Abstract:
An object of the present invention is to provide a method for conveniently producing reduced pyrroloquinoline quinone from oxidized pyrroloquinoline quinone without needing expensive equipment, and a method for stabilizing reduced pyrroloquinoline quinone. According to the present invention, high-quality reduced pyrroloquinoline quinone can be obtained conveniently and efficiently in a manner suitable for industrial-scale production by mixing pyrroloquinoline quinone and ascorbic acid in a solvent.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application enjoys the benefit of Japanese Patent Application No. 2010-31637, filed on Feb. 16, 2010. The disclosure of this earlier application is incorporated herein by reference in its entirety. 
       TECHNICAL FIELD 
       [0002]    This invention relates to a production method of reduced pyrroloquinoline quinone and reduced pyrroloquinoline quinone obtained thereby. The present invention also relates to a stabilization method of reduced pyrroloquinoline quinone. 
       BACKGROUND ART 
       [0003]    Pyrroloquinoline quinone (hereinafter sometimes referred to as “PQQ”) has been proposed and has attracted much attention as a possible new vitamin (Non-patent document 1). Moreover, PQQ is present not only in bacteria but also in eukaryotic molds and yeasts and plays an important role as a coenzyme. Also, PQQ has been found to have many physiological activities such as cell growth-promoting activity, anti-cataract activity, hepatic disease-preventing and therapeutic activity, wound healing activity, antiallergic activity, reverse transcriptase-inhibiting activity, glyoxalase I-inhibiting activity-anticancer activity, and the like. The PQQs can be produced by processes such as organic chemical synthesis (Non-patent document 2) and fermentation (Patent document 1). 
         [0004]    Reduced PQQ is reported to exhibit extremely higher antioxidative activity than that exhibited by the conventional PQQ (Non-patent document 3), and is a useful compound as foods with nutrient function claims, foods for specified health uses, nutrients, nutraceuticals, beverages, feedstuff, veterinarian medicine, cosmetics, pharmaceuticals, therapeutics, prophylactics, etc. Reduced PQQ is reported to be obtained by subjecting oxidized PQQ to reduction with common reducing agents such as sodium borohydride and sodium hyposulfite, reduction with hydrogen on a platinum catalyst, and reduction with glutathione (Non-patent document 3, 4, and 5). These common reducing agents are, however, likely to exhibit toxicity to living organisms. This requires a further step of removing such agents. Moreover, when a catalyst is used, special equipment is required because hydrogen, which readily escapes and is highly explosive, is used. The reduction with glutathione is safer and more easily used, but has a disadvantage in that glutathione is costly. There is therefore a need for a safe, convenient and low-cost method. 
         [0005]    Ascorbic acid is well known as a substance that has reducing action on certain substances. Therefore, a combination of PQQ with ascorbic acid may be proposed as a composition that exhibits antioxidative activity. The reduction with ascorbic acid, however, has been thought to be difficult because the reduced form of PQQ exhibits greater antioxidative activity than that exhibited by ascorbic acid, as described in Non-patent document 3. Furthermore, reduced PQQ is readily oxidized to oxidized PQQ by molecular oxygen. This requires certain measures for storage of reduced PQQ. 
       PRIOR ART DOCUMENTS 
     Patent Document 
       [0000]    
       
         Patent document 1: Japanese Patent Application Laid-Open Publication N0.1-218597 
         Non-patent document 
         Non-patent document 1: Nature, vol. 422, 24, April, 3003, 832 
         Non-patent document 2: JACS, vol. 103, 5599-5600 (1981) 
         Non-patent document 3: J. Agric. Food Chem. 2009, 57, 450-456 
         Non-patent document 4: Bull. Chem. Soc. Jpn, 59, 1911-1914 (1986) 
         Non-patent document 5: Eur. J. Biochem. 118, 395-399 (1981) 
       
     
       SUMMARY OF THE INVENTION 
     Problem to be Solved by the Invention 
       [0013]    The present inventors have found that reduced pyrroloquinoline quinone is obtained in high yield from a solution having a pH of 3.5 or less obtained by mixing a solution of pyrroloquinoline quinone having a pH of 4 or less with a solution of ascorbic acid. The present invention is based on this finding. 
         [0014]    An object of the present invention is to provide a safe, convenient and low-cost production method of reduced pyrroloquinoline quinone and reduced pyrroloquinoline quinone obtained thereby. Also, another object of the present invention is to provide a stabilization method of reduced pyrroloquinoline quinone. 
       Means for Solving Problem 
       [0015]    According to the present invention, the following inventions are provided: 
         [0000]    (1) a production method of reduced pyrroloquinoline quinone, comprising a step of mixing a solution of pyrroloquinoline quinone or a salt thereof having a pH adjusted to 4 or less with an ascorbic acid analog to obtain a solution having a pH of 3.5 or less comprising reduced pyrroloquinoline quinone;
 
