JP2009269826A - Method for producing pseudorotaxane - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a pseudorotaxane in which one molecule of a raw material is clathrated by the several molecules of a cyclodextrin (CyD), by using the CyD with a compound having an isoprenoid structure which is a raw material of an antioxidant used in health food applications. <P>SOLUTION: This method for producing the pseudorotaxane is provided by mixing the compound having the isoprenoid structure in an aqueous solution added with the CyD of an amount of not less than its saturation solubility, and agitating by churning to clathrate the compound having the isoprenoid structure by the CyD. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a pseudo-rotaxane in which a compound having an isoprenoid structure is included in a cyclodextrin (hereinafter referred to as CyD).

Squalene, tocotrienol, coenzyme Q10 (hereinafter referred to as CoQ10, which is simply referred to as CoQ10 or coenzyme Q10 indicates an oxidized form) and the like, which are known as raw materials having antioxidant activity in the health food field. There are problems such as deterioration due to light and ultraviolet rays, and poor handling such as handling of raw materials.
Patent Document 1 (Japanese Patent Laid-Open No. 2005-2005) discloses that at least one of CoQ10 and vitamins is a stabilized CoQ10-containing composition that is included in CyD.

Patent Document 2 (Japanese Patent Application Laid-Open No. 2006-249050) discloses a stabilized complex by including farnesol, menaquinone, tocopherol, and coenzyme Q10 in γ-cyclodextrin. It is stated that it applies to pharmaceutical products. Patent Document 2 also describes that only β-CyD is described in Examples in Patent Document 1 as a conventional example of the background art, and high storage stability was not obtained.
In both Patent Documents 1 and 2, a kneading method is used as a manufacturing method.

  Patent Document 3 (Japanese Patent Publication No. 2003-520827) describes a method for producing CoQ10 and a γ-CyD complex. As an inclusion method, a method using collision by homogenization, melting by heat, or a method of adding CoQ10 to a concentrated γ-CyD solution and homogenizing is shown. However, since stirring is performed at a considerably high speed, there is a concern about deterioration due to the generation of heat in raw materials that are vulnerable to heat.

Patent Document 4 (2003-238402) describes a method of forming a complex by strongly stirring or kneading using α-tocopherol and concentrated aqueous β or γ-CyD. , Reaction time takes more than 1 hour.
In the production method of the present invention, it has been confirmed that a complex is formed with tocopherol and the similar compound tocotrienol in a short reaction time of about 5 minutes.

Japanese Patent Laid-Open No. 2005-2005 JP 2006-249050 A Japanese translation of PCT publication No. 2003-520827 JP 2003-238402 A

  The present invention uses cyclodextrin (CyD) for compounds having an isoprenoid structure, such as squalene, tocotrienols, and reduced CoQ10, which are antioxidant raw materials used in health food applications, so that there are several It is an object of the present invention to provide a method for producing a pseudo-rotaxane which is included by molecular CyD.

  The present invention, by contacting a compound having an isoprenoid structure such as squalene, tocotrienols, and reduced CoQ10, which are antioxidant raw materials used in health food applications, with a solubility method at a molecular level, This is a method for producing a pseudo-rotaxane powder having improved handling properties relating to improvement of physical properties such as stability of the raw material itself and handling of the raw material. Furthermore, pseudo rotaxane powder having excellent properties can be produced by adding steps such as solvent removal, centrifugation, and drying.

(1) A pseudorotaxane is produced by mixing a compound having an isoprenoid structure with an aqueous solution to which cyclodextrin having a saturation solubility or higher is added and stirring the mixture so that the compound having the isoprenoid structure is included in the cyclodextrin. Method.
(2) As the amount of cyclodextrin in excess of the saturation solubility, the molar ratio of cyclodextrin is higher than the molar ratio of forming pseudorotaxane to the molar ratio of the compound having an isoprenoid structure that forms pseudorotaxane and cyclodextrin. The method for producing a pseudo-rotaxane according to (1), wherein
(3) The method for producing a pseudo-rotaxane according to (1) or (2), wherein ultrasonic treatment is performed before or in the initial stage of the shake stirring process as the stirring process.
(4) After shaking and stirring, the precipitate is centrifuged, the supernatant is removed, and dried under reduced pressure to obtain a pseudorotaxane dry powder. (1) to (3) A method for producing a pseudo-rotaxane.
(5) A method for producing a pseudo-rotaxane, wherein the pseudo-rotaxane according to any one of (1) to (4) is a material for ingestion.

