheep blood on mown agar. Mowed agar was incubated at 37 ° C for 3 to 5 days under microaerophilic conditions in anaerobic jars (5-10% carbon dioxide; CampyPakPlus, BBL). When good growth was established at the bottom of the mowed agar, the broth was used to inoculate additional mowed TSA agar with sheep blood. Due to the decrease in viability during prolonged cultivation, the broth collected from mowed agar was combined and stored at 80 ° C. Cultures for analysis were used directly from frozen bubbles. The S.aureus culture was maintained on TSA mowed agar and transferred to fresh agar 24 hours before use.

A cell suspension of each culture was prepared (H. pylori, 10 ⁸ -10 ⁹ cfu / ml; S.aureus 10 ⁶ -10 ⁷ cfu / ml) and 0.5 ml was dispensed on a TSA plate with 5% sheep blood. Standard assay discs (Difco) were placed in a filter-sterilized serial dilution of the pentamer (23 mg / ml in sterilized water). Test discs and sterile control discs (sterilized water) were placed on grafted plates. Control discs containing 80 μg of metronidazole (inhibitory H. pylori) or 30 μg of vancomycin (inhibitory S.aureus) (BBL Sensidiscs) were also placed on the appropriate plate set. Plates containing H. pylori were incubated under microaerophilic conditions. The S.aureus kit was incubated under anaerobic conditions. Suppression zones were measured by observing external growth.

Table 14
Pentamera bioassay against Helicobacter pylori and Staphylococcus aureus Pentamer
Enriched fraction
(mg / ml) S.aureus
Suppression (mm) H. pylori
Suppression (mm) 0 Ni Ni fifteen 0 10 31 10 10 62 eleven eleven 125 13 13 250 fifteen 13 Vancomycin Standard fifteen - Metronidazole standard - eleven Pentamer 96% Purity fifteen eleven NI = no suppression

EXAMPLE 31

NO-dependent hypertension in guinea pigs

The effect of five procyanidin fractions on guinea pig blood pressure was investigated. Briefly, guinea pigs (approximately 400 g body weight; males and females) received anesthesia by injection of 40 mg / kg sodium phenobarbital. The carotid artery was catheterized to control intra-arterial blood pressure. Each of the five fractions of cocoa procyanidins was administered intravenously (dose range 0.1 mg / kg - 100 mg / kg) through the jugular vein. Changes in blood pressure were recorded on a polygraph. In these experiments, the role of NO was established using L-N-methylarginine (1 mg / kg) ten minutes before cocoa procyanidin fractions were used.

Cocoa procyanidin fractions were prepared and analyzed according to the procedures described in US Pat. No. 5,554,645, incorporated herein by reference.

Fraction A represents the fraction obtained by preparative HPLC, including oligomers from monomers to tetramers. HPLC analysis showed the following composition:

Monomers 47.2% Dimers 23.7 Trimers 18.7 Tetramers 10.3

Fraction B is a fraction obtained by preparative HPLC, including oligomers from pentamers to decamers. HPLC analysis showed the following composition:

Pentamers 64.3% Hexamers 21,4 Heptamers 7.4 Octamers 1.9 Nonamers 0.9 Decamers 0.2

Fraction C is a fraction enriched in cocoa procyanidins suitable for preparing fractions A and B above. HPLC analysis showed the following composition:

Monomers 34.3% Dimers 17.6 Trimers 16,2 Tetramers 12.6 Pentamers 8.5 Hexamers 5.2 Heptamers 3,1 Octamers 1.4 Nonamers 0.7 Decamers 0.3

Fraction D is a procyanidin extract prepared from milk chocolate. HPLC analysis showed a composition similar to that presented in table 12 for brand 8. In addition, 10% caffeine and 6.3% theobromine were present.

Fraction E is an extract of procyanidins obtained from dark chocolate prepared with an alkalized liquid cocoa mass. HPLC analysis showed a composition similar to that shown in table 12 for brand 12. In addition, 16.0% caffeine and 5.8% theobromine were present.

In three separate experiments, the effects of using 10 μg / kg of cocoa procyanidin fractions on the arterial blood pressure of guinea pigs under anesthesia were investigated. Intravenous administration of the procyanidin A and E fractions caused a decrease in blood pressure of approximately 20%. This decrease was only slightly different from that obtained using a solvent (DMSO) as a control (15 ± 5%, n = 5). In contrast, the procyanidin fractions B, C, and D (10 mg / kg) caused a marked decrease in blood pressure, up to 50-60% for fraction C. In these experiments, the degree of manifestation of the hypotensive effect was as follows: C> B> D >> A = E.

A typical blood pressure pattern after administration of procyanidin fractions is shown in Fig. 50A for fraction A and in Fig. 50B for fraction C. Fig. 51 illustrates the comparative effects of these fractions on blood pressure.

The possible participation of NO in the development of hypotension in guinea pigs caused by the use of fraction C was analyzed using L-N-methylarginine (L-NMMA). This pharmacological agent slows down the production of NO without inhibiting NO synthase. L-NMMA was used at a dose of 1 mg / kg ten minutes before the administration of cocoa procyanidin fractions. As shown in FIG. 52, treatment of animals with L-NMMA completely blocked hypotension caused by procyanidin C fractions. Indeed, upon subsequent treatment with this inhibitor, the change in blood pressure caused by fraction C was similar to that observed with a single solvent.

EXAMPLE 32

The effect of cocoa procyanidin fractions on NO production in human umbilical vein endothelial cells

Human umbilical vein endothelial cells were obtained from Clonetics, and cultures were maintained according to the manufacturer's specifications. HUVEC cells were plated at 5000 cells / cm ² in a 12-well plate (Falcon). After the third transplant, under the same conditions, they were allowed to reach the merger stage. The supernatant was replaced with a new medium containing certain concentrations of bradykinin (25, 50 and 100 nm) or a fraction of cocoa procyanidins AE (100 μg / ml), as described in example 31. Cultivation was continued for 24 hours and supernatant-free cells were collected and stored frozen before evaluating the NO content as described below. In separate experiments, an NO synthase antagonist (NOS), Nω-nitro-L-arginine methyl ester (L-NAME, 10 μM) was added to evaluate the participation of NOS in the observed NO production.

