EP2197437A1

EP2197437A1 - Use of oleocanthal for the treatment of lipid metabolism disorders

Info

Publication number: EP2197437A1
Application number: EP08851263A
Authority: EP
Inventors: Cyril Estanove; François PRUVOST; Jean-Claude Allart; Frédéric SALDMANN
Original assignee: Bc Development Sa; Sprim
Current assignee: Bc Development Sa; Sprim
Priority date: 2007-09-06
Filing date: 2008-09-05
Publication date: 2010-06-23
Also published as: FR2920668A1; WO2009066021A1; FR2920668B1

Abstract

The invention relates to a novel use of extracts of olive oil comprising derivatives of general formula (A) in which R is a hydrogen atom or a hydroxyl group, in the preparation of a medicament or of a functional food for the treatment of lipid metabolism conditions such as excess weight and obesity.

Description

USE OF OLEOCANTHAL FOR THE TREATMENT OF LIPID METABOLIC DISORDERS

The present invention relates to a new use of oleocanthal, more particularly olive oil extracts containing oleocanthal and derivatives, for the preparation of a medicament useful in therapeutics and nutraceuticals for the treatment of disorders of lipid metabolism.

Obesity is now a major public health problem worldwide, particularly in North America, due to genetic predisposing factors, sedentary lifestyles and unbalanced diets. According to the World Health Organization (WHO, 2002) one billion people worldwide are overweight, including 300 million in obesity, and these numbers are increasing in many countries. Obesity is known to significantly increase the risk of type 2 diabetes, hyperlipidemia, insulin resistance, osteoarthritis and cardiovascular disease, including high blood pressure and atherosclerosis. Overweight can lead to the same risks, although they are lower, but can also escalate into obesity. It is also known that overeating in obese or overweight people can lead to significant lipid and protein damage. Obesity and overweight also have a negative impact on the psychological behavior of those who are affected.

The links between obesity and the inflammatory response have been the subject of many studies. Thus, it has been found that overeating and the consumption of certain nutrients can trigger mediators involved in the mechanism of inflammation. Obesity, insulin resistance, and type 2 diabetes are related to inflammation. Chronic malformation characterized by an abnormal production of proinflairanatory cytokines such as TNFα, which is present at a high level in adipose tissue of the obese. A metabolic signal such as a lipid excess food causes the awakening of the adipocytes, resulting in an increase in fat mass, which triggers an inflammatory response in the endoplasmic reticulum and the production of TNFα which, after macrophagic infiltration, causes a disturbance. ^¬ bation of the body's regulatory mechanisms and an increase in metabolic stress, which in turn leads to a chronic inflammatory reaction. A therapeutic management of these disorders must take into account these mechanisms.

The prescription of physical exercises and appropriate dietary regimes is generally the first way to combat obesity and overweight. Various drugs have also been proposed to treat obesity, for example anorectics such as sibutamine hydrochloride and rimonabant, or gastrointestinal lipase inhibitors. These drugs, however, have side effects that limit its use.

It is therefore desirable to develop new drugs and foods that may be involved in the treatment of overweight and obesity. Olive oil, especially virgin olive oil, contains especially monounsaturated fatty acids

(oleic acid) as well as phenolic compounds with antioxidant properties which have the effect of retarding the oxidation of LDL lipoproteins. Among these phenolic compounds, there are phenolic alcohols such as tyrosol and hydroxytyrosol, benzoic acids, caffeic and elenolic acids esterified with tyrosol and hydroxytyrosol, as well as flavonoids such as flavones and flavonols. Epidemiological studies have shown the health benefits of mediterranean dietary regimes. containing a high intake of fiber, fruit, vegetables, and olive oil which is the main source of fat. For example, RW Owen et al., Lancet Oncol. (Oct. 2000) 107-12. P. Andrewes et al., J. Agric. Food Chem. 51: 5, 1415-1420

(2003), have shown that olive oil contains a particular phenolic derivative, namely deacetoxy ligstroside aglycone, which is responsible for its typical taste. G. K.

