WO2014048888A1

WO2014048888A1 - Dihydroferulic acid and/or dihydrocaffeic acid for use in the treatment of metabolic diseases

Info

Publication number: WO2014048888A1
Application number: PCT/EP2013/069765
Authority: WO
Inventors: Corinne BRENNER; Christian Darimont-Nicolau; Mathieu Renouf; Ornella AVANTI NIGRO
Original assignee: Nestec SA
Current assignee: Nestec SA
Priority date: 2012-09-28
Filing date: 2013-09-24
Publication date: 2014-04-03
Anticipated expiration: 2015-03-28

Description

Dihydroferulic acid and/or dihydrocaffeic acid for use in the treatment of metabolic diseases

Technical field of the invention

The present invention relates to compositions comprising dihydroferulic acid (DHFA) and/or dihydrocaffeic acid (DHCA) for use in the treatment of metabolic diseases such as type 2 diabetes. In addition, the invention relates to food ingredients and food products comprising dihydroferulic acid and/or dihydrocaffeic acid.

Background of the invention

The current diabetes pandemic is a serious problem confronting the health care system. Interestingly, several studies in the course of the last decade have revealed that regular coffee consumption may reduce the risk of developing type 2 diabetes. Nonetheless, the mechanisms and specific compounds responsible for this protective effect are still uncertain.

Hence, an improved prevention and/or treatment of type 2 diabetes would be advantageous, and in particular a more efficient and/or reliable treatment comprising e.g. coffee or grain constituents would be advantageous.

Summary of the invention

In the present study the potential action of coffee on gluconeogenesis has been investigated for a range of pure synthetic coffee constituents such as chlorogenic acids and their metabolites for their potential ability to inhibit hepatic

gluconeogenesis in vitro. Furthermore, the constituents and metabolites have been measured in blood plasma after consumption of coffee.

Thus, an object of the present invention relates to the provision of chlorogenic acids and their metabolites which inhibit hepatic gluconeogenesis. In particular, it is an object of the present invention to provide coffee constituents or metabolites which will be present at relevant plasma concentration.

Thus, one aspect of the invention relates to a composition comprising a compound of the formula I for use as a medicament:

Formula I wherein Rl is methyl or hydrogen, and wherein R2 is methyl or hydrogen.

Another aspect of the present invention relates to a composition comprising a compound of the Formula I for use in the treatment or prevention of metabolic diseases.

Yet another aspect of the present invention is to provide a composition comprising a compound of the Formula I for use in the treatment or prevention of type 2 diabetes in a subject.

Still another aspect of the present invention is to provide a food ingredient comprising a compound of the Formula I.

Yet another aspect relates to a process for enriching or providing a food ingredient with a compound of the Formula I, said method comprising

- providing a sample comprising chlorogenic acids and/or phenolic acids;

- subjecting said sample to enzymatic treatment comprising - incubating the sample with an esterase and/or carbohydrase + feruloyl esterase;

- optionally, incubating the sample with an O-methyltransferase; and

- incubating the sample with a reductase; providing a sample enriched in the compound of the Formula I;

- optionally, mixing or assembling said sample enriched in the compound of the Formula I with a food ingredient; and - providing a food ingredient enriched with a compound of the Formula I.

Brief description of the figures

Figure 1

Figure 1 shows the metabolic fate of chlorogenic acids in humans. For regular coffee consumers, coffee provides a major dietary source of chlorogenic acids. Chlorogenic acids are extensively metabolized following coffee ingestion, with some compounds being absorbed in the stomach/duodenum, while others are absorbed in the small or large intestine. Figure 2

Figure 2 shows the Effect of chlorogenic acids on hepatic gluconeogenesis. H4IIE rat hepatoma cells were incubated for 6 hours in glucose production media (DMEM without glucose, 20 mM lactate, 2 mM pyruvate) with or without chlorogenic acids at indicated concentrations. 10 mM of metformin was included as a control to inhibit gluconeogenesis. Results are expressed as means (+ SEM) from at least three different experiments, each done in duplicate. For each experiment, data were first normalised before pooling. */**/*** p < 0.05/0.01/0.001 as determined by a one-way ANOVA with Dunnetts multiple comparison test, whereby the last three columns were compared to the negative control (= untreated cells stimulated with lactate/pyruvate).

