WO2004096240A1

WO2004096240A1 - Anthocyanins useful for the treatment of diabetes, cardiovascular disorders and to lower the risk of adverse effects of hormone replacement therapy

Info

Publication number: WO2004096240A1
Application number: PCT/GB2004/001919
Authority: WO
Inventors: Leiv Sandvik
Original assignee: Medpalett Pharmaceuticals As; Beacham, Annabel, Rose
Priority date: 2003-05-02
Filing date: 2004-05-04
Publication date: 2004-11-11

Abstract

Anthocyanins lower levels of C-reactive protein (CRP) in a subject, and hence may be useful in the prevention or treatment of type II diabetes and cardiovascular problems, and to lower the risk of adverse side effects for women receiving hormone replacement therapy.

Description

ANTHOCYANINS USEFUL FOR THE TREATMENT OF DIABETES, CARDIOVASCULAR DISORDERS AND TO LOWER THE RISK OF ADVERSE EFFECTS OF HORMONE REPLACEMENT THERAPY

The present invention relates to medical uses of 5 anthocyanins, in particular of a composition comprising a mixture of at least four anthocyanins, at least one of which is a disaccharide and at least one of which is a ^'monosaccharide . In particular, it relates to use of one or more anthocyanins for lowering levels of C-Reactive 10 protein (CRP) in patients with elevated CRP levels.

Elevated levels of C-Reactive protein (CRP) have recently been demonstrated to be a predictor of risk for the development of type 2 diabetes and of risk of cardiovascular events (P.M. Ridker et al, N Engl J Med 15 347: 1557-1565, 2002, and D.J. Freeman et al, Diabetes 51(5): 1596-600, 2002). Reduction of CRP levels may therefore be beneficial in lowering the risk of developing type 2 diabetes and/or the risk of adverse cardiovascular events.

20 Anthocyanins are glycosides of flavylium salts.

Each anthocyanin thus comprises three component parts: the hydroxylated core (the aglycone) ; the saccharide unit; and the counter ion. Anthocyanins are naturally occurring pigments present in many flowers and fruit and 25 individual anthocyanins are available commercially as the chloride salts, e.g. from Polyphenols Laboratories AS, Sandnes, Norway.

As individual compounds, anthocyanins have been proposed for use as antioxidants (e.g. as free radical

30 scavengers) for treatment of the vascular system.

We have now surprisingly found that administration of at least one anthocyanin is effective in lowering CRP levels in humans. One or more anthocyanins may therefore be useful in the prevention and/or treatment 35 of cardiovascular problems and diseases, for example myocardial infarction, ischemic stroke, arteriosclerosis, coronary revasculisation and circulatory problems, and type 2 diabetes, especially when administered orally. In this context, prevention of a disease or condition includes lowering the risk of said disease or condition. Thus viewed from one aspect the invention provides a method of lowering C-Reactive protein levels in a subject (patient) , the method comprising administering at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof to the subject.

The invention further provides a method for the treatment or prevention of type 2 diabetes, the method comprising administering at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof to a subject.

The invention further provides a method for the treatment or prevention of cardiovascular problems and diseases, including myocardial infarction, arteriosclerosis, ischemic stroke, coronary revasculisation, peripheral artery disease and circulatory problems, the method comprising administering at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof to a subject. As used herein, a subject may be human or a non- human animal (e.g. mammalian), preferably a human.

The invention further provides use of at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof in the manufacture of a medicament for lowering CRP levels, for the treatment or prevention of type 2 diabetes, and/or for the treatment or prevention of cardiovascular problems and diseases (including myocardial infarction, ischemic stroke, coronary revasculisation, arteriosclerosis and circulatory problems) .

The invention further provides use of at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof in the manufacture of a cardioprotective medicament .

The invention also provides at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof, and preferably a composition comprising at least four anthocyanins, at least one of which is a monosaccharide and at least one of which is a disaccharide, for use in lowering CRP levels, or in the treatment or prevention of type 2 diabetes or cardiovascular problems and diseases (including myocardial infarction, ischemic stroke, coronary revasculisation, arteriosclerosis and circulatory problems) .