HUVEC NO production was evaluated by measuring the concentration of nitrites in the culture supernatant using the Griess reaction. The Griess reagent was 1% sulfanilamide, 0.1% N- (1-naphthyl) ethylenediamine dihydrochloride. Briefly, 50 μl portions were removed from the various supernatants of four duplicates and incubated with 150 ml of Griess reagent. The absorption at a wavelength of 540 nm was determined in a multiscan setup (Labsystems Multiskans MCC / 340). Sodium nitrate at certain concentrations was used to obtain standard curves. Absorption of the medium containing no cells (empty) was subtracted from the value obtained by examining the supernatant containing the cells.

Fig. 53 illustrates the effect of bradykinin on NO production from HUVEC, where dose-dependent release of NO is monitored. The L-NAME inhibitor completely slowed down bradykinin-induced NO release.

Fig. 54 illustrates the effect of cocoa procyanidin fractions on NO production by HUVEC cells.

Fractions B, C, and D caused the production of a moderate but significant amount of NO in HUVEC. Moreover, fraction C most effectively induced the production of NO, as was estimated using nitrites, while fraction E was almost ineffective. The effect of fraction C on NO production was sharply reduced in the presence of L-NAME. It is interesting to note that fractions B, C and D contained more procyanidin oligomers than fractions A and E. A certain difference between fractions D and E was that E was prepared from dark chocolate, in which, as part of the recipe, alkalized liquid cocoa mass. Alkalization results in base-catalyzed polymerization of procyanidins, which rapidly depletes the levels of these compounds. An analytical comparison of the levels of procyanidins found in these types of chocolate is presented in table 12, where grade 12 is dark chocolate made with alkalized liquid cocoa mass, and grade 11 is plain milk chocolate. Thus, extracts obtained from milk chocolate contain high proportions of procyanidin oligomers that are capable of inducing NO. The addition of an L-NMMA NO inhibitor to the sample of fraction C apparently led to inhibition of NO. The results obtained in the study of procyanidin fractions were compatible with those observed in experiments with NO induction by bradykinin (see Fig. 53).

As in the case of the results with HUVEC, fraction C of cocoa procyanidins showed the greatest hypotensive effect in guinea pigs, while fractions A and E were the least effective. And this time, the presence of high molecular weight procyanidin oligomers was involved in modulating NO production.

EXAMPLE 33

The effect of cocoa procyanidin fractions on NO production by macrophages

Fresh human heparinized blood (70 ml) was added to an equal volume of phosphate buffered saline (PBS) at room temperature. The Ficoll-Hypaque solution was layered under a blood-PBS mixture using a dilution of 3 ml of Ficoll-Hypaque to 10 ml of a blood-PBS mixture. The tubes were centrifuged for 30 minutes at 2000 rpm at 18-20 ° C. The top layer containing plasma and platelets was discarded. The mononuclear cell layer was transferred to another centrifuge tube and the cells were washed twice in Hanks balanced physiological saline. Mononuclear cells were resuspended in complete RPMI 1640 supplemented with 10% fetal calf serum, counted, and trypan blue exclusion determined. Cell granulate was resuspended in complete RPMI 1640 supplemented with 20% fetal calf serum to a final concentration of 1x10 ⁶ cells / ml. Certain amounts of cell suspension were placed in a 96-well culture plate, washed RPMI 1640 with 10% fetal calf serum three times, and non-adherent cells (lymphocytes) were removed.

These cells were incubated for 48 hours in the presence or absence of the five fractions of procyanidin described in Example 31. At the end of the incubation period, the culture medium was collected, centrifuged and the cell-free supernatants stored frozen to determine nitrates.

Macrophage NO production was determined by measuring the nitrite concentration using the Greiss reaction. Greiss reagent was 1% sulfanilamide, 0.1% N- (1-naphthyl) ethylenediamine dihydrochloride. Briefly, 50 μl aliquots were removed from the supernatant in four duplicates and incubated with 150 μl Greiss reagent. The absorption at 540 nm was determined on a multiscan setup (Labsystem Multiskans MCC / 340). Sodium nitrate was used at certain concentrations to determine standard curves. Absorption of cell-free medium (blank control) was subtracted from the value obtained by examining the supernatant containing the cells.

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

55 shows that only the procyanidin fraction C at 100 μg / ml can stimulate the production of NO by monocytes / macrophages. The initial NO production by these cells could not be determined, and nitrites could not be detected in any of the procyanidin fractions at 100 μg / ml. 56 shows that the procyanidin fractions A and D did not increase LPS-induced NO production by monocytes / macrophages primed with gamma interferon. The procyanidin fraction C was slightly effective when LPS-stimulated monocytes / macrophages cultured in the absence of procyanidin fractions produced only 4 μmol / 10 ⁵ cells / in 48 hours. Interferon gamma alone was ineffective in stimulating NO.

Together, these results demonstrate that mixtures of the compounds of the invention used at specific concentrations are capable of inducing NO production by monocytes / macrophages, both independently and independently of LPS or cytokine stimulation.

From the foregoing, it is clear that cocoa extracts and polyphenols, especially the compounds of the invention, as well as the compositions, methods and kits of the invention, have significant and numerous fields of application.

The antitumor utility is clearly demonstrated by data obtained in vivo and in vitro and shows that the compounds of the invention can be used in place of or in conjunction with conventional anti-blastoma agents.

