WO2019121583A1

WO2019121583A1 - Cocoa extract and uses

Info

Publication number: WO2019121583A1
Application number: PCT/EP2018/085337
Authority: WO
Inventors: Elsa Hélène Marie HEYMAN; Lieselot DECROIX; Romain MEUSSEN; Pascale Elisabeth Renée FANÇA-BERTHON; Leila Denise FALCAO; Antoine Charles BILY; Simona BIRTIC
Original assignee: Naturex Sa; L'universite De Lille 2; Vrije Universiteit Brussel
Priority date: 2017-12-18
Filing date: 2018-12-17
Publication date: 2019-06-27
Also published as: GB201721213D0

Abstract

The present invention relates to cocoa extracts comprising polyphenols such as flavan-3-ols, procyanidins, and derivatives thereof, processes for providing such extracts, compositions comprising such extracts, uses of the extracts and compositions, such as in food-stuffs, pharmaceuticals, nutraceuticals and supplements, such as food supplements and sports supplements.

Description

Cocoa Extract and Uses

The present invention relates to cocoa extracts comprising polyphenols such as flavan-3- ols, procyanidins, and derivatives thereof, processes for providing such extracts, compositions comprising such extracts, uses of the extracts and compositions, such as in food-stuffs, pharmaceuticals, nutraceuticals and supplements, such as food supplements and sports supplements.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The use of cocoa beans has evolved during centuries to what is currently called chocolate (processed cocoa beans with different additional component such as sugars and milk among others). Studies of chocolate components have been carried out by means of liquid chromatography and it has been observed that the main purified monomeric compounds contained in chocolate are the flavanols, catechin and epicatechin (J.F. Hammerstone et al., "Identification of procyanidins in cocoa (Theobroma cocoa) and chocolate using high- performance liquid chromatography/mass spectrometry", J. Agric. Food Chem., 47, 490- 496 (1999)).

Polyphenols occur in a variety of plants and in some cases, they represent an important class of compounds for the human diet. Cocoa flavonols (CF) are a subgroup of polyphenols.

Several sports such as skiing, mountaineering, and sometimes cycling and running involve exercise at altitude. The lower barometric pressure at altitude reduces the partial pressure of inspired oxygen, which results in reductions of 0₂ delivery to the active muscles and the brain (Subudhi A. W., et al., J. Appl. Physiol. 2007, 80918, 177-183) and elicits the formation of reactive oxygen species (ROS)(McGinnis G., et al., Int. J. Sport Nutr. Exerc. Metab., 2014, 24, 684-693). This leads to a faster development of peripheral and central fatigue, resulting in decreased exercise performance (Verges S., et al., AJP Regul. Integr. Comp. Physiol., 2012, 302, R903-916). Thus, enhancing O2 delivery by improving blood flow at altitude could improve tolerance to physical exercise and recovery thereafter.

The present inventors have surprisingly found that cocoa extracts comprising polyphenols are effective at restoring hypoxia-induced decline in prefrontal oxygenation and enhancing muscular oxygenation during hypoxia. This may be useful for treating disorders resulting from hypoxia within body tissue, such as diabetes with microvascular complications, pulmonary pathologies like chronic obstructive pulmonary disease (COPD), asthma, carbon monoxide poisoning, anaemic hypoxia, cyanosis and altitude sickness.

The present inventors have also found that cocoa extracts comprising polyphenols, as used in the present invention, do not affect nitrogen oxide (NO) production. This is surprising considering the effects that the extracts can have on restoring hypoxia-induced decline in prefrontal oxygenation and enhancing muscular oxygenation during hypoxia as NO is known to induce vasodilation and therefore increase blood flow and tissue oxygenation.

Extract

The present invention provides a cocoa extract comprising polyphenols, such as flavan-3- ols, procyanidins, and derivatives thereof, which may be referred to hereinafter as the “extract of the invention”.

In particular, the present invention provides a water soluble, hydro-alcoholic cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract.

For example, a water soluble, hydro-alcoholic cocoa extract, wherein the extract has a solubility in water at room temperature, such as about 20°C of more than 10g/I.

All references herein to the extract of the invention will typically refer to extracts obtained from or obtainable from the seeds of the cocoa plant fruit. The seed may be fermented or unfermented. It is preferred that the seeds are unfermented.

As will be appreciated by the person skilled in the art, as used herein the term“obtainable from” means that the extract may be obtained from a plant or may be isolated from the plant, or may be obtained from an alternative source, for example by chemical synthesis or enzymatic production. Whereas the term“obtained” as used herein, means that the extract is directly derived from the plant source.

For the avoidance of doubt, preferences, options, particular features and the like indicated for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all other preferences, option, particularfeatures and the like as indicated for the same or other aspects, features and parameters of the invention.

Unless otherwise stated herein, the weight percentages listed are based on the total weight of the extract, for example the total weight of the dry extract.

The term“about” as used herein, e.g. when referring to a measurable value (such as an amount of weight of a particular component in the composition or reaction mixture), refers to variations of ±20%, ±10%, ±5%, ±1 %, ±0.5%, or particularly, ±0.1 %, of the specified amount.

The extract of the invention may be extracted from the seeds using water only. This extract may be referred to as the water or aqueous extract.

The extract of the invention may be extracted from the seeds using an alcohol, such as ethanol. This extract may be referred to as the alcohol extract, such as the ethanolic extract.

The extract of the invention may be extracted from the seeds using a mixture of alcohol and water (i.e. a hydroalcoholic solvent), such as a mixture of ethanol and water (i.e. a hydroethanolic solvent). This extract may be referred to as the hydroalcoholic extract, such as the hydroethanolic extract. The ratio of alcohol to water may range from about 15% alcohol (v/v) (e.g. ethanol) to about 100% alcohol (e.g. ethanol), the preferred mixture being from about 15% to about 70% ethanol and from about 85% to about 30% water (v/v), such as from about 70%/60%/50% alcohol (i.e. ethanol) / 30%/40%/50% water (v/v) to about 10%/15%/20% alcohol (i.e. ethanol) / 90%/85%/80% water (v/v).

The extract of the invention may be subjected to more than one extraction using a hydroalcoholic solvent (i.e. a hydroethanolic solvent). In some cases, the extract may be extracted one, two or three times using a hydroalcoholic solvent (i.e. a hydroethanolic solvent). The hydroalcoholic solvent (i.e. a hydroethanolic solvent) used in each extraction may have the same ratio of alcohol and water (i.e. ethanol and water), or may have a different ratio of alcohol and water (i.e. ethanol and water). For example, a solvent in which more than 50% is alcohol (i.e. ethanol) may be used, followed by a solvent in which more than 50% water is used or vice versa, such as a solvent comprising 60%/70%/80% alcohol to 40%/30%/20% water (v/v) solvent (i.e. a hydroethanolic solvent) then extracted using a 10%/15%/20% alcohol to 90%/85%/80% water (v/v) solvent (i.e. a hydroethanolic solvent). For example, the extract of the invention may be extracted using a hydroalcoholic solvent comprising 70% of ethanol / 30% of water then re-extracted using a hydroalcoholic solvent comprising 15% ethanol / 85 % water.

The extract of the invention may be extracted from the seeds using other organic solvents, such as acetone, acetonitrile, cyclohexane, ethyl acetate, heptane, and/or hexane. This extract may be referred to as the organic extract.

The extract of the invention (for example, the aqueous or alcoholic or hydroalcoholic extract) may have a polyphenol content of greater than or equal to 35% (dry weight)/total weight of the extract. For example, a polyphenol content from about 35% to about 100% or from about 40% to about 90% or from about 50% to about 80% or from about 60% to about 70%, such as from about 35% to about 60% or to about 55%.

As previously explained, polyphenols include flavan-3-ols and procyanidins.

As used herein, the term “flavan-3-ol” refers to monomers such as catechin and epicatechin.

As used herein, the term“procyanidin” refers to oligomers of catechin and epicatechin.

Any reference to polyphenol herein should be understood to also apply to flavanols and procyanidin, in combination or individually.

The extract of the invention (for example, the aqueous or alcoholic or hydroalcoholic extract) may comprise total levels of flavanols from about 15% to about 50% by weight, such as from about 20% to about 40% by weight, and total levels of monomers from about 3% to about 25% or 30% by weight, such as from about 5% to about 15% or 25% by weight, with levels of catechin of from about 0.01 % to about 10% by weight, such as from about 0.2% to about 5% by weight of the extract; epicatechin from about 2.5 to about 20% by weight, such as from about 5% to about 10% by weight of the extract; theobromin from about 2% to about 20% by weight, such as from about 4% to about 10% by weight of the extract; and caffein from about 0.05% to about 5% by weight, such as from about 0.5% to about 3% by weight of the extract.

For example, the extract of the invention may comprise from about 80 to about 350 mg/g; procyanidin B1 from about 1 to about 10 mg/g; and procyanidin B2 from about 30 to about 125 mg/g or, the extract of the invention may comprise from about 5 to about 70 mg/g catechin; from about 50 to about 350 mg/g epicatechin; from about 0.1 to about 10 mg/g procyanidin B1 , such as from about 0.5 mg/g to about 5 mg/g; and from about 30 to about 125 mg/g procyanidin B2, such as from about 30 mg/g to about 100 mg/g.

Examples of components found in the extract of the invention are shown below in Table 1.

The extract of the invention may be purified or semi-purified depending on the extraction steps used to obtain the extract.

Where the extract is not purified or semi-purified, the extract may be obtained using steps a to k as described in the method of the invention below. A semi-purified extract may be obtained using steps a to n as described in the method of the invention below.

A purified extract may be obtained using steps a to p as described in the method of the invention below.

The purification or semi-purification of the extract may modify the amount of polyphenols present in the extract. For example, a semi-purified extract may have a polyphenol content greater than 20% (dry weightytotal weight of the extract. For example, a polyphenol content of from about 20% to about 100% or from about 30% to about 90% or from about 40% to about 80% or from about 50% to about 70%, such as from about 20% to about 45% or to about 55%, such as about 35% and levels of catechin from about 10 to about 50 mg/g; epicatechin from about 50 to about 250 mg/g; procyanidin B1 from about 1 to about 8 mg/g; and procyanidin B2 from about 25 to about 90 mg/g.

A purified extract may have a polyphenol content greater than 60%% (dry weightytotal weight of the extract. For example, a polyphenol content of from about 60% to about 100% or from about 70% to about 90%, such as from about 60% to about 90%, such as about 85% and levels of catechin from about 40 to about 100 mg/g; epicatechin from about 200 to about 500 mg/g; procyanidin B1 from about 5 to about 20 mg/g; and procyanidin B2 from about 80 to about 250 mg/g.

The extract of the invention may be subjected to an additional extraction to improve aqueous solubility and preferably provide a water-soluble extract. This water-soluble extract may also be referred to as the extract of the invention.

Typically, the water-soluble extract is provided by extracting the defatted cocoa beans (for example the cocoa beans after steps e, f or g) using a hydro-alcoholic solvent, such as a hydro-ethanolic solvent comprising greater than or equal to 50% alcohol (i.e. ethanol) such as 60%/70%/80% ethanol/40%/30%/20% water) and then subjecting the extract to a second optional extraction using a hydro-alcoholic solvent comprising less than or equal to 50% alcohol (i.e. ethanol), such as 10%/15%/20% ethanol/90%/85%/80% water). This extraction provides a water-soluble extract of the invention.

