Method for production of oil enriched in fatty hydroxyoctadecadienic acids (hode) or esters thereof from oil mixture containing linoleic acid or esters thereof

FIELD: improved method for oil production.

SUBSTANCE: target oil, enriched in HODE, or esters thereof is obtained by controlled oxidation of linoleic acid and/or linolenic acid or esters thereof in presence of oxidation catalyst. Oxidation is stopped when total HODE or ester content is more than 5 %, and/or content of isomeric 9-hydroxy-10,12-octadecadienic acid (9-HODE) or esters thereof is more than 1,5 %; and hydroperoxides formed in oxidation process are reduced with reducing agent in presence of antioxidant. Invention is also relates to oil enriched in 9-HODE or esters or salts thereof having an lipolytic action; to drug or food additive for obesity treatment; cosmetic for local treatment of cellulite. Compound for controlling of adipocyte lipolytic activity and hydrolysis of triglycerides accumulated in adipocytes is also disclosed.

EFFECT: novel pharmaceutical composition for obesity treatment.

11 cl, 1 ex

The present invention relates to a method for hydroxyoctadecadienoic acids, in particular 9-hydroxy-10,12-octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid and their mixtures, and their isomers, such as 9 (R/S)-hydroxy-10E, 12Z-octadecadienoic acid, if necessary, in the form of their esters, in particular in the form of ethyl ether.

In the following description hydroxyoctadecadienoic acid called HODE.

The invention relates to a method for HODE by controlled oxidation of linoleic acid and/or linolenic acid, and then recovery.

The invention relates also to a method for HODE rich in linoleic acid and/or linolenic acid triglycerides or of linoleic acid and/or linolenic acid in the form of a free fatty acid or in the form of ester or salt, such as ethyl ether.

The invention relates also to a medicinal product, in particular for the treatment of obesity containing as an active start HODE and pharmacologically acceptable carriers.

The invention relates also to contain HODE food additive, in particular for the treatment of obesity.

The invention relates also to a cosmetic preparation containing HODE and kosmetologicheskii acceptable carriers, in particular for the localized treatment of excess weight and its consequences in relation to the appearance of the skin, such as cellulite, enlarged pores, fatty deposits on the hips.

As a rule, assume that the increasing incidence of overweight and obesity in developed countries is caused by improper diet, either due to too high total inflow calories, and due to the high proportion of lipids in the influx of calories. This is very significant proportion of lipids in the diet (estimated at approximately 50% of the energy contribution) may be reduced under low-calorie diets, which greatly reduced the fat content.

However, the lipids can not be reduced to less than 30% of the energy contribution due to the fact that they not only play an important role in the texture and taste perception of food, but also by the fact that the minimum number is required in the diet, in particular for the biosynthesis of essential fatty acids and hormones and update of membrane phospholipids.

The main thrust of research in the treatment of obesity, conducted over several decades, therefore, is the selection of the desired lipids, i.e. lipids, which do not accumulate in the body, but are used as an energy source. Under this context, it is established that the nature of the fatty acid components of lipids, plays a fundamental role in their metabolism. As the second example, fatty acids with short-chain are more thermogenic than fatty acids with long chain.

Managed to establish that the chain length of fatty acids have a direct impact on energy consumption and, therefore, their accumulation in the body.

One of the purposes of the invention is the identification of molecules that allow the regulation of lipolytic activity adipocyte, is able to hydrolyze accumulated in adipocyte triglycerides to free fatty acids and glycerine.

It is known that adrenaline and caffeine are two molecules, known for its lipolytic action. This, in particular, molecules that increase the levels of cyclic adenosine monophosphate (camp), according to the first stage activation of lipolysis.

One of the purposes of the invention, therefore, is the identification of a molecule that activates lipolysis stronger than caffeine or adrenaline, due to stimulation depozitarnoj production of camp.

Another aim of the invention is the detection of lipolytic molecules, simply and efficiently synthesized from commonly occurring products.

The inventors have unexpectedly found that derivatives of fatty hydroxyoctadecadienoic acid (HODE) have interest thermogenic properties, without causing toxicity, and, in particular, 9-hydroxycut decadienoate acid in the form of a free acid or in the form of esters or salts.

