KR100292673B1 - Intermediate Compounds for the Preparation of Water-soluble Phytosterol Derivatives for Lowering Cholesterol Level - Google Patents

Abstract

The present invention relates to a sterol intermediate compound for the preparation of a water-soluble sterol substituent having a cholesterol lowering effect. The inventors of the present invention not only have a mechanism of inhibiting metabolism of cholesterol in the human body through the structural similarity to cholesterol of phytosterol, but also does not have side effects that can be caused by taking drugs such as cholesterol lowering agents, phytosterol itself In view of its limitation in formulation due to physical limitations that are insoluble in both water and fats and oils, phytosterols are combined with water-soluble macromolecules by chemical methods, which are effective in reducing various cholesterol levels and are safe for the human body. Sterol intermediate compounds were prepared to prepare substituents. Unlike the conventional fat-soluble phytosterol, the sterol intermediate of the present invention not only dissolves easily in water, but also reduces absorption of LDL-cholesterol, which is harmful to the human body, and lowers blood cholesterol levels. It can be used for the preparation of water-soluble sterol substituents that do not involve any side effects such as liver dysfunction.

Description

Intermediate Compounds for the Preparation of Water-soluble Phytosterol Derivatives for Lowering Cholesterol Level

The present invention relates to an intermediate compound for preparing a water-soluble sterol derivative having a cholesterol lowering effect. More specifically, the present invention relates to sterol intermediate compounds for the preparation of water-soluble sterol derivatives that 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. And a sterol intermediate as a highly reactive intermediate, which can be used as a food additive as an intermediate, and is combined with a hydrophilic polymer carrier that is safe for the human body to prepare a water-soluble substituent of phytosterol. The solubility of water in water was not less than 10 mg (weight of the sterol part of the substituent) / ml, regardless of temperature change. Make sure they have the same effect It was completed the people.

After all, the main object of the present invention is to provide a sterol intermediate compound for the preparation of 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 also safe for human body.

In the present invention, succinic anhydride or glutaric anhydride is added to phytosterol, a water-insoluble sterol, in a molar ratio of 1: 1.0 to 1: 1.3, toluene, methylene chloride, tetrahydrofuran, benzene. Or a basic catalyst such as 4-dimethylaminopyridine (DMAP: 4-dimethylaminopyridine), pyridine or triethylamine (TEA) in a nonpolar organic solvent such as diethylether to react with a hydrophilic polymer carrier. The sterol intermediate compound represented by the following formula which is highly reactive with respect to is obtained.

Where

m is 2 or 3.

The hydrophilic carrier and the sterol intermediate compound prepared above at a molar ratio of 1 to 2 times are then subjected to 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3 in a nonpolar organic solvent under a basic catalyst. -(3'-dimethylaminopropyl) carbodiimide (1-ethyl-3- [3'-dimethylaminopropyl] carbodiimide), oxalyl chloride, carbonyl diimidazole, 2-chloropyridium Esterification by addition of a binder such as (2-chloropyridium), 2,2'-dipyridyl disulfide or 2-imidazoyl disulfide The water-soluble sterol substituent shown is manufactured. At this time, the phytosterol used as the water-insoluble sterol is a generic name for the alcohol compound having a steroid structure found in higher plants, the above-described stigmasterol, spinasterol, campesterol and cytosterol And the like, and preferably include α, β, and γ type cytosterols. 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;

m is 2 or 3; 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 sterol intermediate (I)

& 1st process-Reaction of phytosterol and succinic anhydride

The reaction for obtaining the sterol intermediate (I) by the 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 sterol intermediate (I). do.

& 1st-2nd process &: Reaction of phytosterol and glutaric anhydride

The reaction for obtaining the sterol intermediate (I) by reaction of phytosterol with glutaric anhydride is carried out under the following conditions using a basic catalyst such as 4-dimethylaminopyridine (DMAP), pyridine or triethylamine (TEA). . Phytosterol and glutaric 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 such as, the basic catalyst was treated and heated to the boiling point of 40 to 150 ° C., most preferably the used organic solvent for 2 to 30 hours, preferably 4 to 12 hours, and β- The -OH group of the cytosterol 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 glutaric anhydride, and the organic solvent was evaporated to sterol intermediate (I). To obtain.

& 2nd process |: of water-soluble sterol substituent (II) by the coupling reaction 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 wash thoroughly with normal-hexane to prepare a water-soluble sterol substituent (II).

& Water-soluble sterol substituent (II) &

Where

R is -H or ego;

m is 2 or 3; 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 Sterol 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%.

&Amp; Example 1-2: &

A sterol intermediate (I) was obtained in the same manner as in 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 1-3: &

A sterol intermediate (I) was obtained in the same manner as in Example 1-1, except that β-sitosterol and glutaric anhydride were reacted for 6 hours at a molar ratio of 1: 1.20, and the yield was 60%.

