WO2010114339A2

WO2010114339A2 - Water-soluble sterol derivatives capable of lowering cholesterol levels and preparation method thereof

Info

Publication number: WO2010114339A2
Application number: PCT/KR2010/002049
Authority: WO
Inventors: 정대원; 노승권
Original assignee: Chung Dae-Won; Noh Seung-Kwon
Priority date: 2009-04-03
Filing date: 2010-04-02
Publication date: 2010-10-07
Also published as: KR20100110598A; WO2010114320A2; WO2010114339A3; WO2010114320A3

Abstract

The present invention relates to water-soluble sterol derivatives capable of lowering cholesterol levels and a preparation method thereof. The preparation method of water-soluble sterol derivatives according to the present invention comprises: in the presence of a basic catalyst in a non-polar organic solvent, mixing a phytosterol intermediate and polyethylene glycol (PEG) having a weight average molecular weight of 200 to 1,000 at a molar ratio ranging from 1:2 to 1:6, followed by adding a coupling agent to allow a coupling reaction to occur. This method of the present invention using a PEG oligomer as a carrier and controlling the molecular structure of a product can therefore provide water-soluble sterol derivatives capable of lowering cholesterol levels, in which the derivatives are soluble in water at a high concentration of at least about 3.0% by weight with respect to sterol and safer to use for the body and thus useful as a water-soluble food or as a material of medicine.

Description

Water-soluble sterol derivative having cholesterol lowering effect and preparation method thereof

The present invention relates to a water-soluble sterol derivative having a cholesterol lowering effect and a method for producing the same. More specifically, the present invention relates to a water-soluble sterol derivative having high water solubility and human stability, 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, or when ingested excessively, cholesterol is accumulated in blood vessels and causes 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 the use of cholesterol-lowering agents is extremely rare because of the side effects of liver dysfunction due to the inhibition of cholesterol synthase. It is limited.

Chitosan, phytosterol, inositol, and pectin are known as substances that lower blood cholesterol in the human body, but substances other than phytosterol have no clear effect or metabolic mechanism. . Phytosterol, a plant sterol, has been shown to exhibit a mechanism of inhibiting the metabolism of cholesterol in the body through competition with harmful low density lipoprotein (LDL) cholesterol due to its adsorption to the intestinal wall and its structural similarity. Therefore, it has been approved by the FDA as a food additive that has no effect on cholesterol biosynthesis and does not involve any of the above side effects.

Phytosterol is a generic name for alcohol compounds having a steroid structure found in higher plants, and may be classified into stigmasterol, spinasterol, and sitosterol. For example, sitosterol also has α, β, γ types and the like exist. The cholesterol-lowering effect of β-sitosterol (24-ethyl-5α-cholester-3β-ol), the most representative of the various phytosterol compounds, has been confirmed in experiments in male rats and humans. (J. Nutr., 107: 2011-2019 (1977)). In addition, β-sitosterol ester compounds in which β-sitosterol is substituted with fatty acids have been reported to exhibit almost the same effect (J. Nutr. 107: 1139-1146 (1977)). For example, an adult male has reported a 33% reduction in blood cholesterol levels when 5 g of β-sitosterol oleate is administered daily for five days (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 is limited in its formulation due to physical limitations, such as its insoluble in both water and fats and oils, and the development of food materials is insufficient due to the fact that simple refined nutrient types remain commercially available. It is a state.

Recently, in order to make up for the disadvantages of β-sitosterol, β-sitostanol ester compounds have been prepared by reacting β-sitostanol, a solid form of β-sitosterol, with fatty acids. In addition, it has been found that the β-cytostanol ester compound can be used as an additive in solid dairy products such as butter and margarine to lower blood cholesterol (see WO 92/19640). In addition, according to the need to change the phytosterol to water-soluble, and to develop a water-soluble food material that can be easily ingested by food and beverage, by reacting the phytosterol with succinic anhydride to obtain a highly reactive intermediates, polyethylene glycol (usable as a food additive) Water-soluble derivative of phytosterol was prepared by combining and reacting PEG with polyethylene glycol) as a carrier, and the solubility of water in water was investigated in 10 mg regardless of temperature change. It was confirmed that the weight of the sterol) / ml and at the same time having a cholesterol lowering effect equivalent to phytosterol (see Korean Patent No. 292672).

