Method for producing pyrroloquinoline quinone derivatives

Abstract

To provide a method for promoting the derivatization of pyrroloquinoline quinone or its salts. [Solution] The method includes a reaction step of reacting pyrroloquinoline quinone or a salt thereof with an amino group-containing compound in the presence of an emulsifier and / or a calcium compound. The emulsifier comprises at least one selected from the group consisting of long-chain alkylpyridinium salts, long-chain alkylbenzylammonium salts, and polyoxyethylene long-chain alkyl ethers. The calcium compound comprises at least one selected from the group consisting of apatite, calcium carboxylate, calcium sulfonate, calcium phosphonate, and calcium phosphate. A method for producing the pyrroloquinoline quinone or a derivative thereof.

Description

Technical Field

[0001] The present invention relates to a method for producing and analyzing a derivative of pyrroloquinoline quinone or a salt thereof. Further, the present invention relates to a method for promoting the reaction between pyrroloquinoline quinone or a salt thereof and an amino group-containing compound. Furthermore, the present invention relates to a promoter for the above reaction.

Background Art

[0002] Pyrroloquinoline quinone (PQQ) is a substance having an o-quinone structure, which is a condensate of a pyrrole ring and a quinoline ring. PQQ is known to function as an electron transfer agent, and by being incorporated into aminoadipic semialdehyde dehydrogenase (AASDH) involved in the metabolism of the essential amino acid lysine, AASDH can perform oxidation-reduction reactions. That is, it is considered to be a coenzyme of AASDH, and from this, it is regarded as the third oxidation-reduction coenzyme after nicotinamide (pyridine nucleotide) and flavin, and has the potential to become a new vitamin.

[0003] In addition, PQQ is said to have many important physiological activities such as a cell growth promoting effect, a life extension effect, a cognitive function improvement effect, an aging suppression effect, a fat accumulation suppression effect, and a nutritional improvement effect, and the industrial importance of PQQ is increasing.

[0004] [[ID=第20]]PQQ was known to be widely present in bacteria and fungi such as molds and yeasts, but in recent years, it has been reported that it is widely present not only in bacteria but also in plants such as rice and mammals. Although its detection has been reported from various tissues and organs in mammals, mammals do not have a PQQ synthesis pathway, so it is considered that they ingest PQQ from food.

[0005] In the functional food labeling system that utilizes functionality, it is required to calculate accurate measurement data of PQQ in food. In many foods, various substances are provided in a mixed state, but PQQ easily reacts with many food components and is easily interfered with during analysis.

[0006] When considering the quantitative analysis of functional foods containing a wide variety of coexisting components, HPLC-based methods offer excellent quantitative accuracy. However, PQQ analysis is susceptible to interference from interfering substances, which can impair its quantitative accuracy. To address this problem, PQQ has been derivatized. Patent Document 1 discloses a method for quantitatively analyzing PQQ by reacting PQQ with glycine to form a more stable imidazopyrroloquinoline (IPQ) and then analyzing the IPQ.

[0007] Patent Document 2 discloses a method for reacting PQQ with an amino group-containing compound in the presence of a food protein in order to promote the formation of PQQ derivatives. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2019 / 138817 [Patent Document 2] Japanese Patent Publication No. 2024-117387 [Overview of the project] [Problems that the invention aims to solve]

[0009] The inventors investigated and found that when using the method described in Patent Document 2, the presence of gelatin in the analytical sample hinders the derivatization of PQQ.

[0010] The present invention provides a novel method for promoting the derivatization of pyrroloquinoline quinone or its salts. [Means for solving the problem]

[0011] As a result of diligent research by the present inventors, it was found that the reaction between pyrroloquinoline quinone or its salt and an amino group-containing compound is promoted by a predetermined emulsifier or calcium compound.

