JP2009501161A - Heat treated cocoa products useful for improving vascular health - Google Patents

Abstract

  The present invention relates to compositions comprising polyphenols, such as flavanols, procyanidins, and derivatives thereof, obtained by heat treating polyphenols in an aqueous solution, and compositions comprising epimers of flavanols, procyanidins, and derivatives thereof, and The present invention relates to treatment or prevention treatment of NO-responsive diseases or disorders using the same. The present invention is also characterized by the properties of modified monomers or oligomers in which the total amount of epicatechin and catechin does not change and there is a new dimer and no higher oligomers than the trimer. The invention relates to heat-treated cocoa powder and cocoa extract.

Description

Explanation of related applications

  This application is an international patent claiming priority from US Provisional Patent Application No. 60 / 695,361, filed June 29, 2005 as “Formulations for Vascular Health Improvement”. It is an application.

  The present invention relates to a composition comprising polyphenols, such as flavan-3-ols, procyanidins and their derivatives, obtained by heat treating a polyphenol-containing product, a composition comprising epimers of flavan-3-ols and procyanidins, and The present invention relates to a method for treating and preventing a NO-responsive disease or disorder.

  Polyphenols are a diverse group of compounds (Non-Patent Document 1). Some are widely produced in various plants and some are incorporated into the food chain. Sometimes they mean an important group of compounds in human food. For example, flavanols and procyanidins have been shown to be effective in nitric oxide (NO) (for example, Patent Document 1). NO is known to inhibit platelet aggregation, monocyte adhesion and chemotaxis, and vascular smooth muscle tissue diffusion that is critically involved in atherogenesis. When production is inhibited by a pathological condition such as an inhibitor or atherosclerosis, and the blood concentration of NO decreases, the vascular muscles do not relax moderately. The resulting vasoconstriction increases blood pressure and may be involved in certain types of hypertension. Hypertension is a major cause of vascular disease, including cerebral infarction, heart attack, heart failure, and renal failure.

Considering the large number of people suffering from NO-responsive diseases or disorders, there is great interest in finding various treatments to prevent and treat these conditions. Applicants have found that certain compounds described herein can be used for these applications.
US Pat. No. 6,670,390 Ferriera et al, Tetrahedron, 48:10, 1743-1803, 1992

  The present invention relates to heat-treated flavanols (monomers) and procyanidins (oligomers) and / or epimers of flavanols and procyanidins, compositions containing these compounds, and methods for treating and preventing NO-responsive diseases or disorders. .

  In one aspect, the present invention relates to a pharmaceutical, food, food additive, or dietary supplement comprising a compound according to the present invention or a derivative thereof in an amount effective for a NO responsive disease or disorder. The composition may optionally include additional therapeutic agents or health agents, or may be administered in combination with another therapeutic agent or health agent. Packaged products containing the above compositions, labels and / or instructions for use in the treatment or prevention of NO responsive diseases / disorders are also within the scope of the invention.

In one aspect, the present invention provides a method of treating or preventing a vascular disease or disorder by administering a beverage comprising (-)-catechin to a subject in need thereof, wherein The subject is a human or a veterinary animal. (-) - catechin, as monomeric compounds, or by heat-treating oligomeric compounds defined oligomeric compounds and / or described herein, which is defined as the structural formula E _n herein as structural formula A _n As a monomer unit which is a constituent component of the obtained compound, it may be present in a beverage. Typically, the beverage is a cocoa or chocolate-containing beverage or a chocolate flavored beverage.

  In another aspect, the invention relates to a method of preventing or treating a NO responsive disease or disorder in a mammal, such as a human or veterinary animal, by administering an effective amount of a compound according to the invention.

  In yet another aspect, methods are provided for preparing the compounds described herein.

  In yet another aspect, the present invention comprises (±) -catechin and (±) -epicatechin, and their procyanidin oligomers, wherein the total CP content relative to the defatted powder is at least 15 mg / g and has been heat treated. It relates to a heat treated, partially or fully defatted high CP cocoa powder having modified HPLC properties of monomers and oligomers compared to a non-high CP cocoa powder.

  The present invention also provides a dry or liquid, heat treated, having a total CP content for the dried cocoa extract of at least 175 mg / g and having modified properties compared to the unheated cocoa extract. High CP cocoa extract.

  In yet another aspect, the present invention relates to a heat treated cocoa product, such as a cocoa beverage, having modified properties compared to an unheat treated cocoa product. The product has high CP cocoa ingredients such as cocoa powder, liquid or dry cocoa extract, and / or cocoa mass (chocolate liquor). If the product has a low moisture content, the product contains at least about 6 mg / g total cocoa polyphenol (CP) relative to the product. The product preferably has a ratio of epicatechin to catechin 1 of 1 or more. If the product is a high moisture content product such as a beverage, the product contains about 0.2 mg / g total cocoa polyphenols to the product.

High CP cocoa powder with modified properties, high CP cocoa extract, high CP cocoa product, aqueous dispersion of partially or completely defatted cocoa powder, or unfermented or poorly fermented cocoa beans A dry cocoa extract prepared from is prepared by heating. The cocoa component is preferably heated at about 37 ° C. to about 200 ° C. for a time and pH sufficient to epimerize (−)-epicatechin, for example, from about 0.5 minutes to several days. They are preferably heated at about 72 ° C. to about 160 ° C. for about 1 minute to about 6.0 hours. More preferably, they are heated at about 100 ° C. to about 140 ° C. for about 1 minute to about 4 hours.
All patents, patent applications and cited references cited herein are hereby incorporated by reference. In the event of a conflict, the disclosure herein will apply.

  The present invention relates to heat treated flavanols (monomers) and procyanidins (oligomers), and / or epimers of flavanols and procyanidins, compositions comprising such compounds, and methods for treating / preventing NO-responsive diseases or disorders. About.

  In accordance with current nomenclature, the term “flavanol” as used herein refers to monomers such as catechin, epicatechin and the like. Catechin and epicatechin oligomers are called procyanidins. However, for purposes of the present disclosure, these terms may be used interchangeably. Furthermore, it will be appreciated that references to polyphenols herein apply to the combination of flavanols and procyanidins, or individually for them.

The present invention includes a first compound, the second compound having the following structural formula A _n, or pharmaceutical-acceptable possible salts or derivatives (e.g., oxidation products, methyl derivatives, glucuronic acid derivative) heat treating the With respect to a composition comprising an effective amount of a first compound obtainable or obtainable by:

here,
n is an integer of 2 to 18,
R and X each have an α or β type stereochemistry,
R is OH, O-sugar or O-gallate,
The C-4, C-6, C-8 substituents are X, Z, Y, respectively, and the bonding of the monomer units occurs at C-4, C-6 or C-8;
If none of C-4, C-6 or C-8 is linked to another monomer unit, each X, Y or Z is hydrogen or sugar;
The sugar is optionally substituted with a phenol moiety at any position, such as via an ester bond.

  In this specification, the phrase “compound obtained by heat treatment”, “compound obtainable by heat treatment” or “compound is heat treated” means that the compound is sufficiently heated in the presence of water for a sufficient amount of time, It means that the chemical structure and / or function of the product is / are altered. The lower the temperature, the longer the heating time required to cause the change. For example, the change may be maximized by increasing the heating temperature from ambient temperature to 140 ° C., increasing the pH to about 3.8 to about 7, and / or increasing the heating time. In order to reach equilibrium, the temperature and / or pH must be increased. These temperatures and times are at least 40 ° C., more preferably at least 50 ° C., at least 10 hours, more preferably at least 24 hours, and even more preferably at least 48 hours. I will. Or at least 60 ° C, at least 70 ° C, at least 80 ° C, at least 90 ° C, at least 100 ° C, at least 110 ° C, or at least 120 ° C, respectively, for at least 5 minutes, or at least 10 minutes, 15 minutes, or 20 minutes. Will. For example, the compound may be treated at 120 degrees for 10 minutes. Another heat treatment example is described in Example 1. Other temperature / time / pH combinations are also effective, and one of ordinary skill in the art will determine them without undue experimentation using the guidance provided herein. Known techniques such as HPLC / MS analysis may be used to monitor compound changes as described, for example, in Example 1.

  The monomer unit in the above structural formula may be a bond formed by connecting 4 → 6 and 4 → 8. The oligomer having only the linkage of (4 → 8) is linear, and when at least one (4 → 6) bond is present, it becomes a branched-chain oligomer. Heat treated oligomers comprising at least one of the non-natural linkages (6 → 6), (6 → 8), (8 → 8) are also within the scope of the present invention. Oligomers having such linkages can be prepared as described in US Pat. No. 6,156,912, which is incorporated herein by reference.

  The sugar can be selected from the group consisting of glucose, galactose, rhamnose, xylose, and arabinose. The sugar is preferably a monosaccharide or a disaccharide. The phenol moiety is selected from the group consisting of caffeic acid, cinnamic acid, coumaric acid, ferulic acid, gallic acid, hydroxybenzoic acid and sinapinic acid.