Beauchamp et al. Nature, vol. 437, 45-46 (Sept. 2005), have shown that deacetoxy ligstroside aglycone, also called oleocanthal, contained in virgin olive oil obtained by first cold pressing, has inhibitory properties of COX1 and COX2 enzymes. , that is to say anti-inflammatory properties similar to those of ibuprofen, a molecule used for many years as a non-steroidal anti-inflammatory drug. These properties of oleocanthal are also described in WO 2006/122128 which contemplates a variety of conditions that can be treated, given these anti-inflammatory properties. These properties essentially show that oleocanthal could be considered in the indications of ibuprofen, that is to say as a general anti-inflammatory, orally, likely to be useful in rheumatology.

These properties have prompted research to prepare oleocanthal by synthesis to obtain an isolated and purified product free of any secondary product. Thus, the application WO 2007/081808 describes synthetic schemes for the oleocanthal obtained in the form of an isolated and purified enantiomer whose hormone-sensitive lipase inhibitory activity (anti-HSL) is demonstrated and compared with that insulin, which should allow use in the treatment of non-insulin-dependent diabetes. WO 2007/081808 also contemplates a large number of conditions where isolated and purified oleocanthal, as well as certain derivatives, could be useful because of this anti-HSL property, by Examples include osteoarthritis, hypercholesterolemia, heart failure, thrombosis, hyperglycemia, prostate cancer, seborrheic dermatitis, etc. These hypotheses, however, remain to be verified.

Oleocanthal, or deacetoxy-ligstroside aglycone, has been detected in olive oils of various origins, especially in virgin olive oil obtained by first cold pressing, at very low concentrations which generally vary from at 200 ppm (mass), ie from 0.002 to 0.02% by weight, depending on the source of the olive oil.

Oleocanthal may be represented by the following general formula (I):

Oleuropein, which is found in fruits and mainly in olive leaves, is a glycoside represented by the following general formula (II):

)

The use of oleuropein in food or pharmaceutical compositions for stimulating bone mineralization in humans or animals has been proposed in WO 2004091591. The antiviral activity of certain oleuropein derivatives has also been described in US Pat. US Patent 6,455,580. Derivatives such as oleuropein aglycone, having antioxidant properties, and its dialdehyde form can also be extracted from olive oil.

Other derivatives of interest contained in olive oil include the aldehyde forms of oleuropein aglycone and ligstroside aglycone, represented by formula (III) below

wherein R is a hydrogen atom or a hydroxy group, and the dialdehyde form of the decarboxymethyl oleuropein aglycone represented by the formula (IV).

Pharmaceutical use of these compounds requires the production of relatively concentrated fractions. In addition, oleocanthal is a very fragile product, easily destroyed by heat and oxidation, as are most of the other polyphenolic compounds naturally present in vegetable oils.

These derivatives, as well as oleocanthal, participate in the useful properties of olive oil and for this reason various extraction processes have been developed to isolate them or to produce enriched extracts in one or the other. other one or more of these derivatives.

The studies carried out by the Applicant have surprisingly shown that olive oil extracts containing these derivatives, and especially oleocanthal and certain derivatives, have interesting properties. allowing them to be used in therapeutics and nutraceutics to regulate or restore lipid metabolism.

The subject of the present invention is therefore the use of olive oil extracts containing derivatives of general formula (A) below.

wherein R represents a hydrogen atom or a hydroxy group, as well as extracts containing them, for the preparation of a medicament for the treatment of disorders of lipid metabolism.

The invention more particularly relates to the use of the oleocanthal represented by the general formula (A) above, wherein R is a hydrogen atom.

The subject of the invention is also the use of an olive oil extract containing a derivative of general formula (A) above, more particularly the use of an extract enriched in oleocanthal, for the preparation of a drug or a drug, especially for the treatment of overweight and obesity. The extract used in the invention may contain not only oleocanthal but also an oleuropein derivative, especially the dialdehyde form of decarboxymethyl oleuropein aglycone represented by formula (A) above where R is a hydroxy group.