The invention relates therefore to a method for producing oil, enriched with fatty hydroxyoctadecadienoic acid (HODE) and, in particular, 9-hydroxyoctadecadienoic acid (9-HODE), or its ester or its salts, of the oil mixture containing linoleic acid and/or linolenic acid or their esters or their salts, characterized in that conduct controlled oxidation of linoleic acid and/or linolenic acid, or their esters, in the presence of the oxidation catalyst, and the oxidation stops when the total content of HODE or its esters is higher by at least 5% and/or the content of isomeric 9-hydroxy-10,12-octadecadienoic acid (9-HODE) or its esters is higher than at least 1.5%, and restore with the help of a reducing agent formed during the oxidation of the hydroperoxides.

Original oil mixture is a vegetable oil comprising more than 40 wt.% linoleic acid and/or linolenic acid in the form of triglycerides, such as corn oil, borage oil, safflower oil, soybean oil, angrove oil, sunflower oil, oil from black currant seed, wheat germ oil, hemp oil, pumpkin seed oil, linseed oil.

Original oil mixture is an oil mixture containing more than 40 wt.% linoleic acid and/or linolenovoi kilty, preferably, containing 75% ethyl-linoleate.

The final content in the enriched oil all HODE and 9-HODE is at least 5 wt.%, accordingly, 1.5 wt.%.

Preferably, the final content in the enriched oil all HODE and 9-HODE is, accordingly, 10-12 wt.% and 2.2-2.5 wt.%.

The oxidation catalyst is a halide of iron, copper, preferably Fl₃.

The reducing agent is preferably NaBH₄.

The invention relates also to the oil, rich in fatty hydroxyoctadecadienoic acid (HODE) or its esters, or its salts, obtained as described above.

The invention relates also to a medicinal product for the treatment of obesity containing as an active start oily 9-hydroxyoctadecadienoic acid (9-HODE) or its pharmacologically acceptable esters or salts, preferably in the form of enriched oils obtained as described above.

The invention relates also to a food additive for the treatment of obesity containing as an active start oily 9-hydroxyoctadecadienoic acid (9-HODE) or its pharmacologically acceptable esters or salts, preferably in the form of enriched oils obtained as described above.

The invention relates also to a cosmetic treatment for obesity, containing the active is achala fat 9-hydroxyoctadecadienoic acid (9-HODE) or kosmetologicheskii acceptable esters or salts, preferably in the form of enriched oils obtained as described above.

According to the invention, hydroxyoctadecadienoic acid (HODE) is obtained by controlled oxidation of linoleic acid and/or linolenic acid. These fatty acids are present in many vegetable oils.

For example, the following vegetable oils include more than 40% of linoleic acid in the form of triglycerides: borage oil, safflower oil, soybean oil, angrove oil, sunflower oil, oil from black currant seed, wheat germ oil, hemp oil, pumpkin seed oil, linseed oil.

According to the first variant of the invention, HODE can be obtained from enriched in linoleic and/or linolenic acid triglycerides. This method is of interest due to the fact that it allows you to use natural vegetable oil, which is well adapted for use by mouth, but has the disadvantage that it significantly restricts the final content of the isomer of 9-HODE. In fact, the fact that linoleic and/or linolenic acid is in the form of glycerides, limits the effect of oxidation, which represents the first phase in obtaining 9-HODE, however, mainly linoleic acid content of about 40-50% of the aggregate fatty acids the oil restricts the final content of 9-HODE.

The structure of the original product is the triglyceride of linoleic acid or ester of linoleic acid following General formula (I):

in which X means triglyceridemia balance or ethyl residue, or any other lower alkyl residue with 1-6 carbon atoms.

This compound contains a double bond in position 9, 10 and the double bond at position 12, 13. By controlled oxidation receive, according to the invention, the compounds of the following General formulas (II) and (III):

which is literily or alkilany ester of 9-hydroxy-10,12-octadecadienoic acid,

or

which is literily or alkilany ether 13-hydroxy-9,11-octadecadienoic acid.

By controlled oxidation of compounds of formula (I) receive, therefore, a mixture of isomers of formulae (II) and (III).

Linolenic acid exists in two forms: α-linolenic acid (or (Z,Z,Z)-9,12,15-octadecatrienoic acid) and γ-linolenic acid (or (Z,Z,Z)-6,9,12-octadecatrienoic acid).

