Crystal form of pyrroloquinoline quinone disodium salt, preparation method therefor and use thereof

Abstract

Provided are a crystal form of a pyrroloquinoline quinone disodium salt, and a preparation method therefor. The pyrroloquinoline quinone disodium salt crystal compound measured with Cu-Kα rays has diffraction characteristic peaks at least at the following 2θ angles: 8.279, 16.599, 22.558, 23.219, 25.621, 27.26 and 47.499, the error of the 2θ angles being ± 0.2°. The crystal form compound has high crystallinity, high purity, high compression density, high stability and almost no hygroscopicity, and is easy to process, suitable for large-scale production and free of solvent residues, and the preparation method is environmental-friendly and pollution-free.

Description

Pyrroloquinoline quinone disodium salt crystal form and preparation method and application thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application No. 202311264114.2 filed with the State Intellectual Property Office of China on September 27, 2023, entitled “Pyrroloquinoline quinone disodium salt crystal form, preparation method and application thereof”, the entire contents of which are incorporated by reference into the present invention and constitute a part of the present invention for all purposes. Technical Field

[0003] The present application relates to the field of organic compound preparation, and specifically to a pyrroloquinoline quinone disodium salt crystal form, a preparation method, and an application thereof. Background Art

[0004] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0005] Pyrroloquinoline quineone (PQQ), whose chemical name is 4,5-dihydro-4,5-dioxo-1-H-pyrrolo(2,3-f)quinoline-2,7,9-tricarboxylic acid, also known as pyrroloquinoline quinone, was originally discovered to be produced by Gram-negative bacteria. It has a wide range of nutritional effects on microorganisms, animals and plants, and is a nutrient element with antioxidant properties.

[0006] In vivo, pyrroloquinoline quinone is a cofactor involved in redox reactions, serving as a coenzyme for many enzymes, such as dehydrogenases, oxidases, hydratases, and decarboxylases. Pyrroloquinoline quinone contains an o-quinone structure that can directly participate in redox reactions. These enzyme proteins can catalyze the oxidation of non-phosphorylated substrates, such as alcohols, aldehydes, and aldoses. Existing research indicates that pyrroloquinoline quinone can protect the heart from oxidative damage, prevent liver damage, promote nerve growth and protect the nervous system, and prevent acetaldehyde overdose.

[0007] Pyrroloquinoline quinone can be produced through chemical synthesis and fermentation. Pyrroloquinoline quinone disodium salt is regulated as a "Generally Recognized as Safe" (GRAS) substance in the United States and can be used as an ingredient in foods such as energy drinks, sports drinks, and electrolyte drinks. It is also approved as a dietary supplement or natural health food in the European Union and Canada. Currently, chemically synthesized pyrroloquinoline quinone has been approved as a national new food ingredient and can be used in beverages for all populations except infants, pregnant women, and lactating women. The safety assessment materials for pyrroloquinoline quinone disodium salt have been reviewed and approved, and its application as a new food ingredient has been publicly accepted. Commercial products are currently on the market. Pyrroloquinoline quinone disodium salt obtained by existing methods generally contains two waters of crystallization. Existing crystalline forms are unstable and highly hygroscopic. Within the room temperature storage range, humidity fluctuates significantly, resulting in an unsatisfactory solid state and unsuitable processing, which hinders the application and storage of pyrroloquinoline quinone sodium salt.

[0008] Summary of the Invention

[0009] Therefore, the purpose of this application is to provide a crystalline form of pyrroloquinoline quinone disodium salt and a preparation method thereof. The crystalline form of pyrroloquinoline quinone disodium salt provided by the present invention has the advantages of high crystallinity, high purity, high stability, high compacted density, and almost no hygroscopicity. It is easy to process and suitable for large-scale production, has no solvent residue, and the preparation method is environmentally friendly and pollution-free.

[0010] Specifically, the present invention provides the following technical solutions.

[0011] In a first aspect of the present invention, a pyrroloquinoline quinone disodium salt crystalline compound is provided, which has diffraction characteristic peaks at at least the following 2θ angles measured by Cu-Kα rays: 8.279, 16.599, 22.558, 23.219, 25.621, 27.26, 47.499, wherein the error of the 2θ angle is ±0.2°.