[0012] The present invention includes the following embodiments. [1] The process includes a reaction step of reacting pyrroloquinoline quinone or a salt thereof with an amino group-containing compound in the presence of an emulsifier and / or a calcium compound. The emulsifier comprises at least one selected from the group consisting of long-chain alkylpyridinium salts, long-chain alkylbenzylammonium salts, and polyoxyethylene long-chain alkyl ethers. The calcium compound comprises at least one selected from the group consisting of apatite, calcium carboxylate, calcium sulfonate, calcium phosphonate, and calcium phosphate. A method for producing the pyrroloquinoline quinone or a derivative thereof. [2] The method for producing an amino acid according to [1], wherein the amino group-containing compound is an amino acid or a salt thereof. [3] The method for producing the product according to [1] or [2], wherein the emulsifier comprises at least one selected from the group consisting of cetylpyridinium chloride, polyoxyethylene lauryl ether, and benzyldimethyltetradecylammonium chloride. [4] The method for producing calcium compounds according to any one of [1] to [3], wherein the calcium compound comprises at least one selected from the group consisting of apatite, calcium pantothenate, calcium citrate, calcium lactate, calcium stearate, calcium lauryl sulfate, and calcium hydrogen phosphate. [5] The manufacturing method according to any one of [1] to [4], wherein the reaction is carried out in the presence of an aqueous solvent. [6] The manufacturing method according to any one of [1] to [5], wherein the reaction is carried out in the presence of gelatin. [7] An analytical method comprising an analytical step of analyzing the derivative produced by any of the manufacturing methods described in [1] to [6]. [8] The analysis method according to [7], wherein the analysis step includes analyzing the derivative to quantify the pyrroloquinoline quinone or a salt thereof.

Advantages of the Invention

[0013] According to the present invention, the reaction between pyrroloquinoline quinone or a salt thereof and an amino group-containing compound can be promoted.

Modes for Carrying Out the Invention

[0014] Hereinafter, embodiments of the present invention will be specifically described. However, the present invention is not limited to these, and various modifications are possible without departing from the gist thereof.

[0015] <Production Method and Promotion Method> One embodiment of the present invention relates to a method for producing a derivative of pyrroloquinoline quinone or a salt thereof, including a reaction step of reacting pyrroloquinoline quinone or a salt thereof with an amino group-containing compound in the presence of a predetermined emulsifier and / or calcium compound. The production method according to the above embodiment can also be expressed as a method for promoting the reaction of reacting pyrroloquinoline quinone or a salt thereof with an amino group-containing compound in the presence of a predetermined emulsifier and / or calcium compound.

[0016] As a reason why the emulsifier or calcium compound promotes the reaction between pyrroloquinoline quinone or a salt thereof and the amino group-containing compound, it is presumed that some interaction occurs between the emulsifier or calcium compound and pyrroloquinoline quinone or a salt thereof. However, the present invention is not limited by the above presumed reason at all.

[0017] In the production method according to the present embodiment, since the food protein in Patent Document 2 is not used, it is possible to avoid spoilage (growth of miscellaneous bacteria) that reduces the accuracy or stability of PQQ analysis. It is also possible to avoid health risks due to spoilage.

[0018] [Pyrroloquinoline Quinone (PQQ)] PQQ is a compound represented by the following formula (I). [ka]

[0019] Examples of salts of PQQ include alkali metal salts, alkaline earth metal salts, and ammonium salts. The salt of PQQ is preferably a sodium salt, potassium salt, or lithium salt, more preferably a sodium salt, and even more preferably a disodium salt. The disodium salt of PQQ is represented by the following formula (II). Note that the position of sodium may vary due to recrystallization, etc., but its position is not particularly limited. [ka]

[0020] PQQ and its salts shall also include their solvates. Examples of solvates include hydrates. The solvates of PQQ and its salts are preferably 1 to 5 hydrates.

[0021] The structures in formulas (I) and (II) above represent the oxidized form of PQQ, which is a quinone structure, but PQQ may also be the reduced form. The reduced form of PQQ is represented by formula (III) below. Since PQQ is readily convertible between its oxidized and reduced forms, these can be treated as substantially equivalent. [ka]

[0022] Hereafter, when "PQQ" is simply referred to, it includes PQQ (oxidized and reduced forms), its salts, and their solvates, unless otherwise specified.

