EDIBLE COMPOSITION

Field of the invention

The present invention relates to an edible composition capable of reducing fructose uptake.

Background of the invention

Fructose, or fruit sugar, is a simple ketonic monosaccharide found in many plants, where it is often bonded to glucose to form the disaccharide sucrose. It is one of the three dietary monosaccharides, along with glucose and galactose, that are absorbed directly into the bloodstream during digestion. Pure, dry fructose is a very sweet, white, odorless, crystalline solid and is the most water-soluble of all the sugars. Fructose is found in honey, tree and vine fruits, flowers, berries, and most root vegetables. Commercially, fructose is frequently derived from sugar cane, sugar beets, and maize. Crystalline fructose is the monosaccharide, dried, ground, and of high purity. High- fructose corn syrup is a mixture of glucose and fructose as monosaccharides. Sucrose is a compound with one molecule of glucose covalently linked to one molecule of fructose. All forms of fructose, including fruits and juices, are commonly added to foods and drinks for palatability and taste enhancement, and for browning of some foods, such as baked goods.

Research indicates that excessive fructose consumption is a cause of insulin resistance, obesity, elevated LDL cholesterol and triglycerides, leading to metabolic syndrome, type 2 diabetes and cardiovascular disease. The European Food Safety Authority has stated that fructose noted that "high intakes of fructose may lead to metabolic complications such as dyslipidaemia, insulin resistance and increased visceral adiposity".

Eating fructose causes a post-prandial spike in fructose levels and while the body is able to cope with a certain level of fructose, above this level deleterious effects such as those listed may occur. It is therefore desirable to "flatten the fructose spike" in order to bring fructose down to the level that the body can cope with such that the deleterious effects are prevented. In essence, what is required is that the uptake of fructose is slowed. There is therefore a need for compositions that provide such an effect.

US 20140128585 discloses a fructose absorption inhibitor comprising a hydrolyzable tannin as an active component. WO12008474 discloses a fructose absorption inhibitor that has a monoterpene as the active ingredient, and a preventative or therapeutic agent that is for symptoms or maladies caused by overconsumption of fructose.

We have now surprisingly found that if certain fructose uptake inhibitors are provided in combination with a certain glucose uptake inhibitor then the combination delivers synergistically enhanced reduction of fructose uptake. Summary of the invention

Accordingly in a first aspect the present invention provides an edible composition comprising from 100 to 500mg of the glucose inhibitor Hesperetin and 100-500mg of Eucalyptol. Preferably the edible composition comprises from 125 to 450mg of Hesperetin, more preferably 150 to 400mg, even more preferably 175 to 350mg, yet more preferably 200 to 300mg, more preferably still 225 to 250mg.

Preferably the edible composition comprises from 125 to 450mg of Eucalyptol, more preferably 150 to 400mg, even more preferably 175 to 350mg, yet more preferably 200 to 300mg, more preferably still 225 to 250mg.

Preferably the molar ratio of Hesperetin to Eucalyptolis in the range 4:1 to 1 :4, preferably 3:1 to 1 :3, most preferably 2:1 to 1 :2, even more preferably 1.5: 1 to 1 : 1.5, most preferably 1.25:1 to 1 :1.25. Preferably the edible composition comprises up to 75 wt% fructose, more preferably up to 50 wt% fructose, even more preferably up to 40 wt% fructose, yet more preferably up to 30 wt% fructose, more preferably still up to 20 wt% fructose. Preferably the edible composition comprises at least 1 wt% fructose, more preferably at least 2 wt% fructose, even more preferably at least 5 wt% fructose, yet more preferably at least 10 wt% fructose, more preferably still at least 15 wt% fructose.

Preferably the edible composition is a frozen confection such as an ice cream, or a beverage.

The edible composition may be in the form of a packaged beverage comprising no more than 99.95 wt% water. The edible composition may also be in the form of a dry powder contained in a sachet, the dry powder suitable for addition to a meal.

