Title:
APPLES WITH HIGH POLYPHENOLIC CONTENT
Kind Code:
A1


Abstract:
The invention relates to apples comprising high concentrations of polyphenols, to processes for obtaining said apples and to uses of apples as a method of treatment or prophylaxis of cardiovascular disease, colon cancer and digestive health.



Inventors:
Wood, Richard (Herefordshire, GB)
Barnett, William (Herefordshire, GB)
Application Number:
12/376770
Publication Date:
06/09/2011
Filing Date:
08/08/2007
Assignee:
Coressence Limited
Primary Class:
Other Classes:
514/456, 549/399, 800/269, 800/315
International Classes:
A61K36/73; A01H1/02; A01H5/08; A23L5/40; A23L19/00; A23L33/00; A61K31/352; A61P1/00; A61P9/00; A61P35/00; C07D311/20
View Patent Images:



Other References:
Gorinstein et al. COMPARATIVE CONENTS OF DIETARY FIBER, TOTAL PHENOLICS AND MINERALS IN PERSIMMONS AND APPLES; J. Agric. Food Chem., 2001, 49, 952-957.
Lachman et al. POLYPHENOL CONTENT AND ANTIRADICAL ACTIVITY IN DIFFERENT APPLE VARIETIES; Hort. Sci (Prague), 33, 2006 (3): 95-102
Linn et al.: POSTZYGOTIC ISOLATING FACTOR IN SYMPATRIC SPECIATION IN RHAGOLETIS FILES: REDUCED RESPONSE OF HYBRIDS TO PARENTAL HOST-FRUIT ODORS; Proceedings of the National Academy of Sciences of the United States of America (2004) Volume 101, Number 51, pp. 17753-17758 (STN abstract provided only, 2 pages).
Primary Examiner:
LEITH, PATRICIA A
Attorney, Agent or Firm:
FOLEY & LARDNER LLP (WASHINGTON, DC, US)
Claims:
1. An apple comprising a total polyphenolic content in excess of 10 000 mg/kg.

2. An apple as defined in claim 1, comprising a total polyphenolic content between 10 000 and 100 000 mg/kg.

3. An apple as defined in claim 1 or claim 2, comprising a total polyphenolic content between 10 000 and 50 000 mg/kg.

4. An apple as defined in claim 1, wherein said apple comprises a red-cut through apple.

5. An apple as defined in claim 1, wherein said polyphenols comprise flavonoids.

6. An apple as defined in claim 5, wherein said flavonoids comprise flavan-3-ols and proanthocyanidins.

7. An apple as defined in claim 6, wherein said flavan-3-ols comprise compounds of formula (V)a: embedded image wherein R1 represents hydrogen or an OH group.

8. An apple as defined in claim 6, wherein said proanthocyanidins comprise compounds of formula (X)a: embedded image wherein R2 represents hydrogen or an OH group; and n=2 to 30.

9. An apple as defined in claim 1, wherein low molecular weight polyphenols comprise greater than 22% of the total polyphenolic content.

10. An apple as defined in claim 1, wherein low molecular weight polyphenols comprise between 22% to 40% of the total polyphenolic content.

11. An apple as defined in claim 10, wherein the low molecular weight polyphenols comprise between 22% to 40% of the total polyphenolic content, wherein at least 85%, such as at least 90%, for instance at least 95%, such as for instance at least 99% of the low molecular weight polyphenols comprise compounds of formula (V)a.

12. An apple extract comprising polyphenols prepared from an apple as defined in any one of claims 1, 10 and 11.

13. A polyphenol isolate prepared from an apple as defined in any one of claims 1, 10 and 11.

14. A method of providing vasodilation or for the prophylaxis or treatment of cardiovascular disease or colon cancer in a subject in need of such vasodilation or treatment comprising administering to the subject an effective amount of an apple, an apple extract, or a polyphenol isolate as defined in any one of claims 1, 10 and 11.

15. A process for obtaining an apple as defined in any one of claims 1, 10 and 11 which comprises the step of crossing a red-through pome fruit with an apple.