[0012] Furthermore, the pyrroloquinoline quinone disodium salt crystalline compound of the present invention has diffraction characteristic peaks at at least the following 2θ angles when measured by Cu-Kα rays: 8.279, 16.599, 18.722, 19.136, 22.558, 23.219, 25.621, 26.061, 27.2629.721, 30.28, 33.579, 37.581, 38.819, 42.143, 47.499, wherein the error of the 2θ angle is ±0.2°.

[0013] Furthermore, the X-ray powder diffraction pattern of the pyrroloquinoline quinone disodium salt crystalline compound of the present invention is shown in FIG1 .

[0014] According to the TGA curve of the pyrroloquinoline quinone disodium salt crystalline compound of the present invention, it loses weight twice in the range of 80-400° C., and its decomposition peak temperature is 308.8° C.

[0015] Furthermore, the TGA curve of the pyrroloquinoline quinone disodium salt crystalline compound of the present invention is shown in FIG2 .

[0016] The pyrroloquinoline quinone disodium salt crystalline compound of the present invention can be confirmed by the above-mentioned data such as X-ray powder diffraction patterns. Those skilled in the art will understand that the experimental error depends on the conditions of the instrument, the preparation of the sample and the purity of the sample. In particular, it is well known to those skilled in the art that, for example, the X-ray powder diffraction pattern will usually change with the conditions of the instrument. In addition, the experimental error of the peak angle is usually 5% or less, and the error of these angles should also be taken into account, and an error of ±0.2° is usually allowed. In addition, due to the influence of experimental factors such as sample height, the overall deviation of the peak angle will be caused, and a certain deviation is usually allowed. Therefore, those skilled in the art will understand that any crystal form having a pattern that is the same or similar to the characteristic peaks in the spectrum of the present invention falls within the scope of the present invention.

[0017] In a second aspect of the present invention, a method for preparing the pyrroloquinoline quinone disodium salt crystalline compound described in the first aspect is provided, comprising: mixing pyrroloquinoline quinone acid and water, adding a base at 25-30°C, heating to 70-80°C, and filtering after complete dissolution; cooling the filtrate to below 30°C, adjusting the pH to 3.4-4.5 while stirring, stopping the reaction, allowing to stand, and filtering to obtain a solid wet product, adding purified water for recrystallization, cooling to below 30°C, filtering, and drying to obtain the solid.

[0018] Alternatively, in some embodiments of the present invention, the method comprises: mixing pyrroloquinoline quinone disodium salt and water, adding alkali at 25-30°C, heating to 70-80°C, and filtering after complete dissolution; cooling the filtrate to below 20°C, adjusting the pH to 3.4-4.5 while stirring, stopping the reaction, standing and filtering to obtain a solid wet product, adding purified water for recrystallization, cooling to below 30°C, filtering and drying to obtain the product.

[0019] In some embodiments of the present invention, the base is selected from one or more of sodium hydroxide, sodium bicarbonate and sodium carbonate.

[0020] In some embodiments of the present invention, the pH is adjusted to 3.4-4.5 by adding dry ice or introducing carbon dioxide under stirring; in these embodiments, the pH can be adjusted to 4.0-4.2, 3.4-4.0, 3.5-4.0, 3.8-4.0 or 3.5-4.5, etc.

[0021] In some embodiments of the present invention, the drying temperature is 50-70°C and the vacuum degree is 0.06-0.08 MPa; for example, in some embodiments, the drying temperature may be 50-60°C, 55-65°C or 60-70°C.

[0022] In the third aspect of the present invention, a composition is provided, which comprises the pyrroloquinoline quinone disodium salt crystalline compound described in the first aspect.

[0023] In a fourth aspect of the present invention, a product is provided, which comprises the pyrroloquinoline quinone disodium salt crystalline compound described in the first aspect or a composition comprising the pyrroloquinoline quinone disodium salt crystalline compound.

[0024] In some embodiments of the present invention, the products include medicines, foods, health products, and cosmetics.

[0025] In some embodiments of the present invention, the pyrroloquinoline quinone disodium salt crystalline compound can be used as a food additive, a cosmetic additive, etc., and added to food, health products, and cosmetics.

[0026] The food of the present invention is particularly a functional food, such as a beverage, which can be a solid beverage or a liquid beverage.

