The present invention relates to an oil containing hydroxytyrosol and other biophenols as an additional ingredient. The term "oil" refers to olive oil obtained by pressing the fruit of the olive tree (Olea europaea), and to all fats found in the liquid state at room temperature. PRIOR ART

The Mediterranean diet has a proven ability to prevent disorders of the cardiocirculatory apparatus (heart attacks and thrombosis) and, almost certainly, other illnesses too. Attention was drawn to the Mediterranean diet in the 1950s by American nutritionist Ancel Keys, who had noticed the very low incidence of coronary disease in the inhabitants of Campania and the island of Crete, despite their high consumption of olive oil, and theorised that this was due to the diet typical of that geographical area. This observation was followed by the famous Seven Countries Study (published in the book "Eat well and stay well, the Mediterranean way"), based on a comparison of the diets of 12,000 people aged 40 to 59, living in seven countries on three continents (Finland, Japan, Greece, Italy, the Netherlands, USA and Yugoslavia). The results demonstrated that mortality due to ischaemic heart disease (heart attack) is much lower among the Mediterranean populations than in countries like Finland where the diet is rich in saturated fats. Despite differences in cuisine, the common denominator of inhabitants of the Mediterranean is the use of foods such as pasta, vegetables, fruit, fish, herbs and above all, olive oil (produced almost exclusively in the Mediterranean basin). The favourable effects on the health of the diet in question are mainly a lower risk of contracting coronary disease, a lower risk of colon, breast and lung cancer, increased life expectancy, better control of the triglyceride level in the blood and, according to recent research, less serious forms of autoimmune diseases and rheumatoid arthritis. According to current knowledge, said effects are attributable to various ingredients present in the foods traditionally used in the Mediterranean diet, namely monounsatured fatty acids (especially oleic acid), antioxidants and fibres. High quantities of monounsatured fatty acids are contained in extra virgin olive oil. A good example of how useful monounsatured fatty acids are in maintaining good health is a natural food like breast milk, which is rich in fats whose composition resembles that of olive oil. When taken in the correct quantities, it reduces the levels of LDL ("bad" cholesterol) but increases or leaves unchanged the level of HDL ("good" cholesterol). The danger of LDLs lies in their ability, if oxidised by free radicals, to trigger a mechanism that leads to gradual occlusion of the coronaries and consequent heart attack. Antioxidants, which are widespread throughout the plant world, are substances produced by plants to defend their own structures. The antioxidants present in olive oil, for example, protect the oil against oxidation, thus preventing it from becoming rancid. The antioxidants taken with the oil perform the same protective actions in the human body as in plants. In particular, they combat the oxidation produced by free radicals, a by-product of the chemical reactions that take place in the body. Free radicals are harmless in themselves (for example, they represent a natural defence against infection), but harmful if produced in excess: oxidation of the LDLs, caused by free radicals, is the first stage on the route leading to occlusion of the coronaries (and other vessels). The best-known antioxidants contained in olive oil are hydroxytyrosol and oleuropein, which belong to the class of phenols.

3,4-Dihydroxyphenylethanol or hydroxytyrosol (hereinafter called "HT") is a polyphenol with valuable biological activity, as demonstrated in numerous publications [1] [2] [3] [4]. In particular, it has been demonstrated that HT: effectively counteracts the cytotoxic effects of reactive oxygen species in human cell systems [5] [6];

- permeates the cell membranes by means of a passive mechanism [7]; - at concentrations of 2 g/kg of body weight does not cause toxicity or evident alterations in the internal organs of mammals (unpublished proprietary studies);

- delays the oxidation of Low-Density Lipoproteins (LDLs), one of the main mechanisms underlying atherosclerotic processes [4]. It has been demonstrated that hydroxytyrosol is one of the most active antioxidants currently known [6].

Due to its dual hydrophilic and lipophilic nature, HT is stored in the cell membranes, where it performs a protective action against oxidative damage caused by free radicals. It has been demonstrated that HT protects epithelial cell monolayers against the damage caused by hyperoxidation, which involves a reduction in cell viability and an abnormal increase in membrane permeability, leading to inflammation and oxidation of the polyunsaturated acids of the cell membranes. The direct or mediated consequences of the protective effect against free radicals are a reduction in cardiovascular diseases (especially those connected with atherosclerosis) due to the prevention of oxidation of the LDLs and the proliferation of cholesterol, inflammatory disorders, aging, cell degeneration and angiogenesis with consequent tumours, microbial infections and diabetic conditions.

It has also been demonstrated (Manna [6]) that HT performs its protective action in vitro even at very low concentrations (0.75 μg/ml).

A process for preparing hydroxytyrosol with high purity suitable for use as an ingredient in the pharmaceutical, food and cosmetic industries is disclosed, for example, in EP 1623960.

DESCRIPTION OF THE INVENTION

An object of the present invention is to provide an edible vegetable oil that includes HT as a beneficial additional component, so as to provide a healthy, natural food product with additional health-giving and beneficial characteristics, which in particular provides nutritional prevention against damage caused by oxidative stress, inflammation, angiogenesis processes, hypercholesterolaemia, atherosclerotic processes and tumoral processes, and a general effect of nutritional prevention of aging.

