KR100439015B1 - Removing method of cholesterol using solid substrate bound with cyclodextrin derivatives - Google Patents

Abstract

The present invention relates to a method for removing cholesterol by using a fixture in which a cyclodextrin derivative is bound. More specifically, the main molecule (A), which is a derivative of cyclodextrin, in which a hydroxy group is converted into an amine group or an aldehyde group, is bound to a solid substrate (C) in which a support (B) of an aldehyde group or an amine group is introduced by silanization reaction. It relates to a method for removing cholesterol contained in the liquid sample using. In addition, the cholesterol is collected by contacting the organic acid and the alcohol mixture solution to contact the mixture is separated, and then characterized in that it is recycled and reused repeatedly.

The present invention is economical because it can remove cholesterol efficiently and can regenerate and reuse the fixed body.

Description

Removing method of cholesterol using solid substrate bound with cyclodextrin derivatives

The present invention relates to a method for removing a substance such as cholesterol contained in a liquid sample by using a fixture in which a cyclodextrin derivative is bound to a solid substrate.

Cyclodextrins (hereinafter referred to as CyDs) and their derivatives are known to have the property of capturing a wide variety of chemicals by acting as master molecules, and α-CyD, β-CyD, and γ-CyD are particularly effective. Because of their size and characteristics, research on them has been the most active.

Β-CyD of CyD is a cyclic polysaccharide composed of seven glucose, similar to donut, and the central part of the molecule is similar in diameter to cholesterol and hydrophobic circular space, so it can bind to cholesterol, which is a nonpolar molecule. It has a structure.

In addition to cholesterol, β-CyD can bind or capture various other substances having molecular size and nonpolar similarity to cholesterol.

Since β-CyD is not only inexpensive but also has high safety for humans and is allowed to be used as a food additive in many countries, an attempt has been made to remove cholesterol in liquid samples using β-CyD.

In Japanese Patent Laid-Open No. 04168198, 1.2 g of β-CyD was added to 20 g of a cream containing 50% water, stirred at 30 ° C. for 60 minutes, warmed and centrifuged to recover a cream layer. The maximum removal rate of cholesterol was 84.9 Was%. In addition, as a result of treatment of creams by other researchers with β-CyD 0.1M, agitation temperature of 40 ° C and agitation time for 120 minutes, up to 83% of cholesterol could be removed (J. of Dairy Science, Vol. 76, Supplement 1 , Abst. No. D158).

Korean Patent Application Nos. 1997-18599, 1997-37127, and 1997-37128 also describe methods for removing cholesterol from β-CyD from liquid samples such as milk or cream.

In these inventions, the study was made to suggest the optimal conditions for removing cholesterol by conducting research to improve the shortcomings of the previous method for removing cholesterol. In the present invention, the main method is to add β-CyD to the liquid sample and then stir. It is a technique to remove cholesterol bound to β-CyD through centrifugation, and to collect customer molecules such as cholesterol and remove them from a liquid sample by using a host molecule such as β-CyD as described above.

However, since these methods perform the collection reaction of the host molecule and the guest molecule directly in the liquid sample, in order to actually remove the combination of the host molecule-guest molecule conjugate from the liquid sample, it has to go through a cumbersome process such as centrifugation. There is a disadvantage.

In addition to being cumbersome in this procedure, the quality of the original liquid sample may be impaired as a result of the centrifugal action, and a large amount of liquid sample (such as milk or cream) must be centrifuged at the production site. Given that, the process of centrifugation is a very complicated and cumbersome process and needs to be improved.

In addition, in the process of recycling the host molecule by separating the host molecule-guest molecule combination from the liquid sample and then detaching the host molecule and the guest molecule, the procedure is cumbersome and inconvenient.

Therefore, a method of removing cholesterol in milk or an aqueous emulsion solution by using a column by attaching β-CyD to silica has been studied. However, this method does not go through centrifugation, so the process is simplified, but after removing cholesterol, fine particles of silica may be eluted in milk or an aqueous emulsion, which is a concern in terms of food safety.