Figure 3

Figure 3 shows the effect of free phenolic acids (caffeic acid (CA) or ferulic acid (FA)) on hepatic gluconeogenesis. H4IIE rat hepatoma cells were incubated for 6 hours in glucose production media (DMEM without glucose, 20 mM lactate, 2 mM pyruvate) with or without phenolic acids at indicated concentrations. 10 mM of metformin was included as a control to inhibit gluconeogenesis. Results are expressed as means (+/- SEM) from at least three different experiments, each done in duplicate. For each experiment, data were first normalised before pooling . */**/*** p < 0.05/0.01/0.001 as determined by a one-way ANOVA with Dunnetts multiple comparison test, whereby the last three columns were compared to the negative control (= untreated cells stimulated with lactate/pyruvate) . Figure 4

Figure 4 shows the effect of proposed colonic metabolites on hepatic

gluconeogenesis (DHCA and DHFA). H4IIE rat hepatoma cells were incubated for 6 hours in glucose production media (DMEM without glucose, 20 mM lactate, 2 mM pyruvate) with or without dihydrophenolic acids at indicated concentrations. 10 mM of metformin was included as a control to inhibit gluconeogenesis. Results are expressed as means (+/- SEM) from at least three different experiments, each done in duplicate. For each experiment, data were first normalised before pooling . */**/*** p < 0.05/0.01/0.001 as determined by a one-way ANOVA with Dunnetts multiple comparison test, whereby the last three columns were compared to the negative control (= untreated cells stimulated with lactate/pyruvate).

Figure 5

Figure 5 shows the effect of sulfated forms of caffeic acids on hepatic

gluconeogenesis. H4IIE rat hepatoma cells were incubated for 6 hours in glucose production media (DMEM without glucose, 20 mM lactate, 2 mM pyruvate) with or without compounds at indicated concentrations. 10 mM of metformin was included as a control to inhibit gluconeogenesis. Results are expressed as means (+/- SEM) from at least three different experiments, each done in duplicate. For each experiment, data were first normalised before pooling . */**/*** p <

0.05/0.01/0.001 as determined by a one-way ANOVA with Dunnetts multiple comparison test, whereby the last three columns were compared to the negative control (= untreated cells stimulated with lactate/pyruvate) . Figure 6

Figure 6 shows the effect of sulfated forms of ferulic acids on hepatic

gluconeogenesis. H4IIE rat hepatoma cells were incubated for 6 hours in glucose production media (DMEM without glucose, 20 mM lactate, 2 mM pyruvate) with or without compounds at indicated concentrations. 10 mM of metformin was included as a control to inhibit gluconeogenesis. Results are expressed as means (+ SEM) from at least three different experiments, each done in duplicate. For each experiment, data were first normalised before pooling. */**/*** p <

0.05/0.01/0.001 as determined by a one-way ANOVA with Dunnetts multiple comparison test, whereby the last three columns were compared to the negative control (= untreated cells stimulated with lactate/pyruvate).

Figure 7

Figure 7 shows the effect of methylated forms of ferulic acids on hepatic gluconeogenesis. H4IIE rat hepatoma cells were incubated for 6 hours in glucose production media (DMEM without glucose, 20 mM lactate, 2 mM pyruvate) with or without compounds at indicated concentrations. 10 mM of metformin was included as a control to inhibit gluconeogenesis. Results are expressed as means (+ SEM) from at least three different experiments, each done in duplicate. For each experiment, data were first normalised before pooling. */**/*** p <

0.05/0.01/0.001 as determined by a one-way ANOVA with Dunnetts multiple comparison test, whereby the last three columns were compared to the negative control (= untreated cells stimulated with lactate/pyruvate). Figures 8-10

Figure 8-10 shows the plasma kinetics of different coffee metabolites after ingestion of low dose (0.5% instant soluble coffee; 2g soluble coffee in 400ml_ water), medium dose (1% instant soluble coffee; 4g soluble coffee in 400ml_ water) and high dose (2% instant soluble coffee; 8g soluble coffee in 400ml_ water).

Figure 11

Figure 11 shows a schematic overview of how DHCA and DHFA may be

enzymatically produced from coffee or wheat bran. The present invention will now be described in more detail in the following.

Detailed description of the invention

In the present study the potential action of coffee on gluconeogenesis has been investigated for a range of pure synthetic coffee constituents or metabolites for their potential ability to inhibit hepatic gluconeogenesis in vitro. Furthermore, the constituents have been measured in blood plasma after consumption of coffee.