Anthocyanins may also be useful for the treatment or prevention of other conditions in which CRP levels are elevated. For example, it has been suggested that some of the undesirable side effects associated with hormone replacement therapy (HRT) may be due to elevated CRP levels in women on HRT. Thus, the risk of adverse side effects, and in particular of adverse cardiovascular events, might be lowered if HRT was combined with treatment with one or more anthocyanins .

The invention therefore further provides a method of lowering the risk of adverse side effects of HRT, in particular cardiovascular events, said method involving administering at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof to a subject receiving HRT.

The invention further provides use of at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof in the manufacture of a medicament for lowering the risk of adverse side effects of HRT, in particular cardiovascular events.

The anthocyanin may be isolated or synthetic (i.e. produced by chemical reaction) . By isolated in this context is meant a naturally occurring anthocyanin which has been substantially separated from the carbohydrates and lipids which surround it in its natural environment, i.e. the endogenous carbohydrates and lipids, e.g. to an endogenous carbohydrate and/or lipid content of no more than 10 wt.%, preferably no more than 5 wt.%, especially no more than 2 wt.%, and particularly no more than 1 wt.%. Following such separation, which renders the anthocyanin more bioavailable, it can of course be mixed with carbohydrates (e.g. sugars and celluloses) or lipids (e.g. plant oils) in the course of being formulated for administration.

CRP levels in a subject may be measured and monitored using techniques known in the art. They are measured in the blood. Typical values for a non smoker are between about 1.63 mg/1 and about 2.24 mg/1. CRP levels may rise to 100 mg/1 or even 150 mg/1 with strong bacterial infections like pneumonia. However, the present invention is concerned particularly with the reduction of slightly elevated CRP levels, for example levels in a smoker of about 2.78 mg/1 to about 4.31 mg/1 or levels in a person with diabetes of about 2.15 mg/1 to about 6.25 mg/1.

As used herein, an anthocyanin analogue is a compound in which the saccharide unit of an anthocyanin has been replaced with another lipophilic or hydrophilic moiety, for example a polyoxyalkyl chain. Preferably, the hydrophilic moiety is a polyoxyalkyl chain of formula - (OCH₂CH₂)_nOH, wherein n denotes an integer from 1 to 30, preferably from 1 to 10.

As used herein, a derivative is an anthocyanin produced by derivatization of a natural anthocyanin, e.g. a salt, a compound from which some or all of the saccharide component is cleaved, or an acyl derivative.

References to anthocyanins hereinafter include derivatives and analogues thereof . Preferably, the at least one anthocyanin will be administered to a patient orally.

Highly preferably, the at least one anthocyanin is in the form of a composition, more preferably an orally ingestible composition, e.g. a nutritional supplement or a pharmaceutical composition. Particularly preferably, the at least one anthocyanin is in the form of a composition, preferably an orally ingestible composition, comprising at least four anthocyanins, at least one of which is a monosaccharide and at least one of which is a disaccharide.

Synergistic effects may be obtained through administration of a combination of different anthocyanins, in particular a combination comprising both mono-and disaccharide anthocyanins. It is thought that the synergistic effect arises at least in part from the different solubilities and different uptake profiles of the different anthocyanins.

Preferably, the at least one anthocyanin is in the form of a coated spray-dried material.

Compositions for use in the invention preferably contain at least three monosaccharide anthocyanins . Moreover the compositions preferably contain at least one monosaccharide anthocyanin in which the saccharide is arabinose.

The compositions preferably contain at least one disaccharide anthocyanin in which the disaccharide is rutinose (i.e. 6-rhamnosyl-glucose) .

Moreover the compositions for use in the invention preferably contain anthocyanins with at least two different aglycones, more preferably at least four.

Especially preferably the compositions contain anthocyanins in which the aglycone units are cyanidin, peonidin, delphinidin, petunidin, malvidin and optionally also pelargonidin.