  Examples of derivatives include esters, oxidation products, methyl derivatives, and glucuron oxidation products. The oxidation product may be prepared as described in US Pat. No. 5,554,645, which is hereby incorporated by reference. Esters such as, for example, esters with gallic acid may be prepared as described in US Pat. No. 6,420,572, which is hereby incorporated by reference. Methyl derivatives such as 3′O-methyl-, 4′O-methyl-, and 3′O, 4′O-dimethyl-derivatives are described, for example, in Cren-Olive et al., 2002, J. Chem. Soc. Perkin Trans. 1, 821-830. Glucuronidation products are described in Yu et al., “A novel and effective procedure for the preparation of glucuronides.” Organic Letters, 2 (16) (2000) 2539-41 and Spencer et al. 2001, Free Radical Biol. Med. 31 (9), 1139-46.

  The above disclosure regarding linkages, sugars, phenol moieties, and derivatives is applicable to all compounds disclosed herein, but is not repeated here for the sake of brevity.

For the compounds of formula _{A n} or _{E n} described herein, examples of these compounds, the integer n is 3～18,2～12,3～12,2～5,4～12,5～ 12, 3-10, 4-10, or 5-10 compounds. Compounds _An or En _where n is 2 are also within the scope of the invention.

In one embodiment, the present invention is the first compound, and were obtained by heat-treating the second compound having the structural formula A _n or the first available compounds or can be their pharmaceutical-acceptable, For a composition comprising an effective amount of a salt or derivative (including oxidation products, methyl derivatives, glucuronic acid derivatives):

here,
n is an integer of 2 to 18,
R and X each have an α or β type stereochemistry;
R is OH;
The substituents of C-4, C-6, and C-8 are X, Z, and Y, respectively, and the bonding of monomer units occurs at C-4, C-6, and C-8,
When none of C-4, C-6, C-8 is bonded to another monomer unit, each X, Y, Z is hydrogen.

In certain embodiments, the compound is a dimer or trimer procyanidin heat treated, i.e., in the structural formula A _n, a n = 2 or n = 3. The monomer units of these dimers or trimers before heat treatment are all (-)-epicatechin. Examples of these dimers include (−)-epicatechin- (4β → 8)-(−)-epicatechin and (−)-epicatechin- (4β → 6)-(−)-epicatechin.

Dimers or trimers or other oligomers used for heat treatment can be prepared by isolation and purification, or synthesized by methods known in the art, or, for example, flavanols and / or other May be treated as a mixture with procyanidins. These mixtures may be, for example, plant extracts (eg cocoa extracts), dimers or other oligomeric fractions isolated from plant sources (eg cocoa) or cocoa materials such as cocoa powder. The cocoa extract can be prepared, for example, as described in US Pat. No. 5,554,645, and the cocoa powder can be prepared as known in the art, with flavanol / procyanidins. May be prepared as described in US Pat. No. 6,015,913. When heat treating individual purified dimers, dimer mixtures or synthetic compounds, their purity is, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about It may be 90%, at least about 92%, at least about 95%, at least about 98%, or at least about 99%. The above purity may be used for compounds of structural formula _An , and salts and derivatives thereof described herein.

Also within the scope of the present invention are the dimers disclosed in Example 1 (see, eg, FIG. 4) and compositions comprising these dimers, such as beverages such as cocoa or chocolate beverages. Referring to FIG. 4, the extracted ion chromatogram (EIC) of the 575 m / z pseudo-molecular ion shows the dimer detected in the processed beverage. The elution times for the new dimer are 12.6, 14.5 (maximum peak), 16.9, 18.8, and 22.6 minutes. The HPLC method effectively used for the above separation is as follows:
Stationary phase: Hypersil ODS 100 × 4.6 mm Particle size 5 μm
Mobile phase A: 0.1% HOAc / water Mobile phase B: 0.1% HOAc / MeOH
Flow rate: 1.0 ml / min

  Heat-treated (+)-catechin and (−)-epicatechin, and their derivatives (excluding gallic acid derivatives), and compositions containing flavanols, such as beverages such as cocoa or chocolate beverages, are also included in the present invention. It is in the range.

The present invention is further a compound having the structural formula E _n, and effective composition comprising an amount, or pharmaceutically acceptable acceptable salt or composition comprising a derivative thereof (oxidation product of the compound, For methyl derivatives and glucuronic acid derivatives):

here,
n is an integer of 2 to 18,
R and X each have an α or β type stereochemistry;
R is OH, O-sugar or O-gallate,
The substituents of C-4, C-6, and C-8 are X, Z, and Y, respectively, and the bonding of monomer units occurs at C-4, C-6, or C-8;
If none of C-4, C-6 or C-8 is linked to another monomer unit, each X, Y or Z is hydrogen or sugar;
The sugar is optionally substituted with a phenol moiety at any position, such as with an ester linkage,
At least one of the monomer units has the following structural formula:

Have

In the compounds of formula A _n, the monomer units in the above structural formula may be coupled by a connecting above. In the compounds of the formula A _n, sugar, phenol moiety, derivatives are as described above.

In another embodiment, the present invention provides compounds or their pharmaceutical-acceptable possible salts or derivatives having the structural formula E _n (oxidation products, methyl derivatives, glucuronic acid derivative), effective the compound For a composition comprising a sufficient amount:

here,
n is an integer of 2 to 18,
R and X each have an α or β type stereochemistry;
R is OH;
The substituents of C-4, C-6, and C-8 are X, Z, and Y, respectively, and the bonding of monomer units occurs at C-4, C-6, and C-8,
If none of C-4, C-6 or C-8 is bonded to another monomer unit, each X, Y, Z is hydrogen;
At least one of the monomer units has the following structural formula:

Have

Examples of compounds of formula _{E n,} include compounds integer n is 3～18,2～12,3～12,2～5,4～12,5～12,4～10 or 5-10, . In some embodiments, the compound is a dimer or trimer of procyanidins having at least one monomer unit in the form of an epimer. In one embodiment, the compound _{E n} is dimer or trimer, that is, n = 2 or n = 3.

For example, the invention provides compounds or their pharmaceutical-acceptable possible salts or derivatives have the following structural formulas E _n (oxidation products, methyl derivatives, glucuronic acid derivative), and effective amount of the compound Regarding the composition comprising:

here,
n is 2,
R and X each have an α or β type stereochemistry;
R is OH, O-sugar or O-gallate,
The substituents of C-4, C-6, and C-8 are X, Z, and Y, respectively, and the bonding of monomer units occurs at C-4, C-6, or C-8;
If none of C-4, C-6 or C-8 is linked to another monomer unit, each X, Y or Z is hydrogen or sugar;
The sugar is optionally substituted at any position with a phenol moiety, for example by an ester bond,
At least one of the monomer units is (−)-catechin.

In another example, the present invention provides compounds or their pharmaceutical-acceptable possible salts or derivatives having the structural formula E _n (oxidation products, methyl derivatives, glucuronic acid derivative), and effective the compound Regarding the composition comprising by quantity:

here,
n is 2,
R and X each have an α or β type stereochemistry;
R is OH;
The substituents of C-4, C-6, and C-8 are X, Z, and Y, respectively, and the bonding of monomer units occurs at C-4, C-6, and C-8,
If none of C-4, C-6 or C-8 is bonded to another monomer unit, each X, Y, Z is hydrogen;
At least one of the monomer units is (−)-catechin.

  As is known in the art, epimers are diastereoisomers in which only one of two or more tetrahedral stereocenters is reversed, such as one of two or more asymmetric carbon atoms. It is the body. For the types of compounds described herein, the epimer has a configuration in which one of the asymmetric carbon centers C-2 and C-3 is reversed.

  As used herein, the term epimer applies to compounds in which the C-2 carbon atom of the ring is inverted such that the stereochemistry of the C-2 carbon atom is β. In naturally occurring flavanols and procyanidins, the C-2 carbon atom typically has a stereochemistry of α.

  The present invention relates to carbon 2 (C-2) epimers of (+)-catechin and (−)-catechin of flavanols, ie, (+)-catechin and (−)-catechin, and derivatives thereof (eg, methyl derivatives) , Oxidation products excluding glucuronic acid derivatives and gallic acid) and compositions comprising these compounds.

Methods of Use Any of the compounds and / or compositions described herein may be performed according to the methods described herein.

  As used herein, “vascular disease or disorder” refers to a disease or disorder that affects the vascular system, including the heart and brain. Examples of these conditions include atherosclerosis, thrombosis, hypertension (eg secondary and pulmonary hypertension), cardiovascular disease (CVD), coronary artery disease (CAD) (myocardial ischemia, myocardial infarction) , Including stable and unstable angina, acute obstruction or restenosis), diabetes (type I and type II) (eg, vascular complications of diabetes), cognitive impairment or cognitive impairment and / or cardiovascular disorder (Including for the brain), heart failure, cerebrovascular disorder (cerebral infarction, early or recurrent transient ischemic attack, or ischemic, eg after coronary angioplasty or percutaneous coronary intervention) Complications), congestive heart failure, renal failure, kidney disease, peripheral vascular disorders, and more specifically diseases related to vasoconstriction of peripheral blood vessels such as Raynaud's disease (eg, arms or legs) Disease, peripheral arterial injury, intermittent claudication, vasculitis (eg, microvascular), vasospasm, venous thrombosis, venous dysfunction, lymphatic gland dysfunction, severe ischemic limb, acute ischemic limb, atheroembolism And lower limb arterial insufficiency.