Medical use

Thus, an aspect of the present invention relates to a composition comprising a compound of the formula I for use as a medicament:

Several advantages of the compounds according to the present invention have been identified :

1) DHFA and DHCA are naturally occurring metabolites of coffee which may be produced by the gut microbiota after coffee consumption. This is in contrast to the synthetic drug metformin.

2) DHFA and DHCA show anti-hyperglycaemic effect through inhibition of the gluconeogenesis (Example 4). 3) High plasma levels of DHFA and DHCA have been observed after coffee consumption, which is not the case for other similar metabolites (example 6). In the present context it is to be understood that the compounds according to the present invention also covers tautomers, enantiomers and diastereomers of the compounds. Tautomers are isomers (structural isomers) of organic compounds that readily interconvert by a chemical reaction called tautomerization. This reaction commonly results in the formal migration of a hydrogen atom or proton, accompanied by a switch of a single bond and adjacent double bond. The concept of tautomerizations is called tautomerism. Because of the rapid interconversion, tautomers are generally considered to be the same chemical compound .

In the present context it is to be understood that the compounds according to the invention may be enantiomers, diastereomers, as well as tautemers of the compounds according to the invention. Thus, in an embodiment the compounds are enantiomers, diastereomers, or tautemers of the compounds according to the invention. In an embodiment the compound is of the formula I is

wherein Rl is methyl or hydrogen. In another embodiment the compound is dihydroferuiic acid (DHFA), illustrated by Formula II:

Formula II

In yet an embodiment the compound is dihydrocaffeic acid (DHCA), illustrated by Formula III:

The compounds according to the present invention may find different medical uses. Thus, another aspect of the present invention relates to a compound of the Formula I for use in the treatment or prevention of metabolic diseases.

Type 2 diabetes is also a metabolic disease. Thus, in an aspect the invention relates to a composition comprising a compound of the Formula I for use in the treatment or prevention of type 2 diabetes in a subject. The presented data show that compounds of the formula I (and formula II and III) may inhibit

gluconeogenesis as a cause for fasting hyperglycemia. Thus, in an aspect the invention relates to a composition comprising a compound of the Formula I for use in the treatment or prevention of fasting hyperglycemia in a subject. Fasting hyperglycemia is defined as a blood sugar greater than 130 mg/dl (milligrams per deciliter) after fasting for at least 8 hours. The compounds according to the invention may function through inhibition of gluconeogenesis. Thus, an aspect relates to a composition comprising a compound of the Formula I for use in the inhibition of gluconeogenesis in a subject. In an embodiment the gluconeogenesis is hepatic gluconeogenesis. Gluconeogenesis is a metabolic pathway that results in the generation of glucose from non-carbohydrate carbon substrates such as lactate, glycerol, and glucogenic amino acids.

Since the compounds (or composition) according to the present invention are metabolites of compounds present in food products and food ingredients, such ingredients may be enriched with the compounds according to the present invention. Thus, in an embodiment the composition forms part of a food

ingredient. In another embodiment the composition forms part of a food product. The composition according to the present invention may be part of different food products. Thus in an embodiment the food product is selected from the group consisting of beverages, petfoods, and food supplements. In yet an embodiment the beverage is selected from the group consisting of coffee, such as green coffee, tea, milk products, such as skimmed milk and infant milk formulations.

Furthermore, the food or beverage product may be any food or beverage product known in the art. In a preferred embodiment the food or beverage product is a coffee beverage; pure soluble coffee; a soft drink; a dietary supplement; a dairy product; a cereal product; a fruit or vegetable juice product; or a confectionary product, such as a chocolate product, e.g. a chocolate drink. A soluble coffee product may be produced by concentrating and drying the extract of the invention. Before drying, the extract may be mixed with coffee extract that has not been treated to transform chlorogenic acids, e.g. extract of roasted coffee beans, green coffee beans, or both. Methods for producing a soluble coffee product from a coffee extract are well known in the art. When the extract is used for the production of a coffee product, the beans to be extracted may have been subjected to stripping to remove volatile aromas before extraction, e.g. as described in EP-A-1078576. The volatile aromas may then be added back to the extract after the treatment to hydrolyse chlorogenic acids, e.g . after drying, to produce an aromatised soluble coffee product. A soluble coffee product produced from a coffee extract of the invention may be sold as such, or may e.g. be mixed with a creamer and/or sweetener and sold to prepare a coffee beverage comprising creamer and/or sweetener, e.g. cappuccino or cafe latte. As mentioned above the compositions according to the present invention may also find use in petfoods. Thus, in an embodiment the petfood is selected from the group consisting of kibbles and pellets.