However, since the molecular weight of PEG used as a water-soluble carrier in the Republic of Korea Patent No. 292672 is very high molecular weight of 1,500 or more, the weight ratio of sterols in the derivative is 25% or less. Solubility of up to 10 mg / ml by weight of sterols in the derivatives was insufficient for commercialization with various types of products. In addition, considering that only PEG molecular weight of 2,000 or less is recognized as a food additive, and that a lower molecular weight is generally safer, a phytosterol derivative obtained by using PEG having a molecular weight of 1,500 as a carrier may be difficult to obtain as a food additive. The method disclosed in Korean Patent No. 292672 has not yet reached commercialization.

Thus, the present inventors intensively increased the solubility of the water-soluble sterol derivatives and developed a safer water-soluble derivative that is safer to the human body. As a result, the conventional water-soluble derivative is a form in which sterols are bonded to both ends of PEG, which are water-soluble carriers Form "), a sterol-bound form (hereinafter referred to as" mono form ") at one end, and a mixture of unreacted PEG, while the die form significantly reduces the solubility of the entire derivative. It turned out to be a factor. In addition, the presence of unreacted PEG also plays a role in lowering the weight ratio of sterols in all derivatives. As a water-soluble carrier, a low molecular weight PEG oligomer which is easy to obtain a license for food additives is used. By controlling the molar ratio of these two substances in the coupling reaction to induce the monoform formation alone, the weight ratio of sterol in the derivative is increased to 38%, while the solubility in water is about 30 mg (weight of sterol in derivative) regardless of temperature change. It was confirmed that it can be maintained at /) / ml or more, the present invention was completed.

Accordingly, an object of the present invention is to provide a method for preparing a water-soluble sterol derivative that is useful for the treatment of hypercholesterolemia, as well as for the prevention of some cardiovascular diseases and hypertension, and has high water solubility and human safety. It is.

Another object of the present invention is to provide a water-soluble sterol derivative obtained by the above method.

In order to achieve the above object, the present invention

Mixing a phytosterol intermediate and polyethylene glycol (PEG) with a weight average molecular weight of 200 to 1,000 in a molar ratio of 1: 2 to 1: 6 in the presence of a basic catalyst in a nonpolar organic solvent, and adding and binding a binder to react.

Provided are methods for preparing a water soluble sterol derivative.

The present invention also provides a water-soluble sterol derivative having a phytosterol intermediate bonded to one end of polyethylene glycol (PEG) having a weight average molecular weight of 200 to 1,000 obtained by the above method.

According to the method of the present invention, it is possible to prepare a water-soluble sterol derivative having a cholesterol lowering effect, which is soluble in water at a high concentration of about 3.0 wt% or more on a sterol basis and is more safe for human body, which is useful as a water-soluble food or pharmaceutical material. In addition, the water-soluble sterol derivatives of the present invention reduce the absorption of LDL-cholesterol, which is harmful to the human body, while lowering blood cholesterol levels, but do not have any side effects such as liver dysfunction caused by taking drugs such as conventional cholesterol lowering agents. Therefore, the water-soluble sterol derivative of the present invention can easily satisfy the adult daily intake of phytosterol as a food and beverage additive with various types of products, and is easily licensed as a water-soluble carrier. The use of oligomers can be used not only for the treatment of hypercholesterolemia but also for the prevention of some cardiovascular diseases and hypertension, and is useful as a safe water-soluble food material for the human body.

1 is a sterol derivative of the mono form synthesized in <Example 2-1> (Fig. 1 (C)), a mixed sterol derivative synthesized in <Comparative Example 2-3> (Fig. 1 (B) And ultra high vacuum matrix-assisted laser desorption ionization system ("Maldi") analysis of PEG itself (FIG. 1A) having a weight average molecular weight of 600.

In the preparation method according to the present invention, in the presence of a basic catalyst in a non-polar organic solvent, in the ester-bonding reaction of a highly reactive non-aqueous phytosterol intermediate and a polyethylene glycol (PEG), which is a hydrophilic carrier, using a binder, a weight average molecular weight of 200 to 1,000 , Preferably between 200 and 800, more preferably between 400 and 800, and even more preferably between 600 and at the same time the phytosterol intermediate and PEG are 1: 2 to 1: 6, preferably 1: 2 to 1: It is used in the molar ratio of 3 to prepare a water-soluble sterol derivative of the mono form.