The compounds of the invention have antioxidant activity similar to that of BHT and BHA, as well as oxidative stability. Thus, the invention can be used instead of or together with BHT or BHA in cases of known uses of BHA and BHT, such as antioxidants, for example, as antioxidants, food additives.

The invention may also find use instead of or together with topoisomerase II inhibitors, in cases of its currently known use.

The compounds of the invention can be used in the preparation or preservation of food, as well as in the prevention of diseases of bacterial origin. Simply put, the compounds of the invention can be used as antimicrobial agents.

The compounds of the invention can also be used as modulators of cyclooxygenase and / or lipoxygenase, NO or NO synthase, blood glucose or in vivo, and are thus useful in the treatment or prevention or modulation of pain, fever, inflammation, coronary circulation disorders, ulceration , mechanisms of carcinogenesis, vasodilation, as well as as analgesic, anticoagulating, anti-inflammatory agents and modulators of the immune response.

In addition, the compound includes the use of compounds or extracts as carriers for the preparation of pharmaceutical preparations. Accordingly, there are many compositions and methods provided by the invention. For example, antioxidant and preservative compounds; topoisomerase II inhibiting compositions; methods of preserving food products or any desired objects, for example, from oxidation and methods of inhibiting topoisomerase II. Compositions may contain compounds of the invention. The methods may include contacting food, other objects, or topoisomerase II with the expected composition or with the compounds of the invention. Other compositions, methods and embodiments of the invention will be apparent from the subsequent discussion.

From this point of view, it can be noted that the invention has an edible source and that in vitro activity may show at least some of the activity shown in vivo; Based on the data of in vitro and in vivo experiments presented here, doses of the route of administration and formulations can be determined without undue experimentation.

EXAMPLE 34

Micellar electrokinetic capillary chromatography of cocoa procyanidins

A quick method has been developed to determine procyanidin oligomers using micellar electrokinetic capillary chromatography (MESS). This method is a modification of the method described by Delgado et al., 1994. When using the MESS method, it only takes 12 minutes to obtain the same separation as that obtained in 70 minutes when analyzed by normal phase HPLC. Fig. 57 is a MECC separation of cocoa procyanidins obtained in Example 2.

Mass conditions

Cocoa procyanidin extract was prepared by the method described in example 2 and dissolved in a concentration of 1 mg / ml in a MESS buffer containing 200 mM boric acid, 50 mM sodium dodecyl sulfate (electrophoretically pure) and NaOH adjusting the pH to 8.5.

A sample was passed through a 0.45 μm filter and subjected to electrophoresis using a Hewlett Packard HP-3D CZE system operating under the following conditions:

Input buffer: working buffer as described above.

Output buffer: working buffer as described above.

Capillaries with an inner diameter of 50 cm x 75 microns from fused quartz without coating.

Detection: 200 nm, with a diode array detector.

Introduction: 50 mbar for 3 seconds (150 mbar / s).

Voltage: 6 watts.

Amperage: system limit (<300 μA).

Temperature: 25 ° C.

Capillary conditions: 5 minutes of quick warm-up before and after each separation. This method can be changed by profiling the temperature, pressure and voltage parameters, as well as by including organic modifiers and chiral-selective agents in the working buffer.

EXAMPLE 35

MALDI-TOF / MS analysis of procyanidin oligomers with metal salt solutions

A number of MALDI-TOF / MS studies were performed on trimers combined with various metal salt solutions to determine the possibility of detecting oligomer cation adducts. The significance of the experiment was the proof that oligomers of procyanidins have physiological significance in sequestration in vitro and in vivo or in the delivery of metal cations, which are important in the development of physiological processes and disease states.

This method was used as described in Example 15. Briefly, 2 μl of 10 mM solutions of zinc dihydrogen sulfate, calcium chloride, magnesium sulfate, iron chloride hexahydrate, iron sulfate heptahydrate and copper sulfate were individually combined with 4 μl trimer (10 mg / ml ), purified to apparent uniformity as described in Example 14, and 44 μl of DHB was added.

The results (Fig. 58A-F) showed [metal trimer + H] ⁺ ions for copper and iron (ferrous and ferric), whose m / z values corresponded to ± l amu standard deviation for theoretical calculated masses. [Metal trimer + H] ⁺ masses for calcium and magnesium cannot be unambiguously determined on the basis of [metal trimer + H] ⁺ masses for sodium and potassium, whose m / z values did not exceed ± 1 amu standard deviation . [Zn ⁺² trimer + H] ⁺ ion could not be detected. Since several of these cations are multivalent, it is possible to form a metal-oligomer and / or multi-metal oligomer ligand species. However, the scan for these adducts at their predicted masses was unsuccessful.

The results shown above for copper, iron, calcium, magnesium and zinc can be used as general instructions for subsequent analysis of the reaction between other metal ions and the compounds of the invention, taking into account facts such as the oxidation state and the relative position of the ion under discussion in the Periodic table.

EXAMPLE 36

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

An analytical examination was performed on GPC eluents associated with high molecular weight procyanidins prepared as described in Example 3, Method A. Its purpose was to determine whether procyanidin oligomers with n> 12 were present. If present, then these oligomers are additional compounds of the invention. The regulation of existing methods of isolation, separation and purification embodied in the invention can be carried out to obtain these oligomers with the aim of their subsequent assessment in vitro and in vivo for anticancer, antitumor or antineoplastic activity, antioxidant activity, inhibition of the enzymatic activity of DNA topoisomerase II, inhibition of oxidative damage to DNA for antimicrobial activity and activity as modulators of NO or NO synthase, apoptosis, platelet aggregation, blood glucose, or in vivo, as well as its effectiveness as non-steroidal anti-inflammatory agents.