The oxidation reaction is carried out in the presence of an oxidation catalyst, such as a halide of iron, copper, preferably Fl₃. The reaction has a duration of 12 to 78 hours, and the temperature of reaction is AI ranges from -20° With up to 40°C, at atmospheric pressure and/or under reduced pressure. The degree of oxidation is controlled by using different criteria:

- quantification of the formed hydroxides by gas chromatography;

- change of the absorption coefficient in the ultraviolet region at a wavelength of 235±5 nm;

the change in the refractive index.

When the mass percentage of the compounds of formula (II) (9-HODE) or mass percent of a mixture of compounds of the formula (II) and (III) reaches 1-20%, the mixture restore and it restores the hydroperoxides formed during the oxidation reaction. Use ethanol solution of NaBH₄or ethanolic solution of any other reducing agent, which is added by portions to the reaction mixture. The temperature of the mixture maintained within the range of 4-25°C. restore the finish, when the temperature becomes constant. The reaction mixture is neutralized to a pH value of 6-7, by adding an acid solution, preferably a solution of the pharmacologically and/or kosmetologicheskii acceptable acid, for example citric acid.

The compounds of formula (II) and (III) is extracted with hexane or any other organic solvent which can dissolve. The organic phase is washed with water until neutral.

Then the organic phase is dried over anhydrous self the volume of anhydrous sodium or magnesium sulfate.

After that, the filtrate is evaporated in vacuum.

The resulting product is an oily liquid light yellow color, with a slightly aromatic odor. The total content of compounds of formulas (II) and (III) (determined by gas chromatography) it is above 5 wt.% and the content of the compounds of formula (II) [9(R/S)-hydroxy-10E, 12Z-octadecadienoic acid or ester 9 (R/S)-hydroxy-10E, 12Z-octadecadienoic acid] is higher than 1.5 wt.%.

According to the second variant of the invention, which is preferred, the initial product is an oil mixture containing 75 wt.% ethyllinoleate.

The principle of this variant is the same as described in the case of the first variant embodiment of the invention. Carry out selective oxidation ethyllinoleate, then recovery of the formed hydroperoxides and purification by sequential leaching. Get the oil light yellow color with a specific smell, containing about 2.5 wt.% 9-hydroxy-10E, 12Z-octadecadienoic acid (ethyl ester) and 12% of a mixture of compounds of formulas (II) and (III) (as ethyl esters). The hydroxyl group may be in position 9 or 13 double bond in conjugated form can be in a position 10,12 (TRANS, TRANS and CIS, TRANS) and in position 9,11 (TRANS, TRANS and CIS, TRANS).

An example item is produce enriched HODE oil from the mixture, containing 75% ethyllinoleate

1. Oxidation

In thermostatted reactor introduced 500 g of 75%ethyllinoleate. Add 0,500 g anhydrous Fl₃(oxidation catalyst), bring the mixture to a temperature close to 25-27°and a mixture of support with constant stirring.

In the reaction mixture is bubbled with oxygen (technical quality) with filter from stainless steel or bronze with a porosity of 2-4 µm (type of filter for HPLC or filter for particles in the case of gas). This "thin" sparging support at the rate of 1 liter/min for 48 to 72 hours.

Oxidation control

or by measuring the refractive index at a temperature of 20°With crude product from the reaction mixture;

or by measuring the absorption coefficient in the ultraviolet region at a wavelength of 234 nm (detection of conjugated dienes);

by quantitative determination using a vapor-phase chromatography after recovery of the sample taken from the reactor.

The oxidation stops when the content of 9-HODE and/or all HODE is, respectively, about 2.2-2.5% and 10-12%.

2. Recovery

In hydroprocessing the mixture was added 0.16 g of the antioxidant type Coviox T 70. Prepare a 2% (weight/volume) solution of NaBH₄in ethanol and cool it to a temperature of about 4°C. This regenerating solution is antenna and several servings poured to hydroprocessing mixture, maintaining the temperature of the reaction medium below 25°With (about 20°).

The recovery phase finishes when the temperature becomes constant, while the stirring is continued at room temperature (approximately 1 hour).

During the reduction reaction of secreted hydrogen.

