KR20000012167A - Water-soluble Sterol Derivatives for Lowering Cholesterol Level and Process for Preparing the Same - Google Patents

Abstract

PURPOSE: A phytosterol substituents is prepared by combining a phytosterol with a macromolecular polymer. CONSTITUTION: Phytosterol and succinic anhydride in the mole ratio of 1:1.0- 1:1.3 are reacted in a nonpolar solvent such as toluene, methylene chloride, tetrahydrofuran, benzene or diethyl ether in the presence of alkali catalysts such as 4-dimethylaminopyridine, pyridine or triethylamine to give sterol intermediates. The sterol intermediates and a hydrophilic carrier are esterified with a binder such as 1,3-dicyclohexylcarboimide, 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide(1-ethyl-3-£3'-dimethylaminopropyl|carbodiimide), oxalyl chloride, carbonyl diimidazole, 2-chloropyridinum, 2,2'-dipyridyl disulfide or 2-imidazoyl disulfide to give the phytosterol(formula 1; R= H or phytosterol group; n=30- 95).

Description

Water-soluble Sterol Derivatives for Lowering Cholesterol Level and Process for Preparing the Same}

The present invention relates to a water-soluble sterol substituent having a cholesterol lowering effect and a method for producing the same. More specifically, the present invention relates to a water-soluble sterol substituent and a method for preparing the same, which can be used not only for the treatment of hypercholesterolemia but also for the prevention of some cardiovascular diseases and hypertension.

Cholesterol is a nutrient necessary for the human body such as being used as a constituent of a biological membrane and used as a starting material for hormonal synthesis, and when ingested too much cholesterol, it is known to accumulate in blood vessels and cause heart disease. So far, there is no way to prevent this except low-cholesterol diet, and taking drugs such as cholesterol-lowering drugs are effective, but they are only used on a very limited basis for causing side effects of liver dysfunction due to inhibition of cholesterol synthase. I can't.

Chitosan, phytosterol, inositol, and pectin are known substances that lower blood cholesterol in the human body, but substances other than phytosterol have no clear effect or metabolic mechanism. not. Phytosterol, a plant sterol, is known to have a mechanism of action that inhibits the absorption of cholesterol in the body through competition with low density lipoprotein (LDL) cholesterol, which is adsorbed on the intestinal wall and its structural similarity. It is FDA approved as a food additive that does not affect any biosynthetic mechanisms and does not involve any of the side effects mentioned above.

Phytosterol is a generic name for alcohol compounds having a steroid structure found in higher plants, and may be classified into stigmasterol, spinasterol, campesterol and cytosterol, for example. For example, α, β, and γ types also exist in cytosterol. The cholesterol-lowering effect of β-sitosterol (24-ethyl-5α-cholestene-3β-ol), the most representative of the various phytosterol compounds, has been confirmed in experiments in male rats and humans ( See J. Nutr., 107: 2011-2019 (1977). It has also been reported that β-sitosterol ester compounds in which β-sitosterol is substituted by fatty acids have almost the same effect (J. Nutr., 107: 1139-1146 (1977)). For example, an adult male reported that a daily administration of 2 g of β-sitosteryl oleate reduced his blood cholesterol levels by 33% (see 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 itself is limited in formulating due to physical limitations insoluble in water and fats and oils, and the commercialization is currently limited to 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-cholestene-3β-ol), which is a solid form of β-sitosterol, with a fatty acid It has been found that β-sitostanol ester compounds can lower blood cholesterol when used as additives in solid dairy products such as butter and margarine (WO 92/19640). However, β-cytostanol ester compounds have the disadvantage that they are applicable only to solid dairy products, which are relatively in low demand, and do not dissolve in liquid oil products. In addition, it is difficult to hydrogenate β-sitosterol by an organic chemical method to synthesize β-sitostanol and then react with fatty acids. Therefore, the inventors pay attention to the fact that the unsaturated fatty acid or its ester compound is significantly lower in melting point than the saturated fatty acid or its ester compound and most of them are liquid at room temperature, so that β-sitosterol is reacted with the unsaturated fatty acid methyl ester to be dissolved in liquid oil. As a result of clinical trials of the phytosterol fat-soluble substituents described above, it was found that the cholesterol-lowering effect was excellent. However, the fat-soluble substituents were limited to oil-type foods and the like (reference: Patent Application No. 98). -7535).

Therefore, the need to develop a water-soluble food material that can be easily ingested by changing the phytosterol to water-soluble and added to food and beverage has been constantly emerging.

