JP2741627B2 - Inclusion compound of β- and / or γ-cyclodextrin and method for separating and purifying β- and / or γ-cyclodextrin - Google Patents

Description

The present invention relates to novel inclusion compounds of β- and / or γ-cyclodextrin. Furthermore, the present invention relates to a method for efficiently separating and purifying β- and / or γ-cyclodextrin from various cyclodextrin mixtures containing β- and / or γ-cyclodextrin.

[Prior Art] Cyclodextrins (hereinafter abbreviated as CDs)
It has been known for a long time that starch is produced by the action of cyclodextrin glucanotransferase (hereinafter abbreviated as CGTase) produced by Bacillus macerans or Bacillus stearothermophilus. The resulting CDs include α-cyclodextrin (hereinafter abbreviated as α-CD) in which 6 glycols are α-1-4 linked cyclically, and β-cyclodextrin in which 7 glycols are linked cyclically. Cyclodextrin (hereinafter abbreviated as β-CD) and γ-cyclodextrin (hereinafter abbreviated as γ-CD) in which eight glucoses are bound in a cyclic manner are included. In addition, when CGTase is applied to waxy maize or branched dextrin that is rich in aminopectin, branched cyclodextrins are generated and the normal CD as described above is used.
Kinds are also created.

These CDs generally have the property of enclosing various substances, but the selectivity of the substance (guest) to be taken in depends on the type of CD (host), and the size of the cavity of the CD matches the steric size of the guest molecule. It is believed that it stabilizes when exposed and is easy to be included. CDs have a function of solubilizing poorly water-soluble substances,
Stabilization function for unstable substances, volatilization prevention function for volatile substances,
It exerts off-odor masking function, chemical reaction catalysis function, etc., and these functions are thought to be related to the inclusion function in which lipophilic substances are wrapped in the three-dimensional cavities of CDs. For this reason, depending on the desired expression function, various CDs often interfere with each other.
In such a case, it is ideal to use a high-purity CD alone.

β-CD and γ-CD are useful, for example, in pharmaceutical-related applications, such as stabilization, solubilization, promotion of absorption in the body, prevention of deliquescence, and powdering of oil bases, etc. It is useful for solubilization of drugs, promotion of penetration into skin and suppression of irritation, stabilization of perfume, and powdering of oil base. However, in order to exhibit such a function more effectively, as described above, β-CD or γ-
Naturally, it is desirable for the CD to have high purity.

By the way, the reaction mixture obtained by reacting CGTase with the starch as described above contains various CDs and acyclic dextrins as described above, and β-CD
And γ-CD alone are difficult to separate because β-CD has a lower water solubility than γ-CD, but γ-CD is a substance that has high solubility in water and is difficult to crystallize. High cost was required to increase the purity by using such methods.

The following methods are known as typical methods for separating CDs from the above reaction mixture.

(1) A method in which an organic solvent such as acetone is added to a sugar solution to precipitate CDs (see Japanese Patent Publication No. 52-8385).

(2) Purification method using anion exchange resin
No. 46-9223).

(3) A method using a hydrophobic synthetic absorbent resin composed of a porous polymer (see JP-A-56-805).

(4) A method of fractionating with an alkali metal salt of a strongly acidic ion exchange resin (see JP-A-57-30702).

[Problems to be Solved by the Invention] However, the above methods (1), (2), (3) and (4) are somewhat effective when CDs are separated from other oligosaccharides or dextrins. However, these methods are expensive and cannot be used for the purpose of industrially separating only β-CD and / or γ-CD with high purity.

The present invention solves the above-mentioned problems of the prior art and provides various CDs.
It is an object of the present invention to apply a method capable of easily separating and purifying high-purity β-CD and / or γ-CD from a class mixture. In addition, the present invention provides, for example, β-CD and / or γ-CD as an intermediate of β-CD and / or γ-CD as a final product in the above-mentioned method for separating and purifying β-CD and / or γ-CD.
Another object of the present invention is to provide an inclusion compound.

