JP2013112677A - Pyrroloquinoline quinone disodium crystal - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a crystal of very pure and stable PQQ (pyrroloquinoline quinone) sodium salt and a simple method for producing the same.SOLUTION: The pyrroloquinoline quinone disodium salt crystal including three or less molecules of water in a crystal lattice is produced by suspending a trisodium salt of PQQ in water, then adding water-soluble organic solvent to the suspension to reduce the solubility of the PQQ trisodium so that the pH falls in the range between 2 and 5. There is also provided a method of reproducing the crystal by placing broken crystals in an environment of 20-100% humidity for 15 minutes or longer.

Description

The present invention relates to a novel crystal of pyrroloquinoline quinone sodium salt and a method for producing the same.

The oxidized pyrroloquinoline quinone represented by the formula (1) is generally called PQQ, and has been attracting attention because it has been proposed as a new vitamin.

PQQ exists not only in bacteria but also in eukaryotic molds and yeasts, and plays an important role as a coenzyme. In addition, PQQ has been shown to promote cell proliferation, anti-cataract action, liver disease prevention and treatment action, wound healing action, anti-allergic action, reverse transcriptase inhibitory action and glyoxalase I inhibitory action-anticancer action, nerve fiber. Many physiological activities such as regenerative action have been clarified.

PQQ can be produced by an organic chemical synthesis method or a fermentation method. However, PQQ obtained by these methods has a high content of water and impurities, and a technique for obtaining a stable and highly pure PQQ crystal has been demanded. So far, PQQ sodium salt crystals showing single crystals in X-ray crystal structure analysis have been obtained by evaporating and concentrating the disodium salt of PQQ in a phosphate buffer (Non-patent Document 1). However, this method is not suitable for mass production and is not industrially and economically advantageous.
Further, the salting-out method is mixed with a solid in which a salt such as NaCl to be used in a large amount is deposited, so that an operation for removing the existing salt is necessary, and for ensuring stable quality for the salt. There was a drawback that analysis was difficult. In addition, there are many water molecules contained in the crystal lattice, and there are disadvantages in that it is suitable for the growth of microorganisms such as mold, causing deterioration in quality.

As a method for obtaining PQQ crystals that do not have such disadvantages, a method of recrystallization by adding a water-soluble organic solvent has been reported (Patent Document 1). However, there is no information on the crystal form in this document, and further, a cooling facility is required for precipitating the solid, and there is a disadvantage that the processing cost is high. Moreover, it is difficult to handle the crystal without destroying the structure, and when it breaks, it usually does not return.
Accordingly, there is a need for stable crystals and methods for regenerating them.

Japanese Patent Publication 7-113024

JACS, 111, 6682-6828 (1989)

An object of the present invention is to provide a crystal of a highly pure and stable PQQ sodium salt and a simple production method thereof.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have devised conditions for crystallization of PQQ sodium salt as described below, so that the sodium salt of PQQ that can solve the above-mentioned problems The present invention was completed by successfully obtaining new crystals with high purity.
[1] A disodium salt crystal of pyrroloquinoline quinone represented by the formula (1),

A pyrroloquinoline quinone disodium salt crystal comprising 3 molecules or less of water per molecule of pyrroloquinoline quinone disodium salt.
[2] The pyrroloquinoline quinone disodium salt crystal according to [1], which satisfies one or more of (a) to (d).
(A) It has the crystal structure shown in FIGS.
(B) It has a crystal structure defined by the coordinates in FIG. 3 in this specification.
(C) It has crystal lattice constants at a = 7.60, b = 10.09, c = 11.43 Å, α = 72.85, β = 88.01 °, and γ = 82.62 °.
(D) It has a crystal structure belonging to a crystal system space group such as P-1 (# 2).
[3] A method for regenerating a crystal by placing the crystal according to [1] or [2] or a broken crystal in an environment having a humidity of 20 to 100% for 15 minutes or more.
[4] A functional food comprising the pyrroloquinoline quinone disodium salt crystal according to [1] or [2].
[5] A medicament comprising the pyrroloquinoline quinone disodium salt crystal according to [1] or [2].

According to the present invention, it is possible to provide a crystal of a highly pure and stable PQQ sodium salt and a simple production method thereof.

The crystal structure obtained in Example 1. The crystal structure obtained in Example 1. Arrangement of atoms. Powder X-ray diffraction. DSC-XRD measurement results.

Crystal 1 of the present invention is a crystal of disodium trihydrate of oxidized pyrroloquinoline quinone (PQQ) represented by the formula (1).

