JPH0413701A - Inclusion compound of beta-and/or gamma-cyclodextrin and separating purification of beta-and/or gamma-cyclodextrin - Google Patents

Abstract

PURPOSE:To inexpensively obtain the title product, by blending a cyclodextrin mixture with polypropylene glycol in the presence of water to form an insoluble cyclodextrin inclusion compound and removing the polypropylene glycol. CONSTITUTION:A mixture of various cyclodextrins containing beta- and/or gamma- cyclodextrin is blended with polypropylene glycol in the presence of water to form an insoluble beta- and/or gamma-clclodextrin inclusion compound. Then the polypropylene glycol is removed from the inclusion compound to give the objective inclusion compound.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to novel β- and/or γ-cyclodextrin clathrate compounds. 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 cyclodextrin glucanotransferase (hereinafter abbreviated as CGTase) produced by Bacillus macerans and Bacillus stearothermophilus acts on Wl powder to produce it. In these generated CDs, six glucose molecules are cyclically α
-1-4 linked α-cyclodextrin (hereinafter referred to as α
-Abbreviated as CD. ) etc., as well as β-cyclodextrin (hereinafter referred to as β-C
It is abbreviated as D. ) and γ-cyclodextrin (hereinafter abbreviated as γ-CD) in which eight glucose molecules are linked in a cyclic manner. Furthermore, when CGTase is applied to waxy corn or branched dextrin containing a large amount of amylopectin, not only branched cyclodextrins are produced, but also ordinary CDs such as those mentioned above are produced.

These CDs generally have the property of inclusion of various substances, but there is selectivity of the substances (guests) that are taken in depending on the type of CD (host), and the size of the CD cavity and the steric size of the guest molecule are compatible. It is thought that it becomes stable and is easily included. CDs exhibit functions such as solubilizing poorly water-soluble substances, stabilizing unstable substances, preventing volatilization of volatile substances, masking off-odor functions, and acting as a catalyst for chemical reactions. It is thought that this is related to the inclusion effect in which lipophilic substances are wrapped up in the steric cavity of . For this reason, depending on the desired expression function, various CDs often inhibit each other's functions. In such cases, it is ideal to use a single highly purified CD.

For example, β-CD and γ-CD are useful in pharmaceutical applications such as stabilizing drug retention, solubilization, promoting absorption in the body, preventing deliquescent properties, and powdering oil and fat bases. It is useful for solubilizing drugs, promoting skin penetration and suppressing irritation, stabilizing fragrances, and powdering oil and fat bases. However, in order to exhibit this function more effectively, it is necessary to use β-CD and γ-
It goes without saying that it is desirable for CDs to have high purity.

By the way, the reaction mixture obtained by reacting starch with CGTase as described above contains various CDs and acyclic dextrins as described above, among which β-CD and γ- Isolating only CD is
D has lower water solubility than γ-CD, but γ-CD is a substance that has high solubility in water and is difficult to crystallize, so it is extremely difficult.
Furthermore, high costs were required to increase the purity by using solvents and the like.

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

(1) A method in which CDs are precipitated by adding an organic solvent such as acetone to a sugar solution (see Japanese Patent Publication No. 8385/1985).

(2) A method of purification using an anion exchange resin (see Japanese Patent Publication No. 4B-8223).

(3) A method using a hydrophobic synthetic adsorption resin made of a porous polymer (see @Kokai No. 56-805).

(4) A method of fractionation using an alkaline earth 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 not effective to some extent when separating CDs from other oligosaccharides, dextrins, etc. 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 problems of the prior art and
An object of the present invention is to provide a method that allows easy separation and purification of highly pure β-CD and/or γ-CD from a mixture of similar substances. Further, the present invention provides 1, for example, the final product β-CD and/or γ-CD separation and purification method described above.
Another object of the present invention is to provide an inclusion compound of β-CD and/or γ-CD as an intermediate of -CD and/or γ-CD.

[Means for Solving the Problems] In view of the actual state of the art as described above, the present inventors have conducted extensive research to find an efficient and practical method for separating β-CD and/or γ-CD. As a result of repeated experiments, it was found that polypropylene glycol selectively includes only β-CD and γ-CD. That is, the present inventors studied the interactions between various water-soluble polymers and CDs, in view of the fact that research on the inclusion effects of CDs is mostly limited to interactions with low-molecular compounds. Table 1 shows that 20 mg of each water-soluble polymer was added to 5 l of a saturated aqueous solution of β-CD in the case of β-CD, 1 l of a saturated aqueous solution of γ-CD in the case of γ-CD, and 10 mg at about 25°C. The yield of the clathrate compound of the polymer and β-CD or γ-CD when subjected to ultrasonication for one minute and left to stand for one hour is listed.