(2) the production method of (1), wherein the solution of the pyrroloquinoline quinone or salt thereof is an aqueous solution;
 
(3) the production method of (1) or (2), wherein the solution of the pyrroloquinoline quinone or salt thereof has a pH of 2 to 3.5;
 
(4) the production method of (1), wherein the molar ratio of the ascorbic acid analog with respect to the pyrroloquinoline quinone or salt thereof is 1:0.5 to 1000;
 
(5) the production method of (1), wherein the ascorbic acid analog is selected from the group consisting of ascorbic acid, rhamno-ascorbic acid, arabo-ascorbic acid, gluco-ascorbic acid, fuco-ascorbic acid, glucohepto-ascorbic acid, xylo-ascorbic acid, galacto-ascorbic acid, gulo-ascorbic acid, allo-ascorbic acid, erythro-ascorbic acid, 6-desoxyascorbic acid, and an ester and a salt thereof;
 
(6) the production method of (1), further comprising a step of separating the reduced pyrroloquinoline quinone from the solution;
 
(7) reduced pyrroloquinoline quinone produced by the production method of any of (1) to (6);
 
(8) the reduced pyrroloquinoline quinone of (7), which is provided as a solution comprising the ascorbic acid analog;
 
(9) a stabilization method of reduced pyrroloquinoline quinone, comprising bringing reduced pyrroloquinoline quinone or a salt thereof together with an ascorbic acid analog in a solvent.
 
       Effect of the Invention 
       [0016]    Advantageously, according to the present invention, reduced pyrroloquinoline quinone can be produced safely, conveniently and at low cost, and no expensive equipment is required in producing reduced pyrroloquinoline quinone. Also advantageously, according to the present invention, reduced pyrroloquinoline quinone can be stored stably. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0017]    According to the present invention, reduced pyrroloquinoline quinone can be produced by mixing a solution of pyrroloquinoline quinone or a salt thereof having a pH of 4 or less with an ascorbic acid analog to obtain a solution having a pH of 3.5 or less. 
         [0018]    The term “reduced pyrroloquinoline quinone” as used herein refers to the compound represented by formula (1). 
         [0000]    
       
                 
         
             
             
         
       
     
         [0019]    Pyrroloquinoline quinone (in the free form) used in the present invention refers to the compound represented by formula (2). 
         [0000]    
       
                 
         
             
             
         
       