[0027] In a fifth aspect of the present invention, there is provided a use of the pyrroloquinoline quinone disodium salt crystalline compound or a composition comprising the pyrroloquinoline quinone disodium salt crystalline compound described in the first aspect in the preparation of a product having at least one of the following functions:

[0028] 1) Promote microbial growth;

[0029] 2) Promote plant growth;

[0030] 3) Antioxidant, including but not limited to scavenging free radicals, activating antioxidant enzymes, increasing the synthesis of antioxidant substances, inhibiting lipid peroxidation, etc.;

[0031] 4) Protect the heart from oxidative damage;

[0032] 5) Prevent and treat liver damage;

[0033] 6) Promote nerve growth and protect the nervous system;

[0034] 7) Prevent acetaldehyde poisoning; and

[0035] 8) Chelate metal ions, such as iron ions.

[0036] In some embodiments of the present invention, the products include but are not limited to food additives and foods containing the food additives, cosmetic additives and cosmetics containing the cosmetic additives, health products, drug carriers (for example, as carriers of some drugs, delivering and releasing drugs through coordination with pyrroloquinoline quinone disodium salt crystals), chemical analysis reagents (capable of performing chemical analysis such as metal ion detection), feed additives and feed containing the feed additives, etc.

[0037] In a sixth aspect of the present invention, a method is provided, comprising administering to a subject the pyrroloquinoline quinone disodium salt crystalline compound or a composition or product comprising the pyrroloquinoline quinone disodium salt crystalline compound described in the first aspect above, to achieve at least one of the following therapeutic or ameliorative effects:

[0038] 1) Promote microbial growth;

[0039] 2) Promote plant growth;

[0040] 3) Antioxidant, including but not limited to scavenging free radicals, activating antioxidant enzymes, increasing the synthesis of antioxidant substances, inhibiting lipid peroxidation, etc.;

[0041] 4) Protect the heart from oxidative damage;

[0042] 5) Prevent and treat liver damage;

[0043] 6) Promote nerve growth and protect the nervous system; and

[0044] 7) Prevent acetaldehyde poisoning.

[0045] The subject of the present invention is a plant, a microorganism or an animal, the animal is a mammal, especially a human; the plant is a food crop, an ornamental plant, and the like.

[0046] In the present invention, the effective amount required to achieve the above-mentioned functions can be routinely determined by those skilled in the art, taking into account their own knowledge, the state of the art, and other factors. For example, such amount can be determined or demonstrated through in vivo, in vitro, or ex vivo experiments, and then used to better implement the methods described herein. For example, in some embodiments of the present invention, when the pyrroloquinoline quinone disodium salt crystalline compound of the present invention is used as a food, its recommended dosage is ≤20 mg / day; for example, when used as a beverage, its added amount is ≤40 mg / kg.

[0047] Compared with the prior art, the advantages of the present invention include:

[0048] The present invention provides a new crystal form of pyrroloquinoline quinone disodium salt. The crystal form compound has the advantages of high crystallinity and high purity. In particular, compared with the existing pyrroloquinoline quinone disodium salt, it also has the advantages of high stability and high compaction density. It is almost non-hygroscopic, easy to process and suitable for large-scale production, has no solvent residue, and the preparation method is environmentally friendly and pollution-free. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The drawings constituting part of the present application are provided to provide a further understanding of the present application. The illustrative embodiments and their descriptions of the present application are provided to explain the present application and do not constitute an undue limitation on the present application. The following describes the implementation scheme of the present application in detail in conjunction with the drawings, wherein:

[0050] Figure 1: X-ray powder diffraction pattern of the pyrroloquinoline quinone disodium salt crystalline compound in Example 1.

[0051] Figure 2: Thermogravimetric analysis (TGA) diagram of the pyrroloquinoline quinone disodium salt crystalline compound in Example 1.

[0052] Figure 3: Micrographs (A, B, C) of the crystalline compound of pyrroloquinoline quinone disodium salt in Example 1. Images A, B, and C all show that the compound is in the form of flaky crystals.

[0053] Figure 4: X-ray powder diffraction pattern of the pyrroloquinoline quinone disodium salt crystalline compound in Comparative Example 1.

[0054] Figure 5: X-ray powder diffraction pattern of the pyrroloquinoline quinone disodium salt crystalline compound in Comparative Example 2.

[0055] Figure 6: X-ray powder diffraction pattern of the pyrroloquinoline quinone disodium salt crystalline compound in Comparative Example 3. DETAILED DESCRIPTION

[0056] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or as recommended by the manufacturer.

[0057] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in this area or in accordance with the product instructions. In addition, any methods and materials similar to or equivalent to those described herein can be applied to the present method. The preferred embodiments and materials described herein are for demonstration purposes only.