Powders that form pseudorotaxanes with improved stability, storage, and handling properties by inclusion of compounds with isoprenoid structures such as squalene, tocotrienols, and reduced CoQ10 in cyclodextrin (CyD) using the solubility method Can be manufactured.
It was possible to provide a method capable of efficiently producing a pseudo-rotaxane powder containing an isoprenoid compound and cyclodextrin in a short time.
According to the present invention, a pseudo-rotaxane powder capable of reducing deterioration of a compound having an isoprenoid structure due to factors such as heat, light, ultraviolet light, and air oxidation can be produced.
In the case of a liquid raw material such as squalene, it can be produced into a dosage form that can improve handling properties such as raw material handling by pulverization. In particular, since squalene is unstable to heat and light, a refrigerator or a cool and dark place is recommended as a storage method. However, a simple storage method can be taken by preparing the product of the present invention. The product of the present invention can also be applied to a storage container, not a brown bottle but a transparent container. In addition, it can be carried out at room temperature without special consideration of the operating conditions.
In the present invention, when blended with other materials, for health foods and the like that often contain multiple raw materials containing isoprenoid compounds at the time of commercialization, the quality degradation due to a decrease in the content of isoprenoid compounds is suppressed. An effect with excellent stability can be expected. Applicable dosage forms of the obtained composite include powders, granules, and tablets used for pharmaceuticals and health foods.

Hereinafter, the present invention will be described in detail.
The “pseudorotaxane” as used in the present invention refers to a supramolecular complex that becomes a precipitate derived from a “pseudorotaxane” or a complex similar to “pseudorotaxane”.
The “pseudo-rotaxane” of the present invention is a structure in which the opening (rotator) of a cyclic molecule is clasped by a linear molecule (axis) and is stable. It can be a processed raw material or processed powder with improved properties. It can also be called a supramolecular complex. Rotaxane is a structure in which bulky sites are formed at both ends of the shaft, which is a linear molecule that penetrates, and the ring of the cyclic molecule cannot be removed from the shaft due to steric hindrance. The pseudo-rotaxane was considered to be easy to use by separating the guest compound as a shaft from the host cyclic compound, and the present invention was studied. Further, in order to form rotaxane, a reaction reagent that cannot be used in the food and health food fields must be used, and therefore, it is not suitable for the present invention.

<CyD>
CyD used in the present invention is a kind of cyclic oligosaccharide having a cyclic structure in which several molecules of glucose are linked by α (1 → 4) glucoside bond (see FIG. 1). Different α (6), β (7), and γ (8) types can be used depending on the number of glucose. In particular, β and γ forms are suitable for the present invention.

  CyD is known to have a hydrophobic cavity in the molecule and to form an inclusion complex by incorporating various guest molecules depending on the cavity diameter. The supramolecular inclusion properties of CyD include solubilization and stability improvement of poorly water-soluble drugs, controlled release, improved bioavailability, reduced bitterness / odor and local irritation, oily or low melting point powder It is used in many fields such as food, cosmetics, and pharmaceuticals, such as bodyization and prevention of volatility.

<Isoprenoid compound>
In the present invention, a compound having an isoprenoid structure (hereinafter sometimes referred to as “isoprenoid compound”) such as reduced CoQ10, squalene, and tocotrienol is used as a guest compound.
CoQ10 used in the present invention is a reduced type and is a crystalline powder exhibiting white to pale yellow. Reduced CoQ10 changes to an oxidized form when left in the air, so care must be taken for maintenance. Squalene used in the present invention is a viscous, colorless and transparent liquid. Further, the tocotrienol used in the present invention is a viscous liquid exhibiting yellow to reddish brown containing 65% or more of total tocotrienol and 92% or more as total tocopherols.

Reduced CoQ10
Squalene
Tocotrienol

<Production method>
A solubility method is used as a method of including the isoprenoid compound raw material used in the present invention in CyD. The solubility method refers to the inclusion body by preparing a solution of CyD, adding a certain amount of a guest compound, that is, an isoprenoid compound raw material, and mixing by stirring to obtain a precipitate, followed by drying after removing the liquid components. Is a method of isolating. When the CyD solution is a saturated solution, it can also be called a saturated solution method or a saturated aqueous solution method. Further, when using the solubility method, by applying ultrasonic waves for a short time immediately after the isoprenoid compound raw material is added to the saturated solution, the dispersibility can be improved and the reaction can be facilitated.
The solubility method has disadvantages such as using a relatively large amount of water and taking steps that require the precipitate to be isolated from the aqueous solution, but the isolated precipitate uses a large amount of water. Thus, using water as a reaction medium, there is an advantage that a drug and CyD can easily interact with each other, and a powder having a good complex forming property can be obtained in a short time. It is also effective for compounds that do not generate heat and are sensitive to heat. In addition, compared with the kneading method, this production method hardly prevents the drug from being oxidized during preparation because the drug hardly comes into contact with air (oxygen). In the case of the compound used in the present invention, it is considered that it can contribute to the suppression of the decomposition of the isoprenoid chain.
In the present invention, the stability can be further improved by having a plurality of cyclodextrins involved in the inclusion.
Further, in the present invention, by using the solubility method, it was possible to obtain a powder having a high pseudorotaxane formation property as compared with the kneading method employed in Patent Document 1 or 2.
The kneading method is a method of obtaining a composite by adding a small amount of water to cyclodextrin and a certain amount of guest compound, forming a paste using a stirrer such as a mixer, and removing moisture by drying or the like. Since this kneading method uses a small amount of water as a reaction medium, it prevents the disadvantages caused by hydrolysis reaction for drugs that are unstable to water as much as possible. This is a production method in which an inclusion complex is formed by an action such as shearing force. However, heat is generated by stirring, and the amount of water that is easily decomposed by this heat and the amount of water used are small. However, there are also many disadvantages that the yield of the complex is poor, or the complex is difficult to form. Moreover, since it is a paste when collect | recovering from a mixer, handling property is also bad.