[Means for Solving the Problems] In view of the above-mentioned situation in the art, the present inventors have
As a result of intensive studies to find an efficient and practical method for separating β-CD and / or γ-CD, it was found that polypropylene glycol selectively included only β-CD and γ-CD. That is, the present inventors have studied the interaction between various water-soluble polymers and CDs in view of the fact that studies on the inclusion function of CDs are mostly limited to interactions with low molecular weight compounds. . Table 1 shows 20 of each water-soluble polymer.
mg in the case of β-CD is added to 5 ml of β-CD saturated aqueous solution, and γ-C
In the case of D, add 1 ml of a saturated aqueous solution of γ-CD, ultrasonically stir at about 25 ° C for 10 minutes, and leave the polymer and β-
It is a list of the yields of inclusion compounds of CD or γ-CD.

Furthermore, as a result of the same experiment as shown in Table 1, oligosaccharides and polysaccharides including CDs (including branched CDs) other than β-CD and γ-CD, and monosaccharides were included in polypropylene glycol and inclusion compounds. No complex was formed.

The present invention has been completed based on the above findings.

That is, the present invention provides an inclusion compound of β- and / or γ-cyclodextrin in which a polypropylene glycol molecule is included in β- and / or γ-cyclodextrin molecules constituting an inclusion lattice. is there.

Further, the present invention provides a mixture of various cyclodextrins containing β- and / or γ-cyclodextrin and polypropylene glycol to form an insoluble inclusion compound of β- and / or γ-cyclodextrin, The present invention further provides a method for separating and purifying β- and / or γ-cyclodextrin, which comprises removing polypropylene glycol from the β- and / or γ-cyclodextrin inclusion compound.

 Hereinafter, the present invention will be described in detail.

The polypropylene glycols capable of forming the inclusion compound of β-CD and / or γ-CD of the present invention are limited in their minimum molecular weight. Table 2 shows that 20 mg of each of polypropylene glycols having different average molecular weights was added to 5 ml of a β-CD saturated aqueous solution for β-CD, and γ-CD for γ-CD.
Lists the yields of inclusion compounds of polypropylene glycol (PPG) and β-CD or γ-CD when added to 1 ml of a saturated aqueous solution, ultrasonically stirred at about 25 ° C. for 10 minutes, and allowed to stand for 1 hour. It is.

The polypropylene glycol used had an average molecular weight of 400, 425 and 725 and was obtained from Aldrich Japan Ink and had an average molecular weight of 1000, 2000 and 30.
Those having a molecular weight of 00 and 4000 (the average molecular weight was determined from the hydroxyl value) were obtained from Wako Pure Chemical Industries, Ltd.

Table 2 shows that β-CD and γ-CD do not form inclusion compounds with those having a low molecular weight such as dipropylene glycol and tripropylene glycol.
The minimum molecular weight that the polypropylene glycol must have to form an inclusion compound with β-CD or γ-CD is, for example, an average molecular weight of 400 even though the average molecular weight is 400 above and below the average. Since it is common to obtain only polypropylene glycol having a molecular weight distribution having the following formula, it cannot be specified accurately in the above-described experiment. However, in the case of β-CD, from Table 2, the inclusion compound yield was 1% for polypropylene glycol having an average molecular weight of 400 and the inclusion compound yield was 27% for polypropylene glycol having an average molecular weight of 425. , Molecular weight 425
It is speculated that there will be the required minimum molecular weight around. In addition, in the case of γ-CD, from Table 2, the inclusion compound yield is 0% for tripropylene glycol having a molecular weight of 192 and the inclusion compound yield is 76% for polypropylene glycol having an average molecular weight of 400. It is assumed that there will be a required minimum molecular weight between 200 and 400.
From the above, the β-CD and / or γ-
The minimum molecular weight of polypropylene glycol in the inclusion compound of CD is specified only as the minimum molecular weight necessary for forming an inclusion compound with β-CD and / or γ-CD.