Fig. 59 is a MALDI-TOF mass spectrum of a sample of the GPC eluent described above. It is obvious that [M + Na] ⁺ and / or [M + K] ⁺ and / or [M + 2Na] ⁺ ions characterize procyanidin oligomers from tetramers to octadecameras.

It was determined that acid and heat treatment cause hydrolysis of procyanidin oligomers. Therefore, the invention includes controlled hydrolysis of oligomers of high molecular weight procyanidins (for example, where n is 13-18), as a method of producing lower molecular weight procyanidin oligomers (for example, where n is 2-12).

EXAMPLE 37

Dose-response relationship between procyanidin oligomers and canine and feline cell lines

The dose-response relationship of procyanidin oligomers was evaluated against several canine and feline cell lines obtained from the Waltham Pet Food Center, Waltham on-the-Wolds, Melton Mowbray, Leicestershire, UK. These cell lines were

normal canine GH kidney cell line;

normal canine kidney cell line MDCK;

normal feline CRFK kidney cell line and

feline lymphoblastic cell line FeA producing leukemia virus, which were cultured under the conditions described in example 8, method A.

The procyanidin monomers and oligomers, where n is 2-10, were purified as described in Example 14, Method D. The oligomers were also tested using normal phase analytical HPLC as described in Example 14, Method C, and the following results were obtained:

Procyanidins % purity after HPLC Monomers 95.4 Dimers 98.0 Trimmers 92.6 Tetramers 92.6 Pentamers 93.2 Hexamers 89.2 (contain 4.4% pentamers) Heptamers 78.8 (contain 18.0% hexamers) Octamers 76.3 (contain 16.4% heptamers) Nonamers 60.3 (contain 27.6% octamers) Decamers 39.8 (contains 22.2% of nonamers,
16.5% octamers and 13.6% heptamers)

In such cases, when the purity of the oligomers is <90%, the methods embodied in the invention are used for their repeated purification.

Each cell line was connected with monomers and each of the procyanidin oligomers at doses of 10 μg / ml, 50 μg / ml and 100 μg / ml, and the results are shown in Figs. 60-63. As can be seen in the figures, when applying high doses (100 μg / ml) of individual oligomers, the same inhibitory effect was obtained on the feline lymphoblastic FeA cell line and on the feline normal kidney cell line CRFK. In these cases, cytotoxicity appeared with the tetramer, and higher oligomers showed increasingly higher cytotoxic results. In contrast, when exposed to high doses (100 μg / ml) of individual oligomers on the canine cell lines of the normal kidney GH and MDCK, higher oligomers were required to exhibit cytotoxicity. In canine normal kidney GH cell line, cytotoxicity appeared with the use of a pentamer. On the canine cell line of the normal kidney, MDCK cytotoxicity appeared with hexamer. In both of these cases, the use of higher oligomers caused an increase in the level of cytotoxicity.

EXAMPLE 38

Tablet Formulations

A tablet formulation was prepared using solid cocoa prepared by the methods described in US Application Serial Number 08/709406, filed September 6, 1996, incorporated herein by reference. Briefly, this edible material was prepared using a process that increases the natural content of the compound of the invention compared to their level in cocoa obtained by the traditional method, so that the ratio of the initial amount of the compounds of the invention found in unprocessed beans to their content after preparation is less than 2 or equal to 2. For simplicity, the cocoa solids material is referred to herein as CP-cocoa solids. The compound or compounds of the invention, for example, in isolated and / or purified form, can be used in tablets, as described in the example, instead of or in combination with a CP-cocoa solid.

The finished tablet formulation includes the following components (expressed as a percentage by weight):

Solid CP-Cocoa 24.0% 4x Natural Vanilla Extract (Bush Boake Allen) 1,5 Magic stearate (dry lubricant) (AerChem, Inc.) 0.5% Dipac Tablet Sugar (Amstar Sugar Corporation) 37.0% Xylitol (American Xyrofin, Inc.) 37.0% 100.0%

The CP-cocoa solids and vanilla extract are mixed together in a food processor for 2 minutes. Sugar and magnesium stearate are carefully mixed, followed by combining with a dry substance mixture of CP-cocoa / vanillin. The material is passed through a tablet press (VZV) at maximum pressure and compaction to obtain round tablets (15 mm x 5 mm) with a mass of 1.5-1.8 grams. Another type of tablet with the above composition is prepared using commercially available natural cocoa powder with low fat content (11% fat) instead of CP-cocoa solids (11% fat). Both types of tablets are obtained with the corresponding aromatic characteristics and texture properties.

The analysis of the two tablet formulations was carried out using the procedure described in example 4, method 2. In this case, the analyzes focused on the concentration of the pentamer and the overall level of monomers and compounds of the invention, where n is 2-12, are presented below.

Sample pills Pentamer (mcg / g) General
Qty Pentamer (mcg / 1.8 g conservation) Total qty (mcg / 1.8 g conservation) Solid tablets
CP Cocoa 239 8277 430 14989 Low Fat Commercial Cocoa Powder Tablets undefined 868 undefined 1563

These data showed a higher content of pentamers and the total number of compounds of the invention in tablets prepared on the basis of solid CP-cocoa, compared to other tablet forms. Thus, tablet forms formulated with CP-cocoa solids are ideal for oral delivery of a compound of the invention in pharmaceutical use as food additives and in food.

A qualified specialist in this field can easily prepare other tablet forms, including a wide range of flavors, colorants, excipients, vitamins, minerals, OTC medicines, sugar fillers, UV protective agents (e.g. titanium dioxide, colorants, etc. .), binders, hydrogels and the like, with the exception of polyvinylpyrrolidone, which would irreversibly bind the compounds of the invention or combinations thereof. The amount of sugar fillers can be adjusted, which allows you to change the dosage of the compounds according to the invention or their combination.

It is self-evident that there are many possible variations of the above without departing from the essence and scope of this example.