[0023] PQQ may be provided as a sample containing other optional components. Samples are not particularly limited, but examples include foods (including beverages), pharmaceuticals, and quasi-drugs. Examples of foods include general foods and functional foods (e.g., foods for specified health uses, foods with functional claims, and foods with nutritional function claims). More specific examples of samples include oral medications and supplements such as capsules, tablets, powders, and granules; beverages; jellies; gummies; and other foods such as retort foods. In addition to the above, samples may also include cosmetics, cleansers, and other topical preparations that are not food or beverages, and can encompass all products containing PQQ. While not particularly limited, the advantages of the manufacturing method according to this embodiment are especially evident when the sample contains gelatin.

[0024] [Amino group-containing compounds] The amino group-containing compound used for PQQ derivatization is preferably one that can form a stable PQQ derivative. Examples of amino group-containing compounds include amino acids, alkylamines, alkanolamines, arylamines, hydroxylamines, hydrazides, peptides, and polyamines, with amino acids being preferred.

[0025] Examples of amino acids include isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, tyrosine, cysteine, aspartic acid, asparagine, serine, glutamic acid, glutamine, proline, glycine, alanine, and arginine, with glycine, arginine, and leucine being preferred.

[0026] Amino acids may also be in the form of salts. Examples of amino acid salts include alkali metal salts, alkaline earth metal salts, and ammonium salts.

[0027] From the viewpoint of analyzing PQQ derivatives, it is preferable to use one type of amino group-containing compound, and it is more preferable to use glycine to form IPQ.

[0028] [Emulsifiers and calcium compounds] The reaction between PQQ and an amino group-containing compound can be accelerated by using a specified emulsifier and / or calcium compound.

[0029] By using emulsifiers and / or calcium compounds instead of food proteins, spoilage problems can be avoided.

[0030] The emulsifier and / or calcium compound may be sterilized before use. In the case of sterilization by heat, the sterilization temperature is preferably 40 to 140°C, more preferably 50 to 120°C, even more preferably 60 to 100°C, and particularly preferably 70 to 90°C.

[0031] The amount (total amount) of the emulsifier and / or calcium compound in the reaction system is preferably 0.001 to 20% by mass, and more preferably 0.01 to 5% by mass.

[0032] (emulsifier) Examples of emulsifiers include long-chain alkylpyridinium salts, long-chain alkylbenzylammonium salts, and polyoxyethylene long-chain alkyl ethers.

[0033] The long-chain alkylpyridinium salt is preferably a compound represented by the following formula (1). [ka] [In formula (1), R is an alkyl group having 10 to 22 carbon atoms, preferably an alkyl group having 12 to 20 carbon atoms, more preferably an alkyl group having 14 to 18 carbon atoms, and even more preferably an alkyl group having 16 carbon atoms. X is a halogen, preferably a chloride.

[0034] The long-chain alkylpyridinium salt is preferably cetylpyridinium chloride.

[0035] The long-chain alkylbenzylammonium salt is preferably a compound represented by the following formula (2). [ka] [In formula (2), R 1 This is an alkyl group having 8 to 20 carbon atoms, preferably an alkyl group having 10 to 18 carbon atoms, more preferably an alkyl group having 12 to 16 carbon atoms, and even more preferably an alkyl group having 14 carbon atoms. R 2 and R 3 Each of these is independently an alkyl group having 1 to 3 carbon atoms, preferably an alkyl group having 1 or 2 carbon atoms, and more preferably an alkyl group having 1 carbon atom. X is a halogen, preferably a chloride.

[0036] The long-chain alkylbenzylammonium salt is preferably benzyldimethyltetradecylammonium chloride.

[0037] The polyoxyethylene long-chain alkyl ether is preferably a compound represented by the following formula (3). R-(OCH2CH2) n -OH (3) [In formula (3), R is an alkyl group having 8 to 16 carbon atoms, preferably an alkyl group having 10 to 14 carbon atoms, and more preferably an alkyl group having 12 carbon atoms. n is an integer between 5 and 50, preferably between 10 and 40, and more preferably between 20 and 30.