In Table 2, both β-CD and γ-CD are average molecular weights.
An inclusion compound is formed with 400 or more polypropylene glycols.In the yield, β-CD is easier to form an inclusion compound with polypropylene glycol having an average molecular weight of 725 to 2000, whereas γ-CD 400 to 1
It can be seen that it is easy to form an inclusion compound with polypropylene glycol having an average molecular weight somewhat lower than 000. From this, it is understood that separation of β-CD and γ-CD is possible by using propylene glycol having different average molecular weights properly.

Next, as a result of studying the formation of an inclusion compound of β-CD and polypropylene glycol having an average molecular weight of 1000, the amount of the generated inclusion compound increased with the increase in the amount of polypropylene glycol added to the β-CD aqueous solution, and the reaction was observed. Reached a saturated state, that is, a state where it could no longer react, and a stoichiometric inclusion reaction was found.

As a result of quantitative study of the formation of the inclusion compound, one molecule of β-CD
Was found to be bound. This was also confirmed by the ¹ H-NMR spectrum. It has also been found that the length of two molecules of propylene glycol corresponds to the length of the cavity of β-CD. FIG. 1 is a model diagram of the structure of an inclusion compound of polypropylene glycol and β-CD estimated from the results. In FIG. 1, the hatched portion may be considered to be a cross section of β-CD. The same is presumed for the inclusion compound of polypropylene glycol and γ-CD.

FIG. 2 is a diagram showing a powder X-ray diffraction pattern of an inclusion compound of polypropylene glycol having an average molecular weight of 1000 and β-CD. In FIG. 2, the abscissa θ indicates the diffraction angle, and the ordinate CPS indicates the reflection intensity (count / sec). As shown in FIG. 2, this clathrate is crystalline, and its powder X-ray diffraction pattern is similar to that of a low molecular weight compound such as propionic acid or propanol.
This is different from the powder X-ray diffraction pattern of the inclusion complex with CD, which indicates that the inclusion compound of polypropylene glycol and β-CD is not a so-called cage-shaped structure and is not an isomorphous crystal.

In thermogravimetric analysis, β-CD melted and decomposed at a temperature lower than 310 ° C., whereas the inclusion compound of polypropylene glycol having an average molecular weight of 1000 and β-CD was 32.
Decomposition temperatures above 0 ° C were noted.

FIG. 3 is a diagram showing a powder X-ray diffraction pattern of an inclusion compound of polypropylene glycol having an average molecular weight of 1000 and γ-CD. The abscissa and ordinate in FIG. 3 are the same as in FIG. FIG. 3 shows that this clathrate compound is crystalline.

Next, the method for separating and purifying β-CD and / or γ-CD of the present invention will be described in detail. The method includes, for example, various CDs,
Further, a sugar solution containing acyclic dextrin or the like is contact-mixed with polypropylene glycol to form a precipitate of an insoluble clathrate compound of β-CD and / or γ-CD with polypropylene glycol. Is separated from the mixture by solid-liquid separation, polypropylene glycol is released by an operation described later, and β-CD and / or γ-CD are separated.

The mixing ratio between the sugar solution and the polypropylene glycol as described above may be usually 2 mol or more as a propylene glycol unit per 1 mol of β-CD and / or γ-CD. It is not preferable to use a large amount in terms of cost.

Solid-liquid separation can be performed by a general separation means such as filtration, centrifugation, or membrane separation using an ultrafiltration membrane.

The rate at which β-CD and / or γ-CD forms an inclusion compound with polypropylene glycol depends on the molecular weight (and thus the average molecular weight) of the polypropylene glycol. That is, if the average molecular weight of the polypropylene glycol is small, the inclusion reaction is generally fast, and a reaction time of several minutes is sufficient. When the average molecular weight of the polypropylene glycol increases, the reaction rate decreases.For example, after stirring for about 10 minutes by ultrasonic waves, if left at about 25 ° C. for about 1 hour, the clathration reaction is almost completed, The clathrate precipitates.
In addition, the ultrasonic stirring may be separated into two layers when the polypropylene glycol is added to the sugar solution, and thus has the significance of making the dispersion uniform.