Oleocanthal containing extracts are particularly useful in the treatment of overweight and resulting metabolic disorders, including conditions such as obesity and related diseases, particularly type 2 diabetes mellitus, insulin resistance, atherosclerosis. As discussed in detail below, the studies conducted by the applicant showed that the compositions based on ¹ oleocanthal the present invention exert a direct action on lipid metabolism, resulting in a decreased uptake of fats, more particularly circulating fatty acids, by the adipocytes.

The studies carried out have shown that oleocanthal, at concentrations of 0.3 and 1.0 μg / ml in the extract of the invention, over a period of 24 hours, has the effect of reducing the release of the oleate on the lower side of the intestinal barrier, with an inhibition rate of 43% and 45% respectively. This shows that oleocanthal, in vivo, could decrease the passage of lipids ingested in the blood.

In addition, studies have shown that the oleocanthal enriched extract does not cause inhibition of the formation of new adipocytes, nor the neosynthesis of lipids by these cells. On the other hand, at concentrations of 0.3 and 1.0 μg / ml, it limits the storage of lipids by decreasing the incorporation of circulating fatty acids into the adipocytes. Since this phenomenon is the major mechanism of accumulation of subcutaneous fat, it appears that oleocanthal can be effective as a component of drug or food administered as part of a slimming treatment.

Finally, the studies have demonstrated an action of oleocanthal on the inflammatory mechanisms involved in the metabolic disorders characteristic of obesity, insulin resistance and type 2 diabetes.

These studies thus demonstrate the interest of the action of oleocanthal on genes induced by TNFα, allowing to limit the harmful effects by a modulation of the kinase system whose role is crucial in the action of the insulin and glucose uptake. These results show that Oléocanthal can be used advantageously in treatments intended to control the consequences of disorders. in obese subjects or those with a tendency to become obese.

The oleocanthal of the invention may be used in the form of olive oil extract with a high oleocanthal content, for example of the order of 10% to 30% by weight, as a diet dietary supplement intended for subjects who are overweight. Extracts with a higher concentration of oleocanthal, of the order of 50 to 90% by weight, can be prepared for specific applications. The dialdehyde concentration of the decarboxymethyl oleuropein aglycone, when the extract contains, is generally between 1 and 15%.

The studies carried out have also demonstrated the absence of influence of the relative humidity rate on the stability over time of the oléocanthal enriched olive oil extract of the invention and a moderate influence of the temperature. according to the composition of the extract, which facilitates its preservation and that of the compositions containing it.

The present invention also has the advantage of allowing an effective treatment of the lipid metabolism disorders mentioned above by means of an oleocanthal enriched extract whose preparation cost is considerably lower than that of the isolated and purified oleocanthal obtained by synthesis. The oleocanthal compositions according to the present invention can be administered orally using suitable formulations, and more particularly by means of drops, capsules or soft capsules. The capsules or capsules may be gelatin-based, prepared according to conventional techniques.

The unit doses used may be of the order of 0.1 to 50 mg, and preferably between 1 and 25 mg of derivative of formula (A), especially oleocanthal.

The dosage is chosen by the practitioner according to the condition of the patient and the intended purpose, but is generally between 5 and 25 mg of oleocanthal per day for an adult. Thus, a treatment adapted to an average case may consist in administering one to three capsules of 25 mg of 10% oleocanthal extract per day. The duration of the treatment is adapted to the state of the patient and for example, for a patient having an established overweight with tendency to obesity, the treatment may consist in administering a capsule of 25 mg of 10% extract. oleocanthal daily for 1 to 2 months, followed by maintenance treatment at a rate of 1 to 3 capsules per week for 2 to 3 months. The treatment can be strengthened and extended for an obese patient, and it can be for example 2 or 3 capsules

(containing 10 or 30% extracts depending on the condition of the patient) per day for 2 to 3 months, followed by one capsule per day for 3 to 6 months. For a diabetic patient, the treatment is usually one capsule per day with no time limit. In the latter case, such treatment with oleochal may limit or delay or even avoid conventional insulin treatment. Several examples of formulations adapted to the invention and experimental results are given below without limitation.

Example 1

A 10% oleocanthal extract is prepared from a virgin olive oil, first cold pressed, of Pietra Pinta origin whose oleocanthal content is about 48 mg / kg.