When the product of the present invention is prepared using the solubility method, the use ratio of the isoprenoid compound raw material and CyD is set such that the molar ratio of CyD is higher than the molar ratio of forming pseudorotaxane (increasing the amount of CyD). This makes it possible to easily react with the isoprenoid compound raw material, and to keep the amount of CyD dissolved in the solution even during the reaction, thereby efficiently maintaining the pseudorotaxane formation reaction.
In the solubility method of the present invention, the formation of pseudorotaxane was observed even when the stirring (shaking) time was as short as 5 minutes, and the pseudorotaxane was formed in an extremely short time than the generally performed shaking time of about 5 days. It was confirmed that it could be formed.

  As a post-treatment of the precipitate obtained by the reaction, it is preferable to obtain a dry powder by drying under reduced pressure. The freeze-drying method may break the pseudorotaxane structure.

<Pseudo-rotaxane confirmation means>
Whether or not the obtained complex is a pseudorotaxane can be confirmed using powder X-ray diffraction or NMR spectrum. Strictly speaking, it is necessary to prove it by a single crystal X-ray structural analysis or an atomic force microscope in order to conclude that it is a pseudorotaxane. Moreover, pseudo rotaxane is highly likely to be obtained as a precipitate due to its properties, and therefore can be predicted to some extent by visual observation.
The presence or absence of pseudorotaxane structure formation is confirmed by comparing a diffraction peak indicating a pseudorotaxane-like supramolecular complex with another known pseudorotaxane by powder X-ray diffraction. The NMR spectrum is used by the complex formed to calculate the number of CyD inclusions per guest compound molecule. That is, to confirm the composition and stoichiometry of the pseudorotaxane. However, for the isoprenoid compound raw material that is a single compound such as squalene or reduced CoQ10, the method for calculating the number of inclusions of CyD by NMR spectrum is effective, but a raw material such as a mixture of tocotrienols and tocopherols was used. In some cases, the method of calculating from the quantitative results by high performance liquid chromatography (HPLC) is effective.

<Uses of pseudorotaxane powder>
The product of the present invention can be used as one of the formulation formulation ingredients in a dosage form such as a capsule, a tablet, a powder, a liquid, an ointment, or a cream depending on the intended use. In particular, it is suitable for oral intake, percutaneous absorption, and external preparation for skin from the viewpoint of safety.
Since the product of the present invention can obtain a pseudo-rotaxane powder having a relatively higher purity than that of the kneading method, the powder has superior temporal stability compared to the kneaded body, and the powder is not encapsulated or pseudo-rotaxane. It is considered that the partial deterioration of the drug is unlikely to occur in the parts that are not. As a result, long-term storage stability can be ensured (long-term setting of the shelf life of quality), and drug deterioration can be suppressed by combining with other raw materials (simplification of drug formulation).
In addition, the powder having high pseudorotaxane formation can be an important factor in designing a pharmaceutical formulation such as controlled release of a drug from pseudorotaxane.

  In particular, when used as one of the formulation of tablets, it is suitable for the direct powder compression method (direct compression method) as a method for producing tablets. The direct hitting method is an industrially inexpensive manufacturing method in terms of the manufacturing process, compared with the wet granule compression method (ordinary so-called wet method).

1. Samples and solvents (1) Isoprenoid compounds Reduced CoQ10 (manufactured by Kaneka Co., Ltd.), squalene (manufactured by Kishimoto Special Liver Oil Industry Co., Ltd.), Tocotrienol (manufactured by Tocotrienol) Eisai Food Chemical Co., Ltd. Tocolit 92) was used.
(2) CyD (cyclodextrin)
β-CyD and γ-CyD (Nihon Shokuhin Kako Co., Ltd.) were used.
(3) Solvent H ₂ O as a solvent was obtained by distilling ion-exchange purified water twice. As other reagents and solvents, commercially available special grade products were used.

2. Preparation of isoprenoid compound / CyDs complex
2-1. Preparation of reduced CoQ10 / γ-CyD kneaded body (kneading method)
The experimental operation was performed under light shielding. Kneaded in an agate mortar while adding water to reduced CoQ10 and γ-CyD (molar ratio 1: 3 to 1: 5), and dried under reduced pressure for several hours to overnight to obtain a kneaded body. The kneading time was 5 minutes and 1 hour, respectively, and the amount of H ₂ O added was set to 4.2 ml for 1: 3, 5.6 ml for 1: 4, and 7 ml for 1: 5. The compounding amount of the sample is shown below.
[Reduced CoQ10: γ-CD = 1: 3] Reduced CoQ10 powder 863.34 mg, γ-CD powder 3.891 g
[Reduced CoQ10: γ-CD = 1: 4] Reduced CoQ10 powder 863.34 mg, γ-CD powder 5.188 g
[Reduced CoQ10: γ-CD = 1: 5] Reduced CoQ10 powder 863.34 mg, γ-CD powder 6.485 g