The crystals of the present invention have the crystal structure shown in FIGS. 1 and 2; and / or have the crystal structure defined by the coordinates in Table 1 herein; and / or a = 7.60, b = 10.09 C = 11.43Å, α = 72.85, β = 88.01 °, γ = 82.62 °; and / or
It has a crystal structure belonging to a crystal space group such as P-1 (# 2).
This substance is identified by single crystal X-ray diffraction, but its presence can be confirmed by a general powder X-ray diffractometer equipped with a monochromator.

The water contained in this crystal is three molecules, but the crystal structure can be maintained even if one molecule is reduced. The crystal 2 reduced by one molecule can be confirmed by a general powder X-ray diffractometer, and the results are 9.1 °, 10.3 °, 13.8 °, 17.7 °, 18.3 °. There is a peak at 24.0 ° (± 0.2 °).
In the case of PQQ disodium salt obtained by the conventional ethanol recrystallization method, many diffraction peaks did not appear by measurement with a powder X-ray diffractometer, and it was considered that the solid was low in crystallinity.
The crystal of the present invention is crystallographically different from the PQQ disodium salt reported in Non-Patent Document 1, and exists at a distance at which quinone oxygen and carboxylic acid oxygen ion-bond with sodium. In addition, the surface spacing of the aromatic rings is small and high density. Further, as a feature of the crystal of the present invention, the hydrated water becomes three molecules in a high humidity state, and even if water is removed from the crystal lattice, it has an advantage that it can be restored to its stable quality.

As a method for producing the PQQ disodium salt crystal of the present invention, for example, the trisodium salt of PQQ as a raw material is suspended in water. At this time, it is preferable to add 10-80 g of trisodium salt of PQQ per liter of water. Next, a water-soluble organic solvent is added to the suspension so as to have a concentration of 10 to 90 (v / v)%, preferably 20 to 80 (v / v)%, thereby reducing the solubility of the trisodium salt of PQQ. . And the method of crystallizing by adjusting pH to the range of 2-5 is mentioned.

Alternatively, after suspending (partially dissolved) PQQ trisodium salt in a water-soluble organic solvent, the concentration of the water-soluble organic solvent is 10 to 90 (v / v)%, preferably 20 to 80 (v / v) After adding water to a concentration of%, it may be crystallized by adjusting the pH to be in the range of 2-5. At this time, it is preferable to add 0.5 to 800 g of trisodium salt of PQQ per liter of the total amount of the water-soluble organic solvent and water added.

Alternatively, the trisodium salt of PQQ is suspended (may be partially dissolved) in an aqueous medium containing 10 to 90 (v / v)%, preferably 20 to 80 (v / v)% of a water-soluble organic solvent. Then, the pH may be adjusted to a range of 2 to 5 for crystallization. At this time, it is preferable to add 0.5 to 800 g of trisodium salt of PQQ per liter of the aqueous medium.

The trisodium salt of PQQ used as a raw material is an organic chemical synthesis method (for example, JACS, Vol. 103, pages 5599-5600 (1981)) or a fermentation method (for example, JP-A No. 1-218597 and Japanese Patent No. 2692167). Etc.). The trisodium salt of PQQ used as a raw material may be crystalline or amorphous. Further, impurities may be included.

Specific examples of water-soluble organic solvents that can be used include methanol, ethanol, n-propanol, isopropanol, ethylene glycol, propylene glycol, methoxyethanol, diethylene glycol, methoxydiethylene glycol, glycerin, methoxypropanol, acetone, methyl ethyl ketone, and acetonitrile. , Ethyl lactate, methyl hydroxyisobutyrate and the like. Of these, alcohol is more preferable, and ethanol is particularly preferable.
A water-soluble organic solvent is used as a poor solvent to lower the solubility of PQQ trisodium salt, and the concentration thereof may be appropriately set within the above range according to the initial amount of PQQ trisodium salt.

In a general crystal production method, after dissolving a solid in a solvent, a poor solvent is added to precipitate the crystal. In the present invention, crystallization can be performed even in a suspended state where a solid is present. It is preferable that the operation can be performed in a suspended state because the volume of the apparatus to be used can be reduced and the discharged waste water can be reduced.

The temperature of both liquids when adding a water-soluble organic solvent to an aqueous solution and / or suspension of a trisodium salt of PQQ is not particularly limited as long as the liquid does not freeze. Therefore, −30 ° C. to 80 ° C. is preferable.
After adding the water-soluble organic solvent, the temperature of the solution or suspension when the acid is added to adjust the pH to 2 to 5 is not particularly limited as long as the liquid does not freeze. Since efficiency falls and the rate of crystallization falls if it makes it low temperature, 5 to 80 degreeC is preferable.

