MXPA99010678A - Phytosterol and / or phytostate derivatives - Google Patents

Abstract

The phytosterol and / or phytostanol esters of polyunsaturated fatty acids from 18 to 22 carbon atoms and at least three carbon-carbon double bonds are effective for the reduction of both cholesterol and triglycerides of the dream

Description

DERIVATIVES OF FTTOESTEROL AND / OR FTTOESTANOL FIELD OF THE INVENTION The present invention relates to polyunsaturated fatty acid esters of phytosterols and / or phytostanols and to their use.

BACKGROUND OF THE INVENTION Phytosterols are found among plant sterols, for example in small amounts in vegetable oils such as corn oil, seed oil or oils from other oily plants, where they are found as free sterols, esters of fatty acids and glycosides. Phytosterols are structurally similar to cholesterol, with the main differences in the carbon skeleton of their side chains. In nature you will find a good number of different phytosterol structures. The most common are campesterol, beta-sitosterol and stigmaesterol. The reduction of phytosterols provides saturated phytosterols, called phytostanols, such as campestanol or sitostanol, which are also found in nature in small amounts. A normal human diet R E P. : 3200? it typically leads to the ingestion of less than half a gram per day of said substances in various forms.

It is known that the ingestion of phytosterols and / or phytostanols in defined amounts, for example of several grams per day or more, can reduce cholesterol levels in blood serum. It is assumed that free phytosterols and phytostanols inhibit the absorption of dietary cholesterol and bile cholesterol through the displacement of cholesterol. However, only modest reductions in serum cholesterol levels have been generally observed by adding phytosterols or free phytostanols to the diet.

Arteriosclerosis is a cause that causes death in many parts of the Western world. It has been shown that low density lipoprotein (LDL) cholesterol is directly associated with the development of cardiovascular diseases, while high density lipoprotein (HDL) cholesterol has an inverse relationship with the development of cardiovascular diseases. People with combined hyperlipidemia are even more at risk for heart disease. High levels of cholesterol in blood serum and high levels of triglycerides are generally accepted, both as causes and as indicators of the progression of cardiovascular diseases. Thus, the decrease of cholesterol and the decrease of triglycerides are considered as desirable targets as main strategies for intervention. Many methods have been proposed to lower serum cholesterol, among which are the use of certain pharmaceutical agents and the ingestion of phytosterols in various forms. Similarly, many methods have been proposed to decrease serum triglycerides, among which are the ingestion of polyunsaturated fatty acids (PUFA) in various forms.

Physical properties are especially important in food applications. The properties of the ingredients allow and limit the ways in which the products can be supplied, for example in oils or shortenings. In addition, properties such as solubility and melting point can affect the acceptability of a food product by changing texture, taste or mouth taste, by complicated, unpredictable ways. A problem with the use of free phytosterols has been its crystalline nature and its limited solubility in oils. Generally, large amounts have been used to achieve an effect on cholesterol levels, but as a consequence, resulting physical problems have appeared. So, other forms have been sought.

WO 96/38047 discloses a food product based on fats, which contains natural fatty components that have a diminishing effect on blood cholesterol and where at least one component of tocotrienol is physically present., oryzanol and phytosterol, preferably mixed with at least one PUFA triglyceride component. The phytosterols present are mainly in the form of free phytosterol at low, defined, and relatively insoluble concentrations. The resulting products are semi-solid. Much higher proportions of PUFA triglycerides are used than phytosterols. The effects of mixtures on triglyceride levels are not described.

US Patent 4,588,717 describes phytosterol fatty acid esters as vitamin supplements or as diet pills, said esters being obtained from a phytosterol and a fatty acid of 18 to 20 carbon atoms. Included among these fatty acids are also unsaturated acids, linolenic acid, linoleic acid and arachidonic acid. It is generally known that these acids have almost no effect on triglyceride levels.

The patent WO 97/42830 describes the preparation and use of gels consisting of partially crystallized mixtures of natural edible oils with low concentrations of sterols and esters of sterol (especially sitosterol and aryzanol) and optionally monoglycerides, in certain proportions as a means to give firmness to liquid edible fats. Because of the low content of sterol and sterol ester, said necessity products require considerable volumes and an additional caloric content, to supply phytosterols and phytosterol esters in sufficient quantities to effectively lower cholesterol.

