WO2007002851A2 - Thermally-processed cocoa products useful for vascular health improvement - Google Patents

Abstract

The invention relates to compositions containing polyphenols such as flavanols, procyanidins, and derivatives thereof obtainable by thermally treating the polyphenols in an aqueous solution, and to compositions comprising epimers of flavanols, procyanidins, and derivatives thereof, and methods of therapeutic or prophylactic treatment of NO-responsive diseases or disorders using the same. The invention also relates to thermally-processed cocoa powders and cocoa extracts characterized by an altered monomelic and oligomeric profile where the total amount of epicatechin and catechin is unchanged, where new dimers are present, and where oligomers higher than the trimers are absent.

Description

THERMALLY-PROCESSED COCOA PRODUCTS USEFUL FOR VASCULAR HEALTH IMPROVEMENT

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This is application is a PCT application which claims priority to provisional application Serial No. 60/695,361 filed June 29, 2005 for "Formulations for Vascular Health Improvement". FIELD OF THE INVENTION

[0002] The invention relates to compositions containing polyphenols such as flavan-3-ols, procyanidins, and derivatives thereof which are obtainable by thermally treating the polyphenol-containing products, compositions comprising epimers of flavan-3-ols, and procyanidins, and to methods of therapeutic or prophylactic treatment of NO-responsive diseases or disorders using the same. BACKGROUND OF THE INVENTION

[0003] Polyphenols are a diverse group of compounds (Ferriera et al,

Tetrahedron, 48:10, 1743-1803, 1992). They widely occur in a variety of plants, some of which enter into the food chain. In some cases they represent an important class of compounds for the human diet. For example, flavanols and procyanidins have been shown to have an effect on nitric oxide (NO) (e.g. U.S. Pat. No. 6,670,390). NO is known to inhibit platelet aggregation, monocyte adhesion and chemotaxis, and proliferation of vascular smooth muscle tissue which are critically involved in the process of atherogenesis. When circulating concentrations of NO are reduced, either because production is blocked by an inhibitor, or in pathological states, such as atherosclerosis, the vascular muscles do not relax to the appropriate degree. The resulting vasoconstriction increases blood pressure and may be responsible for some forms of hypertension. Hypertension is a leading cause of vascular diseases, including stroke, heart attack, heart failure, and kidney failure.

[0004] Given the large number of people suffering from NO-responsive diseases or disorders, there is considerable interest in finding various therapeutic ways to prevent and treat these conditions. Applicants have discovered that certain compounds described herein can be utilized for such applications. BRIEF SUMMARY OF THE INVENTION

[0005] The invention relates to thermally treated flavanols (monomers) and procyanidins (oligomers), and/or flavanol and procyanidin epimers, compositions comprising such compounds, and methods of treatment and prophylaxis of NO-responsive diseases or disorders. [0006] In one aspect, the invention relates to a composition, such as a pharmaceutical, a food, a food additive, or a dietary supplement comprising the compounds of the invention or derivatives thereof in the amount effective for therapeutic or prophylactic treatment of NO-responsive diseases or disorders. The composition may optionally contain an additional therapeutic or beneficial-to-health agent, or may be administered in combination with another therapeutic or beneficial-to-health agent. Packaged products containing the above-mentioned compositions and a label and/or instructions for use for treatment or prophylaxis of NO-responsive diseases/disorders are also within the scope of the invention.

[0007] In one aspect, the invention provides a method of treating or preventing vascular disease or disorder by administering to a subject in need thereof a beverage comprising (-)-catechin, wherein the subject is a human or a veterinary animal. The (-)-catechin may be present in the beverage as a monomeric compound or as a constituent monomeric unit of an oligomeric compound of formula E_n as defined herein and/or of a compound obtained by thermally treating an oligomeric compound of formula A_n as defined herein. Typically the beverage is a cocoa- or chocolate-containing beverage or a cocoa- or chocolate-flavored beverage.

[0008] In another aspect, the invention relates to methods for preventing or treating an NO-responsive disease or disorder in a mammal, such as a human or a veterinary animal, by administering an effective amount of the compound of the invention.

[0009] In yet another aspect, methods of making the compounds described herein are provided.

[0010] In yet another aspect, the invention relates to a thermally-processed, partially defatted or fully defatted high CP cocoa powder which comprises (±)-catechin and (±)-epicatechin and procyanidin oligomers thereof, which has a total CP content of at least 15 milligrams per gram of the defatted powder, and which has an altered monomeric and oligomeric HPLC profile compared to a high CP cocoa powder that has not been thermally processed.

[0011] It also relates to thermally-processed high CP cocoa extracts, dry or liquid, which have a total CP content of at least 175 milligrams per gram of the dry cocoa extract and also have altered profiles compared to cocoa extracts that have not been thermally-processed. [0012] In yet another aspect, the invention relates to thermally-processed cocoa products such as cocoa beverages having an altered profile compared to cocoa products that have not been thermally processed. The products contain high CP cocoa ingredients such as cocoa powder, liquid or dry cocoa extract, and/ or chocolate liquor. When the product is a low moisture product, the product contains at least about 6 milligrams of total cocoa polyphenols per gram of the product. Preferably, the product has an epicatechin to catechin ratio of 1 to greater than 1. When the product is a high moisture content product such as a beverage, the product contains at least about 0.2 milligrams of total cocoa polyphenols per gram of the product.

[0013] The high CP cocoa powder, high CP cocoa extracts, and high CP cocoa products with the altered profiles are prepared by heating an aqueous dispersion of partially or fully defatted cocoa powder or a dry cocoa extract prepared from unfermented or underfermented cocoa beans. Preferably the cocoa ingredients are heated at about 37° to about 200°C for a time and at a pH sufficient to epimerize the (-)-epicatechin, e.g., from about 0.5 minutes to several days. Preferably, they are heated at about 72°C to about 16O⁰C for about 1 minute to about 6.0 hours. More preferably, they are heated at about 100° to about 140°C for about 1 minute to about 4 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 represents NP-HPLC-MS data for Cocoa Drink A and the cocoa powder used to prepare the drink.

[0015] Figure 2 represents NP-HPLC-MS data for Cocoa Drink B and the cocoa powder used to prepare the drink.

[0016] Figure 3 represents RP-HPLC-MS data for cocoa power used to prepare Cocoa Drink A, and EICs for monomers, dimers and trimers.

[0017] Figure 4 represents RP-HPLC-MS data for Cocoa Drink A, and EICs for monomers, dimers and trimers.

[0018] Figure 5 represents RP-HPLC-MS data for cocoa power used to prepare Cocoa Drink B, and EICs for monomers, dimers and trimers.

[0019] Figure 6 represents RP-HPLC-MS data for Cocoa Drink B, and EICs for monomers, dimers and trimers.

[0020] Figure 7 represents a scheme of a bioassay guided fractionation of

Cocoa Drink A.

[0021] Figure 8 represents a dose-dependent relaxation mediated by test compounds in pre-contracted aortic rings. [0022] Figure 9 represents an effect of Cocoa Drink A fractions on precontracted aortic rings.

[0023] Figure 10 represents NP-HPLC/FLD data for the uncooked high CP cocoa powder.

[0024] Figure 11 represents NP-HPLC/FLD data for the cooked high CP cocoa powder.

[0025] Figures 12 A to D represent NP-HPLC/FLD data for high CP cocoa powder cooked for 30 min, 7.75 hours, and 24 hours.

[0026] Figure 13 represents HPLC data for the monomeric fraction isolated from the cooked high CP cocoa powder.

[0027] Figures 14 A to D represent RP-HPLC data for synthetic epimerized

B2 dimer (A), uncooked high CP cocoa powder (B), cooked high CP cocoa powder (C), and spiked, cooked high CP cocoa powder (D). DETAILED DESCRIPTION OF THE INVENTION

[0028] All patents, patent applications and references cited in this application are hereby incorporated herein by reference. In case of any inconsistency, the present disclosure governs.

[0029] The invention relates to thermally treated flavanols (monomers) and procyanidins (oligomers), and/or flavanol and procyanidin epimers, compositions comprising such compounds, and methods of treatment and prophylaxis of NO-responsive diseases or disorders.

[0030] According to the most recent nomenclature, and as used herein, the term "flavanol" refers to monomers, e.g. catechin and epicatechin. Oligomers of catechin and epicatechin are referred to as procyanidins. However, for purposes of the present disclosure, these terms may be used interchangeably. Moreover, any reference to polyphenol herein should be understood to also apply to flavanols and procyanidin, in combination or individually.

[0031] The present invention relates to a first compound, and a composition comprising an effective amount of the first compound obtained by, or obtainable by, thermally treating a second compound having the following formula A_n, or a pharmaceutically acceptable salt or derivative thereof (e.g. oxidation products, methylated derivatives, glucuronidated derivatives): wherein

n is an integer from 2 to 18;

R and X each have either α or β 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 bonding of monomeric units occurs at C-4, C-6 or C-8; when any C-4, C-6 or C-8 are not bonded to another monomeric unit, each X, Y or Z is a hydrogen or a sugar; the sugar is optionally substituted with a phenolic moiety at any position, for instance, via an ester bond.