     
         [0020]    According to the present invention, reduced PQQ can be produced by reducing pyrroloquinoline quinone with an ascorbic acid analog. 
         [0021]    Pyrroloquinoline quinone used in the present invention can be pyrroloquinoline quinone (in the free form) or a salt of pyrroloquinoline quinone. 
         [0022]    The “salt of pyrroloquinoline quinone” used in the present invention includes an alkali metal salt, an alkaline-earth metal salt, and an ammonium salt of pyrroloquinoline quinone, with alkali metal salt being preferred. 
         [0023]    Pyrroloquinoline quinone used in the present invention can be in the free form or in the form of any alkali metal salt. Readily available pyrroloquinoline quinone, in the free form and in the form of disodium and dipotassium salts, among others, is easily used. 
         [0024]    The alkali metal salt of pyrroloquinoline quinone used in the present invention includes salts of sodium, potassium, lithium, cesium, rubidium, and the like. Preferably, sodium and potassium salt are more preferred because they are readily available. Pyrroloquinoline quinone may be substituted with one to three atoms of alkali metals to form an alkali metal salt thereof, which may be any of a monoalkali metal salt, a dialkali metal salt and a trialkali metal salt, preferably a dialkali metal salt. The alkali metal salt of pyrroloquinoline quinone is especially preferably disodium and dipotassium salts. 
         [0025]    Pyrroloquinoline quinone or a salt thereof used in the present invention may be commercially available or produced by known methods. 
         [0026]    Pyrroloquinoline quinone or a salt thereof used in the present invention can be used in a solution. Any solvent that allows the reaction to proceed may be used. Pyrroloquinoline quinone or a salt thereof can be dissolved in a solvent such as water, alcohol, and dimethyl sulfoxide to form a solution, and preferred is water as the solvent (i.e., an aqueous solution of pyrroloquinoline quinone or a salt thereof) in that it is not highly problematic even when it remains in the product. 
         [0027]    The solution of pyrroloquinoline quinone or a salt thereof can be prepared in a concentration of, for example, 0.001 to 30 g/L, preferably 0.01 to 15 g/L, more preferably 0.1 to 5 g/L. 
         [0028]    The pH of the solution of pyrroloquinoline quinone or a salt thereof can be adjusted to 4 or less in order to efficiently obtain reduced PQQ. Also, the pH can be adjusted to 1 or more in order to increase the solubility of pyrroloquinoline quinone. The pH of the solution of pyrroloquinoline quinone or a salt thereof is preferably 1 to 4, more preferably 1 to 3.5, even more preferably 2 to 3.5, and even more preferably 2 to 2.5. To adjust the pH of the solution, an acidic substance (for example, hydrochloric acid) or an alkaline substance (for example, sodium hydroxide) can be used. 
         [0029]    Non-limiting examples of the ascorbic acid analog may include ascorbic acid, and those analogous to ascorbic acid such as rhamno-ascorbic acid, arabo-ascorbic acid, gluco-ascorbic acid, fuco-ascorbic acid, glucohepto-ascorbic acid, xylo-ascorbic acid, galacto-ascorbic acid, gulo-ascorbic acid, allo-ascorbic acid, erythro-ascorbic acid, 6-desoxyascorbic acid as well as esters and salts thereof (for example, palmitate, stearate, sodium salt, and calcium salt). Moreover, these ascorbic acid analogs may be in L- (for example, L-ascorbic acid and sodium L-ascorbate), D- (for example, D-arabo-ascorbic acid, sodium D-arabo-ascorbate), or racemic form. 
         [0030]    More specifically, the analogs may include, for example, L-ascorbic acid, L-ascorbic acid palmitate, L-ascorbic acid stearate, and D-arabo-ascorbic acid. 
         [0031]    Any of the ascorbic acid analogs mentioned above can be suitably used in the production of reduced pyrroloquinoline quinone, and among the ascorbic acid analogs mentioned above, particularly suitably used are water-soluble analogs in consideration of the ease of separation from the reduced pyrroloquinoline quinone produced. Most preferred are the analogs in the free form such as L-ascorbic acid and D-arabo-ascorbic acid from the viewpoint of availability and cost. 
         [0032]    The ascorbic acid analog used in the present invention may be commercially available or produced by known methods. 
         [0033]    The ascorbic acid analog used in the present invention can be used neat or in a solution. When used in solution, the ascorbic acid analog can be dissolved in a solvent such as water, alcohol, and dimethyl sulfoxide to form a solution. Preferably, the ascorbic acid analog is used as an aqueous solution. 
         [0034]    The solution of the ascorbic acid analog can be prepared in a concentration of, for example, 0.1 to 500 g/L, preferably 0.5 to 100 g/L. 
         [0035]    In the production method according to the present invention, the step of “mixing a solution of pyrroloquinoline quinone or a salt thereof with an ascorbic acid analog to obtain a solution having a pH of 3.5 or less comprising reduced pyrroloquinoline quinone” can be accomplished by reacting pyrroloquinoline quinone or a salt thereof with an ascorbic acid analog in a solvent, such as, for example, by mixing a solution of pyrroloquinoline quinone or a salt thereof with a solution of an ascorbic acid analog, or adding an ascorbic acid analog to a solution of pyrroloquinoline quinone or a salt thereof to reduce the pyrroloquinoline quinone, resulting in reduced pyrroloquinoline quinone. 
         [0036]    In the step mentioned above, also included is an embodiment where a solution comprising pyrroloquinoline quinone or a salt thereof and an ascorbic acid analog is orally administered to a mammal, and the reaction is allowed to occur in vivo (for example, oral cavity, stomach, and intestine). 