The oil is extracted with an ethanol / water mixture, the hydroalcoholic phase is washed with hexane, preconcentrated and extracted with ethyl acetate. The ethyl acetate phase is then concentrated to dryness.

An extract containing approximately 10% oleocanthal in the oily phase, comprising in particular:

- oléocanthal approx. 10% - Oleuropein derivative approx. 15%

- pinoresinols approx. 10%

The oleuropein derivative indicated above is constituted by the dialdehyde form of decarboxymethyl oleuropein aglycone. The extract still contains a few percent of oleocanthal (molecular weight 362) and oleuropein derivatives.

(molecular weight 378).

This extract is packaged in gelatin-based soft-walled capsules according to a conventional technique, each capsule containing 25 mg of extract.

Example 2

A 30% oleocanthal extract is prepared from a first cold pressed virgin olive oil of Campione origin with an oleocanthal content of about 212 mg / kg.

The oil is extracted with an ethanol / water mixture, the hydroalcoholic phase is washed with hexane, preconcentrated and extracted with ethyl acetate. The ethyl acetate phase is then concentrated to dryness. An extract containing approximately 30% oily phase oléocanthal, comprising in particular:

- oléocanthal approx. 30%

- Oleuropein derivative approx. 10%

- pinoresinols approx. The oleuropein derivative indicated above is the same as in Example 1. The extract still contains a few percent of derivatives of oleocanthal (molecular weight 362) and oleuropein (molecular weight 378).

This extract is packaged in 25 mg capsules as in Exemplel. Example 3

A sample to 50% ¹ oleocanthal is prepared from the extract of Example 2, and purified by preparative chromatography. This extract, which contains about 10% of oleuropein derivative and less than 10% of pinoresinols, is packaged in soft capsules according to the same technique as in Example 1, in order to prepare capsules with a high oleocanthal content, intended for treatment of obese patients.

Example 4

The effects of oleocanthal extracts according to the invention were studied on the differentiation of adipocytes, on lipogenesis and on the storage of circulating lipids.

The tests were carried out using three extracts whose oleocanthal content was respectively 1 μg / ml, 0.3 μg / ml and 0.1 μg / ml.

The reference molecules used are TNFα, which is an inhibitor of adipocyte differentiation, and cerulenin, which is an inhibitor of lipogenesis. The cells used are pre-adipocytes of mouse of line 3T3-L1 grown at confluence in DMEM growth medium. At high confluence, the culture medium was replaced by differentiation medium (DMEM containing differentiating molecules). Control cultures were carried out throughout the duration of the experiment (undifferentiated controls). After differentiation, the cells were transferred to post-differentiation medium (growth medium + insulin) and then incubated for 48 h at 37 ^° C. and 5% CO ₂ .

For the measurement of adipocyte differentiation, after incubation, the post-differentiation medium was removed, and the cells were incubated in DMEM growth medium for 24 h and 5% CO ₂ . After removal of the culture supernatants, the cells were rinsed in PBS and intracellular lipids were stained by Adipored (BioWhittaker PT-7009).

For the measurement of the storage of circulating lipids, the same manipulations are performed but after incubation, the post-differentiation medium was removed, and the cells were incubated in the DMEM growth medium supplemented with the [ ¹⁴ C] oleate radioactive marker. (Biotrend CFA243) for 48 hours and 5% CO ₂ . At the end of the incubation, the adipocytes were washed and the lipids were extracted according to the Bligh and Dyer protocol, dried under nitrogen, and the radioactivity was measured by liquid scintillation (LKB Rackbeta counter).

The measurements were also made simultaneously on a control (without oleocanthal). Undifferentiated cells were found to be weakly labeled, unlike differentiated cells stained with Adipored. Many fat vesicles characteristic of the differentiation of adipocytes were present. The effects on circulating lipid storage were evaluated by measuring the incorporation of [ ¹⁴ C] oleate into differentiated adipocytes.

As shown by the results in Table 1 below, treatment with the reference product, 20 μM cerulin, significantly decreased the uptake of Oleate into the adipocytes. Oleocanthal, at 1 and 0.3 μg / ml, was also found to significantly inhibit oleate incorporation in a dose-dependent manner.