16. A process as defined in claim 15 wherein the red-through pome fruit is hawthorn and the apple is crabapple.

17. A prebiotic composition comprising one or more of: an extract isolated from an apple comprising a total polyphenolic content in excess of 10 000 mg/kg; and/or an extract isolated from an apple wherein low molecular weight polyphenols comprise greater than 22% of the total polyphenolic content of the apple; and one or more prebiotic agents.

18. A dosage form comprising an apple extract comprising phenols and/or polyphenols isolated from an apple as defined in claim 1.

19. A method for treating cardiovascular disease in a subject in need of such treatment comprising administering to the subject an effective amount of a dosage form of a polyphenol isolate prepared from an apple as defined in any one of claims 1, 10 and 11 which comprises one or more low molecular weight polyphenols, or a pharmaceutically acceptable salt, or prodrug thereof, and one or more pharmaceutically acceptable carriers, excipients, or diluents.

20. A method for treating colon cancer in a subject in need of such treatment comprising administering to the subject an effective amount of a dosage form of a polyphenol isolate prepared from an apple as defined in any one of claims 1, 10 and 11 which comprises one or more high molecular weight polyphenols, or a pharmaceutically acceptable salt, or prodrug thereof, and one or more pharmaceutically acceptable carriers, excipients, or diluents.

21. A dietary composition comprising an apple extract comprising polyphenols or a polyphenol isolate prepared from an apple as defined in any one of claims 1, 10 and 11.

22. A dietary composition as defined in claim 21, which additionally comprises other nutrients, e.g. vitamins, minerals, and prebiotics and probiotics.

23. A dietary composition as defined in claim 21 and optionally other nutrients, e.g. vitamins, minerals, and prebiotics and probiotics, which is a drink, such as a fruit juice, sports drink, yoghurt drink and a milk drink or a solid foodstuff, e.g. a food snack bar, such as a fruit bar, nut bar and cereal bar, a cereal and a dessert.

24. A dietary composition as defined in claim 23, which is formulated as a liquid dosage form.

25. A dietary composition as defined in claim 24, wherein the liquid dosage form additionally comprises thickeners, tonicity adjusting agents, buffering agents.

26. A method of providing vasodilation or for the prophylaxis or treatment of cardiovascular disease or colon cancer in a subject in need of such vasodilation or treatment comprising administering to the subject an effective amount of a dietary composition as defined in claim 21.

27. A method of providing vasodilation in a subject in need of such vasodilation comprising administering to the subject an effective amount of a dosage form of a polyphenol isolate prepared from an apple as defined in any one of claims 1, 10 and 11 which comprises one or more high molecular weight polyphenols, or a pharmaceutically acceptable salt, or prodrug thereof, and one or more pharmaceutically acceptable carriers, excipients, or diluents.

28. A method of providing vasodilation in a subject in need of such vasodilation comprising administering to the subject an effective amount of a dosage form of a polyphenol isolate prepared from an apple as defined in any one of claims 1, 10 and 11 which comprises one or more high molecular weight polyphenols, or a pharmaceutically acceptable salt, or prodrug thereof, and one or more pharmaceutically acceptable carriers, excipients, or diluents.

Description:

The invention relates to apples comprising high concentrations of polyphenols, to processes for obtaining said apples and to uses of apples as a method of treatment or prophylaxis of cardiovascular disease, colon cancer and digestive health.

Cardiovascular disease (CVD) and cancer are two leading causes of death worldwide.

In the European Union, CVD is the main source of morbidity and mortality, costing 169 billion Euros annually (Leal et al. (2006) Eur. Heart J. 27(13): 1610-9), whilst in the US, the American Heart Association has estimated that 71,300,000 Americans have one or more forms of CVD.

According to the World Health Organisation, cancer kills about 7.6 million (or 13%) people worldwide every year. In particular, cancers of the lung, stomach, liver, colon and breast are responsible for over half these deaths.

Epidemiological evidence seems to suggest that a diet high in fruits and vegetables offers a significant protective effect against these chronic diseases. Much of this protective effect has been attributed to a major class of phytochemicals found commonly in fruits and vegetables called polyphenols.

Polyphenols have been referred to as nature's biological response modifiers because of strong experimental evidence demonstrating their ability to modify the body's reaction to allergens, viruses, and carcinogens. They show anti-allergic, anti-inflammatory, anti-microbial and anti-cancer activity. In addition, polyphenols act as powerful antioxidants, protecting against oxidative and free radical damage, and help to prevent various diseases associated with oxidative stress.