For example, the defatted cocoa beans (for example the ground cocoa beans, such as ground cake or meal) may be extracted using a hydro-alcoholic solvent, such as a hydro- ethanolic solvent comprising greater than or equal to 50% alcohol (i.e. ethanol) such as 60%/70%/80% ethanol/40%/30%/20% water), concentrated and the concentrate dried, followed by the dried concentrate obtained being extracted using a hydro-alcoholic solvent comprising less than or equal to 50% alcohol (i.e. ethanol), such as 10%/15%/20% ethanol/90%/85%/80% water).

It has been found by the present inventors, that multiple extraction of the cocoa beans after steps e, f or g as described in the method of the invention below using a hydro- alcoholic solvent, such as a hydro-ethanolic solvent comprising greater than or equal to 50% alcohol (i.e. ethanol) such as 60%/70%/80% ethanol/40%/30%/20% water), followed by a second extraction using a hydro-alcoholic solvent comprising less than or equal to 50% alcohol (i.e. ethanol), such as 10%/15%/20% ethanol/90%/85%/80% water) removes a wax family macro-composition which increases the solubility of the extract without affecting the levels of polyphenols present.

Typically, the water-soluble extract of the invention comprises less than 20% Ci₆ and Cie fatty acids, by weight of the extract, such as less than 10% or less than 5% or less than 1 %.

As used herein, the term“water soluble” is intended to mean that at least 80% by weight of the extract will dissolve in water at room temperature, such as about 20°C, such as at least 85% or at least 90% or at least 95%, or that when dissolved in water at room temperature, such as about 20°C, less than 20% by weight of the extract remains in solid form, such as less than 15% or less than 10% or less than 5%, or that the extract of the invention has a solubility in water at room temperature, such as about 20°C of more than 10g/I, such as more than 15g/l or more than 20g/l.

As used herein, the term“room temperature” refer to a temperature from about 15°C to about 25°C, such as about 20°C.

The skilled person will understand that the extract of the invention may typically be provided in solid form but may be provided in liquid form as a solution depending on the type of extraction process used to obtain the extract. By solid form, it is included that the composition may be provided as an amorphous solid, or as a crystalline or part-crystalline solid.

The extract of the invention or the water soluble extract of the invention may comprise less than about 0.0001 % of carotenoid. For example, the extract of the invention or the water soluble extract of the invention may comprise less than about 0.001 mg of carotenoid compounds per gram of extract or the extract of the invention or the water soluble extract of the invention may contain no carotenoid compounds.

Process for Obtaining the Extract The extract of the invention may be obtained or is obtainable by the extract and isolation processes as generally described herein below, or routine modifications thereof.

Typically, the extract of the invention may be obtained or be obtainable by a process comprising:

a. Peeling the fruit (removing from its natural casing)

b. Depulping the seeds

c. Blanching the seeds

d. Drying the seeds

e. Defatting by means of pressing

f. Stabilizing the defatted product

g. Grinding

h. Solid-liquid extraction

i. Solid-liquid separation

j. Concentrating the liquid extract

k. Obtaining the extract obtained by drying the concentrate obtained in (j)

L. Liquid-liquid extraction of the extract concentrated in (j) and separating the aqueous and organic phases

m. Concentrating the aqueous phase obtained in (I)

n. Obtaining the semi-purified extract by drying the concentrate obtained in (m) o. Concentrating the organic phase obtained in (I)

p. Obtaining the purified extract by drying the concentrate obtained in (o).

For example, the extract may be obtained using steps a to k;

a. Peeling the fruit (removing from its natural casing)

b. Depulping the seeds

c. Blanching the seeds

d. Drying the seeds

e. Defatting by means of pressing

f. Stabilizing the defatted product

g. Grinding

h. Solid-liquid extraction

i. Solid-liquid separation

j. Concentrating the liquid extract

k. drying the concentrate obtained in (j).lf a semi-purified extract is required, then steps a to n may be used and if a purified extract is required, then steps a to p may be used. If a water-soluble extract is required, then step h may be modified to include the additional extract steps described above. For example, step h may be modified so that the ground product from g. is extracted using a hydro-alcoholic solvent comprising greater than or equal to 50% alcohol, followed by a second extraction using a hydro-alcoholic solvent comprising less than or equal to 50% alcohol.

Each of the phases (a) to (p) of the process is described in detail below: a) Peeling the fruit or removing it from its natural casing

This phase of the process comprises opening the cocoa pods and extracting the fresh seeds from their original casing. This phase may be carried out manually maintaining the necessary hygienic conditions to prevent the cocoa beans from being contaminated as much as possible. Fresh cocoa seeds are obtained in this step.

The fresh seeds may be cooled for the purpose of preserving the polyphenols present in the fresh cocoa seeds, as well as the partial elimination of the pulp covering them. If the seeds are cooled, this may be carried out by immersing the seeds in water, for example, immersing the seeds in water at a temperature of from about 1 °C to about 25°C. b) Depulping the seeds

The object of this phase of the process is to completely or partially remove the pulp covering the fresh cocoa seeds for the purpose of optimizing the yield of the drying step and preventing the seeds sticking together once they are dry.

The depulping may be carried out using a stainless steel depulping machine, such as a Cl Talsa, model D500, adapted for this type of processing. Alternatively, other depulping methods may be used as determined by those skilled in the art. c) Blanching the seeds

This phase of the process comprises blanching the seeds with water. The internal seed temperature may be from about 85°C to about 100°C. The blanching may be conducted for a time period of from about 3 minutes to about 15 minutes. For example, the blanching may be conducted at a temperature of about 95°C for about 5 minutes. The main objectives of this phase are to carry out a complete or partial inactivation of the activity of the polyphenol oxidase enzyme present in cocoa beans, which aids in preserving the initial polyphenol content present in the seed.

Typically, a continuous blanching system for example is used to carry out this step. When a continuous blanching system is used the beans pass directly from the depulping machine to a cylindrical mesh where they are longitudinally conveyed through the cylinder by a worm screw; the beans are then submersed in boiling water to achieve an internal bean temperature of about 95°C, with a contact time of about 5 minutes. The water is heated by means of a steam jacket and the contact time of the beans is controlled by changing the speed of the worm screw.

Alternatively, a batch blanching system can also be used. This system may comprise of submersing limited amounts of cocoa beans in a stainless steel tank provided with water heating resistances. A cylinder manufactured with a stainless steel mesh allowing the inlet of hot water and preventing the exit of the beans is used to submerse the beans. Once the contact time has been completed, the cylinder with the beans is removed. d) Drying the seeds

The purpose of this phase is to reduce the moisture content of the blanched unfermented seed to minimize the microbial fermentation therein. The moisture of the seeds after drying may be from about 1 % to about 12% expressed in weight/weight. The drying may be carried out at room temperature drying the seeds in the sun or in dryers at a temperature of not more than about 80°C.

The beans may be dried in a step dryer with an air circulation system. For example, in this dryer the moisture is removed by means of a process involving several heating steps which can be carried out in independent chambers. Heating parameters changing the drying speed of each of the steps are applied in each chamber, thus optimizing the bean drying and preventing the closure of the pores of said beans. A cooling step may be included after the last drying step, in which the seed temperature is lowered until reaching about 30°C. The air is heated at the inlet of the system and its temperature is controlled throughout the entire process by means of arranging sensors in each of the chambers. At the outlet of the dryer, the moisture contained in the air is removed by means of a condensation process, such that it can be recirculated. Alternatively, the drying may be carried out in a batch dryer in drying platforms. e) Defatting by means of pressing

The objective of the pressing phase is to partially remove the fat present. This process may be carried out by extruding the cocoa beans.

A screw press, also called expeller or extruder, may be used to carry out this phase of the process. This equipment comprises a continuous mechanical extractor in which the beans are pressed and the cocoa butter is extracted by pressure.

This equipment comprises a hopper in which the cocoa beans are unloaded, subsequently passing to a vat in which they are heated at a temperature of not less than about 35°C; this material is pressed by a helical screw rotating inside a cylinder, such that the fat is ejected and drained. The product obtained in this phase is called cake if it is from shelled beans, or meal if it is from beans with shell. The temperature of this product when coming out of the press must be less than about 80°C.

A partially defatted inactivated unfermented cocoa cake is obtained in this phase of the process. The fat content of this cake may be from about 8% to about 30% expressed in a percentage of dry weight, for example, about 12%. f) Stabilizing the defatted product

The object of this phase of the process is to reduce the temperature of the cake (or meal) obtained in the previous step to a temperature of less than about 35°C so as to facilitate the subsequent grinding step. The stabilization process may be carried out with a cooler, provided internally with paddles stirring the product. This equipment can be provided with a jacket through which cold water is circulated to facilitate the reduction of the temperature in the product. g) Grinding

The object of this phase of the process is to reduce the particle size of the cake or meal for the purpose of improving the contact surface at the time of carrying out the extraction. In this step, a cocoa product is obtained in which at least about 80%, preferably at least about 95%, and still more preferably at least about 99% of the cocoa particles, have a particle size of less than about 0.5 mm.

The grinding may be carried out with a hammer mill provided with a sieve, or with any other grinding system which allows reaching the desired particle size. h) Solid-liquid extraction

The purpose of this phase is to carry out a selective extraction of the polyphenols present in the cocoa cake or meal. The migration of the polyphenols of the cocoa cake or meal to the extraction solvent depends on a set of factors such as the solid-liquid ratio, the particle size of the material to be extracted, the extraction mixture, the extraction temperature and the extraction time.

The solid/liquid ratio may be more than 1/3, for example 1/5.

This extraction is preferably carried out using ground cake or meal because the particle size reduction increases the contact surface and facilitates the extraction of polyphenols.

This step of the process may be carried out using a polar extraction solvent, such as water, alcohol or a mixture of water and alcohol, such as ethanol and water. When used the ratio of alcohol to water may range from 30% alcohol (e.g. ethanol) and 70% water (v/v), to 100% alcohol (e.g. ethanol), the preferred mixture being 70% ethanol and 30% water (v/v).

The extraction temperature may be from about 40°C to about 80°C, for example about 70°C.

The extraction time used may be more than about 30 minutes, for example about 1 and a half hours.

This step may be carried out in stainless steel extractors provided with hot water or steam jackets to maintain the desired extraction temperature. Typically, the mixture is frequently stirred with stainless steel paddles which are actuated by means of motors provided with devices for changing the speed (mechanical or electrical) for the purpose of optimizing the extraction process. i) Solid-liquid separation

The purpose of this phase is to separate the half-depleted solid (solid phase) and the polyphenol-rich polar solvent extract (liquid phase).

Depending on the required concentration of the polyphenol content in the extract, the half- depleted solid can be subjected to an additional extraction step (if a higher concentration of polyphenols is required) or be eliminated as a residue.

Ideally, only two extraction steps with the same raw material and same solvent are carried out because further extractions typically do not significantly increase the concentration of polyphenol significantly.

The mass yield corresponds to the percentage ratio between the amount of solids recovered in the polar solvent extract and the initial amount of cake or meal.

The polar solvent extract obtained in the first extraction step typically comprises a dry residue of from about 5 to about 100 g/L, for example about 25 g/L. The dry residue expresses the solid content in the extract. This value can decrease if a washing step is carried out during the separation. In the event of successive extraction, they can be carried out in the same conditions described above. The extract obtained in the second extraction step can be combined with the extract obtained in the first step.

The separation can be carried out using any suitable equipment known in the art; for example, using a decanter, a vacuum filtration system with the optional addition of filtering earth, or through a plate filter. j) Concentrating the liquid extract

The objective of this phase is to remove some or all of the polar solvent.

The process may be carried out in stainless steel concentrators and may done in one, two or three steps. The concentration temperature may be from about 30°C to about 70°C depending on the vacuum pressure reached in the equipment.