2-2. Preparation Method of Reduced CoQ10 / γ-CyD Solubility Method Preparation Product 232 mg / mL γ-CyD aqueous solution 10 mL was added to 221 mg of reduced CoQ10 powder and stirred. Ultrasonic waves were applied for several tens of seconds to several minutes, and shaken (100 rpm) using a shaker for a fixed time. The reaction solution was centrifuged at 7000 rpm for 10 minutes, the supernatant was removed, and unreacted γ-CyD was removed. Furthermore, in order to remove unreacted reduced CoQ 10, the product of the present invention was obtained by washing with diethyl ether and drying under reduced pressure for several hours to overnight. The shaking was performed under the setting conditions of 5 minutes and 1 hour, respectively.

2-3. Preparation Method of Squalene / γ-CyD and Squalene / β-CyD Solubility Method Preparation The experimental operation was performed under nitrogen substitution and in the dark. 5.8 mL of 232 mg / mL γ-CyD aqueous solution was added to 85.8 mg of squalene, or 32 mL of 18.5 mg / mL β-CyD aqueous solution was added to 42.9 mg of squalene, and vigorously stirred. Ultrasonic waves were applied for several tens of seconds, and the mixture was shaken (100 rpm) for a certain period of time using a shaker. The reaction solution was centrifuged at 7000 rpm for 10 minutes, the supernatant was removed, and unreacted β-CyD or γ-CyD was removed. The obtained precipitate was dried under reduced pressure for several hours to overnight to obtain the product of the present invention. In addition, each shaking was implemented on the setting conditions for 5 minutes and 1 day.

2-4. Preparation of tocotrienol / β-CyD kneaded body (kneading method)
The experimental operation was performed under light shielding. Tocotrienol and β-CyD (molar ratio of about 1: 2 to 1: 3) were kneaded in an agate mortar while adding H ₂ O and dried overnight under reduced pressure to obtain a kneaded body. The kneading time is 5 minutes each, and the amount of H ₂ O added is 3.3 ml for tocotrienol / β-CyD (molar ratio about 1: 2), and for tocotrienol / β-CyD (molar ratio about 1: 3). The test was carried out under 5 ml setting conditions. The compounding amount of the sample is shown below.
[Tocotrienol / β-CyD (molar ratio about 1: 2)] Tocotrienol 854.00 mg, β-CyD powder 4.540 g
[Tocotrienol / β-CyD (molar ratio about 1: 3)] Tocotrienol 854.00 mg, β-CyD powder 6.810 g

2-5. Preparation of Tocotrienol / γ-CyD and Tocotrienol / β-CyD Solubility Method Preparations The experimental operation was performed under nitrogen substitution and in the dark. Tocotrienol (98 mg) was added with 232 mg / mL γ-CyD aqueous solution (5.1 mL), or tocotrienol (24.5 mg) was added with 18.5 mg / mL β-CyD aqueous solution (12.8 mL) and stirred vigorously. The ultrasonic wave was applied for several tens of seconds and shaken for a certain time (100 rpm). The reaction solution was centrifuged at 7000 rpm for 10 minutes, the supernatant was removed, and unreacted β-CyD or γ-CyD was removed. The product of the present invention was obtained by drying under reduced pressure for several hours to overnight. In addition, each shaking was implemented on the setting conditions for 5 minutes.

3. Macroscopic observation of precipitate formation Generally, pseudo-rotaxanes of CyD form hydrogen bonds between adjacent CyDs, making them unable to form hydrogen bonds with water molecules and becoming poorly water-soluble. In this experiment, reduced CoQ10, squalene and tocotrienol were added to various CyD saturated aqueous solutions prepared in advance, and the solutions prepared according to the preparation conditions of the solubility method were observed with the naked eye and photographed. Incidentally, it was also observed in the state added only in H ₂ O as compared solution and photographed.
FIG. 2 shows the result of visual observation of the solution at the end of preparation of the isoprenoid compound / CyD supramolecular complex.

  (1) From FIG. 2, in the system in which only each compound raw material was added to water, a state in which the affinity with water was not obtained and separation was observed. However, in the solubilized preparation with CyD added, a white to white yellow precipitate was obtained, and by shaking, it was observed that it was uniformly dispersed in the solution in a short time. Therefore, it was suggested that supramolecular complexes similar to pseudorotaxane were formed in reduced CoQ10 in γ-CyD system and in squalene and tocotrienol in β- and γ-CyD systems.

  (2) Squalene and Tocotrienol alone separated into oil in water. On the other hand, white precipitates were confirmed in the system to which β- and γ-CyD aqueous solutions were added. Since squalene and tocotrienol are usually liquids, this allows storage and processing in powder form.