[0032] As used herein, the phrases "compound obtained by thermally treating," "compound obtainable by thermally treating," or "compound is thermally treated" means that the compound was/is exposed to sufficient heat for a sufficient period of time in the presence of water to cause alterations in the compound's chemical structure and/or function. The lower the temperature, the longer the exposure required to cause the changes. For example, changes may be maximized by increasing the heating temperature from ambient up to 140°C, by increasing the pH from about 3.8 to up to about 7, and/or by increasing the heating time. In order to reach equilibrium one must increase the temperature and/or the pH. Such temperatures and times may be at least 40°C, more preferably at least 50°C, for at least 10 hours, more preferably at least 24 hours, or more preferably at least 48 hours; or at least 60°C, at least 7O⁰C, at least 8O⁰C, at least 9O⁰C, at least 100⁰C, at least HO⁰C, or at least 12O⁰C, each for at least five, or at least 10, 15 or 20 minutes. For example, the compound may be treated at 12O⁰C for 10 minutes, or 120°C for 20 minutes. Another example of thermal treatment is described in Example 1. Other temperature/time/pH combinations are also effective and the skilled artisan may determine such without undue experimentation using guidance provided herein. Known techniques, such as HPLC/MS analysis may be used to monitor the compound's changes as described, for example, in Example 1. [0033] Monomelic units in the above formula may be bonded via 4→6 and

4— >8 linkages. Oligomers with exclusively (4-»8) linkages are linear; while the presence of at least one (4— >6) bond results in a branched oligomer. Also within the scope of the invention are thermally treated oligomers comprising at least one non-natural linkage (6— >6), (6— »8), and (8— >8). Oligomers having such linkages may be prepared as described in US Pat. No. 6,156,912, hereby incorporated herein by reference.

[0034] The sugar can be selected from the group consisting of glucose, galactose, rhamnose, xylose, and arabinose. The sugar is preferably a monosaccharide or di- saccharide. The phenolic moiety is selected from the group consisting of caffeic, cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.

[0035] Examples of derivatives include esters, oxidation products, methylated derivatives and glucuronidated products. Oxidation products may be prepared as disclosed in U.S. Pat. No. 5,554,645, the relevant portions of which are incorporated herein by reference. Esters, for example esters with gallic acid, may be prepared using known esterification reactions, and for example as described in US Pat. No. 6,420,572, the disclosure of which is hereby incorporated herein by reference. Methylated derivatives, such as 3'O-methyl-, 4'O- methyl-, and 3'O, 4'O-dimethyl- derivatives may be prepared, for example, as described in Cren-Olive et al., 2002, J. Chem. Soc. Perkin Trans. 1, 821-830. Glucuronidated products may be prepared as 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.

[0036] The above disclosure relating to linkages, sugars, phenolic moieties, and derivatives is applicable to all compounds disclosed herein, and for brevity, is not repeated.

[0037] Referring to any compound of formula A_n or E_n described herein, examples of such compounds are those wherein the integer n is 3 to 18; 2 to 12; 3 to 12; 2 to 5; 4 to 12; 5 to 12; 3 to 10; 4 to 10; or 5 to 10. Also within the scope of the invention is the compound A_n or E_n wherein n is 2.

[0038] In one embodiment, the invention relates to a first compound, and the composition comprising an effective amount the first compound, obtained by, or obtainable by, thermally treating a second compound having the formula A_n, or a pharmaceutically acceptable salt or derivative thereof (including oxidation products, methylated derivatives, glucuronidated derivatives):

wherein n is an integer from 2 to 18;

R and X each have either α or β stereochemistry;

R is OH; the substituents of C-4, C-6 and C-8 are X, Z, and Y, respectively, and bonding of monomelic units occurs at C-4, C-6 and C-8; when any C-4, C-6 or C-8 are not bonded to another monomelic unit, X, Y, and Z are hydrogen.

[0039] In certain embodiments, the compound is a thermally treated procyanidin dimer or trimer i.e., n=2 or n=3 in the formula A_n. The monomelic units of such dimer or trimer prior to thermal treatment may be all (-)-epicatechin. Examples of such dimers are (-)-epicatechin-(4β— >8)-(-)-epicatechin and (-)-epicatechin-(4β→6)-(-)- epicatechin.

[0040] The dimer or trimer or other oligomers used for thermal treatment may be isolated and purified or synthetically prepared as is known in the art, or may be treated as a mixture with, for example, flavanols and/or other procyanidins. Such a mixture may be, for example, a plant extract (e.g. cocoa extract), a dimer or other oligomer fraction isolated from a plant source (e.g. cocoa), or a cocoa ingredient such as cocoa powder. Cocoa extract may be prepared, for example, as described in US Pat No. 5,554,645, cocoa powders may be prepared as is known in the art, and cocoa powders having conserved levels of flavanols/procyanidins may be prepared as is described in US Pat. No. 6,015,913. When purified individual dimers, dimer mixtures or synthetic compounds are used for thermal treatment, the degree of their purity may be, for example, at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 92%, or at least about 95%, or at least about 98%, or at least about 99%. The above degrees of purities may be utilized for any compound of the formula A_n, its salts and derivatives described herein.

[0041] Also within the scope of the invention are dimer disclosed in Example

1, see e.g. Figure 4, and compositions comprising such dimers, for example, a beverage such as a cocoa or chocolate beverage. Referring to Figure 4, the Extracted Ion Chromatogram (EIC) for the pseudomolecular ion of 577 m/z represents dimers detected in processed beverage. The elution times for the new dimers are 12.6, 14.5 (largest peak), 16.9, 18.8, and 22.6 minutes. The HPLC method used to effect the above separation was as follows: Stationary phase: Hypersil ODS 100 x 4.6 mm 5μm particle size Mobile phase A: 0.1% HOAc in water Mobile phase B: 0.1% HOAc in MeOH Flow rate: l.O ml/min Gradient Time % B

0 15

20 25

30 75

45 100

[0042] Also within the scope of the invention are thermally treated (+)- catechin and (-)-epicatechin and their derivatives (except gallated derivatives), and compositions comprising such flavanols, for example, a beverage such as a cocoa or chocolate beverage.

[0043] The present invention further relates to a compound, and a composition comprising an effective amount of the compound, having the following formula E_n, or a pharmaceutically acceptable salt or derivative thereof (including oxidation products, methylated derivatives, glucuroni dated derivatives):

n

w erein n is an integer from 2 to 18;

R and X each have either α or β 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 bonding of monomeric units occurs at C-4, C-6 or C-8; when any C-4, C-6 or C-8 are not bonded to another monomeric unit, each X, Y or Z is a hydrogen or a sugar; the sugar is optionally substituted with a phenolic moiety at any position, for instance, via an ester bond; and at least one of the monomeric units has the following formula:

[0044] Monomeric units in the above formula may be bonded via linkages described above for the compounds of formula A_n. Sugars, phenolic moieties, and derivatives are as described above for the compounds of formula A_n.

[0045] In another embodiment, the invention relates to a compound, and the composition comprising an effective amount the compound, having the formula E_n, or a pharmaceutically acceptable salt or derivative thereof (including oxidation products, methylated derivatives, glucuronidated derivatives):

n wherein n is an integer from 2 to 18;

R and X each have either α or β stereochemistry;

R is OH; the substituents of C-4, C-6 and C-8 are X, Z and Y, respectively, and bondi monomelic units occurs at C-4, C-6 and C-8; when any C-4, C-6 or C-8 are not bonded to another monomelic unit, X, Y a hydrogen; and ng of at least one of the monomeric units has the following formula:

[0046] Examples of the compounds of formula E_n are those wherein integer n is 3 to 18; 2 to 12; 3 to 12; 2 to 5; 4 to 12; 5 to 12; 4 to 10; or 5 to 10. In sc embodiments, the compound is a procyanidin dimer or a trimer with at least one moi unit in an epimer form. In certain embodiments, the compound E_n is a dimer or trim the n=2 or n=3. >me

[0047] For example, the present invention relates to a compound, anc nomeric composition comprising an effective amount of the compound, having the following ier i.e., E_n, or a pharmaceutically acceptable salt or derivative thereof (including oxidation p methylated derivatives, glucuronidated derivatives): 1 a

, formula iroducts,

wherein n is 2;

R and X each have either α or β 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 bonding of monomeric units occurs at C-4, C-6 or C-8; when any C-4, C-6 or C-8 are not bonded to another monomeric unit, each X, Y or Z is a hydrogen or a sugar; the sugar is optionally substituted with a phenolic moiety at any position, for instance, via an ester bond; and at least one of the monomeric units is (-)-catechin.

[0048] In another example, the invention relates to a compound, and the composition comprising an effective amount the compound, having the formula E_n, or a pharmaceutically acceptable salt or derivative thereof (including oxidation products, methylated derivatives, glucuronidated derivatives):

wherein n is 2;

R and X each have either α or β stereochemistry;

R is OH; the substituents of C-4, C-6 and C-8 are X, Z and Y, respectively, and bonding of monomeric units occurs at C-4, C-6 and C-8; when any C-4, C-6 or C-8 are not bonded to another monomeric unit, X, Y and Z are hydrogen; and at least one of the monomeric units is (-)-catechin. [0049] As is known in the art, epimers are diastereoisomers that have the opposite configuration at only one of two or more tetrahedral stereogenic centers, for instance at one of two or more asymmetric carbon atoms. With respect to the type of compounds described herein, an epimer has inverted stereochemical configuration at one of the asymmetric carbon centers C-2 and C-3.