The solvents removed from the extract can be recovered and separated in a rectification tower to be reused in future extraction processes. When an alcohol/water mixture is used as the polar solvent, the alcohol is preferably eliminated completely from the extract and water is eliminated partially, such that the extract is in the aqueous medium.

This concentrate in an aqueous medium can be used to obtain three end products. It can be directly dried to obtain the extract or may be purified with an organic solvent, such as ethyl acetate, to obtain a purified extract or a semi-purified extract. k) Obtaining the extract obtained by drying the concentrate obtained in (j)

The objective of this phase is to reduce the moisture content of the concentrate obtained in (j) until it has powder characteristics. This step may be carried out using a spray-dryer, or a dryer with an alternative vacuum system for example. The drying temperatures may be from about 40°C to about 100°C.

Other equipment can be used for the purpose of reaching the desired particle size in the end product, such as mills.

The extract obtained may have a moisture content of less than about 10% expressed in weight/weight, preferably less than about 5%, for example about 3%.

The extract obtained may have a particle size of less than about 500 microns, preferably less than about 200 microns, for example less than about 50 microns.

The extract obtained (for example, the aqueous or alcoholic or hydroalcoholic extract) may comprise a polyphenol content greater than 35% (dry weight)/total weight of the extract. For example, a polyphenol content from about 35% to about 100% or from about 40% to about 90% or from about 50% to about 80% or from about 60% to about 70%, such as from about 35% to about 60% or to about 55%.

The total polyphenol content was measured according to the Folin-Ciocalteau method.

The extract obtained may comprise flavanols from about 15% to about 50% by weight and total levels of monomers from about 3% to about 25% by weight with levels of catechin of from about 0.1 to about 10% by weight of the extract; epicatechin from about 2.5 to about 20% by weight of the extract; theobromin from about 2% to about 20% by weight of the extract; and caffein from about 0.05% to about 5% by weight of the extract.

For example, the extract of the invention may comprise levels of catechin of from about 5 or about 10 to about 70 mg/g; epicatechin from about 50 or about 80 to about 350 mg/g; procyanidin B1 from about 0.1 or about 1 to about 10 mg/g; and procyanidin B2 from about 30 to about 125 mg/g.

The determination of the flavonol content in each of the extracts was carried out by column chromatography using an Alginet 1 100 chromatograph with C18 column packing and eluting with acidified water and acetonitrile with a UV diode array at a wavelength at 200 nm. The standards solutions were provided by Sigma Aldrich (catechin and epicatechin), and Extrasynthese (Procyanidins B1 and B2).

I) Liquid-liquid extraction of the extract concentrated in (j) when a hydroalcoholic solvent is used and separating the aqueous and organic phases

Purification of the concentrate obtained in (j) by liquid-liquid extraction. The purpose of this phase is to obtain the purified extract and the semi-purified extract from the concentrate.

A liquid-liquid extraction with an organic solvent, such as ethyl acetate, is carried out for the purification. The purification occurs by the migration of one part of the polyphenols of the concentrate obtained in (j) to an organic solvent, such as ethyl acetate.

When the concentrate obtained in (j) is in an aqueous medium, upon adding an organic solvent, such as ethyl acetate, due to the difference in polarity, both of them form two non- miscible phases corresponding to an aqueous phase (semi-purified) and an organic solvent phase (purified).

The purification process may be carried out in a counter-current system at a temperature of preferably from about 20°C to about 70°C; for example at about 50°C.

The phases are then separated by difference in polarity. Once the phases are separated, these are concentrated and dried, two different products being obtained. These are the semi-purified extract obtained from the aqueous fraction and the purified extract obtained from the organic solvent fraction. m) Concentrating the aqueous extract obtained in (I)

The objective of this phase is to remove the water from the semi-purified extract.

The concentration may be carried out in stainless steel concentrators which may be of one, two or three steps. The concentration temperature can change from about 30°C to about 70°C according to the vacuum pressure reached by the system. The extract may have a solid concentration from about 15 % to about 40 % weight/weight; for example about 20% weight/weight. n) Obtaining the semi-purified extract by drying the concentrate obtained in (m)

The objective of the drying phase is to reduce the moisture content until the extract has powder characteristics. This step may be carried out by using a spray-dryer, or a dryer with an alternative vacuum system for example. The drying temperatures may be from about 40°C to about 80°C.

The semi-purified extract obtained may have a particle size of less than about 500 microns, preferably less than about 200 microns, for example less than about 50 microns.

The semi-purified extract obtained may have a moisture content of less than about 10% expressed in weight/weight, preferably less than about 5 %, for example about 3%.

The semi-purified extract obtained may comprise a polyphenol content greater than 20%(dry weightytotal weight of the extract. For example, a polyphenol content from about 20% to about 100% or from about 30% to about 90% or from about 40% to about 80% or from about 50% to about 70%, such as from about 20% to about 45% or to about 55%, such as about 35%.

The semi-purified extract obtained may comprise levels of catechin from about 10 to about 50 mg/g; epicatechin from about 50 to about 250 mg/g; procyanidin B1 from about 0.1 or about 1 to about 8 mg/g; and procyanidin B2 from about 25 to about 90 mg/g. The polyphenol profile analysis is carried out with the same one as described previously in step (k). o) Concentrating the organic phase obtained in (I)

The objective of this phase is to remove the organic solvent, such as ethyl acetate, from the purified extract.

The concentration may be carried out in stainless steel concentrators which may be of one, two or three steps. The concentration temperature may be from about 30°C to about 70°C according to the vacuum pressure reached by the system. The extract may have a solid concentration of from about 15% to about 40% weight/weight; for example about 20% weight/weight. p) Obtaining the purified extract by drying the concentrate obtained in (o)

The objective of the drying phase is to reduce the solvent content until the extract has powder characteristics. This step may be carried out by using a spray-dryer, or a dryer with an alternative vacuum system for example. The drying temperatures may be from about 40°C to about 80°C.

The purified extract obtained may have a particle size of less than about 500 microns, preferably less than about 200 microns, for example less than about 50 microns.

The purified extract obtained may have a moisture content of less than about 10% expressed in weight/weight, preferably less than about 5 %, for example about 3%.

The semi-purified extract obtained may comprise a polyphenol content greater than 60%(dry weightytotal weight of the extract. For example, a polyphenol content from about 60% to about 100% or from about 70% to about 90%, such as from about 60% to about 90%, such as about 85%.

The total polyphenol content was measured according to the Folin-Ciocalteau method. The semi-purified extract obtained may comprise levels of catechin from about 40 to about 100 mg/g; epicatechin from about 200 to about 500 mg/g; procyanidin B1 from about 5 to about 20 mg/g; and procyanidin B2 from about 80 to about 250 mg/g.

The polyphenol profile analysis is carried out with the same one as described previously in step (k).

Optional phases

- Cleaning

The cleaning phase is optional, and its objective is to eliminate the remains of foreign substances accompanying cocoa seeds. This cleaning may be carried out in a Bulher equipment, consisting of a sieving system with a mesh size comprised from about 2 to about 10 mm, and provided with an optional air suction system allowing the separation of foreign substances.

- Shelling

The shelling phase is optional and its objective is to partially remove the shell surrounding the cocoa seeds. This process is carried out mechanically. Dry unfermented cocoa seeds pass through an automated system where they are fragmented, the released shell is removed by a suction process, thus obtaining cocoa nibs (the term nib refers to the shell- free cocoa fragments). Equipment such as those manufactured by Martin Lloveras, Bauermeister, Lehmann and others may be used, reaching yields of 2% by weight of residual shell on the nib.

Cocoa nibs with a residual shell content less than 7%, and preferably more than 2% are obtained in this phase.

As explained above, in a particular aspect of the invention, the defatted cocoa beans may be extracted using a hydro-alcoholic solvent, such as a hydro-ethanolic solvent comprising greater than or equal to 50% alcohol (i.e. ethanol) such as 60%/70%/80% ethanol/40%/30%/20% water) followed by a second optional extraction using a hydro- alcoholic solvent comprising less than or equal to 50% alcohol (i.e. ethanol), such as 10%/15%/20% ethanol/90%/85%/80% water). This extraction may be conducted after step e, f or g, for example instead of the solid/liquid extraction described in step h above. This extraction provides a water-soluble extract of the invention.

For example, the water-soluble extract of the invention may be prepared using a method comprising:

a. Peeling the fruit (removing from its natural casing);

b. Depulping the seeds;

c. Blanching the seeds;

d. Drying the seeds;

e. Defatting by means of pressing;

f. Stabilizing the defatted product;

g. Grinding;

h. Solid-liquid extraction, wherein the ground product from g. is extracted using a hydro- alcoholic solvent comprising greater than or equal to 50% alcohol, followed by a second extraction using a hydro-alcoholic solvent comprising less than or equal to 50% alcohol; i. Solid-liquid separation;

j. Concentrating the liquid extract;

k. drying the concentrate obtained in (j).

In an aspect of the invention, the extraction process described above maybe conducted using a solvent that is not ethyl acetate.

Accordingly, the present invention provides a cocoa extract obtained by the processes described herein.

Compositions and administration

The extract of the invention may be provided in the form of a pharmaceutical composition (which may also be referred to as a pharmaceutical formulation), veterinary composition orfunctional food composition, such as a food, drink, feed or pet food or a food, drink, feed or pet food supplement, comprising the extract of the invention and optionally a pharmaceutically/veterinary acceptable excipient or (functional) food acceptable ingredient, as appropriate.

As used herein, references to pharmaceutically or veterinary acceptable excipients may refer to pharmaceutically or veterinary acceptable adjuvants, diluents and/or carriers as known to those skilled in the art. Food acceptable ingredients include those known in the art (including those also referred to herein as pharmaceutically acceptable excipients) and can be natural or non-natural, i.e. their structure may occur in nature or not. In certain instances, they can originate from natural compounds and be later modified (e.g. maltodextrin).

By“pharmaceutically or veterinary acceptable” we mean that the additional components of the composition are generally safe, non-toxic, and neither biologically nor otherwise undesirable. For example, the additional components are generally sterile and pyrogen free. Such components must be“acceptable” in the sense of being compatible with the extract of the invention and not deleterious to the recipients thereof. Thus, “pharmaceutically acceptable excipients” includes any compound(s) used in forming a part of the formulation that is intended to act merely as an excipient, i.e. not intended to have biological activity itself.

The skilled person will understand that extracts of the invention (e.g. in the form of compositions, such as pharmaceutical or veterinary compositions) may be administered to a patient or subject (e.g. a human or animal patient or subject) by any suitable route, such as by the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal, intraperitoneal, and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route.

Extracts of the invention may be administered orally. In such instances, pharmaceutical or veterinary compositions according to the present invention may be specifically formulated for administration by the oral route.

Pharmaceutical or veterinary compositions for oral administration include solid dosage forms such as hard or soft capsules, tablets, troches, dragees, pills, lozenges, powders and granules. Where appropriate, they can be prepared with coatings such as enteric coatings, or they can be formulated so as to provide controlled release of the active ingredient, such as sustained or prolonged release, according to methods well known in the art.

Liquid dosage forms for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups and elixirs. Compositions (e.g. pharmaceutical or veterinary or food compositions) described herein, such as those intended for oral administration, may be prepared according to methods known to those skilled in the art, such as by bringing the components of the composition into admixture.

The compositions of the invention may contain one or more additional components as food ingredients or pharmaceutical components, such as sweetening agents, flavouring agents, colouring agents and preserving agents. The compositions of the invention may contain the active ingredient(s) in admixture with non-toxic pharmaceutically acceptable excipients (or ingredients) which are suitable for the manufacture of tablets. These excipients (or ingredients) may, for example, be: inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, maltodextrin or alginic acid; binding agents, for example, starch, gelatine or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.

