KR100278194B1 - Method for Manufacturing Fat-soluble Ester Compound of Phytosterol or Phytostanol Unsaturated Fatty Acid for Lowering Cholesterol Level - Google Patents

Abstract

The present invention relates to a method for producing an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in liquid oil that can be used for the treatment of hypercholesterolemia, as well as for the prevention of some cardiac diseases and hypertension and the like, and a functional food comprising the same. The present invention is obtained by dissolving phytosterol or phytostanol and unsaturated fatty acid in a nonpolar organic solvent and adding a basic catalyst, followed by esterification reaction by adding a carboxyl group activating substance dissolved in the nonpolar organic solvent, and in an electrical process. A method for producing an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in a liquid oil, which comprises a step of removing the solvent under reduced pressure from one reactant, followed by stirring and precipitation with methanol or a methanol / acetone mixed solvent. In the present invention, the unsaturated fatty acid ester of phytosterol or phytostanol prepared by the above method is not easily dissolved in liquid oils such as corn oil, unlike conventional phytosterol or phytostanol, and LDL-cholesterol which is harmful to human body. It has been found that the absorption of Hg lowers blood cholesterol levels, while not affecting the absorption of HDL-cholesterol. Therefore, the unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in the liquid oil prepared by the present invention is widely used as an active ingredient in foods that can be used not only for the treatment of hypercholesterolemia but also for the prevention of some heart disease and high blood pressure. Could be applied.

Description

Method for manufacturing fatty-soluble ester compounds of fat-soluble phytosterols or phytostanols having a cholesterol-lowering effect {Method for Manufacturing Fat-soluble Ester Compound of Phytosterol or Phytostanol Unsaturated Fatty Acid for Lowering Cholesterol Level}

The present invention relates to a process for producing unsaturated fatty acid esters of phytosterols or phytostanols soluble in liquid oils having a cholesterol lowering effect. More specifically, the present invention provides a method for preparing an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in liquid oil that can be used not only for the treatment of hypercholesterolemia but also for the prevention of some cardiovascular diseases and hypertension and the like, and a food comprising the same. It is about.

Cholesterol is a nutrient necessary for the human body, such as being used as a constituent of biological membranes and as a starting material for hormonal synthesis, but when it is ingested excessively, cholesterol is accumulated in blood vessels and causes heart disease. So far, there is no preventive method other than low cholesterol diet, and taking drugs such as cholesterol-lowering drugs are effective, but their use is extremely limited because of causing side effects such as liver dysfunction due to inhibition of cholesterol synthase. Therefore, the development of foods with a cholesterol-lowering action is required.

Chitosan, phytosterol, inositol, pectin, etc. are known as substances that lower blood cholesterol in the human body, but the effects or metabolic mechanisms are not clear when phytosterol is excluded. Phytosterol, a plant sterol, has a known mechanism of inhibiting metabolism of cholesterol in the body through competition with low density lipoprotein (LDL) -cholesterol, which is harmful to human body due to its structural similarity to cholesterol. It has been approved by the FDA.

On the other hand, phytosterol collectively refers to an alcohol compound having a steroid structure in higher plants, and may be classified into stigmasterol, spinasterol, campesterol, and cytosterol, for example, cytosterol. Α-, β-, and γ- types also exist. Cholesterol-lowering effect of β-sitosterol (24-ethyl-5α-cholestene-3β-ol), which is the most representative substance among various phytosterol compounds, was confirmed in an experiment in male rats and human bodies (Sugano, M et al., J. Nutr., 107: 2011-2019, 1977). In addition, β-sitosterol ester compounds in which β-sitosterol is substituted with fatty acids show almost the same effect (Mattson, F. H. et al., J. Nutr. 107: 1139-1146, 1977). For example, when 2 g of β-sitosteryl oleate was administered to adult men daily for 5 days, blood cholesterol levels were 33% lower (Mattson, FH et al., Am. J. Clin. Nutr., 35: 697-700, 1982).

In addition to the cholesterol lowering effect, β-sitosterol is also known as a major component of Zea mays L., which is a treatment for periodontal disease and gingivitis. However, β-sitosterol by itself is limited in formulation due to physical limitations insoluble in water and fats and oils, and commercialization remains at the level of simple tablets of nutrient type and development as a food material is insufficient. .

Recently, in order to compensate for the disadvantage of β-sitosterol, β-sitostanol obtained by reacting β-sitostanol (24-ethyl-5α-cholestane-3β-ol), which is a solid form of β-sitosterol, with a fatty acid It is known that when ester compounds are used as additives in solid dairy products such as butter, margarine and the like, they can lower blood cholesterol (see WO 92/19640). However, the β-cytostanol ester compound is applicable only to solid dairy products, which are relatively in low demand in Korea, and are not dissolved in liquid oil products, and the β-cytosterol is hydrogenated by an organic chemical method, thereby producing β-cyto. There is a difficulty in reacting with fatty acid after synthesizing stanol.