3. Neutralization to pH=6-7

To the reaction mixture slowly and with stirring, add 100 ml of 25%aqueous solution of citric acid in ethanol. Can happen hydrogen gas and sometimes precipitation of sodium citrate.

4. Extraction, decantation, washing and drying

To the reaction mixture with rapid stirring, 500 ml of n-hexane, and then 500 ml of water and left to settle. After the separation of the two phases Recuperat the organic phase.

This organic phase is washed until neutral wash water, then dried by filtering through about 140 g anhydrous sodium sulfate.

5. Evaporation

To the organic phase added 0.16 g of antioxidant (Coviox T 70) and n-hexane is evaporated in vacuum at a temperature of about 50-60°C.

6. The conditioning and storage

The oil obtained is stored in a vessel made of dark glass or vessel in the form of a barrel in a nitrogen atmosphere and at a temperature below 10°C.

Pharmacological properties

The aim of the invention is the identification of molecules with and the efficiency in relation to lipolysis, i.e. capable of facilitating hydrolysis accumulated in adipocyte triglyceride, and thus the released fatty acids are then metabolized.

Pharmacological study using human adipocytes

The purpose of this study is to investigate the influence of 9-HODE on the production of cyclic adenosine monophosphate (camp), which is an activator of lipolysis in human adipocytes. Studying the effects of 9-HODE compared with that of adrenaline and caffeine, which are two molecules, known for its stimulating effects on lipolysis.

It is found that the concentration of 10^-4M 9-HODE causes an increase in the content of camp, equivalent achieved using 10^-3M caffeine, and above increase, whatever the concentration obtained at the expense of adrenaline.

At a concentration of 10^-3M 9-HODE concentration camp 10 times higher than obtained with the help of caffeine (7940 femtomole camp/ million adipocytes against 799).

These results allow us to conclude that 9-HODE in vitro is an effective activator of lipolysis due to stronger stimulation depozitarnoj production of camp compared with molecules of caffeine and adrenaline.

Studies on the rat

Conduct research using twelve rats, which at first is subjected to limitation the food (modeling diet) for two weeks and then get a normal diet (weight gain) in combination or not with the introduction of sunflower oil, processed according to the method according to the invention and containing 2 wt.% 9-HODE. Use a control group that receives sunflower oil without 9-HODE. After treatment for eleven days in both groups, define fat mass.

The results obtained in the group receiving containing 2 wt.% 9-HODE sunflower oil, show that fat mass was significantly (p<0.001) and 25%less than defined in the placebo group (21.8 g, compared to 29.5 g in the control group).

Introduction oral and containing 2 wt.% 9-HODE sunflower oil thus allow for a statistically meaningful way to reduce fat mass in rats receiving vegetable oil.

The study of skin penetration

Carry out a study of dermal penetration in vitro using human skin cells FRANZ. These cells include the surface of the human skin the size of 1.76 cm²that put 800 mcg 9-HODE. After exposure for 6 hours carry out quantitative determination of 9-HODE by gas chromatography on the sample surface of human skin, the epidermis, dermis and receptor phase.

The distribution of 9-HODE following:

- the surface of the skin 33%

the epidermis 11%

- dermis 32%

receptor phase 24%.

Thus, 9-HODE has the ability to significant skin penetration, allowing e which directly affect the level of adipocytes.

CLINICAL studies of 9-HODE

Clinical study of the efficacy of cosmetic gel containing 0.5% 9-HODE

Conduct a clinical study of twenty women with excess body fat on the hips ("fat deposits on the hips"). The test was applied on each hip in a month, twice a day, the gel containing 0.5% HODE. The criterion of effectiveness is the value of the circumference of the hips at two levels.

The results obtained testify to the effectiveness of the product, because watching circumference reduction of 6 mm at the top level and 5.4 mm on the lower level of the hips, and these two values decrease statistically very significant.

Clinical study of the efficacy of cosmetic gel containing 1.5% of 9-HODE

Another study conducted in 21 women with excess fat on the hips. The investigational product was a cosmetic solution containing 1.5% of HODE, and this study helped to demonstrate a statistically significant reduction in hip circumference 7 mm, starting from the twelfth day of application, and 10 mm after application for 24 days. On the other hand, 80% of women observed a clear improvement in skin elasticity and reducing the effects of cellulite and enlarged pores.