In a second aspect, the present invention provides an edible composition comprising from 100 to 500mg of Hesperetin and 100-500mg of Eucalyptol or Vanillin or a combination thereof for use in reducing the uptake of fructose. Preferably the edible composition for use in the second aspect comprises from 125 to 450mg of Hesperetin, more preferably 150 to 400mg, even more preferably 175 to 350mg, yet more preferably 200 to 300mg, more preferably still 225 to 250mg.

Preferably the edible composition comprises from 125 to 450mg of Eucalyptol or Vanillin or a combination thereof, more preferably 150 to 400mg, even more preferably 175 to 350mg, yet more preferably 200 to 300mg, more preferably still 225 to 250mg.

Preferably the molar ratio of Phloretin to Eucalyptol or Vanillin or a combination thereof is in the range 4:1 to 1 :4, preferably 3:1 to 1 :3, most preferably 2:1 to 1 :2, even more preferably 1 .5:1 to 1 :1 .5, most preferably 1 .25:1 to 1 :1.25.

Detailed description of the invention Fructose absorption occurs in the small intestine via the GLUT-5 (fructose only) transporter, the GLUT2 transporter, for which it competes with glucose and galactose, and potentially a number of GLUT transporters of similar structure. Over-consumption of fructose, inhibition of GLUT2, GLUT5 and other transporters of similar structure by phytochemicals, such as flavonoids, or other issues, may result in delivery of unabsorbed fructose into the large intestine, which will cause more water to be drawn into the large intestine through the process of osmosis causing diarrhoea. In addition, the excessive fructose becomes a source of nutrients for the gut flora resulting in a higher production of short chain fatty acids, hydrogen, carbon dioxide and other gases due to fermentation. This increase of gas causes gastrointestinal side effects that mimic irritable bowel syndrome. For this reason, it is desirable to slow the uptake of fructose rather than prevention.

Excess fructose consumption has also been hypothesized to be a cause of insulin resistance, obesity, elevated LDL cholesterol and triglycerides, leading to metabolic syndrome. In preliminary research, fructose consumption was correlated with obesity and encouraged visceral adipose tissue deposition in humans.

Studies indicate that there may be an increased risk of cardiovascular disease from a high intake of fructose. Studies have also associated high fructose consumption with increased incidence of hypertension, both acutely and in the long term in subjects without a history of hypertension.

Yet another study in humans concluded that fructose and sucrose "produced significantly higher fasting plasma triglyceride values than did the glucose diet in men" and "...if plasma triacylglycerols are a risk factor for cardiovascular disease, then diets high in fructose may be undesirable". A study later confirmed this by showing that consuming beverages with high levels of high-fructose corn syrup caused heightened levels of LDL cholesterol, non-HDL cholesterol, apolipoprotein B, all of which are lipid/lipoproteins risk factors for cardiovascular disease.

Excessive fructose consumption may also contribute to the development of non-alcoholic fatty liver disease. A 2008 study found a risk of incident gout associated with high consumption of fructose or fructose-rich foods. Compared with consumption of high glucose beverages, drinking high-fructose beverages with meals results in lower circulating insulin and leptin levels, and higher ghrelin levels after the meal. Since leptin and insulin decrease appetite and ghrelin increases appetite, some researchers suspect that eating large amounts of fructose increases the likelihood of weight gain.

For all these reasons, it is evident that it is desirable that the uptake of fructose from the diet be reduced or at least slowed down such that the body can process the fructose with a minimisation of the deleterious effects associated with elevated consumption.