Table 1 Polymer (average molecular weight) Yield 1 β-CD γ-CD Polyvinyl alcohol c Polymerization degree: 5oo)
0 0/l (degree of polymerization +2
000) OO polyacrylamide
(10000) OO polyvinyh pyrrolidoshi (+0000)
OO polyethylene sidilycol (+00
0) 00 Voliv D Biresig v Co-h (100
0) 98 80 Furthermore,
Results of experiments similar to those in Table 1 show that β-CD and γ-
It has been found that oligosaccharides, polysaccharides, and monosaccharides containing CDs other than CDs (including branched CDs) do not form complexes such as clathrate compounds with polypropylene glycol.

The present invention was completed based on the above findings.

That is, the present invention provides β- and/or γ-
- Provides a β- and/or γ-cyclodextrin inclusion compound in which a polypropylene glycol molecule is included in a cyclodextrin molecule.

Furthermore, the present invention involves mixing a mixture of various cyclodextrins containing β- and/or γ-cyclodextrin with polypropylene glycol to produce an insoluble β- and/or γ-cyclodextrin inclusion compound; The present invention provides a method for separating and purifying β- and/or γ-cyclodextrin, which comprises removing polypropylene glycol from the β- and/or γ-cyclodextrin clathrate compound.

The present invention will be explained in detail below.

The polypropylene glycol that can form the β-CD and/or γ-CD clathrate of the present invention is limited in its minimum molecular weight. Table 2 shows that 20 mg each of polypropylene glycols with different average molecular weights were added to β-C
In the case of D, add 5 ml of β-CD saturated aqueous solution and add γ-CD
In the case of polypropylene glycol (PPG) and β-CD or γ-C, add 1 liter of saturated aqueous solution of γ-CD, sonicate for 10 minutes at about 25°C, and let stand for 1 hour.
The yields of the clathrate compounds of D are listed.

The polypropylene glycol used had an average molecular weight of 400.425 and 725, which were obtained from Aldrich Japan Inc., and an average molecular weight of 1000.
．． 2000.3000 and 4090 (average molecular weight is
It was determined from the hydroxyl value. ) is manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.
It was obtained from.

Table 2 Average Molecular Weight Yield (z) of PPG β-CD γ-CD Dipropylene Glycol Tripropylene Glycol From Table 2, β-CD and γ-CD are compounds with molecular weights such as dipropylene glycol and tripropylene glycol. It can be seen that small ones do not form clathrate compounds. The minimum molecular weight that polypropylene glycol must have in order to form an clathrate compound with β-CD and γ-CD is, for example, even if the average molecular weight is 400, it must be in a range that exceeds 100 above and below the average value. Generally speaking, only polypropylene glycol with a molecular weight distribution of For polypropylene glycol with an average molecular weight of 400, the clathrate compound yield is 1%, and for polypropylene glycol with an average molecular weight of 425, the clathrate compound yield is 27%, so it is assumed that there is a required minimum molecular weight near the molecular weight of 425. be done. Also, in the case of γ-CD.

From the ts2 table, the clathrate yield is 0% for tripropylene glycol with a molecular weight of 192, and the average molecular weight is 400.
For polypropylene glycol, the clathrate yield was 76
%, it is assumed that there is a required minimum molecular weight between 200 and 400. Based on the above, the minimum molecular weight of polypropylene glycol in the clathrate compound of β-CD and/or γ-CD of the present invention is set at the minimum molecular weight of polypropylene glycol that can form a clathrate compound with β-CD and/or γ-CD. Specify only the minimum molecular weight required.

In Table 2, both β-CD and γ-CD form clathrate compounds with polypropylene glycol having an average molecular weight of 400 or more, but in terms of yield, β-CD has an average molecular weight of 725 to 2000. γ-CD tends to form clathrate compounds with polypropylene glycol having a somewhat lower average molecular weight of 400 to 1000. From this, it is understood that separation of β-CD and γ-CD is possible by properly using propylene glycols having different average molecular weights.

Next, as a result of research on the formation of clathrate compounds between β-CD and polypropylene glycol with an average molecular weight of 1000,
The amount of the clathrate compound produced increases as the amount of polypropylene glycol added to the β-CD aqueous solution increases, and the reaction reaches a saturation state, that is, a state where no further reaction is possible, and the clathrate is stoichiometrically formed. I found it to be responsive.

