The present invention is related to a water-olive extract product.

The olive oil is known for its benefits to health. In general, the concentration of the phenolics in the olive oil ranges from 50 to 1000 μg/g of oil depending on the olive oil variety. This amount of antioxidants in the olive oil is about 1 to 2% of the available pool of antioxidants. The rest is lost with the by-products of the olive fruit processing (e.g. wastewater and pomace).

Compositions derivable from vegetation water, rich in hydroxytyrosol have been described. The US 2011/0257117 is related to such a composition, which is used for treating an inflammatory condition. Enrichment of food compositions with hydroxytyrosol and tyrosol may be accomplished by the use of other approaches. For example, the US 6746706 deals with processed food compositions, comprising 20- 100% of an aqueous phase, characterized by an enhanced content of tyrosol and hydroxytyrosol. The aqueous phase preferably results from exposing olive oil under hydrolytic conditions to an aqueous phase, so that lipophilic phenolic compounds will hydrolyze and will migrate as hydrophilic polyphenols to the aqueous phase.

The object of the present invention is to provide a water-olive extract capable of i) ameliorating metabolic parameters and ii) having a cardioprotective effect. This object is solved by a water-olive extract, comprising tyrosol, ligstroside and eleuropein aglycones, elenolic acid and ligstroside dialdehyde.

In the present invention, a water-olive extract is disclosed. Said water-olive extract comprises tyrosol, ligstroside and eleuropein aglycones, elenolic acid and ligstroside dialdehyde. The water-olive extract comprises polyphenols. Herein, the "total polyphenol content" means the total amount of olive polyphenols being present in the water-olive extract. Preferably, the water-olive extract comprises total olive polyphenols in an amount of 0.1-10 gr/lt. Preferably, said extract does not contain hydroxytyrosol as the principal component or as one main component. Preferably, hydroxytyrosol is present in the water-olive extract in an amount less than 45% (w/v), preferably less than 35% (w/v), more preferably less than 25% (w/v), even more preferably less than 15% (w/v) and most preferably less than 5% (w/v), based on the total phenolic content (w/v) or the total polyphenol content. More preferably, said extract contains tyrosol as the principal component. Even more preferably, the extract contains tyrosol and ligstroside degradation products as the principal components. Preferably, tyrosol and ligstroside degradation products, together are present in the water-olive extract in an amount more than 5%(w/v), preferably more than io%(w/v), more preferably more than 20%(w/v), even more preferably more than 30% (w/v) and most preferably more than 40%(w/v), based on the total phenolic content(w/v) or the total polyphenol content.

In a further embodiment, the water-olive extract is used as a medicament. In another embodiment, the extract is used to improve at least one of the following metabolic syndrome and/or cardioprotective parameters: to decrease LDL- cholesterol concentration (mg/dl), to increase HDL-cholesterol concentration (mg/dl), to increase the HDL/LDL index or to revert insulin hypersecretion and/or resistance in overweighted individuals.

In another embodiment, the invention comprises a pharmaceutical composition comprising the water-olive extract and a pharmaceutically acceptable carrier. In one embodiment, this extract is encapsulated. The water-olive extract of the present invention can be produced from olive mill wastewater or olive extracts. In particular, the extract is derivable, for example, by the process described in WO 2005/003037. An exemplary method for obtaining said water-olive extract is the following: Olive mill waste water (OMWW) is treated by passing through a system of filters, composed of a combination of sub stratums of natural products selected from the group consisting of turf, sand, and sawdust, and optionally one or more filters of resins selected from the group consisting of cationic, mixed-bed, and PVPP resins. According to a further aspect, the water-olive extract may be used as a means to confer a beneficial health effect. The beneficial effects of said water-olive extract are attributed to the totality of its phenolic constituents. The water-olive extract is administered orally to the subjects.

According to a preferred embodiment, the water-olive extract of the present invention may be administered in the form of a capsule or a micro-capsule. Preferably, the capsule wall is such that permits the delivery of the capsule content in specific areas of the gastrointestinal tract of the subject (for example stomach,duodenum). Examples of suitable capsule wall materials include: gums, starches, poly vinyl alcohol (PVA), Shellac or hydroxypropylmethylcellulose (HPMC), alginates, methylcellulose.