In one preferred embodiment, the composition also contains at least one trisaccharide anthocyanin. Individual anthocyanins are available commercially as the chloride salts, e.g. from Polyphenols Laboratories AS, Sandnes, Norway. A particularly preferred anthocyanin composition for use in the invention is available from Medpalett Pharmaceuticals AS of Sandnes, Norway, under the trade name MEDOX.

Examples of anthocyanins suitable for use in the invention include: cyanidin-3-O-β-glucoside; cyanidin-3- O-β-galactoside; cyanidin-3-O-α-arabinoside; cyanidin-3- O-β-xyloside; cyanidin-3-0- (6"-0-α-rhamnosyl-β- glucoside) ; cyanidin-3-O- (2"-0-β-glucosyl-β-galactoside) ; cyanidin-3-O- (2"-0-β-glucosyl-β-glucoside) ; cyanidin-3-0- (2"-0-β-xylosyl-β-glucoside) ; cyanidin-3-0- (2" -O-β- xylosyl-β-galactoside) ; cyanidin-3, 5-di-O-β-glucoside; cyanidin-3-O-β-galactoside-5-O-β-glucoside; cyanidin-3-0- α-arabinoside-5-0-β-glucoside; cyanidin-3-0- (2"-0-β- xylosyl-β-glucoside) -5-0-β-glucoside; cyanidin-3-0- (2"-0- β-xylosyl-6"-0-β-glucosyl-β-galactoside) ; pelargonidin-3- O-β-glucoside; pelargonidin-3-0- (6"-0-α-rhamnosyl-β- glucoside) ; pelargonidin-3, 5-di-O-β-glucoside; peonidin- 3-O-β-glucoside; peonidin-3-O-α-arabinoside; peonidin- 3, 5-di-0-β-glucoside; delphinidin-3 -O-β-glucoside; delphinidin-3-0- (6" -0-α-rhamnosyl-β-glucoside) ; delphinidin-3-0- (2" -O-β-xylosyl-β-glucoside) ; petunidin- 3-0-β-glucoside; malvidin-3-0-β-glucoside; malvidin-3 , 5- di-O-β-glucoside,- and malvidin-3-0-α-arabinoside-5-0-β- glucoside. If desired, one or more hydroxy groups, especially on the saccharide unit, in the anthocyanins may be acylated, e.g. carrying a C^^, more especially a C₃.₉ saturated or unsaturated acyl group, for example a mono- or dicarboxylic acid residue, e.g. a malonyl, p-coumaryl or feruloylyl group. Thus for example such acylated compounds include cyanidin-3-0- (6"-0- (E-p-coumaryl) -2"- O-β-xylosyl-β-glucoside) ; cyanidin-3-0- (6"-0- (E-p- coumaryl) -2 "-O-β-xylosyl-β-glucoside) -5-0-β-glucoside; cyanidin-3-0- (2"-0-β-xylosyl-6"-0- (E-feruloyl-β- glucosyl) -β-galactoside) ; cyanidin-3-0- (2"-0-β-xylosyl- 6"-0- [E-coumaryl-β-glucosyl] -β-galactoside) ; and petunidin-3-O- (6"-0- (4" ' -O-E-coumaryl) -α-rhamnosyl-β- glucoside) -5-0-β-glucoside. References herein to anthocyanins and derivatives thereof include such acylated compounds .

The counter ion in the anthocyanins for use in the invention may be any physiologically tolerable counteranion, e.g. chloride, succinate, fumarate, malate, maleate, citrate, etc. Preferably however the counter ion is a fruit acid anion, in particular citrate, as this results in the anthocyani (s) having a particularly pleasant taste. Particularly suitable sources for the anthocyanins for use in the invention are fruits such as cherries, bilberries, blueberries, blackcurrants, redcurrants, grapes, cranberries, strawberries, and apples and vegetables such as red cabbage. Bilberries, in particular Vaccinium myrtillus, and blackcurrants, in particular Ribes nigrum, are especially suitable. The berries of V. myrtillus contain fifteen monosaccharide anthocyanins, namely the aglycone : saccharide combinations of cyanidin, peonidin, delphinidin, petunidin and malvidin and glucose, galactose and arabinose. The currants of R. nigrum contain four anthocyanins, namely the 3-glucosides and 3-rutinosides of cyanidin and delphinidin.