The average molecular weight of the polypropylene glycol used in the method for separating and purifying β-CD and / or γ-CD of the present invention is as follows.
As can be seen from the table, about 425 to about 4 for β-CD
000 is preferred, about 725 to about 2000 is more preferred, while about 400 to about 4000 is preferred for γ-CD,
More preferably, about 400 to about 1500.

When it is desired to separate β-CD and γ-CD from a mixture (sugar solution) containing both, first, polypropylene glycol having an average molecular weight of about 425 or less, preferably about 400, is added to the mixture, and γ-CD is substantially isolated. Alternatively, a γ-CD rich inclusion compound is produced, the inclusion compound is subjected to solid-liquid separation, and then the polypropylene glycol is removed from the inclusion compound. If the substance thus obtained is substantially pure γ-CD, the separation and purification of γ-CD will be completed in one step of the above procedure, but if the substance also contains β-CD, Substantially pure γ-CD if the above procedure is repeated as necessary for the substance
Can be separated. However, industrially, it is possible that β-CD may be contained to a certain extent, so that the purity of γ-CD may be set to a necessary level according to the application.

In the above-mentioned case, β-CD is separated from β-CD and the inclusion compound in the mixture (sugar solution) obtained by substantially separating γ-CD or the γ-CD-rich inclusion compound by the above operation. Select and add polypropylene glycol with an average molecular weight that is easy to produce,
A substantially pure or β-CD rich inclusion compound of β-CD is produced, the inclusion compound is subjected to solid-liquid separation, and then the polypropylene glycol is removed from the inclusion compound. If the remaining mixture (sugar solution) still contains γ-CD, the above γ-CD separation procedure is repeated to substantially
May be used as a residual mixture (sugar solution) which no longer contains. However, industrially, there may be no problem even if γ-CD is contained to a certain extent, so that the purity of β-CD may be set to a necessary level according to the application.

Next, an operation of removing polypropylene glycol from an inclusion compound of polypropylene glycol and β-CD and / or γ-CD will be described. The precipitate of the clathrate separated and washed from the reaction mixture is dried, if necessary, and then an appropriate organic solvent is added thereto.When the mixture is agitated, the polypropylene glycol is released from the clathrate and dissolved in the solvent. , Β-CD and / or γ-CD remain as precipitate. When the solvent containing polypropylene glycol is removed from the suspension by a general solid-liquid separation means such as filtration, centrifugation, or membrane separation using an ultrafiltration membrane, β-CD and / or γ- CD crystals are obtained. Specific examples of the organic solvent include lower alcohols such as methanol, ethanol, and propanol, and halogenated hydrocarbons such as dichloromethane. As another method, when the precipitate of the clathrate is suspended in water and heated to a temperature of 80 ° C. or more, the clathrate is weakened and the polypropylene glycol is liberated, and the polypropylene glycol and β-CD and / or γ- Since both of the CDs are dissolved in water, the resulting aqueous solution can be subjected to, for example, column chromatography at a relatively high temperature, or concentrated and then subjected to hot filtration to separate the two. As yet another method, the precipitate of the inclusion compound is suspended in water, and benzoic acid, phenol, or another substance having a higher inclusion power with β-CD and / or γ-CD than polypropylene glycol is added. It is replaced by the polypropylene glycol included in β-CD and / or γ-CD and is included, and the polypropylene glycol is released. Since the polypropylene glycol and the new inclusion compound are often dissolved in water, the resulting aqueous solution can be separated by, for example, column chromatography or concentration and filtration. If the new clathrate has a useful application, there is no need to further separate and purify β-CD and / or γ-CD, which is reasonable.

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples, but it goes without saying that the present invention is not limited to Examples.

Example 1 10% by weight of a CD mixture (composition: α-CD 30% by weight, β-CD
(CD 15% by weight, γ-CD 5% by weight, dextrin 50% by weight)
An aqueous solution containing 15% by weight of polypropylene glycol (average molecular weight: 400) is added to 100 ml of an aqueous solution containing
After ultrasonic stirring and mixing for 1 minute, and left for 1 hour, an inclusion compound of polypropylene glycol and γ-CD was precipitated. The resulting suspension was filtered with a filter using a filter paper having a pore size of 0.45 μm to separate the precipitate of the clathrate, and then washed with 200 ml of pure water.