Patent WO / 98/06405 describes a method for reducing cholesterol in the bloodstream by administering beta-sitostanol with campestanol in certain proportions, such as esters of fatty acids derived from vegetable oils.

Patent US 5,502,045 describes the reduction of cholesterol absorption in the bloodstream by the administration of beta-sitostanol esters of acids of 2 to 22 carbon atoms derived from vegetable oils.

In the Journal of Lipid Research (1933, 34, 1535-1544), experiments are described and referenced experiments with feed mixtures of human subjects, of esters of sitostanol obtained from fatty acids of rapeseed oil. It was found that phytostanol esters reduced serum LDL cholesterol more effectively than free phytosterols, even though they were hydrolyzed during the intestinal passage.

In the European Journal of Clinical Nutrition ("European Journal of Clinical Nutrition") 1998, 52, 334-343, results of human trials with margarines enriched with phytosterols and phytosterol esters. It was shown that plasma concentrations of total cholesterol and LDL cholesterol had been reduced by sterol esters mixed with margarines, compared to controls with similar fatty acid profiles. All materials contained esters of unsaturated fatty acids, especially those of oleic, linoleic or linolenic acid. No effect on the concentration of triglycerides in plasma was seen, with the margarines enriched with sterol.

DESCRIPTION OF THE INVENTION It has now been discovered that phytosterol and / or phytostanol esters obtained from phytosterols and / or phytostanols with certain omega-3 polyunsaturated fatty acids (n-3 fatty acids) are surprisingly effective in reducing both cholesterol and cholesterol. serum triglycerides. Said polyunsaturated fatty acids are, for example, eicosapentaenoic acid (EPA) with five unsaturated carbon-carbon double bonds, or docosahexaenoic acid (DHA) with six unsaturated carbon-carbon double bonds. These esters significantly decreased both plasma cholesterol and triglyceride levels, whereas phytosterol combined with vegetable oil only decreased plasma cholesterol levels. Consequently, these esters can be used as a combined agent for cholesterol reduction and as an agent to lower triglycerides, and thus positively affect two of the major risk factors for cardiovascular diseases.

The effects described above have been shown in rats and the methods employed and the results obtained are described summarized below.

Animal treatment Thirty male Fischer rats, with a weight of 177 ± 14 g, they were kept on a high fat diet (table 1) during the two weeks preceding the treatment. They were randomly divided into five experimental groups of six animals each. The control group (group 1) continued with the high fat diet used during the two weeks of the pretreatment period. For the other experimental diets, in order to have isocaloric diets and an equal amount of fat in all the experimental diets, 2% (p /?) Of the fat content of the control diet was replaced (1% oil). coconut and 1% corn oil) for 2% (w / w) of the following lipids: Group 2: 2% mixture of sitosterol / oleic oil rich in sunflower (TRISUN 80) (in the ratio 1: 1); Group 3: 2% of the ester sitostanol-DHA; Group 4: 2% of the ester stigmaesterol-EPA: Group 5: 2% mixture of sitosterol + EPA / DHA ester (in the ratio 1: 1).

The fatty acid compositions of the experimental diets are shown in Table 2. The rats were given free access to water and diet, and were kept in a 12 hour light / dark cycle. The diet of the cages was renewed daily, all unconsumed material was discarded and the food intake was measured. Blood samples (1 ml) were collected by retro-orbital puncture at the beginning of the experimental period (week 0) and after 2 weeks of treatment (week 2). After 4 weeks, the animals were sacrificed by withdrawing blood from the vena cava, with isoflurane anesthesia. The blood was collected in tubes containing EDTA as an anticoagulant.

Lipid analysis __ Plasma was prepared from heparinized blood by immediate centrifugation at 1600 g for 10 minutes at 4 ° C. The plasma cholesterol, triglycerides and HDL cholesterol assays (precipitation method) were determined enzymatically on a COBASFARA analyzer (Roche Diagnostica, Switzerland). Non-HDL cholesterol was calculated by difference. The fatty acid composition of the diets was analyzed by gas chromatography.

Statistical analysis All data are expressed as means ± SD for the animals of each diet group. Differences in the means between dietary groups were analyzed by one-way analysis of the covariance (ANCOVA) with the subsequent Dunnet test for multiple comparison against the control group (group 1 and / or group 2). The adjusted covariant was the value of the corresponding parameter at the beginning of the treatment period (week 0). All trials were performed at the 5% level and the 95% confidence intervals were calculated.