However, it has been estimated that most people do not consume sufficient quantities to obtain the health benefits offered by polyphenols. For example, the average current US daily intake of epicatechin is 2 to 4 times lower than the projected dose required to achieve a CVD risk reduction (Gu et al. (2004) J Nutr 134(3): 613-7; Prior and Gu (2005) Phytochemistry 66(18): 2264-80). As a result, increasing intake through the diet has emerged as an important health goal.

Apples can be a significant source of polyphenols, providing approximately 10% of the projected dose required to achieve a CVD risk reduction (Gu et al. (2004) supra). However, the concentration and distribution of polyphenols varies dramatically in each variety (see Table 1). Indeed, many modern dessert apples can have a polyphenol content less than 1000 mg/kg.

It is therefore an object of this invention to provide apples with increased levels of polyphenols.

Thus, according to one aspect of the invention, there is provided an apple comprising a total polyphenolic content in excess of 5 000 mg/kg.

According to a first aspect of the invention, there is provided an apple comprising a total polyphenolic content in excess of 10 000 mg/kg.

In one embodiment, the polyphenolic content is in the range of 10 000 to 100 000 mg/kg. In a further embodiment, the polyphenolic content is in the range of 10 000 to 50 000 mg/kg. Therefore, these apples comprise a much greater concentration of polyphenols than any other reported fresh weight source. As such, they can supply a large source of polyphenols in people's diets, and consequently provide consumers with the health benefits associated with an increased intake of polyphenols. It will be appreciated that references herein to “polyphenolic content” refer to fresh weight content. Equivalent ranges for dry weight content would be approximately 5 to 7 times higher than the fresh weight content presented.

In one embodiment, the apple comprises a “red-cut through” apple. It will be appreciated that the term “red-cut through” is well known in the art and refers to an apple having red flesh.

When used herein, the term polyphenol refers to a group of chemicals characterised by the presence of more than one phenol group per molecule. In one embodiment, the polyphenols include the sub-group flavonoids.

Over 5000 naturally occurring flavonoids have been characterised from various plants. They have been classified according to their chemical structure and can be categorised into various sub-classes. In one embodiment, the flavonoids include:

(a) flavanones (e.g. Hesperetin, Naringenin, Eriodictyol), which are based on the chemical skeleton shown in formula (I):

embedded image

(b) isoflavanones (e.g. Genistein, Daidzein, Glycitein), which are based on the chemical skeleton shown in formula (II):

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(c) flavones (e.g. Luteolin, Apigenin, Tangeritin), which are based on the chemical skeleton shown in formula (III):

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(d) flavonols (e.g. Quercetin, Kaempferol, Myricetin, Isorhamnetin, Pachypodol, Rhamnazin), which are based on the chemical skeleton shown in formula (IV):

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(e) flavan-3-ols (e.g. Catechin, Gallocatechin, Epicatechin, Epigallocatechin), which are based on the chemical skeleton shown in formula (V):

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(f) flavan-3,4-diol, which are based on the chemical skeleton shown in formula (VI):

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(g) dihydroflavonols, which are based on the chemical skeleton shown in formula (VII):

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(h) anthocyanidins (e.g. Cyanidin, Delphinidin, Malvinidin, Pelargonidin, Peonidin, Petunidin), which are based on the chemical skeleton shown in formula (VIII):

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(i) anthocyanins, which are based on the chemical skeleton shown in formula (IX):

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wherein sugar may be selected from glucose, arabinose, galactose or the like.

In one embodiment, the flavonoids include proanthocyanidins. Proanthocyanidins are biopolymers composed of flavan-3-ol subunits. Polymers are linked principally through the 4 and 8 positions and 4 and 6 positions. In a further embodiment, the proanthocyanidin is a 4,8 linked polymer, having the structure shown in Formula (X):

embedded image

In one embodiment, the flavonoids include anthocyanidins, proanthocyanidins, flavanols, flavonols, flavones, flavanones and isoflavones. In a further embodiment, the flavonoids include flavanols and proanthocyanidins.

In one embodiment, the flavan-3-ols have the structure shown in formula (V)a:

embedded image

wherein R1 represents hydrogen or an OH group.