The present invention has surprisingly found that an edible composition with specific combinations of fructose inhibitors Eucalyptol or Vanillin with glucose inhibitor Hesperetin is capable of retarding the uptake of fructose. Hesperetin is the 4'-methoxy derivative of eriodictyol, and has the following structure:

Eucalyptol is a natural organic compound known by a variety of synonyms: 1 ,8-cineol, 1 ,8-cineole, cajeputol, 1 ,8-epoxy-p-menthane, 1 ,8-oxido-p-menthane, eucalyptol, eucalyptole, 1 ,3,3-trimethyl-2-oxabicyclo[2,2,2]octane, cineol, and cineole with the following structure:

Vanillin is a phenolic aldehyde with the following structure:

Although it may have been observed that one of Hesperetin or Eucalyptol or Vanillin may have an effect on glucose or fructose uptake, the combination of Hesperetin with Eucalyptol or Hesperetin with Vanillin actually provides a synergistic improvement in fructose uptake inhibition. That is to say that the fructose uptake inhibition achieved by Hesperetin with Eucalyptol in combination is greater than the inhibition that would be expected merely from the additive effect of these compounds. The same is true for Hesperetin with Vanillin.

As will be seen below, the synergistic effects have been demonstrated in vivo and through consideration of absorption, distribution, and metabolism of Hesperetin, Eucalyptol, and Vanillin the edible composition of the invention therefore comprises from 100 to 500mg of Hesperetin and 100-500mg of Eucalyptol or Vanillin or a combination thereof.

Preferably the edible composition comprises from 125 to 450mg of Hesperetin, more preferably 150 to 400mg, even more preferably 175 to 350mg, yet more preferably 200 to 300mg, more preferably still 225 to 250mg.

Preferably the edible composition comprises from 125 to 450mg of Eucalyptol or Vanillin or a combination thereof, more preferably 150 to 400mg, even more preferably 175 to 350mg, yet more preferably 200 to 300mg, more preferably still 225 to 250mg. Preferably the molar ratio of Hesperetin to Eucalyptol or Vanillin or a combination thereof is in the range 4:1 to 1 :4, preferably 3:1 to 1 :3, most preferably 2:1 to 1 :2, even more preferably 1 .5:1 to 1 :1 .5, most preferably 1 .25:1 to 1 :1.25. Since the edible composition addresses issues associated with the uptake of fructose the composition itself may comprise high fructose for example up to 75 wt% fructose, more preferably up to 50 wt% fructose, even more preferably up to 40 wt% fructose, yet more preferably up to 30 wt% fructose, more preferably still up to 20 wt% fructose. Preferably the edible composition comprises at least 1 wt% fructose, more preferably at least 2 wt% fructose, even more preferably at least 5 wt% fructose, yet more preferably at least 10 wt% fructose, more preferably still at least 15 wt% fructose.

Any form of edible composition may be suitable for the present invention. The edible composition of the invention could be consumed as a supplement to a high fructose meal to retard fructose uptake. Alternatively, the edible composition of the invention could be comprised as part of another food product. Preferably the edible composition is a frozen confection such as an ice cream, or a beverage. It will be appreciated that the edible composition is intended to be consumed completely in a single sitting, i.e. as a single meal or similar in order to deliver the required levels of Hesperetin and Eucalyptol and/or Vanillin

The edible composition may also be in the form of a packaged beverage comprising no more than 99.95 % w/w water. The edible composition can be in the form of a dry powder contained in a sachet, the dry powder suitable for addition to a meal.

The invention also provides an edible composition comprising from 100 to 500mg of Hesperetin and 100-500mg of Eucalyptol or Vanillin or a combination thereof for use in reducing the uptake of fructose. The edible composition for use in reducing the uptake of fructose can comprise from 125 to 450mg of Hesperetin, more preferably 150 to 400mg, even more preferably 175 to 350mg, yet more preferably 200 to 300mg, more preferably still 225 to 250mg. The edible composition for use in reducing the uptake of fructose can comprise from 125 to 450mg of Eucalyptol or Vanillin or a combination thereof, more preferably 150 to 400mg, even more preferably 175 to 350mg, yet more preferably 200 to 300mg, more preferably still 225 to 250mg.

The molar ratio of Hesperetin to Eucalyptol or Vanillin or a combination thereof in the edible composition for use in reducing the uptake of fructose may be in the range 4:1 to 1 :4, preferably 3:1 to 1 :3, most preferably 2:1 to 1 :2, even more preferably 1 .5:1 to 1 :1 .5, most preferably 1.25:1 to 1 :1 .25.