Accordingly, the present inventors have succinic anhydride as a result of earnest research to develop a hydrophilic polymer carrier that is safe for the human body by combining a phytosterol having a lowering effect of cholesterol, which is one of the main inducers of various adult diseases, with a water-soluble macromolecule and chemical method. To obtain highly reactive intermediates, and to combine the intermediates and the food additives and use a hydrophilic polymer carrier that is safe for the human body to prepare a water-soluble substituent of phytosterol, and then to the water of the electrical As a result of the solubility test, it was confirmed that the solubility in water was not less than 10mg (the weight of the sterol part of the substituent) / ml regardless of the temperature change. After confirming that the present invention has been completed .

After all, it is an object of the present invention to provide a sterol substituent as a water-soluble food material that can be used not only for the treatment of hypercholesterolemia but also for the prevention of some cardiovascular diseases and hypertension and is safe for humans.

Another object of the present invention is to provide a method for preparing an electrosoluble sterol substituent.

In the present invention, succinic anhydride is added to phytosterol, which is a water-insoluble sterol, in a molar ratio of 1: 1.0 to 1: 1.3, toluene, methylene chloride, tetrahydrofuran, benzene, or diethylether. Reaction in a nonpolar organic solvent with a basic catalyst such as 4-dimethylaminopyridine (DMAP: 4-dimethylaminopyridine), pyridine or triethylamine (TEA) to give a sterol intermediate that is highly reactive to a hydrophilic polymeric carrier, The hydrophilic carrier and the sterol intermediate in a molar ratio of 1 to 2 times therewith were subjected to 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3'-dimethylaminopropyl) in a nonpolar organic solvent under a basic catalyst. Carbodiimide (1-ethyl-3- [3'-dimethylaminopropyl] carbodiimide), oxalyl chloride, carbonyl diimidazole, 2-chloropyridium water-soluble sterol represented by the following formula by adding a binder such as ridium), 2,2'-dipyridyl disulfide, or 2-imidazoyl disulfide Prepare a substituent. In this case, phytosterol used as a water-insoluble sterol is a generic name for alcohol compounds having a steroid structure found in higher plants, such as stigmasterol, spinasterol, campesterol, and cytosterol. It includes, and preferably comprises the α, β, γ type cytosterol. As the hydrophilic polymer carrier, polyethylene glycol (PEG), xanthan gum, guar gum, sodium alginate, dextrin, oligosaccharide, chitosan or carboxymethyl cellulose are used. Particularly, polyethylene glycol has an average molecular weight. Preferably 1,500 to 4,000, more preferably 2,000 to 3,000, most preferably 2,000.

Where

R is -H or ego; And,

n is an integer from 30 to 95.

Hereinafter, the method for producing a water-soluble sterol substituent as a food material of the present invention will be described in more detail by dividing the process by the step of using β-sitosterol as a phytosterol and PEG as a hydrophilic polymer carrier.

First Step: Obtaining Intermediate (I)

The reaction for obtaining intermediate (I) by reaction of phytosterol with succinic anhydride is carried out under the following conditions using a basic catalyst such as 4-dimethylaminopyridine (DMAP), pyridine or triethylamine (TEA). Phytosterol and succinic anhydride are added in a molar ratio of 1: 1.0 to 1: 1.3, most preferably in a molar ratio of 1: 1.3, and toluene, methylene chloride, tetrahydrofuran, benzene or diethylether After dissolving in a non-polar organic solvent, the basic catalyst was treated and heated to the boiling point of 40 to 150 DEG C, most preferably the used organic solvent for 2 to 30 hours, preferably 4 to 9 hours, to obtain β-sitosterol. The -OH group is converted to -COOH to volatilize the organic solvent when the reaction is completed. The remaining solid was dissolved in methylene chloride, extracted with distilled water, and then the organic solvent layer dried with MgSO ₄ was filtered and cooled to crystallize and remove the unreacted succinic anhydride, and the organic solvent was evaporated to obtain a sterol substituent (I). do.

Second Step: Interaction of Water-soluble Sterol Substitutes (II) by the Interaction of Intermediate (I) and PEG

Produce

The water-insoluble sterol intermediate (I) obtained in the first step and the hydrophilic polymer carrier, preferably polyethylene glycol (PEG), are dissolved in the nonpolar organic solvent in a molar ratio of 1: 1 to 2: 1, and the reaction is stirred at room temperature. 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide (1-ethyl-3- [3'-dimethyl-aminopropyl] carbodiimide) as a binder , Oxalyl chloride, carbonyl diimidazole, 2-chloropyridium, 2,2'-dipyridyl disulfide or 2- The reaction is performed for 5 to 15 hours by adding an imidazoyl disulfide and a basic catalyst. After completion of the reaction, the reaction is filtered to remove DCC-urea to terminate the reaction. Then, precipitate in normal hexane (n-hexane) to filter out the precipitate and washed sufficiently with normal-hexane to prepare a water-soluble sterol substituent (II).