Examples of the pH adjustment method include addition of an acid or a buffer. When adding an acid, the type of acid used is not particularly limited, but inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid, nitric acid, sulfuric acid, formic acid, acetic acid, propionic acid, butyric acid, trichloro Examples thereof include organic acids such as acetic acid, methanesulfonic acid, and benzenesulfonic acid, among which hydrochloric acid is preferable.

What is necessary is just to stir as needed during or after adjustment of pH. Moreover, you may leave still. The stirring time can be, for example, 5 minutes to 7 days. The standing time can be, for example, 5 minutes to 15 days.
In the state where the pH is stable and constant at a value between pH 2 and 5, the crystals precipitated by filtration or centrifugation can be separated to obtain crystals.

Crystal 1 is a three-hydrated substance, but there is no problem even if the moisture content is different as long as it has the crystal structure of the present invention. The water content varies depending on the drying conditions. Although the crystal structure may be broken by reducing the amount of hydration, the crystal of the present invention can be returned to the original crystal structure by contacting with water after breaking. Contact with water is possible by immersing in a solution containing water in which crystals containing a water-soluble organic solvent are difficult to dissolve, or by placing in a humid environment for 15 minutes or more. It is easy and preferable for the operation of the crystal to be in contact with a high humidity environment. Here, the high humidity indicates 20% or more, and it is more preferable that the contact is performed at a humidity of 70-100%.

The PQQ disodium crystal obtained in the present invention can be used as food, functional food, pharmaceuticals or quasi drugs for humans or animals.
Specific examples include, but are not limited to, powders, external preparations for skin, injections, oral preparations, suppositories, capsules, tablets, chewable tablets, and drinks.

As additives used in the preparation, saccharides such as water, fructose and glucose, oils such as falling raw oil, soybean oil and olive oil, and glycols such as polyethylene glycol and polypropylene glycol are used as the liquid agent. it can. As an excipient for solid preparations such as tablets, capsules, granules, etc., sugars such as lactose, sucrose, mannitol, etc., kaolin, talc, magnesium stearate etc. as lubricants, starch, sodium alginate as disintegrants, Examples of the binder include polyvinyl alcohol, cellulose, and gelatin, examples of the surfactant include fatty acid esters, and examples of the plasticizer include glycerin and the like, but are not limited thereto. You may add a dissolution accelerator, a filler, etc. as needed.

Functional food means foods taken for the purpose of maintaining nutrition or supplementing nutrition, such as health foods, nutritional supplements, functional nutritional foods, and nutritional health foods. When commercialized as a functional food, additives used in food, such as sweeteners, coloring agents, preservatives, thickening stabilizers, antioxidants, coloring agents, bleaching agents, antibacterial and antifungal agents, gum bases Bitterings, enzymes, brighteners, acidulants, seasonings, emulsifiers, fortifiers, manufacturing agents, fragrances, spice extracts and the like can be used. In general, it can also be added to ordinary foods such as miso, soy sauce, instant miso soup, ramen, fried noodles, curry, corn soup, merbaudofu, marvo eggplant, pasta sauce, pudding, cake, bread and the like.

The PQQ disodium crystal of the present invention can be used alone or in combination with other materials. Materials that can be combined include vitamins such as vitamin B group, vitamin C and vitamin E, amino acids, astaxanthin, carotenoids such as α-carotene and β-carotene, ω3 fatty acids such as docosahexaenoic acid and eicosapentaenoic acid, Although omega-6 fatty acids, such as arachidonic acid, are illustrated, it is not limited to these.

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

In the examples, the following conditions were used.
Powder X-ray diffraction: M18XCE device manufactured by Mac Science Co., Ltd. or RINT2500 manufactured by Rigaku Co., Ltd.
X-ray: Cu / tube voltage 40 kV / tube current 100 mA
Scan speed: 4.000 ° / min
Sampling width: 0.020 °
The moisture content was measured by the Karl Fischer method.

Reference Example 1 Preparation of PQQ Trisodium Salt as Raw Material Based on Example 1 of Japanese Patent No. 2692167, the culture solution obtained by culturing Hyphomicrobium DSM1869 is centrifuged to remove cells, and PQQ A culture supernatant containing was obtained. The strain can be obtained from DSM (Deutsche Sammlung von Microorganisms (German Collection of Microorganisms and Cell Cultures)).
The culture supernatant is passed through a Sephadex G-10 (Pharmacia) column so that PQQ is adsorbed and eluted with an aqueous NaCl solution to obtain a PQQ aqueous solution with a pH of 7.5 and a NaCl concentration of 60 g / L. And cooled to obtain PQQ trisodium salt. The purity of the obtained PQQ trisodium salt by UV absorption of high performance liquid chromatography was 99.0%.