Preferably, the composition is used to reduce post-prandial fructose uptake. Preferably the use is for reduction of post-prandial fructose uptake in a non-diabetic person.

The invention may also provide a method of reducing post-prandial fructose uptake in a non-diabetic person comprising the steps of:

(a) oral administration of the composition of the first aspect of the invention to the non-diabetic person; and

(b) oral administration of a composition comprising fructose to the non-diabetic person;

wherein step (a) is simultaneous with, precedes by 0 to 90, preferably 0 to 60 minutes, or follows by 0 to 30 minutes step (b).

Similarly, the invention may provide a method for treating a person in need thereof for type 2 diabetes comprising the steps of:

(a) oral administration of the composition of the first aspect of the invention to the person in need thereof; and

(b) oral administration of a composition comprising fructose to the person in need thereof;

wherein step (a) is simultaneous with, precedes by 0 to 90, preferably 0 to 60 minutes, or follows by 0 to 30 minutes step (b). In a final aspect the invention could provide a composition according to the first aspect of the invention for use in the treatment of dyslipidaemia, insulin resistance, increased visceral adiposity, or type 2 diabetes. Examples

Fructose uptake model

To identify potential natural plant phytochemicals which inhibit the uptake of fructose, a model was used based on total cumulative fructose transport across differentiated Caco- 2 monolayers seeded onto a trans-well permeable inserts. The model was modified from the paper 'New and better protocols for a short-term Caco-2 cell culture system' by Yamashita et. al. (2002) to optimise for GLUT5, the presumed major fructose gut transporter. Caco-2 cells were seeded into cell culture inserts (2.5x105 cells/ well for 24 well plates and 1 x105 for

96 well plates) coated with collagen II matrix in growth medium:

DMEM+Glutamax-1 (contains 4.5g/L D-Glucose

+ 25mM Hepes) [Invitrogen #32430027]

- + 10% (100x) Non Essential Amino Acids[lnvitrogen #1 1 14035]

+ 10% 100mM Sodium pyruvate solution[Sigma S8636]

+ 10% Fetal Bovine Serum[Sigma F7524]

30ml of Growth medium was added to the feeder plate below the inserts. The cells were left to attach over 24hrs at 37oC 5% C02. Following a gentle wash (both inserts and feeder plate) in PBS, the cells were incubated in Differentiation Medium:

BD Entero-STIM™ Enterocyte Differentiation Medium, [BD Biosciences #05495] +(1000x) MITO+™ Serum Extender solution, [BD Biosciences #356007] for a further 48hrs, followed by 72hrs in growth medium.

Assay

Cell monolayers were washed gently in PBS(+) and the inserts transferred to a new standard tissue culture plate. The cells were incubated with fresh PBS(+) for 30-60mins at 37°C 5% C02. Cell inserts were then transferred to a new plate, and potential actives or treatments were added with 25mM fructose and 25mM glucose in PBS(+) to the cell/inserts and PBS was added to the collection well for 120-150min. PBS(+) from the collection well was then analysed for glucose and fructose content.

Lucifer yellow transport as a measure of monolayer integrity and insert membrane integrity was measured by addition Lucifer yellow [Sigma L0144]. The cell inserts were transferred to a new plate, the supernatant gently aspirated from the cells and replaced with 100uM Lucifer Yellow solution, PBS(+) added to the collection well and incubated at 37oC 5% C02 for 1 hr.

Permeability of the membranes to Lucifer Yellow was checked by measuring the fluorescence of the samples at 485 excitation & 530 emission. Fructose and glucose uptake assay

The fructose and glucose assay was based on Campbell et al. (1999) "Cost-effective colorimetric microtitre plate enzymatic assays for sucrose, glucose and fructose in sugarcane tissue extracts", J Sci Food Agric 79: 232-236. Glucose and fructose were first converted to glucose-6-phosphate and fructose-6- phosphate respectively in the presence of 1.25mg/ml ATP (Sigma, A26209) and 1 .6U/m Ihexokinase (Sigma, H6380).