Water Soluble Sterol Substitute (II)

Where

R is -H or ego; And,

n is an integer from 30 to 95.

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, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1: Obtaining Intermediate (I)

Example 1-1:

Β-sitosterol and succinic anhydride were dissolved in toluene in a molar ratio of 1: 1.15 in a flask fitted with a reflux tube and Dean-Stark, and then DMAP was added. The mixture was heated to 140 ° C. for 9 hours to maintain the reflux state of toluene, and toluene was volatilized after completion of the reaction by checking the progress of the reaction by TLC. The solid was dissolved in methylene chloride and evaporated again to recrystallize to afford sterol intermediate (I), with a yield of 70%.

Example 1-2:

Intermediate (I) was obtained by the same method as Example 1-1, except that β-sitosterol and succinic anhydride were reacted for 4 hours at a molar ratio of 1: 1.20, and the yield was 68%.

Example 2: Preparation of Water-soluble Sterol Substituent (II) by Coupling Intermediate (I) and PEG

Produce

After dissolving the water-insoluble sterol intermediate (I) and PEG in a small amount of methylene chloride in a molar ratio of 1.5: 1, DCC as a binder and DMAP as a catalyst were added while stirring the reaction at room temperature. After the reaction was completed after 10 hours by TLC, the reaction was completed by filtration to remove DCC-urea, and the filtrate was precipitated in normal-hexane. The precipitate was obtained by filtration and then washed thoroughly with normal-hexane to obtain the final product, water-soluble sterol substituent (II), and the structure of the electrical material was confirmed by ¹ H-NMR.

Example 3: Optimization of Preparation Conditions for Water-Soluble Sterol Substitutes (II)

Example 3-1: Relationship between PEG molecular weight and reactivity

As shown in Table 1 below, a water-soluble sterol substituent (II) was prepared by the method described in Example 2 using a sterol intermediate (I) and PEG each having a different average molecular weight as a reactant, and the average of the sterols in the electrical material. Degree of substitution (DS) was measured using ¹ H-NMR.

TABLE 1 Reaction Conditions of PEG and Intermediate (I) and Average Degree of Substitution of Sterols

Molecular weight of PEG Molar ratio of reactants ([PEG]: [I]) Response time (hr) DS ^* 1,000 1: 1.5 10 0.97 2,000 1: 1.5 14 1.09 3,000 1: 1.5 14 1.08

DS (degree of substitution): Average degree of substitution of sterols in water-soluble sterol substituents (II)

As shown in the results of Table 1, the reactivity confirmed by the average degree of substitution of the sterol in the water-soluble sterol substituent (II) was found to be not significantly affected by the molecular weight of PEG.

Example 3-2: Relationship between PEG molecular weight and solubility

The solubility of the water-soluble sterol substituent (II) mentioned in Table 1 above was examined by the method of leaving the samples at each concentration for 2 days at each temperature.

Table 2 Solubility of Water-soluble Sterol Substitutes (II) According to the Molecular Weight of PEG

PEG molecular weight DS ^* Temperature (℃) Maximum dissolved concentration (wt%) ^** Water Soluble Sterol Substitute (II) Standard Sterol Residue Criteria 1,000 0.97 35 Insoluble at 0.3% - 2,000 1.09 35 13.2 3.0 Room temperature 10.0 2.2 4 10.0 2.2 4,000 1.08 35 14.0 1.8 Room temperature 14.0 1.8 4 14.0 1.8

DS (degree of substitution): Average degree of substitution of sterols in water-soluble sterol substituents (II)

** Yield = number of moles of water soluble sterol substituent (II) / number of moles of PEG

As shown in Table 2, the sterol substituent (II) prepared using PEG with a molecular weight of 1,000 was hardly dissolved in water, and the sterol substituent (II) prepared with PEG having a molecular weight of 2,000 and a PEG having a molecular weight of 4,000. When the substituents (II) were compared with each other, the sterol substituents (II) were dissolved up to a higher concentration for the molecular weight of 4,000. However, the solubility of the molecular weight of 2,000 is better if the solubility is converted based on the sterol residue, so that the molecular weight of PEG as a carrier is fixed to 2,000 in the following examples.

Example 3-3: Measurement of Change in Average Degree of Substitution with Scale of Binding Reaction

The molecular weight of PEG used as a carrier was fixed at 2,000, and the binding reaction scale was expanded by the method described in Example 2, and various kinds of water-soluble sterol substituents (II) -1, -2, -3 having different average substitution degrees , -4 was prepared, and the synthesis results thereof are summarized in Table 3.