  “NO-responsive disease or disorder” refers to a health condition that is responsive to treatment with NO. Examples of these conditions include, but are not limited to, NO-mediated or NO-dependent diseases and disorders, where the pathology of the disease / disorder is due to dysfunction of the NO pathway. For example, as a medical condition, hypertension (eg, primary, secondary, pulmonary hypertension, etc.), cardiovascular disease, coronary artery disease, diabetes (type I and type II) (eg, vascular complication of diabetes), sickle cell Anemia, cognitive dysfunction or cognitive impairment, and / or cardiovascular disorders (including those for the brain), heart failure, cerebral infarction, congestive heart failure, renal failure, kidney disease. Hypertension increases the risk of heart failure, cerebral infarction, congestive heart failure, renal failure, so use of the above compounds that reduce vascular tone alone or with drugs that protect other vasculature (including cardiovascular) Thus, these medical conditions can be suppressed. Particularly preferred subjects include hypertension and diabetes, obesity, adverse lipid status (eg, high cholesterol) and / or a combination of smokers, who have some risk of heart failure and cerebral infarction. Doubled. In general, subjects having at least one risk factor for cardiovascular disease (as recognized by the American Heart Association) may be treated as described herein.

  As used herein, “treatment” refers to, for example, cardiovascular disease by slowing disease progression, extending survival, reducing the risk of death, and / or improving disease parameters to some extent. It means improving the existing medical condition.

  The term “preventing” means reducing the risk associated with the development of a disease, including reducing the onset of the disease.

  As used herein, the term “agent that protects or treats the vasculature” refers to agents other than the compounds of the present invention that are effective in treating or protecting the vasculature. Examples of these agents include antiplatelet therapeutics (eg, COX inhibitors such as aspirin), NO regulators, cholesterol-reducing agents (eg, sterols, stanols).

Thus, the present invention provides (i) a method of treating or preventing a vascular disease or disorder, or (ii) a method of treating or preventing a NO-responsive disease or disorder, or (iii) any of those described herein. compounds (e.g., formula a _n or compounds of the E _n, or their pharmaceutically acceptable possible salts or derivatives (oxidation products, methyl derivatives, glucuronic acid derivative)) and the therapeutic in an effective amount The present invention relates to a method for treating antiplatelet by administering it to a subject in need thereof.

For example, the method no problem in practice by administering one of the n structural compounds of A _n is 2.

  The compounds according to the invention are also suitable for use in combination therapy with other vascular / cardiac protective agents and / or NO modulators. Examples of these agents will be apparent to those skilled in the art and include, for example, type B procyanidins and type A procyanidins. These compounds may be administered as a mixture with the compound according to the present invention or separately.

  Type A procyanidins can be naturally derived or prepared synthetically. For example, Form A procyanidins are described in Lou et al., Phytochemistry, 51: 297-308 (1999) or Karchesy and Hemingway, Agric, Food Chem., 34: 966-970 (1986). As such, it may be isolated from peanut peel, which is incorporated herein by reference for each relevant location. Other sources of the above compounds include, for example, Foo et al., J. Nat. Prod., 63: 1225-1228, Praia, which is incorporated herein by reference in their respective respective places. (Prior) et al., J. Agricultural Food Chem., 49 (3): 1270-76 (2001), rubber casks (Ecdysanthera utilis), which is incorporated herein by reference, respectively. (Lie-Chwen et al., J. Nat. Prod., 65: 505-8 (2002)), Aesculus hippocastanum (US Pat. No. 4,863,956). 1,1-diphenyl-2-picrylhydra under the neutral conditions described in Kondo et al., Tetrahedron Lett, 41: 485 (2000), incorporated herein by reference, respectively. An A-type compound may be obtained from B-type procyanidins through oxidation using a dill (DPPH) radical.

  Methods for obtaining natural and synthetic B-type procyanidins are well known in the art, eg, Romanczyk et al., US Pat. No. 6,670,390, Romantik et al., US Pat. No. 6,207, 842, Romantik et al., US Pat. No. 6,420,572, and Romantik et al., US Pat. No. 6,156,912.

  The method of the present invention may be used on humans or animal animals such as dogs, cats, horses and the like.

  Accordingly, the following applications are within the scope of the present invention. (I) treatment or prevention of vascular diseases or disorders, or (ii) treatment or prevention of NO-responsive diseases, or (iii) antiplatelet treatment drugs, foods, supplements or dietary supplements. Use of any of the compounds described herein, pharmaceutically acceptable salts or derivatives thereof in the manufacture. All of the specific conditions cited herein are within the scope of these applications.

  Effective amounts for use in the above methods may be determined by one of ordinary skill in the art using the guidance provided herein and common general knowledge in the art. For example, an effective amount can be to achieve a physiologically relevant concentration in the mammalian body (eg, blood). These physiologically relevant concentrations can be at least about 20 nanomolar (nM), preferably at least about 100 nM, and more preferably at least 500 nM. In one embodiment, the blood concentration of a mammal such as a human reaches at least about 1 micromolar. Compounds of structural formula An as defined herein can be administered at about 50 mg / day to about 1000 mg / day, preferably about 100-150 mg / day to about 900 mg / day, and about 300 mg / day to about 1000 mg / day. Most preferred is 500 mg / day. However, it is possible to use more than the above amount.

  The compound can be administered acutely, or therapeutic / prophylactic administration can be effected on a dosing schedule, ie, an effective period of time, eg daily, once every month, once every two months, once every six months, once a year, etc. Alternatively, it may be continued based on other dosing schedules, such as a skilled practitioner determines these periods as needed. Dosing may be continued for at least as long as needed for the therapeutic / preventive effect to appear. The composition is preferably administered daily to maintain an effective concentration of the compound in the mammal's body, and most preferably administered a few times a day, such as morning and evening. To obtain the most beneficial results, the composition can be administered for at least about 30 days, or at least about 60 days. These dosing schedules may be repeated periodically. Based on the guidance provided herein and common general knowledge in the art, one skilled in the art can select compounds suitable for acute and / or chronic administration. For example, compounds that are effective in a single dose and / or immediately after administration (eg, 2 hours after administration) may be used when an immediate acute phase response is required. Compounds that show an effect after repeated administration can be used accordingly. Thus, dosage forms that are compatible with such administration (eg, acute, chronic) are within the scope of the invention.

  Assays to determine the minimum dosage required for treatment or the optimal dosage for use in the above therapeutic methods are also within the scope of the present invention. The methods described in the Examples or other known dose response methods that predict the effectiveness of a compound for the treatment of the diseases or disorders cited herein may be used. Also within the scope of the invention are dosage forms that are adapted to deliver at least the minimum therapeutically effective dose, or an optimal amount.

Compositions and dosage forms The present invention relates to compositions described herein that may be formulated as pharmaceuticals, foods, food additives, and dietary supplements.

  As used herein, “pharmaceutical” refers to a medicinal drug. See Merriam- Webster's Collegiate Dictionary, 10th edition (1993). A medicinal product may be referred to as a medicament. As used herein, a “nutritional supplement” is a product (other than tobacco) intended for supplementation with or containing one or more of the following food ingredients: vitamins, minerals, herbs or other plants, amino acids, Substances in foods used by humans, or concentrates, metabolites, constituents, extracts or combinations of these ingredients intended to supplement foods by increasing the total daily intake. “Food” is a substance containing proteins, carbohydrates and / or fats that is utilized within the body of an organism that maintains growth, repairs, life processes and provides energy. The food may also contain auxiliary substances such as minerals, vitamins and spices. See Merriam Webster College English Dictionary 10th Edition (1993). The term food includes beverages adapted for human or animal consumption. “Food additives” are defined by the US Food and Drug Administration (FDA) 21 C.F.R. 170.3 (e) (l) and include direct and indirect additives.

  The composition may include a substrate, diluent, or excipient.

  Depending on the intended use, the substrate, diluent or excipient may be selected to be suitable for human or veterinary applications, foods, food additives, dietary supplements or pharmaceutical applications. The composition may optionally include additional cancer and / or cardiovascular therapeutics. The composition may be co-administered or continuously with these additional agents, taking into account factors such as age, gender, weight, genetic characteristics, individual subject or patient condition, and route of administration. it can.