Glucose-6-phosphate was converted to NADH in the presence of 0.54mg/ml NAD+ (Sigma, N6522) and 0.72U/ml glucose-6-phosphate dehydrogenase (Sigma, G8529) in the same reaction mix.

After 15 min the concentration of NADH is measured at 340nm to give a measure of glucose when compared to a glucose standard curve. Fructose-6-phosphate was then converted to NADH by the addition of 5.4U/ml phosphoglucose isomerase (Sigma, P5381 ). After 15 min the concentration of NADH is measured at 340nm to give a measure of glucose + fructose. Fructose concentration is determined by subtracting the glucose concentration and compaing to a fructose compared to a standard curve. Test Compounds

Initially, 14 phytonutrients were tested for their ability to inhibit fructose uptake using the model described above. These phytonutrients were:

- gallic acid; ellagic acid; pyrogalol; astilbin; quercetin-3-glucoside; cinnamaldehyde; eucalyptol; vanillin; lectin glycine; aribinogalactan; phloretin; luteolin; phloridzin; luteolin-7-glucoside.

Of these 14 phytonutrients, 6 were found to have some inhibitory activity on fructose uptake at 150-300uM. These 6 fructose uptake inhibitors were then tested for synergistic activity with four known phytonutrient glucose transporter GLUT2 inhibitors. The test compounds are therefore given in Table 1.

Table 1 - Test compounds

Synergistic activity was tested for in a cross-wise design such that each glucose uptake inhibitor was tested with each fructose uptake inhibitor with 2-3 repeats over several experiments. Each compound was solubilised in DMSO at 150mM and used at a working dilution of 150uM. DMSO at 0.1 % was also tested as a vehicle control and used as the threshold value of fructose uptake - i.e. DMSO was deemed to be a notional 100% and so if a test compound, or pair of test compounds, caused a fructose uptake of greater than 100% this was an increase in fructose uptake, and if a test compound, or pair of test compounds caused a fructose uptake of less than 100% this was a decrease in fructose uptake.

The results are shown in Table 2 in which:

Fructose uptake is shown as a percent fructose uptake vs the DMSO control The Glucose Inhibitor is shown by "(G)"

The Fructose Inhibitor is shown by "(F)"

Synergistic combinations are shown in bold and denoted by "(S)" in the "Fructose uptake (%)" column.

o Combinations are defined as synergistic when the fructose uptake inhibition achieved by (G) in pairwise combination with (F) is greater than the inhibition that would be expected merely from the additive inhibitory effect of (G) + (F).

Compounds tested Concentrations Fructose Standard of compound uptake error

tested (%)

Phloretin (G) 150uM 1 12.1 29.6

Vanillin (F) 150uM 84.9 16.7

Phloretin + Vanillin 150uM + 150uM 46.4 (S) 1 .95

Hesperetin (G) 150uM 104.6 27.8

Vanillin (F) 150uM 84.9 16.7

Hesperetin + Vanillin 150uM + 150uM 45.3 (S) 18.5

Hesperetin (G) 150uM 104.6 27.8

Cinnamaldehyde (F) 150uM 130.6 64.8

Hesperetin + Cinnamaldehyde 150uM + 150uM 89.3 64.8

Luteolin (G) 150uM 108.6 37.9

Cinnamaldehyde (F) 150uM 130.6 64.8

Luteolin + Cinnamaldeyde 150uM + 150uM 128.8 53.8

Resveratol (G) 150uM 99.1 58.7

Cinnmaldehyde 150uM 130.6 64.8

Resveratol + Cinnamaldehye 150uM + 150uM 85.6 42.1

Luteolin (G) 150uM 108.6 37.9

Eucalyptol (F) 150uM 1 17.2 52.3

Luteolin + Eucalyptol 150uM + 150uM 43.4 (S) 24.2

Resveratol (G) 150uM 99.1 58.7

Eucalyptol (F) 150uM 1 17.2 52.3

Resveratol + Eucalyptol 150uM + 150uM 131.1 77.4

Phloretin (G) 150uM 121.9 39.2

Cinnamaldehyde (F) 150uM 127.9 58.7

Phloretin + Cinnamaldehyde 150uM + 150uM 140.4 52.4 Phloretin (G) 150uM 121.9 39.2