In one embodiment, the flavan-3-ols have the structure shown in formula (V)b:

embedded image

wherein R1 is as defined above.

Compounds of formula (V)b wherein R1 represents hydrogen are known as epicatechin and compounds of formula (V)b wherein R1 represents an OH group are known as epigallocatechin.

In one embodiment, the flavan-3-ols have the structure shown in formula (V)c:

embedded image

wherein R1 is as defined above.

Compounds of formula (V)c wherein R1 represents hydrogen are known as catechin and compounds of formula (V)c wherein R1 represents an OH group are known as gallocatechin.

In one embodiment, the anthocyanidins include cyanidin, having the structure shown in formula (VIII)a:

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In one embodiment, the flavonoids include delphinidin, having the structure shown in formula (VIII)b:

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In one embodiment, the proanthocyanidins have the structure shown in formula (X)a:

embedded image

wherein R1 is as defined above; and
n=2 to 30.

In one embodiment, n>10 (i.e. 11-30). In a further embodiment, the compounds of formula (X)a have a molecular weight in excess of 1000. In a yet further embodiment, the compounds of formula (X)a have a molecular weight >1000 and <9000 (e.g. >3000 and <9000).

Proanthocyanidins include the subgroups of procyanidins and prodelphinidins and upon acid hydrolysis these yield cyanidin and delphinidin respectively.

Methods of Treatment

The polyphenols of the invention have been demonstrated to show numerous health benefits. For example, epidemiological studies have suggested that higher flavonoid intake is associated with a decreased risk of CVD, (Arts et al (2001) Epidemiology 12(6): 668-675; Sesso et al. Am J Clin Nutr 77: 1400-8; Mink et al. (2007) Am J Clin Nutr 85(3): 895-909), which may be a result of their ability to improve endothelial function and inhibit platelet aggregation in humans (Keen et al. (2005) Am J Clin Nutr 81 (1 Suppl): 298S-303S; Vita J A (2005) Am J Clin Nutr 81(Suppl): 292S-7S; Heptinstall et al. (2006) J Cardiovasc Pharmacol 47 Suppl 2: S197-205).

However, recent evidence suggests that molecules with a molecular weight above 600 are rarely absorbed into the bloodstream in the small intestine, as they are too large to pass through the intestinal wall. For example, the Tmax values for catechin and epicatechin observed in human studies indicate that these low molecular weight compounds are absorbed mainly in the small intestine (Donovan et al., (1999), J. Nutr., 129(9), 1662-1668; Richelle et al. (1999) Eur. J. Clin. Nutr. 53(1): 22-26; Lee et al. (2002), Cancer Epidemiol. Biomark. Prev., 11(10 Pt 1): 1025-1032). Furthermore (+)-catechin monomers are shown to possess relatively low bioavailability compared to (−)-epicatechin monomers (Shoji T et al. (2006) J Agric Food Chem. 54(3): 884-92).

Oligomeric and polymeric proanthocyanidins do not appear to be significantly absorbed in the small intestine, although procyanidins B1 (epicatechin-(4β-8)-catechin) and B2 (epicatechin-(4β-8)-epicatechin) have both been detected at low levels in human plasma. The dimer B5 (epicatechin-(4β-6)-epicatechin) could not be detected in plasma after chocolate consumption (Holt et al., (2002), Am. J. Clin. Nutr., 76, 798-804). No studies have detected proanthocyanidins with degrees of polymerization larger than the tetramers in plasma. As such, procyanidin oligomers and polymers (compounds of formula (X)a, where n>2) account for under 10% of the polyphenols absorbed in the small intestine and are therefore unlikely to contribute to the reduced risk of CVD (Déprez et al., (2001), Antioxidants and Redox Signalling, 3, 957-967; Shoji et al. (2006) Supra). Indeed recent trials showed no consistent link between CVD risk reduction and the consumption of proanthocyanidins (Williamson et al (2005) Am J Clin Nutr. 81(1 Suppl), 243S-255S).