According to a further embodiment, the water-olive extract is used as a food additive. For example, the said water-olive extract may be incorporated in a food matrix. Preferably, the water-olive extract is encapsulated in a matrix or shell prior to its addition in a food composition. As such, the water-olive extract may be added by direct addition in a food matrix. Examples of suitable food matrices for said encapsulated water-olive extract include meat and dairy products. The invention is further illustrated by the following example without restricting the scope of the invention, and by Figures 1 to 14.

The figures show: Figure 1: 500 Hz lD NMR spectrum of OMWW polyphenols extract

Figure 2: Final body weight (gr) in Groups A-E , after the 16 weeks period

Figure 3: Body weight gaining (gr) in Groups A-E, in the course of the first 12 weeks of diet intervention.

Figure 4: Total Antioxidant Capacity in the rat serum for Groups A-E, after the 16 weeks period.

Figures 5 A-B: Glucose (mg/dl) and Insulin levels in subjects of Groups A-E, after the 16 weeks period.

Figures 6: Total cholesterol levels (mg/dl) and total triglycerides (mg/dl) in the subjects of Groups A-E, after the 16 weeks period. Figures 7 A-C: HDL (mg/dl), LDL levels (mg/dl) and the relative ratio thereof, in the subjects of Groups A-E, after the 16 weeks period.

Figure 8: Histological analysis of liver and kidney tissue of subjects of Groups A-E, after the 16 weeks period.

Figure 9: The design of the cross-over human study.

Figure 10: Blood glucose levels (mg/dL) in human samples after the intake of the water-olive extract product.

Figure 11: Insulin levels (uU/mL) in human samples after the intake of the water-olive extract product.

Figure 12: The lipid profile of the participants in the human study, before and after the intake of the water-olive extract product.

Figure 13: The anti-atherogenic ratio (HDL/LDL) one month after the intake of the water-olive product.

Figure 14: Biochemical analytes in the blood, reflecting hepatic (GOT, GPT) or renal damage(urea, creatinine).

Example 1:

1. Olive mill wastewaters (OMWW) were treated according to method described in W0200 00 Q y. The olive-water extract was NMR analyzed for the determination of phenolic compounds. Analysis showed the presence of eleuropein and ligstroside degradation products. The main products, detected by lD NMR (Figure 1) were tyrosol, ligstroside and eleuropein aglycones, elenolic acid (monomeric and dialdehyde forms) and ligstroside dialdehyde. Together tyrosol and ligstroside were detected in levels of between 5-6 g/L, while hydroxytyrosol was found in levels lower than 0.2 g/L.

This polyphenolic extract was microencapsulated, using different technologies, compatible for their use in food products for human consumption, at a concentration of

2. Animal study

The experiment used 8 groups of Spague-Dawley rats (Groups A-E). The water-olive extract was administered to Groups C-E, according to the scheme below: Group A: Normal diet (T.2018S.12), ad libitum;

Group B: High fat (60% animal fat-lard-) diet (TD 06414), ad libitum;

Group C: High fat (60% animal fat-lard-) diet (TD 06414), ad libitum, fortified witho.85 g encapsulated product/kg food;

Group D: High fat (60% animal fat-lard-) diet (TD 06414), ad libitum, fortified with8,5 g encapsulated product/kg food;

Group E: High fat (60% animal fat-lard-) diet (TD 06414), ad libitum, fortified g encapsulated product/kg food.

Rats were weighted weekly and sacrificed after 16 weeks.

2.1 Body weight

All rat groups fed with a high fat diet had a significantly higher body weight (Figure 2) after the 16 weeks of diet intervention, compared to the Control group.

The administration of the water-olive extract did not have a differential effect on the rats' body weight, as shown in Figure 3.

All the following biochemical/metabolic parameters were determined following the 16 weeks period and the sacrifice of the subjects.

2.2. Absorption

Previous data (J. Nurt. 132, 409-417, 2002) suggest a small intestinal absorption of olive polyphenols. In this study, estimation of absorption was made indirectly, by measuring changes in the Total Antioxidant Capacity in the animal serum. The TAC was determined according to WO 20144068140 and Figure 4 shows the TAC results, measured in Trolox equivalent/ ml(Figure 4).