PQQ sodium salt was analyzed under the following analysis conditions.
(PQQ analysis)
PQQ concentration and purity were measured under the following conditions.
Apparatus: Shimadzu Corporation, high performance liquid chromatography, LC-20A
Column: YMC-Pack ODS-TMS (5 μm), 150 × 4.6 mm D.
Measurement temperature: 40 ° C
Detection: Absorbance at 260 nm Eluent: 100 mM CH ₃ COOH / 100 mM CH ₃ COONH ₄ (30/70, pH 5.1)
Elution rate: 1.5 mL / min

(Na analysis)
Na concentration was measured under the following conditions.
Pump: Shimadzu Corporation, cation chromatography LC-6A
Column oven: Shimadzu Corporation, HIC-6A
Measurement temperature: 40 ° C
Detector: Tosoh Corporation, conductivity meter CM-8000
Column: Showa Denko KK, Shodex, IC Y-521
Eluent: 4 mM HNO ₃
Elution rate: 1.0 mL / min
From the respective concentrations of PQQ and Na obtained, the substance amount ratio of PQQ and Na contained in the PQQ sodium salt was determined. The PQQ disodium salt of the present invention has a substance amount ratio of 2.0 ± 0.2, and the PQQ trisodium salt has a substance amount ratio of 3.0 ± 0.2.

Example 1 Crystal of PQQ disodium salt 12 g of PQQ trisodium salt prepared in Reference Example 1 was added to 500 g of ion-exchanged water and 500 mL of a mixed solution. At this time, it became suspended. While measuring with a pH meter, hydrochloric acid was added dropwise to adjust the pH to 3.6, followed by stirring at room temperature for 1 hour. Stored at 50 ° C. and left for more than 10 days. The crystals in this solution were taken out and measured by single crystal X-ray diffraction.
Red single crystal C14H10N2Na2011 0.100 x 0.100 x 0.100 mm Fixed to glass fiber, using Rigaku single crystal X-ray structure analyzer (VariMax with RAPID system), CuK ・ (λ = 1.54187Å) 40 kV 30 mA It measured using -180 degreeC (use of a spraying low temperature apparatus) Cu-K line | wire. The results are shown in FIGS.
From this single crystal data, peaks at 9.1 °, 10.3 °, 13.8 °, 17.7 °, 18.3 °, 24.0 ° (± 0.2 °) are shown by powder X-ray diffraction. I understood that. The amount of water was 12.6%.

Example 2 PQQ trisodium as a precursor crystal raw material was a water-containing solid obtained in the same manner as in the reference example. 60 g of a hydrated PQQ trisodium salt containing 20 g of PQQ was added to a mixture of 500 ml of ion-exchanged water and 500 ml of ethanol. At this time, the solid is not melted. Hydrochloric acid was added thereto at room temperature to adjust the pH to 3.5. Hydrochloric acid was added slowly dropwise over about 2 hours. Stir for 2 days. Filtration yielded hydrous PQQ disodium crystals in 99 mol% yield. The color was light red. The water content was 26.5%.
The crystal was placed in an environment of 40 ° C. and RH 75% for more than one night, and the moisture content was measured. As a result, it was 11.5%. The peak at 2θ was 9.1, 10.3, 13.8, 17.7, 18.3, 24.0 ± 0.2 °.
Even when water was present in excess, it returned to the trihydrate composition and maintained its crystal structure.

Example 3 Moisture content 11.4%
The precursor crystal obtained in Example 2 was placed in a vacuum dryer and the pressure was reduced as much as possible with a diaphragm vacuum pump, followed by drying at 50 ° C. for 1 hour. As a result of measuring the water content, it was 11.4%. The color was light red. The peak at 2θ was 9.1, 10.3, 13.8, 17.7, 18.3, 24.0 ± 0.2 °. The powder X-ray diffraction data is shown in FIG.
The crystals were placed in an environment of 40 ° C. and RH 75% for more than one night, and the water content was measured. As a result, it was 11.7%. The amount of hydration corresponds to 2.7, and the structure as a trihydrate is maintained. The result of the powder X-ray with high humidity was the same, and the structure was maintained.