Next, an aqueous solution containing 15% by weight of polypropylene glycol (average molecular weight: 1000) was added to the filtrate obtained by the above-mentioned filtration operation, mixed by ultrasonic stirring at room temperature for 10 minutes, and allowed to stand for 1 hour. And a β-CD-rich inclusion compound was precipitated. The obtained suspension was filtered in the same manner as described above, and the precipitate was separated and washed with 200 ml of pure water.

The two precipitates thus obtained were transferred to separate beakers, and 100 ml of 99.5% ethanol was added to each,
When the mixture is stirred, substantially only the polypropylene glycol dissolves in the ethanol and is released. Therefore, each of the obtained suspensions is filtered, and the residue is washed with 99.5% ethanol to obtain substantially pure γ. It was possible to obtain β-CD having considerably high purity with respect to -CD.

[Effect] The inclusion compound of β-CD and / or γ-CD of the present invention can be used as an intermediate for preparing pure β-CD and / or γ-CD. And / or a mixture of various cyclodextrins containing γ-CD and polypropylene glycol can be easily prepared as a precipitate by mixing in the presence of water.

According to the method for separating and purifying β-CD and / or γ-CD of the present invention, after preparing the above-mentioned inclusion compound of β-CD and / or γ-CD, polypropylene glycol is easily converted from the inclusion compound. Since it can be removed, high-purity β-CD and / or γ-CD can be produced at low cost.

[Brief description of the drawings]

FIG. 1 shows the relationship between the polyethylene glycol of the present invention and β-CD
FIG. 3 is a model diagram of the estimated structure of an inclusion compound with a propylene glycol unit included in the hatched β-CD cavity. FIG. 2 is a diagram showing a powder X-ray diffraction pattern of the inclusion compound of the present invention comprising polypropylene glycol having an average molecular weight of 1000 and β-CD. FIG. 3 is a diagram showing a powder X-ray diffraction pattern of the inclusion compound of the present invention comprising polypropylene glycol having an average molecular weight of 1000 and γ-CD.

Claims (6)

(57) [Claims] 1. An inclusion compound of β- and / or γ-cyclodextrin in which polypropylene glycol molecules are included in β- and / or γ-cyclodextrin molecules constituting an inclusion lattice. 2. A mixture of various cyclodextrins containing β- and / or γ-cyclodextrin and polypropylene glycol are mixed in the presence of water to form an insoluble β- and / or γ-cyclodextrin clathrate. A method for separating and purifying β- and / or γ-cyclodextrin, which comprises producing the product and then removing the polypropylene glycol from the β- and / or γ-cyclodextrin inclusion compound. 3. The method according to claim 2, wherein said polypropylene glycol has an average molecular weight of about 400 to about 4,000. 4. The mixture of various cyclodextrins contains β- and γ-cyclodextrin, the average molecular weight of the polypropylene glycol is about 425 or less, and the formed inclusion compound is substantially the same as that of γ-cyclodextrin. Claim 2 wherein the compound is a clathrate compound alone or in a γ-cyclodextrin-rich state.
Item 4. The method for separation and purification according to Item 3 or 3. 5. The mixture of various cyclodextrins according to claim 4, except that the mixture of substantially cyclodextrin or the clathrate compound rich in γ-cyclodextrin is described in claim 4. A mixture of the remaining various cyclodextrins, wherein the average molecular weight of the polypropylene glycol is about 425 to about 4000.
3. The method for separation and purification according to claim 2, wherein the clathrate compound produced is substantially a β-cyclodextrin alone or a β-cyclodextrin-rich clathrate compound. 6. An organic solvent is added to the β- and / or γ-cyclodextrin clathrate to obtain the β- and / or γ-cyclodextrin inclusion compound.
The method according to any one of claims 2 to 5, wherein the polypropylene glycol is removed from the-and / or γ-cyclodextrin inclusion compound.