Examples of food supplements according to the present invention are capsules and pills.

It is of course to be understood that the compositions according to the present invention are to be provided to a subject. Thus, in an embodiment the subject is a mammal, such as a human, a cat, a dog or a horse.

Food ingredient

As previously mentioned the compounds according to the present invention are metabolites of constituents present in e.g. coffee or grains. However, the compounds according to the present invention are not naturally present in food product but are metabolites which may be formed after consumption e.g . by the gut microbiota. Thus an aspect of the present invention relates to a food ingredient comprising a compound of the Formula I. In another aspect the invention relates to a food ingredient enriched with a compound of the Formula I. Such ingredient may find medical use as described above.

The specific concentration of the compounds of formula I may vary in the food ingredient. Thus, in an embodiment the level of the compound of the Formula I is in the range 0.0001 μΜ - 100 μΜ, such as 0.001 μΜ - 10 μΜ, such as 0.01 μΜ - 10 μΜ, or such as 0.1 μΜ - 10 μΜ.

The compounds according to present invention which may form part of a food ingredient may be provided by different methods. Thus, in an embodiment at least part of the compound of the Formula I is chemically and/or enzymatically produced . Figure 11 illustrates how such compounds may be enzymatically produced from a natural source. Food product

The food ingredient according to the present invention may form part of a food product. Thus, in an aspect the invention relates to a food product comprising the food ingredient according to the present invention. In yet an aspect the invention relates to a composite food product, wherein at least one part of the composite food products comprises the food ingredient according to the present invention. This may be the case where a food product is constituted of multiple independent parts (composite), where e.g. only one of the parts comprises the food ingredient according to the invention.

Process

The compounds according to the present invention may be synthetically produced . Thus, an aspect of the present invention relates to a process for enriching or providing a food ingredient with a compound of the Formula I, said method comprising

- providing a sample comprising chlorogenic acids and/or phenolic acids;

- subjecting said sample to enzymatic treatment comprising

- incubating the sample with an esterase and/or carbohydrase +

feruloyl esterase;

- optionally, incubating the sample with an O-methyltransferase; and

Figure 11 shows a schematic overview of how DHCA and DHFA may be

enzymatically produced from coffee or wheat bran. Thus, in an embodiment coffee such as 5-CQA is present in the sample and the enzymes are esterase and a reductase and the enriched product comprises DHCA. As illustrated in figure 11 part of the sample may be modified to DHFA by the addition of 0- methyltransferase. It is to be understood that the enzymes may be added sequentially or simultaneously as illustrated in figure 11. In another embodiment whole grains such as wheat grains and/or ferulic acid is present in the sample and the enzymes are carbohydrase (such as xylanase), feruloyl esterase and reductase and the enriched product comprises DHFA. In another embodiment the carbohydrase is xylanase.

As mentioned above the sample may be a natural source such as grains or coffee. Thus, in an embodiment the sample is provided from a natural source. In yet an embodiment the natural source is whole grain, such as wheat grain or coffee, such as green coffee, roasted coffee or a polyphenol extract. In yet another

embodiment the sample the natural source is selected from the group consisting of blackberry, raspberry, black currant, strawberry, blueberry, kiwi, cherry, plum, aubergine, apple, pear, chicory, artichoke, potato, corn flour, flour: wheat, rice, oat, cider, and coffee. All these sources are known to comprise chlorogenic acids.

To make the process more efficient the sample may be pre-treated before enzymatic treatment. Thus, in an embodiment the sample is pre-treated before enzymatic treatment. In yet another embodiment the pre-treatment is

solubilisation.

As mentioned above the metabolites according to the present invention may be processed by the gut flora after consumption of a food product comprising precursors. Such microorganisms may be used to process e.g. coffee by providing the relevant enzymes required to produce the compounds according to the present invention. Thus, in an embodiment the enzymatic steps are performed by micro-organisms. In yet another embodiment the micro-organisms are selected from the group consisting of bacteria, yeast, and fungi.