EXAMPLE 39

Encapsulated Finished Formulation

An embodiment of Example 38 for oral administration of the compounds of the invention is in the form of detachable capsules made of gelatin, as well as soft, hermetically sealed capsules made of gelatin and a plasticizer such as glycerol. Detachable capsules contain a compound of the invention or a combination of compounds, or a CP-cocoa solid, as described in Examples 38 and 40, in the form of a powder that can optionally be mixed with excipients such as lactose or sucrose, which allows for varying dosages of the compounds of the invention. In soft capsules, the compounds of the invention, or a combination of the compounds, or a solid of CP-cocoa, are in a suitable liquid such as fatty oils or cocoa butter, or combinations thereof. Since the compound or compounds of the invention can be very sensitive, for example sensitive to UV radiation, the capsule may contain UV protective agents such as titanium dioxide or suitable colorants. Capsules may also contain the excipients described in the previous example. It is self-evident that there are many possible variations of the above without departing from the essence and scope of this example.

EXAMPLE 40

Finished formulations in the form of input (SOI) and non-input (non-SOI) in the Identity Standard for dark and milk chocolate

Formulations of the compounds of the invention or combinations of compounds obtained by the methods embodied in the invention may be in the SOI or non-SOI form of dark and milk chocolate, representing a means for delivering the compound to humans and animals. Reference is made to the pending U.S. application serial number 08/709406, filed September 6, 1996, incorporated herein by reference. US application 08/709406 relates to a method for producing cocoa butter and / or cocoa solids while maintaining the level of compounds of the invention as in cocoa beans, which uses a unique combination of cooking steps. Briefly, the edible cocoa solid obtained by this method preserves the natural content of the compounds of the invention, in contrast to the levels that are stored in traditionally processed cocoa, namely, the ratio of the initial amount of the compounds of the invention in unprocessed beans to that obtained after manufacture is less than two or equal to 2. For simplicity, this cocoa solids material is referred to herein as CP-cocoa solids. The CP-Cocoa solids are used as a powder or liquid to make SOI or non-SOI chocolate, drinks, snacks, baked goods, and culinary ingredients. The term “SOI chocolate” as used herein refers to any chocolate used in the United States as a food product. , which complies with the Identity Standard established by the US Food and Drug Administration in accordance with the Federal Food, Drug, and Cosmetic Act means. Definitions and standards for various types of chocolate are well established. The term “non-SOI chocolate” as used herein refers to any non-standardized types of chocolate that contain compositions that fall outside the ranges specified for standardized chocolate.

Examples of non-standardized types of chocolate are obtained when cocoa butter or milk fat is partially or completely replaced or when a sweetener in the form of carbohydrates with a high nutritional value is partially or completely replaced, or in the case of adding flavorings that mimic milk, butter, cocoa powder or chocolate, or in the case of other additions or exceptions to the recipe that are outside the scope of the Federal Identity Standard for chocolate or its various types.

As a confectionery product, chocolate can be in the form of solid lump chocolate, for example in the form of tiles or new forms, and can also be a component of other, more complex confectionery products, where chocolate is optionally combined with any substances of the Association producing flavors and extracts, with natural juices, spices, herbs and extracts that are included in the category of natural flavors, with substances identical to natural, with artificial flavors defined in the FEMA list GRASS, FEMA & FDA, on the list of the European Council (CoE), the International Organization for the Production of Food Additives (IOFI), which adopt the FAO / WHO food standards program, food code and food code, as well as other foods such as caramel, nougat, fruit slices, nuts, water and the like. These foods have microbiological stability at 65-85 ° F (18-28 ° C) under normal atmospheric conditions. Other complex confectionery products are obtained by pouring soft chocolate of various inclusions, such as "drunk cherries" or peanut butter. Other complex confectionery is made from chocolate-coated ice cream or other chilled or frozen desserts. In general, it can be said that chocolate used to coat or fill products must be more liquid than chocolate to make tiled solid chocolate or new forms of it.

In addition, chocolate may also be low-fat chocolate containing fats and non-fat solid substances, having sweeteners in the form of carbohydrates with high nutritional value and edible emulsifiers. For chocolate with reduced fat content, reference is made to US Patent No. 4810516, 4701337, 5464649, 5474795 and WO 99/19923.

Dark chocolate gets its dark color due to a certain amount of liquid cocoa mass or alkalized liquid cocoa mass, or solid cocoa, or alkalized solid cocoa used in any formulation. However, the use of alkalized dry cocoa solids or liquid cocoa mass would not be used in the dark chocolate formulations of this invention, as Example 27, Table 13, shows that a compound of the invention is lost due to the alkalization process.

Examples of formulations of dark and milk chocolate SOI and non-SOI formulations are listed in Tables 16 and 17. In these formulations, the amount of compounds of the invention represented by CP-cocoa solids was compared with compounds of the invention presented in commercially available cocoa solids.

The following description presents the steps for preparing these finished chocolate formulations.

The process of making non-SOI dark chocolate

1. Keep all paddle and rotary mixers closed during the whole process to avoid light.

2. Download a batch of all ingredients except 40% free fat (cocoa butter and dry milk fat), maintaining a temperature of 30-35 ° C.