[Table 3] Change of Average Substitution Degree by Expansion of the Coupling Reaction

Water Soluble Sterol Substitute (II) Amount of PEG Used (g) Molar ratio of reactants ([PEG]: [I]) Response time (hr) DS ^* Amount (g) of water soluble sterol substituent (II) Yield (%) ^** One 30 1: 1.5 14 1.09 24.4 63.4 2 30 1: 1.5 14 0.98 28.2 75.1 3 100 1: 1.5 18 0.92 86.2 69.7 4 100 1: 1.5 23 1.33 86.7 64.6

DS (degree of substitution): Average degree of substitution of sterols in water-soluble sterol substituents (II)

** Yield = number of moles of water soluble sterol substituent (II) / number of moles of PEG

As can be seen in Table 3, the DS value of the water-soluble sterol substituent (II) prepared by the expansion of the reaction is slightly changed, but if the scale, the molar ratio of the reactants and the reaction time are kept constant, the DS value can be kept almost constant. It was considered to be present.

Example 4 Determination of Solubility with Temperature of Water-Soluble Sterol Substitute (II)

Since the water-soluble sterol substituent prepared by the present invention should be able to be added to food and beverage, the change in solubility of the water-soluble substituent (II) was investigated in accordance with the temperature change in consideration of the fact that general food and beverage is sold in a refrigerated state.

As a result of measuring the solubility with temperature using the water-soluble sterol substituent (II) -1, -2, -3, -4 prepared in Example 3-3, the solubility is generally cholesterol in 1 can (250 ml) Sufficiently available at or above 10 mg (partial weight of sterol in water-soluble sterol substituents) / ml (corresponding to 1 wt%), which contains 2.7 g of sterol, the daily intake of adults recognized by the FDA as being effective in lowering In addition, it was confirmed that the change of solubility with the change of temperature was also small (see Table 4).

TABLE 4 Solubility of Water-soluble Sterol Substitutes (II) in Water

Water Soluble Sterol Substitute (II) DS ^* Temperature (℃) Maximum concentration dissolved (wt%) ^** Water Soluble Sterol Substitute (II) Standard Sterol Residue Criteria One 1.09 35 13.2 2.8 Room temperature 10.0 2.2 4 10.0 2.2 2 0.98 35 10.0 2.0 Room temperature 10.0 2.0 4 10.0 2.0 3 0.92 35 20.0 3.8 Room temperature 20.0 3.8 4 20.0 3.8 4 1.33 35 10.0 2.6 Room temperature 10.0 2.6 4 10.0 2.6

DS (degree of substitution): Average degree of substitution of sterols in sterol substituents

** Concentration (wt%) = (g number of sterol substituent (II) / g number of water) X 100

Example 5: Confirmation of Cholesterol Lowering Effect Through Animal Experiment

After stabilizing healthy SD (Sprague-Dawley) male rats for three weeks, four rats of each experimental group were orally administered ¹⁴ C-cholesterol and the sterol substituent (II) -3 of Example 4 simultaneously for three days. The dose of ¹⁴ C-cholesterol was administered by dispersing 0.004 mg (1 X 10 ^-8 moles) in 0.5 ml of corn oil per day, and in the experimental group, 2 moles (2 X 10 ^-8 moles) of the ¹⁴ C-cholesterol dose. ) And an aqueous solution of sterol substituent (II) -3 in moles (3.9 X 10 ^-5 moles) corresponding to 15 mg of cholesterol (groups 2 and 3, respectively) and ¹⁴ C-cholesterol as a control group. (Group 1: (-) control), the administration of phytosterol with ¹⁴ C-cholesterol (Group 4: (+) control) was used (see Table 5).

TABLE 5 Administration method and amount of water-soluble sterols and phytosterols

Group 1 Group 2 Group 3 4th group ¹⁴ C-cholesterol Dosage 0.004mg / day Dosing method Oral administration in 0.5ml of corn oil sample Sample Type - Water Soluble Sterol Substitute (II) -3 Water Soluble Sterol Substitute (II) -3 Phytosterol Sample amount - ¹⁴ moles of C-cholesterol Confiscation equivalent to 15 mg of cholesterol Sample administration method - Aqueous solution, oral administration Aqueous solution, oral administration Dispersed in corn oil, oral administration

After administering the sample for 3 days, 6 ml of blood was collected from the rat's heart, divided into 2 each of 3 ml to make 8 samples for each group, and then mixed with 2 ml of scintillation measurement solution and ¹⁴ C-cholesterol. The average and standard deviation of the six samples, except for the maximum and minimum values, were calculated.