Sterols according to the present invention refers to phytosterols collectively referred to alcohol compounds having a steroid structure found in higher plants, specific examples thereof include stigmasterol, spinasterol and cytosterol. Preferably, α, β, γ type cytosterol can be used.

Hereinafter, the method for preparing a water-soluble sterol derivative of the present invention will be described in more detail with reference to an example in which β-sitosterol is employed as the phytosterol.

According to the method of the present invention, the water-insoluble phytosterol intermediate and the PEG oligomer which is a hydrophilic carrier are dissolved in the nonpolar organic solvent in a molar ratio of 1: 2 to 1: 6, and the binder is mixed with a basic catalyst while stirring the reaction mixture at room temperature. After the reaction is carried out for 15 to 15 hours, the reaction product is filtered to terminate the reaction by removing unreacted PEG and byproducts. To prepare a hexane (n -hexane) and isopropanol mono form of water-soluble sterol derivative the desired crystallized with (isopropanol) solvent mixture consisting of - Then, the resulting product extracted with distilled water and n.

The phytosterol intermediate used in the method of the present invention may be represented by the following formula (1), and the mono-type water-soluble sterol derivative prepared by the method of the present invention may be represented by the following formula (2):

Formula 1

Formula 2

Wherein n is an integer from 5 to 20.

The nonpolar organic solvent used in the present invention may be toluene, methylene chloride, dichloroethane, tetrahydrofuran, benzene, diethyl ether or mixtures thereof.

The binder used in the present invention is 1,3-dicyclohexylcarbodiimide (DCC), ethylene dichloride, oxalyl cholride, carbonyl diimidazole, 2-chloropyri 2-chloropyridium, 2,2-dipyridyl disulfide, 2-imidazoyl disulfide or mixtures thereof.

The basic catalyst used in the present invention may be 4-dimethylaminopyridine (DMAP), pyridine, triethylamine (TEA) or mixtures thereof.

The binder and basic catalyst may be used in amounts of 1.2 to 1.6 molar equivalents and 0.05 to 0.1 molar equivalents, respectively, based on 1 molar equivalent of phytosterol intermediate.

The phytosterol intermediate represented by Formula 1 may be prepared based on a conventionally known method. Specifically, phytosterol and succinic anhydride are 1: 1 to 1:10, most preferably in a nonpolar organic solvent at a molar ratio of 1: 1.15 to 1: 1.2, followed by addition of a basic catalyst and 40 to 150 ° C. for 2 to 30 hours, preferably 4 to 9 hours, most preferably Preferably, by heating to the boiling point of the used organic solvent, the -OH group of the phytosterol is converted to the -COOH group to obtain a phytosterol intermediate with high binding reactivity with PEG.

The resulting phytosterol intermediate can then be obtained by purification according to conventional methods. For example, when the reaction is completed, the organic solvent is volatilized and the remaining solid is dissolved in methylene chloride, washed with distilled water and dried over MgSO ₄ . MgSO ₄ is removed via filtration and then cooled to crystallize unreacted succinic anhydride. The crystals are removed via filtration and methylene chloride is evaporated to yield phytosterol intermediates.

As described above, in the present invention, a monomolecular form is produced by using a low molecular weight PEG oligomer which is easily obtained as a food additive as a water-soluble carrier and controlling the molar ratio of these two substances in the coupling reaction between PEG and sterol. By inducing, a water-soluble sterol derivative having a phytosterol intermediate bonded to one end of a PEG having a weight average molecular weight of 200 to 1,000 is obtained.

The water-soluble sterol derivative of the mono form according to the present invention can increase the weight ratio of sterol in the derivative to 38% and maintain solubility in water at about 30 mg (weight of sterol in the derivative) / ml or more regardless of temperature change, In addition to the treatment of hypercholesterolemia, it can be used for the prevention of some cardiovascular diseases and hypertension, and is useful as a safe water-soluble food and pharmaceutical material for the human body.

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 Preparation of Phytosterol Intermediates of Formula 1

In a flask with a reflux tube, β-sitosterol and succinic anhydride were dissolved in methylene chloride at a molar ratio of 1: 5, and then DMAP was added as a catalyst, and heated to 50 ° C. for 23 hours to maintain reflux by TLC. The progress of the reaction was confirmed. After the reaction was completed, the mixture was extracted with distilled water at room temperature to remove DMAP. Then, the methylene chloride layer dried over MgSO ₄ was filtered and cooled to crystallize and remove the unreacted succinic anhydride, and methylene chloride was evaporated to give the phytosterol intermediate of the formula (1) as a title compound (yield 79%).