Results The growth of the rats was similar in all the dietary groups during the 4 weeks of the feeding period. The average food intake during the 4-week period of the five dietary regimens was 12 g / day / rat. Dietary treatment had no significant effect on body weight and food consumption.

Plasma cholesterol was significantly lower from 28% to 46% in all four groups treated with phytosterols compared to control and 46% to 66% with respect to the pretreatment period (week 0) (Table 3) .

HDL cholesterol levels were almost unaffected by the phytosterol treatment (table 4). Therefore, non-HDL cholesterol (VLDL cholesterol + LDL cholesterol) was decreased mainly by phytosterol treatment.

Plasma triglyceride levels were significantly decreased from 18% to 39% in the groups treated with phytosterol combined with n-3 fatty acids compared to the control group and from 15% to 41% compared to the period of pretreatment (week 0) (table 5), while phytosterol combined with vegetable oil (group 2) did not significantly decrease plasma triglycerides.

Table 1 Composition of the diet3 rich in fats of rats.

The diet contained 0.5% by weight of cholesterol, 1% by weight of sodium cholate and the standard mixture of vitamin and mineral standard, according to the specifications for the rats. aThe main fats consisted of coconut almond (18% by weight), coconut oil (2.5% by weight) and corn oil (2.5% by weight). r f in s (_p O i abla 2 Fatty acid composition of experimental diets (* molar) Group 1 Group 2 Group 3 Group 4 Group 5 Fatty acids Control 2% (mixture of 2% of ester sito- 2% of ester stig- 2% of mixture of esters sitoesterol + Trisun) eswnol-DH? maesterol-EPA sitoesterol EPA / DHA? Saturated 57.73 56.57 57.62 56.16 56.86 Monoenos 18.84 25.35 15.59 15.62 16.34 PUFAs 23.43 18.08 26.79 27.98 26.81 Sum n -6 22,08 16,76 16,85 16,92 17,47 Sum n-3 1,21 1,15 9,91 10,89 9,20 C 14 33,91 33,63 34,99 34,05 34.04 C 15 17.84 16.84 16.76 16.58 16.66 C 18 5.38 5.64 5.33 5.26 5.42 • C 18: 1-9 17.99 24.39 15,08 15,02 15.16 C 18: 1-7 0.55 0.67 0.41 0.42 0.56 C 18: 2-6 21.91 16.54 16.31 16.56 16.74 C 18: 3-3 1.21 1.15 1.17 1.21 1.25 C 20: 5-3 0.00 0.00 0.11 9.52 4.56 C 22: 6-3 0.00 0.00 8.58 0.13 2.76 The results are expressed as percent methyl esters of fatty acids (molar I).

I-1 Lp O Ol O in Tt la 3 Effects of phytosterol esters on total cholesterol in plasma, in rats in Change in percent of pretreatment. Significantly different from the control at week 2 or week 4 (P <0.05). Significantly different from group 2 (mixture of sitosterol + trisun) at week 2 or week 4 (P <0.05). t in in or in or TPblfl 4 Effects of phytosterol esters on lipoproteins in rats * Significantly different from the control at week 2 or week 4 (P <0.05). Significantly different from group 2 (mixture of sitosterol + trisun) at week 2 or week 4 (P <0.05). to t in or in or in Table 5 Effects of phytosterol esters on plasma triglycerides in rats l- > Change in percent of pretreatment. Significantly different from the control at week 2 or week 4 (P <0.05). Significantly different from group 2 (mixture of sitosterol + trieun) at week 2 or week 4 (P <0.05).

The physical properties of organic compounds such as physical state, melting point and solubility can not be predicted with certainty, from chemical structures. These properties significantly influence the acceptability of the food product since they affect the texture, taste or taste of mouth by complex and unpredictable pathways. In the context of the present invention, EPA and DHA esters were synthesized with sitoesterol, sitostanol and stigmaesterol in pure form as well as mixtures of these sterols with other sterols and with mixtures of said acids with other fatty acids. Some of the compounds and mixtures were liquid while others were partially solid at room temperature or lower. All were significantly more soluble in edible oil than the corresponding phytosterols or phytostanols. For comparison, sitoestanol esters were also synthesized with mixtures of fatty acids containing significant levels of unsaturated fatty acids of 16 to 20 carbon atoms, especially the linolenic acid obtained from rapeseed oil, and it was found that the mixtures obtained were very crystalline. ambient and lower temperature. Much more edible oil was needed to completely dissolve these esters compared to esters prepared with EPA or DHA.