The disaccharide anthocyanins are more water- soluble than the monosaccharides; moreover cyanidin and delphinidin anthocyanins are amongst the most water- soluble anthocyanins .

Besides the at least one anthocyanin, the compositions for use in the invention may desirably contain further beneficial or inactive ingredients, e.g. vitamins (in particular vitamin C) , flavones, isoflavones, anticoagulants (e.g. maltodextrin, silica, etc.), desiccants, inert diluents, excipients etc. Desirably however the at least one anthocyanin constitutes at least 50% by weight of the composition, excluding any coating material.

The compositions used according to the invention may be administered enterally or parenterally; however administration into the gastrointestinal tract, especially oral administration, is preferred. Suitable administration forms include powders, sprays, solutions, syrups, tablets, dispersions and, especially, capsules.

While the use of a combination of different anthocyanins is important in the present invention, it also represents a problem to produce due to their different physicochemical properties. The commercially available individual anthocyanins do not readily mix to form a free-flowing powder such as would be particularly suitable for tableting or filling into capsules. Normal fruit extracts containing anthocyanins cannot be spray- dried to a free-flowing powder yielding instead a sticky mess. We have found that for spray drying to be successful, the anthocyanin solution must be freed of free sugars and acids, and plant-derived lipids and macromolecules, in particular lectins . Anthocyanin mixtures for use in the invention are preferably prepared by a process comprising: loading an aqueous anthocyanin solution (e.g. a liquid plant extract) onto a cation exchange resin; flushing the resin with water; applying an acidic alkanol eluant to said resin until anthocyanin begins to elute therefrom whereafter applying a non-acidic alkanol eluant to said resin; collecting anthocyanin-containing eluate from said resin; evaporating off alkanol from said eluate; adding water and if necessary an anticoagulant to the eluant to produce an aqueous anthocyanin solution, preferably a solution containing at least two anthocyanins; spray-drying said solution; and optionally coating the spray dried product (optionally after mixing with further physiologically tolerable components, e.g. vitamin C, further anthocyanins, etc. and/or after tableting) . The spray-drying is preferably effected using by spraying into an inert atmosphere, e.g. a nitrogen atmosphere, with inlet temperatures of 130 to 160°C and flow rates of 5 to 12 L/hour. If the spray-dried product is sticky, then increasing flow rate and inlet temperature and/or increasing anticoagulant content and/or increasing atomiser rotation rate will result in a non-sticky product. The optimum temperature, flow- rate, etc. can be determined in this way for each separate anthocyanin source. The anthocyanin solution to be spray-dried desirably is an aqueous solution containing anthocyanins at 5 to 15% wt, more preferably 8 to 12% wt, dry solids basis.

The anthocyanin composition for use in the invention is preferably coated. This is important due to the hygroscopic nature of the anthocyanins. Coating may be by conventional coating techniques following tableting of the spray dried product; alternatively, and preferably, coating is achieved by filling the spray dried product into capsules. While conventional gelatin capsules may be used, it is preferred to use cellulose capsules, such as Vcaps from Capsugel SA, Belgium.

The coated compositions for use in the invention preferably contain 50 to 250 mg anthocyanin per dose unit (e.g. capsule) , more preferably 70 to 160 mg. The dosage for a human patient is preferably 50 to

1000 mg anthocyanin per day, for example 50 to 250 mg, especially 100 to 500 mg per day, particularly 250 to 350 mg per day, e.g. one to five, preferably two to four, 75 mg anthocyanin capsules per day. The dose is preferably administered one to four times each day, e.g. twice . This corresponds approximately to the anthocyanin content of one or two cups full of fresh berries . The exact dosage will depend on the individual patient, and on the condition being treated.