[0038] The polyoxyethylene long-chain alkyl ether is preferably polyoxyethylene lauryl ether.

[0039] (Calcium compounds) Examples of calcium compounds include apatite, calcium carboxylate, calcium sulfonate, calcium phosphonate, and calcium phosphate.

[0040] The calcium compound is preferably apatite, calcium pantothenate, calcium citrate, calcium lactate, calcium stearate, calcium lauryl sulfate, or calcium hydrogen phosphate, and more preferably apatite, calcium pantothenate, calcium citrate, calcium lactate, or calcium hydrogen phosphate.

[0041] [Reaction Process] The reaction step of the manufacturing method according to this embodiment is a step of reacting PQQ with an amino group-containing compound in the presence of an emulsifier and / or a calcium compound to form a derivative of PQQ.

[0042] The PQQ derivative is not particularly limited as long as it is formed by bonding an amino group-containing compound to PQQ. The structure of the PQQ derivative varies depending on the amino group-containing compound used, but when the amino group-containing compound is glycine, the PQQ derivative is imidazopyrroloquinoline (IPQ) of the following formula (IV). [ka]

[0043] PQQ and glycine are presumed to react through the following mechanism. [ka]

[0044] The reaction step is preferably carried out in the presence of a solvent. Examples of solvents include water, organic solvents, and mixed solvents thereof, with water being preferred. A buffer solution may also be used as the solvent.

[0045] Water-soluble organic solvents are preferred. Examples of water-soluble organic solvents include methanol, ethanol, propanol, acetone, acetonitrile, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, methylformamide, and formamide.

[0046] The reaction conditions (e.g., temperature, pressure, and time) can be appropriately determined depending on the type of amino group-containing compound, emulsifier, and / or calcium compound. Examples of suitable temperatures include 0-130°C, 10-80°C, and 20-40°C. Examples of pressures include 0.01 to 100 MPa and atmospheric pressure. In terms of time, for batch reactions, examples include 0.01 to 240 hours and 0.1 to 24 hours. In terms of time, for continuous reactions, examples include 0.001 to 24 hours and 0.01 to 12 hours.

[0047] The amount of amino group-containing compound used is not particularly limited, but is preferably 0.5 to 10 times the mass of PQQ. 9 It is twice as much, more preferably 10 to 10 8 It is double.

[0048] The amount (total amount) of emulsifier and / or calcium compound used is not particularly limited, but is preferably 1 to 10 times the mass of PQQ. 8 It is twice as much, more preferably 10 to 10 7 It is double.

[0049] When derivatizing PQQ in a sample containing optional components, it is preferable to add an amino group-containing compound, an emulsifier, and / or a calcium compound to the sample from an external source.

[0050] The reaction process may be carried out in the presence of gelatin. The advantages of the manufacturing method according to this embodiment are particularly evident when gelatin is present.

[0051] <Analysis method> One embodiment of the present invention relates to an analytical method that includes an analytical step of analyzing a PQQ derivative produced by the above manufacturing method. The analytical step preferably includes analyzing the PQQ derivative to quantify PQQ.

[0052] In the analytical method according to this embodiment, PQQ efficiently derivatized with an emulsifier and / or a calcium compound is the target of analysis, thus enabling accurate quantification of PQQ. The analytical method according to this embodiment is particularly advantageous when quantifying PQQ at low concentrations (preferably greater than 0 mg / L and 20 mg / L or less).

[0053] The analytical method is not particularly limited as long as it can analyze PQQ derivatives; therefore, known methods can be employed. Examples of known methods include liquid chromatography, gas chromatography, ultraviolet-visible absorption spectroscopy, and nuclear magnetic resonance spectroscopy. Preferably, it is liquid chromatography, and more preferably, high-performance liquid chromatography (HPLC).

[0054] The specific analytical conditions can be set appropriately depending on the type of PQQ derivative, the analytical method, etc. For example, the analytical conditions disclosed in International Publication No. 2019 / 138817 may be adopted.