It is thought, instead, that these molecules pass to the colon with minimal breakdown (Rios, L. et al., (2002) Am. J. Clin. Nutr., 76, 1106-1110). At this stage, they may be digested by intestinal micro flora, for which they act as an effective prebiotic, or they may inhibit colon cancer. In support of this, it has been shown that apple procyanidins (compounds of formula (X)a) significantly reduced the number of pre-cancerous lesions in the colons of laboratory animals (American Association for Cancer Research, Third Annual International Conference), whilst apple procyanidins (compounds of formula (X)a, where n>9) gave rise to an 82% reduction in colon cancer cell survival (Kroon et al. (2004) Am J Clin Nutr 80(1): 15-21).

Therefore, based on this finding it is appropriate to separate polyphenols into low molecular weight polyphenols and high molecular weight polyphenols.

In one embodiment, the low molecular weight polyphenols comprise monomers (compounds of formula (I) to (IX)) and dimers (compounds of formula (X)a, where n=2), having a molecular weight below 600. In a further embodiment, the low molecular weight polyphenols comprise monomers (compounds of formula (I) to (IX)), having a molecular weight below 300.

In one embodiment, the concentration of low molecular weight polyphenols comprise greater than 22% of the total polyphenolic content. In one embodiment, the low molecular weight polyphenols comprise between 22% to 40% of the total polyphenolic content. In a further embodiment, the low molecular weight polyphenols comprise between 22% to 40% of the total polyphenolic content, wherein at least 85%, such as at least 90%, for instance at least 95%, such as for instance at least 99% of the low molecular weight polyphenols comprise compounds of formula (V)a.

In a yet further embodiment, low molecular weight polyphenols comprise between 22% to 40% of the total polyphenolic content, wherein at least 85%, such at least 90%, for instance at least 95%, such as for instance at least 99% of the low molecular weight polyphenols comprise compounds of formula (V)b wherein R1 represents hydrogen (e.g. epicatechin alpha isomers).

In one embodiment, the high molecular weight polyphenols comprise oligomers and polymers (compounds of formula IV, where n>2), having a molecular weight above 600. In a further embodiment, the high molecular weight polyphenols comprise oligomers and polymers (compounds of formula IV, where n>1), having a molecular weight above 300.

Thus, it is believed that low molecular weight polyphenols may represent an effective prophylaxis or treatment of cardiovascular disease. Furthermore, it is also believed that high molecular weight polyphenols may constitute an effective prophylaxis or treatment for colon cancer.

It will be appreciated that as the apples, as hereinbefore defined, comprise such high quantities of both types of such polyphenols, it is likely that they will have the ability to be effective in the prophylaxis or treatment of cardiovascular disease or colon cancer.

It will also be appreciated that numerous apple extracts can be prepared from said apples, for example apple slices, apple pulp, apple juice, and the like, as well as various dried extracts, by processes well known to those skilled in the art. These resulting apple extracts will therefore typically comprise a high concentration of polyphenols. Thus in one aspect of the invention, there is provided an apple extract, prepared from an apple as hereinbefore defined, comprising polyphenols. Such an apple extract is likely to have similar therapeutic effects to the apple.

It will also be appreciated that one or more polyphenols may be isolated from said apples by processes well known to those skilled in the art. Such a polyphenol isolate will have a therapeutic effect as described hereinbefore.

Thus, according to a further aspect, there is provided an apple, an apple extract or a polyphenol isolate as hereinbefore defined for use in the prophylaxis or treatment of cardiovascular disease or colon cancer.

In a further aspect, there is provided a use of an apple, an apple extract or a polyphenol isolate as hereinbefore defined in the manufacture of a medicament for use in the prophylaxis or treatment of cardiovascular disease or colon cancer.

In a yet further aspect of the invention there is provided a method of treatment or prophylaxis of cardiovascular disease or colon cancer, which comprises administration of an apple, an apple extract or a polyphenol isolate as hereinbefore defined.

In a yet further aspect of the invention there is provided a pharmaceutical composition comprising an apple, an apple extract or a polyphenol isolate for use in the prophylaxis or treatment of cardiovascular disease or colon cancer.

It will be appreciated that the term “treatment” and “treating” as used herein means the management and care of a patient for the purpose of combating a condition, such as a disease or a disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications. The patient to be treated is preferably a mammal, in particular a human being, but it may also include animals, such as dogs, cats, cows, sheep, horses and pigs.