         [0037]    “Mixing” as used herein can be accomplished by adding one object to be mixed to the other object to be mixed, or adding the objects to be mixed to a separate vessel. 
         [0038]    For “adding” as used herein, an additive may be added at once or gradually to an object to which the additive is to be added. 
         [0039]    In this reaction, the molar ratio of pyrroloquinoline quinone or a salt thereof to an ascorbic acid analog may vary depending on the content of the desired reduced form of PQQ, assuming an equimolar reaction of pyrroloquinoline quinone or a salt thereof with an ascorbic acid analog. In practical terms, the ascorbic acid analog is preferably used in an amount of 0.5 to 1000-fold moles with respect to PQQ, and in the case of isolation of reduced PQQ, preferably 0.9 to 10-fold moles. When the ascorbic acid analog is used in an amount lower than this range, the content of the reduced form of PQQ is low, so that no desired effect can be produced. Using the ascorbic acid analog in an amount beyond the range may not cause trouble but leads to higher cost. 
         [0040]    In the production method according to the present invention, the molar ratio of an ascorbic acid analog with respect to pyrroloquinoline quinone or a salt thereof may be, in a solvent, 1:0.5 to 1000, preferably 1:1.5 to 1000, more preferably 1:1.5 to 100, and even more preferably 1:1.5 to 10. 
         [0041]    In the production method according to the present invention, the reaction can be carried out at any temperature, for example, a temperature of −10° C. to 180° C., and preferably of 0° C. to 100° C. The reaction proceeds even at room temperature. Preferably, if the rate of reaction is to be increased, then the temperature is raised, and in order to increase the throughput, preferred is a temperature of 50° C. or more, due to higher solubility at higher temperature. 
         [0042]    In the production method according to the present invention, the reaction can be carried out for any period of time, for example, for 0.2 to 48 hours, and preferably for 0.5 to 24 hours. 
         [0043]    The reaction may be carried out in any solvent that allows the reaction to proceed, and preferably in an aqueous solution that is not highly problematic even when it remains in the product. The reaction is carried out particularly in an aqueous solution having a pH of 5 or less, and more preferably of 4 or less, since the reaction may not proceed easily under alkaline conditions. It is not particularly problematic that acid or alkaline is used to adjust the pH of the solution. Acid or alkaline may be used, if necessary. 
         [0044]    In the production method according to the present invention, the reaction is not particularly limited, but is preferably carried out in a low oxygen atmosphere. To prevent oxygen in the air from converting reduced PQQ into oxidized PQQ, the operation mentioned above can be carried out in an inert gas atmosphere such as nitrogen and argon according to conventional methods. Reduction of the oxidation reaction can be achieved by replacement with inert gas, reduced pressure, boiling, or any combination thereof. It is advisable to at least use replacement with inert gas, i.e. an inert gas atmosphere. The inert gas mentioned above can include, for example, nitrogen gas, helium gas, argon gas, hydrogen gas, carbonic acid gas and the like, and preferred is nitrogen gas. 
         [0045]    In the production method according to the present invention, the pH of a solution (reaction solution) obtained by reacting a solution of pyrroloquinoline quinone or a salt thereof with an ascorbic acid analog can be adjusted to 3.5 or less by adjustment of the pH of the solution of pyrroloquinoline quinone or a salt thereof and the amount of the ascorbic acid analog, or through a pH adjustment step using an acidic substance (for example, hydrochloric acid) or an alkaline substance (for example, sodium hydroxide). 
         [0046]    The pH of the resultant solution can be adjusted to 3.5 or less, preferably 3 or less, more preferably 2.8 or less, and even more preferably 2.6 or less. 
         [0047]    More specifically, the production method according to the present invention may be carried out as follows: 
         [0048]    Pyrroloquinoline quinone is prepared in an aqueous solution having a concentration of 0.01 g/L to 15 g/L. This concentration range is described as a range in which pyrroloquinoline quinone is dissolved, and beyond this range, the reaction tends to proceed in suspension. The reaction is completed by a simple procedure of adding the ascorbic acid in powder or solution thereto. The reaction temperature is usually from 0° C. to 100° C. The reaction time varies depending on the reaction temperature, and a reaction time of about 0.2 to about 48 hours is easily used. 
         [0049]    At this time, acid or base may be added if the pH is to be adjusted. 
         [0050]    In the solution obtained through the reaction, the reduced form of PQQ is eventually precipitated. The resultant precipitate can be reduced pyrroloquinoline quinone. 
         [0051]    The term “precipitate” as used herein refers to a solid phase (solid) formed from a liquid phase (solution). 
         [0052]    The precipitate can be separated from the solution. The precipitate (the reduced form of PQQ precipitated) can be obtained by filtration, centrifugation, or decantation. In addition, the obtained precipitate may also be washed with water or alcohol. It may also be then dried under reduced pressure to obtain a solid product. Alternatively, the precipitate can be provided as is, without such separation operations. 
         [0053]    In addition, in the case where a purer substance is required, recrystallization enables the purity of the substance to be improved. Recrystallization may be carried out by operations of dissolving the substance in a good solvent such as dimethyl sulfoxide, and reducing the solubility of the solution, for example, by lowering the temperature of the solution, adding a poor solvent to the solution, or concentrating the solution. In addition, the substance can also be purified by column chromatography. 