[0050] As used herein, the term epimer applies to a compound having an inversion at the C-2 ring carbon atom such that the stereochemical configuration at the C-2 carbon atom is beta. Naturally occurring flavanols and procyanidins typically have alpha stereochemistry at the C-2 carbon atom.

[0051] The invention also encompasses the carbon 2 (C-2) epimers of flavanols (+)-catechin and (-)-epicatechin, i.e., (+)-eρicatechin and (-)-catechin, and their derivatives (e.g. methylated derivatives, glucuronidated derivatives and oxidation products except gallated derivatives) and compositions comprising these compounds.

Methods of Use

[0052] Any compound and/or composition described herein may be used to practice the methods described in the present application.

[0053] As used herein, "vascular disease or disorder" refers to any disease or disorder affecting the vascular system, including the heart and the brain. Examples of such conditions include 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) (e.g. vascular complications of diabetes), cognitive dysfunction or disorder and/or vascular circulation disorders (including those of the brain), heart attack, cerebrovascular disease (including stroke, initial and/or recurrent transient ischemic attack, or ischemic complications e.g. after coronary angioplasty or percutaneous coronary intervention), congestive heart failure, kidney failure, renal disease; diseases and/or disorders associated with vasoconstriction of peripheral blood vessels (e.g. blood vessels located in arms and legs) such as peripheral vascular disease and more specifically Raynaud's disease, peripheral artery disease (PAD), intermittent claudication, vasculitis (e.g. of small blood vessels), vasospasm, venous thrombosis, venous insufficiency, lymphatic insufficiency, critical limb ischemia, acute limb ischemia, atheroembolism, and lower extremity ischemia

[0054] An "NO-resρonsive disease or disorder" refers to a health condition which responds to treatment with NO. Examples of such conditions include, but are not limited to NO-mediated or NO-dependent diseases and disorders, in which the pathology of the disease/disorder is caused by abnormal functioning of the NO pathway. For example, the conditions include hypertension (e.g. primary, secondary and pulmonary hypertension), cardiovascular disease, coronary artery disease, diabetes (type I and type II) (e.g. vascular complications of diabetes), sickle cell anemia, cognitive dysfunction or disorder and/or vascular circulation disorders (including those of the brain), heart attack, stroke, congestive heart failure, kidney failure, and renal disease. Because high blood pressure increases the risk of heart attack, stroke, congestive heart failure, and kidney failure, the compounds described above which cause vasorelaxation can be utilized, alone or in combination with other vascular (including cardiovascular)-protective agents, to prevent these conditions. Particularly suitable subjects include subjects with high blood pressure in combination with diabetes, obesity, adverse lipid profile (e.g. high cholesterol levels) and/or smokers, in which patients the risk of heart attack and stroke increases several times. Generally, any subject having at least one of the cardiovascular disease risk factors (as recognized by the American Heart Association) may be treated as described herein.

[0055] As used herein, "treatment" means improving an existing medical condition, such as cardiovascular disease, for example by slowing down the disease progression, prolonging survival, reducing the risk of death, and/or providing a measurable improvement of disease parameters.

[0056] The term "preventing" means reducing the risks associated with developing a disease, including reducing the onset of the disease.

[0057] As used herein, the terms "vascular-protective or therapeutic agent" refers to an agent other than a compound of the invention which is effective to treat or protect the vascular system. Examples of such agents are anti-platelet therapy agents (e.g. COX inhibitors, such as aspirin); NO-modulating agents, cholesterol reducing agents (e.g. sterol, stanol).

[0058] Thus, the invention relates to (i) a method of treating or preventing a vascular disease or disorder, or (ii) a method of treating or preventing an NO-responsive disease or disorder, or (iii) a method of anti-platelet therapy by administering to a subject in need thereof an effective amount of any compound described herein (e.g. the compound of the formula A_n or E_n or a pharmaceutically acceptable salt or derivative thereof (including oxidation products, methylated derivatives and glucuronidated derivatives).

[0059] For example, the above methods may be practiced by administering any compound of the formula A_n, wherein n is 2. [0060] The present compounds are also suitable for use in combination therapy with other vascular/cardioprotective and/or NO-modulating agents. Examples of such agents will be apparent to persons of skill in the art, and may include B-type procyanidins as well as A-type procyanidins. Such compounds may be administered in admixture with the compounds of the invention or separately.

[0061] A-type procyanidins may be of natural origin or synthetically prepared.

For example, A-type procyanidins may be isolated from peanut skins as described in Lou et al, Phytochemistry, 51: 297-308 (1999), or Karchesy and Hemingway, /. Agric, Food Chem., 34:966-970 (1986), the relevant portions of each being hereby incorporated herein by reference. Other sources of the above compounds are cranberries as described, for example in Foo et al, J. Nat. Prod., 63: 1225-1228, and in Prior et al., J. Agricultural Food Chem., 49(3): 1270-76 (2001), the relevant portions of each being hereby incorporated herein by reference, and Ecdysanthera utϊlis (Lie-Chwen et al, J. Nat. Prod., 65:505-8 (2002)) and Aesculus hippocastanum (U.S. Pat. No. 4,863,956), the relevant portions of each being hereby incorporated herein by reference. A-type compounds may also be obtained from B- type procyanidins via oxidation using l,l-diphenyl-2-pycrylhydrazyl (DPPH) radicals under neutral conditions as described in Kondo et al, Tetrahedron Lett, 41: 485 (2000), the relevant portions of which are hereby incorporated herein by reference.

[0062] Methods of obtaining natural and synthetic B-type procyanidins are well known in the art and are described, for example, in U.S. Pat. Nos. 6,670,390 to Romanczyk et al ; 6,207,842 to Romanczyk et al; 6,420,572 to Romanczyk et al. ; and 6,156,912 to Romanczyk et al

[0063] The methods of the invention may be used in a human or a veterinary animal, such as a dog, a cat, and a horse.

[0064] Thus, the following uses are within the scope of the invention. Use of any compound described herein or a pharmaceutically acceptable salt or derivative thereof in the manufacture of a medicament, food, nutraceutical or dietary supplement for use for (i) treating or preventing a vascular disease or disorder; (ii) treating or preventing an NO- responsive disease; or (iii) as anti-platelet therapy. All specific conditions recited herein are within the scope of such uses.

[0065] The effective amount for use in the above methods may be determined by a person of skill in the art using the guidance provided herein and general knowledge in the art. For example, the effective amount may be such as to achieve a physiologically relevant concentration in the body (e.g. blood) of a mammal. Such a physiologically relevant concentration may be at least about 20 nanomolar (nM), preferably at least about 100 and more preferably at least 500 nM. In one embodiment, at least about one micromole in the blood of the mammal, such as a human, is achieved. The compounds of formula An as defined herein may be administered at from about 50 mg/day to about 1000 mg/day, preferably from about 100-150 mg/day to about 900 mg/day, and most preferably from about 300 mg/day to about 500 mg/day. However, amounts higher than stated above may be used.

[0066] The compounds may be administered acutely, or treatment/preventive administration may be continued as a regimen, i.e., for an effective period of time, e.g., daily, monthly, bimonthly, biannually, annually, or in some other regimen, as determined by the skilled medical practitioner for such time as is necessary. The administration may be continued for at least a period of time required to exhibit therapeutic/prophylactic effects. Preferably, the composition is administered daily, most preferably two or three times a day, for example, morning and evening to maintain the levels of the effective compound in the body of the mammal. To obtain the most beneficial results, the composition may be administered for at least about 30, or at least about 60 days. These regiments may be repeated periodically. Based on the guidance provided herein and general knowledge in the art, a person of skill in the art can select compounds that are suitable for acute and or/chronic administration. For example, compounds that show effect after single dose administration and/or shortly after administration (e.g. two hours post administration) may be used when quick, acute response is required. Compounds that show effect after repeated administration may be used accordingly. Thus, dosage forms adapted for such administration (e.g. acute, chronic) are within the scope of the invention.

[0067] Also within the scope of the invention are assays for determining a minimum therapeutically required dosage amount or an optimal dosage amount for use in the above therapeutic methods. Methods described in the examples, or any other dose response methods known to be predictive of compound effectiveness to treat the disease or disorder recited herein may be used. Dosage forms adapted to deliver at least a minimum therapeutically effective amount, or an optimal amount, are within the scope of the invention.

Compositions and Formulations

[0068] The invention relates to compositions described herein which may be formulated as pharmaceuticals, food, food additives or dietary supplements.

[0069] As used herein, a "pharmaceutical" is a medicinal drug. See Merriam-

Webster's Collegiate Dictionary, 10th Edition, 1993. A pharmaceutical may also be referred to as a medicament. As used herein, a "dietary supplement" is a product (other than tobacco) that is intended to supplement the diet that bears or contains the one or more of the following dietary ingredients: a vitamin, a mineral, an herb or other botanical, an amino acid, a dietary substance for use by man to supplement the diet by increasing the total daily intake, or a concentrate, metabolite, constituent, extract or combination of these ingredients. A "food" is a material containing protein, carbohydrate and/or fat, which is used in the body of an organism to sustain growth, repair and vital processes and to furnish energy. Foods may also contain supplementary substances such as minerals, vitamins and condiments. See Merriam- Webster's Collegiate Dictionary, 10th Edition, 1993. The term food includes a beverage adapted for human or animal consumption. A "food additive" is as defined by the FDA in 21 C.F.R. 170.3(e)(l) and includes direct and indirect additives.