An object of the present invention is to solve the above problems and to provide a method for simply removing cholesterol in a liquid sample and a method for recycling cyclodextrin by using a fixed body to which a cyclodextrin derivative is bound.

1 is a view schematically showing a fixture used in the present invention.

2 is a diagram showing a state in which cyclodextrin introduced to the solid substrate surface of the fixture used in the present invention traps cholesterol.

<Explanation of symbols for the main parts of the drawings>

A: Owner molecule B: Support

C: solid substrate

The present invention is characterized by a method for removing cholesterol from a liquid sample by using a fixed fixture combined with a cyclodextrin derivative.

In the fixed body used in the present invention, the main molecule (A), which is a derivative of the cyclodextrin in which the hydroxy group is converted into an amine group or an aldehyde group, is a solid substrate in which a support (B) of an aldehyde group or an amine group is introduced through a silanization reaction ( It is characterized in that it is coupled to C).

Of the fixed bodies used in the present invention, the main molecule (A) is a substance selected from α-CyD, β-CyD and γ-CyD. Alternatively, Calix [n] -arenes or Cucurbituril may be used instead of CyD.

In the fixed body used in the present invention, the support (B) is a substance that serves to attach the host molecule (A) to the solid substrate (C) as a linker (linker). At this time, the support (B) is composed of 1 to 15 elements of the main skeletal chain except for the side chain (side chain), can be selected by combining one or more than two components.

As the solid substrate (C) in the fixture used in the present invention, a material selected from glass, silicon wafer, or quartz, which is mainly composed of silicon oxide (SiO 2), may be used. Mono-6-O- (p-tosylsulfonyl) -β-cyclodextrins (Tetrahedron Letter, Vol. 25, No. 31, pp 3331-3334, 1984) and mono-6-deoxy-6-, which are cyclodextrin derivatives Amino-β-cyclodextrins and Cyclodextrin monoaldehydes (Ref .; Tetrahedron Letter, Vol. 36, No 46, pp. 8371-8374, 1995) were synthesized, and silanized on glass substrates, followed by electric Doa-β- Mono-6-deoxy-6-amino-β-cyclodextrins (CyD) and cyclodextrin monoaldehyde (CyDMA) are introduced to imine groups or aldehyde groups to obtain a fixture in which a cyclodextrin derivative and a glass substrate are combined.

Hereinafter, the present invention will be described in detail.

In general, reactions that collect steroid-based molecules such as cholesterol are reversible reactions that can trap or dissociate guest molecules by adjusting solvents, pH, and the like. Therefore, the process of collecting the guest molecules by using the fixed body is not only very simple, the fixed body fixed to the owner molecule has the advantage that can be repeatedly used.

In the present invention, the main molecule (A), which is a derivative of a cyclodextrin in which a hydroxy group is converted to an amine group or an aldehyde group, is bonded to a solid substrate (C) in which a support (B) of an amine group or an aldehyde group is introduced by a silanization reaction. After placing the CyD fixtures in the milk solution of the Stain Jar container and shaking them for 10-30 minutes at the refrigeration temperature, the cholesterol in the milk is collected in the CyD fixtures to remove cholesterol.

The glass fixture, which formed the collection complex, was placed in a mixed solution of alcohols and acids, shaken at 40 to 60 ° C for 1 to 3 hours, and then the fixed body was taken out, washed with alcohol and distilled water, and dried to remove from the CyD fixture. Cholesterol can be released to easily regenerate the CyD fixture.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are intended to illustrate the present invention but are not intended to limit the scope of the present invention.

<Example 1>: Cholesterol removal in the milk using (beta) -CyD fixation body

Five sheets of β-CyD fixtures prepared as described above (7.5 cm x 2.5 cm) were fixed in a container (Stain Jar, Fisher Inc.) and 50 ml of homogenized milk having a cholesterol content of about 6.5 mg was placed in a container and refrigerated. After shaking with a shaker for 30 minutes at a temperature (4-10 ° C.), all five β-CyD fixtures were taken out. The β-CyD solids contained in the above-mentioned cholesterol were added to a mixed solvent mixed with acetic acid and n-butanol 3: 1 (v / v), and the cholesterol was β-CyD high using a shaker at 50 ° C. for 2 hours. After separation from the suspension, the amount of cholesterol was measured by gas chromatography. At this time, GC conditions are as follows.