As mentioned above, the compositions for use in the invention preferably also contain vitamin C, e.g. 10 to 1000, preferably 20 to 200, mg per gram anthocyanin.

Besides anthocyanins, the compositions for use in the invention will generally also contain a anticoagulant, e.g. maltodextrin, lactose or silica, added to the anthocyanin solution to be spray dried to prevent coagulation in the spray drying process.

Typically the anticoagulant will be present at 0.3 to 0.8g, especially about 0.5g, per gram anthocyanin.

Where anthocyanins from more than one plant source are to be included in the compositions for use in the invention, they are preferably mixed after spray-drying. Thus, for example, the compositions for use in the invention preferably contain anthocyanins from V. myrtillus and R. nigrum mixed after spray-drying, preferably in a weight ratio of 0.5:1 to 1:0.5, especially about 1:1.

As mentioned above, the individual anthocyanins available commercially and used in research and as the active components of medicaments are not readily formulated into the sorts of free-flowing powders that are particularly suitable for tableting or capsule filling. This is inconvenient for handling and formulation, and the spray drying procedure described above may be used to transform such compounds into an easily handleable free-flowing powder which is suitable for tableting or capsule filling.

Analogues of anthocyanins may be synthesised using analogous methods to those known in the art for the synthesis of anthocyanins but with the saccharide moiety replaced with a suitable hydrophilic moiety. Known synthetic routes to flavylium ions usually involve the coupling together of two halves, the so-called "eastern" and "western" portions of the molecule (see for example, R. Doering, Justus Liebigs Ann . Chem, 1957, 606, 137;

A. Katrizky, P. Czerney, J. Levell, W. Du, Eur. J. Org.

Chem . 1998,2623; C. Michaelis, R. Wizinger, Helv. Chim.

Acta 1951, 34, 1761; K. Kokkinos, R. Wizinger, Helv.

Chim . Acta 1973, 56, 983; F. Herstein, S. Von

Kostanecki, Ber. Dtsch . Chem . Ges . 1899, 32, 318; A.

Roque, C. Lodeiro, F. Pina, M. Maestri, R. Ballardini, V. Balzani, Eur. J. Org. Chem . 2002, 2699; and R. A.

McCelland, G. H. McGall, J. Orgr. Chem . 1982, 47, 3730) .

In the synthesis of anthocyanins, the western half contains the saccharide moiety, which is introduced before the two halves are coupled together.

A representative generalised synthetic scheme for anthocyanin analogues is shown in Figure 1. Analogous routes may be used for the synthesis of other anthocyanin analogues. If necessary, suitable protecting groups as known in the art may be used during the synthesis .

"western" half coiφling "eastern" half reaction

eac R' independent^ = H, OMe or OH Figure 1

A preferred anthocyanin analogue based on cyanidin is shown in Figure 2, wherein n may denote an integer from 1 to 30, preferably 1 to 10.

X is a counterion, for example chloride

Figure 2

Analogues of anthocyanins in which the saccharide moiety of an anthocyanin has been replaced with another hydrophilic moiety form a further aspect of the invention. Preferred such compounds comprise an anthocyanin aglycone unit, for example a cyanidin, peonidin, delphinidin, petunidin, malvidin or pelargonidin aglycone unit, coupled to a hydrophilic moiety, for example a polyoxyalkyl chain, preferably a chain of formula - (OCH₂CH₂)_nOH, wherein n denotes an integer from 1 to 30, preferably from 1 to 10.

In a further aspect, the invention also provides a method of lowering C-Reactive protein levels in a patient, the method comprising administering at least one flavone or isoflavone to the patient.

The invention further provides a method for the treatment or prevention of type 2 diabetes, the method comprising administering at least one flavone or isoflavone to a patient.