[0070] The compositions may contain a carrier, a diluent, or an excipient.

Depending on the intended use, the carrier, diluent, or excipient may be chosen to be suitable for human or veterinary use, food, additive, supplement or pharmaceutical use. The composition may optionally contain an additional cancer treating agent and/or a cardiovascular therapeutic agent. The compositions can be co-administered or sequentially administered with such additional agent taking into consideration such factors as the age, sex, weight, genetics and condition of the particular subject or patient, and, the route of administration.

[0071] Examples of compositions of the invention for human or veterinary use include edible compositions for oral administration, such solid or liquid formulations, for instance, capsules, tablets, pills and the like, as well as chewable solid or beverage formulations, (e.g., cocoa or chocolate flavored solid or liquid compositions); liquid preparations for orifice, e.g., oral, nasal, anal, vaginal etc., administration such as suspensions, syrups or elixirs (including cocoa or chocolate flavored compositions); and, preparations for parental, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions. However, the active ingredient in the compositions may complex with proteins such that when administered into the bloodstream, clotting may occur due to precipitation of blood proteins; and, the skilled artisan should take this into account. In such compositions the active cocoa extract may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, DMSO, ethanol, or the like.

[0072] The inventive compounds may be prepared by thermally treating compounds derived from different sources, for example procyanidins of natural origin (e.g. genus Theobroma, genus Herrania) or those synthetically prepared. [0073] Methods of preparing the starting staring material (flavanols and procyanidins) for use in the present invention are well known in the art (e.g. US Pat. Nos. 5,554,645; 6,420,572; 6,156,912; 6,476,241; and 6,864,377, the relevant portions of which are hereby incorporated herein by reference. The polyphenols may be of natural origin, for example, from a cocoa bean or another natural source of polyphenols, or from polyphenols prepared synthetically. A person of skill in the art may select natural or synthetic polyphenol based on availability or cost.

[0074] Flavanol and/or procyanidin derivatives may also be useful as a starting material. These include esters of monomer and oligomers such as the gallate esters (e.g. epicatechin gallate and catechin gallate); compounds derivatized with a saccharide moiety such as mono- or di-saccharide moiety (e.g. β-D-glucose), for example at positions X, Y, and/or Z in the above formulas; glycosylated monomers and oligomers, and mixtures thereof; metabolites of the procyanidin monomers and oligomers, such as the sulphated, glucuronidated, and methylated forms except for the enzyme cleavage products of procyanidins generated by colonic microflora metabolism. The derivatives may be from natural sources or prepared synthetically.

[0075] The compounds of the invention may be prepared by thermally treating

(in an aqueous solution) flavanols, procyanidins or their derivatives having alpha stereochemistry at the C-2 atom to cause rotation about the C2 atom resulting in beta stereochemistry at the C-2 atom. This approach is particularly suitable for preparing flavanol epimers, for example, epimers of (-)-epicatechin and (+)-catechin.

[0076] Preparation of flavanol epimers may be conducted according to the following scheme 1 (and as described in 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, the disclosures of which are hereby incorporated herein by reference): Scheme 1. Epimerization of flavan-3-ols at C-2 in aqueous media (Freudenberg et al., 1922; Freunberg and Purmann,1924).

(+)-epicatechin, [2S, 3S] mainly [errt-epicatechin]

(-)-catechin, [2S, 3R]

(-)-epicatechin, [2R, 3R]* mainly [enf-catechin]

*known to occur in cocoa

[0077] The lower the temperature, the longer the exposure required to cause epimerization. For examples, such temperatures and times may be at least 4O⁰C, more preferably at least 5O⁰C, for at least 10 hours, more preferably at least 24 hours, or more preferably at least 48 hours; or at least 60⁰C, at least 7O⁰C, at least 80⁰C, at least 9O⁰C, at least 100⁰C, at least 110°C, or at least 12O⁰C, each for at least five, or at least 10, 15 or 20 minutes. For example, the compound may be treated at 12O⁰C for 10 minutes, or 12O⁰C for 20 minutes. Other temperature/time combinations are also effective and the skilled artisan may determine such without undue experimentation using general knowledge in the art and the guidance provided herein. Known techniques, such as HPLC/MS analysis may be used to monitor the success of the reaction.

[0078] The epimeric compounds may be prepared by thermally treating compounds derived from different sources, for example procyanidins of natural origin (e.g. genus Theobroma, genus Herrania) or those synthetically prepared (obtained as is well known in the art, see e.g. US Pat. Nos. 5,554,645, 6,420,572, 6,156,912, 6,476,241, 6,864,377) or by purchasing flavanols from commercial sources.

[0079] Epimers of procyanidins may be prepared according to the methods described in US Pat Nos. 6,420,572; 6,156,912; 6,476,241; and 6,864,377; and International Appl. Publication WO04/030440 (the disclosures of which are hereby incorporated herein by reference) using flavanol epimers as starting building blocks. [0080] For example, the following building blocks may be used in the synthesis of procyanidin epimers:

[0081] The procyanidin epimers having a 4β-8 linkage (such as those described in Table 1) may then be prepared according to the following scheme (the steps of benzylating, hydroxyethoxylating, coupling, and debenzylating are as described in the US patents recited above). Depending on the starting building blocks, the approach may be taken to prepare various combinations of epimeric procyanidins.

Step i

Benzylate

(-)-Epicatechin Tetra - O-benzyl-(-)-epicatechin

(+)-Epicatechin Tetra-O-benzyl-(+)-epicatechin

Step 2

4-Hydroxyethoxy tetra - O-benzyl-(-)-epicatechin

Tetra-O-benzyl-(+)-epicatechin

4-hydroxyethoxy tetra-O-benzyl-(+)-epicatechin Step 3

8)-

4-Hydroxyethoxy tetra-O-benzyl-(+)-epicatechin

Debenzylate

Tetra-O-benzyl- (+)-epicatechin- (4β, 8)- (+)-Epicatechin- (4β, 8)-(-)-epicatechin tetra-O-benzyl-(-)-epicatechin

[0082] A person of skill in the art will envision other methods of making the compounds of the invention using the knowledge in the art and the guidance herein.

[0083] The foods comprising the compounds described herein, and optionally another cardio/vascular protective agent may be adapted for human or veterinary use, and include pet foods. [0084] The foods may be a low moisture food such as a confectionery, or a high moisture food such as a cocoa beverage. Typically, the foods include up to 25% of a protein, preferably 5-20%, most preferably 1-15%, up to 60% of a fat, preferably 1 to 50%, most preferably 5-45%, and up to 70% of a carbohydrate, preferably 1-60%, most preferably 5-50%. Some foods will not always contain proteins, fats, and carbohydrates. The water activity of the food is about 0.2-0.95, preferably 0.4 to about 0.85.

[0085] The food may be a confectionery such as a standard of identity (SOI) and non-SOI chocolate, such as milk, sweet and semi-sweet chocolate including dark chocolate, low fat chocolate and a candy which may be a chocolate covered candy. Other examples include a baked product (e.g. brownie, baked snack, cookie, biscuit) a condiment, a granola bar, a toffee chew, a meal replacement bar, a spread, a syrup, a powder beverage mix, a cocoa or a chocolate flavored beverage, a pudding, a rice cake, a rice mix, a savory sauce and the like. If desired, the foods may be chocolate or cocoa flavored. Food products may be chocolates and candy bars, such as granola bars, containing nuts, for example, peanuts, walnuts, almonds, and hazelnuts.

[0086] A drink, such as a cocoa drink, comprising (-)-catechin and/or dimers described herein is also within the scope of the invention. For example, the drink can comprise more (-)-catechin than (-)-epicatechin, e.g. the ratio of (-)-epicatechin to (-)- catechin may be at least 1:2 or at least 1:3. Cocoa Ingredients

[0087] Thermally processed cocoa ingredients are used in the high CP food products. When the products are a low moisture content product, they contain at least about 6 milligrams, preferably about 8, and more preferably about 10 milligrams of cocoa polyphenols per gram of the product, and the epicatechin to catechin ratio in the product is 1 to greater than 1. Preferably they contain at least about 10 milligrams, more preferably about 12, and most preferably about 14 milligrams of cocoa polyphenols per gram of the product, and the epicatechin to catechin ratio in the product is 1.0 to greater than 0.66. More preferably they contain at least about 12 milligrams, more preferably about 14, and most preferably about 16 milligrams of cocoa polyphenols per gram of the product, and the epicatechin to catechin ratio in the product is 1.0 to greater than 0.54. Even more preferably, they contain at least about 13 milligrams, more preferably about 15, and most preferably about 17 milligrams of cocoa polyphenols per gram of the product, and the epicatechin to catechin ratio in the product is 1.0 to greater than 0.42. [0088] The high CP cocoa ingredients include a thermally-processed, partially defatted or fully defatted high CP cocoa powders which comprise (±)-catechin and (±)- epicatechin, and procyanidin oligomers thereof, which have a total CP content of at least about 25 milligrams, preferably about 12 to about 25 milligrams of cocoa polyphenols per gram of the defatted cocoa powder.