Combination Therapies

Many diseases are treated using more than one medicament in the treatment, either concomitantly administered or sequentially administered. It is therefore within the scope of the invention to use the apples, extracts or polyphenol isolates of the invention in therapeutic methods for the treatment of one of the above mentioned diseases in combination with one another, or as an adjunct to, or in conjunction with, other established therapies normally used in the treatment said disease. In one embodiment, there is provided a pharmaceutical composition comprising an apple, extracts or polyphenol isolate as hereinbefore defined in combination with one or more additional therapeutic agent.

By analogy, it is also within the scope of the invention to use the apples, extracts or polyphenol isolates of the invention in combination with other therapeutically active compounds normally used in the treatment of one of the above-mentioned diseases in the manufacture of a medicament for said disease.

The combination treatment may be carried out in any way as deemed necessary or convenient by the person skilled in the art and for the purpose of this specification, no limitations with regard to the order, amount, repetition or relative amount of the compounds to be used in combination is contemplated.

Methods of Synthesis

Accordingly, the invention also provides a process for obtaining an apple as hereinbefore defined which comprises the step of crossing a red-through pome fruit with an apple.

It will be appreciated that references to “red-through pome fruit” refer to a fruit having red flesh and which is produced by flowering plants in the subfamily maloideae of the family rosaceae. Examples of such red-through pome fruits include crabapple (Malus) and hawthorn (Crataegus; eg. Crataegus oxyacantha). In one embodiment, the red-through pome fruit is hawthorn (Crataegus; eg. Crataegus oxyacantha). The advantage of preparing apples of the invention from hawthorn is that hawthorn fruits are typically less than 20 mm in diameter, have a high surface area:volume ratio and are therefore easier to freeze dry. Hawthorn fruits are therefore capable of providing a high concentration of polyphenols in the resultant apples and have consequently been proposed as a natural remedy for cardiovascular disease (Walker, A. F. et al., (2002) Phytother Res. 16, 48-54).

It will be appreciated that the red-through pome fruit may be crossed with any suitable apple. The choice of apple will depend largely upon the desired properties of the resultant apple (e.g. sweetness, firmness and the like). In one embodiment, the red-through pome fruit is crossed with a crabapple (Malus).

Typically, the process of the invention will require intervention at each stage. For example, following the crossing step, polyphenol profiling should generally be performed on resultant apples until they can be categorised into one of the following three categories:

(1) fruits characterised by a total polyphenol content in the range 10,000 to 20,000 mg/kg, more typically 12,000 to 14,000 mg/kg (fresh weight basis);
(2) fruits characterised by a total polyphenol content in the range 20,000 to 100,000 mg/kg, more typically 15,000 to 40,000 mg/kg (fresh weight basis); and
(3) fruits characterised by a total polyphenol content up to 10,000 mg/kg, more typically 9,000 to 10,000 mg/kg (fresh weight basis).

Typically, once the apples can be categorised into one of the above mentioned three categories, the process of the invention will then require a propagation step to yield viable apples.

Compositions

In one embodiment, the high molecular weight polyphenols of the invention may be used in combination with other prebiotic agents, for example monosaccharides, disaccharides and polysaccharides. Thus, according to a further aspect of the invention, there is provided a prebiotic composition, comprising one or more high molecular weight polyphenols and one or more monosaccharide, disaccharide or polysaccharide. A suitable saccharide is oligofructose which has the structure shown in formula (XI):

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wherein n is typically in the range 2 to 14, suitably 2 to 10, e.g. 2 to 8. In a further embodiment, the higher molecular weight polyphenols of the invention may be used in combination with the apple extracts, which provides various monosaccharides, disaccharides and polysaccharides.

When the polyphenols are used in combination with other prebiotic agents or apple extracts, the components may be administered either sequentially or simultaneously by any convenient route. This combination provides the advantage of stimulating the micro flora, which helps to improve gut health, gut function, calcium absorption and immune response, as well as further reducing the risk of colon cancer (Rios, L. et al., supra).

In one aspect, the invention provides a dosage form; which comprises the apple extracts or polyphenol isolates and/or the combinations referred to above (hereinafter referred to as the compositions of the invention).

The compositions of the invention may conveniently be administered alone or in combination with pharmaceutically acceptable carriers or excipients. The compositions of the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.