Therefore, from the viewpoint of prophylaxis that not only treatment of hypercholesterolemia, but also prevention of some heart disease and high blood pressure, economical method for preparing unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in liquid oil such as vegetable oil There is a constant need for development.

Accordingly, the inventors have focused on the fact that the unsaturated fatty acid or its ester compound has a significantly lower melting point than the saturated fatty acid or its ester compound and most of them are liquid at room temperature, so that phytosterol or phytostanol is combined with unsaturated fatty acid. A method for preparing an unsaturated fatty acid ester compound was developed, and it was confirmed that the obtained material had the same effect as phytosterol or phytostanol and was soluble in liquid oil, thereby completing the present invention.

After all, the main object of the present invention is to provide an economical method for preparing unsaturated fatty acid ester compounds of phytosterol or phytostanol, which are soluble in liquid oil, which can not only treat hypercholesterolemia but also prevent some cardiovascular diseases and hypertension.

Another object of the present invention is to provide a food comprising an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in the liquid oil prepared by the electric preparation method.

The method for preparing an unsaturated fatty acid ester compound of phytosterol or phytostanol having a cholesterol lowering effect of the present invention includes phytostanol, which is a hydrogenation form of phytosterol or an electrical substance, toluene, methylene chloride, tetrahydrofuran, After dissolving in a nonpolar organic solvent such as benzene or diethylether, and adding a basic catalyst such as 4-dimethylaminopyridine (DMAP), pyridine or triethylamine (TEA), and then unsaturated Fatty acids with 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide (EDC: 1-ethyl-3- [3'-dimethylaminopropyl] carbodiimide) , Oxalyl chloride, carbonyl diimidazole, 2-chloropyridium, 2,2'-dipyridyl disulfide or 2- Imidajoildisulfa Esterification by addition of a carboxyl activating material such as 2- (imidazoyl disulfide), and the reaction product obtained in the above step is filtered and the solvent is removed, followed by methanol or methanol / acetone (8: 2, v / v). ) Stirring and precipitation in a mixed solvent. At this time, the phytosterol used as a reactant collectively refers to an alcohol compound having a steroid structure found in higher plants, and includes the above-described stigmasterol, spinasterol, camphorsterol and cytosterol, preferably α-, β -, γ- type cytosterol, phytostanols include the cytosubstanol, campestanol, which is a hydrogen-substituted form of phytosterol described above. On the other hand, as unsaturated fatty acid, the C4-C22, Preferably it is 12-20, More preferably, it is 16-18, The thing with an unsaturated degree uses the range of 1-3.

In addition, the present invention provides a food comprising an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in the liquid oil produced by the electric preparation method. In this case, the electric food includes, but is not limited to, cooking oil, salad oil, cooking oil, mayonnaise, margarine, salad dressing, butter or shortening.

Hereinafter, a method for preparing an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in a liquid oil having a cholesterol-lowering effect of the present invention is divided into steps based on the example employing β-sitosterol as phytosterol and oleic acid as unsaturated fatty acid. It will be described in more detail.

Step 1: esterification of phytosterols with unsaturated fatty acids

β-sitosterol and unsaturated fatty acids are added in a molar ratio of 1: 1 to 1: 3, preferably 1: 2, and dissolved in a nonpolar organic solvent such as toluene, methylene chloride, tetrahydrofuran, benzene or diethyl ether. , Basic catalyst such as DMAP, pyridine or TEA is added and stirred at 0-70 ° C., preferably 22-28 ° C., most preferably 25 ° C. to completely dissolve all particles. Carboxyl activating materials such as DCC, EDC, oxalylchloride, carbonyldiimidazole, 2-chloropyridium, 2,2'-dipyridyldisulfide or 2-imidazoyldisulfide at the temperature of electricity are dissolved in an organic solvent. The molar ratio of 1: 1.1 to 1: 3, preferably 1: 1.3 to 1: 1.6, and most preferably 1: 1.4 relative to β-sitosterol, and slowly dropwise, raising the temperature to 30 to 34 ° C, and 1 to After stirring for 5 hours, preferably 2 to 4 hours, most preferably 3 hours, the end point of the reaction is determined using TLC.