The compositions of the invention, such as pharmaceutical compositions (which may also be referred to as a pharmaceutical formulations), veterinary compositions or functional food compositions, such as a food, drink, feed or pet food or a food, drink, feed or pet food supplement, comprising the extract of the invention and optionally a pharmaceutically/veterinary acceptable excipient or (functional) food acceptable ingredient, may comprise less than about 0.0001 % of carotenoid. For example, the compositions of the invention may comprise less than about 0.001 mg of carotenoid compounds per gram of composition or the compositions of the invention may contain no carotenoid compounds.

The compositions of the invention may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

Suitable pharmaceutical or veterinary carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, maltodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid, arabic gum, modified starch and lower alkyl ethers of cellulose, saccharose, silica. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Moreover, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, maltodextrin, dextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, magnesium hydroxide; stearic acid, arabic gum, modified starch and lower alkyl ethers of cellulose, saccharose, silicon dioxide. Examples of liquid carriers are syrup, vegetables oils, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Moreover, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

The term“carrier” as used herein, may refer to a natural product or a product originating from nature that has been transformed or modified so that it is distinct from the natural product from which it originated.

Depending on the disorder, and the subject, to be treated, as well as the route of administration, extracts of the invention may be administered at varying doses (i.e. therapeutically effective doses, as administered to a patient in need thereof). In this regard, the skilled person will appreciate that the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.

The pharmaceutical or veterinary orfood compositions comprise an extract of the invention in a therapeutically effective amount. As used herein, the term “effective amount” is synonymous with“therapeutically effective amount”,“effective dose”, or“therapeutically effective dose” and when used in the present invention refers to the minimum dose of the extract of the invention necessary to achieve the desired therapeutic effect and includes a dose sufficient to reduce a symptom associated with inflammation. Effectiveness in treating the diseases or conditions described herein can be determined by observing an improvement in an individual based upon one or more clinical symptoms, and/or physiological indicators associated with the condition. An improvement in the diseases or conditions described herein also can be indicated by a reduced need for a concurrent therapy.

The appropriate effective amount of the extract of the invention to be administered to an individual for a particular disease or condition can be determined by a person of ordinary skill in the art by taking into account factors, including, without limitation, the type of disease or condition, the location of the disease or condition, the cause of the disease or condition, the severity of the disease or condition, the degree of relief desired, the duration of relief desired, the particular dosage of extract of the invention that is used, the rate of excretion of the extract of the invention used, the pharmacodynamics of the extract of the invention used, the nature of other compounds that may be included in the composition, the particular formulation, the particular route of administration, the particular characteristics, history and risk factors of the patient, such as, e.g., age, weight, general health and the like, or any combination thereof.

Additionally, where repeated administration of the extract of the invention is used, an effective amount of the extract of the invention will further depend upon factors, including, without limitation, the frequency of administration, the half-life of the extract of the invention, or any combination thereof.

In the use or method of the invention the extract or composition of the invention may be administered to provide cocoa flavonols in an amount of from about 100mg/day to about 4000mg/day, or from about 500mg/day to about 2000mg/day, or about 1765mg/day. If the extract is administered in the form of a pharmaceutical or veterinary or food, feed or pet food supplement or food, feed or pet food composition comprising the extract, the extract would be present in an amount to provide the above dosages of extract. For example, the food composition may comprise from about 100mg to about 4000mg or from about 500mg to about 2000mg, or about 1765mg/day of the extract of the invention and the pharmaceutical composition may comprise 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 10Omg, 250mg, 500mg, 10OOmg, 1500mg or 2000mg/day of the extract of the invention, such that the pharmaceutical composition may be administered one or more times per day in order to provide from about 100mg to about 4000mg or from about 500mg to about 2000mg, or about 1765mg of the extract of the invention. In the use or method of the invention the extract or composition of the invention may be administered to provide from about 20 mg to about 200 mg epicatechin per day, such as from about 50 mg to about 150 mg, or about 100 mg of epicatechin per day.

When included within a composition (e.g. a pharmaceutical or veterinary composition or a food composition), the extract is typically present in an amount from about 1 % by weight to about 100% by weight, for example, from about 10% by weight to about 90% by weight or about 20% by weight to about 80% or from about 30% by weight to about 70% or from about 40% by weight to about 60% by weight.

The compositions of the invention, such as pharmaceutical or veterinary or food compositions may consist of or consist essentially of the extract of the invention and pharmaceutical or veterinary or food composition.

For the avoidance of doubt, in this specification when we use the term“comprising” or “comprises” we mean that the extract or composition being described must contain the listed ingredient(s) but may optionally contain additional ingredients. When we use the term“consisting essentially of” or“consists essentially of” we mean that the extract or composition being described must contain the listed ingredient(s) and may also contain small (for example up to 5% by weight, or up to 1 % or 0.1 % by weight) of other ingredients provided that any additional ingredients do not affect the essential properties of the extract or composition. When we use the term“consisting of” or“consists of” we mean that the extract or composition being described must contain the listed ingredient(s) only.

Therapeutic uses

The extract of the invention may have particular biological effects, which may be useful in the treatment of medical conditions.

Tissues require oxygen for survival. Oxygen passively diffuses in the lung alveoli according to a pressure gradient. Oxygen diffuses from the breathed air, mixed with water vapour, to arterial blood, where its partial pressure is around 100 mmHg (13.3 kPa).

In the blood, oxygen is bound to hemoglobin, a protein in red blood cells. The binding capacity of hemoglobin is influenced by the partial pressure of oxygen in the environment, as described in the oxygen-hemoglobin dissociation curve. In peripheral tissues, oxygen again diffuses down a pressure gradient into cells and their mitochondria, where it is used to produce energy in conjunction with the breakdown of glucose, fats, and some amino acids.

Delivery depends on adequate ventilation, gas exchange, and circulatory distribution. Tissue hypoxia typically occurs within 4 minutes of failure of any of these systems because the oxygen reserves in tissue and lung are relatively small. The physiological and pathological mechanisms that result in tissue hypoxia can be classified into two main groups:

1 ) those causing arterial hypoxaemia, such as low inspired oxygen partial pressure (high altitude), alveolar hypoventilation (sleep apnoea, opiate overdose), ventilation- perfusion mismatch (acute asthma, atelectatic lung zones), and right to left shunts; and

2) those causing failure of the oxygen-haemoglobin transport system without arterial hypoxaemia, such as inadequate tissue perfusion; low haemoglobin concentration; abnormal oxygen dissociation curve (haemoglobinopathies, high carboxyhaemoglobin) and histotoxic poisoning of intracellular enzymes (cyanide poisoning, septicaemia).

More than one mechanism may contribute to tissue hypoxia, and predicting the response to supplemental oxygen requires careful evaluation of these functions.

Hyperventilation due to carotid chemoreceptor stimulation becomes pronounced when the arterial partial pressure of oxygen (PaC ) falls to 5.3 kPa. Peripheral vasodilation with consequent systemic hypotension and eventually coma occurs if the PaOå falls below 4 kPa.

Thus, hypoxia can be associated with a large, unrelated group of disorders which underlay a variety of diseases and disorders.

It has been surprisingly and unexpectedly found by the present inventors that in situations where the partial pressure of inspired oxygen is low (for example, in situations caused by certain diseases, at altitude or during exercise), the extract of the invention may restore this hypoxia-induced decline in oxidation of the prefrontal cerebral cortex (also referred to as prefrontal oxidation).

The extract of the invention may restore the hypoxia-induced decline in prefrontal oxidation and/or may enhance muscular oxygenation during hypoxia. The present invention therefore provides a cocoa extract or a composition comprising a cocoa extract for use in treating or preventing for treating disorders resulting from low oxygen levels (hypoxia) within body tissue, such as diabetes with microvascular complications, pulmonary pathologies like chronic obstructive pulmonary disease (COPD), asthma, carbon monoxide poisoning, anaemic hypoxia, cyanosis and altitude sickness.

The present invention also provides a cocoa extract comprising a polyphenol content greater than 35% (dry weight)/total weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids for use in treating or preventing for treating disorders resulting from hypoxia within body tissue, such as diabetes with microvascular complications, pulmonary pathologies like chronic obstructive pulmonary disease (COPD), asthma, carbon monoxide poisoning, anaemic hypoxia, cyanosis and altitude sickness.

The present invention also provides the use of a cocoa extract or a composition comprising a cocoa extract in the manufacture of a medicament for treating or preventing disorders resulting from low oxygen levels (hypoxia) within body tissue.

The present invention also provides the use of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids in the manufacture of a medicament for treating or preventing disorders resulting from hypoxia within body tissue.

The present invention also provides a method of treating or preventing disorders resulting from low oxygen levels (hypoxia) within body tissue comprising the administration of a therapeutically effect amount of a cocoa extract or a composition comprising a cocoa extract to a subject in need thereof.

The present invention also provides a method of treating or preventing disorders resulting from hypoxia within body tissue comprising the administration of a therapeutically effect amount of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weight)/total weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids to a subject in need thereof.

As used herein, the term“hypoxia” means a condition in which the body or a region of the body is deprived of adequate oxygen supply at the tissue level.

Hypoxia may be the result of a decrease in the amount of oxygen present in the arteries (arterial hypoxaemia) caused by altitude. For example, such altitudes where the partial pressure of inspired oxygen is 80% or less than that of the pressure at sea level, for example 70% or less, or 60% or less or 50% or less.

The hypoxia may be the result of diseases or conditions that result in a decrease in the amount of oxygen present in the arteries (arterial hypoxaemia) reaching the body tissue, such as sleep apnoea, opiate overdose, acute asthma, atelectatic lung zones or exercise.

The hypoxia may also be the result of a combination of both altitude and the conditions previously mentioned.

The cocoa extracts or compositions of the present invention may be used to treat a patient suffering from hypoxia or susceptible to hypoxia due altitude and/or participating in exercise.

The present invention also provides a cocoa extract or composition comprising a cocoa extract for use in enhancing tolerance to and/or capacity to physical exercise in hypoxic conditions.

The present invention also provides a cocoa extract comprising a polyphenol content greater than 35% (dry weight)/total weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weight)/total weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids for use in enhancing tolerance and/or capacity to physical exercise in hypoxic conditions.

The present invention also provides the use of a cocoa extract or a composition comprising a cocoa extract in the manufacture of a medicament for enhancing tolerance to and/or capacity to physical exercise in hypoxic conditions. The present invention also provides the use of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids in the manufacture of a medicament for enhancing tolerance and/or capacity to physical exercise in hypoxic conditions.

The present invention also provides a method of enhancing tolerance to and/or capacity to physical exercise in hypoxic conditions comprising the administration of a therapeutically effect amount of a cocoa extract or a composition comprising a cocoa extract to a subject in need thereof.

The present invention also provides a method of enhancing tolerance and/or capacity to physical exercise in hypoxic conditions comprising the administration of a therapeutically effect amount of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids to a subject in need thereof.

The present invention also provides a cocoa extract or composition comprising a cocoa extract for use in enhancing recovery after exercise in hypoxic conditions.

The present invention also provides a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids for use in enhancing recovery after exercise in hypoxic conditions.

The present invention also provides the use of a cocoa extract or a composition comprising a cocoa extract in the manufacture of a medicament for enhancing recovery after exercise in hypoxic conditions.

The present invention also provides the use of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids in the manufacture of a medicament for enhancing recovery after exercise in hypoxic conditions.