It was further discovered that the compounds of the present invention offer unique physical advantages. The compounds have a higher solubility in edible oils than other phytosterol esters described so far, which is an advantage for their incorporation into a wide variety of food products. These materials allow the supply of phytosterols and / or phytostanols and selected PUFAs, in their ester form in the highest possible concentration per unit volume. This is advantageous for incorporation into products where small volumes are important, such as dispersible formulations in water or when additional non-essential edible oils are not desirable. This presents physical advantages over simple mixtures or formulations of other phytosterols / phytostanols and / or their fatty esters with PUFAs and their ester or triglyceride forms normally available.

Accordingly, an object of the present invention are phytosterol and / or phytostanol esters obtained from phytosterols and / or phytostanols with PUFAs having 18 to 22 carbon atoms and at least three unsaturated carbon-carbon double bonds. Another object of the present invention is the use of said phytosterol and / or phytostanol esters or mixtures thereof in the diet and human dietetic food for the purpose of lowering serum cholesterol levels and serum triglyceride levels., in humans. The compounds according to the present invention are preferably used in a total amount with a content of 1 to 10 grams per day of phytosterol ester and / or phytostanol ester. Still another object of the present invention is the use of these phytosterol esters and / or phytostanol or mixtures thereof, in formulations in suitable physical forms such as capsules, etc., as dietary supplements or as ingredients in foods as well as also these formulations per se.

The preferred phytosterols are beta-sitosterol or stigmasterol and campesterol or mixtures thereof.

More preferred are beta-sitosterol and stigmasterol or mixtures thereof. The most preferred is beta-sitosterol. The preferred phytostanols are beta-sitostanol and campestanol or mixtures thereof. The most preferred is beta-sitostanol. Preferred PUFAs are EPA and DHA.

It is readily understood that the esters of the present invention do not need to be used in the pure state. The mixture of these esters can be used. Mixtures of these esters with other fatty esters of phytosterol / phytostanols can also be used. The proportions of phytosterol and / or phytostanols used can vary with the origin thereof. Likewise, the proportions of PUFA and other fatty acids may vary. It is also understood that the products may contain some free phytosterols / phytostanols and / or glycerides or PUFA esters. The physical properties can vary as a consequence, from those having a high proportion of phytosterol / stanol polyunsaturated esters and then they are perfectly soluble liquids in edible oils, up to those of the mixture having lower ratios of unsaturation, and then they are semi-solid or waxy The compounds according to the present invention can be prepared according to known methods. For example they can be obtained by esterification of a phytosterol / phytostanol with an n-3 PUFA in a known manner. Alternatively, they can be prepared preferably by interesterification of free phytosterols / phytostanols with n-3 PUFAs esters, heating in the presence of an interesterification catalyst, wherein (i) the interestinication is effected solvent-free, (ii) the fatty esters they include suitable esters and triglycerides of 1 to 4 carbon atoms, (iii) the catalyst is a sodium alkoxide of an alcohol of 1 to 4 carbon atoms. The reaction is suitably carried out by heating the mixture to 80-140 ° C at a pressure of 133-6650 Pa and carrying out the reaction preferably with a stoichiometric amount to an excess of PUFA ester. The following examples illustrate the invention in more detail: Example 1 To a mixture of 0.91 g of docosahexaenoic acid (purity: 90%), 1.03 g of stigmaterol (purity: 95%) and dimethylaminopyridine (50 mg) in 18 ml of anhydrous dichloromethane, a solution of dicyclohexylcarbodiimide was added. (0.63 g) in 5 ml of dichloromethane. After 4 hours of stirring at room temperature, the reaction was terminated. Then, methanol (0.5 g) and acetic acid were added (0.25 g), and the mixture was stirred for an additional hour. The mixture was cooled to 0 ° C, filtered and the solids were washed with exano (3 x 25 mL). The solvent was removed under reduced pressure and the residue subjected to flash chromatography on silica gel obtaining a pure fraction of 1.0 g of docosahexaenoate of stigmaterol in the form of a colorless oil consistent with the NMR and IR data. This substance was kept in oil form when stored for several weeks at room temperature and when cooling for several weeks at -20 ° C.