Second Step: Obtaining Phytosterol-Unsaturated Fatty Acid Ester Compounds

After the esterification reaction of phytosterol and unsaturated fatty acid is completed, the reaction product is filtered and the solvent is removed under reduced pressure to obtain an oily substance. The obtained oily substance was stirred vigorously by adding methanol or methanol / acetone (8: 2, v / v) mixed solution at 30 to 50 ° C, preferably 35 to 45 ° C, most preferably 40 ° C, and 0 Cooling and precipitation at 1-4 [deg.] C. for 1 hour to 30 hours, preferably 3 hours to 27 hours, most preferably 5 hours to 24 hours, a phytosterol-unsaturated fatty acid ester compound in the form of a wax is obtained.

The β-sitosterol-unsaturated fatty acid ester compound obtained in the present invention was confirmed to have an effect of lowering cholesterol in animal experiments in male rats, and was dissolved in oils such as corn oil and sesame oil. There was no change in properties in terms of cooking oil and physical properties.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention. Particularly, the following examples show examples of employing β-sitosterol as phytosterol, but the range is applicable to stigmasterol, spinasterol, campestrol or cytosterol in accordance with the spirit of the present invention, and to unsaturated fatty acids. Although oleic acid with an unsaturation of 1 is targeted, it is clear that the present invention is also applicable to unsaturated fatty acids such as linoleic acid with 2 unsaturation and linolenic acid with 3 unsaturation. Although cooking oil (corn oil) was used as an oil, it is possible to apply the present invention to other oils such as sesame oil, cottonseed oil, soybean oil, wheat germ oil, rice bran oil, safflower oil, rapeseed oil and sunflower oil according to the purpose of the present invention. Will be more obvious.

Example 1: esterification of β-sitosterol with oleic acid

30 g (72 mmol) of β-sitosterol and 41.45 g (147 mmol) of oleic acid were dissolved in 125 mL of methylene chloride in a round flask, and then 2.65 g (22 mmol) of DMAP, a basic catalyst, was added and stirred vigorously in a water bath at 25 ° C. Thereby completely dissolving all particles. After dissolving 22.39 g (109 mmol) of DCC, a carboxyl activating material, in 75 mL of methylene chloride, the DCC solution was added dropwise at a rapid rate and the temperature was raised to 32 ° C., and then the dropping rate was decreased and the temperature was maintained. After the dropping was finished, the base wall was washed with 25 mL of methylene chloride and stirred for 3 hours, and then TLC confirmed the end point of the reaction.

Example 2: Obtaining β-Sitosteryl-Olate Compound

Example 2-1 Precipitation Using Methanol

Dicyclohexylurea precipitate formed during the reaction was removed by filtration, and the solvent was evaporated under reduced pressure to obtain an oily substance. 400 mL of methanol was added to the obtained oily material, stirred at 40 ° C. for 1 hour, and then stored in a refrigerator at 4 ° C. for 5 hours to solidify the product in wax form, and then remove the solvent. After 300 mL of methanol was poured again, the above process was repeated once more, and the TLC was used to check the content of impurities (unreacted oleic acid) in the product. It was. Through the above process, β-sitosteryl oleate was obtained in 90% yield.

Example 2-2 Precipitation Using Methanol / Acetone Mixture

The dicyclohexylurea precipitate produced during the reaction was filtered off and the oily material was obtained by volatilizing the solvent under reduced pressure. The oily substance thus obtained was dissolved in 20 mL of methylene chloride, and then slowly added dropwise to precipitated methanol / acetone (8: 2, v / v) mixed solvent which was vigorously stirred. After standing at 4 ° C. for at least 24 hours, it was filtered to obtain β-sitosteryl oleate in the form of a wax in 51% yield.

Example 3:

Thirty three-week-old Sprague-Dawley male rats bred at the experimental animal breeding center of Seoul National University were used to homogenously mix cholesterol and cholic acid in a normal diet at a concentration of 1% and 0.5%, respectively, for 1 week. By feeding, the blood cholesterol level was raised. While feeding the same feed as described above, the beta -cytosteryl oleate in the wax form synthesized in Examples 1 and 2 was mixed and fed. The dose of the sample was 0.16 g / kg / day based on β-sitosteryl oleate, and the total blood cholesterol level, high density lipoprotein (HDL) -cholesterol and low density lipoprotein (LDL) -cholesterol levels after 1 week were measured. Statistical processing was performed and an equal variance test was performed.