3. Grind to 20 microns.

4. Dry conching for 1 hour at 35 ° C.

5. Add all lecithin and 10% cocoa butter at the beginning of the wet conching cycle; wet conching for 1 hour.

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

7. Temper, mold and pack the chocolate.

The process of making SOI dark chocolate

1. Load batches of ingredients, with the exception of milk fat, at a temperature of 60 ° C.

2. Grind to 20 microns.

3. Dry conching for 3.5 hours at 60 ° C.

4. Add lecithin and milk fat and conduct wet conching for 1 hour at 60 ° C.

5. Standardize if necessary and mix for one hour at 35 ° C. Temper, mold and pack chocolate.

Non-SOI Chocolate Process

1. Keep all paddle and rotary mixers closed during the whole process to avoid light.

2. Load sugar, whole milk powder, malt milk powder and 66% cocoa butter, conch for 2 hours at 75 ° C.

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

4. Grind to 20 microns.

5. Add the remainder of cocoa butter, conduct dry conching for 1.5 hours at 35 ° C.

6. Add dry milk fat and lecithin, conduct wet conching for one hour at 35 ° C.

7. Standardize, temper, mold and pack chocolate.

Milk Chocolate SOI Process

1. Download a batch of all ingredients except 65% cocoa butter and milk fat at a temperature of 60 ° C.

2. Grind to 20 microns.

3. Carry out dry conching for 3.5 hours at 60 ° C.

4. Add lecithin, 10% cocoa butter and dry milk fat; conduct wet conching for 1 hour at 60 ° C.

5. Add the rest of the cocoa butter, standardize if necessary and mix for 1 hour at 35 ° C.

6. Temper, mold and pack the chocolate.

The CP-cocoa solids and commercial liquid chocolate masses used in the finished formulations were analyzed to determine the pentamers and the overall level of monomers and compounds of the invention in which n is 2-12, as described in method 2 of example 4, before they are included in the finished preparative forms. These values were then used to calculate the expected levels in each chocolate recipe, as shown in Tables 16 and 17. In the cases of non-SOI dark chocolate and non-SOI milk chocolate, the products thereof were similarly analyzed to determine pentamers and the overall level of monomers and compounds inventions where n is 2-12. The results are presented in tables 16 and 17.

The results of studies of the finished formulations conducted in these examples show that SOI and non-SOI dark and milk chocolate, prepared on the basis of the solid substance CP-cocoa, contain approximately 6.5 times more pentamers and 3.5 times more than the total number of monomers and compounds of the invention than expected in SOI and non-SOI dark chocolate; approximately 4.5; 7.0 times the expected number of pentamers and 2.5; 3.5 times the expected total in SOI and non-SOI milk chocolate, respectively.

Studies of some chocolate products have not been conducted, since the difference between the expected levels of the compounds of the invention in finished chocolate products prepared with CP-cocoa solids was significantly higher than in those prepared according to the same recipes, but with commercially available dry cocoa. However, preparation results were evaluated for non-SOI products of dark milk chocolate. As shown in the tables, there is a 25-50% loss of pentamers, while only slight discrepancies are observed in general levels. Not wishing to associate this with any theory, we believe that these losses occur due to heating and / or due to the low-chain fatty acids in milk (for example, acetic acid, propionic acid and butyric acid), which can lead to hydrolysis oligomers (for example, trimers can be hydrolyzed to monomers and dimers). Alternatively, a lengthy, stepwise manufacturing process may preserve the oxidative or irreversible binding of the compounds of the invention to protein sources within the scope of this recipe. Thus, the invention includes alternative methods for preparing finished chocolate formulations and processes to prevent or minimize these losses.

Numerous variations of these examples are understood by those skilled in the art to cover a wide range of recipes, ingredients, cooking methods, and mixtures in order to rationally control the natural levels of the compounds of the invention for various chocolate applications.

Table 16
Finished dark chocolate formulation prepared with non-alkalized cocoa ingredients non-SOI CP-Cocoa solids dark chocolate SOI dark chocolate based on solid cocoa solids SOI dark chocolate based on commercial dried cocoa Ready preparative form Ready preparative form Ready preparative form 41.49% sugar 41.49% sugar 41.49% sugar 3% whole milk powder 3% whole milk powder 3% whole milk powder 26% solid CP cocoa 52.65% CP-liquid 52.65% commercial liquid 4.5% commercial liquid 2.35% dry milk. fat 2.35 dry milk fat 21.75% cocoa butter 0.01% vanillin 0.01% vanillin 2.75% dry milk fat 0.5% lecithin 0.5% lecithin 0.01% vanillin 0.5% lecithin Total fat: 31% Total fat: 31% Total fat: 31% Particle size: 20 microns Particle size: 20 microns Particle size: 20 microns The expected levels of pentamers and the total level of oligomeric procyanidins (monomers and oligomers with n = 2-12; in μg / g) Pentamer: 1205 Pentamer: 1300 Pentamer: 185 Total: 13748 Total: 14646 Total: 3948 Actual levels of pentamers and all oligomeric procyanidins (monomers and oligomers with n = 2-12, in μg / g) Pentamers: 561 Total: 14097 Not carried out Not carried out

Table 17
Finished milk chocolate formulation prepared with unsweetened cocoa ingredients non-SOI CP-Cocoa solids dark chocolate SOI dark chocolate based on solid cocoa solids SOI dark chocolate based on commercial dried cocoa Ready preparative form Ready preparative form Ready preparative form 46,99.65 sugar 46,99.65 sugar 46,99.65 sugar 15.5% whole milk powder 15.5% whole milk powder 15.5% whole milk powder 4,5 solid CP cocoa 13.9% CP-liquid 13.9% of sales liquid 5.5% commercial liquid 1.6% dry milk fat 1.6% dry milk fat 24.4% cocoa butter 0.035% vanillin 0.0035% vanillin 1.6% dry milk fat 0.5% lecithin 0.5% lecithin 0.035% vanillin 17.5% cocoa butter 17.5% cocoa butter 0.5% lecithin 4.0% malt. milk powder 4.0% malt. milk powder 4.0% malt milk powder Total fat: 31.75% Total fat: 31.75% Total fat: 31.75% Particle size: 20 microns Particle size: 20 microns Particle size: 20 microns The expected levels of pentamers and the total level of oligomeric procyanidins (monomers and oligomers with n = 2-12; in μg / g) Pentamer: 225 Total: 2734 Pentamer: 343
Total: 3867 Pentamer: 49
Total: 1042 Actual levels of pentamers and all oligomeric procyanidins (monomers and oligomers with n = 2-12, in μg / g) Pentamers: 163 Total: 2399 Not carried out Not carried out

EXAMPLE 41

Hydrolysis of procyanidin oligomers

Example 14, method D, describes normal phase HPLC performed to purify the compounds of the invention. Oligomers were obtained as fractions dissolved in the mobile phase. The solvent was then removed using standard vacuum distillation (20-29 inches Hg, 40 ° C) on a rotary evaporator. It was observed that the loss of certain oligomers increased with decreasing oligomer size, with an increase in residence time in a vacuum distiller, or when a temperature of> 40 ° C was applied.