         [0054]    In the production method of reduced pyrroloquinoline quinone according to the present invention, the reaction proceeds well at near room temperature. Therefore, a composition containing 0.5 to 1000-fold moles of an ascorbic acid analog with respect to the number of moles of the pyrroloquinoline quinone or salt thereof can be provided to produce reduced pyrroloquinoline quinone in a vessel including a cup immediately before intake, or in the oral cavity, stomach, and intestine in vivo. 
         [0055]    A composition containing 0.5 to 1000-fold moles of an ascorbic acid analog with respect to the number of moles of the pyrroloquinoline quinone or salt thereof can be made to use for the purposes mentioned above, since the reaction proceeds even at room temperature, which is consistent with the object of the present invention. If this composition, whether in a solid form or a solution form, is orally administered, then the composition turns to a solution state and reacts. 
         [0056]    According to a preferred embodiment of the production method of the present invention, there is provided a production method of reduced pyrroloquinoline quinone, comprising a step of mixing a solution of pyrroloquinoline quinone or a salt thereof having a pH of 2 to 3.5 with an ascorbic acid analog or a solution thereof to obtain a solution having a pH of 3 or less comprising reduced pyrroloquinoline quinone. 
         [0057]    According to a more preferred embodiment of the production method of the present invention, there is provided a production method of reduced pyrroloquinoline quinone, comprising a step of mixing a solution of pyrroloquinoline quinone or a salt thereof having a pH of 2 to 2.5 with an ascorbic acid analog or a solution thereof to obtain a solution having a pH of 2.6 or less comprising reduced pyrroloquinoline quinone. 
         [0058]    According to the present invention, there is provided reduced pyrroloquinoline quinone produced by the production method according to the present invention. 
         [0059]    Reduced pyrroloquinoline quinone is susceptible to oxidation, and strict control to prevent its oxidation is therefore necessary for storage after separation and purification. It may be stored in a reducing atmosphere to prevent oxidation. However, conventional reducing agents are often toxic and are unable to be used. In the present invention, oxidation can be prevented by forming a composition containing ascorbic acid in molar ratios of 0.5 to 1000 with respect to reduced pyrroloquinoline quinone, which enables stable storage without performing purification and provision as it is. This is one of advantages of the production method of the present invention. 
         [0060]    According to the present invention, to store reduced pyrroloquinoline quinone stably, reduced pyrroloquinoline quinone can be provided as a solution containing an ascorbic acid analog. 
         [0061]    Also, after the separation operation, an ascorbic acid analog can be added to form a composition. This composition can be provided in a state of a solid, or suspension or solution, and the composition in suspension in water can be easily provided based on the reaction conditions. 
         [0062]    According to the present invention, there is provided a stabilization method of reduced pyrroloquinoline quinone. The stabilization method according to the present invention is characterized by protecting reduced pyrroloquinoline quinone from oxidation by molecular oxygen. The stabilization method according to the present invention can stabilize reduced pyrroloquinoline quinone by making reduced pyrroloquinoline quinone or a salt thereof present in a solvent together with an ascorbic acid analog. 
         [0063]    In the stabilization method according to the present invention, the molar ratio of an ascorbic acid analog with respect to reduced pyrroloquinoline quinone can be 1:0.5 to 1000, and preferably 1:1 to 500 in a solvent. 
         [0064]    The reduced pyrroloquinoline quinone obtained by the present invention may take any dosage form, which can be selected appropriately depending on use applications. A composition for oral ingestion of the present invention can be used for humans or animals as foods, functional foods, pharmaceuticals or quasi drugs. Functional foods as used here mean foods taken for the purpose of maintenance of health or nutrition as an alternative to meals, such as health foods, nutrients, foods with nutrient function claims, and nutrient health food. Specific dosage forms include, but are not limited to, capsules, tablets, chewables, tablets, and drinks. 
         [0065]    In the commercialization as functional foods, additives used for foods, for example, sweeteners, colorants, preservatives, thickening agents, antioxidants, color fixatives, bleaching agents, antibacterial and antifungal agents, gum bases, bittering agents, enzymes, glazing agents, acidifiers, seasoning, emulsifiers, enrichments, manufacturing agents, flavoring agents, spice extracts and the like can be used. It is generally possible to add to usual foods, for example, miso, soy sauce, instant miso soup, ramen, yakisoba, curry, corn soup, mabo-dofu, mabo-nasu, pasta sauce, pudding, cake, bread and the like. 
         [0066]    According to the present invention, there is provided a composition to produce reduced PQQ. 
         [0067]    According to the present invention, there is provided a composition to produce reduced PQQ which works strongly against oxidation, and can be stored stably. 
         [0068]    According to the present invention, the following inventions are also provided: 
         [0000]    (1) a production method of reduced pyrroloquinoline quinone, comprising mixing pyrroloquinoline quinone with ascorbic acid in a solvent;
 