  Examples of compositions according to the invention for humans or veterinary animals include, for example, edible compositions for oral administration such as capsules, tablets, pills and the like solid or liquid, and chewable solids or drinks ( Liquid formulations (e.g., cocoa or chocolate-flavored solid or liquid compositions), suspensions, syrups, or elixirs for administration into openings such as mouth, nose, anus, vagina, etc. Or a chocolate-flavored composition) and formulations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (eg injectable administration) such as sterile suspensions or emulsions It is done. However, the active ingredient in the composition will form a complex with the protein, such as clotting, which occurs when the protein in the blood precipitates when administered to the bloodstream. A skilled technician should take this into account. In these compositions, the active cocoa extract can be mixed with a suitable substrate, diluent or excipient such as sterile water, saline, glucose, DMSO, ethanol.

  Compounds according to the present invention are prepared from heat-treated compounds derived from different sources, such as procyanidins of natural origin (eg, Theobroma, Herrania) or synthetically prepared. It does not matter.

  Methods of preparing the starting materials (flavanols and procyanidins) used in the present invention are well known in the art (eg, US Pat. No. 5,554,645, incorporated herein by reference). 6,420,572, 6,156,912, 6,476,241, and 6,864,377). The polyphenol can be a natural source such as cocoa beans, or another natural source polyphenol, or a synthetically prepared polyphenol. One skilled in the art may select natural or synthetic polyphenols based on availability or cost.

  Flavanol and / or procyanidin derivatives are also useful as starting materials. These include monomeric or oligomeric esters, such as gallic acid esters (eg epicatechin gallate and catechin gallate), such as X, Y and / or Z positions in the above structural formula where the monosaccharide or disaccharide moiety ( Compounds derivatized with sugar moieties such as β-D-glucose), glycosylated monomers and oligomers and mixtures thereof, sulfation excluding the enzymatic cleavage products of procyanidins produced by metabolism of colonic flora, Examples include metabolites of procyanidin monomers and oligomers such as glucuronidated and methylated forms.

  The compound of the present invention is converted from flavanol, procyanidin or a derivative thereof having a C-2 atom stereochemistry such that the C-2 atom stereochemistry becomes β by rotating around the C-2 atom. It may be prepared by heat treatment (in aqueous solution). This approach is particularly suitable for the preparation of flavanol epimers such as (−)-epicatechin and (+)-catechin.

The preparation of flavanol epimers is depicted in the following scheme 1:

(And incorporated herein by reference), Freudenberg, K. and Purrmann, L. (1924). Raumisomere Catechin IV. Liebig's Annalen, 437, 472-85; Fredenberg, K , Bohme, L. and Purrmann, L. (1922). Raumisomere Catechin II. Ber. Dscht. Chem. Ges., 55, 1734-47).

  Epimerization occurs at lower temperatures and longer exposures. For example, these temperatures and times can be at least 40 ° C., more preferably at least 50 ° C., at least 10 hours, more preferably at least 24 hours, or more preferably at least 48 hours, or at least 60 At least 70 ° C., at least 80 ° C., at least 90 ° C., at least 100 ° C., at least 110 ° C., or at least 120 ° C., for at least 5 minutes, or at least 10 minutes, 15 minutes, or 20 minutes, respectively. . For example, the compound can be treated at 120 ° C. for 10 minutes, or 120 ° C. for 20 minutes. Other temperature / time combinations are also effective, and one of ordinary skill in the art can determine these without undue experimentation using general knowledge in the art and the guidance provided herein. Absent. Known techniques such as HPLC / MS analysis may be utilized to monitor the success of the reaction.

  Epimerized compounds are obtained, for example, from naturally occurring (eg, Theobroma, Herrania) or synthetically (obtained as is well known in the art, US Pat. No. 5,554). , 645, 6,420,572, 6,156,912, 6,476,241 and 6,864,377) procyanidins, etc. It can be prepared from heat-treated compounds from another source or procured by purchasing flavanols from a commercial source.

  Starting from the flavanol epimer, procyanidin epimers were prepared from US Pat. Nos. 6,420,572, 6,156,912, 6,476,241, and 6,864,377. And the methods described in WO 04/030440, which is hereby incorporated by reference, the disclosures of which are incorporated herein by reference.

For example, the following starting materials may be utilized for the synthesis of procyanidin epimers:

  Procyanidin epimers with 4β-8 linkages (described in Table 1) may then be prepared according to the following scheme (benzylation, hydroxyethoxylation, coupling, and debenzylation steps are As described in U.S. Pat. Various combinations of epimerized procyanidins can be prepared, depending on the starting material and various combinations.

  One of ordinary skill in the art, using knowledge in the art and guidance herein, will envision other methods of preparing the compounds of the present invention.

  Foods comprising the compounds described herein, and optionally other cardio / vascular protective agents, can be suitable for human or veterinary use, including pet foods.

  The food may be a food with a low water content such as confectionery or a food with a high water content such as a cocoa beverage. Typically up to 25% protein, preferably 5-20%, most preferably 1-15%, fat up to 60%, preferably 1-50%, most preferably 5-45%. Carbohydrate up to 70%, preferably 1-60%, most preferably 5-50%. Some foods may not necessarily contain protein, fat, and carbohydrates. The water activity of the food is about 0.2 to 0.95, preferably 0.4 to about 0.5.

  Foods include US Identity Standard (SOI) and Non-US Identity Standard (non-SOI), including milk, sweet, and semi-sweet chocolate, including black chocolate, low-fat chocolate, and chocolate-coated candy It can be a confectionery such as chocolate. Other examples include baked goods (eg brownies, baked snacks, cookies, biscuits), spices, granola bars, toffee chew, meal replacement bars, spreads, syrups, mixed powder drinks, Examples include cocoa, chocolate flavored beverages, pudding, rice cakes, mixed rice, and savory sauce. If necessary, the food may be flavored with chocolate or cocoa. The food product can be a candy bar, such as a chocolate and granola bar, containing nuts such as peanuts, walnuts, almonds, hazelnuts.

  Beverages, such as cocoa drinks, comprising (−)-catechin and / or dimers described herein are also within the scope of the present invention. For example, the beverage may contain more (−)-catechin than (−)-epicatechin, such as a ratio of (−)-epicatechin to (−)-catechin, such as at least 1: 2 or at least 1: 3. it can.

Cocoa material The heat-treated cocoa material is used for high CP foods. For products with low moisture content, these products contain at least about 6 mg of cacaopolyphenol per gram of product, preferably about 8 mg, more preferably about 10 mg, relative to the catechin of epicatechin in the product. The ratio is 1 while it is greater than 1. They preferably contain at least about 10 mg cocoa polyphenol per gram of product, more preferably about 12 mg, most preferably about 14 mg, whereas the ratio of epicatechin to catechin in the product is greater than 0.66. 1 They preferably contain at least about 12 mg cocoa polyphenol per gram of product, more preferably about 14 mg, most preferably about 16 mg, whereas the ratio of epicatechin to catechin in the product is greater than 0.54. 1 More preferably, they contain at least about 13 mg cocoa polyphenol per gram of product, more preferably about 15 mg, most preferably about 17 mg, and the ratio of epicatechin to catechin in the product is greater than 0.42. Is 1.

  The high CP cocoa material is a heat treated, partially or fully defatted high CP cocoa powder comprising (±) -catechin and (±) -epicatechin and their procyanidin oligomers, wherein the defatted cocoa powder The total CP content per gram is at least 25 mg, preferably about 12 to about 25 mg of cocoa polyphenol.

  If the product is a food product with a high water content, such as a beverage, they contain at least about 0.2 mg of total cocoa polyphenol per gram of product, preferably 0.2-0.4 mg. It is more preferable that 4-0.8 mg is contained, and it is most preferable that 0.8-1.2 mg is contained.

  Epicatechin content relative to catechin in foods with high moisture content varies depending on the cocoa polyphenol content of the product. Typically, a product containing about 0.2-0.4 mg has a ratio of 1 to greater than 1, and a product containing about 0.4-0.8 mg has a ratio of 0.42. 1 for a product containing about 0.8 to 1.2 mg and about 1 for about 0.54 and about 0 for products containing about 1 to 1.2 mg or more. Has a ratio of about 1 to .66.

  The material includes a dry or liquid heat treated high CP cocoa having a total CP content per gram of dry cocoa extract of at least about 200 mg, preferably about 250 to about 500 mg, most preferably about 350 to about 500 mg. Extracts are also included. The extract has improved properties compared to the cocoa extract that has not been heat treated. Ingredients also include a heat-treated cocoa product, such as a cocoa beverage having modified properties compared to a non-heat-treated cocoa beverage product. The product comprises cocoa powder and / or high CP cocoa materials such as liquid or dry cocoa extract and / or cocoa mass. If the product is a product with a high water content, such as a beverage, the product contains at least about 0.2 mg of total cocoa polyphenol per gram of product. The cocoa polyphenol content is preferably about 0.2 to 5 mg, more preferably about 0.6 to about 2.0 mg, and most preferably about 0.6 to about 2.0 mg.