[0055] <Reaction accelerator> One embodiment of the present invention relates to a reaction accelerator between PQQ and an amino group-containing compound, comprising an emulsifier and / or a calcium compound. The reaction accelerator according to this embodiment may further contain an amino group-containing compound. Details of PQQ, the amino group-containing compound, the emulsifier, and the calcium compound are as described in the <Manufacturing Method and Acceleration Method> section above. [Examples]

[0056] The present invention will be described in more detail below using examples and comparative examples, but the technical scope of the present invention is not limited thereto.

[0057] <Analysis method> The samples used for the analysis are as follows. Unless otherwise specified, the reagents used in the examples are special grade reagents manufactured by Wako Pure Chemical Industries, Ltd. · Pyrroloquinoline quinone disodium salt (manufactured by Mitsubishi Gas Chemical Company, Na / pyrroloquinoline quinone molar ratio 1.70 - 2.10, water content < 12%, HPLC purity 100%) · HPLC eluent (100 mM CH3COOH / 100 mM CH3COONH4 = 30 / 70 (pH 5.1)): 6.0 g of CH3COOH was dissolved in distilled water, and made up to a total volume of 1 L to prepare 100 mM CH3COOH (solution 1). Separately, 7.71 g of CH3COONH4 was dissolved in distilled water, and made up to a total volume of 1 L to prepare 100 mM CH3COONH4 (solution 2). Then, 300 mL of solution 1 and 700 mL of solution 2 were mixed to obtain an HPLC eluent and a buffer solution used as a carbonate buffer solution. The pH of the obtained buffer solution was 5.1 ± 0.2.

[0058] [HPLC Conditions] Liquid delivery unit: LC - 10AD (manufactured by Shimadzu Corporation) Column: YMC - Pack ODS - A (manufactured by YMC, length 150 mm, inner diameter 4.6 mm, particle size 5 μm) Detector: UV259 nm HPLC eluent: as described above Column temperature: 40 °C Eluent flow rate: 1.5 mL / min Injection volume: 3 μL Analysis time: 30 min

[0059] <Derivatization of PQQ> [Comparative Example A1] 1 mL of 2 g / L pyrroloquinoline quinone disodium salt and 40 mL of 10% by mass aqueous glycine solution were added to a 100 mL plastic container (AS ONE, I - Boy), and stirred at 50 °C for 2 hours. From the HPLC analysis under the above conditions, the yield of imidazopyrroloquinoline (IPQ) was 47%.

[0060] [Example A1] 0.40 g of cetylpyridinium chloride, 1 mL of 2 g / L pyrroloquinoline quinone disodium salt, and 40 mL of 10% by mass glycine aqueous solution were added to a 100 mL plastic container (AS ONE, iBoy) and stirred at 50°C for 2 hours. HPLC analysis under the above conditions showed a yield of 65% for imidazopyrroloquinoline (IPQ).

[0061] [Examples A2-A7 and Comparative Examples A2-A3] The reaction was carried out in the same manner as in Example A1, except that the additives listed in Table 1 below were used instead of the cetylpyridinium chloride used in Example 1. The results are shown in Table 1.

[0062] [Table 1]

[0063] <Derivatization of PQQ in the presence of gelatin> [Examples B1-B2 and Comparative Examples B1-B2] 10 mg of pyrroloquinoline quinone disodium salt, 5 g of gummy candy (manufactured by Kabaya Foods, product name: Tough Gummy), and 200 mL of a 10% by mass glycine aqueous solution containing 1% of the additives listed in Table 2 below were added to a 500 mL plastic container (AS ONE, iBoy) and stirred at 50°C for 2 hours. The yield of imidazopyrroloquinoline (IPQ) was measured by HPLC analysis under the above conditions. The results are shown in Table 2.

[0064] [Table 2]

[0065] Even in the presence of gummy candy (gelatin), the derivatization of PQQ could be promoted by using a specified emulsifier or calcium compound. On the other hand, when skim milk, a food protein, was used, no promoting effect was observed due to interference by gelatin.