The invention further provides a method for the treatment or prevention of cardiovascular problems, myocardial infarction, ischemic stroke, coronary revasculisation and circulatory problems, the method comprising administering at least one flavone or isoflavone to a patient.

The invention further provides use of at least one flavone or isoflavone in the manufacture of a medicament for lowering CRP levels, for the treatment or prevention of type 2 diabetes, and/or for the treatment or prevention of cardiovascular problems (including myocardial infarction, ischemic stroke, arteriosclerosis, coronary revasculisation and circulatory problems) .

The invention further provides use of at least one flavone or isoflavone in the manufacture of a cardioprotective medicament .

The invention also provides at least one flavone or isoflavone for use in lowering CRP levels, or in the treatment or prevention of type 2 diabetes or cardiovascular problems (including myocardial infarction, ischemic stroke, coronary revasculisation, arteriosclerosis and circulatory problems) .

EXAMPLE 1: Anthocyanin extraction

As raw material is used the cakes of fruit skin produced as the waste product in fruit juice pressing from V. myrtillus and R. nigrum. Fruit juice producers generally add lectinases to the fruit before pressing to release anthocyanins into the juice produced; however the cake still contains a high anthocyanin content.

200L methanol, with 0.1% wt . HC1 content, is added to 120 kg fruit skin cake. The mixture is allowed to stand for 24 hours at ambient temperature (20-25°C) whereafter the methanol is drained off. 150L methanol (0.1% HC1) is then added to the fruit skin. The mixture is allowed to stand for 24 hours at ambient temperature (20-25°C) whereafter the methanol is drained off. A further 150L methanol (0.1% HC1) is then added to the fruit skin. The mixture is allowed to stand for 24 hours at ambient temperature (20-25°C) whereafter the methanol is drained off. The methanolic solutions are combined and concentrated to 30% on a nanofilter membrane (e.g. such as are available from Osmonics) with an exclusion limit of 300 D.

If desired, ethanol may be used in place of methanol and a lectinase may be added to the methanol for the soaking step.

The concentrated methanolic solutions are further concentrated to 5% wt. dry solids content on a rotary evaporator .

Ion-exchanged water (5 to 10L) is added to the concentrate to produce an essentially aqueous solution which is then mixed thoroughly in about 1:1 volume ratio with ethyl acetate. The phases are separated and traces of ethyl acetate are evaporated from the anthocyanin containing aqueous phase.

The aqueous phase is then loaded onto an ion exchange column (e.g. Amberlite XAD7) which is flushed with ion exchanged water.

Methanol, 0.25% wt . citric acid, is then added to the column until anthocyanins begin to appear in the eluate whereafter elution is continued with pure methanol. In this way the eluate collected is essentially free of free citric acid.

The eluate is evaporated down on a rotary evaporator to a dry solids content of 10% wt. Water is added and the remaining methanol is evaporated off to a dry solids content of 10% wt. Maltodextrin is then added to bring the dry solids content up to 15% wt.

EXAMPLE 2 : Spray-drying

The aqueous solution produced in Example 1 is spray- dried in a pharmaceutical spray dryer (Type SD-4-R-CC) from Niro A/S, Copenhagen, Denmark in a nitrogen atmosphere, using a rotary atomizer, with an inlet temperature of 130-160°C and with a flow rate of 5 to 12 L/hour.

The product is a free flowing deep purple powder which should be stored in a dry atmosphere.

EXAMPLE 3 : Capsules

The spray-dried products from V. myrtillus and R. nigrum, produced in Example 2 (with a water content of less than 5% wt . ) are mixed in a 1:1 weight ratio and filled into cellulose capsules (V caps) to produce an anthocyanin content per capsule of 75 mg or 150 mg.

The capsules are then packed in blister packs.