  In this high CP cacao powder, cacao extract and cacao product, the total amount of epicatechin and catechin is not substantially changed by heat treatment, but the ratio of (+)-catechin to (−)-catechin is From 90:10 to 4:96, the cocoa extract changes from 90:10 to 34:66. Interestingly, the ratio of (−)-epicatechin to (+)-epicatechin does not change or only slightly changes with heat treatment.

  High CP cocoa powder, high CP cocoa extract, and high CP cocoa products with modified properties are preferably partially or fully defatted, prepared from unfermented or under-fermented cocoa beans It is prepared by heating an aqueous dispersion of cocoa powder or dried cocoa. The cocoa material or cocoa product is heated at a pH and temperature sufficient for epimerization of (−)-epicatechin. Typically, the material or product is heated at a pH of about 3.8 to about 8 and a temperature of about 37 ° C. to about 200 ° C. for about 0.5 minutes to several days. They are preferably heated at a pH of about 5 to about 7.5 and a temperature of about 72 ° C. to about 160 ° C. for about 1 minute to about 6 hours. Most preferably, they are heated at a pH of about 6 to about 7.4 and a temperature of about 100 ° C. to about 140 ° C. for about 1 to about 4 hours.

  The material may also include heat-treated cocoa mass. The cocoa mass contains at least about 10 mg, preferably about 20 to about 50 mg, more preferably about 13 to about 17 mg of cocoa polyphenol per gram of defatted cocoa liquor.

Various methods, including oral, sublingual, buccal, nasal, rectal, intravenous, parenteral, topical, etc., of a medicament comprising a compound according to the present invention optionally together with another NO-modulator or cardiac / vascular therapeutic agent Can be administered. Those skilled in the art will be able to determine dosage forms suitable for compounds of formula A _n, and optionally the transport of different cardiac / vascular protective agent maximized. Thus, dosage forms suitable for various administrations are within the scope of the present invention and include tablets, capsules, gelatin capsules (gel caps), powders or granules for mass or unit dosage, emulsions, suspensions, pastes. Including solid, liquid and semi-solid dosage forms such as creams, gels, foams or jellies. Sustained release dosage forms are also within the scope of the present invention. Suitable pharmaceutically acceptable substrates, diluents or excipients are generally known in the art and can be readily determined by one skilled in the art. For example, a tablet can include an effective amount of a polyphenol-containing composition and optionally a substrate such as sorbitol, lactose, cellulose, or dicalcium phosphate.

  Nutritional supplements containing cocoa flavanols and / or procyanidins (and optionally other NO regulators or cardio / vascular therapies) can be prepared using methods known in the art, for example Nutrients such as calcium diphosphate, magnesium stearate, calcium nitrate, vitamins, and minerals may be included.

  Described for purposes of indicating the presence of a composition according to the present invention (eg, a food, a dietary supplement, a pharmaceutical) and a compound according to the present invention and / or treating heart / vascular problems, or as described herein As a prophylactic treatment, articles of manufacture such as packaged products containing labels indicating the use of the composition are also within the scope of the present invention. The label and / or instructions for use may refer to any method of use described herein.

  The present invention manufactures a product comprising any of the compositions described herein, packages the composition into a product, and uses the composition / product for any of the uses described herein. On how to teach or advertise. Such instructions, instructions or promotions include advertisements.

  A product (packaged) adapted for use in combination therapy comprising at least one compound according to the invention and at least one additional chemotherapeutic and / or cardio / vascular agent (ie other than a compound according to the invention) Products or kits) are also within the scope of the present invention, where the chemotherapeutic and / or cardio / vascular agents can be provided in a separate package or mixed with a compound of the present invention.

  The following procedure was used for the preparation and testing of high CP products.

Sample Preparation In Example 1, the beverage was lyophilized and extracted twice with an acetone / water / acetic acid mixture (CH ₂ COCH ₃ : H ₂ 0: HOAc = 79.5: 20: 0.51) It was sonicated at 50 ° C. for 15 minutes and centrifuged at 583.3 / s (35000 rpm) for 6 minutes. The solvent was recovered from the collected supernatant under reduced pressure or vacuum and lyophilized. The resulting material was used for phase HPLC analysis.

  In Examples 3 and 4, the preparation of cocoa beverage samples was modified to provide better analysis of products with high moisture content. Sample preparation consisted of taking 32-35 g of beverage, transferring it quantitatively to a 100 ml volumetric flask, adding 0.5 ml of glacial acetic acid and adding acetone. This approach yields a solution comparable to the extraction solvent used for other cocoa samples consisting of 70: 29.5: 0.5 acetone: water: acetic acid (v / v / v). The beverage sample was not defatted prior to analysis. The water in the beverage was used to make a water fraction of the extraction solvent.

Normal Phase Chromatography—HPLC / MS Analysis—Adamson et al. Method In Example 1, the published normal phase HPLC method of Adamson et al. (J. Agric. Food Chem., 1999, 47 pp. 4184-4186). ) Was used. The conditions were as follows:
a) Column: Phenomenex Lichrosphere silica
Size: 25cm x 4.6mm
Particle size: 5μm
Pore size: 100mm
b) Mobile phase:
A. Dichloromethane B. Methanol C.I. Water: acetic acid (1: 1)
Gradient condition:
Initial: 82% A / 14% B / 4% C
Time = 30 minutes 67.6% A / 28.4% B / 4% C
Time = 50 minutes 53.2% A / 42.8% B / 4% C
Time = 51 minutes 10% A / 86% B / 4% C
Time = 56 minutes 82% A / 14% B / 4% C
Re-equilibration-7 minutes c) Flow rate: 1.0 ml / min d) Column temperature: 37 ° C
e) Injection volume: 5.0 ml
f) Detection: Fluorescence: Excitation wavelength 276 nm: Emission wavelength -316 nm
Normal phase chromatography-diol Method Other embodiments normal phase chromatography was used was generally not used halogen termed diol method (Halogen free) methods. This method is described by MA Kelm et al., “High Performance Liquid Chromatography Separation and Purification of Cacao (Theobroma cacao L.) Procyanidins According to Degree of Polymerization Using a Diol Stationary Phase” (J. Agr. & Food Chem. 2006 Mar 8; 54 (5): 1571-6).

The conditions were as follows:
Analytical normal phase HPLC Name: CPDIOL-3.M
Column: Intersil Diol 250 × 4.6mm
Mobile phase A 98: 2 Acetonitrile: Acetic acid
Mobile phase B 95: 3: 2 Methanol: H ₂ O: acetic acid Flow rate:

The column used was 250 × 4.6 mm, id, 5 μm Develosil diol (Phenomenex (Trans, Calif., USA)). The two-component mobile phase consisted of (A) CH ₃ CN: HOAc (98: 2, v / v) and (B) CH ₃ OH: H ₂ O: HOAc (95: 3: 2). Fractionation was performed at 30 ° C. and a flow rate of 1.0 ml / min with a linear gradient as follows: 0-35 min, 0-40% B; 35-45 min, 40% B Held at 45% to 46 minutes, 40% to 0% B, 0% B for 4 minutes. The eluent was monitored by fluorescence detection at excitation 276 nm and emission 316 nm.

Reversed Phase High Pressure Liquid Chromatography-C18 Method Photodiode Array, Agilent 1100 LC Instrument and Quadrapole MS Integrated with Fluorescence Detectors for Separation and Detection of Monomers and Procyanidins, and Unfermented Cocoa It was used to determine the ratio of epicatechin to catechin in beans, cacao extract, uncooked and cooked cocoa powder, and cocoa beverages. A Hypersil ODS (C18, 100 × 4.6 mm, 5 μm) column was used. The mobile phase consists of A (1% acetic acid / water) and B (0.1% acetic acid / methanol), using a linear gradient, 10-25% B (v / v) for 20 minutes, then B was increased to 100% in 10 minutes and 100% B was maintained for 10 minutes. The flow rate was set at 1.0 ml / min. The column temperature was set to 20 ° C. The peak intensity was recorded with the UV detector set at 280 nm and the UV spectrum was recorded for 200-600 nm. The ionization technique was performed by electrospray (ESI) and all mass spectral data were acquired in the form of negative ions. As a quantitative task, this chromatography and FLD detection was used to establish a calibration curve. The eluent was monitored by fluorescence detection at excitation 276 nm and emission 316 nm.

Preparative high pressure liquid chromatography (HPLC) to diol method The preparative HPLC method was used for physical separation of the monomer and dimer fractions of Example 5. An improved method for the separation and elution of individual flavan-3-ol monomers and / or procyanidin oligomers introduces (A) a liquid containing monomers and / or oligomers into a polar bonded diol stationary phase And (B) separating individual monomers and / or oligomers based on the degree of polymerization, passing the two-component mobile phases A and B through the column, and (C) separating the monomer and dimer fractions. Each step of eluting the individual fractions to be wiped.