[0089] When the products are high moisture content foods such as a beverages, they contain at least about 0.2, preferably 0.2 to 0.4, or more preferably 0.4 to 0.8. or most preferably 0.8 to 1.2 milligrams of total cocoa polyphenols per gram of the product.

[0090] As with the low moisture foods, the epicatechin to catechin content of the high moisture foods varies depending upon the cocoa polyphenol content of the product. Typically, products which contain about 0.2 to 0.4 milligrams have a ratio of 1 to greater than 1, the products which contain about 0.4 to about 0.8 milligrams have a ratio of 1 to 0.42, products which contain about 0.8 to about 1.2 milligrams have a ratio of about 1 to about 0.54, and the products which contain about 1 to greater than 1.2 milligrams to about 0.66 have a ratio of about 1 to about 0.66.

[0091] The ingredients also include thermally-processed high CP cocoa extracts, dry or liquid, which have a total CP content of at least about 200 milligrams, preferably about 250 to about 500, most preferably about 350 to about 500, per gram of the dry cocoa extract. The extracts also have altered profiles compared to cocoa extracts that have not been thermally-processed. The ingredients also include thermally-processed cocoa products such as cocoa beverages having an altered profiled compared to cocoa products that have not been thermally processed. The products contain high CP cocoa ingredients such as cocoa powder and/or liquid or dry cocoa extract and/or chocolate liquor. When the products are a high moisture product such as a beverage, the products contain at least about 0.2 milligrams of total cocoa polyphenols per gram of the product. Preferably, the cocoa polyphenol content is 0.2 to 5 milligrams, more preferably about 0.6 to about 2.0 milligrams, most preferably about 0.6 to about 2.0 milligrams.

[0092] In this high CP cocoa powder, cocoa extract, and cocoa products, the total amount of epicatechin and catechin is substantially unchanged by the thermal processing but the ratios of (+)-catechin to (-)-catechin is changed from 90:10 to 4:96 for the cocoa solids and from 90:10 to 34:66 for the cocoa extract. Interestingly, the ratio of (-)-epicatechin to (+)-epicatechin is unchanged or only slightly changed by the thermal processing.

[0093] The high CP cocoa powder, high CP cocoa extracts, and high CP cocoa products with the altered profiles are prepared by heating an aqueous dispersion of partially or fully defatted cocoa powder or dry cocoa, preferably prepared from unfermented or underfermented cocoa beans. The cocoa ingredients or cocoa products are heated for a time and at a pH and temperature sufficient to epimerize the (-)-epicatechin. Typically, the ingredients or products are heated at about pH 3.8 to about 8 and at about 37° to about 200°C for about 0.5 minutes to several days. Preferably, they are heated at about pH 5 to about pH 7.5 at about 72° to about 16O⁰C for about 1 minute to about 6 hours. Most preferably they are heated at about pH 6 to about pH 7.4 and at about 100° to about 140°C for about 1 to about 4 hours.

[0094] The ingredients also include thermally-processed chocolate liquor.

The chocolate liquor contains at least about 10 milligrams of cocoa polyphenols per gram of the defatted cocoa liquor, preferably about 20 to about 50 milligrams, more preferably about 13 to about 17 milligrams.

[0095] Pharmaceuticals containing the inventive compounds, optionally in combination with another NO-modulating agent, or cardio/vascular therapeutic agent, may be administered in a variety of ways such as orally, sublingually, bucally, nasally, rectally, intravenously, parenterally and topically. A person of skill in the art will be able to determine a suitable mode of administration to maximize the delivery of the compound of formula A_n, and optionally another cardio/vascular protective agent. Thus, dosage forms adapted for each type of administration are within the scope of the invention and include solid, liquid and semi-solid dosage forms, such as tablets, capsules, gelatin capsules (gelcaps), bulk or unit dose powders or granules, emulsions, suspensions, pastes, creams, gels, foams or jellies. Sustained-release dosage forms are also within the scope of the invention. Suitable pharmaceutically acceptable carriers, diluents, or excipients are generally known in the art and can be determined readily by a person skilled in the art. The tablet, for example, may comprise an effective amount of the polyphenol-containing composition and optionally a carrier, such as sorbitol, lactose, cellulose, or dicalcium phosphate.

[0096] The dietary supplement containing cocoa flavanol and/or procyanidin, and (optionally another NO-modulating agent, or cadio/vascular therapeutic), may be prepared using methods known in the art and may comprise, for example, nutrient such as dicalcium phosphate, magnesium stearate, calcium nitrate, vitamins, and minerals.

[0097] Further within the scope of the invention is an article of manufacture such as a packaged product comprising the composition of the invention (e.g. a food, a dietary supplement, a pharmaceutical) and a label indicating the presence of the inventive compounds and/or directing use of the composition to treat cardio/vascular problems, or as preventive therapies described herein. The label and/or instructions for use may refer to any of the methods of use described in this application.

[0098] The invention also relates to methods of manufacturing the article of manufacture comprising any of the compositions described herein, packaging the composition to obtain an article of manufacture and instructing, directing or promoting the use of the composition/article of manufacture for any of the uses described herein. Such instructing, directing or promoting includes advertising.

[0099] Also within the scope of the invention is an article of manufacture

(such as a packaged product or kit) adapted for use in combination therapy comprising at least one compound of the invention and least one additional chemotherapeutic and/or cardio/vascular agent (i.e., other than the compound of the invention), which chemotherapeutic and/or cardio/vascular agent may be provided as a separate composition, in a separate container, or in admixture with the compound of the invention.

[0100] The following procedures were used for the preparation and testing of the high CP products. Preparation of Samples

[0101] In Example 1, the drinks were freeze dried and extracted twice with an acetone/water/acetic acid mixture (CH₂COCH₃ :H₂0:HO Ac, 79.5:20:0.51), sonicated for 15 min at 50°C, centrifuged for 6 min at 35000 rpm. The solvents were recovered from the collected supernatants under reduced pressure or under vacuum and freeze dried. The resulting material was used for the phase HPLC analysis.

[0102] In Examples 3 and 4, sample preparation for the cocoa drinks was modified to better accommodate the analysis of high moisture products. Sample preparation consisted of taking 32-35 grams of the drink and quantitatively transferring it to a 100 mL volumetric flask, adding 0.5 mL of glacial acetic acid, and adding acetone. This approach yields a solution that is comparable to the extraction solvent used on the other cocoa samples which consisted of 70:29.5:0.5-acetone:water:acetic acid (v/v/v). Drink samples were not defatted prior to analysis. The water of the drink was utilized to make up the aqueous portion of the extraction solvent. Normal Phase Chromatography-HPLC/MS Analysis - Adamson et al. method

[0103] For Example 1 , the published normal phase HPLC method of

Adamson et al. (J. Agric. Food Chem., 1999, 47 pp. 4184-4186) was used. Conditions were as follows: a) Column:

Phenomenex Lichrosphere Silica Size: 25 cm x 4.6 mm Particle size: 5 micron Pore Size: 100 Angstrom b) Mobile Phase:

A. Methylene Chloride

B. Methanol

C. Water: Acetic Acid (1:1)

Gradient Conditions:

Initial: 82% A / 14% B / 4% C

Time = 30 mins. 67.6% A / 28.4% B / 4% C Time = 50 mins. 53.2% A / 42.8% B / 4% C Time = 51 mins. 10% A / 86% B / 4% C Time = 56 mins. 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 microliters f) Detection:Fluorescence: Excitation Wavelength 276 nm:Emission Wavelength - 316 nm

Normal Phase Chromatography - Diol Method

[0104] For the other examples, the normal phase chromatography employed was a halogen free method generally referred to as the DIOL method. The method is disclosed in "High Performance Liquid Chromatography Separation and Purification of Cacao (Theobroma cacao L.) Procyanidins According to Degree of Polymerization Using a Diol Stationary Phase" by M.A. KeIm, et al., (J. Agr. & Food Chem. 2006 Mar 8;54(5):1571- 6).

The Conditions were as follows: Analytical Normal-Phase HPLC method Name: CPDIOL-3.M Column: Intersil Diol 250 x 4.6mm Mobile Phase A 98:2 acetonitrile: acetic acid Mobile Phase B 95:3:2 methanol:H2O: acetic acid Flow Rate:

Gradient Time (min) %B 0 0

35 40

45 40

46 100 50 100

The column used was a 250 x 4.6-mm, i.d., 5 μm Develosil diol (Phenomenex, Torrance, CA). The binary mobile phase consisted of (A) CH₃CN:HOAc, (98:2, v/v) and (B) CH₃OH:H₂O:HOAc (95:3:2). Separations were effected by a linear gradient at 30°C with a 1.0 mL/min flow rate as follows: 0-35 min, 0-40% B; 35-45 min, 40% B isocratic; 45-46 min, 40-0%B, 4 min hold at 0%B. Eluent was monitored by fluorescence detection with excitation at 276 nm and emission at 316 nm.