Clinical study to improve thermogenesis after a single oral administration of 9-HODE

Studies is the study conducted on ten healthy subjects, which were consumed in random order the following products:

- sunflower oil 20 g

- sunflower oil 20 g, containing 2% 9-HODE.

Each subject receives a week of each period products in random order (cross-Protocol or "cross over").

Subjects spend two hours in the calorimetric cell (basal energy expenditure), then absorb one of the two products during standard eating 800 calories and remain in a period of 6 hours in the calorimetric chamber (energy consumption after processing).

The average energy expenditure (kJ/min) in the case of two products:

The difference in power consumption after treatment statistically significantly (p<0,01). It increases thermogenesis, which is close to 8%, especially of interest for the treatment of obesity.

Therefore, according to the invention, it is possible to treat obesity through the mouth and localized overweight local path. In the case of oral route, 9-HODE can be introduced in the form of the acid or in the form of triglycerides. In SL the tea local path 9-HODE is preferably introduced in the form of the free acid,

9-HODE can be entered as a food additive in the form of gelatin capsules, tablets, potable solution, pastilles, chewing gum, etc. and/or in food. Can be used pure or diluted 9-HODE in creams, gels, cosmetic pencils, lipstick, etc.

Printed on zone mass excess, it can have a direct effect on adipocytes. 9-HODE can be entered as a drug systemic or local path for the treatment of obesity. Input dose, for example, by oral constitute 50-5000 mg/day, and the local way of 0.1-100%.

1. The method of obtaining oil, enriched with fatty hydroxyoctadecadienoic acid (HODE) or their esters from the oil mixture containing linoleic acid or its esters, characterized in that conduct controlled oxidation of linoleic acid and/or linolenic acid or their esters in the presence of the oxidation catalyst, and the oxidation stop when the contents of all HODE or their esters is higher than 5% and/or the content of isomeric 9-hydroxy-10,12-octadecadienoic acid (9-HODE) or its esters is higher than 1.5%, and restore using the reducing agent in the presence of an antioxidant formed during the oxidation of the hydroperoxides.

2. The method according to claim 1, Otley is audica fact, the original oil mixture is a vegetable oil comprising more than 40 wt.% linoleic acid and/or linolenic acid in the form of triglycerides, such as corn oil, borage oil, safflower oil, soybean oil, angrove oil, sunflower oil, oil from black currant seed, wheat germ oil, hemp oil, pumpkin seed oil, linseed oil.

3. The method according to claim 1, characterized in that the starting oil mixture is an oil mixture containing more than 40 wt.% linoleic acid and/or linolenic acid, in the form of ethyl ester, preferably containing 75% ethyllinoleate.

4. The method according to any one of claims 1 to 3, characterized in that the final content in the enriched oil all HODE and 9-HODE is at least 5 wt.%, accordingly, 1.5 wt.%.

5. The method according to any one of claims 1 to 4, characterized in that the final content in the enriched oil all HODE and 9-HODE is 10-12 wt.%, accordingly, the 2.2-2.5 wt.%.

6. The method according to any one of claims 1 to 5, characterized in that the oxidation catalyst is a halide of iron, copper, preferably Fl_C.

7. The method according to any one of claims 1 to 6, characterized in that the reducing agent is preferably NaBH₄.

8. Enriched fat 9-hydroxyoctadecadienoic acid (9-HODE) or its ester or its salt, the oil, the region is non lipolytic action, content HODE above 5% and/or with the content of 9-HODE higher than 1.5%, which can be obtained using the method according to any one of claims 1 to 7.

9. Drug for the treatment of obesity containing as an active start oily 9-hydroxyoctadecadienoic acid (9-HODE) or its pharmacologically acceptable esters or salts, preferably in the form of enriched oil on item 8.

10. Dietary Supplement for the treatment of obesity containing as an active start oily 9-hydroxyoctadecadienoic acid (9-HODE) or its pharmacologically acceptable esters or salts, preferably in the form of enriched oil on item 8.

11. Cosmetic for localized treatment of obesity and its consequences in relation to the appearance of the skin, such as cellulite; enlarged pores, fatty deposits on the hips, containing as an active start oily 9-hydroxyoctadecadienoic acid (9-HODE) or kosmetologicheskii acceptable esters or salts, preferably in the form of enriched oil on item 8.