  (3) These results suggest that reduced CoQ10 may form supramolecular complexes with γ-CyD and squalene and tocotrienol with β- or γ-CyD. As to which CyD and pseudorotaxane-like supramolecular complex form with each isoprenoid compound, it is necessary to confirm in addition to visual observation, but the position of the double bond of the isoprenoid side chain and the spacing of the methyl groups are different. Since the isoprenoid compounds differ from each other, it is expected that the flexibility and thickness of the molecules are related to the possibility of formation of a pseudorotaxane-like supramolecular complex. it can.

4. Structure of isoprenoid compound / CyDs complex 4-1. Powder X-ray diffractometer When powder sample is used by powder X-ray diffractometry, powder X with diffraction angle on the horizontal axis and diffraction intensity on the vertical axis A line diffractogram can be created. Powder X-ray diffraction is used for sample identification, evaluation of crystallinity, and measurement of crystallinity etc. This time it is mainly used for sample identification.
Powder X-ray diffraction was measured by using a RINT 2500 VL type automatic X-ray diffractometer manufactured by Rigaku Denki Co., Ltd., and fixing the sample to a glass cell. The measurement conditions are as follows. Cu-Ka line (1.542 A); tube voltage: 40 kV; tube current: 40 mA; scan speed: 1 ° / min; diffraction angle: 5 ° to 35 °; slit: 1 ° -1 ° -0.15 mm
In general, the structure of a CyD composite crystal is classified into three types: cage type, layered type, and cylindrical type. Of these, the crystal structure of CyD pseudorotaxane is known to form a cylindrical structure. In order to confirm the solid-state interaction between CyD and the isoprenoid compound in the supramolecular complex, powder X-ray diffraction measurement was performed.

4-2. ¹ H-NMR spectrum In order to confirm the composition and stoichiometry of the solubilized preparation, ¹ H-NMR spectrum was measured.
Reduced CoQ10 / γ-CyD solubility preparation, squalene / β-CyD solubility preparation, and squalene / γ-CyD solubility preparation were dissolved in d ₆ -DMSO, respectively, and JMN-A manufactured by JEOL Ltd. Measurements were made using a 500 nuclear magnetic resonance apparatus (external magnetic field: 500 MHz).

4-3. Continuous change method A fixed amount of isoprenoid compounds (tocotrienol, squalene) and γ-CyD aqueous solution (0, 58, 116, 174, 232 mg / mL) or β-CyD aqueous solution (0, 4.625, 9.25) , 13.875, 18.5 mg / mL), and the same operation as in the preparation of the above-described isoprenoid compound / CyD supramolecular complex was performed. The shaking time was all 1 day. The yield was calculated from the weight of the obtained precipitate.

5. 5. Analysis result of structure of isoprenoid compound / CyDs complex 5-1. Powder X-ray diffraction results From powder X-ray diffraction patterns of solid samples of reduced CoQ10 / γ-CyD complex, squalene / β- and γ-CyD complex, tocotrienol / β- and γ-CyD complex, β- And γ-CyD included the isoprenoid side chain of these compounds, suggesting that the CyD supramolecular complex forms a cylindrical structure. As the sample, the sample obtained by the above-mentioned 2 was used as the sample obtained by the solubility method and the sample obtained by the kneading method.

(1) Reduced CoQ10 / γ-CyD solubility method preparation
In order to confirm the interaction in the solid state between reduced CoQ10 and γ-CyD in the powder of the present invention, powder X-ray diffraction measurement was performed. As comparative materials, the following shows diffraction patterns of reduced CoQ10 alone, γ-CyD alone, reduced CoQ10 / γ-CyD kneaded body, and PPG / γ-CyD composite in which formation of pseudorotaxane is confirmed. FIG. 3 is a comparison diagram using a preparation for 1 hour of shaking, and FIG. 4 is a comparative diagram using a preparation for 5 minutes of shaking.

From the powder X-ray diffraction pattern results (shaking time: 1 hour) in FIG. 3, reduced CoQ10 alone has characteristic peaks around 19.0 ° and 23.0 °, and γ-CyD alone has 12.4 °, 18.8 ° and It can be seen that it has a characteristic peak around 20.5 °. In addition, PPG / γ-CyD has characteristic peaks in the vicinity of 7.4 °, 14.1 °, 14.9 °, 15.8 °, 16.6 °, and 27.0 °. The kneaded body (molar ratio 1: 5) has peaks around 12 ° and 19 ° derived from reduced CoQ10 and γ-CyD, and peaks around 14 ° like PPG / γ-CyD composites. Is not observed, it is considered that reduced CoQ10 and γ-CyD are simply physically mixed, and the complex-forming property is low. Next, in the kneaded body (molar ratio 1: 4, 1: 3), a peak around 23 ° seen in the reduced form Q10 was observed, but the peak around 19 ° disappeared, and PPG / γ-CyD composite Since the peaks around 7 ° and 26 ° seen in the body were also observed at the same time, there was also a physical mixture of reduced CoQ10 and γ-CyD, but a complex was formed than in the case of 1: 5 Was suggested.
In the solubilized preparation, characteristic peaks are observed around 7 ° and 14 °, as in PPG / γ-CyD, and the peak around 19 ° seen in reduced CoQ10 is not observed. It is considered that a pseudorotaxane having high formability, that is, high purity, has been obtained. The molar ratio of the reduced CoQ10 / gγ-CyD complex of the solubility preparation product was 1: 3.6 from ¹ H-NMR spectrum measurement, so that the complex was formed predominantly over the kneaded body. I can say that.
Polypropylene glycol (PPG), which has a structure similar to isoprenoid chain, is known to form a supramolecular inclusion complex polypseudorotaxane with CyDs. can do.