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 as a catalyst. 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 a trace of unreacted succinic anhydride was removed by crystallization to afford the title compound phytosterol intermediate (yield 70%).

Obtaining the phytosterol intermediate (yield 68%) of the title compound in the same manner as in <Example 1-2>, except that β-sitosterol and succinic anhydride were reacted for 4 hours at a molar ratio of 1: 1.20. It was.

Example 2: Preparation of Water-soluble Sterol Derivatives of Formula 2

<Example 2-1> Mono form was prepared using PEG having a weight average molecular weight of 600 as a water-soluble carrier.

The phytosterol intermediate (10 g) prepared in Example 1 and PEG (58.4 g) having a weight average molecular weight of 600 were dissolved in methylene chloride (about 80 ml) in a molar ratio of 1: 5, and then the reaction mixture was stirred as a binder at room temperature. DCC (5.22 g) and DMAP (0.19 g) as catalyst were added. When the reaction was completed after 10 hours by tracing the progress of the reaction by TLC, the reaction was filtered to remove DCC-urea and the reaction was terminated. With distilled water (160ml) extracted four times and the resulting product was distilled under reduced pressure an organic solvent, n-hexane (n -hexane) and isopropanol (isopropanol) 2: The title compound was crystallized in the form of the mono to a mixed solvent consisting of a 1 9.7 g of a sterol derivative were prepared.

Example 2-2 Production of Mono Form Using PEG with Weight Average Molecular Weight 1,000 as Water-Soluble Carrier

The phytosterol intermediate (10 g) prepared in Example 1 and PEG (97.3 g) having a weight average molecular weight of 1,000 were dissolved in methylene chloride (200 ml) at a molar ratio of 1: 5, and then the reaction mixture was stirred at room temperature as a binder as a DCC. (5.22 g) and DMAP (0.19 g) were added as catalyst. When the reaction was completed after 4 hours by tracking the progress of the reaction by TLC, the reaction was filtered to remove DCC-urea and the reaction was terminated. The obtained product was extracted four times with distilled water (400 ml) and crystallized with isopropanol to prepare 16.8 g of the title compound mono sterol derivative.

Comparative Example 2-3 When a PEG having a weight average molecular weight of 600 was used as a water soluble carrier to prepare a mixture of mono form, die form and unreacted PEG

The phytosterol intermediate prepared in Example 1 (10 g) and PEG (11.7 g) having a weight average molecular weight of 600 were dissolved in methylene chloride (50 ml) in a molar ratio of 1: 1, and then DCC was used as a binder while stirring the reaction mixture at room temperature. (5.22 g) and DMAP (0.19 g) were added as catalyst. When the reaction was completed after 24 hours by tracking the progress of the reaction by TLC, the reaction was filtered to remove DCC-urea and the reaction was terminated. The resulting product was extracted with distilled water (50ml) 4 times, and n-hexane (n -hexane) and isopropanol (isopropanol) is 4: it was crystallized with a mixed solvent consisting of 13 times with a mono-form, di-form and the unreacted PEG mixture 7.5 g of a sterol derivative was prepared.

Example 3: Structural Analysis of Water Soluble Sterol Derivatives

Chemical structural analysis of the water-soluble sterol derivative prepared in Example 2 can be analyzed by H-NMR using CDCl ₃ as a solvent, but this shows an average value, and the actual distribution of PEG, mono form and die form is ultra-vacuum. This can be confirmed by analysis of an ultra high vacuum Matrix-Assisted Laser Desorption Ionization system (hereinafter referred to as "Maldi").

Maldi is an analyzer that measures the molecular weight of a substance and can accurately grasp the distribution of the three components in the product. 1, a sterol derivative synthesized in <Example 2-1> (FIG. 1C), a sterol mixed with unreacted PEG, mono form and die form synthesized in <Comparative Example 2-3> Maldi of the derivative (FIG. 1B) and PEG itself (FIG. 1A) having a weight average molecular weight of 600 is shown for comparison. When one molecule of sterol is bound to PEG, the molecular weight theoretically increases by 499. From FIG. 1, the mixed sterol derivative synthesized in <Comparative Example 2-3> has a relatively high molecular weight in PEG in an unreacted form. It remains to be seen that the groups with an increase in molecular weight of about 500 (ie mono form) and the groups with an increase of about 1000 (ie die form) are mixed. On the other hand, the water-soluble sterol derivative synthesized in <Example 2-1> appears in the same form where the molecular weight is increased by about 500 compared to PEG, and at the same time, there is no peak determined as unreacted PEG or die form. It can be seen that it consists only of the mono form.