(2) the production method of (1), wherein the solvent is water.
 
(3) the production method of (1) or (2), wherein the mixed solution has a pH of 5 or less;
 
(4) the production method of any one of (1) to (3), wherein 0.5 to 1000-fold moles of ascorbic acid are used with respect to the number of moles of the pyrroloquinoline quinone;
 
(5) a composition containing ascorbic acid in molar ratios of 0.5 to 1000 with respect to pyrroloquinoline quinone;
 
(6) the composition of (5), wherein pyrroloquinoline quinone is in the reduced form.
 
       EXAMPLES 
       [0069]    The present invention will now be described more specifically with reference to the following examples and comparative examples, but is not intended to be limited thereto. In addition, all percentages in the context of the present invention are by weight, unless otherwise stated. 
         [0070]    In Examples and Comparative Examples, the  13 C-NMR measurements were performed on a JEOL 500 MHz NMR, JNM-ECA500 Spectrometer at room temperature. 
         [0071]    In Examples and Comparative Examples, the UV measurements were performed on a HITACHI U-2000 Spectrophotometer. 
       Confirmatory Experiment for Reduction Reaction 
     Example 1 
       [0072]    A reagent (trade name: BioPQQ) from MITSUBISHI GAS CHEMICAL COMPANY, INC. was used as a raw material pyrroloquinoline quinone disodium. L-ascorbic acid was available from Wako Pure Chemical Industries, Ltd. 
         [0073]    In 500 g of water, 1.53 g of pyrroloquinoline quinone disodium was dissolved to a concentration of 0.01 mol/L. In 100 g of water, 3.51 g of L-ascorbic acid was dissolved to a concentration of 0.2 mol/L. 
         [0074]    20 g of a pyrroloquinoline quinone disodium solution having a concentration of 0.01 mol/L (pH 3.5) was mixed with 20 g of an L-ascorbic acid solution having a concentration of 0.2 mol/L (pH 2.5). The molar ratio of L-ascorbic acid is 20 with respect to pyrroloquinoline quinone. These two solutions were mixed at room temperature, and reacted at 70° C. for 2 hours (pH 2.5). After the reaction, a solid was precipitated. To this solution, hydrochloric acid was added to adjust the value of pH to 1 or less. This was centrifuged at 1000 rpm for 10 minutes, and the supernatant was removed to obtain a solid. The resultant solid was washed with a degassed aqueous hydrochloric acid solution, and dried under nitrogen gas stream. Heavy dimethyl sulfoxide was added thereto, and then this solution was transferred to an NMR tube under nitrogen gas stream, and the  13 C-NMR measurements were performed at room temperature. 
         [0075]    The results obtained were as follows: 105.7, 111.0, 119.4, 122.9, 123.6, 128.1, 131.3, 134.2, 137.8, 140.9, 142.6, 162.2, 165.5, 170.1 ppm (DMSO-d6: reference 39.5 ppm). 
         [0076]    These values are in agreement with those for the reduced form described in non-patent document 5 and formation of the reduced form was confirmed. Moreover, peaks at 173.3 and 178.0 ppm attributable to the quinone structure were absent among these measured data. 
       Comparative Example 1 
       [0077]    To a saturated aqueous solution of pyrroloquinoline quinone disodium, hydrochloric acid was added to adjust the value of pH to 1 or less. There was a red solid precipitated, which was passed through a filter to obtain pyrroloquinoline quinone in the free form. Heavy dimethyl sulfoxide was added thereto, and then this solution was transferred to an NMR tube, and the  13 C-NMR measurements were performed at room temperature. 
         [0078]    The results obtained were as follows: 113.5, 124.5, 126.4, 127.6, 129.2, 134.3. 136.3, 146.8, 148.7, 160.9, 164.9, 168.7, 173.3, 178.0 ppm (DMSO-d6: reference 39.5 ppm). Unlike the peaks in Example 1, no peaks were observed that should appear for the reduced PQQ. 
       Example 2 
       [0079]    20 g of a pyrroloquinoline quinone disodium solution having a concentration of 0.01 mol/L (pH 3.5) was mixed with 10 g of an L-ascorbic acid solution having a concentration of 0.2 mol/L (pH 3). The molar ratio of L-ascorbic acid is 10 with respect to pyrroloquinoline quinone. These two solutions were mixed and stirred at room temperature. The color of the mixture immediately changed, and a solid was precipitated. After allowing the solution to stand at room temperature overnight, it had a pH of 3 as measured with a pH test strip. The solid was separated through centrifugation (1000 rpm, 10 minutes), and dried at reduced pressure to obtain 0.073 g of a solid of the reduced form containing water free from the oxidized form. Analysis of UV spectra showed that the solid was in the reduced state. 
       Example 3 
       [0080]    20 g of a pyrroloquinoline quinone disodium solution having a concentration of 0.01 mol/L (pH 3.5) was mixed with 5 g of an L-ascorbic acid solution having a concentration of 0.2 mol/L (pH 3). The molar ratio of L-ascorbic acid is 5 with respect to pyrroloquinoline quinone. These two solutions were mixed and stirred at room temperature. The color of the mixture immediately changed, and a solid was precipitated. After allowing the solution to stand at room temperature overnight, it had a pH of 3 as measured with a pH test strip. The solid was separated through centrifugation (1000 rpm, 10 minutes), and dried at reduced pressure to obtain 0.044 g of a solid of the reduced form. The isolated crude yield was 66%. 