Example 4 Moisture content 7%
The precursor crystal obtained in Example 2 was placed in a vacuum dryer and the pressure was reduced as much as possible with a diaphragm type vacuum pump, and dried at 50 ° C. for 22 hours. As a result of measuring the water content, it was 7%. The solid color was brown.
As a result of the powder X-ray, it was a mixture of crystals different from Example 2. The crystals were placed in an environment of 40 ° C. and RH 75% for one night or more, and the water content was measured. As a result, the hydration amount was changed to 2.5% at 10.8%. The powder X-ray diffraction at this time was a peak at 2θ of 9.1, 10.3, 13.8, 17.7, 18.3, 24.0 ± 0.2 °. The color at this time was bright red.
When the moisture content was reduced to 7%, the crystal structure was broken, but when the moisture was applied, it changed to the original crystal. The color also became the original crystal color.

Example 5 Moisture content 0.7%
The precursor crystal obtained in Example 2 was placed in a vacuum dryer and the pressure was reduced as much as possible with a diaphragm type vacuum pump, and dried at 50 ° C. for 77 hours. As a result of measuring the water content, it was 0.7%. The color at this time was black purple. As a result of powder X-rays, the crystals were different from those in Example 2. The crystals were placed in an environment of 40 ° C. and RH 75% for more than one night, and the water content was measured. The powder X-ray diffraction at this time was a peak at 2θ of 9.1, 10.3, 13.8, 17.7, 18.3, 24.0 ± 0.2 °. The color at this time was bright red.
When water decreased, the crystal structure was broken, but it changed to the original crystal by applying humidity. The color also became the original crystal color.
A stable structure can be obtained by using the crystal of the present invention. In addition, the crystal structure can be easily recovered.

Example 6 DSC-XRD Measuring Device Rigaku SmartLab (Sample Horizontal Multipurpose X-ray Diffraction) Differential Scanning Calorimetry-X-ray Diffraction Simultaneous Measuring Device (DSC-XRD) High Speed One-Dimensional Detector D / teX Ultra
Condition X-ray source Cu-Kα (40 kV-50 mA) scanning axis 2θ / θ scanning speed 5 deg./min measuring range 3-60 deg. (2 / θ)
The powder of Example 2 was measured. When the temperature was raised to 180 ° C. in nitrogen, all the original crystals disappeared. Even if the temperature was lowered, it did not return to the original structure. From this state, the humidity was gradually increased, and thermal analysis and XRD measurement were simultaneously performed. The result is shown in FIG.
Heat generation was observed at 20% humidity, and XRD changed to the original trihydrate structure after 2 hours.
The crystals of the present invention could be restored even at a humidity of 20%.

Comparative Example 1 Solid Precipitation of PQQ Disodium Salt by Ethanol Recrystallization Method All the PQQ trisodium salt prepared in Reference Example 1 was dissolved in ion-exchanged water, and 800 g of a solution containing 10 g / L of PQQ was prepared. Hydrochloric acid was added to adjust the pH to 3.5, and 200 mL of ethanol was added thereto. At this time, a red solid precipitated. After stirring at room temperature for 5 hours, the mixture was allowed to stand at 5 ° C. for 24 hours to precipitate a solid. Solids were collected by continuous centrifugation and dried under reduced pressure at 50 ° C.
The powder X-ray diffraction spectrum of the obtained solid was measured. The obtained solid had a low crystallinity with almost no peaks other than a peak on the low angle side. Analysis by gas chromatography under the same conditions as in Example 2 revealed that ethanol remained in 0.02% solids.

The PQQ disodium salt crystal of the present invention is useful in the fields of foods, functional foods, pharmaceuticals or quasi drugs for humans or animals.

Claims (5)

A disodium salt crystal of pyrroloquinoline quinone represented by the formula (1),

A pyrroloquinoline quinone disodium salt crystal comprising 3 molecules or less of water per molecule of pyrroloquinoline quinone disodium salt. The pyrroloquinoline quinone disodium salt crystal according to claim 1, satisfying one or more of (a) to (d).
(A) It has the crystal structure shown in FIGS.
(B) It has a crystal structure defined by the coordinates in FIG. 3 in this specification.
(C) It has crystal lattice constants at a = 7.60, b = 10.09, c = 11.43 Å, α = 72.85, β = 88.01 °, and γ = 82.62 °.
(D) It has a crystal structure belonging to a crystal system space group such as P-1 (# 2). A method for regenerating a crystal by placing the crystal according to claim 1 or 2 in which the crystal is broken in an environment having a humidity of 20 to 100% for 15 minutes or more. A functional food comprising the pyrroloquinoline quinone disodium salt crystal according to claim 1 or 2. A medicament comprising the pyrroloquinoline quinone disodium salt crystal according to claim 1 or 2.