Eucalyptol (F) 150uM 1 15.9 50.1

Phloretin + Eucalyptol 150uM + 150uM 34.4 (S) 1 1.7

Hesperetin (G) 150uM 95.4 37.0

Eucalyptol (F) 150uM 1 15.9 50.1

Hesperatin + Eucalyptol 150uM + 150uM 37.2 (S) 25.9

Luteolin (G) 150uM 104.3 35.1

Vanillin (F) 150uM 106.9 46.2

Luteolin + Vannilin 150uM + 150uM 1 1 1.1 6.8

Resveratol (G) 150uM 97.8 55.9

Vanillin (F) 150uM 106.9 46.2

Resveratol + Vanillin 150uM + 150uM 96.9 31.4

Phloretin (G) 150uM 81.6 29.4

Lectin glycine (F) 150uM 82.8 9.2

Phloretin + Lectin glycine 150uM + 150uM 100.6 32.7

Phloretin (G) 150uM 81.6 29.4

Quercetin-3-glucoside (F) 150uM 83.9 33.0

Phloretin + Quercetin-3-glucoside 150uM + 150uM 100.6 32.7

Phloretin (G) 150uM 81.6 29.4

Catechin (F) 150uM 80.8 14.4

Phloretin + Catechin 150uM + 150uM 124.5 1 12.9

Luteolin (G) 150uM 99.6 33.8

Lectin glycine (F) 150uM 82.8 9.2

Luteolin + Lectin glycine 150uM + 150uM 52.8 (S) 12.4

Luteolin (G) 150uM 99.6 33.8

Catechin (F) 150uM 80.8 14.3

Luteolin + Catechin 150uM + 150uM 1 12.5 22.5

Hesperetin (G) 150uM 120.1 54.9

Lectin glycine (F) 150uM 82.8 9.2

Hesperetin + Lectin glycine 150uM + 150uM 84.4 29.9

Hesperetin (G) 150uM 120.1 54.9

Quercetin-3-glucoside (F) 150uM 83.9 33.0

Hesperetin + Quercetin-3- 150uM + 150uM 1 16.8 41.3 glucoside

Hesperetin (G) 150uM 120.1 54.9

Catechin (F) 150uM 80.8 14.3 Hesperetin + Catechin 150uM + 150uM 103.7 44.0

Luteolin (G) 150uM 70.5 7.7

Quercetin-3-glucoside (F) 150uM 60.0 26.8

Luteolin + Quercetin-3-glucoside 150uM + 150uM 26.1 5.1

Resveratol (G) 150uM 95.3

Quercetin-3-glucoside (F) 150uM 80.4

Resveratol + Quercetin-3- 150uM + 150uM 78.8

glucoside

Resveratol (G) 150uM 95.3

Lectin glycine (F) 150uM 88.1

Resveratol + Lectin glycine 150uM + 150uM 85.7

Resveratol (G) 150uM 95.3

Catechin (F) 150uM 83.5

Resveratol + Catechin 150uM + 150uM 81.5

Table 2 - Fructose Uptake Inhibition Results

As can be seen from the results in Table 2, the combination of Hesperetin with both Eucalyptol and Vanillin results in synergistic inhibition of fructose uptake. It can also be seen that these pairwise combinations must be specifically selected from the 24 potential pairwise combinations of the test compounds because the results also show that 18 of the combinations did not result in a synergistic inhibition and, in fact, 10 of the combinations caused fructose uptake was even greater than the DMSO control.