The process according to the present application may comprise further steps. Thus, in an embodiment the food ingredient enriched in a compound of the Formula I is mixed or assembled with a food product, thereby providing a food product enriched in a compound of the Formula I. The wording "assembling" relates to the situation where the food product and food ingredient are not mixed, but instead assembled, e.g . in the case the ingredient is a topping or part of a layered product.

The food ingredient according to the present invention may be produced by a process according to the present invention. Thus, an aspect relates to a food ingredient obtainable by a process according to the present invention. Similarly the invention relates to a food product obtainable by a process according to the present invention It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention .

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples. Examples

Example 1

Background experimental protocols and setup.

The aim of this study was to investigate the inhibitory activity of coffee-derived compounds, as well as their metabolites, on hepatic gluconeogenesis.

Background

Regular coffee consumption is associated with a lower risk of type 2 diabetes. Paradoxically, it has been found that ingestion of caffeine may lead to an acute elevation in blood glucose and insulin levels, whereas the consumption of decaffeinated coffee may reduce the blood glucose response. This finding indicates that compounds other than caffeine may be responsible for the beneficial effect of coffee on glucose homeostasis. Apart from caffeine, coffee contains a complex profile of several potentially bioactive compounds, including chlorogenic acids, trigonelline, or cyclic dipeptides (= diketopiperazines).

Chlorogenic acids

Chlorogenic acids are the second major components in coffee after caffeine, accounting for 6-10% of dry matter. They are a family of esters which are formed by the linkage of a hydroxycinnamate (e.g. CA, FA) to a quinic acid . The hydroxycinnamate can be attached to a 3-, 4- or 5-position of the quinic moiety and depending on the hydroxycinnamate, the resulting structure is called caffeoylquinic acid (CQA) or feruloylquinic acid (FQA), respectively (Figure 1, (1)). The different isoforms of CQAs and FQAs can be detected in human plasma as early as 30 minutes and up to 1 hour following coffee consumption, with a maximal plasma concentration (Cmax) of approximately 50 nM (example 6).

Without being bound by theory, it is believed that during this early absorption, which probably occurs in the stomach/small intestine (Figure 1,(2)), only a small proportion of chlorogenic acids enter the bloodstream. The majority is passed on to the small intestine (Figure 1,(3)), where the ester bond of CQAs and FQAs is cleaved by esterases into phenolic acids and quinic acids. The released free phenolic acids (e.g. CA, FA) may enter the circulation where they can be detected at ~1 hour after ingestion with a Cmax of 150 - 300 nM. Nonetheless, it is estimated that 70% of ingested chlorogenic acids finally end up in the large intestine (Figure 1, (4)), where they undergo extensive metabolism through interaction with the colonic microbiota. As shown in example 6 the metabolites DHCA and DHFA start appearing in human plasma after ~ 4 hours and can be detected up to 24 hours following ingestion of a single cup of coffee, with a Cmax of < 1 μΜ.

Once in the bloodstream, chlorogenic acids and their metabolites are subjected to phase II metabolism in the liver (Figure 1, (5)), which leads to inactivation and urinary excretion of the compounds. This biotransformation involves methylation, sulfation or glucuronidation steps.

METHODOLOGY AND TRIALS :

Cell model :

All experiments were done using the H4IIE rat hepatoma cell line. Chemicals and reagents

H4IIE cells: ATCC, ref. CLR-1548

EMEM : ATCC, ref. 30-2003

Glucose-free DMEM : Milan analytica AG, ref. P04-01548

Metformin : Merck; Metformine-HCI; Merck Sante, Centre de production de Calais, Lot no C13294

D-glucose: Merck, ref. 1.08337

Sodium pyrufate: SIGMA P5280

Sodium lactate: SIGMA L7022

Cytotoxicity detection kit (LDH) : Roche 11644793001

Protocol :

A cellular bioassay was established according to a method described by Okamoto with some modifications. Briefly, H4IIE cells pre-cultured at high density were seeded into a 12-well plate at a density of 3 x 105 cells/cm2 and cultured for 24 h in EMEM supplemented with 10% FCS. Thereafter, cells were washed three times with PBS, followed by pre-incubation in phenol red-free DMEM containing 1 g/L glucose for another 16 h. The assay was started by exchanging the medium for glucose-free DMEM in the presence or absence of gluconeogenesis-inducing substrates (2 mM sodium pyruvate and 20 mM lactate). Coffee compounds were added at different concentrations (1 μΜ, 5 μΜ, 10 μΜ) and tested in duplicate. 10 mM of metformin was included as a positive control for inhibition of

gluconeogenesis. The conditioned media was removed after 6 h of incubation to assay glucose content using a fluorometric glucose assay kit (BioVision). The same time, LDH enzyme activity was determined by a cytotoxicity detection kit to exclude the possibility of cytotoxic effects. Components were tested in at least three independent experiments performed at different cell passages. Compounds:

The following classes of compounds were tested :

1) Chlorogenic acids absorbed in stomach/small intestine: 5-Caffeoylquinic acid (5-CQA); 3-Feruloylquinic acid (3-FQA), 4-FQA, 5-FQA

2) Chlorogenic acid derivatives absorbed in the stomach/small intestine:

Caffeic acid (CA), Ferulic acid (FA) 3) Chlorogenic acid metabolites absorbed in the colon : Dihydroferulic acid (DHCA), Dihydrocaffeic acid (DHFA)

4) Phase II liver metabolites of chlorogenic acids: CA 3-0-sulfate,CA-4-0- sulfate, DHCA 3-O-sulfate, FA 4-O-sulfate, DHFA 4-O-sulfate, Me-FA, Me- DHFA

Abbreviations:

CA Caffeic acid

CQA Caffeoylquinic acid

DHCA Dihydrocaffeic acid

DHFA Dihydroferulic acid

FA Ferulic acid

FQA Feruloylquinic acid Analysis

Results are expressed as means ± SEM from at least three independent experiments, whereby each experiment was done in duplicate. For each experiment, data were separately normalised to untreated, pyruvate/lactate- stimulated control cells before pooling . Statistical analysis was performed using a one-way ANOVA with Dunnetts multiple comparison, whereby pyruvate/lactate- stimulated cells pre-treated with test compounds were compared to untreated cells that were only stimulated with the pyruvate/lactate mixture.

Example 2

Test of the glucose-suppressing effect of pure chlorogenic acid isomers including 5-CQA, 3 -FQA, 4 -FQA and 5-FQA.

These isomers also appear in human plasma following coffee consumption Example 6. Metformin, a widely used anti-diabetic agent for type 2 diabetes, was included as a positive control.

Experiments were performed as described in example 1. Results

As shown in Fig 2, stimulation of untreated cells with 2 mM pyruvate and 20 mM lactate resulted in an approximately 3-fold increase in gluconeogenesis. As expected, the induction of gluconeogenesis was blocked with the biguanide drug metformin. On the other hand, pre-treating the cells with 5-CQA led to a reduction of 65 - 85 % at concentrations of 5- 10 μΜ, while FQAs were found to be less effective in suppressing glucose output (Fig 2). Nevertheless, 3-FQA and 4-FQA still caused a moderate reduction in glucose production when used at a

concentration of 10 μΜ.

Conclusion

These compounds have a glucose-suppressing effect. However since the plasma levels are low they are not considered suitable in a treatment protocol (see example 6).

Example 3

Test of the glucose-suppressing effect of caffeic acid (CA) or ferulic acid (FA). Experiments were performed as described in example 1.

Phenolic acid equivalents such as CA and FA can be detected in human plasma where they peak at approximately one hour following coffee ingestion (example 6). Results

When adding caffeic acid (CA) or ferulic acid (FA) to the cell culture assay, an inhibitory effect on gluconeogenesis at concentrations of 5 - 10 μΜ was observed (Fig 3). Conclusion

Caffeic acid (CA) and ferulic acid (FA) have a glucose-suppressing effect. However since the plasma levels are low they are not considered suitable in a treatment protocol (see example 6). Example 4

Tests on DHCA and DHFA.

Experiments were performed as described in example 1.

Results

As shown in Fig 4, DHCA and DHFA significantly inhibited gluconeogenesis at all concentrations tested. Importantly, these metabolites are already effective at their observed physiological concentration around 1 μΜ (20-25% inhibition) which stands in contrast to the previous compounds tested (see example 6).

Conclusion

DHCA and DHFA have a glucose-suppressing effect.

Example 5

Effect of hepatic chlorogenic acid metabolites

Following ingestion, chlorogenic acids and their derivatives are subjected to phase II metabolism which leads to sulfated, methylated or glucuronidated structures.