Losses of certain oligomers associated with a decrease in the size of the oligomer were attributed to the time-temperature factor of acid hydrolysis by residual acetic acid present in the mobile phase solution. This observation was confirmed by the following experiment, in which 100 mg of hexamer was dissolved in 50 ml of a mobile phase containing methylene chloride, acetic acid, water and methanol (see Example 14, Method D, for solvent proportions) and subjected to temperature / time-dependent distillation. Certain portions were taken at specific times for analytical normal phase HPLC as described in Example 4, Method 2. The results are illustrated in FIGS. 64 and 65, where the levels of hexamers decreased with increasing temperature / time.

Fig. 65 illustrates the appearance of one of the hydrolysis products (trimer) as a function of temperature / time. The monomer and other oligomers (from dimer to pentamer) also showed a temperature / time dependence.

These results indicated that extreme caution should be exercised and safety precautions should be taken while handling the polymer compounds of the invention.

The results presented above, together with the results obtained in examples 5, 15, 18, 19, 20 and 29, show that the method described above can be used to complement other methods embodied in the invention in order to identify any given oligomer of the invention .

For example, the complete hydrolysis of any of the above oligomers, which gives exclusively (+) - catechin or (-) - epicatechin, excludes the possibility of obtaining an oligomer structure based on "mixed monomers" and reduces the possibility of stereochemical bonds that are characteristic of each monomer constituting any from the above oligomers.

Further, the complete hydrolysis of any given oligomer, which gives both (+) - catechin and (-) - epicatechin in certain proportions, provides the specialist with information about the monomer composition of this oligomer and, therefore, gives a stereochemical characterization of the possible bonds for each oligomer included monomer.

It will be appreciated by a qualified person that acid catalyzed epimerization of individual monomers can occur and that appropriate control experiments and mild hydrolysis conditions (e.g. using organic acids such as acetic acid instead of concentrated HCl, HNO ₃ , etc.) should be provided.

Thus, although the present invention is described in detail in its preferred embodiments, it should be understood that the invention defined by the proposed claims should not be limited to the specific details given above, since their obvious changes are possible without departing from the scope and essence of the presented invention .

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Claims (21)

1. A method of treating a patient in need of treatment with an NO modulating agent, comprising orally administering to the patient a composition comprising a polymer compound of formula A _n in an amount effective for the treatment, wherein A is a monomer of the formula

where n represents an integer from 2 to 18,
R and X each have α or β stereochemistry,
R represents OH,
substituents C-4, C-6 and C-8 represent X, Z and Y, respectively, and the linking of monomer units occurs with C-4, C-6 or C-8, and if any of C-4, C-6 or C-8 is not connected to another monomer unit, then X, Y and Z represent hydrogen, provided that Y and Z of the first monomer unit represent hydrogen when the second monomer unit is connected to C-4 of the first unit named
or its derivative and pharmacologically acceptable carrier or excipient. 2. A method of treating a mammal in need of treatment with NO synthase modulating agents, comprising orally administering to the mammal a composition comprising a polymer compound of the formula A _n , where A is a monomer of the formula

where n represents an integer from 2 to 18,
R and X each have α or β stereochemistry,
R represents OH,
the substituents C-4, C-6 and C-8 represent X, Z and Y, respectively, and the binding of monomer units occurs with C-4, C-6 or C-8,
and if any of C-4, C-6 or C-8 is not connected to another monomer unit, then X, Y and Z represent hydrogen, provided that Y and Z of the first monomer unit represent hydrogen when the second monomer unit is associated with C-4 of the named first unit,
or its derivative,
and a pharmacologically acceptable carrier or excipient. 3. The method according to claim 1 or 2, characterized in that it increases the synthesis of nitric oxide. 4. The method according to claim 3, characterized in that the synthesis of nitric oxide is enhanced by inducing inducible nitric oxide synthase (iNOS) in a mammal. 5. The method according to any one of claims 1 to 4, characterized in that the treatment subject suffers from NO-dependent hypercholesterolemia. 6. The method according to any one of claims 1 to 4, characterized in that the treatment subject suffers from atherosclerosis, thrombosis, heart attack, stroke, or blood circulation problems. 7. A method for modulating platelet aggregation comprising administering to a mammal in need thereof a platelet aggregation modulating composition comprising a polymer compound of the formula A _n , where A is a monomer of the formula

where: n represents an integer from 2 to 18,
R and X each have α or β stereochemistry,
R represents OH,
the substituents C-4, C-6 and C-8 represent X, Z and Y, respectively, and the binding of monomer units occurs with C-4, C-6 or C-8,
and if any of C-4, C-6 or C-8 is not connected to another monomer unit, then X, Y and Z represent hydrogen, provided that Y and Z of the first monomer unit represent hydrogen when the second monomer unit is associated with C-4 of the named first unit,
or its derivative,
in a pharmacologically acceptable carrier or excipient. 8. A method of treating, preventing, or ameliorating atherosclerosis or re-stenosis in a mammal, comprising administering to the mammal a composition comprising a polymer compound of the formula A _n , wherein A is a monomer of the formula:

where n represents an integer from 2 to 18,
R and X each have α or β stereochemistry,
R represents OH,
the substituents C-4, C-6 and C-8 represent X, Z and Y, respectively, and the binding of monomer units occurs with C-4, C-6 or C-8,
and if any of C-4, C-6 or C-8 is not connected to another monomer unit, then X, Y and Z represent hydrogen, provided that Y and Z of the first monomer unit represent hydrogen when the second monomer unit is associated with C-4 of the named first unit,
or its derivative,
in a pharmacologically acceptable carrier or excipient. 9. A method for modulating thrombosis in a mammal, comprising administering to that mammal a thrombosis modulating composition comprising a polymer compound of the formula A _n , wherein A is a monomer of the formula:

where n represents an integer from 2 to 18,
R and X each have α or β stereochemistry,
R represents OH,
the substituents C-4, C-6 and C-8 represent X, Z and Y, respectively, and the binding of monomer units occurs with C-4, C-6 or C-8,
and if any of C-4, C-6 or C-8 is not connected to another monomer unit, then X, Y and Z represent hydrogen, provided that Y and Z of the first monomer unit represent hydrogen when the second monomer unit is associated with C-4 of the named first unit,
or its derivative,
in a pharmacologically acceptable carrier or excipient. 10. A method of reducing arterial hypertension in a mammal in need of such treatment, comprising administering to a patient a composition comprising a polymer compound of the formula A _n in an amount effective for the treatment, where A is a monomer of the formula

where n represents an integer from 2 to 18,
R and X each have α or β stereochemistry,
R represents OH,
the substituents C-4, C-6 and C-8 represent X, Z and Y, respectively, and the binding of monomer units occurs with C-4, C-6 or C-8,
and if any of C-4, C-6 or C-8 is not connected to another monomer unit, then X, Y and Z represent hydrogen, provided that Y and Z of the first monomer unit represent hydrogen when the second monomer unit is associated with C-4 of the named first unit,
or its derivative,
in a pharmacologically acceptable carrier or excipient. 11. The method according to any one of claims 1 to 10, characterized in that the subject of treatment is a person. 12. The method according to any one of claims 1 to 10, characterized in that the subject of treatment is a veterinary animal. 13. The method according to any one of claims 1 to 12, characterized in that n represents a number from 5 to 12. 14. The method according to any one of claims 1 to 13, characterized in that n represents the number 5. 15. The method according to any one of claims 1 to 14, characterized in that in the compound of formula A _{n the} binding occurs between C-4 and C-6 of adjacent monomers or between C-4 and C-8 of adjacent monomers;
each of X, Z and Y represents H or an adjacent monomer, provided that if X and Y are adjacent monomers, Z represents H, and if X and Z are adjacent monomers, Y represents H and at least one of the two terminal monomers linked to the adjacent monomer in C-4 and, optionally, Y and Z, each, are hydrogen. 16. The method according to any one of claims 1 to 12, characterized in that the compound is selected from the group consisting of a trimer selected from the group consisting of [EC- (4β → 8)] ₂ -EC, [EC- (4β → 8)] ₂ -C and [EC- (4β → 6)] ₂ -EC, a tetramer selected from the group consisting of [EC- (4β → 8)] _3- EC, [EC- (4β → 8)] ₃ -C and [EC- (4β → 8)] ₂ -ES- (4β-6) ₂ -C, a pentamer selected from the group consisting of [EC- (4β → 8)] ₄ -EC, [EC- (4β → 8)] ₃ -ES- (4β → 6) -EC, [EC- (4β → 8)] ₃ -ES- (4β → 8) -C and [EC- (4β → 8)] ₃ - EC- (4β → 6) -C, a hexamer selected from the group consisting of [EC- (4β → 8) ₅ -EC, [EC- (4β → 8) ₄ -ES- (4β → 6) -EC, [EC- (4β → 8)] ₄ -ES- (4β → 8) -C and [EC- (4β → 8)] ₄ -ES- (4β → 6) -C, a heptamer selected and the group consisting of [EC- (4β → 8)] _6-EU, [EC- (4β → 8)] ₅ -EC- (4β → 6) -EU, [EC- (4β → 8)] _5-EU - (4β → 8) -C and [EC- (4β → 8)] ₅ -ES- (4β → 6) -C, an octamer selected from the group consisting of [EC- (4β → 8)] ₇ -ES , [EC- (4β → 8)] ₆ -EC- (4β → 6) -EC, [EC- (4β → 8)] _6- EC- (4β → 8) -C and [EC- (4β → 8 )] ₆ -ES- (4β → 6) -C, nonamer selected from the group consisting of [EC- (4β → 8)] ₈ -EC, [EC- (4β → 8)] ₇ -ES- (4β → 6) -EC, [EC- (4β → 8)] ₇ -ES- (4β → 8) -C and [EC- (4β → 8)] ₇ -ES- (4β → 6) -C, and a decamera selected from the group consisting of [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 EC is an epicatechin group and C is a catechin group. 17. The method according to any one of claims 1 to 16, characterized in that the compositions are selected from the group consisting of liquid, suspension, delayed-release polymer system, tablets, capsules, chocolate, corresponding to the Identity Standard, and chocolate, not meeting the Identity Standard . 18. The method according to any one of claims 1 to 17, characterized in that the said polymer compound is essentially a pure compound obtained from Theobroma and Herrania species or from their hybrids from intraspecific or interspecific crosses. 19. The method according to any one of claims 1 to 17, characterized in that the radical R of the terminal monomer unit of the named polymer compound is O-R ', where R' represents the remainder of gallic acid. 20. The method according to any one of claims 1 to 6, characterized in that it further includes the introduction of at least one additional agent that modulates NO or NO synthase. 21. The method according to claim 10, characterized in that it further provides for the introduction of at least one additional agent that reduces arterial hypertension.

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