Similarly, 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 pharmaceutical compositions formed by combining the compositions of the invention and the pharmaceutically acceptable carriers are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration. The formulations may conveniently be presented in unit dosage form by methods known in the art of pharmacy.

Thus, in a further aspect, there is provided a dosage form comprising one or more low molecular weight polyphenols, or a pharmaceutically acceptable salt, or prodrug thereof, and one or more pharmaceutically acceptable carriers, excipients, or diluents for use in the treatment of cardiovascular disease.

Thus, in a further aspect, there is provided a dosage form comprising one or more high molecular weight polyphenols, or a pharmaceutically acceptable salt, or prodrug thereof, and one or more pharmaceutically acceptable carriers, excipients, or diluents for use in the treatment of colon cancer.

The dosage forms may be formulated for administration by any suitable route such as the oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), transdermal, intracisternal, intraperitoneal, vaginal and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route, the oral route being preferred. It will be appreciated that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient chosen.

For topical use, sprays, creams, ointments, jellies, gels, inhalants, dermal patches, implants, solutions of suspensions, etc., containing the compounds of the present invention are contemplated. For the purpose of this application, topical applications shall include mouthwashes and gargles.

Compositions for oral administration include solid dosage forms, such as hard or soft capsules, tablets, troches, dragees, pills, lozenges, powders, granules, and liquid dosage forms such as solutions, emulsions, aqueous or oily suspensions, syrups and elixirs, each containing a predetermined amount of the compositions of the invention, and which may include a suitable excipient. Compositions intended for oral use may be prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

Aqueous suspensions may contain the compositions of the invention in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide such as lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the compositions of the invention in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as a liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the compositions of the invention in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. They may also contain buffering agents such as citrate and phosphate buffers, effervescent agents formed from carbonates, e.g. bicarbonates such as sodium or ammonium bicarbonate, and a solic acid, for example citric acid or an acid citrate salt. Additional excipients, for example, sweetening, flavouring, and colouring agents may also be present.

The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example a liquid paraffin, or a mixture thereof. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known methods using suitable dispersing or wetting agents and suspending agents described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as solvent or suspending medium. For this purpose, any bland fixed oil may be employed using synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

In a further embodiment, the polyphenol containing fractions and polyphenol isolates can be dried, for example spray-dried or dried under vacuum at a temperature less than 30° C., e.g. less than 20° C. and optionally freeze-dried, and formulated into a solid dosage form.

Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example, starch, gelatine or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.

The tablets 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.

Formulations for oral use may also be presented as hard gelatine capsules where the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or a soft gelatine capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Dosage forms for parenteral administration include sterile aqueous and non-aqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. The aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art. Depot injectable formulations are also contemplated as being within the scope of the present invention.

The compositions for rectal administration of the compounds may also be in the form of suppositories. These compositions can be prepared by mixing the compositions of the invention with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will thus melt in the rectum to release the compositions of the invention. Such materials include cocoa butter and polyethylene glycols, for example.

The compounds of the invention, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. The compound(s) may be administered therapeutically to achieve therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the systems associated with the underlying disorder. Therapeutic benefit also includes halting or slowing the progression of the disease, regardless of whether improvement is realised.

The exact dosage will depend upon the frequency and mode of administration, the sex, age, weight and general condition of the subject treated, the nature and severity of the condition treated and any concomitant diseases to be treated and other factors evident to those skilled in the art. Determination of the effective dosage is well within the capabilities of those skilled in the art.

When a compound of the invention or a pharmaceutically acceptable salt, solvate or prodrug thereof is used in combination with a second therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

In a further aspect, the invention provides a dietary composition, e.g. a drink, such as a fruit juice, sports drink, yoghurt drink, a milk drink, tea and the like or a solid foodstuff, e.g. a food snack bar, such as a fruit bar, nut bar and cereal bar, a cereal, a dessert, a chocolate (e.g. milk and dark) bar and the like, which comprises the apple extracts, and/or polyphenol isolates and/or the combinations referred to above.

In one embodiment, the dietary composition may additionally comprise other nutrients, e.g. vitamins, minerals, and prebiotics, such as oligofructose and apple fibres, and probiotics.