As shown in Figure 4, TAC is decreased following a period of high fat dietary intervention in Group B, but is normalized in Groups C-E. A calculated 50% normalization was obtained by the addition of 1.27 g/kg food. 2.3. Blood glucose-Insulin Fasting blood glucose was not significantly modified by the high fat diet (Figure 5A). However, insulin levels were significantly higher in the high fat group, suggesting changes compatible with the establishment of metabolic syndrome. Addition of polyphenols in the diet normalized the insulin levels, in spite of the high fat diet (Figure 5B).

2.4. Blood lipids

Total cholesterol was not significantly modified in the high fat diet (Figure 6A), although a tendency of lowering total cholesterol levels was observed after the addition of polyphenols in the rat diet. However, the level of triglycerides increased significantly (Figure 6B).

It was surprisingly found that the addition of olive polyphenols in the diet, resulted in a significant increase of HDL-C and a profound decrease of LDL-C (Figure 7). Interestingly enough, the cardioprotective HDL/LDL index is dose-dependently increased by the addition of polyphenols, with a maximal value at the suggested concentration (Figure 7 A-C).

2.5. Toxicity

Histological sections of liver and kidney did not reveal any sign of toxicity (Figure 8) even in the high dose of polyphenols in the food, after the 16 weeks period of administration in rats. Similar findings apply for the levels of urea and creatinine in the serum . It has to be noted that this high dose is 10 times higher than the recommended dose in humans. Thus, in conclusion, by administering the herein disclosed dosage for 16 weeks to rats fed with a high fat diet (60% total fat), a cardioprotective effect was observed. In specific, by administering the herein disclosed dosage for 16 weeks to rats fed with a high fat diet (60% total fat), an improvement in at least one of the following metabolic syndrome and/or cardioprotective parameters was monitored: decrease of LDL- cholesterol concentration (mg/dl), increase of HDL-cholesterol concentration (mg/dl), increase of the HDL/LDL index and reversion of insulin hypersecretion in overweighted individuals. Moreover, no toxicity was apparent even at ιοχ doses of the product, after the completion of the 16 weeks period.

3. Human study

Humans were administered in the food (parizer) 30 mg of microencapsulated OMWW concentrated polyphenols, in a Maltodextrin (Cargil) vehicle, containing 7-10 total concentrated polyphenols, as assayed by the Lowry method, for 4 weeks. This dose is the recommended one from the European and the American Society of Cardiology (corresponding to the intake of 20-25 ml olive oil, and reported to exert the maximum effect on blood lipid oxidation. They were fed ad libitum and basic anthropometric and biochemical parameters were assayed at the beginning and the end of the study. The design of the cross-over study is shown in Figure 9:

In phase 1, half of the volunteers were administered the microencapsulated polyphenol- containing food, while the other half was administered the same food without polyphenols. After a two-week wash-out period, the two groups were reversed.

3.1. Anthropometric measures

No modifications of body weight, cardiac or other function was observed during the 10 weeks of this study.

3.2. Glucose-Insulin

Blood glucose levels were slightly decreased (in a non-significant level) after the intake of polyphenols. It is shown in Figure 10. Insulin, on the other hand, was significantly decreased, after the intake of the preparation, suggesting a beneficial effect of the administered polyphenols on glucose homeostasis and subsequently on the metabolic syndrome, in pre-diabetic cases, as shown in Figure 11:

3.3. Lipid profile

The lipid profile of the participants is presented in Figure 12. HDL plasma concentration was not modified by the intake of microencapsulated polyphenols. However, it is to note that, as the study was performed in rural Crete, HDL plasma concentration was already very high at the beginningof the study. Therefore, the effect of polyphenols on HDL might not be perceptible. In contrast, LDL plasma concentration was significantly decreased after polyphenolintake, both as an absolute plasma concentration (left panel) and as a decrease, as compared to initial levels (right panel). As a consequence, the anti- atherogenic ratio (HDL/LDL was significantly increased, after one month of polyphenol intake (Figure 13).

3.4. Toxicology of the product

As stated above, no modificastion of body weight, pulse rate or blood pressure was observed after intake of encapsulated polyphenols. However, as these compounds are metabolizedin the liver and excreted through the kidney, we have assayed biochemical analytes in the blood, reflecting hepatic (GOT, GPT) or renal damage (urea, creatinine). As presented in Figure 14, no change of any analyte was observed, verifying the absence of chronic toxicity of the product, in parallel with our data in animal studies.