From the powder X-ray diffraction pattern results (shaking time: 5 minutes) in FIG. 4, reduced CoQ10 alone has characteristic peaks around 19.0 ° and 23.0 °, and γ-CyD alone has 12.4 °, 18.8 ° and It can be seen that it has a characteristic peak around 20.5 °. PPG / γ-CyD has characteristic peaks around 7.4 °, 14.1 °, 14.9 °, 15.8 °, 16.6 °, and 27.0 °. The kneaded body (molar ratio 1: 5) has a peak around 23 ° as with reduced CoQ10 and 15 ° to 18 ° like γ-CyD. Conceivable. The kneaded body (molar ratios 1: 4, 1: 3) also has a peak near 19 ° as in the case of γ-CyD. Therefore, it is considered that the complex-forming property is low even at this molar ratio. This indicates that the powder prepared by the kneading method is in a state where the reduced CoQ10 and γ-CyD hardly interact and are simply physically mixed. On the other hand, in the solubilized preparation, a characteristic peak of PPG / γ-CyD complex is observed around 7 ° and 14 °, and the peak around 19 ° seen in reduced CoQ10 is not observed, Since the diffraction pattern is generally similar to the PPG / γ-CyD complex, it is considered that a high-purity pseudorotaxane having a high complex formation property, that is, a high purity is obtained.
From this result, it was recognized that the solubility method preparation formed a pseudo-rotaxane-like complex even in a short time of 5 minutes.

(2) Squalene / β-, γ-CyD Solubility Preparation Products In order to confirm the solid state interaction between squalene and β- and γ-CyD in the powder of the present invention, powder X-ray diffraction measurement was performed. . As comparative raw materials, diffraction patterns of β-CyD alone, γ-CyD alone, and PPG / β-CyD complex and PPG / γ-CyD complex confirmed to form pseudorotaxane are shown below. FIG. 5 is a comparison diagram of squalene / γ-CyD, and FIG. 6 is a comparative diagram of each of the squalene / β-CyD solubility method preparations (shaking time: 5 minutes, 1 day).

  From the powder X-ray diffraction pattern results of FIGS. 5 and 6, the squalene / γ-CyD solubility preparation showed a diffraction pattern characteristic of a cylindrical structure similar to the PPG / γ-CyD composite. It is considered that a pseudo-rotaxane-like complex is formed even in a short time of 5 minutes. Similarly, the squalene / β-CyD solubility preparation also showed a diffraction pattern characteristic of the cylindrical structure similar to that of the PPG / β-CyD composite. It is thought that the body is formed. In addition, there was no difference between the β-system and the γ-system between the shaking time of 5 minutes and 1 day.

(3) Tocotrienol / β-, γ-CyD Solubility Method Preparation To confirm the solid state interaction between tocotrienol and β- and γ-CyD in the powder of the present invention, powder X-ray diffraction measurement was performed. . As comparative materials, diffraction patterns of β-CyD alone, γ-CyD alone, tocotrienol / β-CyD kneaded body, and PPG / β-CyD complex and PPG / γ-CyD complex confirmed to form pseudorotaxane Is shown below (FIGS. 7 and 8). The product of the present invention and the kneaded body are each a powder prepared by shaking or kneading for 5 minutes. FIG. 7 is a comparison diagram of tocotrienol / γ-CyD, and FIG. 8 is a comparison diagram of tocotrienol / β-CyD system.

    From the results of the powder X-ray diffraction pattern in Fig. 7, the tocotrienol / γ-CyD solubility method preparation showed a diffraction pattern characteristic of the cylindrical structure similar to that of the PPG / γ-CyD composite. The product is considered to form a supramolecular complex.

From the powder X-ray diffraction pattern results of FIG. 8, β-CyD alone has characteristic peaks around 10.7 °, 12.5 ° and 19.5 °, and PPG / β-CyD around 6.6 to 7.0 ° and around 11.7 °. It can be seen that it has a characteristic peak. In the kneaded body (molar ratio 1: 2, 1: 3), characteristic peaks around 10.7 °, 12.5 ° and 19.5 ° derived from β-CyD were observed. This indicates that the kneaded body contains a large amount of tocotrienol and unreacted β-CyD.
On the other hand, in the solubilized product, the characteristic peaks around 12.5 ° and 19.5 ° derived from β-CyD were not observed, but the characteristic peaks around 6.6 to 7.0 ° and 11.7 derived from PPG / β-CyD were observed. Remarkably recognized and the overall diffraction pattern is similar, so the solubility-processed product has a high formation of a pseudo-rotaxane-like complex, that is, the target complex with high purity is obtained. Conceivable. The molar ratio of the tocotrienol / β-CyD complex of the solubility method preparation was almost 1: 2 by HPLC measurement, so that the complex was formed over the kneaded body. I can say.