Example 4: Determination of Solubility of Water-soluble Sterol Derivatives

Since the water-soluble sterol derivatives prepared by the present invention should be able to be added to food and beverages, the change in solubility of the water-soluble derivatives with temperature changes was investigated in consideration of the fact that general food and beverages are sold in a refrigerated state. Solubility experiments were observed after dissolving the sterol derivative at various concentrations at 35 ℃, and then stored at room temperature or 4 ℃ for 60 days.

Table 1 shows the results of measuring solubility according to temperature using the water-soluble sterol derivatives prepared in <Example 2-1>, <Example 2-2> and <Comparative Example 2-3>.

Table 1

* Concentration (% by weight) = (g number of water-soluble sterol derivatives / g number of water) X 100

** Concentration (% by weight) = (g number of sterols / g number of water in water-soluble sterol derivatives) X 100

From Table 1, when comparing Examples 2-1 and 2-2, the derivative of Example 2-2 using PEG having a large molecular weight as a carrier shows slightly higher solubility on the basis of the derivative, but on the basis of the sterol residue It can be seen that the derivative of Example 2-1 using PEG having a weight average molecular weight of 600 shows a higher solubility and is a more preferable substance. In addition, when comparing Example 2-1 and Comparative Example 2-3, the solubility of the derivative present in the mono form is significantly improved compared to the derivative present in the mixed form, despite the use of the same water-soluble carrier It can be seen.

As such, the water-soluble sterol derivative synthesized in Example 2-1 can dissolve up to 3.0 wt% (30 mg / ml) on a sterol basis even at 4 ° C., which is effective in lowering cholesterol of adult adults recognized by the FDA. It is possible to include a daily intake of 2.7 g of sterol in 100 ml containers of food and beverage and pharmaceutical products, so that it can be widely applied to various types of products.

Claims

A water-soluble sterol comprising mixing the phytosterol intermediate and polyethylene glycol (PEG) with a weight average molecular weight of 200 to 1,000 in a molar ratio of 1: 2 to 1: 6 in the presence of a basic catalyst in a nonpolar organic solvent, and adding a binder to bind the reaction. Process for the preparation of derivatives.
The method of claim 1,

Method for producing a water-soluble sterol derivative, characterized in that the phytosterol intermediate is a compound represented by the following formula (1):

[Formula 1]
The method of claim 2,

The phytosterol is stigmasterol, spinasterol or cytosterol, characterized in that the method for producing a water-soluble sterol derivative.
The method of claim 1,

The nonpolar organic solvent is selected from the group consisting of toluene, methylene chloride, dichloroethane, tetrahydrofuran, benzene, diethyl ether, and mixtures thereof.
The method of claim 1,

The basic catalyst is selected from the group consisting of 4-dimethylaminopyridine, pyridine, triethylamine and mixtures thereof.
The method of claim 1,

Method for producing a water-soluble sterol derivative, characterized in that the polyethylene glycol has a weight average molecular weight of 200 to 800.
The method of claim 1,

Method for producing a water-soluble sterol derivative, characterized in that the coupling reaction is carried out at room temperature for 5 to 15 hours.
The method of claim 1,

The binder is 1,3-dicyclohexylcarbodiimide (DCC), ethylene dichloride (ethylene dichloride), oxalyl cholride, carbonyl diimidazole (carbonyl diimidazole), 2-chloropyridium (2- chloropyridium, 2,2-dipyridyl disulfide, 2-imidazoyl disulfide, and mixtures thereof, wherein the water-soluble sterol derivative is selected from the group consisting of chloropyridium, 2,2-dipyridyl disulfide, and 2-imidazoyl disulfide. Manufacturing method.
A water-soluble sterol derivative having a phytosterol intermediate bonded to one end of a polyethylene glycol (PEG) having a weight average molecular weight of 200 to 1,000 obtained by the method of any one of claims 1 to 8.
The method of claim 9,

A water-soluble sterol derivative, characterized in that the water-soluble sterol derivative is a compound represented by the following formula (2):

[Formula 2]

Where

n is an integer of 5-20.