Reversed Phase High Pressure Liquid Chromatography - Cl 8 Method

[0105] An Agilent 1100 LC instrument coupled with photodiode array, fluorescence detector, and quadrapole MS was used for the separation and detection of the monomers and procyanidins, as well as the determination of epicatechin to catechin ratios in the unfermented cocoa beans, cocoa extracts, uncooked and cooked cocoa powder, and the cocoa drink. A Hypersil ODS (C 18, 100 x 4.6 mm, 5 μm) column was employed. The mobile phase consisted of A (1% acetic acid in water) and B (0.1% acetic acid in methanol) using linear gradients of 10-25% B (v/v) for 20 min followed by an increase to 100% B for 10 min and up to 100% B for 10 min. The flow rate was set to 1.0 mL/min. The column over temperature was set at 2O⁰C. The UV detector was set at 280 nm to record peak intensity, and UV spectra were recorded from 200-600 nm. The ionization technique was electrospray (ESI) and the mass spectrum data was all acquired in negative ion mode. For the quantitative work, the calibration curves were established using this chromatography and FLD detection. Eluent was monitored by fluorescence detection with excitation at 276 nm and emission at 316 nm. Preparative High Pressure Liquid Chromatography (HPLC) - Diol Method

[0106] A preparative HPLC method was used for the physical isolation of the monomelic and dimeric fractions of Example 5. The improved process for separating and eluting individual flavan-3-ol monomer(s) and/or procyanidin oligomer(s) comprises the steps of (A) introducing a liquid sample containing the monomer(s) and/or oligomer(s) into a liquid chromatography column packed with a polar bonded diol stationary phase; (B) separating the individual monomer(s) and/or oligomer(s), based on the degree of polymerization, passing binary mobile phases A and B through the column; and (C) eluting individual fractions containing the monomer and dimer fractions. The stationary phase has a particle size from about 3 μm to about 10 μm. A preparative diol column with the following slightly modified conditions were used: Mobile phase A:99:l acetonitrile: acetic acid Mobile phase B 95:4:1 methanol: water: acetic acid Column: Devosil lOODIOL-10 Flow Rate: 55 mL/min

Gradient composition:

Time (min) % B

0 0

35 30

40 80

41 0

50 0

EXAMPLES

Example 1: LCMS investigation of procyanidin chemistry of cocoa drinks and testing of drink fractions in aortic ring assay

[0107] The example describes Liquid Chromatography Mass Spectrometry

(LCMS) analyses of two cocoa beverages (prepared according to different processes) and their respective cocoa powders. Qualitative differences with particular attention paid to the monomers, dimers, and trimers were assessed in the various samples. Aortic ring assay was used to identify functional fractions/compounds using drink extracts as a starting material.

[0108] Two drinks were prepared according to the following processes.

[0109] High polyphenol cocoa powder prepared according to a method described in U.S. Pat. No. 6,015,913 to Kealey et al, was used for the preparation of both drinks.

[0110] Representative HPLC profiles of the cocoa powders, and their monomer, dimer and trimer fractions, are shown in Figures 1, 2, 3, and 5. [0111] Cocoa Drink A was prepared as follows: (i) cocoa powder was mixed with water at 80°C for 20 minutes (this step revives any remaining spores in the powder and allows for their destruction during UHT); (ii) the mixture was subjected to UHT treatment at the temperature of 14O⁰C for the period of 6-7 seconds; (iii) the mixture was packaged into an 85ml container and subjected to retort at the maximum water temperature of 115°C for 10 minutes (total treatment of 19 minutes), and maximum pressure of 2.6 bars. Rotation was applied to help heat transfer. Any variation of the process that can accomplish the functional and/or structural effects described herein can be used.

[0112] Cocoa Drink B was prepared as follows. High polyphenol cocoa powder, milk powder, sugar, thickeners, emulsifiers, vitamin mix, vanilla flavor and bottled water (at 22⁰C) were blended and mixed at ambient temperature, homogenized using high pressure pump, UHT treated (16O⁰C for 15 seconds) and aseptically packaged. Exemplary equipment for use in the preparation of this drink includes a Breddo Likwifier (Division of American Ingredients Co., Kansas City, Missouri) with connecting piping through a Waukesha positive displacement pump and the Silverson 275/400 mixer (Silverson Machines Ltd., Waterside, Chesham Bucks, England). The resulting drink contained about 170 mg of flavanols and procyanidins.

[0113] Extracts of both drinks and their respective cocoa powders were prepared as shown in Figure 7. In summary, the drinks were freeze dried and extracted twice with acetone/water/acetic acid mixture (CH₃COCH₃:H₂O:HOAc, 79.5:20:0.5), sonicated for 15 min at 50⁰C, centrifuged for 6 min at 3500 rpm, and the solvents were removed from the collected supernatants under reduced pressure or under vacuum, and freeze drying. The resulting material was used for HPLC analysis.

LCMS Results

[0114] Extracts from each of the beverages and their respective cocoa powders were subjected to both NP and RP LCMS studies. NP-HPLC chromatograms of tested samples are represented in Figures 1 and 2.

[0115] Referring to Figure 1, the NP-HPLC chromatogram of Cocoa Drink A differed significantly from that observed for the cocoa powder from which the drink was prepared. While the cocoa powder contained discreet groupings of monomers through decamers (Figure 1, top panel), the NP-HPLC chromatogram of Cocoa Drink A contained mostly monomers through trimers (Figure 1, bottom panel).

[0116] Thus, in Cocoa Drink A, the peak areas for the epicatechin and catechin were inverted in comparison to its cocoa powder, i.e., Cocoa Drink A contained more catechin than epicatechin in contrast to the cocoa powder which contained 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 observed in Cocoa Drink A was approximately 1:3, and a chiral separation indicated that most of catechin was in the form of (-) catechin, a ratio of (+) catechin to (-) catechin was on the order of 1:44. (+) epicatechin was not observed.

[0117] Referring to Figure 2, Cocoa Drink B also contained more catechin than the cocoa powder from which it was prepared, however, the ratio of epicatechin to catechin observed in Cocoa Drink B was approximately 1:1.

[0118] Differences in dimers are also quite apparent. Extracted ion chromatograms (EIC) shown in Figures 1 and 2 support the identity of these peaks as dimers (m/z 577). The EICs (Figures 3 and 5) of cocoa powders used in preparation of Cocoa Drinks A and B show typical dimers present in cocoa (B2 and B5 dimers). Referring to Figure 4 and 6, the peak for dimer B5 has all but disappeared in both beverages. However, most importantly, Cocoa Drink A contained a number of dimers (-4-5) not found in the starting cocoa material (Figure 4). Most noteworthy is a large peak at 14.5 min whose fragmentation pattern (not presented) is consistent with other B-type dimers. Cocoa Drink B also contained additional dimer peaks albeit smaller.

Aortic Ring Assay Results

[0119] Cocoa Drink A extracts were subjected to bioassay guided fractionation as diagramed in Scheme 1. Fractions 96A-96E were tested as described below. Five fractions (9A-9E) were obtained from the bioactive fraction 96E using preparative RP HPLC and await assay. Fractions 9A, 9B, 9C, 9D, and 9E have been tentatively characterized by LCMS as being composed of non-procyanidin/B-type trimer/A-type dimer, B-type trimer (major)/B-type tetramer (trace), B-type dimer (major)/B-type trimer (trace), B- type dimer/B-type trimer, and B-type trimer, respectively.

[0120] Aortic rung cultures were obtained as previously described in Karim

M., McCormick K., Kappagoda CT., "Effects of Cocoa Procyanidins on Endothelium Dependent Relaxation", Journal of Nutrition 2000; 130:2105S-2108S. After the rings were mounted in 10 mL of oxygenated Krebs' buffer, and allowed to stabilize, the rings were first precontracted with norepinephrine (10^"6 M) then endothelium viability was tested using acetylcholine (10^"6 M). The rings were then washed and allowed to return to baseline tension. Once at baseline, the rings were contracted again with norepinephrine (10^"6 M). When the contraction reached a steady state, the rings were given catalase (400 U / mL), and after an additional period of stabilization, cumulative concentrations of the test compounds (10 μg/ml to 500 μg/ml) were added. Two separate rings were also tested as a time control (acetylcholine, 10^"9M to 10^'4M) and as a vehicle control (ethanol or DMSO). When appropriate the effect of N^ω-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, was added (10^~5 M) prior to addition of catalase and the test compound. Also, select rings were denuded of endothelium prior to addition of the test compounds, in order to test for endothelium dependent relaxation.

[0121] Relaxation of the aortic rings was measured as a decrease in tension

(g), and is expressed as a percent of the contraction response to norepinephrine. All drug concentrations are expressed as the final concentration in the tissue bath buffer. Group data are expressed as means ± SEM of n experiments.

[0122] Acetylcholine achieved maximal relaxation at 10^"6M. DMSO did produce a relaxation response at higher concentrations (800 - 1000 μl of 50% or 100% DMSO); these are concentrations that are associated with the 400 μg/ml and 500 μg/ml doses of the test compounds. All test compounds produced a relaxation response at 500 μg/ml (n=l) that was to a similar or lesser extent then the corresponding vehicle control, as a result, the relaxation observed with the test compounds at this dose was considered as producing no response. For concentrations ranging from 10 μg/ml to 300 μg/ml, no significant relaxation was observed with either the 50% or 100% DMSO (n = 2) vehicle control. In the remainder of the experiments (n = 2), the test compounds were made in 50% Ethanol. No relaxation response was observed when the vessels were given 50% ethanol equivalents as a vehicle control.