5.2 Quantitation by HPLC (High Performance Liquid Chromatography) “Tocolit 92” is a mixed solution of tocotrienol and tocopherol, containing 65% or more of internal tocotrienol and a mixture containing 92% or more of the total tocopherol. It is a solution. In the case of such a multi-component mixed solution, it is considered that quantitative measurement by HPLC is more appropriate than ¹ H-NMR spectrum measurement, and the tocotrienol / γ-CyD complex and tocotrienol / We decided to derive the approximate composition of the β-CyD complex. Tocotrienol and tocopherol have similar structural formulas, and their molecular weights are relatively close, so an accurate numerical value cannot be calculated, but an approximate numerical value can be calculated.

<Tocotrienol / β-CyD and γ-CyD solubility preparations HPLC analysis conditions>
Tocotrienol was measured according to the Tocotrienol Quantification Method in the 8th edition of the Food Additives Official Document.
The amount corresponding to about 0.025 g of the total tocotrienol of the test product was precisely measured, 5 mL of water and 10 mL of hexane were added, and the mixture was vigorously stirred for 5 minutes and centrifuged at 3,000 rpm for 5 minutes, and the hexane layer was recovered. The test solution was made exactly 100 mL with N-hexane. Separately, weigh accurately about 0.05 g each of d-α-tocopherol for quantification, d-β-tocopherol for quantification, d-γ-tocopherol for quantification, and d-δ-tocopherol for quantification, and put each in a brown volumetric flask. Was added to make exactly 100 mL to obtain a standard stock solution. A standard stock solution was accurately weighed and mixed so that the composition ratio of the tocotrienol homologue in the sample and the composition of the corresponding tocopherol homologue were almost the same, and used as a standard solution. 10 μL each of the test solution and the standard solution were weighed, and liquid chromatography was performed under the following operating conditions.

Column Shiseido Silica SG120 (5μm 4.6mmID × 250mm)
Column temperature 40 ° C
Detector Ultraviolet absorptiometer (UV295nm)
Mobile phase hexane: dioxane: 2 propanol 985: 10: 5
Flow rate 1mL / min

  As a result of the quantitative test by HPLC, the obtained product of the present invention was found to have about 2 molecules of CyD per one molecule of tocotrienol.

5-3 ¹ H- results of NMR spectra to confirm the composition and stoichiometry of the present invention product, was analyzed by ¹ H-NMR spectrum.
^{From the 1} H-NMR spectrum, it was found that the number of γ-CyD in the reduced CoQ10 / γ-CyD solubility preparation was about 3.6 per molecule of reduced CoQ10. In addition, the β-CyD number of the squalene / β-CyD solubility preparation was about 3 per squalene molecule, and the γ-CyD number of the squalene / γ-CyD supramolecular complex was about 2.7 per squalene molecule. Obtained.

(1) Reduced CoQ10 / γ-CyD Solubility Method Preparation FIG. 9 shows the ¹ H-NMR spectrum of the reduced CoQ10 / γ-CyD solubility method preparation.
FIG. 9 is a ¹ H-NMR spectrum after dissolving a reduced CoQ10 / γ-CyD solubility method preparation in DMSO. The γ-CyD 2,3-position proton was observed around 5.8 ppm, and the reduced side chain-CH ₂ proton peak of reduced CoQ10 was observed around 1.9-2.1 ppm. Regarding this spectral peak, the composition ratio was calculated from the integrated value of γ-CyD and reduced CoQ10, and as a result, the number of γ-CyD bound to the linear side chain of one molecule of reduced CoQ10 was about 3.6. Obtained.

(2) The ¹ H-NMR spectra of the squalene / CyD solubility method preparation squalene / γ-CyD and the squalene / β-CyD solubility method preparation are shown in FIGS. FIG. 10 shows squalene / γ-CyD, and FIG. 11 shows squalene / β-CyD.
The upper figure shows squalene / γ-CyD and squalene / β-CyD solubility preparations (shaking time: one day), ¹ H-NMR spectra after each was dissolved in DMSO. In this spectrum, the protons in the 2,3-position of γ-CyD around 5.7 ppm, the protons in the 2,3-position of β-CyD around 5.6 ppm, the proton of the vinyl group of squalene around 5.1 ppm, 1.99 to 2.03 A -CH ₂ proton peak of squalene was observed near ppm, and a -CH ₃ proton peak was observed near 1.6 ppm. With regard to this spectral peak, the composition ratio was calculated from the integrated values of β-, γ-CyD and squalene. As a result, the number of β-CyD bonded to the squalene molecule was 2.96 and the number of γ-CyD was 2.68. Results were obtained.