EXAMPLE 4: CRP pilot study

Design: Ten healthy subjects were included in the study. Each participant took four 75mg blueberry capsules (Medox^®, Medpallett Pharmaceuticals, Norway) daily for four weeks. Blood samples were taken twice: before the subject began to take blueberry capsules, and three-four weeks after the subject had taken four blueberry capsules daily. S-CRP were measured in these blood samples . Statistical Analysis:

To demonstrate whether the average change from before to after three-four weeks intake of blueberry capsules was statistically significant, a two-sided Wilcoxon sign test with 5% level of significance was used.

Description of the subjects:

Five woman and five men, aged 23 - 60 years, all healthy.

None of the subjects reported side effects or displeasure with the capsules .

Table 1 - Average S-CRP levels (mg/L)

p-value p=0.020

Discussion:

Table 1 shows that average S-CRP was reduced from 1.90 mg/L to 1.38 mg/L after three-four weeks daily intake of blueberry capsules and that this reduction was statistically significant (p=0.020). This finding gives a strong indication that four weeks daily intake of four blueberry capsules reduces S-CRP to a considerable extent in healthy persons .

Claims

1. A method of lowering C-Reactive protein levels in a patient, the method comprising administering at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof to the patient.

2. A method for the treatment or prevention of type 2 diabetes, the method comprising administering at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof to a patient.

3. A method for the treatment or prevention of cardiovascular problems, the method comprising administering at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof to a patient .

4. A method according to claim 3 wherein the cardiovascular problem is a myocardial infarction, ischemic stroke, arteriosclerosis, coronary revasculisation or a circulatory problem.

5. a method of lowering the risk of adverse side effects of HRT, said method involving administering at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof to a subject receiving HRT.

6. Use of at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof in the manufacture of a medicament for use in lowering C-Reactive protein levels in a patient.

7. The invention further provides use of at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof in the manufacture of a medicament for lowering the risk of adverse side effects of HRT.

8. Use of at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof in the manufacture of a medicament for use in the treatment or prevention of type 2 diabetes or cardiovascular problems and diseases (including myocardial infarction, ischemic stroke, coronary revasculisation, arteriosclerosis and circulatory problem) .

9. Use of at least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof in the manufacture of a cardioprotective medicament .

10. A method or use as claimed in any one of claims 1 to 9 wherein at least four anthocyanins, at least one of which is a monosaccharide and at least one of which is a disaccharide, are used.

11. A method or use as claimed in any one of claim 1 to 10 wherein at least three monosaccharide anthocyanins are used.

12. A method or use as claimed in any one of claims 1 to 11 wherein at least one monosaccharide anthocyanin in which the saccharide is arabinose is used.

13. A method or use as claimed in any one of claims 1 to 12 wherein at least one disaccharide anthocyanin in which the disaccharide is rutinose is used.

14. A method or use as claimed in any one of claims 1 to 13 wherein anthocyanins with at least two different aglycones are used.

15. A method or use as claimed in any one of claims 1 to 14 wherein anthocyanins with at least four different aglycones are used.

16. A method or use as claimed in any one of claims 1 to 15 wherein anthocyanins with cyanidin, peonidin, delphinidin, petunidin and malvidin aglycone units are used.

17. A method or use as claimed in any one of claims 1 to 16 wherein anthocyanins with cyanidin, peonidin, delphinidin, petunidin, malvidin and pelargonidin aglycone units are used.