& Example 2: Preparation of Water-soluble Sterol Substitute (II) by Coupling Reaction of Intermediate (I, m = 2) and PEG

The water-insoluble sterol intermediate (I, m = 2) and PEG were dissolved in a small amount of methylene chloride in a molar ratio of 1.5: 1, then DCC as binder and DMAP as 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 manufacturing conditions of water-soluble sterol substituent (II, m = 2)

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

As shown in Table 1 below, a sterol intermediate (I, m = 2) and PEG, each having a different average molecular weight, were used as a reactant to prepare a water-soluble sterol substituent (II, m = 2) by the method described in Example 2. In addition, the degree of substitution (DS) of the sterol in the electrical material was measured using ¹ H-NMR.

& [Table 1] Reaction conditions of PEG and intermediate (I) and average degree of substitution of sterol &

Molecular weight & of PEG & Molar ratio && ([PEG]: [I]) of reactant & & Reaction 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 sterol in water-soluble sterol substituent (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 substituent (II) according to molecular weight of PEG &

& PEG molecular weight & & DS && ^* & Temperature (℃) & Maximum concentration dissolved (wt%) && ^** & & Water soluble sterol substituent (II) standard & & Sterol residue standard & & 1,000 & & 0.97 & & 35 & & Does not dissolve even in & 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 substitution degree of sterol in water-soluble sterol substituent (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 substitution degree due to scale-up 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 in average substitution degree due to scale-up of binding reaction

& Water-soluble sterol substituent (II) & & Amount (g) of PEG which we used & Molar ratio of reactant && ([PEG]: [I]) & & Reaction 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 sterol in water-soluble sterol substituent (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 & solubility measurement according to the temperature of the water-soluble sterol substituent (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 & solubility in water of water-soluble sterol substituent (II)

& Water-soluble sterol substituent (II) & & DS && ^* & Temperature (℃) & & Maximum concentration (wt%) dissolved && ^** & & Water soluble sterol substituent (II) standard & & Sterol residue standard & &One& & 1.09 & & 35 & & 13.2 & & 2.8 & & Normal temperature & 10.0 & & 2.2 & &4& & 10.0 & & 2.2 & &2& & 0.98 & & 35 & & 10.0 & & 2.0 & & Normal temperature & 10.0 & & 2.0 & &4& & 10.0 & & 2.0 & & 3 & & 0.92 & & 35 & & 20.0 & & 3.8 & & Normal temperature & 20.0 & & 3.8 & &4& & 20.0 & & 3.8 & &4& & 1.33 & & 35 & & 10.0 & & 2.6 & & Normal temperature & 10.0 & & 2.6 & &4& & 10.0 & & 2.6 &

& * DS (degree of substitution): Average degree of substitution of sterol in sterol substituent &

& ** Concentration (wt%) = (g number of sterol substituents (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 sterol and phytosterol

& Group 1 & 2 group & & 3 group & & 4 group & & ¹⁴ && C-cholesterol & & Dose & 0.004mg / day & How to administer & Disperse in 0.5ml of corn oil orally &sample& & Sample type & &-& & Water-soluble sterol substituent (II) -3 & & Water-soluble sterol substituent (II) -3 & & Phytosterol & & Sample amount & &-& & ¹⁴ && C-cholesterol double confiscation & Confiscation that corresponds to & 15% of cholesterol & Sample & & administration method &-& & Aqueous solution, & & oral administration & Aqueous solution, oral administration & Dispersion in corn, && 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 of each group 3 days after administration (unit: dpm) &

& Sample number & &medium& &Standard Deviation& & Group 1 & 6 & & 460.6 & & 154.9 & & 2 group & & 6 & & 416.6 & & 77.4 & & 3 group & & 6 & ＆ 274.7 ＆＆ ^* ＆ & 55.3 & & 4 group & & 6 & ＆ 258.9 ＆＆ ^* ＆ & 33.2 &

& * &&& P &&& <0.01 vs one group &

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 sterol intermediate compound for preparing a water-soluble sterol substituent having a cholesterol lowering effect. Unlike the conventional fat-soluble phytosterol, the sterol intermediate of the present invention not only dissolves easily in water, but also reduces absorption of LDL-cholesterol, which is harmful to the human body, and lowers blood cholesterol levels. It can be widely used in the preparation of water-soluble sterol substituents that do not involve any side effects such as liver dysfunction.

Claims (1)

Sterol intermediate compounds for the preparation of water-soluble phytosterol derivatives represented by the following formula: Where Phytosterols are stigmasterol, spinasterol, camphorsterol or cytosterol; And, m is 2 or 3.