Total cholesterol in the blood sample Before sample administration (mg / dl) 1 week after sample administration (mg / dl) *% Reduction rate Control 206.40 ± 50.81 136.30 ± 15.00 β-sitosteryl oleate 217.20 ± 67.44 97.60 ± 23.73 ** 28%

*: The rate of decrease of total cholesterol level compared to the control group after 1 week of sample administration

**: Significant difference from control (p <0.05)

HDL-cholesterol level in blood sample Before sample administration (mg / dl) 1 week after sample administration (mg / dl) Control 23.00 ± 5.66 25.40 ± 10.00 β-sitosteryl oleate 22.00 ± 5.83 26.00 ± 6.52

LDL-cholesterol level in blood Before sample administration (mg / dl) 1 week after sample administration (mg / dl) *% Reduction rate Control 192.67 ± 43.23 110.90 ± 23.58 β-sitosteryl oleate 195.20 ± 69.73 71.60 ± 26.53 ** 35%

*: Reduction rate of LDL-cholesterol level compared to control group 1 week after sample administration

**: Significant difference from control group (p <0.05)

As shown in Tables 1, 2, and 3, β-sitosteryl oleate, like β-sitosterol, reduces the absorption of harmful LDL-cholesterol through the competitive absorption mechanism with LDL-cholesterol, thereby reducing blood cholesterol. While lowering, it did not affect the absorption of HDL-cholesterol.

Example 4:

The solubility of the β-cytosteryl oleate obtained in Example 1 was investigated (see Table 4). The solubility is the result of checking whether or not the precipitate was formed after leaving the sample of each concentration (% (w / v)) for 3 days at each temperature. In Table 4, ○ indicates that no precipitate was formed and This is the case when generated. As shown in Table 4, it was confirmed that β-sitosteryl oleate was easily dissolved in liquid oil. Compared with general cooking oil by the standard, there was no significant difference in quality as summarized in Table 5.

Solubility of β-Sitosteryl Olate in Cooking Oil One% 2% 3% 4% 5% 7% 10% 4 ℃ ○ ○ ○ ○ ○ ○ 25 ℃ ○ ○ ○ ○ ○ ○ 37 ℃ ○ ○ ○ ○ ○ ○ ○

Solubility of β-Sitosteryl Olate in Cooking Oil One% 2% 3% 4% 5% 7% 10% 4 ℃ ○ ○ ○ ○ ○ ○ X 25 ℃ ○ ○ ○ ○ ○ ○ X 37 ℃ ○ ○ ○ ○ ○ ○ ○

Quality Comparison of Cooking Oil and Cooking Oil Added 1% of β-Sitosteryl Olate Static specification cooking oil Added Cooking Oil Acid 0.6 or less 0.085 0.085 Peroxide strand 1.0 or less 0.1 0.12 Swordsman 187-195 or less 192 194 Unfinished 2.0 or less 0.5 2.08 Iodine 103-130 127 127.4

Claims (8)

To phytosterol or phytostanol, a carboxyl activating material dissolved in a nonpolar organic solvent by dissolving phytosterol or phytostanol and an unsaturated fatty acid in a molar ratio of 1: 1 to 1: 3 and adding a basic catalyst, Gradually dropping at a molar ratio of 1: 1.1 to 1: 3 and stirring the mixture at 30 to 34 ° C. for 1 to 5 hours for esterification; And, The unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in a liquid oil comprising the step of filtering the reactant obtained in the above process, removing the solvent under reduced pressure, adding a methanol or methanol / acetone mixed solvent, stirring and precipitation. Manufacturing method. The method of claim 1, Phytosterols are characterized in that stigmasterol, spinasterol, camphorsterol or cytosterol A process for preparing an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in liquid oil. The method of claim 1, Phytostanol is characterized in that it is cytosethanol or campestanol A process for preparing an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in liquid oil. The method of claim 1, Unsaturated fatty acids have 4 to 22 carbon atoms, and the degree of unsaturation is 1 to 3 A process for preparing an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in liquid oil. The method of claim 1, The nonpolar organic solvent is toluene, methylene chloride, tetrahydrofuran, benzene or diethyl ether, characterized in that A process for preparing an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in liquid oil. The method of claim 1, The basic catalyst is characterized in that 4-dimethylaminopyridine (DMAP), pyridine or triethylamine (TEA) A process for preparing an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in liquid oil. The method of claim 1, Carboxyl group activators include 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide (EDC), oxalylchloride, carbonyldiimidazole, 2- Chloropyridium, 2,2'-dipyridyldisulfide or 2-imidazoyldisulfide, characterized in that A process for preparing an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in liquid oil. The method of claim 1, The liquid oil is one or more selected from the group consisting of corn oil, cottonseed oil, soybean oil, wheat germ oil, rice bran oil, safflower oil, rapeseed oil, sunflower oil and sesame oil A process for preparing an unsaturated fatty acid ester compound of phytosterol or phytostanol soluble in liquid oil.