TABLE 6 Average value of ¹⁴ C-cholesterol in each group after 3 days of administration (unit: dpm)

Number of samples medium Standard Deviation Group 1 6 460.6 154.9 Group 2 6 416.6 77.4 Group 3 6 274.7 ^* 55.3 4th group 6 258.9 ^* 33.2

* P <0.01 vs Group 1

As shown in Table 6 above, the amount of ¹⁴ C-cholesterol in group 3 administered with cholesterol plus a mole of water-soluble sterol substituent (II) -3 corresponding to 15 mg of cholesterol was 274.7 ± 55.3. -) Significantly lower than 460.6 ± 154.9 of the control group, and the amount of ¹⁴ C-cholesterol measured in the 4 group ((+) control group) in which a mole of phytosterol equivalent to 15 mg of cholesterol was administered together with cholesterol. Compared with (258.9 ± 33.2), the cholesterol lowering effect was almost the same. In other words, the sterol substituent (II) incorporating phytosterol to PEG, which is a water-soluble polymer, has the same cholesterol lowering effect as phytosterol, and is thought to have completely improved the water-insoluble property which acted as the biggest obstacle to the development of phytosterol.

As described and demonstrated in detail above, the present invention provides a water-soluble sterol substituent having a cholesterol lowering effect and a method for preparing the same. Unlike conventional fat-soluble phytosterols, the water-soluble sterol substituents of the present invention not only dissolve easily in water, but also reduce the absorption of LDL-cholesterol, which is harmful to the human body, while lowering blood cholesterol levels, while inducing a conventional drug such as a cholesterol lowering agent. There are no side effects such as liver dysfunction. Therefore, the water-soluble sterol substituent of the present invention easily satisfies the daily intake of phytosterols, which the FDA recognizes as having an effect of lowering cholesterol as a food and beverage additive, thereby preventing not only the treatment of hypercholesterolemia but also some heart disease and high blood pressure. It can be expected to play a role as a water-soluble food material that can be used and safe for humans.

Claims (10)

Reacting phytosterol with succinic anhydride in a nonpolar organic solvent under a basic catalyst to obtain a highly reactive sterol intermediate; And, A method for producing a water-soluble sterol substituent comprising the step of binding the sterol intermediate obtained in the above step and the hydrophilic polymer carrier by adding a binder under a basic catalyst in a non-polar organic solvent. The method of claim 1, Phytosterols are characterized in that stigmasterol, spinasterol, camphorsterol or cytosterol Method for preparing a water-soluble sterol substituent. The method of claim 1, In the reaction of phytosterol and succinic anhydride, the nonpolar organic solvent is toluene, methylene chloride, tetrahydrofuran, benzene or diethylether, characterized in that Method for preparing a water-soluble sterol substituent. The method of claim 1, In the reaction of phytosterol with succinic anhydride, the basic catalyst is 4-dimethylaminopyridine (DMAP), pyridine or triethylamine (TEA). Method for preparing a water-soluble sterol substituent. The method of claim 1, The reaction of phytosterol and succinic anhydride is characterized in that the phytosterol and succinic anhydride are carried out at a molar ratio of 1: 1.0 to 1: 1.3 for 2 to 30 hours at a temperature of 40 to 150 ° C. Method for preparing a water-soluble sterol substituent. The method of claim 1, The hydrophilic polymer carrier is polyethylene glycol, xanthan gum, guar gum, sodium alginate, dextrin, oligosaccharides, chitosan or carboxymethylcellulose, characterized in that Method for preparing a water-soluble sterol substituent. The method of claim 6, The average molecular weight of polyethylene glycol is characterized in that 1,500 to 4,000 Method for preparing a water-soluble sterol substituent. The method of claim 1, Bonding reaction is characterized in that the polyethylene glycol is carried out at room temperature for 5 to 15 hours in a molar ratio of 1: 1 to 1: 2 with the sterol intermediate, Method for preparing a water-soluble sterol substituent. The method of claim 1, The binder is 1,3-dicyclohexylcarbodiimide, 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide (1-ethyl-3- [3'-dimethylaminopropyl] carbodiimide), oxalyl chloride ( oxalyl chloride, carbonyl diimidazole, 2-chloropyridium, 2,2'-dipyridyl disulfide or 2-imidazoyl disulfide (2,2'-dipyridyl disulfide) 2-imidazoyl disulfide) Method for preparing a water-soluble sterol substituent. A water-soluble sterol substituent represented by the following formula prepared by the method of claim 1: Where R is -H or ego; And, n is an integer from 30 to 95.