Example 2 Analogously to example 1, stigmaesterol eicosapentaenoate was prepared from eicosapentaenoic acid (purity: 90%) and stigmaterol. The stigmasterol eicosapentaenoate (1.46 g) was obtained in the form of a colorless oil that remained liquid at a temperature between 20 ° C and -20 ° C.

Example 3 Analogously to Example 1, a mixture of eicosapentaenoic acid-docosahexaenoic acid esters of stigmaterol was prepared from stigmasterol with a mixture of 49% eicosapentaenoic acid and 27% docosahexaenoic acid. The mixture of stigmasterol esters was obtained in the form of a colorless oil that remained liquid in the temperature range of 20 ° C to -20 ° C.

Example 4 Analogously to example 1, docosahexaenoate of stigma-ethanol was prepared from stigma-ethanol (purity: 95%) and docosahexaenoic acid (purity: 90%). The docosahexaenoate of stigma-ethanol was obtained as a lightly colored oil, which remained liquid between 20 ° C and -20 ° C.

Example 5 Analogously to Example 1, the stigma-ethanol eicosapentaenoate was prepared from stigma-ethanol and eicosapentaenoic acid. Eicosapentaenoate of stigma-ethanol was obtained as a slightly yellowish oil, which remained liquid in the temperature range between 20 ° C and -20 ° C.

Example 6 Analogously to example 1, a mixture of stigma-ethanol esters of eicosapentaenoic acid and docosahexaenoic acid was prepared from stigma-ethanol and a mixture of 49% eicosapentaenoic acid with 27% docosahexaenoic acid. A mixture of stigmaestanol esters of eicosapentaenoic acid and docosahexaenoic acid was obtained in the form of a colorless oil which became turbid when stored at 20 ° C and partially solid at -20 ° C.

Example 7 Analogously to example 1, a mixture of sterol esters of PUFA was prepared from a mixture of beta-sitosterol, campesterol and stigmasterol and a mixture of 49% eicosapentaenoic acid with 27% docosahexaenoic acid. A mixture of sterol PUFA esters was obtained in the form of a turbid oil containing some solids at 20 ° C and partially solid at -20 ° C.

Example 8 Analogously to example 1, a mixture of unsaturated fatty esters of stigma-ethanol was prepared from the stigma-ethanol and a mixture of fatty acids obtained from the basic hydrolysis of a sample of a commercial food of Swiss rapeseed oil (9% of saturated, 61% monounsaturated, 30% polyunsaturated triglycerides). A mixture of unsaturated fatty esters of stigma-ethanol was obtained in the form of a colorless oil which crystallized slowly at room temperature. At -20 ° C the material was essentially solid.

Example 9 A mixture of phytosterols (20.6 g of a commercial mixture of 43% sitosterol, 23 stigmasterol, campesterol 24% with other minor sterols) and 75% ethyl esters of DHA-EPA (16.8 g of a commercial mixture of 43% docosahexaenoate of ethyl and 32% ethyl eicosapentaenoate with other fatty esters), dried at 120 ° C while bubbling with an inert gas stream. To the liquid mixture was added sodium ethoxide (1.03 ml of 21% solution in ethanol). The mixture was stirred at 120 ° C at 15 mbar vacuum for two hours. The light brown mixture was cooled to 80 ° C and the catalyst was paralyzed with dilute acid. The separated oily phase was dehydrated by heating under reduced pressure while bubbling with an inert gas stream. 35.0 g of crude phytosterol sters were obtained, in the form of a turbid, light brown oil, which remained fluid at room temperature. HPLC showed that the conversion to sterol esters was 95%.

Example 10 A mixture of phytosterols (148 g of a commercial mixture of 43% sitosterol, 23 stigmaterol, campesterol 24% with other minor sterols) and fish oil glycerides (141 g of a commercial glyceride mixture with a fatty acid composition of 17% EPA- and 11% DHA), was dehydrated by bubbling at 120 ° C with an inert gas. To the liquid mixture, sodium ethoxide (11.9 ml of 21% solution in ethanol) was added. The mixture was stirred at 120 ° C at 15 mbars vacuum for one hour.