In one embodiment, the dietary composition is formulated as a liquid dosage form as hereinbefore defined. In a further embodiment, the liquid dosage form may additionally comprise thickeners, tonicity adjusting agents and buffering agents.

Examples of suitable tonicity adjusting agents include sugars and sodium chloride, which can be used to provide a solution of a particular strength, for example and isotonic solution. Examples of suitable buffering agents include citrates and phosphates.

Thus, in a further aspect, there is provided a dietary composition as hereinbefore for use in the prophylaxis or treatment of cardiovascular disease and/or colon cancer.

In a further aspect, there is provided a dietary composition as hereinbefore defined comprising in the manufacture of a medicament for use in the prophylaxis or treatment of cardiovascular disease and/or colon cancer.

In a yet further aspect of the invention there is provided a method of treatment or prophylaxis of cardiovascular disease and/or colon cancer, which comprises administration of a dietary composition as hereinbefore defined.

In a yet further aspect of the invention there is provided a pharmaceutical composition comprising a dietary composition as hereinbefore defined for use in the prophylaxis or treatment of cardiovascular disease and/or colon cancer.

The invention will now be illustrated by the following non-limiting Examples.

EXAMPLE 1

Preparation of Apples Having High Polyphenol Content

Fruits obtained from hawthorn (Crataegus oxyacantha) were crossed with crabapples (Malus) as hereinbefore described. The resultant fruits were profiled for polyphenol content and the results are shown in Table 1 wherein the resultant polyphenol contents have been compared with commercially available apples:

TABLE 1
Comparison of polyphenol content of apples of the
invention with commercially available apples
TotalTotal
PolyphenolPolyphenol
ContentContent
(mg/kg)-(mg/kg)-
AppleCodeDry WeightFresh Weight
Traditional Dessert Class432120,5254,527
Traditional Dessert Class422116,7553,327
Traditional Dessert Class022316,2552,928
Traditional Dessert Class425114,3883,067
Traditional Dessert Class492113,7592,773
Traditional Dessert Class527112,6662,458
Traditional Dessert Class424111,4542,339
Modern Dessert Class (Red)42619,9512,232
Modern Dessert Class (Green)52813,506730
Apple A4211189,58533,375
Apple B4201186,81734,701
Apple C333164,95514,750
Apple D310250,31710,161
Apple E436148,75110,290
Apple F423148,7339,920
Apple G330146,96110,348
Apple H439246,50011,249
Apple I435132,7396,883

Sample of results n=420, as an average of three samples consisting each of 7 individual fruits taken from the outside of individual trees (north, east, south and west locations), top, bottom and centre of tree.

It should be noted that Table 1 refers to the total polyphenol content, however, only flavan-3-ol monomers and polymers consisting of (−)-epicatechin, (+)-catechin, dimers, trimers, tetramers, pentamers, hexamers, heptamers and polymers were measured. Therefore, the total polyphenol content is likely to be much greater than the amounts presented in Table 1.

It can be seen from the results shown in Table 1 that apples A-I of the invention possessed greater polyphenol content than commercially available apples.

EXAMPLE 2

Cardiovascular Study with Epicatechin Extracts

10 healthy volunteers were recruited to test the effectiveness of the epicatechin extract obtained from the process of the invention as a means of improving the vascular tone and therefore cardiovascular function. 5 volunteers were given a placebo fruit drink and 5 volunteers were provided with the same test drink, but with 1 mg/kg body weight (based on (−)-epicatechin units) added. Vascular tone was continuously quantified by the reflective index obtained by non-invasive digital photoplethysmography and an algorithm for continuous, investigator-independent, automatic analysis of digital volume pulse. The data were presented as relative Stiffness Index SI (m/s) and correlated with vascular age. Pulse waves were measured by the transmission of red and infrared light through the finger pulp. The local minimum of the first derivative was determined and the corresponding turning point (=inflection point) of the pulse wave was thereby defined. The reflective index was calculated from the mean of the third to the seventh data point after the turning point (=inflection point).

FIGS. 1 and 2 show the results of a 2.5 hour trial. These figures indicate that the arterial stiffness index (SI; FIG. 1) and the Vascular Age (FIG. 2) of the group (n=10) consuming the test drink including the extract was statistically improved (i.e. reduced) when compared to the control.