The stationary phase has a particle size of about 3 μm to about 10 μm. A preparative diol column was used with the following slightly modified conditions:
Mobile phase A: 99: 9 Acetonitrile: Acetic acid Mobile phase B: 95: 4: 1 Methanol: Water: Acetic acid Column: Devosil 100DIOL-10
Flow rate: 55 ml / min

Example 1: LCMS study of procyanidin chemistry of cocoa beverages and testing of beverage fractions in the aortic ring assay This example describes the liquid chromatographic mass of two cocoa beverages (prepared according to different methods) and their respective cocoa powders Analysis (LCMS) is described. A variety of samples were evaluated for qualitative differences focusing on monomers, dimers and trimers. The aortic ring assay was used to identify functional fractions / compounds using beverage extracts as starting materials.

  Two beverages were prepared according to the following method.

  High polyphenol cocoa powder prepared according to the method described in US Pat. No. 6,015,913 to Kealey et al. Was used in the preparation of both beverages.

  Representative HPLC properties of cocoa powder and their monomers, dimers and trimers are shown in FIGS.

Cocoa beverage A was prepared as follows: (i) Mixing cocoa powder with water at 80 ° C. for 20 minutes (this process restores the remaining spores in the powder and destroys them during UHT) (Ii) subjecting the mixture to UHT treatment at 140 ° C. for 6-7 seconds; (iii) filling the mixture into a 85 ml container and 10 minutes at a maximum water temperature of 115 ° C. (total treatment time 19 minutes); Retorting was carried out at a maximum pressure of 2.6 × 10 ⁵ Pa (2.6 bar). Rotation was applied to assist heat transfer. Any of the process variations that can achieve the functional and / or structural effects described herein can be utilized.

  Cocoa beverage B was prepared as follows. High polyphenol, powdered milk, sugar, thickener, emulsion, mixed vitamins, vanilla flavor, and potable water (22 ° C) are mixed at ambient temperature, homogenized using a high-pressure pump, and treated with UHT ( 15 seconds at 160 ° C.) and aseptically packed. Typical equipment used to prepare this beverage includes Breddo Likwifier (Division of American Ingredients Co., Inc., Kansas City, Missouri, USA) with connecting piping through a Waukesha positive displacement pump, and Silverson 275/400 mixer (Silverson Machines, Cheshambucks, Waterside, UK). The resulting beverage contained about 170 mg of flavanols and procyanidins.

Both beverage extracts and their individual cocoa powders were prepared as shown in FIG. In summary, the beverage is lyophilized and extracted twice with an acetone / water / acetic acid mixture (CH ₃ OH: H ₂ O: HOAc = 79.5: 20: 0.5) and 15 minutes at 50 ° C. The solution was sonicated, centrifuged at 58.3 / s (3500 rpm) for 6 minutes, the solvent was removed from the collected supernatant under reduced pressure or under vacuum, and lyophilized. The resulting material was used for HPLC analysis.

LCMS results Extracts from each beverage and their individual cocoa powders were subjected to NP and RP-LCMS studies. The NP-HPLC chromatogram of the test sample is shown in FIGS.

  Referring to FIG. 1, the NP-HPLC chromatogram of cocoa beverage A was significantly different from that observed with the cocoa powder from which the beverage was prepared. The cocoa powder contains individual groups of monomers to 10-mers (FIG. 1, upper panel), but the NP-HPLC chromatogram of cocoa beverage A was mostly monomers to trimers. (Figure 1, lower panel).

  Therefore, in cocoa beverage A, the peak areas of epicatechin and catechin were reversed compared to cocoa powder. That is, cocoa beverage A contained more catechin than epicatechin, in contrast to cacao powder containing more epicatechin than catechin. Typically, in cocoa powder, the ratio of (−)-epicatechin to (+)-catechin is about 9: 1. In contrast, the ratio of epicatechin to catechin found in cocoa beverage A is approximately 1: 3, and the chiral separation indicates that the majority of catechin is (−)-catechin and (+) to (−)-catechin. -The ratio of catechins was shown to be about 1:44. (+)-Epicatechin was not observed.

  Referring to FIG. 2, cocoa beverage B also contains more catechin than the cocoa powder prepared therefrom, but the ratio of epicatechin to catechin found in cocoa beverage B was approximately 1: 1.

  Differences in the dimer are also quite obvious. The extracted ion chromatogram (EIC) shown in FIGS. 1 and 2 suggests the identity of these peaks with the dimer (m / z 577). The EIC (FIGS. 3 and 5) of the cocoa powder used in the preparation of cocoa beverages A and B shows that typical dimers are present in cocoa (B2 and B5 dimers). Referring to FIGS. 4 and 6, the dimer B5 peak is almost extinguished in both beverages. Most importantly, however, cocoa beverage A contained many dimers (˜4-5 mer) not found in the starting cocoa material (FIG. 4). Most noteworthy is the large peak of 14.5 minutes whose decomposition pattern (not shown) is consistent with other B-type dimers. Cocoa beverage B also contained an additional dimer peak, albeit small.

Results of aortic ring assay The cocoa beverage A extract was subjected to the fraction-derived bioassay shown in Scheme 1. Fractions 96A-96E were tested as described below. Five fractions (9A-9E) were obtained from bioactive fraction 96E using preparative RP HPLC and awaited assay. Fraction 9A, 9B, 9C, 9D, and 9E are respectively obtained without procyanidin / B-type trimer / A-type trimer, B-type trimer (main component) / B-type tetramer (trace component), If it is composed of B-type dimer (main component) / B-type trimer (minor component), B-type dimer / B-type trimer, and B-type trimer, it is provisionally determined by LCMS. Characterized.

Aortic ring cultures were obtained as described in Karim M., McCormick K., Kappagoda CT., "Effects of Cocoa Procyanidins on Endothelium Dependent Relaxation", Journal of Nutrition 2000; Mount the aortic ring in 10 ml oxygen-rich Krebs' buffer, keep it constant, first pre-contract the ring with norepinephrine (10 ^-6 M), then endothelial cell activity ( endothelium viability) was tested with acetylcholine (10 ⁻⁶ M). The wheel was then washed and returned to the standard tension. Upon returning to baseline, the rings were contracted with norepinephrine (10 ⁻⁶ M). When contraction reached steady state, catalase (400 U / ml) was given to the ring, and after a further time for stabilization, cumulative concentrations of test compound (10 mg / ml to 500 mg / ml) were added. Another two rings were also tested as temporal controls (acetylcholine, 10 ⁻⁹ M to 10 ⁻⁴ M) and vehicle controls (ethanol or DMSO). N ^omega - To correct the effect of the nitro -L- arginine methyl ester (L-NAME), prior to addition of catalase and the test compound was added NOS inhibitors ⁽¹⁰ -5 M). In addition, for testing of endothelium-dependent relaxation, the selected ring endothelium was exfoliated prior to adding the test compound.

  Aortic ring relaxation was measured as a decrease in tension (g) and expressed as a percentage of the contractile response to norepinephrine. All drug concentrations are expressed as the final concentration in the tissue bath buffer. Group data are expressed as ± SEM of n experiments.

Acetylcholine achieved a maximum relaxation of 10 ⁻⁶ M. DMSO produced a relaxation reaction at high concentrations (800-1000 μl with 50% or 100% DMSO). These are the concentrations from administration of test compounds at 400 μg / ml and 500 μg / ml. All of the test compounds produced a relaxation response at 500 μg / ml (n = 1), which was similar or inferior to the corresponding vehicle control, and as a result, the relaxation obtained with this dose of test compound produced a response. It was considered not. At concentrations ranging from 10 μg / ml to 300 μg / ml, no significant relaxation was observed in either 50% or 100% DMSO (n = 2) vehicle controls. In the remaining experiments (n = 2), test compounds were prepared with 50% ethanol. No relaxation response was observed when the container was given an equal volume of 50% ethanol as a vehicle control.

  The following results include one experiment with a test compound prepared in 50% DMSO and two experiments with 50% ethanol. Only MK-1312-96E produced a marked maximal relaxation response (33.6% ± 3.0, n = 3; p <0.05) at a final bath concentration of 200 μg / ml. , Analysis of variance with Bonferroni correction (ANOVA)). There was also no significant difference between the maximum relaxation response of MK-1312-96E and the maximum relaxation response of acetylcholine (student t-test). Although MK-1312-96D appeared to produce a relaxation response (20.3%, ± 8.7, n = 3) at a laboratory concentration of 200 μg / ml, this effect is not statistically significant. There wasn't. MK-1312-96A (compound dissolved in 100% DMSO), MK-1312-96B, and MK-1312-96C did not react. In vessels where the endothelium was detached or given L-NAME prior to the addition of the test compound, the relaxation response of these compounds was all ineffective or significantly inhibited.

  In conclusion, MK-1312-96E is capable of producing an endothelium-dependent relaxation response in this in vitro acute endothelium function model mediated by nitric oxide.

  The bioassay and fractionation methods described above are repeated until the pure active compound or compounds are separated. NMR and MS are used to achieve structural elucidation of bioactive compounds.