As a result of a quantitative study on the formation of clathrate compounds, it was found that one molecule of β-CD per two monomer units of polypropylene glycol.
was found to be connected. This is IH-NM
It was also confirmed by R spectrum. It is also known that the length of two molecules of propylene glycol corresponds to the length of the β-CD cavity. FIG. 1 is a model diagram of the structure of the clathrate compound of polypropylene glycol and β-CD estimated from these results. In the R1 diagram, the shaded area can be considered to be the cross section of β-CD, and it is presumed that the same holds true for the clathrate 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 and β-CD having an average molecular weight of 1000. In FIG. 2, the abscissa θ represents the diffraction angle, and the ordinate CPS represents the reflection intensity (count 7 seconds). From Figure 2, this clathrate is crystalline, and its powder X-ray diffraction pattern is similar to the powder X-ray diffraction pattern of an inclusion complex of β-CD and a low molecular weight compound such as propionic acid or propatool. This shows that the clathrate compound of polypropylene glycol and β-CD is not an isomorphic crystal with a so-called cylindrical structure.

In addition, in thermogravimetric analysis, β-CD melts and decomposes at temperatures lower than 310°C, while the average molecular weight is 100°C.
The inclusion compound of 0 polypropylene glycol and β-CD exhibited a decomposition temperature of over 320°C.

FIG. 3 is a diagram showing a powder X-ray diffraction pattern of an inclusion compound of polypropylene glycol and γ-CD having an average molecular weight of 1000. The abscissa and ordinate in FIG. 3 are the same as in FIG. From FIG. 3, it can be seen 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 explained in detail.
A sugar solution containing polypropylene glycol and a cyclodextrin is brought into contact and mixed to form a precipitate of an insoluble clathrate of β-CD and/or γ-CD and polypropylene glycol.1. This precipitate is separated into solid and liquid from the mixture, polypropylene glycol is released by the operation described below, and β-CD and/or γ-C
This is a method of separating D.

The mixing ratio of the above-mentioned sugar solution and polypropylene glycol should normally be 2 moles or more in terms of propylene glycol units/ ) per 1 mole of β-CD and/or γ-CD, and polypropylene glycol is not necessary. It is not preferable to use a larger amount than the above because it increases the cost.

Solid-liquid separation can be performed by common separation means such as filtration, centrifugation, and membrane separation using an ultrafiltration membrane or the like.

The rate at which β-CD and/or γ-CD forms clathrates with polypropylene glycol depends on the molecular weight (and therefore the average molecular weight) of the polypropylene glycol. That is, when the average molecular weight of polypropylene glycol is small, the inclusion reaction is generally fast, and a few minutes is sufficient for the reaction time. As the average molecular weight of polypropylene glycol increases, the reaction rate slows down; however, for example, if the polypropylene glycol is subjected to ultrasonic absorption for 10 minutes and then left at about 25°C for about 1 hour, the clathration reaction is almost completed. , the clathrate will precipitate. Furthermore, when an ultrasonic stirrer or polypropylene glycol is added to a sugar solution, it may separate into two layers, so it is important to make the dispersion uniform.

As can be seen from Table 2, the average molecular weight of the polypropylene glycol used in the separation and purification method of β-CD and/or γ-CD of the present invention is preferably about 425 to about 4000 for β-CD. Approximately 725 to approximately 2000
is more preferred, while for γ-CD, about 400 to about 4000 is preferred, and about 400 to about 1500
is even more preferable.

When it is desired to separate β-CD and γ-CD from a mixture (sugar solution) containing both, polypropylene glycol having an average molecular weight of about 425 or less, preferably around 400 is added to the mixture, so that substantially no γ-CD is present. or γ-C
A D-rich clathrate is produced, the clathrate is subjected to solid-liquid separation, and then polypropylene glycol is removed from the clathrate. If the substance obtained in this way is substantially pure γ-CD, the separation and purification of γ-CD will be completed in the above procedure, but if the substance also contains β-CD, Substantially pure γ-CD can be separated by repeating the above procedure as many times as necessary for the substance. However, industrially, there may be cases where it is acceptable even if β-CD is contained to a certain extent, so the purity of γ-CD may be adjusted to a necessary level depending on the intended use.

The separation of β-CD in the above case involves adding β-CD and the clathrate compound to the remaining mixture (sugar solution) from which γ-CD substantially alone or the γ-CD-rich clathrate compound has been separated. Polypropylene glycol with an average molecular weight that is easy to produce is selected and added to produce a clathrate containing substantially only β-CD or β-CD rich, and this clathrate is subjected to solid-liquid separation, and then the polypropylene glycol is added. Removed from clathrates. If the remaining mixture (sugar solution) still contains γ-CD, repeat the γ-CD separation procedure described above to obtain a remaining mixture (sugar solution) that substantially no longer contains γ-CD.
You can use . However, industrially, there may be cases where there is no problem even if γ-CD is included, so β
- The purity of the CD may be adjusted to a necessary level depending on the intended use.