18. A method or use as claimed in any one of claims 1 to 17 wherein at least one trisaccharide anthocyanin is used.

19. A method or use as claimed in any one of claims 1 to 18 wherein the at least one anthocyanin is selected from the group consisting of cyanidin-3-O-β-glucoside; cyanidin-3-O-β-galactoside; cyanidin-3-O-α-arabinoside; cyanidin-3-0-β-xyloside; cyanidin-3-0- (6"-0-α-rhamnosyl- β-glucoside) ; cyanidin-3-0- (2"-0-β-glucosyl-β- galactoside) ; cyanidin-3-0- (2"-0-β-glucosyl-β-glucoside) ; cyanidin-3-0- (2"-O-β-xylosyl-β-glucoside) ; cyanidin-3-0- (2"-0-β-xylosyl-β-galactoside) ; cyanidin-3, 5-di-0-β- glucoside; cyanidin-3-O-β-galactoside-5-O-β-glucoside; cyanidin-3-O-α-arabinoside-5-O-β-glucoside; cyanidin-3 - 0- (2 "-O-β-xylosyl-β-glucoside) -5-0-β-glucoside; cyanidin- 3-0- (2"-0-β-xylosyl-6"-0-β-glucosyl-β-galactoside) ; pelargonidin-3 -O-β-glucoside; pelargonidin-3-0- (6"-0-α- rhamnosyl-β-glucoside) ; pelargonidin-3 , 5-di-0-β- glucoside; peonidin-3 -O-β-glucoside; peonidin-3-O-α- arabinoside; peonidin-3 , 5-di-O-β-glucoside; delphinidin- 3-O-β-glucoside; delphinidin-3-O- (6"-0-α-rhamnosyl-β- glucoside) ; delphinidin-3-O- (2 "-O-β-xylosyl-β-glucoside) ; petunidin-3-O-β-glucoside; malvidin-3-O-β-glucoside; malvidin-3, 5-di-0-β-glucoside; and malvidin-3-O-α- arabinoside-5-O-β-glucoside.

20. A method or use as claimed in any one of claims 1 to 19 wherein an anthocyanin in which at least one hydroxy group is acylated is used.

21. A method or use as claimed in claim 20 wherein an anthocyanin having an acylated hydroxy group substituent on a saccharide unit is used.

22. A method or use as claimed in any one of claims 1 to 21 wherein said anthocyanin is derived from fruits or vegetables .

23. A method or use as claimed in any one of claims 1 to 22 wherein said anthocyanin is derived from bilberries and blackcurrants.

24. A method or use as claimed in claim 23 to wherein the anthocyanins are derived from V. myrtillus and R. nigrum in a weight ratio of 0.5:1 to 1:0.5.

25. A method or use as claimed in any one of claims 1 to 24 wherein the at least one anthocyanin is in the form of a composition.

26. A method or use as claimed in claim 25 wherein the at least one anthocyanin is in dosage unit form containing 50 to 250 mg anthocyanin per dose unit.

27. A method or use as claimed in claim 25 or 26 wherein said at least one anthocyanin constitutes at least 50% by weight of the composition excluding the weight of any coating material.

28. A method or use as claimed in any one of claims 25 to 27, wherein the composition is orally ingestible.

29. A method or use as claimed in any one of claims 25 to 28, wherein the composition is in the form of a coated, spray-dried material.

30. At least one anthocyanin or a pharmaceutically acceptable derivative or analogue thereof, for use in lowering CRP levels in a patient, in the treatment or prevention of type 2 diabetes or cardiovascular problems or diseases, as a cardioprotective agent, or to lower the risk of side effects of HRT.

31. A composition comprising at least four anthocyanins, at least one of which is a monosaccharide and at least one of which is a disaccharide, for use in lowering CRP levels in a patient, in the treatment or prevention of type 2 diabetes or cardiovascular problems, as a cardioprotective agent, or to lower the risk of side effects of HRT.

32. A method of lowering C-Reactive protein levels in a patient, the method comprising administering at least one flavone or isoflavone to the patient.

33. A method for the treatment or prevention of type 2 diabetes, the method comprising administering at least one a flavone or isoflavone to a patient.

34. A method for the treatment or prevention of cardiovascular problems, the method comprising administering at least one flavone or isoflavone to a patient .

35. Use of at least one flavone or isoflavone in the manufacture of a medicament for use in lowering C- Reactive protein levels in a patient.

36. Use of at least one flavone or isoflavone in the manufacture of a medicament for use in the treatment or prevention of type 2 diabetes or cardiovascular problems (including myocardial infarction, ischemic stroke, arteriosclerosis, coronary revasculisation and circulatory problem) .

37. Use of at least one flavone or isoflavone in the manufacture of a cardioprotective medicament.