       Example 4 
       [0081]    20 g of a pyrroloquinoline quinone disodium solution having a concentration of 0.001 mol/L (pH 3.5) was mixed with 10 g of an L-ascorbic acid solution having a concentration of 0.2 mol/L (pH 2). The molar ratio of L-ascorbic acid is 100 with respect to pyrroloquinoline quinone. These two solutions were mixed and stirred at room temperature. The color of the mixture immediately changed, and a solid was precipitated. After allowing the solution to stand at room temperature overnight, it had a pH of 2 as measured with a pH test strip. The solid was separated through centrifugation (1000 rpm, 10 minutes), and dried at reduced pressure to obtain 0.006 g of a solid of the reduced form. 
       Example 5 
       [0082]    0.5 g of a pyrroloquinoline quinone disodium solution having a concentration of 0.01 mol/L (pH 3.5) was mixed with 0.5 g of a sodium L-ascorbate solution having a concentration of 0.5 mol/L. The pH of the solution at this time was in a range of 6 to 7. The molar ratio of L-ascorbic acid is 50 with respect to pyrroloquinoline quinone. These two solutions were mixed at room temperature, and this mixed solution was added to 10 ml of simulated gastric fluid (pH 1.2). After allowing the solution to stand at room temperature overnight, a solid of the reduced form was precipitated. By bringing the solution into acidic conditions, the reaction tends to proceed easily. Also, it is considered that the reduction takes place even in vivo. 
       Example 6 
       [0083]    1 g of a pyrroloquinoline quinone disodium solution having a concentration of 0.01 mol/L (pH 3.5) was mixed with 1 g of an L-ascorbic acid solution having a concentration of 0.011 mol/L (pH 2.5). The molar ratio is 1:1.1 in this case. After the reaction at 30° C. for eight days (pH 2.5), 82% of PQQ reacted and a substance containing the reduced form was obtained. 
       Comparative Example 2 
       [0084]    0.5 g of a pyrroloquinoline quinone disodium solution having a concentration of 0.01 mol/L was mixed with 0.5 g of a sodium L-ascorbate solution having a concentration of 0.5 mol/L at room temperature. The pH of the solution was in the range of 6 to 7 in this case. The molar ratio of L-ascorbic acid is 50 with respect to pyrroloquinoline quinone. When the mixed solution was allowed to stand at room temperature for three days, the solution did not change (pH 7), and the reduced form was not obtained. 
       Storage Test 
     Example 7 
       [0085]    In 500 g of water, 1.53 g of pyrroloquinoline quinone disodium was dissolved to a concentration of 0.01 mol/L. In 100 g of water, 3.51 g of L-ascorbic acid was dissolved to a concentration of 0.2 mol/L. 
         [0086]    20 g of the pyrroloquinoline quinone disodium solution mentioned above having a concentration of 0.01 mol/L (pH 3.5) was mixed with 20 g of an L-ascorbic acid solution having a concentration of 0.2 mol/L at room temperature. The molar ratio of L-ascorbic acid is 20 with respect to pyrroloquinoline quinone. The pH of the solution was 2 as measured with a pH test strip. The mixed solution was allowed to react at room temperature overnight (pH 2). After the reaction, a solid of the reduced form was precipitated. Under this condition, it is believed that at least 15-fold moles of L-ascorbic acid is present, even allowing for the amounts consumed in the reduction reaction and in air oxidization. 
         [0087]    Subsequently, after the solution was stored at room temperature for one week, it was maintained in the reduced state. 
       Comparative Example 3 
       [0088]    The solid of the reduced form obtained in Example 6 was separated through centrifugation. The supernatant was removed, and the solid was washed with water. This process removed L-ascorbic acid. An aqueous sodium hydroxide solution was added thereto to adjust the value of pH to 7. All the reduced form was converted to the oxidized form in 30 minutes. 
       Example 8 
       [0089]    1 g of a pyrroloquinoline quinone disodium solution having a concentration of 0.01 mol/L (pH 3.5) was mixed with 1 g of an L-ascorbic acid solution having a concentration of 0.014 mol/L. When 1 g of this mixed solution was transferred to a 15-ml container and brought into contact with air, the solution included 14% of oxidized PQQ (pH 3). After this oxidized PQQ-containing reduced PQQ was left at 30° C. for one week, 18% of oxidized PQQ was included. 
       Confirmatory Experiment for pH Conditions 
     Example 9 
       [0090]    Pyrroloquinoline quinone disodium was dissolved in water and the concentration was adjusted to 2 g/L. The pH of the aqueous solution was adjusted to 1 or less by using hydrochloric acid and NaOH. Also, L-ascorbic acid was dissolved in water and the concentration was adjusted to 4 g/L. 