Results

As shown in figures 5-7, several sulfated or methylated forms of CA and FA/DHFA were included in the analysis. The inhibitory potential of CA/DHCA and FA/DHFA was removed upon conjugation, with the exception of CA-3-O-sulfate, which still had a minor effect (20% inhibition) on gluconeogenesis at a concentration of 10 μΜ.

Conclusion

Neither of the tested compounds have a major glucose-suppressing effect. Example 6

Plasma levels of the most promising compounds tested in example 2-5 following ingestion of coffee were determined. Methods

Plasma levels of different coffee metabolites were measured after consumption of different doses of soluble coffee Plasma levels of different Coffee doses. Low dose =0.5% coffee in 400 ml, medium dose = 1% coffee in 400 ml and high dose =2% coffee in 400 ml (figures 8-10).

Conclusion

The data provided in figures 8-10 show that only DHFA and DHCA show plasma levels which may be considered as clinical relevant after 10 hours, whereas the level of the other tested metabolites decreases within a shorter period .

Overall conclusion

Example 2 demonstrates that the chlorogenic acids 5-CQA, 3-FQA and 4-FQA have a moderate effect on gluconeogenesis, with 5-CQA having the strongest effect. However, despite being major components in coffee, only trace amounts of CQAs and FQAs appear in the circulation following coffee consumption. Example 6 shows that these isomers can reach concentrations of up to around 50 nM in human plasma following ingestion of 400 ml of 2% soluble instant coffee (figure 8).

Hence, although having observed a moderate effect of chlorogenic acids on gluconeogenesis, the physiological concentration of these compounds is not sufficient to explain the potential beneficial effect that coffee might have on gluconeogenesis.

When investigating the potential of the phenolic acids CA and FA to inhibit glucose production (example 3), a significant effect at concentrations above 5 μΜ was observed . However, as for chlorogenic acids, this concentration were not of physiological relevance since the measured plasma level after coffee intake is much lower (Example 6 and figure 9). The most intriguing finding of this study is that the colonic metabolites DHCA and DHFA inhibit gluconeogenesis at concentrations of > 1 μΜ (example 4). Notably, Example 6 demonstrates that these compounds are present in human plasma in much higher concentration than the parent compounds. Indeed, plasma levels of DHCA and DHFA can be as high as approximately 1 μΜ following coffee

consumption. In addition, the results show that both compounds are relatively stable from a metabolic point of view. Based on the present study, it is proposed that DHCA and DHFA are important mediators of the anti-diabetic effect of coffee via suppression of gluconeogenesis.

Claims

1. A composition comprising a compound of the formula I for use as a

medicament:

2. A composition comprising a compound of the Formula I for use in the treatment and/or prevention of metabolic diseases.

3. A composition comprising a compound of the Formula I for use in the treatment and/or prevention of type 2 diabetes in a subject.

4. The composition according to any of the preceding claims, wherein the compound of the formula I is

wherein Rl is methyl or hydrogen.

5. The composition according to any of claim 1-4, wherein said composition forms part of a food ingredient.

6. The composition according to any of claims 1-5, wherein said composition forms part of a food product.

7. The composition according to claim 6, wherein the food product is selected from the group consisting of beverages, petfood, and food supplements.

8. A food ingredient comprising a compound of the Formula I.

9. The food ingredient according to claim 9, wherein the level of the compound of the Formula I is in the range 0.0001 μΜ - 100 μΜ, such as 0.001 μΜ - 10 μΜ, such as 0.01 μΜ - 10 μΜ, or such as 0.1 μΜ - 10 μΜ.

10. A food product comprising the food ingredient according to claim 8 or 9.

11. A process for enriching or providing a food ingredient with a compound of the Formula I, said method comprising

- providing a sample comprising chlorogenic acids and/or phenolic acids;

- subjecting said sample to enzymatic treatment comprising - incubating the sample with an esterase and/or carbohydrase +

feruloyl esterase;

- optionally, incubating the sample with an O-methyltransferase; and

- optionally, mixing or assembling said sample enriched in the compound of the Formula I with a food ingredient; and providing a food ingredient enriched with a compound of the Formula I.

12. The process according to claim 11, wherein the sample is provided from a natural source.

13. The process according to 12, wherein the natural source is whole grain, such as wheat grain or coffee, such as green coffee roasted coffee or a polyphenol extract.

14. The process according to any of claims 11-13, wherein the enzymatic steps are performed by micro-organisms.

15. A food ingredient obtainable by a process according to any of claims 11-14.