Example 2: LCMS inspection of procyanidin chemistry on partially defatted high CP cocoa powder 500 CP of deionized water in a 1 L round bottom flask equipped with a water cooled condenser. Suspended in (pH 5.3). A mantle heater was used as the heat source and the mixture was refluxed. Samples were placed for 30 minutes, 7.75 hours, 24 hours. A normal phase HPLC / FLD trace of the uncooked high CP cocoa powder is shown in FIG. Separation was performed by the diol method. FIG. 11 shows a normal phase HPLC trace of cooked high CP cocoa powder (Adamson et al. Method). 12A-D show the traces before cooking and after cooking for 30 minutes, 7.75 hours, 24 hours.

  The total CP content of the high CP cocoa powder before treatment was ˜57 mg / g or ˜6%. The CP content was measured using an internal standard chromatographic measurement system. The measured polyphenol is a monomer to a 10mer. Once cooked, the total CP content was reduced to 30 mg / g. The monomer content determined from this data is 13.79 mg / g (1.4 mass% monomer) of the monomer present in the high CP cocoa powder that has not been cooked, The amount of the mass did not change even after cooking, indicating that the amount was 15.8 mg / g (1.6% by mass).

An evaluation method using reverse phase RP-HPLC (C18) was also performed. A calibration curve was established as a standard. The amount of uncooked high CP cocoa powder monomer was properly matched to the amount determined under normal phase conditions. As a control, the monomer fraction was separated from the heat-treated high CP cocoa powder using a preparative diol column. Reversed phase analysis of the purified fraction isolated from the heat treated high CP cocoa powder showed a clean trace of epicatechin and catechin. Table 1 shows the catechin and epicatechin contents in the uncooked and cooked high CP cocoa powder.

Example 3: Investigation of the ratio of epicatechin to catechin The ratio of epicatechin to catechin was measured using the C18 HPLC technique. The various cocoa products tested included unfermented cocoa beans, two high CP cocoa extracts, uncooked and cooked high CP cocoa powder, and cocoa beverage A. Cocoa extract A was prepared by extracting unfermented cocoa beans with aqueous ethanol (25% water / 75% ethanol, v / v). Cocoa extract B was prepared by extracting unfermented cocoa beans with aqueous acetone (20% water / 80% ethanol, v / v). The ratio is shown in Table 2.

For example, in non-heat treated products such as cocoa extract, the epicatechin content is greater than the catechin content, which is consistent with unfermented cocoa beans. For high CP cocoa powder, the ratio of epicatechin to catechin is 79:21. In highly processed samples such as cocoa beverage A, the ratio of epicatechin to catechin reaches ~ 35: 65. Cocoa beverage A received several high temperature treatments during preparation (see Example 1). This ratio of epicatechin to catechin of ~ 35: 65 is the thermodynamic equilibrium of these two diastereomers during the epimerization reaction (catechin is by nature the more stable form). Thus, the degree of processing provides some insight into the degree of conversion of epicatechin to catechin. The ratio of epicatechin to catechin when the degree of processing is small or unprocessed is ˜95: 5. As processing further, especially high temperature processing, the ratio shifts to ˜80: 20, like high CP cocoa powder that has not been cooked. If the degree of processing is high, the ratio reaches an equilibrium point.

Example 4: Investigation of chiral content Chiral chromatography was performed to determine the ratio of stereoisomers. The ratio of (+/−)-catechin was obtained under one set of chromatographic conditions and the ratio of (+/−)-epicatechin was obtained under another set of chromatographic conditions.

Epicatechin and catechin found in various cocoa samples were further analyzed for stereochemical structure. The chiral content is shown in Table 3.

  Catechin is a minor component in cocoa beans, and the natural ratio of (+)-catechin to (−)-catechin is 90:10. For catechins, the dominant type in beans is (+)-catechin. For the two cocoa extracts, the ratio differs from the cocoa bean data, with (+/−)-catechin changing to a ratio of about 40:60. That is, the (−)-stereoisomer increases. Perhaps because the conversion is stereospecific, the source of (−)-catechin is the conversion of (−)-epicatechin to (−)-catechin. Further processing promotes conversion until the dominant mold is (−)-catechin. In highly processed cocoa samples such as cocoa beverage A, the content is increased by processing until (−)-catechin becomes the dominant form. This is consistent with the expected conversion reaction because (−)-catechin is produced by conversion of (−)-epicatechin under the heat treatment conditions for preparing cocoa beverage A. (−)-Epicatechin is the only isomer observed in trace amounts of workpieces. A very small amount of the stereoisomer, (+)-epicatechin, is observed in highly processed cocoa samples. This is consistent with the fact that (+)-epicatechin is expected to be generated from (+)-catechin, and (+)-epicatechin is a more unstable stereoisomer. Chiral chromatography was performed to determine the ratio of stereoisomers. The ratio of (+/−)-catechin was obtained under one series of chromatographic conditions and the ratio of (+/−)-epicatechin was obtained under another series of chromatographic conditions.

  The results are shown for all four stereoisomers present in varying amounts in the processed material, ie, cooked high CP cocoa powder and cocoa beverage A.

Example 5: Separation of monomer and dimer A total of 1.02 g of high CP cocoa powder was added to 15 ml of 75:25 (acetonitrile: acetic acid = 99: 1) :( methanol: water: acetic acid = 95: 4: 1) Dissolved in the solution, centrifuged at 58.3 / s (3500 rpm) for 5 minutes, and filtered through a 0.45 μm filter. After several HPLC operations, the heat-treated cocoa powder was purified (operation 1: injection 2.0 ml, operation 2: injection 5.0 ml, operation 3: injection 5.0 ml, operation 4: injection 4.0 ml) . All monomer peaks eluting at ˜12.4 minutes and all dimer peaks eluting at ˜27.5 minutes were combined, concentrated in vacuo and lyophilized. Monomer and dimer yields were 6 mg and 4 mg, respectively. The sample was dissolved in a mobile phase suitable for subsequent analytical separation. FIG. 13 shows a trace of the monomer fraction separated from the cooked high CP cocoa powder.

Example 6: Comparison of uncooked high CP cocoa powder, cooked high CP cocoa powder, and synthetic (−)-epicatechin- (4β, 8)-(−)-catechin dimer less than 1 mg Synthesis of (−)-epicatechin- (4β, 8)-(−)-catechin dimer was dissolved in a mixture containing 10% ethanol and 0.1% acetic acid in deionized water. The dimer is a natural B2 dimer, ie, an epimer dimer of (−)-epicatechin- (4β, 8)-(−)-epicatechin.

  A total of 0.3 g of cooked high CP cocoa powder was dissolved in a mixture of 10% ethanol and 0.1% acetic acid in 2 ml deionized water, sonicated for 5 minutes, 75.0 / The mixture was centrifuged at s (4500 rpm) for 5 minutes and filtered through a 45 μm filter. A 200 μl sample was then used for LC / MS analysis.

  A total of 0.3 g cooked high CP cocoa powder was dissolved in a mixture containing 10% ethanol and 0.1% acetic acid in 2 ml deionized water and sonicated for 5 minutes, 75.0 / s Centrifuged at 4500 rpm for 5 minutes and filtered through a 45 μm filter. Next, a 200 μl sample was taken and less than 1 mg of synthetic (−)-epicatechin- (4β, 8)-(−)-catechin was added.

  A total of 0.33 g of uncooked high CP cocoa powder was dissolved in a mixture containing 10% ethanol and 0.1% acetic acid in 2 ml of deionized water and sonicated for 5 minutes, 75.0 / The mixture was centrifuged at s (4500 rpm) for 5 minutes and filtered through a 045 μm filter. 200 μl of sample was used for LC / MS analysis.

Basic standard data (MSD) settings are: ionization mode: API-ES, mode: scan, polarity: negative, mass width: 100-1500, fragmentor: 70, threshold: 150, step interval: 0.20 Capillary voltage: 3000, dry gas: 10 L / min, nebulizer pressure 3.45 × 10 ⁵ Pa (50 psig), dry gas temperature: 345 ° C.

  HPLC analysis showed a significant change in the dimer region. See Figures 14A-14D. The retention time of dimer B2 under these conditions was 12.7 minutes, 24.5 minutes for dimer B5 and 6.8 minutes for the dimer of epimerized B2. 14A-D show synthetic epimerized B2 dimer (A), unheated high CP cocoa powder (B), heat treated high CP cocoa powder (C), and enhanced heat treatment. 2 shows a reverse phase separation trace of a high CP cocoa powder (D) produced. In the heat-treated high CP cocoa powder, dimer B5 almost disappears, dimer B2 still exists, and a new dimer exists at a retention time of 6.8 minutes. The enhancement with the synthetic epimerized B2 dimer showed an increase in the region of the same peak (see 14D).

  The dimer fraction was separated from the cooked high CP cocoa powder using a preparative diol column. As expected, RP-HPLC / FLD and MS (single ion monitor mode, m / z = 577) traces of this purified fraction that showed monomer were not shown here. However, the new dimer was clearly shown. The new dimer has a retention time that is inconsistent with B2, B5 and epimerized B2 under these chromatographic conditions. Consideration of possible dimer epimers of B2 and B5 includes 6 additional isomers, and possible dimer epimers of B2 and B5 where new dimers are usually present in cocoa There is.