Next, the precipitate of the clathrate separated and washed from the reaction mixture will be explained as follows: After drying, when an appropriate organic solvent is added and the mixture is stirred, polybutropylene glycol is liberated from the clathrate compound and dissolved into the solvent, while β-CD and/or γ-CD remains as a precipitate. remain. When the solvent containing polypropylene glycol is removed from this suspension by general solid-liquid separation means such as filtration, centrifugation, and membrane separation using an ultrafiltration membrane, α-CD crystals are obtained. . Specific examples of the above-mentioned organic solvents include lower alcohols such as methanol, ethanol and propatool, and halogenated hydrocarbons such as dichloromethane. Alternatively, when the precipitate of the clathrate compound is suspended in water and heated to a temperature of 80°C or higher, the clathrate force weakens and polypropylene glycol is liberated, and the polypropylene glycol and β-CD and/or γ- Since both CDs are soluble in water, they can be separated by subjecting the resulting aqueous solution to, for example, column chromatography at a relatively high temperature or by hot filtration after concentration. Yet another method is to suspend the clathrate precipitate in water and add benzoic acid, phenol, or other substances that have a greater clathration force with β-CD and/or γ-CD than polypropylene glycol. The polypropylene glycol is replaced and included in β-CD and/or γ-CD, and the polypropylene glycol is liberated. Because polypropylene glycol and new clathrate compounds are often soluble in water.

In that case, the resulting aqueous solution can be subjected to column chromatography, for example, or filtered after concentration to separate the two. If this new clathrate compound has a useful use, it will be possible to further develop β-CD and/or γ
-There is no need to separate and purify CD, which is reasonable.

[Examples] Hereinafter, the present invention will be specifically explained with reference to Examples, but it goes without saying that the present invention is not limited to the Examples.

Example 1 10% by weight CD mixture (composition is 30% by weight α-CD)
An aqueous solution containing 15% by weight of polyprolene gellicol (average molecular weight: 400) was added to 100ml of an aqueous solution containing 15% by weight of β-CD, 5% by weight of γ-CD, 50% by weight of dextrin, and subjected to ultrasonication for 10 minutes at room temperature. Mix, 1
After standing for a period of time, an inclusion compound of polypropylene glycol and γ-CD was precipitated. The resulting suspension has a pore size of 0.4
The mixture was filtered using a filter using 5 gm filter paper to separate the precipitate of the clathrate compound, 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 in the above filtration operation, mixed with Zhao sonic stirring for 10 minutes at room temperature, and left to stand for 1 hour. β-C with
The D. rich clathrate was precipitated. The resulting suspension was filtered in the same manner as above to separate the precipitate, and then filtered at 200 m
It was washed with 1 liter of pure water.

Transfer the two precipitates thus obtained to separate beakers, add 100 ml of 99.5% ethanol to each, and stir. Substantially only the polypropylene glycol dissolves in the ethanol and is liberated. Substantially pure γ-C is obtained by filtering each of the obtained suspensions and washing the residue with 99.5% ethanol.
It was possible to obtain β-CD with considerably high purity.

[Effect] The β-CD and/or γ-CD clathrate of the present invention has the following properties:
It can be used as an intermediate for preparing pure β-CD and/or γ-CD, and can be used by combining a mixture of various cyclodextrins containing β-CD and/or γ-CD with polypropylene glycol in the presence of water. It can be easily prepared as a precipitate by mixing with

According to the method for separating and purifying β-CD and/or γ-CD of the present invention, after preparing the above-mentioned β-CD and/or γ-CD clathrate compound, polypropylene glycol can be easily extracted from the clathrate compound. High purity β-
CD and/or γ-CD can be manufactured at low cost.

[Brief explanation of drawings]

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

Claims (6)

[Claims] (1) A β- and/or γ-cyclodextrin inclusion compound in which a polypropylene glycol molecule is 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 produce an insoluble β- and/or γ-cyclodextrin inclusion compound. . A method for separating and purifying β- and/or γ-cyclodextrin, the method comprising: then removing the polypropylene glycol from the β- and/or γ-cyclodextrin clathrate compound. (3) The separation and purification method according to claim 2, wherein the 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 polypropylene glycol has an average molecular weight of about 425 or less, and the clathrate compound produced is substantially solely of γ-cyclodextrin or The separation and purification method according to claim 2 or 3, characterized in that the clathrate is a γ-cyclodextrin-rich clathrate compound. (5) The mixture of various cyclodextrins is obtained in claim 4, except for the γ-cyclodextrin substantially alone or the γ-cyclodextrin-rich clathrate compound described in claim 4. It is a mixture of various residual cyclodextrins, and the average molecular weight of the polypropylene glycol is about 425 to about 4000.
The separation and purification method according to claim 2, wherein the clathrate compound produced is substantially solely β-cyclodextrin or a β-cyclodextrin-rich clathrate compound. (6) A patent characterized in that the polypropylene glycol is removed from the β- and/or γ-cyclodextrin clathrate compound by adding an organic solvent to the β- and/or γ-cyclodextrin clathrate compound. A separation and purification method according to any one of claims 2 to 5.