Column: HP-5 (Crosslinked 5% Ph Me silicone),

Film thickness of the column: 0.25 μm,

-Film length of the column: 30 m,

Film diameter of the column: 0.35 mm,

-Oven temperature: 2 minutes at 190 ℃ → 20 ℃ / min → 3 minutes at 230 ℃ →

25 minutes hold at 255 ℃

Carrier gas: helium (2 ml / min), air (300 ml / min), hydrogen (30 ml / min)

Detector: FID,

Internal standard: Cholestane (1mg / ml)

When the amount of cholesterol measured by the gas chromatography method described above was converted to the unit area of the β-CyD fixture, it was found that cholesterol per 10000 1002 area of the β-CyD fixture was collected at a rate of 20 molecules. . On the other hand, it can be seen that the main molecule capable of capturing cholesterol, that is, β-CyD, is attached at a rate of 20 molecules or more per 10000 Å ² area of the β-CyD fixture through the collection amount of cholesterol.

<Example 2> : Regeneration method of β-CyD fixture

Experiments to find the optimal conditions for dissociation of the capture complexes, ie, separation of the collected cholesterol from the glass fixture with β-CyD attached, using the solvents shown in Table 1 It was.

Put acetic acid and n-butanol in a mixed solvent at 3: 1 (v / v) into a glass fixed body, and shake it with a shaker (shaker) at 50 ° C. for 2 hours. It was taken out, washed with n-butanol, ethanol, distilled water and dried.

In experiments with solvents of several different compositions, the amount of dissolved cholesterol in each solvent was measured by gas chromatography.

Table 1 shows the degree of dissociation of cholesterol from β-CyD fixture according to the composition ratio of each solution.

TABLE 1

menstruum Mixing ratio Nautical degree Acetic acid: butanol 3: 1 1.00 Chloroform: Butanol 2: 1 0.10 Hexane: Butanol 2: 1 0.15 Chloroform: Ethanol 3: 2 0.40

As shown in Table 1, the dissociation of cholesterol was best when acetic acid and n-butanol were used at a ratio of 3: 1 (v / v), and β-CyD fixtures were easily regenerated.

In the present invention, since the cholesterol can be easily removed from the liquid sample without undergoing the centrifugation process using the CyD fixation body, the quality of the liquid sample is not impaired, and the cyclodextrin can be recycled.

Claims (5)

The fixed molecule in which the main molecule (A) of the cyclodextrin derivative whose hydroxy group is converted into an amine group or an aldehyde group is bound to a solid substrate (C) into which the support (B) of the aldehyde group or the amine group is introduced by silanization reaction. Method for removing cholesterol using a fixed body combined with a cyclodextrin derivative, characterized in that to remove cholesterol in the solution by using The method according to claim 1, wherein the β-CyD fixture is placed in a container and contacted at 4-10 ° C. for 10-30 minutes in a solution containing cholesterol to collect cholesterol in the β-CyD fixture, followed by separation. Cholesterol removal method using a fixed body combined with a cyclodextrin derivative [Claim 2] The method of claim 1, wherein the solution containing cholesterol is milk or a liquid containing cholesterol in a solution state. After the cholesterol-fixed fixture was reacted with a mixed solution of an organic acid and an organic solvent at 40-60 ° C. for 1-3 hours, the isolate was separated, washed with alcohol and purified water, and dried to remove cholesterol from the β-CyD fixture. Method for removing cholesterol using a fixed body combined with a cyclodextrin derivative, characterized in that to separate and regenerate the electric fixed body to reuse The organic acid according to claim 4, wherein the organic acid is acetic acid, chloroform, nucleic acid, or citric acid, and the organic solvent is methanol, ethanol, furopanol or butanol. v) A method for removing cholesterol using a fixed body combined with a cyclodextrin derivative, characterized by using a mixed solvent mixed with