[0066] <Derivatization of PQQ on a small scale> [Examples C1-C5 and Comparative Examples C1-C6] 40 μL of 1% by mass aqueous glycine solution, 40 μL of 0.1% by mass additives listed in Table 3 below, and 80 μL of 200 mg / L pyrroloquinoline quinone disodium salt were placed in a 200 μL PCR tube and mixed. The mixture was reacted at 50°C for 1 hour. The mixture was then immediately returned to room temperature, 100 μL of the reaction mixture was added to 96 wells, and the absorbance at 450 nm was measured. To correct for turbidity caused by the additive, the absorbance at 450 nm of 100 μL of a mixture consisting of 40 μL of a 1% by mass glycine aqueous solution, 40 μL of a 0.1% by mass additive, and 80 μL of water was also measured. A calibration curve was prepared using IPQ standards, and the yield was determined. The results are shown in Table 3.

[0067] [Table 3]

[0068] <Storage stability (E. coli inhibition) test> [Examples D1-D4, Comparative Example D1, and Reference Example D1] LB medium (manufactured by Fujifilm Wako Pure Chemical Industries) was sterilized at 121°C for 15 minutes. E. coli DH5α was cultured in LB medium at 37°C for 6 hours. Dimethyl sulfoxide was added to 10% of the medium volume and stored at -80°C. This was then added to PBS pH 7.4 (GIBCO) and the OD was adjusted to 0.1 before use. 100 mg of the additives listed in Table 4 below and 2 mL of water were placed in a 5 mL tube. 50 μL of E. coli solution was then added, and the mixture was left at 30°C for 1 day to prepare the sample. SCD agar medium (Fujifilm Wako Pure Chemical Industries: SCD agar medium "Daigo") was autoclaved and placed in a 9cm polystyrene petri dish to prepare plates for bacterial contamination testing. Samples were diluted to 1 / 100 with PBS, 50 μL of which was spread onto the plates, and incubated at 37°C for 1 day. The number of colonies was then counted. The results are shown in Table 4.

[0069] [Table 4]

[0070] In Comparative Example D1, significant decay progressed. On the other hand, no colonies were observed in Examples D1 and D2. Furthermore, in Examples D3 and D4, the number of colonies was similar to that of Reference Example D1, and no significant decay progress was confirmed. Therefore, accurate and stable analysis of PQQ is possible in these examples.

Claims

1. The process includes a reaction step of reacting pyrroloquinoline quinone or a salt thereof with an amino group-containing compound in the presence of an emulsifier and / or a calcium compound. The emulsifier comprises at least one selected from the group consisting of long-chain alkylpyridinium salts, long-chain alkylbenzylammonium salts, and polyoxyethylene long-chain alkyl ethers. The calcium compound comprises at least one selected from the group consisting of apatite, calcium carboxylate, calcium sulfonate, calcium phosphonate, and calcium phosphate. A method for producing the pyrroloquinoline quinone or a derivative thereof.

2. The amino group-containing compound is an amino acid or a salt thereof. The manufacturing method according to claim 1.

3. The emulsifier comprises at least one selected from the group consisting of cetylpyridinium chloride, polyoxyethylene lauryl ether, and benzyldimethyltetradecylammonium chloride. The manufacturing method according to claim 1 or 2.

4. The calcium compound comprises at least one selected from the group consisting of apatite, calcium pantothenate, calcium citrate, calcium lactate, calcium stearate, calcium lauryl sulfate, and calcium hydrogen phosphate. The manufacturing method according to claim 1 or 2.

5. The above reaction is carried out in the presence of an aqueous solvent. The manufacturing method according to claim 1 or 2.

6. The above reaction is carried out in the presence of gelatin. The manufacturing method according to claim 1 or 2.

7. An analytical method comprising an analytical step of analyzing the derivative produced by the manufacturing method described in claim 1 or 2.

8. The analytical method according to claim 7, wherein the analytical step includes analyzing the derivative to quantify the pyrroloquinoline quinone or a salt thereof.

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