[0123] The following results include one experiment with the test compounds made up in 50% DMSO and two experiments with 50% ethanol: Only MK-1312-96E produced a significant maximal relaxation response (33.6% ± 3.0, n = 3; p<0.05, ANOVA, with Bonferroni correction) at the final bath concentration of 200 μg/ml. There is also no significant difference between the maximal relaxation response of MK-1312-96E and the maximal relaxation response of acetylcholine (student's t-test). MK-1312-96D appeared to produce a relaxation response (20.3 % ± 8.7, n = 3) at the 200 μg/ml bath concentration, but this effect was not statistically significant. MK-1312-96A (compound was dissolved in 100% DMSO), MK-1312-96B, and MK-1312-96C did not respond. All relaxation responses to these compounds were abolished, or significantly inhibited when the vessel was either denuded of endothelium or given L-NAME prior to addition of the test compound. [0124] In conclusion, MK-1312-96E is able to produce endothelium- dependent relaxation responses in this in vitro model of acute endothelium function that is mediated through nitric oxide.

[0125] The above described bioassay and fractionation process are repeated until a pure active compound or compounds are isolated. NMR and MS are used to accomplish structural elucidation of the bioactive compound(s). Example 2: LCMS investigation of procyanidin chemistry in high CP partially defatted cocoa powder

[0126] A high CP cocoa powder (50 g) was suspended in 500 mL of de- ionized water (pH 5.3) in a 1 L round bottom flask equipped with a water cooled condensor. A heat mantel was used as the heat source and the mixture was refluxed. Samples were taken at 30 min, 7.75 hours, and 24 hours. The normal phase HPLC/FLD trace of the original uncooked high CP cocoa powder is shown in Figure 10. Separation was with the diol method. Figure 11 shows the normal phase HPLC trace for the cooked high CP cocoa powder (Method of Adamson, et al.). Figures 12A to D show the traces prior to cooking and after cooking for 30 min, 7.75 hours, and 24 hours.

[0127] The total CP content of the high CP cocoa powder prior to any processing was -57 mg/g or -6%. The CP content was measured using an in-house standard chromatographic measurement system. The polyphenols measured included the monomer through decamer. Once cooked the total CP content was reduced to 30 mg/g. Monomer content determined from this data shows that were 13.79 mg/g of monomers present in the uncooked high CP cocoa powder (1.4% monomer by mass) and that the monomer amount was unchanged after cooldng, with the amount being 15.8 mg/g (1.6% by mass).

[0128] Quantitation using reverse phase RP-HPLC (C 18) was performed as well. Calibration curves were established with authentic standards. The monomer amounts for the uncooked high CP cocoa powder were fairly consistent with the amounts determined under normal-phase conditions. As a control, the monomeric fraction was isolated from the cooked high CP cocoa powder using a preparative diol column. Reverse phase analysis of the purified fraction isolated from the cooked high CP cocoa powder showed a clean trace of epicatechin and catechin. The catechin and epicatechin content in uncooked and cooked high CP cocoa powder is shown in Table 1. Table 1

Catechin Epicatechin Total Monomers Based

(mg/mL) (mg/mL) Monomers on Sample Mass

(mg/mL) (%)

High CP 1.70 x 10^"3 1.01 x 10^'2 1.18 x lO^'2 1.14% cocoa powder 3.13 x 1(T³ 1.21 x 10^"2 1.52 x 10^"2 1.52%

Cooked high CP 9.86 x 10^"3 3.12 X lO^"3 1.3O x IO^"2 1.27% cocoa powder 8.18 x 10^"3 4.8 x 10^"3 1.30 x IQ-² 1.27%

Monomeric fraction 0.323 0.176 0.500 50% isolated from cooked high CP cocoa powder

Example 3: Investigation of ratio of epicatechin to catechin [0129] The ratio of epicatechin to catechin was measured using Cl 8 HPLC methodology. The various cocoa products tested included unfermented cocoa beans, two high CP cocoa extracts, uncooked and cooked high CP cocoa powder, and Cocoa Drink 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% acetone, v/v). The ratios are shown in Table 2.

Table 2

Cocoa Product Epicatechin:Catechin

Based on 100 Unfermented cocoa beans 95 : 5

Cocoa Extract A 96:4

Cocoa Extract B 90:10

Uncooked high CP cocoa powder 79:21

Cooked high CP cocoa powder 33:67

Cocoa drink A 35:65

[0130] In the products that have not been thermally processed, e.g., the cocoa extracts, the epicatechin content is greater than the catechin content, which is consistent with what is observed in unfermented cocoa beans. For the high CP cocoa powder, the epicatechin to catechin ratio is 79:21. For the highly processed samples, such as Cocoa Drink A and the cooked high CP cocoa powder, a ratio of -35:65 epicatechin to catechin is reached. Cocoa Drink A underwent several high temperature steps during its preparation (see Example 1). This -35:65 epicatechin to catechin ratio is the thermodynamic equilibrium of these two diastereomers for the epimerization reaction (catechin is naturally the more stable form). Thus, the degree of processing provides some insight into the degree of conversion of epicatechin to catechin. With minor or no processing, the ratio of epicatechin to catechin is -95:5. With more processing, particularly high temperature processing, the ratio shifts to -80:20 as with the uncooked high CP cocoa powder. With high processing, the ratio reaches the equilibrium point.

Example 4: Investigation of Chiral Content

[0131] In order to determine the ratio of stereoisomers, chiral chromatography was performed. The ratio of (+/-)-catechin was obtained under one set of chromatographic conditions and that of (+/-)-epicatechin were obtained under a different set of chromatographic conditions.

[0132] The epicatechin and catechin observed in the various cocoa samples were further analyzed for stereochemical make up. The chiral content is provided in Table 3.

Table 3

Cocoa product (-)/(+)-Epicatechin (+)/(-)-Catechin

Unfermented cocoa bean 100:0 90:10

Cocoa Extract A 100:0 39:61

Cocoa Extract B 100:0 34:66

Uncooked high CP cocoa powder 100:0 13:87

Cooked high CP cocoa powder 95:5 4:96

Cocoa drink A (freeze-dried) 94:6 3:97

[0133] Catechin is a minor component in the cocoa bean and the naturally occurring ratio of (+)-catechin to (-)-catechin is 90:10. For catechin, the predominant form in the bean is (+)-catechin. In the two cocoa extracts the ratio changes to about 40:60 (+/-)- catechin which differs from the cocoa bean data - there is an increase in the presence of (-)- stereoisomer. Presumably, the source of the (-)-catechin is the conversion of (-)-epicatechin to (-)-catechin since the conversion is stereospecific. Further processing enhances the conversion until the predominant form is (-)-catechin. Processing enhances the (-)-catechin content until it becomes the predominant stereoisomer in the highly processed cocoa samples such as Cocoa Drink A. This is consistent with the expected conversion reaction since (-)- catechin is generated by the conversion of (-)-epicatechin under the heat processing conditions used to prepare Cocoa Drink A. (-)-Epicatechin is the only isomer observed in minorly processed materials. The stereoisomer, (+)-epicatechin is observed in very small amounts in the highly processed cocoa samples. This is consistent with the fact that the (+)- epicatechin is expected to be generated from the (+)-catechin and that (+)-epicatechin is the less stable stereoisomer. In order to determine the ratio of stereoisomers, chiral chromatography was performed. The ratio of (+/-)-catechin was obtained under one set of chromatographic conditions and that of (+/-)-epicatechin were obtained under a different set of chromatographic conditions.

[0134] The results show that all four stereoisomers exist in varying amounts in the processed materials, i.e., the cooked high CP cocoa powder and Cocoa Drink A.

Example 5: Isolation of monomers and dimers

[0135] A total of 1.02 g of the high CP cocoa powder was dissolved in 15 mL of 75:25 (99:1 acetonitrile: acetic acid) : (95:4:1 methanol: water: acetic acid), centrifuged for 5 min at 3500 rpm, and filtered with a 0.45 μm filter. Several HPLC runs were performed to purify the cooked cocoa powder (Run 1: 2.0 mL injection, Run 2: 5.0 mL injection, Run 3: 5.0 mL injection, and Run 4: 4.0 mL injection). All monomelic peaks which eluted at -12.4 minutes and dimeric peaks which eluted at -27.5 minutes were combined, evaporated under reduced pressure, and lypholized. The monomer and dimer yields were 6 mg and 4 mg, respectively. Samples were dissolved in appropriate mobile phases for subsequent analytical separation. Figure 13 shows the trace for the monomelic fraction isolated 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

[0136] Less than 1 mg of synthetic (-)-epicatechin-(4β,8)-(-)-catechin dimer , was dissolved in a mixture containing 10% ethanol and 0.1% acetic acid in de-ionized water. The dimer is an epimer dimer of the naturally occurring B2 dimer, i.e., (-)-epicatechin-(4β,8)- (-)-epicatechin.