(3) Number of CyDs bound to one molecule As a result of the above, the number of CyDs bound to the isoprenoid chain of one molecule of reduced CoQ10 or the number of CyDs bound to one molecule of Squalene obtained from the integrated value of CyD and reduced CoQ10 or Squalene Indicates. The result showed that the number of γ-CyD bound to the isoprenoid chain of the reduced CoQ10 1 molecule was 3.6. The number of β-CyD bound to one squalene molecule was 2.96, and the number of γ-CyD was 2.68.

(4) Supramolecular complex structure “pseudo-rotaxane”
From these facts, it is expected to have a structure as shown in FIGS. This structure forms a structure in which the opening (rotator) of a cyclic molecule is included in a skewered manner by a linear molecule (axis). Applicable.

5-3. Result of pseudorotaxane formation by continuous change method
It was confirmed that the yields of Squalene / CyD supramolecular complex, Tocotrienol / CyD supramolecular complex, and reduced CoQ10 / CyD supramolecular complex increased linearly. From these results, it was suggested that the yield of the supramolecular complex increases in a concentration-dependent manner in any of reduced CoQ10, Squalene, and Tocotrienol. The Squalene / CyD supramolecular complex and Tocotrienol / CyD supramolecular complex are illustrated.

(1) Yield of Squalene / CyD supramolecular complex FIG. 12 shows the effect of the concentration of aqueous CyDs solution on the yield of Squalene / CyD supramolecular complex. In both β- and γ-CyD systems, the yield of supramolecular complexes increases linearly with increasing CyDs concentration, and is approximately 56%, 232 mg / mL γ in 154 mg / mL β-CyD aqueous solution. -CyD aqueous solution reached about 70%.

(3) Yield of Tocotrienol / CyD supramolecular complex
The effect of concentration on the yield of CyD supramolecular complex was investigated using Tocotrienol. (Figure 13) Similar to Squalene, in both β- and γ-CyD systems, the yield of supramolecular complex increases linearly with increasing CyDs concentration, and the 154 mg / mL β-CyD aqueous solution is about 43%, 232 mg / mL γ-CyD reached approximately 80%.

6). Results It was found from the above analytical test that pseudorotaxane powder can be advantageously produced by a production method using a solubility method.
By using the solubility method, reduced CoQ10, Squalene, and Tocotrienol were able to produce a powdered pseudorotaxane encapsulated in cyclodextrin as a guest compound.
When γ-CyD aqueous solution was added to reduced CoQ10 powder and shaken for 5 minutes, a precipitate of reduced CoQ10 / γ-CyD supramolecular complex similar to polypseudorotaxane was formed, and high complex formation was observed even after shaking for 1 hour. A supramolecular complex similar to that of a pseudorotaxane having a high purity was confirmed.
When β- and γ-CyD aqueous solutions were added to Squalene and shaken for 5 minutes or 1 day, a pseudorotaxane-like Squalene / β- and γ-CyD supramolecular complex precipitate was formed.
When β- and γ-CyD aqueous solutions were added to Tocotrienol and shaken for 5 minutes, a precipitate of a pseudocotaxane-like Tocotrienol / β- and γ-CyD supramolecular complex was formed.

Diagram showing cyclodextrin cyclic structure Macroscopic photograph of isoprenoid compound / CyD supramolecular complex preparation solution X-ray diffraction pattern (reduced CoQ10 / γ-CyD, 1 hour) X-ray diffraction pattern (reduced CoQ10 / γ-CyD, 5 minutes) X-ray diffraction pattern (squalene / γ-CyD) X-ray diffraction pattern (squalene / β-CyD) X-ray diffraction pattern (tocotrienol / γ-CyD) X-ray diffraction pattern (tocotrienol / β-CyD) ¹ H-NMR spectrum (reduced CoQ10 / γ-CyD) ¹ H-NMR spectrum (squalene / γ-CyD) ¹ H-NMR spectrum (squalene / β-CyD) Graph showing the yield of squalene / CyD supramolecular complex Graph showing the yield of tocotrienol / CyD supramolecular complex

Claims (5)

  A method for producing a pseudo-rotaxane by mixing a compound having an isoprenoid structure with a cyclodextrin by mixing the compound having an isoprenoid structure with an aqueous solution to which a cyclodextrin having a saturation solubility or higher is added and stirring the mixture.   As the amount of cyclodextrin in excess of the saturation solubility, the molar ratio of cyclodextrin should be higher than the molar ratio of forming pseudorotaxane to the molar ratio of the compound having an isoprenoid structure that forms pseudorotaxane and cyclodextrin. A process for producing a pseudo-rotaxane according to claim 1.   The method for producing a pseudo-rotaxane according to claim 1 or 2, wherein an ultrasonic treatment is performed as the stirring treatment before or in the initial stage of the shaking stirring treatment step.   The pseudorotaxane according to any one of claims 1 to 3, wherein the pseudorotaxane is produced by centrifuging the precipitate after shaking and stirring, removing the supernatant and drying under reduced pressure to obtain a pseudorotaxane dry powder. Method. The method for producing a pseudo-rotaxane, wherein the pseudo-rotaxane according to claim 1 is a material for ingestion.