[0058] Analytical instruments and test conditions:

[0059] 1. XRD: Rigaku Ultma IV, copper target; scanning angle range (°) 5-90; scanning speed 5° / min.

[0060] 2. Thermogravimetric analysis: TGA German Netzsch STA449 F5, the test gas atmosphere is nitrogen, the specific test temperature range (℃) is 30-500, and the heating rate is 10℃ / min.

[0061] 3. Liquid chromatography conditions are as follows:

[0062] Liquid phase detection instrument: Shimadzu LC-20A

[0063] Chromatographic column: TCI Kaseisorb LC ODS 2000 (4.6 mm × 150 mm, 5 μm) or chromatographic column with equivalent performance.

[0064] Column temperature: 30°C

[0065] Flow rate: 1.0 mL / min.

[0066] Wavelength: 250nm.

[0067] Injection volume: 20 μL.

[0068] Mobile phase: Mobile phase A: 10 mmol / L potassium hydrogen phosphate-15 mmol / L tetrabutylammonium bromide mixed solution (pH 7.4); Mobile phase B: acetonitrile.

[0069] The raw materials used in the following examples and comparative examples can be purchased or obtained according to the conventional methods in this area. For example, pyrroloquinoline quinone acid or pyrroloquinoline quinone disodium salt can be prepared by organic chemical synthesis. Pyrroloquinoline quinone acid or pyrroloquinoline quinone disodium salt are crystallized and may contain impurities in the raw materials. For example, 6-amino-5-methoxy-1H-indole-2-carboxylic acid ethyl ester and dimethyl 2-oxopentaconedioate can be used as raw materials, and pyrroloquinoline quinone acid and pyrroloquinoline quinone disodium salt can be prepared by processes such as coupled ring reaction, formation of quinoline ring, oxidation reaction, ester hydrolysis reaction and refining.

[0070] Example 1

[0071] To a reaction flask, add 16.5g of pyrroloquinoline quinone acid and 330g of water. Slowly add 7g of sodium hydroxide at 25-30°C. After completion, heat the flask to 70-80°C and stir for 2h until the material is almost completely dissolved. Filter the filtrate hot with suction to obtain a filtrate. Cool the filtrate to below 30°C and, with stirring, introduce CO2 to the filtrate until the pH reaches 4.0-4.2. Stop the reaction. After standing for 2h, filter the filtrate with suction to obtain 36.2g of a wet solid. Add 362g of purified water to the wet solid for recrystallization. After completion, cool the filtrate to below 30°C and filter the solid to obtain 35.1g of pyrroloquinoline quinone disodium salt. Dry the wet solid in an oven to obtain 15.9g of pyrroloquinoline quinone disodium salt crystals I. Vacuum drying is performed at 60-70°C (vacuum 0.08MPa). The resulting crystals I were tested, and HPLC analysis revealed a purity of 99.9%. Na ion analysis revealed a mass ratio of pyrroloquinoline quinone to sodium of 1:1.98, indicating that the crystals were pyrroloquinoline quinone disodium salt. X-ray powder diffraction patterns, thermogravimetric analysis (TGA) patterns, and micrographs of this crystal form are shown in Figures 1-3 , and X-ray powder diffraction data are shown in Table 1 . Figure 2 shows that the decomposition peak temperature of this crystalline compound is 308.8°C. Figure 3 shows that this crystalline compound is a flaky crystal.

[0072] Table 1 X-ray powder diffraction data of pyrroloquinoline quinone disodium salt crystal Ⅰ

[0073] Example 2

[0074] 16.5g of pyrroloquinoline quinone acid and 330g of water were added to a reaction flask. 7g of sodium hydroxide was slowly added to the reaction flask at 25-30°C. After completion, the temperature was raised to 70-80°C and stirred for 2h. The material was basically completely dissolved and then hot filtered to obtain a filtrate. The filtrate was cooled to below 30°C and dry ice was added to the filtrate with stirring until the pH reached 3.4-4.0. The reaction was stopped. After standing for 2h, the filtrate was filtered to obtain 37.6g of a solid wet product. This wet product was recrystallized by adding 376g of purified water. After completion, the product was cooled to below 30°C and filtered to obtain 36.5g of a solid wet product of pyrroloquinoline quinone disodium salt. The wet product was dried in an oven to obtain 17.3g of pyrroloquinoline quinone disodium salt crystal form II. The vacuum drying temperature was 60-70°C (vacuum degree 0.08MPa). The resulting crystal II was tested and its purity was 99.6% by HPLC. Na ion analysis revealed that the mass ratio of pyrroloquinoline quinone to sodium in the crystals was 1:1.97, indicating that the crystals were pyrroloquinoline quinone disodium salt. The X-ray powder diffraction pattern of this crystal form was consistent with that of Example 1.