[0137] A total of 0.3 g of the cooked high CP cocoa powder was dissolved in

2 mL of a mixture containing 10% ethanol and 0.1% acetic acid in de-ionized water, sonicated for 5 min, centrifuged for 5 min at 4500 rpm, and filtered with a 45 μm filter. A 200 μl sample was then taken for LC/MS analysis.

[0138] A total of 0.3 g of cooked high CP cocoa powder was dissolved in 2 mL of a mixture containing 10% ethanol and 0.1% acetic acid in de-ionized water, sonicated for 5 min, centrifuged for 5 min at 4500 rpm, and filtered with a 45 μm filter. A 200 μl sample was taken and <1 mg of the synthetic (-)-epicatechin-(4β,8)-(-)-catechin was added.

[0139] A total of 0.33 g of uncooked high CP cocoa powder was dissolved in

2 mL of a mixture containing 10% ethanol and 0.1% acetic acid in de-ionized water, sonicated for 5 min, centrifuged for 5 min at 4500 rpm, and filtered with a 45 μm filter. A 200 μl sample was taken for LC/MS analysis.

[0140] MSD Settings were Ion mode: API-ES, Mode: Scan, Polarity:

Negative, Mass Range: 100-1500, Fragmetnor: 70, Threshold: 150, Step size: 0.20, Capillary voltage: 3000, Drying gas 10 1/min, Nebulizer pressure 50 psig, Drying gas temp: 345°C.

[0141] The HPLC analysis showed that the dimeric region underwent significant alterations. See Figures 14A to 14D. The retention time under these conditions for the B2 dimer was 12.7 min, for the B5 dimer it was 24.5 min, and for the epimerized B2 dimer it was 6.8 min. Figures 14 A to D show traces for the reverse phase separation of synthetic epimerized B2 dimer (A), uncooked high CP cocoa powder (B), cooked high CP cocoa powder (C), and spiked, cooked high CP cocoa powder (D). The cooked high CP cocoa powder shows that dimer B5 is nearly gone, that dimer B2 is still present, and that a new dimer with a retention time of -6.8 minutes is present. Spiking the sample with the synthetic epimerized B2 dimer showed an increase in the area of that same peak (see Figure 14D).

[0142] The dimer fraction was isolated from the cooked high CP cocoa powder using a preparative diol column. The RP-HPLC/FLD and MS (single ion monitoring mode m/z = 577) trace of this purified fraction showed the monomers, as expected, were not present. However, new dimers are clearly shown. The new dimers have retention times that are not consistent with B2, B5, and epimerized B2 under these chromatographic conditions. Considering that the possible epimers of the B2 and B5 dimers include a total of six additional isomers, it is possible that the new dimers are epimers of the B2 and B5 dimers normally present in cocoa.

[0143] While 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. It is intended, therefore, for the appended claims to cover all such modifications and changes as may fall within the true spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS

1. A thermally-processed, partially defatted or fully defatted cocoa powder having a total cocoa polyphenol content of at least about 25 milligrams per gram of the defatted cocoa powder, wherein the cocoa polyphenols comprise (±)-catechin, (±)- epicatechin, and procyanidin oligomers thereof, characterized by an altered monomeric profile where the ratio of epicatechin to catechin is about 1 to greater than about 1.

2. The cocoa powder of Claim 1, wherein the total cocoa polyphenol content is about 20 to about 50 milligrams per gram and wherein the ratio is greater than about 1 to about 0.42.

3. A thermally-processed, chocolate liquor having a total cocoa polyphenol content of at least about 10 milligrams per gram, based on defatted cocoa solids, wherein the cocoa polyphenols comprise (±)-catechin, (±)-epicatechin, and procyanidin oligomers thereof, characterized by an altered monomeric profile where the ratio of epicatechin to catechin is about 1 to greater than about 1.

4. The chocolate liquor of Claim 3, wherein the total cocoa polyphenol content is about 12 to about 25 and wherein the ratio is about 1 to greater than about 0.42.

5. A thermally-processed cocoa extract, wherein the total polyphenol content of the extract is at least about 200 milligrams of cocoa polyphenols per gram of the dried extract; wherein the cocoa polyphenols comprise (±)-catechin, (±)-epicatechin, and procyanidin oligomers thereof, characterized by an altered monomeric profile where the ratio of epicatechin to catechin is about 1 to greater than about 1.

6. The extract of Claim 5, wherein the total cocoa polyphenol content is about 250 to about 500 and the ratio is about 1 to about 0.42.

7. A thermally-processed, low moisture content cocoa product containing cocoa ingredients selected from the group consisting of a high CP cocoa powder, a liquid or dry high CP cocoa extract, a high CP chocolate liquor, and mixtures thereof, which product has a total cocoa polyphenol content of at least about 6.0 milligrams per gram of the product, wherein the cocoa polyphenols comprise (±)-catechin, (±)-epicatechin, and procyanidin oligomers thereof, characterized by an altered monomeric profile where the ratios of epicatechin to catechin is 1 to greater than 1.

8. The cocoa product of Claim 7, wherein the product has a total cocoa polyphenol content of up to 49 milligrams.

9. A thermally-processed, high moisture content cocoa product containing cocoa ingredients selected from the group consisting of a high CP cocoa powder, a liquid or dry high CP cocoa extract, a high CP chocolate liquor, and mixtures thereof, which product has a total cocoa polyphenol content of at least about 0.2 milligrams per gram of the product, wherein the cocoa polyphenols comprise (±)-catechin, (±)-epicatechin, and procyanidin oligomers thereof, characterized by an altered monomelic profile where the ratios of epicatechin to catechin is 1 to greater than 1.

10. The product of Claim 9, wherein the product has a total cocoa polyphenol content of up to about 5 milligrams.

11. The cocoa powder of Claim 1, chocolate liquor of Claim 3, cocoa extract of Claim 5, or cocoa product of Claim 7 or 9, wherein the total amount of epicatechin and catechin is substantially unchanged from the amount initially present in the cocoa powder, cocoa extract, chocolate liquor, or cocoa product.

12. The cocoa powder of Claim 1, chocolate liquor of Claim 3, cocoa extract of Claim 5, or cocoa product of Claim 7 or 9, characterized by the presence of four additional dimers which have different elution times than known dimers B2 and B5 under the same chromatographic conditions.

13. The cocoa powder of Claim 1, which is prepared by heating cocoa powder prepared from unfermented or underfermented cocoa beans at about 37° to about 19O⁰C for a time and at a pH sufficient to epimerize the (-)epicatechin.

14. The chocolate liquor of Claim 3, which is prepared by heating chocolate liquor prepared from unfermented or underfermented cocoa beans at about 37° to about 190°C for a time and at a pH sufficient to epimerize the (-)epicatechin.

15. The cocoa extract of Claim 5, which is prepared by dissolving the dry cocoa extract in water or an aqueous organic solvent and heating the solution at about 37° to about 190°C at a pH from about 4.0 to about 8.0 for from about 0.5 minutes to several days.

16. A cocoa drink consisting essentially of water and a cocoa powder, which drink contains more catechin than epicatechin, in contrast to the cocoa powder from which it is prepared, and wherein the ratio of epicatechin to catechin in the drink is approximately 1:3.

17. The cocoa drink of Claim 16, wherein most of the catechin in the drink is (-)- catechin.

18. A method of treating or preventing a vascular disease or disorder by administering to a human or veterinary animal a cocoa beverage comprising (-)-catechin and dimers which have elution times different from dimers B2 and B5 under the same chromatographic conditions and wherein the vascular disease or disorder is selected from the group of 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 H)(e.g., vascular complications of diabetes), cognitive dysfunction or disorder and/or vascular circulation disorders (including those of the brain), heart attack, cerebrovascular disease (including stroke, initial and/or recurrent transient ischemic attack, or ischemic complications, e.g., after coronary angioplasty or percutaneous coronary intervention), congestive heart failure, kidney failure, renal disease, diseases and/or disorders associated with vasoconstriction of peripheral blood vessels (e.g., blood vessels located in arms and legs) such as peripheral vascular disease and more specifically Raynaud's disease, peripheral artery disease (PAD), intermittent claudication, vasculitis (e.g., of small blood vessels), vasospasm, venous thrombosis, venous insufficiency, critical limb ischemia, acute limb ischemia, atheroembolism, and lower extremity ischemia.

19. The method of Claim 18, wherein the beverage is a cocoa beverage.

20. The method of Claim 19, wherein the beverage is a cocoa beverage and wherein the vascular disease or disorder is selected from the group of 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)(e.g., vascular complications of diabetes), cognitive dysfunction or disorder and/or vascular circulation disorders (including those of the brain), heart attack, cerebrovascular disease (including stroke, initial and/or recurrent transient ischemic attack, or ischemic complications, e.g., after coronary angioplasty or percutaneous coronary intervention), congestive heart failure, kidney failure, renal disease, diseases and/or disorders associated with vasoconstriction of peripheral blood vessels (e.g., blood vessels located in arms and legs) such as peripheral vascular disease and more specifically Raynaud's disease, peripheral artery disease (PAD), intermittent claudication, vasculitis (e.g., of small blood vessels), vasospasm, venous thrombosis, venous insufficiency, critical limb ischemia, acute limb ischemia, atheroembolism, and lower extremity ischemia.