The present invention also provides a method of enhancing recovery after exercise in hypoxic conditions comprising the administration of a therapeutically effect amount of a cocoa extract or a composition comprising a cocoa extract to a subject in need thereof.

The present invention also provide a method of enhancing recovery after exercise in hypoxic conditions comprising the administration of a therapeutically effect amount of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids to a subject in need thereof.

As used herein, the phrase“hypoxic conditions” means conditions in which inadequate oxygen is able to reach body tissue, for example, at altitude, such as altitudes where the partial pressure of inspired oxygen is 80% or less than that of the pressure at sea level, for example 70% or less, or 60% or less or 50% or less or due to a condition such as sleep apnoea, opiate overdose, acute asthma, atelectatic lung zones or exercise.

As used herein, the term“exercise” or“physical exercise” means that the heart rate is from about 40% to about 100% of maximum, such as from about 50% to about 90% or from about 60% to about 80%, or about 70%. For example, a 20 year old human would be expected to have a maximum heart rate of 220 - 200 = 200 beats per minute (BPM). Thus, exercise would be considered to be when the heart rate reached a rate from 80 BPM to 200 BPM, or from 100 BPM to 180 BPM or from about 120 BPM to 160 BPM, or about 140 BPM.

As used herein, the term“tolerance” means the ability to continue to exercise at a level from about 70% to 100%, such as from about 80% to 90% of the level that the exercise could be conducted at sea level.

As used herein, the term“recovery” means the heart rate has decreases to a level below 40% of maximum, such as 30% of maximum. For example, the extract of the invention or a composition comprising an extract of the invention may enhance recovery by decreasing the time taken for the heart rate to reach below 40% of maximum after completion of exercise.

The extract of the invention or composition comprising an extract of the invention is typically administered to an individual, for example a human or an animal subject.

Animal subjects that may be treated by the extract or composition of the invention include, but are not limited to, cats, dogs, horses, and cattle (such as sheep and cows).

When the extract or composition of the invention is administered may depend on the purpose for which it is administered or the method used. For example, when the extract of the invention is used to counteract the effects of altitude or to aid exercise recovery, the extract may be taken from about 2 weeks to the same time as the exposure to altitude or exercise, such as from about 1 week to about the same time, or from 7, 6, 5, 4, 3, 2, or 1 day(s) before the exposure to altitude or exercise, or the extract may be taken after exercise, such as just after exercise to about 2 weeks after exercise, such as just after exercise to about 1 week after exercise or from 1 , 2, 3, 4, 5, 6, or 7 day(s) after exercise.

During this time, the extract of the invention may, for example, be taken more than once a day, once a day or may be taken alternate days.

The extract for use, composition for use, use or method as previously defined may be performed on a human subject or an animal subject.

As used herein, the term“treatment” (and, similarly,“treating”) takes its normal meaning in the field of medicine. In particular, the term may refer to achieving a reduction in the severity of one or more symptom associated with the disease or condition (e.g. the arterial hypoxemia), as may be determined using techniques known to those skilled in the art (for example, by a medical physician) and/or to slowing the progression of the disease or condition (i.e. increasing the amount of time taken for the disease or disorder to progress to a more severe state, e.g. when compared to the time expected to be taken in a patient not so treated) and/or to alleviate or lessen the symptoms of the disease or condition.

As used herein, the term“prevention” (and, similarly,“preventing”) includes references to the prophylaxis of the disease or disorder (and vice-versa). In particular, the term may refer to achieving a reduction in the likelihood of the patient (or healthy subject) developing the condition (for example, at least a 10% reduction, such as at least a 20%, 30% or 40% reduction, e.g. at least a 50% reduction).

For the avoidance of doubt, in the context of the present invention, the terms“treating” and “preventing” include the therapeutic, or palliative, treatment of subjects/patients in need of, as well as the prophylactic treatment and/or diagnosis of patients which are susceptible to, the relevant disease states.

As used herein in relation to medical conditions, the term“reducing” may refer to making the observed quantity smaller or decrease in size.

As used herein, the terms“subject” and“patient” may be used interchangeably and include mammalian species (particularly humans).

Non-therapeutic uses

The extract of the invention may have particular effects, which may be useful in the treatment of non-medical conditions.

The extract of the invention may restore the hypoxia-induced decline in prefrontal oxidation and may enhance muscular oxygenation during hypoxia.

The present invention therefore provides the use of a cocoa extract or composition comprising a cocoa extract for enhancing tolerance to physical exercise in hypoxic conditions.

The present invention also provides the use of a cocoa extract or composition comprising a cocoa extract for enhancing recovery after exercise in hypoxic conditions.

Certain aspects of the invention are described in the following numbered paragraphs.

1 . A cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract.

2. A cocoa extract according to paragraph 1 , wherein the extract is a hydro-alcoholic extract. 3. A cocoa extract according to paragraph 1 or 2, wherein the extract is a hydro- ethanolic extract.

4. A cocoa extract according to paragraph 1 or 2, wherein the extract further comprises levels of catechin of from about 10 to about 70 mg/g; epicatechin from about 80 to about 350 mg/g; procyanidin B1 from about 0.1 to about 10 mg/g; and procyanidin B2 from about 30 to about 125 mg/g.

5. A process for providing a cocoa extract comprising:

a. Peeling the fruit (removing from its natural casing);

b. Depulping the seeds;

c. Blanching the seeds;

d. Drying the seeds;

e. Defatting by means of pressing;

f. Stabilizing the defatted product;

g. Grinding;

h. Solid-liquid extraction;

i. Solid-liquid separation;

j. Concentrating the liquid extract;

k. Obtaining the extract obtained by drying the concentrate obtained in (j);

L. Liquid-liquid extraction of the extract concentrated in (j) and separating the aqueous and organic phases;

m. Concentrating the aqueous phase obtained in (I);

n. Obtaining the semi-purified extract by drying the concentrate obtained in (m);

o. Concentrating the organic phase obtained in (I);

p. Obtaining the purified extract by drying the concentrate obtained in (o).

6. A process for providing a cocoa extract according to paragraph 5, wherein the solid-liquid extraction in step h is carried out using a hydro-ethanolic solvent comprising about 70% ethanol/30% water, and then re-extracted using a further hydro-ethanolic solvent comprising about 15% ethanol/85% water.

7. A cocoa extract obtained as defined in Paragraph 6.

8. A composition comprising an extract as defined in Paragraphs 1 to 4 or Paragraph 7. 9. A composition according to paragraph 8, further comprising a carrier.

10. A composition according to paragraphs 8 or 9, which is a pharmaceutical composition, veterinary composition or functional food composition.

1 1 . A composition according to paragraph 10, wherein the functional food composition is a food, feed or pet food or a food, feed or pet food supplement.

12. A cocoa extract according to paragraphs 1 to 4 or 7 or a composition comprising a cocoa extract according to paragraphs 8 to 1 1 for use in treating or preventing for treating disorders resulting from hypoxia within body tissue.

13. The use of a cocoa extract according to paragraphs 1 to 4 or 7 or a composition comprising a cocoa extract according to paragraphs 8 to 1 1 in the manufacture of a medicament for treating or preventing disorders resulting from hypoxia within body tissue.

14. A method of treating or preventing disorders resulting from hypoxia within body tissue comprising the administration of a therapeutically effect amount of a cocoa extract according to paragraphs 1 to 4 or 7 or a composition comprising a cocoa extract according to paragraphs 8 to 1 1 to a subject in need thereof.

15. An extract for use, composition for use, use or method according to paragraphs 12 to 14, wherein the hypoxia is the result of a decrease in the amount of oxygen present in the arteries (arterial hypoxaemia).

16. An extract for use, composition for use, use or method according to paragraph 15, wherein the decrease in the amount of oxygen present in the arteries is due to the partial pressure of inspired oxygen being 80% or less than that of the pressure at sea level.

17. An extract for use, composition for use, use or method according to paragraph 15 or 16, wherein the decrease in the amount of oxygen present in the arteries is due to sleep apnoea, opiate overdose, acute asthma, atelectatic lung zones or exercise.

18. A cocoa extract according to paragraphs 1 to 4 or 7 or composition comprising a cocoa extract according to paragraphs 8 to 1 1 for use in enhancing tolerance and/or capacity to physical exercise in hypoxic conditions. 19. The use of a cocoa extract according to paragraphs 1 to 4 or 7 or a composition comprising a cocoa extract according to paragraphs 8 to 1 1 in the manufacture of a medicament for enhancing tolerance and/or capacity to physical exercise in hypoxic conditions.

20. A method of enhancing tolerance and/or capacity to physical exercise in hypoxic conditions comprising the administration of a therapeutically effect amount of a cocoa extract according to paragraphs 1 to 4 or 7 or a composition comprising a cocoa extract according to paragraphs 8 to 1 1 to a subject in need thereof.

21 . A cocoa extract according to paragraphs 1 to 4 or 7 or composition comprising a cocoa extract according to paragraphs 8 to 1 1 for use in enhancing recovery after exercise in hypoxic conditions.

22. The use of a cocoa extract according to paragraphs 1 to 4 or 7 or a composition comprising a cocoa extract according to paragraphs 8 to 1 1 in the manufacture of a medicament for enhancing recovery after exercise in hypoxic conditions.

23. A method of enhancing recovery after exercise in hypoxic conditions comprising the administration of a therapeutically effect amount of a cocoa extract according to paragraphs 1 to 4 or 7 or a composition comprising a cocoa extract according to paragraphs 8 to 1 1 to a subject in need thereof.

24. The extract for use, composition for use, use or method according to any one of paragraphs 12 to 23, wherein the extract is administered in the form of a pharmaceutical or veterinary composition comprising a cocoa extract as defined in any one of paragraphs 1 to 4 or 7 and optionally a pharmaceutically or veterinary acceptable excipient.

25. The extract for use, composition for use, use or method according to paragraph 24, wherein the pharmaceutical or veterinary composition is administered from about 2 weeks before to at the same time as the exposure to hypoxic conditions or exercise or from just after to about 2 weeks after the exposure to hypoxic conditions or exercise.

26. The extract for use, composition for use, use or method according to paragraph 24 or 25, wherein the pharmaceutical composition is for oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal, intraperitoneal, or parenteral administration. 27. The extract for use, composition for use, use or method according to any one of the paragraphs 12 to 23, wherein the extract is administered in the form of a food, feed or pet food composition or product, a drink product or a food, feed or pet food supplement comprising a cocoa extract as defined in paragraphs 1 to 4 or 7 and optionally food or drink acceptable ingredients.

28. The extract for use, composition for use, use or method according to paragraph 27, wherein the food, feed or pet food composition or product, a drink product or a food, feed or pet food supplement is administered from about 2 weeks before to at the same time as the exposure to hypoxic conditions or exercise or from just after to about 2 weeks after the exposure to hypoxic conditions or exercise.

29. The extract for use, composition for use, use or method according to any one of paragraphs 12 to 28, wherein the extract is administered in an amount from about 10mg/day to about 400mg/day.

30. The extract for use, composition for use, use or method according to paragraphs 12 to 29, wherein the use or method is performed on a human subject.

31 . The extract for use, composition for use, use or method according to Paragraphs 12 to 30, wherein the use or method is performed on an animal subject.

Brief Description of the Figures

Figure 1 shows the proanthocyanidin profile of the water-soluble cocoa extract.

Figure 2 shown the HPTLC profile confirming the macro-composition removed as a wax family.

Figure 3 shows the gas chromatographic profile of wax removed from the water-soluble cocoa extract.