The light brown mixture was quenched with dilute acid, and the separated oil phase was dehydrated under reduced pressure to obtain 249 g of a light brown oil which crystallized slowly in a semi-solid mass. HPLC showed that the conversion was 93%.

'Example 11 The solubilities of the materials obtained according to the procedures described in examples 1-8, as well as the original sterols, were determined in a commercial sample of Swiss rapeseed oil, by the alternative addition of small increments of oil at room temperature. to heavy amounts of sterol esters and stirring for periods of 5 minutes until the solution was achieved. The minimum starting ratio was around 1: 1 and discontinuous tests were carried out until reaching 10: 1.

Material Solubility g of oil / g of material Docosahexaenoate of miscible stigma sterol > 1 Eicosapentao miscible stigmasterol> 1 Esters Mixture EPA-DHA miscible stigma sterol > 1 Docosahexaenoate of miscible stigma-ethanol > 1 Eicosapenta in miscible stigma-ethanoate > 1 Esters blend EPA-DHA of soluble stigma-ethanol > 4 Esters blend EPA-DHA of miscible sitoesterol mixture sterols > 1 Mix of rapeseed esters of Insoluble stigma-ethanol > 10 Insoluble stigma sterol > 10 Insoluble stigma-ethanol > 10 Ethyl ester of docosahexaenoic acid 90% miscible > 1 EPA miscible 90% ethyl ester > 1 It is noted that in relation to this date, the best method known to the applicant, to implement said invention is that which is clear from the manufacture of the objects to which it refers.

Having described the invention as above, the content of the following is claimed as property:

Claims (10)

1. Phytosterol and / or phytostanol esters obtained from phytosterols and / or phytostanols with polyunsaturated fatty acids, characterized in that the polyunsaturated fatty acid has from 18 to 22 carbon atoms and at least three unsaturated carbon-carbon double bonds.

2. Esters according to claim 1, characterized in that the phytosterol is beta-sitosterol, stigmasterol or campesterol or a mixture thereof, preferably beta-sitosterol or stigmaterol or a mixture thereof, more preferably beta-sitosterol.

3. Esters according to claim 1 or claim 2, characterized in that the phytostanol is campestanol or beta-sitostanol or a mixture thereof, preferably beta-sitostanol.

Esters according to any one of claims 1 to 3, characterized in that the polyunsaturated fatty acid is eicosapentaenoic acid or docosahexaenoic acid.

5. Esters according to any one of claims 1 to 4, characterized in that they further comprise in admixture therewith, phytosterol and / or phytostanol esters with fatty acids or the polyunsaturated fatty acids specified in claim 1 or claim 4, and / or which further comprise in admixture therewith, free phytosterols / phytostanols and / or glycerides or PUFA esters.

6. The use of esters according to any one of claims 1 to 5, characterized in that the human diet and dietetic food, in order to decrease serum cholesterol levels and serum triglyceride levels, in humans.

7. The use of esters according to any one of claims 1 to 5, characterized in that in formulations, in suitable physical forms, preferably in capsules, as dietary supplements or as ingredients in foods.

8. Formulations in suitable physical forms, preferably in capsules, of dietary supplements or foods characterized in that they contain esters as claimed in any one of claims 1 to 5.

9. A process for the preparation of esters according to any one of claims 1 to 5, characterized in that it comprises the esterification of a free phytosterol / phytostanol to a mixture thereof, with a polyunsaturated fatty acid n-3 with 18 to 22 atoms carbon and at least three unsaturated carbon-carbon double bonds to a mixture thereof, in a manner known per se.

10. Esters according to any one of claims 1 to 5, characterized in that they have been obtained by interesterification of phytosterols / phytostanols with fatty esters of n-3 polyunsaturated fatty acids, heating in the presence of an interesterification catalyst, wherein (i) the interesterification is effected solvent-free, (ii) the fatty esters include suitable simple triglycerides and esters of 1 to 4 carbon atoms, (iii) the catalyst is a sodium alkoxide of an alcohol of 1 to 4 carbon atoms, wherein the reaction is conveniently carried out by heating the mixture to 80-140 ° C at a pressure of 133-6650 Pa and wherein the reaction is carried out with a stoichiometric amount to an excess of the PUFA ester.