         [0091]    A 0.5 mL aliquot of each of the two aqueous solutions mentioned above was mixed in a 1.5-mL container, and the resultant mixture was reacted at room temperature for 24 hours. The pH after the reaction was 1. Subsequently, the resultant reaction solution was subjected to centrifugation (1000 rpm, 10 minutes), and the supernatant was removed to obtain a solid. The resultant solid was washed using hydrochloric acid having a concentration of about 9%, and dissolved in dimethyl sulfoxide. UV measurements were performed and the yield of the reduced form was calculated to be 68%. 
       Example 10 
       [0092]    Pyrroloquinoline quinone disodium was dissolved in water and the concentration was adjusted to 2 g/L. The pH of the aqueous solution was adjusted to 2.2 by using hydrochloric acid and NaOH. Also, L-ascorbic acid was dissolved in water and the concentration was adjusted to 4 g/L. 
         [0093]    A 0.5 mL aliquot of each of the two aqueous solutions mentioned above was mixed in a 1.5-mL container, and the resultant mixture was reacted at room temperature for 24 hours. The pH after the reaction was 2.6. Subsequently, the resultant reaction solution was subjected to centrifugation (1000 rpm, 10 minutes), and the supernatant was removed to obtain a solid. The resultant solid was washed with hydrochloric acid having a concentration of about 9%, and dissolved in dimethyl sulfoxide. UV measurements were performed and the yield of the reduced form was calculated to be 100%. 
       Example 11 
       [0094]    Pyrroloquinoline quinone disodium was dissolved in water and the concentration was adjusted to 2 g/L. The pH of the aqueous solution was adjusted to 2.6 using hydrochloric acid and NaOH. Also, L-ascorbic acid was dissolved in water and the concentration was adjusted to 4 g/L. 
         [0095]    A 0.5 mL aliquot of each of the two aqueous solutions mentioned above was mixed in a 1.5-mL container, and the resultant mixture was reacted at room temperature for 24 hours. The pH after the reaction was 3. Subsequently, the resultant reaction solution was subjected to centrifugation (1000 rpm, 10 minutes), and the supernatant was removed to obtain a solid. The resultant solid was washed using hydrochloric acid having a concentration of about 9%, and dissolved in dimethyl sulfoxide. UV measurements were performed and the yield of the reduced form was calculated to be 66%. 
       Example 12 
       [0096]    Pyrroloquinoline quinone disodium was dissolved in water and the concentration was adjusted to 2 g/L. The pH of the aqueous solution was adjusted to 3.5 using hydrochloric acid and NaOH. Also, L-ascorbic acid was dissolved in water and the concentration was adjusted to 4 g/L. 
         [0097]    A 0.5 mL aliquot of each of the two aqueous solutions mentioned above was mixed in a 1.5-mL container, and the resultant mixture was reacted at room temperature for 24 hours. The pH after the reaction was 3.5. Additionally, 100 μL of concentrated hydrochloric acid was added to bring the pH of the solution to 1. Subsequently, the resultant mixed solution was subjected to centrifugation (1000 rpm, 10 minutes), and the supernatant was removed to obtain a solid. The resultant solid was washed using hydrochloric acid having a concentration about 9%, and dissolved in dimethyl sulfoxide. UV measurements were performed and the yield of the reduced form was calculated to be 54%. 
       Comparative Example 4 
       [0098]    Pyrroloquinoline quinone disodium was dissolved in water and the concentration was adjusted to 2 g/L. The pH of the aqueous solution was adjusted to 4.3 using hydrochloric acid and NaOH. Also, L-ascorbic acid was dissolved in water and the concentration was adjusted to 4 g/L. 
         [0099]    A 0.5 mL aliquot of each of the two aqueous solutions mentioned above was mixed in a 1.5-mL container, and the resultant mixture was reacted at room temperature for 24 hours. The pH after the reaction was 3.5. Subsequently, the resultant reaction solution was subjected to centrifugation (1000 rpm, 10 minutes), and the supernatant was removed to obtain a solid. The resultant solid was washed using hydrochloric acid having a concentration of about 9%, and dissolved in dimethyl sulfoxide. UV measurements were performed and the yield of the reduced form was calculated to be 1%. 
       Comparative Example 5 
       [0100]    Pyrroloquinoline quinone disodium was dissolved in water and the concentration was adjusted to 2 g/L. The pH of the aqueous solution was adjusted to 7.6 using hydrochloric acid and NaOH. Also, L-ascorbic acid was dissolved in water and the concentration was adjusted to 4 g/L. 
         [0101]    A 0.5 mL aliquot of each of the two aqueous solutions mentioned above was mixed in a 1.5-mL container, and the resultant mixture was reacted at room temperature for 24 hours. The pH after the reaction was 3.5. Subsequently, the resultant reaction solution was subjected to centrifugation (1000 rpm, 10 minutes), and the supernatant was removed to obtain a solid. The resultant solid was washed using hydrochloric acid having a concentration of about 9%, and dissolved in dimethyl sulfoxide. UV measurements were performed and the yield of the reduced form was calculated to be 9%.