Figure 4 shows a photograph illustrating how the water-soluble extract of the invention fully dissolves in an aqueous solution compared to the extract of invention. Figure 5 shows the interventional exercise protocol which was executed twice in hypoxia and twice in normoxia, following 7 days of cocoa flavanol (CF) or placebo (PL) intake.

Figure 6 shows the effect of 7-day cocoa flavanol (CF, black lines) or placebo (PL, grey lines) supplementation in hypoxia (H, dashed lines and normoxia (N, full lines) on plasma epicatechin (A) and plasma nitrite (B) concentrations.

Figure 7 shows the effect of 7-days cocoa flavanol (CF, black lines) or placebo (PL, grey lines) supplementation in hypoxia (H, dashed lines) and normoxia (N, full lines) on exercise-induced changes in plasma arginine:citrulline ratio (A), plasma malondialdehyde concentration (MDA) (pmol/L) (B), plasma trolox equivalent antioxidant capacity (TEAC) (C) and plasma uric acid concentration (UA) (pmol/L) (D).

Figure 8 shows the effect of 7-days cocoa flavanol (CF, black lines) or placebo (PL, grey lines) supplementation in hypoxia (H, dashed lines) and normoxia (N, full lines) on tissue oxygenation (TSI, %) in the M. vastus lateralis (A) and prefrontal cerebral cortex (B).

Examples

The present invention will be further described by reference to the following, non-limiting examples.

Preparation of Cocoa Extract

The cocoa extract of the invention may be prepared using the technique defined in EP2071961 and as described, which is herein incorporated by reference.

To provide the water-soluble extract of the invention, the solid-liquid extraction (step h) was modified to include a step where the defatted cocoa beans were extracted using hydroalcoholic solvent (70% of ethanol / 30% of water) then re-extracted by hydroalcoholic solvent (15% ethanol / 85 % water) which yielded the Water Soluble (WS) product.

The proanthocyanidin composition of the WS product is shown in Figure 1. This is similar to the non-soluble version obtained using the same method but without the optional second step in step h described above. The composition of both the extract and the water-soluble (WS) extract are shown in Tables 1 , 2 and 3 below.

Table 1 : Composition of the cocoa extracts.

Table 2: Procyanidin content of the extract of the invention and water-soluble extract of the invention.

Table 3: Polyphenol content of the extract of the invention and water-soluble extract of the invention. The fraction removed from the extract by the additional of the optional second step in step h was characterized as a wax fraction as shown in Figures 2 and 3 and characterized in Table 4 below.

Table 4: total fatty acid content corrected with dry extract and extraction yield.

The solubility of the WS extract was compared to the original extract by dissolving both extracts (0.5% m/m) in distilled water at 20°C. The results are shown in Figure 4.

Materials and methods

Participants

A sample size calculation, based on the results of Allgrove, Patel and Wiswedel (Patel et al, J. Int. Soc. Sports Nutr., 2015, 12, 7-1 1 ; Allgrove et al., Int. J. Sport Nutr Exerc Metab., 201 1 , 21 , 1 13-123; Wiswedel et al., Free Radio Bio Led., 2004, 37 41 1 ) indicated that 14 subjects were required to detect differences at P value p<0,05 with 90 % power. The recruitment started in January 2016. Subjects were excluded when (i) younger than 20 years or older than 35 years, (ii) smoking or smoking in the past, (Hi) took antioxidant supplementation, (iv) trained less than 10 h per week, (v) had stayed at high altitude (> 2000m) for more than 3 weeks during the last 6 months, or (vi) if the medical examination prior to the experiment revealed they were hypertensive or had cardiovascular disease. Fifteen healthy well-trained male cyclists were selected for participation in this study. One subjects dropped out because of an injury (knee injury). The study was approved by the UZ Brussel Ethics Committee and was in accordance with the declaration of Helsinki. The experimental procedures and potential risks were explained to the participants and a written informed consent was provided and signed before the start of the study. This trial was registered at clinicaltrials.gov as NCT03135314.

Study design

A randomized, placebo controlled, counter-balanced, cross-over study design was used. On the first lab visit, subjects underwent a complete medical screening (including skinfolds measures) and performed a maximal incremental cycle test on an electromagnetically braked cycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands). During this test, initial work rate was set at 80W and work rate was then increased every 3 minutes by 40 W until volitional exhaustion. Maximal oxygen uptake (V0₂max) was determined using the Metalyzer cortex (Biophysik GmbH, Germany) and peak power output (PPO) was determined.

Subsequently, subjects visited the lab once every 2 weeks for 8 weeks (4 visits): each visit was preceded by a 1 -week wash-out (except for the first visit) and a 1 -week nutritional intervention (placebo (PL) or cocoa flavonals (CF)). The sequence of the 4 nutritional interventions was randomly assigned for each participant by using a computer-based randomly permutated block method. The allocation list was generated by CT (co-author), recruitment of participants was conducted by LD (first author) and allocation of participants was conducted by a third author (EL). Participants and researchers involved in data collection, outcome assessment and statistical analysis were blinded to the nutritional intervention. Participants and all researchers, except for LD, were blinded for FIO2.

Subjects performed 4 interventional trials in randomised order: (1 ) exercise in (normobaric) hypoxia (H) (3000 m; 14.3 % O2) preceded by 7 days of CF intake [H-CF], (2) exercise in H (3000 m; 14.3 % O2) preceded by 7 days of PL intake [H-PL] (3) exercise in normoxia (N) (0 m; 21 .0 % O2) preceded by 7 days of CF intake and [N-CF] (4) exercise in N (0 m; 21 % O2) preceded by 7 days of PL intake [N-PL] All experimental trials were conducted in 20° C and relative humidity was kept between 30 and 40 %. Supplemen ta tion

Subjects were asked to consume the provided supplements (PL or CF, Naturex, Avignon, France) every morning at breakfast during the 6 days prior to the testing day. On the testing day, subjects consumed the last dose of supplements upon arrival in the lab. The daily dose of CF consisted of 4 capsules, containing a total of 1765 mg cocoa extract of which 100 mg EP, 23 mg catechin, 1 19 mg theobromine and 17 mg caffeine (Table 5).

Table 5. Composition of cocoa flavanol (CF) and placebo (PL) supplementation (Naturex) (daily dose).

The PL capsule contained 1765 mg maltodextrine and was matched with the CF capsule in colour and shape, theobromine and caffeine content. Subjects were unable to distinguish the 2 interventions. The nutritional intervention was double-blinded and counter-balanced. Subjects were provided with a list of foods rich in polyphenols which they should avoid throughout the 8-week study. They were asked to abstain from caffeine during the last 24 h prior to each intervention trial and to repeat the same nutritional regimen during the last 24 h prior to each intervention trial. Subjects completed a 24h-food recall on 3 random days during the study, to check for a potential influence of polyphenol intake on the measurements.

The four interventional trials

Subjects were asked to keep a training diary for the entire duration of the study and to repeat the same weekly training regimen (volume and intensity) for the duration of the study. They were instructed to abstain from intensive training the last 24 h prior to each intervention trial. On each visit, subjects arrived at the lab at the same time of the day in a 3-h fasted state. The entire protocol is depicted in Figure 4. First, a baseline FMD measurement took place. Subsequently, a catheter was placed in a forearm vein and a first venous blood sample was collected. Subjects then consumed the last dose of their supplementation, together with a carbohydrate rich meal, which was carefully selected by a nutritionist to contain 600 kcal, 85% carbohydrates, 10% proteins and 5% fat. After the meal, subjects entered the isobaric hypoxic chamber, which was pre-set at the desired % 0₂. Subjects were asked to sit down and relax. It was shown that the maximal concentration of plasma flavanols is reached 100 minutes after acute CF intake and the plasma concentration of flavanols remains at a maximum for 50 minutes. Therefore, ninety-five minutes after the last supplement intake, a second blood sample was taken. Subsequently, the participants started a 20-minute steady state (SS) cycling exercise, one hundred minutes after the last dose. During the SS, power output was fixed at 45% of their PPO. SS was followed by five minutes passive rest in seated position and a blood sample was taken. The 20-minute TT then started at 75% of PPO, but subjects were free to increase or decrease their power output as desired from the outset. The goal was‘to perform as much as possible during 20 minutes’. Subjects received information on the time lapsed, but did not receive any feedback regarding power output or heart rate (HR). HR and saturation (Sa0₂) were recorded continuously during the experimental trial using a chest belt and Polar HR monitor and a pulse oximeter which was positioned on the participants’ left index finger (Medlab, Germany). Rate of perceived exertion (RPE) was measured at the start and after 5, 10, 15 and 20 minutes of the SS and the TT. Blood lactate was enzymatically determined in a capillary blood sample from the ear lobe (Ekf, Biosen 5030, Magdeburg, Germany), at the start, after 10 and 20 minutes of the SS, and the TT. During the 20-minute TT, the completed work (kJ) was used as the main outcome parameter of exercise performance. The occurrence of acute mountain sickness (AMS) was assessed using the Lake Louise Questionnaire at the end of each trial, but none of the subjects experienced any symptom of AMS.

Measurements

Primary outcome measures were muscle and cerebral oxygenation, markers of oxidative stress and exercise tolerance and performance. Secondary outcome measures included flavanol metabolites, markers of NO availability, FMD and mean arterial pressure.

Flow-mediated dilation (FMD) and mean arterial pressure (MAP)

Upon arrival at the lab, subjects were instructed to relax in supine position for 10 minutes, during which MAP was measured automatically (Medisana BU510, Kerkrade, Netherlands). Then, arterial endothelial function was assessed by FMD, as described by Theunissen et al. (Eur J Appl Physiol, 2013, 1 13, 2967-2975). Muscle and cerebral oxygenation during exercise

Near-infrared spectroscopy (NIRS) (Portalite continuous-wave NIRS system (Artinis, Elst, Netherlands), a non-invasive optical imaging technique, was used to assess changes in oxygenation status of the prefrontal cerebral cortex and the M. vastus lateralis during exercise. The use of NIRS to assess tissue oxygenation, including its limitations, has been extensively described (Ferrari et al., Can J Appl Physiol, 2004, 29, 463-487).

Upon entrance in the isobaric hypoxic chamber, one emitters/receptor optode pair was positioned over the left prefrontal cortical area between Fp1 and F3, according to the modified international EEG 10-20 system. One emitter/receptor optode pair was attached to the (shaved) skin on the lower third of the belly of the right M. vastus lateralis (middle between the lateral epicondyle and trochanter). Skinfold measures during the medical screening assured that the adipose tissue thickness was well below 1 .5 cm to allow the NIRS photons to penetrate into the muscle. The inter-optode distance for both probes was 4 cm and the probes were covered with a black cloth to minimize intrusion of extraneous light. A dark elastic band was wrapped around the head and the leg to keep the NIRS- optode pairs in place.

NIRS data collection was started 20 minutes prior to the start of exercise. To collect a baseline NIRS value, the mean of a 2-minute period during which subjects sat still without speaking or moving, was calculated. The tissue saturation index (TSI) was determined by spatially resolved spectroscopy and offers a surrogate measure of the fraction of 0₂ saturated haemoglobin and myoglobin, reflecting a tissue oxygenation status in percentage (%). Data were collected at a sampling frequency of 5 Hz and were down- sampled with factor 5 for analysis. During exercise, mean TSI was calculated per 30 second window. NIRS values of the following 30-sec epochs were used for data analysis: 0, 5, 10, 15 and 20 minutes of the SS and TT.