  Although the invention has been described with respect to certain specific embodiments, it will be appreciated that many modifications and changes may be made by those skilled in the art without departing from the invention. Accordingly, it is intended in the appended claims to cover all those modifications and variations that would fall within the true spirit and scope of the present invention.

FIG. 1 represents NP-HPLC-MS data for cocoa beverage A and the cocoa powder used to prepare the beverage. FIG. 2 represents NP-HPLC-MS data for cocoa beverage B and the cocoa powder used to prepare the beverage. FIG. 3 represents NP-HPLC-MS data and monomeric, dimeric, and trimer EICs of cocoa powder used to prepare cocoa beverage A. FIG. 4 represents RP-HPLC-MS data and monomeric, dimeric, and trimer EICs for cocoa beverage A. FIG. 5 presents RP-HPLC-MS data and monomeric, dimeric, and trimer EICs of cocoa powder used to prepare cocoa beverage B. FIG. 6 represents the RP-HPLC-MS data and monomeric, dimeric and trimer EICs for cocoa beverage B. FIG. 7 represents a bioassay scheme with fractions of cocoa beverage A. FIG. 8 represents dose-dependent relaxation mediated by test compounds in pre-contracted aortic rings. FIG. 9 represents the effect of the fraction of cocoa beverage A on the pre-contracted aortic ring. FIG. 10 represents NP-HPLC / FLD data for high CP cocoa powder that has not been cooked. FIG. 11 represents NP-HPLC / FLD data for cooked high CP cocoa powder. Figures 12A-D represent NP-HPLC / FLD data of cooked high CP cocoa powder for 30 minutes, 7.75 hours, 24 hours. FIG. 13 represents the HPLC data of the monomer fraction fractionated from the cooked high CP cocoa powder. 14A-D show synthetically epimerized B22 mer (A), uncooked high CP cocoa powder (B), uncooked high CP cocoa powder (C), and enhanced cooked RP-HPLC data for high CP cocoa powder (D).

Claims (20)

  A heat treated, partially or fully defatted cocoa powder having a total cocoa polyphenol content of at least about 25 mg / g defatted cocoa powder, wherein the cocoa polyphenol is (±) -catechin, (±) -epicatechin And cocoa powders characterized by the nature of modified monomers comprising procyanidin oligomers, and having a ratio of epicatechin to catechin of greater than about 1 to about 1.   The cocoa powder according to claim 1, wherein the total cocoa polyphenol content is from about 20 to about 50 mg / g and the ratio is greater than about 1 to about 0.42.   Heat-treated cocoa mass having a total cocoa polyphenol content of at least about 10 mg / g based on defatted cocoa solids, wherein the cocoa polyphenol is (±) -catechin, (±) -epicatechin, and their procyanidins A cocoa mass characterized by the properties of a modified monomer comprising oligomers and having a ratio of epicatechin to catechin of greater than about 1 to about 1.   4. The cocoa mass of claim 3, wherein the total cocoa polyphenol content is about 12 to about 25, and the ratio is about 1 while greater than about 0.42.   A heat-treated cocoa extract, wherein the total cocoa polyphenol content in the extract is at least about 200 mg per gram of dry extract, and the cocoa polyphenol is (±) -catechin, (±) -epicatechin , And their procyanidin oligomers, characterized by the properties of modified monomers that are about 1 versus a ratio of epicatechin to catechin of greater than about 1.   6. The extract of claim 5, wherein the total cocoa polyphenol content is about 250 to about 500 mg and the ratio is about 1 to about 0.42. A heat-treated cocoa product with a low water content,
A cocoa component selected from the group consisting of high CP cocoa powder, liquid or dry high CP cocoa extract, high CP cocoa mass, and mixtures thereof;
The product has a total cocoa polyphenol content of at least about 6.0 mg / g of product, the cocoa polyphenol comprising (±) -catechin, (±) -epicatechin, and their procyanidin oligomers; A cocoa product characterized by the nature of a modified monomer having a catechin to catechin ratio of greater than 1.   8. The cocoa product of claim 7, wherein the product has a total cocoa polyphenol content of up to about 49 mg. A heat-treated cocoa product with a high water content,
A cocoa component selected from the group consisting of high CP cocoa powder, liquid or dry high CP cocoa extract, high CP cocoa mass, and mixtures thereof;
The product has a total cocoa polyphenol content of at least about 0.2 mg / g of product, the cocoa polyphenol comprising (±) -catechin, (±) -epicatechin, and their procyanidin oligomers; Characterized by the nature of the modified monomer, wherein the ratio of catechin to catechin is 1 versus 1
Cocoa products.   The product of claim 9, wherein the product has a total cocoa polyphenol content of up to about 5 mg.   The cocoa powder according to claim 1, characterized in that the total amount of epicatechin and catechin is not substantially different from the amount initially present in the cocoa powder, cocoa extract, cocoa mass or cocoa product. The cocoa mass according to claim 3, the cocoa extract according to claim 5, or the cocoa product according to claim 7 or 9.   4. The cocoa powder according to claim 1, characterized in that there are four additional dimers with different elution times from the known dimers B2 and B5 under the same chromatogram conditions. A cocoa mass, the cocoa extract according to claim 5, or the cocoa product according to claim 7 or 9.   Cacao powder prepared from unfermented or under-fermented cocoa beans is prepared by heating at about 37 ° C. to about 190 ° C. for a time and pH sufficient to epimerize (−)-epicatechin The cocoa powder according to claim 1.   Prepared by heating cacao mass prepared from unfermented or poorly fermented cacao beans at about 37 ° C. to about 190 ° C. for a time and pH sufficient to epimerize (−)-epicatechin The cocoa mass according to claim 3.   Dissolve the dried cocoa extract in water or an aqueous organic solvent and heat the solution at about 37 minutes to about 190 ° C. for about 0.5 minutes to several days at a pH of about 4.0 to about 8.0. The cocoa extract according to claim 5, wherein the cocoa extract is prepared. A cocoa beverage consisting essentially of water and cacao powder,
A cocoa beverage in which the beverage contains more catechin than epicatechin as opposed to cacao powder to prepare the beverage, and the ratio of epicatechin to catechin in the beverage is approximately 1: 3.   The cocoa beverage according to claim 16, wherein a majority of the catechin in the beverage is (-)-catechin. Diseases of the vasculature by administering to humans or veterinary animals a cocoa beverage comprising (-)-catechin and a dimer having a different elution time from dimers B2 and B5 under the same chromatogram conditions Or a method of treating or preventing a disorder,
The vascular disease or disorder is atherosclerosis, thrombosis, hypertension (eg, primary, secondary and pulmonary hypertension), cardiovascular disease (CVD), coronary artery disease (CAD) Blood, myocardial infarction, stable and unstable angina, including acute obstruction or restenosis), diabetes (type I and type II) (eg, vascular complications of diabetes), cognitive impairment or cognitive impairment and / or Cardiovascular disorders (including those related to the brain), heart failure, cerebrovascular disorders (cerebral infarction, early and / or recurrent transient ischemic attacks, or coronary angioplasty or percutaneous coronary artery Including ischemic complications such as post-vention), congestive heart failure, renal failure, kidney disease, peripheral vascular disorders, and more specifically peripheral vasoconstriction such as Raynaud's disease (eg, arm or leg blood vessels) Related diseases and disorders, peripheral arterial injury (PAD), intermittent claudication, vasculitis (eg, microvascular), vasospasm, venous thrombosis, venous dysfunction, severe ischemic limb, acute ischemic limb, atheroma A therapeutic or prophylactic method characterized by being selected from the group consisting of embolism and arterial insufficiency of lower limbs.   The method of claim 18, wherein the beverage is a cocoa beverage.   The beverage is a cocoa beverage and the vascular disease or disorder is atherosclerosis, thrombosis, hypertension (e.g., primary, secondary and pulmonary hypertension), cardiovascular disease (CVD), Coronary artery disease (CAD) (including myocardial ischemia, myocardial infarction, stable and unstable angina, acute occlusion or restenosis), diabetes (type I and type II) (eg, vascular complications of diabetes), cognition Dysfunction or cognitive impairment and / or cardiovascular disorders (including those about the brain), heart failure, cerebrovascular disorders (cerebral infarction, early and / or recurrent transient ischemic attack, or eg coronary artery Including ischemic complications such as after angioplasty or percutaneous coronary intervention), congestive heart failure, renal failure, kidney disease, peripheral vascular disorders, and more specifically peripheral vasoconstriction such as Raynaud's disease ( Diseases and disorders related to the blood vessels of the arms or legs, etc., peripheral arterial dysfunction (PAD), intermittent claudication, vasculitis (eg microvascular), vasospasm, venous thrombosis, venous dysfunction, severe illness 20. The method of claim 19, wherein the method is selected from the group consisting of a blood limb, an acute ischemic limb, an atheroembolism, and a lower limb arterial insufficiency.

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