The edible vegetable oil is preferably olive oil, in particular extravirgin olive oil. HT is added to the olive oil according to the invention, which already contains percentages of hydroxytyrosol ranging between 0.005 and 0.8% by weight, in such quantities as to reach an HT content exceeding the maximum values found in the olive oils naturally available. The olive oil according to the invention, characterised by the addition to said oil of HT or fractions containing it, will therefore contain HT in percentages by weight of between 0.85 and 10%, preferably between 0.9 and 5%, and more preferably between 1 and 2%. The HT-enriched olive oil of the invention retains the typical organoleptic characteristics of traditional olive oil as perceived by the consumer, at the same time presenting an increased antioxidant action.

Another purpose of the invention is the use of oil with added HT to improve the body resistance to oxidative stress, inflammation, angiogenesis processes, hypercholesterolaemia, atherosclerotic processes and tumoral processes, and a general effect of nutritional prevention of aging.

The oil according to the invention may possibly contain other ingredients selected from those typically contained in the olive, such as oleuropein, gallic acid, trihydroxybenzoic acid, caffeic acid, etc.

The HT added to the extravirgin olive oil supplements the baseline quantity of polyphenols naturally contained in the oil, which is in the

50-800 mg/kg range on average [33, 34], and helps to reduce the initial peroxide value and keep it low over time. This also allows the acidity of the oil to be controlled over time (see examples below).

Similar comments also apply to other vegetable oils to which HT is added.

The oil according to the invention maintains the organoleptic properties, chemico-physical characteristics and content of HT and other polyphenols and, surprisingly, is stable for at least 30 months. By contrast, the vegetable oils normally marketed have a shelf life not exceeding 12-18 months.

Some examples of oil according to the invention, containing hydroxytyrosol in addition to other biophenols and possibly other health-giving ingredients, are set out below.

Example 1

Preparation of an extravirgin olive oil enriched with an 0.1% (w/w) hydroxytyrosol content exceeding 90% purity.

Commercial extravirgin olive oil was used. The acidity (as oleic acid) and peroxide value were tested; the profile of the minor polar compounds was also checked to evaluate their baseline quantity before adding the biophenol components to the oil.

The methods used are those laid down by Commission Regulation (EEC) No. 2568/91 of 11 July 1991 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis in "Annex II" for "Determination of the free fatty acids" and "Annex III" for "Determination of the peroxide value". The profile of the MPCs was developed and designed to define and quantify the profile of said phenol compounds. 1.077 g of hydroxytyrosol was added to 1 kg of oil, taking up 8 ml of an ethanol solution (HT purity > 95%) of 135 mg/ml of HT. This ingredient was produced according to the method described in EP 1623960. The effective dose was calculated for a man weighing 70 kg on the basis of the study by Visioli F. et al. [15]. Consequently, each tablespoonful (13 g) contains approx. 14 mg of HT.

The preparation was divided into aliquots, each placed in a 30 ml dark glass food container with metal crimp cap; one part was left at room temperature and another was placed in a climatic chamber at 30 ⁰ C ± 2°C and 60% RH ± 5% RH [32]. The preparations were monitored for 24 months, and the results of the determinations are set out below.

As will be seen from the graph in Figure 1 , the formulation based on extravirgin olive oil with added hydroxytyrosol retains the quantity of polyphenols for longer than the same oil without added hydroxytyrosol. Moreover, the higher hydroxytyrosol content better preserves the oil against the formation of peroxides, and keeps the acidity low (see graphs in Figures 2 and 3).

Example 2

Preparation of a corn oil enriched with a mixture of 0.1% (w/w) polyphenol and hydroxytyrosol with a purity exceeding 90%.

Commercial corn oil was used. The acidity (as oleic acid) and the peroxide value were tested; the profile of the minor polar compounds was also checked to evaluate their baseline quantity before adding the biophenol components to the .oil. The methods used are those set out in the preceding example. Approx.

2.50 g of a polyphenol mixture enriched with hydroxytyrosol, a product obtained from the vegetation water produced by olive processing, containing hydroxytyrosol at the concentration of 2.52 g/1 and tyrosol at the concentration of 1.52 g/1, was added to 1 kg of oil. This ingredient was produced according to the method described in EP 1623960. The percentage composition of the ingredients (dry weight) is set out below.

Percentage composition of ingredients (dry weight): Hydroxytyrosol 44.6 ± 1.1

Tyrosol 1 1.4 ± 0.6

Trihydroxybenzoic acid 16.7 ± 1.1

Caffeic acid 4.4 ± 1.1

Homovanillic acid 5.3 ± 1.8 Catechol 5.1 ± 0.7

Methyl gallate 1.3 ± 0.2

Hydroxycinnamic acid 1.9 ± 0.4

Veratrol 0.9 ± 0.2

Figure 4 shows an example of typical HPLC-UV chromatogram at 280 nm.

The mixture was produced according to the method described in EP 1623960 so as to obtain an 0.1% hydroxytyrosol concentration. The preparation was divided into aliquots, each of which was placed in a 30 ml dark glass food container with a metal crimp cap; one part was left at room temperature, and another was placed in a climatic chamber at 3O ⁰ C ± 2 ⁰ C and 60% RH ± 5% RH [32]. The preparations were monitored for 12 months, and the results of the determinations are set out below:

Table 1

Figures 5-7 graphically summarize the results obtained in this Example. REFERENCES

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