Blood analyses

Venous blood samples were collected at baseline, and at the start and end of the SS and the TT. Blood was collected into 5 ml EDTA tubes, 5 ml heparinized tubes and 8 ml anticoagulant-free tubes and were centrifuged immediately to obtain plasma or after 30 minutes at room temperature to allow clotting to obtain serum (10 minutes at 3000 rpm, 4° C). Plasma and serum were aliquoted and stored at -80° C until further analyses. Values were corrected for changes in plasma volume using the haematocrit and haemoglobin concentration according to Dill and Costill (J Appl Physio., 1974, 37, 247-248). Serum flavanols

Serum samples were analysed for EP and catechin concentrations as described by Neukam et al (Eur J Nutr, 2007, 46, 53-56).

Plasma nitrite and nitrate

Plasma nitrite and nitrate concentrations were determined by the method described by Wylie et al. (J Appl Physiol, 2013, 1 15, 325-336) , which was slightly adapted as follows: heparinized plasma samples were deproteinized using zinc sulfate (ZnS0₄)/sodium hydroxide (NaOH) precipitation. Aqueous ZnS0₄ [300 pi 5% (w/v)] and 360 mI 0.25 M NaOH were added to 100 mI of sample.

Plasma arginine and citrulline

150 mI_ of internal standard (50 mM arginine, methanol mixture) was added to 10 mI_ of heparinized plasma and centrifuged (13000 rpm, 10 minutes, 4°C) to remove the precipitated proteins. Supernatant was collected and dried under a stream of nitrogen at 70°C. The dried extract was dissolved in 100 mI_ of a butanol solution containing 3N HCI and kept at 70°C for 40 minutes. The solvent was removed by evaporation under nitrogen flow at 70°C. The sample was then dissolved in 2.5 ml. of water-methanol (90:10, v/v) containing 0.1 % formic acid and 5 mI_ was injected into an analytical column (Kinetex C18 (5pm, 2.1x100mm)). Mass spectrometric analysis was performed using an UFLC-XR Shimadzu coupled with an QTRAP® 5500 hybrid system, equipped with a Turbo VTM ion197 source (AB Sciex, Foster City, CA, USA). Multiple reaction monitoring (MRM) measurement was performed using optimal cone and collision energy values. Each run was performed at a flow rate of 500 pL/min at 30°C, lasting 9 minutes in total. A gradient profile consisted of solution A (water with 0.1% (v/v) formic acid) and solution B (methanol with 0.1% (v/v) formic acid). The percentage of organic solution B was changed gradually as follows: 0 min, 2%; 4 minutes, 7%; 6 minutes, 50%; 7 minutes, 2 %; 9 minutes, 2%. Data were acquired with Analyst Software version 1.5.2. Calibration curves, performed in water, were obtained adding increasing concentrations of arginine and citrulline from 12.5 to 125 pM.

Quantification of total antioxidant capacity (TEAC), uric acid (UA), malondialdehyde (MDA) in plasma

Plasma antioxidant capacity was quantified as trolox equivalent antioxidant capacity (TEAC) according to Fischer et al.(29). In this procedure, the decolorization of the preformed green-blue 2,2^'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS^’+) radical by the heparinized plasma within a fixed time reflects the antioxidant capacity of the sample. To correct plasma TEAC values for individual differences in uric acid (UA) concentrations, the most abundant antioxidant in blood, UA plasma concentrations were quantified by HPLC. MDA, a marker of lipid peroxidation as a result of oxidative stress, was quantified in EDTA-plasma samples after derivatization with thiobarbituric acid by using HPLC with fluorometric detection as described by Lepage et al. (Anal Biochem, 1991 , 197, 277-283).

Statistical analyses

Statistical analyses were performed with IBM SPSS Statistics (version 22; IBM Corp, Armonk, USA) and considered significant at p < 0.05. Data are presented as mean ± standard deviation (SD) for n=13, except when otherwise indicated. Normality and sphericity of the data were assessed by the Kolmogorov-Smirnov test and Mauchly’s test. One-way repeated measures ANOVAs (supplement) were used to assess baseline differences in nitrite, nitrate, MAP and FMD after 6 days of supplementation, before the intake of the last dose. Two-way repeated measures ANOVAs (fraction of inspired 0₂ (F1O2) x supplement) were used to assess differences between CF and PL in H and N (i) at baseline (sub chronic effect supplement), (ii) start of SS (acute effect supplement and Fi02) and start of TT. Three-way repeated measures ANOVAs ( 7O2 x supplement x time) were used to assess differences between CF and PL in H and N during exercise for the following outcome parameters: nitrite, nitrate, arginine:citrulline ratio (arg:citr ratio), TEAC, UA, MDA, work performance during TT, muscular and prefrontal TSI. Post-hoc analysis was performed using the Bonferroni correction. RPE was not normally distributed and was therefore analysed by Friedman tests and Wilcoxon signed rank tests. A Pearson correlation was used to assess correlations between baseline concentrations of nitrite and nitrate and relative increases in nitrite and nitrate concentration after 6 days of CF intake. Subject characteristics

The 14 well-trained athletes included in this study were 30.7 ± 3.1 years old, had a height of 1 .80 ± 0.05 m, weight of 73.4 ± 7.4 kg and BMI of 22.5 ± 1 .5. They had a VC>2max of 62.9 ± 5.8 mL/min^*kg and PPO of 366 ± 45 W.

Effects of CF intake on (-)-epicatechin and (+)-catechin

At baseline, there was no significant difference between 6-day CF and PL intake on serum EP concentration. Two-way repeated measures ANOVA showed a significant supplement x time interaction (F=14.70, p<0.001 ). Post-hoc analysis showed that 100 minutes after acute CF intake, serum EP was elevated compared to baseline (= 135 ± 44 %, p<0.05), while there was no increase after PL intake. During SS exercise, a further increase of serum EP after CF intake (+ 256 ± 32 %, p<0.001 compared to baseline) was noticed, while there was no change after PL intake. During the TT, there was no further increase in EP after CF intake (in both N and H), showing that a plateau phase in serum EP was reached (Figure 5A). Serum catechin was not affected by CF intake, compared to PL intake, at any time points and in both N and H (Table 6 for baseline values, other data not shown).

Table 6. Baseline measures following 6-day (before intake of last dose) cocoa flavanol (CF) or placebo (PL) intake (n=14).

MAP: mean arterial pressure, FMD: flow mediated dilation . * p<0.05 between CF and PL. Epicatechin and catechin, were measured in serum, nitrite, nitrate, malonaldehyde (MDA), Arginine (Arg), Citrullin (Citr), Uric acid (UA) and Trolox Equivalent Antioxidant Capacity (TEAC) were measured in plasma.

Effect of CF intake on NO availability during exercise in H

eNOS dependent NO production

eNOS dependent NO synthesis was reflected by plasma nitrite and nitrate concentrations and by the plasma ratio of arg:citr (31 ). Plasma nitrite and nitrate did not change during exercise and were not affected by H (Figure 5B). Arg:citr ratio significantly decreased at the end of exercise compared to pre-exercise (main effect of time: F=14.1 , p=0.003) and was significantly higher in H compared to N (main effect of F1O2. F=10.0, p=0.008)(Figure 6A). Sub chronic and acute CF intake did not significantly change plasma nitrite, nitrate and arg:citr ratio, either at rest, or after exercise. However, a significant negative correlation was found between baseline nitrite (after 6 days of PL intake) and the relative increase in nitrite concentration after 6 days of CF intake ( R² = 0.67, p<0.001 , Pearson correlation). Oxidative stress and antioxidant capacity

Two-way repeated measures ANOVAs at baseline and pre-exercise showed that MDA was not affected by sub chronic (Table 3) nor by acute CF intake in rest. Three-way repeated measures ANOVA during exercise revealed a significant interaction effect of f/me x supplementation (F=7.95 p=0.018): the significant exercise-induced increases in plasma MDA concentrations after PL intake (+ 12.2 ± 5.5 %, p=0.047 in N and +19.0 ± 6.8 %, p=0.016 in H), were suppressed by CF intake in both N (+ 2.9 ± 4.4 %, NS) and H (+ 2.0 ± 4.4 %, NS) (Figure 6B).

Two-way repeated measures ANOVAs at baseline and pre-exercise showed that total plasma antioxidant capacity, measured as TEAC, was neither affected by the intake of 6 day CF intake, nor by acute CF intake and H in rest. Three-way repeated measures ANOVA revealed that the exercise-induced increase in TEAC was larger in H than in N (time x F₁O_2'. F= 4.83 p=0.05), but was not affected by CF intake (Figure 6C). To correct for individual differences in UA, the most abundant plasma antioxidant that contributes to plasma TEAC, UA concentrations were quantified in every sample. Two-way repeated measures ANOVAs showed that UA was neither affected by sub chronic CF intake, nor by acute CF intake at rest. Three-way repeated measures ANOVA showed that UA concentrations were elevated after exercise, and in H compared to N (main effect of time: F=25.26, p<0.001 ; main effect of F₁O₂· F=6.48 p=0.026), while CF did not influence this response (Figure 6D).

Vasoreactivity

At baseline, MAP was not different between 6-day CF and PL intake (Table 3). FMD was significantly increased after 6 days of CF intake compared to PL (main effect of supplement: F=5.59, p=0.042) (Table 3). The change in FMD after 6 days of CF intake compared to 6 days of PL was not correlated with the relative increases in nitrite and (-)- epicatechin concentration after 6 days of CF intake compared to PL.

Muscle oxygenation during exercise

At the start of SS exercise, TSI in the V. lateralis was not affected by the supplement and H. During SS, a significant interaction time x F₁O₂ effect was found for TSI (F=1 1.95 p<0.001 (Figure 7A). Post-hoc Bonferroni corrections showed that TSI decreased during the first 5 minutes and then stabilized. This exercise-induced decrease was aggravated in H, compared to N, while CF intake had no effect. At the start of the TT, TSI was significantly lower in H than in N (main effect of F1O2: F=6.96 p=0.02). Three-way repeated measures ANOVAs showed a main effect of time during the TT for TSI (F=71.65 p<0.001 ): TSI significantly decreased during the first 5 minutes and stabilized during the last 15 minutes. H and CF intake did not influence TSI during the TT.

Prefrontal cortex oxygenation during exercise

At the start of SS exercise, both H and CF influenced TSI (main effect of F1O2. F=7.05, p=0.02, main effect of supplement: F=7.66, p=0.017) (Figure 7B). TSI was significantly lower in H compared to N. TSI was significantly higher after CF intake compared to PL (Figure 7B). During SS, 3-way repeated measures ANOVA showed a significant main effect of supplement (F= 12.28, p=0.004) and a significant FIO2 x time interaction for TSI (F=24.10 p<0.0001 ). CF intake significantly increased prefrontal TSI during SS exercise. TSI significantly decreased in H, but not in N.

At the start of the TT, TSI was significantly lower in H than in N ( effect of F1O2. F=6.43 p=0.026), while CF intake had no significant effect. Three-way repeated measures ANOVA showed a significant F1O2 x supplement x time interaction effect for TSI during the TT (F=4.1 1 p=0.016). In N, TSI decreased significantly for the entire duration of the TT after both PL and CF intake. However, a larger decrease was observed after CF intake, compared to PL (significant supplement x time interaction effect (F=6.38 p<0.001 )). In H, TSI enormously decreased during the first 5 minutes and did not change significantly during the remaining 15 minutes after both PL and CF intake and no interaction effect of supplement x time was found.