Table 1

Inv. = oleocanthal of the invention.

These results show that the oleocanthal according to the present invention does not inhibit the formation of new adipocytes or, significantly, the synthesis of lipids by these cells. On the other hand, at concentrations of 0.3 and 1 μg / ml, it significantly limits the storage of lipids in adipocytes. Since this phenomenon is the major mechanism of accumulation of subcutaneous fat, these results demonstrate the interest of the oleocanthal of the invention in slimming treatments.

Example 5

The effects of the oleocanthal extracts of the invention on the passage of lipids through the intestinal epithelial barrier have been studied as indicated below.

The study was made by measuring the passage kinetics of oleic acid in the presence of Oleocanthal of the invention in the intestinal epithelial barrier model reconstructed from cells of Caco2 line.

Caco2 cells were seeded on Millicell 24 inserts (Millipore) in MEM culture medium. At nearly two weeks of culture, by renewing the inserts and the culture medium daily, the integrity of the epithelial barrier obtained was verified by electrical resistance measurements. A solution of [ ¹⁴ C] oleate at 2 μCi / ml in a 10 mM taurocholate buffer in PBS at pH 7.4 was deposited at the surface of the inserts in the presence of oleocanthal of the invention. The same test without oleocanthal is carried out as a control.

50 μl of the apical compartment (donor compartment) and 50 μl of the basal compartment (receptor compartment) were taken at regular time intervals (0, 30 min, 1 h, 2 h, 4 h, 6 h and 24 h) and the present radioactivity was measured in liquid scintillation.

The results are shown in Table 2 below.

Table 2

30 min. 1 h 2 h

Cpm% Cpm% Cpm%

Witness 2555 100 8525 100 12006 100

1 μg / ml 4624 181 7008 82 10555 88

0.3 μg / ml 4672 183 9015 106 9934 83

0, 1 μg / ml 9045 354 10675 125 13769 115

4 h 6 h 24 h

Cpm% Cpm% Cpm%

Witness 19763 100 20657 100 47413 100

1 μg / ml 18714 95 18697 91 27178 57

0.3 μg / ml 17642 89 16503 80 26058 55

0, 1 μg / ml 17479 88 23739 115 44878 95

The oleocanthal according to the invention was tested at concentrations of 0.1 μg / ml, 0.3 μg / ml and 1 μg / ml, as indicated in the table above.

The percentage indicated is calculated relative to the witness.

These results show a decrease over time of the amount of [ ¹⁴ C] oleate present in the apical compartment, in parallel with an increase in the amount of [ ¹⁴ C] oleate in the basal compartment of the culture inserts. However, it appears that the majority of the cell-penetrating oleate is metabolized and does not show up in the basal compartment. It is found that, under the conditions of the experiment, the oleocanthal of the invention at a concentration of 0.1 μg / ml does not affect the penetration of the oleate into the Caco2 barrier. On the other hand, at concentrations of 0.3 and 1 μg / ml, it tends to reduce the release of oleate from the basal compartment of the barrier after 6 hours of culture.

Claims

1. Use of an olive oil extract comprising at least one derivative of general formula (A) below

wherein R represents a hydrogen atom or a hydroxy group, for the preparation of a medicament for the treatment of disorders of lipid metabolism.

2. Use according to claim 1, characterized in that the derivative is 1 oleocanthal represented by the formula (A) where R is a hydrogen atom.

3. Use according to claim 2, characterized in that the olive oil extract contains 10 to 30% by weight of oleocanthal.

4. Use according to claim 3, characterized in that the extract further contains the dialdehyde form of decarboxymethyl oleuropein aglycone.

5. Use according to any one of the preceding claims, characterized in that unit doses of between 1 and 25 mg of derivative of formula (A) are used.

6. Use according to any one of claims 1 to 5, characterized in that the drug is intended for the treatment of overweight and obesity.

7. Use according to any one of claims 1 to 5, characterized in that the drug is intended for the treatment of type 2 diabetes, insulin resistance, and atherosclerosis.

8. Use of an olive oil extract comprising a derivative of general formula (A) according to claim 1 for the preparation of a medicament for the treatment of overweight.