Exercise tolerance and performance

Steady state

Two-way repeated measures ANOVA showed that at the start of SS, Sa0₂ was significantly lower in H than in N (Table 7). At rest, CF intake did not alter Sa0_2. HR and lactate were similar in H and N and were not different after CF intake, compared to PL. Three-way repeated measures ANOVAs showed a significant F_/0₂ time interaction effect for Sa0_2, HR and lactate during SS. An exercise-induced decrease in Sa0₂ occurred in H, but not in N. The exercise-induced increase in HR was larger in H than N. In N, lactate decreased during SS, but in H, lactate increased during SS. During SS, RPE was significantly higher in H than in N. The (significant) difference in Sa0₂ between CF and PL intake during SS exercise in H (-1 .21 ± .48% in CF vs. PL) was smaller than the accuracy range (2 - 3%) claimed by the distributor of the pulse oximeter used and might thus not be reliable. CF did not influence HR, lactate and RPE during SS exercise.

Table 7. Effect of 7-day cocoa flavanol intake on physiological changes during moderate- and high intensity exercise in hypoxia and normoxia (n=14).

PL: placebo, CF: cocoa flavanol, H: hypoxia, N: normoxia, SS: steady-state, TT: time trial, Sa0₂: peripheral oxygen saturation, HR: heart rate, RPE: rate of perceived exertion. *: p<0.05: main effect of F1O2 (O2₎ (H-PL compared to N-PL and H-CF compared to N-CF); £: p<0.05: main effect of supplement (S) CF compared to PL); $: p<0.05: main effect of time (T) (compared to previous timepoint)

Time trial

TT performance (work performed during the 20 minute-TT) decreased in H compared to N, but CF intake did not influence TT performance (Table 4). Two-way repeated measures ANOVAs at the start of the TT showed that Sa0₂ and lactate were significantly lower (-8 ± 1 %, p<0.001 ) and higher (+0.9 ± 0.2 mmol.L ¹, p<0.001 ) in H compared to N. No significant difference between N and H was observed for HR. CF did not influence SaC>2, lactate or HR. Three-way repeated measures ANOVAs showed a significant F1O2 x time interaction effect for Sa0_2, HR and lactate during the TT. Post hoc analysis showed a larger drop in Sa0₂ and a larger increase in lactate during the TT in H compared to N. Post hoc analysis showed a faster elevation of HR, but lower HR_max at the end of the TT in H than in N. RPE was significantly higher in H than in N during the first half of the TT, but there was no difference during the second half. CF intake did not influence any of these physiological changes.

Claims

1. A water soluble, hydro-alcoholic cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract.

2. A cocoa extract according to claim 1 , wherein the extract has a solubility in water at room temperature of more than 10g/I.

3. A cocoa extract according to claim 1 or 2, wherein the extract is a hydro-ethanolic extract.

4. A cocoa extract according to claim 1 or 2, wherein the extract further comprises levels of catechin of from about 5 to about 70 mg/g; epicatechin from about 50 to about 350 mg/g; procyanidin B1 from about 1 to about 10 mg/g; and procyanidin B2 from about 30 to about 125 mg/g.

5. A process for providing a cocoa extract according to claim 1 comprising:

a. Peeling the fruit (removing from its natural casing);

b. Depulping the seeds;

c. Blanching the seeds;

d. Drying the seeds;

e. Defatting by means of pressing;

f. Stabilizing the defatted product;

g. Grinding;

j. Concentrating the liquid extract;

k. drying the concentrate obtained in (j).

6. A process for providing a cocoa extract according to claim 5, wherein the solid- liquid extraction in step h is carried out using a hydro-ethanolic solvent comprising about 70% ethanol/30% water, and then re-extracted using a further hydro-ethanolic solvent comprising about 15% ethanol/85% water.

7. A cocoa extract obtained as defined in Claim 5 or 6.

8. A composition comprising an extract as defined in Claims 1 to 4 or Claim 7.

9. A composition according to claim 8, further comprising a carrier.

10. A composition according to claims 8 or 9, which is a pharmaceutical composition, veterinary composition or functional food composition.

1 1 . A composition according to claim 10, wherein the functional food composition is a food, feed or pet food or a food, feed or pet food supplement.

12. A cocoa extract according to claims 1 to 4 or 7 or a composition comprising a cocoa extract according to claims 8 to 1 1 for use in treating or preventing for treating disorders resulting from hypoxia within body tissue.

13. The use of a cocoa extract according to claims 1 to 4 or 7 or a composition comprising a cocoa extract according to claims 8 to 1 1 in the manufacture of a medicament for treating or preventing disorders resulting from hypoxia within body tissue.

14. A method of treating or preventing disorders resulting from hypoxia within body tissue comprising the administration of a therapeutically effect amount of a cocoa extract according to claims 1 to 4 or 7 or a composition comprising a cocoa extract according to claims 8 to 1 1 to a subject in need thereof.

15. An extract for use, composition for use, use or method according to claims 12 to 14, wherein the hypoxia is the result of a decrease in the amount of oxygen present in the arteries (arterial hypoxaemia).

16. An extract for use, composition for use, use or method according to claim 15, wherein the decrease in the amount of oxygen present in the arteries is due to the partial pressure of inspired oxygen being 80% or less than that of the pressure at sea level.

17. An extract for use, composition for use, use or method according to claim 15 or 16, wherein the decrease in the amount of oxygen present in the arteries is due to sleep apnoea, opiate overdose, acute asthma, atelectatic lung zones or exercise.

18. A cocoa extract according to claims 1 to 4 or 7 or composition comprising a cocoa extract according to claims 8 to 1 1 for use in enhancing tolerance and/or capacity to physical exercise in hypoxic conditions.

19. The use of a cocoa extract according to claims 1 to 4 or 7 or a composition comprising a cocoa extract according to claims 8 to 1 1 in the manufacture of a medicament for enhancing tolerance and/or capacity to physical exercise in hypoxic conditions.

20. A method of enhancing tolerance and/or capacity to physical exercise in hypoxic conditions comprising the administration of a therapeutically effect amount of a cocoa extract according to claims 1 to 4 or 7 or a composition comprising a cocoa extract according to claims 8 to 1 1 to a subject in need thereof.

21 . A cocoa extract according to claims 1 to 4 or 7 or composition comprising a cocoa extract according to claims 8 to 1 1 for use in enhancing recovery after exercise in hypoxic conditions.

22. The use of a cocoa extract according to claims 1 to 4 or 7 or a composition comprising a cocoa extract according to claims 8 to 1 1 in the manufacture of a medicament for enhancing recovery after exercise in hypoxic conditions.

23. A method of enhancing recovery after exercise in hypoxic conditions comprising the administration of a therapeutically effect amount of a cocoa extract according to claims 1 to 4 or 7 or a composition comprising a cocoa extract according to claims 8 to 1 1 to a subject in need thereof.

24. The extract for use, composition for use, use or method according to any one of claims 12 to 23, wherein the extract is administered in the form of a pharmaceutical or veterinary composition comprising a cocoa extract as defined in any one of claims 1 to 4 or 7 and optionally a pharmaceutically or veterinary acceptable excipient.

25. The extract for use, composition for use, use or method according to claim 24, wherein the pharmaceutical or veterinary composition is administered from about 2 weeks before to at the same time as the exposure to hypoxic conditions or exercise or from just after to about 2 weeks after the exposure to hypoxic conditions or exercise.

26. The extract for use, composition for use, use or method according to claim 24 or 25, wherein the pharmaceutical composition is for oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal, intraperitoneal, or parenteral administration.

27. The extract for use, composition for use, use or method according to any one of the claims 12 to 23, wherein the extract is administered in the form of a food, feed or pet food composition or product, a drink product or a food, feed or pet food supplement comprising a cocoa extract as defined in claims 1 to 4 or 7 and optionally food or drink acceptable ingredients.

28. The extract for use, composition for use, use or method according to claim 27, wherein the food, feed or pet food composition or product, a drink product or a food, feed or pet food supplement is administered from about 2 weeks before to at the same time as the exposure to hypoxic conditions or exercise or from just after to about 2 weeks after the exposure to hypoxic conditions or exercise.

29. The extract for use, composition for use, use or method according to any one of claims 12 to 28, wherein the extract is administered in an amount from about 10mg/day to about 400mg/day.

30. The extract for use, composition for use, use or method according to claims 12 to

29, wherein the use or method is performed on a human subject.

31 . The extract for use, composition for use, use or method according to Claims 12 to

30, wherein the use or method is performed on an animal subject.

32. A cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids for use in treating or preventing for treating disorders resulting from hypoxia within body tissue.

33. The use of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids in the manufacture of a medicament for treating or preventing disorders resulting from hypoxia within body tissue.

34. A method of treating or preventing disorders resulting from hypoxia within body tissue comprising the administration of a therapeutically effect amount of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids to a subject in need thereof.

35. An extract for use, composition for use, use or method according to claims 32 to 34, wherein the hypoxia is the result of a decrease in the amount of oxygen present in the arteries (arterial hypoxaemia).

36. An extract for use, composition for use, use or method according to claim 35, wherein the decrease in the amount of oxygen present in the arteries is due to the partial pressure of inspired oxygen being 80% or less than that of the pressure at sea level.

37. An extract for use, composition for use, use or method according to claim 35 or 36, wherein the decrease in the amount of oxygen present in the arteries is due to sleep apnoea, opiate overdose, acute asthma, atelectatic lung zones or exercise.

38. A cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids for use in enhancing tolerance and/or capacity to physical exercise in hypoxic conditions.

39. The use of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids in the manufacture of a medicament for enhancing tolerance and/or capacity to physical exercise in hypoxic conditions.

40. A method of enhancing tolerance and/or capacity to physical exercise in hypoxic conditions comprising the administration of a therapeutically effect amount of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids to a subject in need thereof.

41 . A cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids for use in enhancing recovery after exercise in hypoxic conditions.

42. The use of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids in the manufacture of a medicament for enhancing recovery after exercise in hypoxic conditions.

43. A method of enhancing recovery after exercise in hypoxic conditions comprising the administration of a therapeutically effect amount of a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids to a subject in need thereof.

44. The extract for use, composition for use, use or method according to any one of claims 32 to 43, wherein the extract is administered in the form of a pharmaceutical or veterinary composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids and optionally a pharmaceutically or veterinary acceptable excipient.

45. The extract for use, composition for use, use or method according to claim 44, wherein the pharmaceutical or veterinary composition is administered from about 2 weeks before to at the same time as the exposure to hypoxic conditions or exercise or from just after to about 2 weeks after the exposure to hypoxic conditions or exercise.

46. The extract for use, composition for use, use or method according to claim 44 or 45, wherein the pharmaceutical composition is for oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal, intraperitoneal, or parenteral administration.

47. The extract for use, composition for use, use or method according to any one of the claims 32 to 43, wherein the extract is administered in the form of a food, feed or pet food composition or product, a drink product or a food, feed or pet food supplement comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the cocoa extract comprises less than about 0.0001 % carotenoids or a composition comprising a cocoa extract comprising a polyphenol content greater than 35% (dry weightytotal weight of the extract, wherein the composition comprises less than about 0.0001 % carotenoids and optionally food or drink acceptable ingredients.

48. The extract for use, composition for use, use or method according to claim 47, wherein the food, feed or pet food composition or product, a drink product or a food, feed or pet food supplement is administered from about 2 weeks before to at the same time as the exposure to hypoxic conditions or exercise or from just after to about 2 weeks after the exposure to hypoxic conditions or exercise.

49. The extract for use, composition for use, use or method according to any one of claims 32 to 48, wherein the extract is administered in an amount from about 10mg/day to about 400mg/day.

50. The extract for use, composition for use, use or method according to claims 32 to

49, wherein the use or method is performed on a human subject.

51 . The extract for use, composition for use, use or method according to Claims 32 to

50, wherein the use or method is performed on an animal subject.