[0075] Example 3

[0076] 16.5g of pyrroloquinoline quinone acid and 330g of water were added to the reaction flask. 8.7g of sodium carbonate was slowly added to the reaction flask at 25-30°C. The temperature was raised to 70-80°C and stirred for 3h. The material was basically completely dissolved and hot filtered to obtain a filtrate. The filtrate was cooled to below 30°C and 3.2g of dry ice was added to the filtrate while stirring. After completion, the pH of the solution was 3.8-4.0. After standing for 2h, the solution was filtered to obtain 34.6g of solid material wet product. After washing with deionized water, the solution was filtered to obtain a solid material wet product of pyrroloquinoline quinone disodium salt. The wet product was added to 346g of purified water for recrystallization. After completion, the solution was cooled to below 30°C and filtered to obtain 32.8g of pyrroloquinoline quinone disodium salt solid material wet product. The wet product was dried in an oven to obtain 14.3g of pyrroloquinoline quinone disodium salt crystal form III. The vacuum drying temperature was 50°C-60°C (vacuum degree 0.08 MPa). The resulting crystal III was tested, and its purity was determined by HPLC to be 99.9%. Na ion analysis revealed that the mass ratio of pyrroloquinoline quinone to sodium in the crystals was pyrroloquinoline quinone:Na = 1:1.96, indicating that the crystals were pyrroloquinoline quinone disodium salt. X-ray powder diffraction of this crystal form was consistent with that of Example 1.

[0077] Example 4

[0078] 16.5 g of pyrroloquinoline quinone acid and 330 g of water were added to a reaction flask. 8.7 g of sodium carbonate was slowly added to the reaction flask at 25-30 ° C. The temperature was raised to 70-80 ° C and stirred for 3 h. The material was basically completely dissolved and then hot filtered to obtain a filtrate. The filtrate was cooled to below 30 ° C. CO2 was introduced into the filtrate with stirring until the pH of the solution was between 3.5 and 4.0. After standing for 2 h, it was filtered to obtain 32.4 g of a solid wet product. After washing with deionized water, it was filtered to obtain a solid wet product of pyrroloquinoline quinone disodium salt. This wet product was added to 324 g of purified water for recrystallization. After completion, it was cooled to below 30 ° C and filtered to obtain 31.9 g of a solid wet product of pyrroloquinoline quinone disodium salt. This wet product was dried in an oven to obtain 13.6 g of pyrroloquinoline quinone disodium salt crystal form IV. The vacuum drying temperature was 50°C-60°C (vacuum degree 0.08 MPa). The resulting crystal IV was tested, and its purity was determined by HPLC to be 99.8%. Based on Na ion analysis, the mass ratio of pyrroloquinoline quinone to sodium in the crystals was pyrroloquinoline quinone:Na=1:1.93, indicating that the crystals are pyrroloquinoline quinone disodium salt. The X-ray powder diffraction pattern of this crystal form was consistent with that of Example 1.

[0079] Example 5

[0080] 16.5 g of pyrroloquinoline quinone acid and 330 g of water were added to a reaction flask. 13.8 g of sodium bicarbonate was slowly added to the reaction flask at 25-30°C. The temperature was raised to 70-80°C and stirred for 3 hours until the material was substantially completely dissolved. The mixture was then hot filtered to obtain a filtrate. The filtrate was cooled to below 30°C and CO2 was introduced into the filtrate with stirring until the pH of the solution was between 3.5 and 4.0. After standing for 2 hours, the filtrate was filtered to obtain 33.5 g of a wet solid. After washing with deionized water, the solid was filtered to obtain a wet solid of pyrroloquinoline quinone disodium salt. This wet solid was recrystallized by adding 335 g of purified water. After completion, the solid was cooled to below 30°C and filtered to obtain 31.4 g of a wet solid of pyrroloquinoline quinone disodium salt. The wet solid was dried in an oven to obtain 12.9 g of pyrroloquinoline quinone disodium salt Form V. The vacuum drying temperature was 50°C-60°C (vacuum degree 0.08 MPa). The resulting crystal V was tested and its purity was 99.9% by HPLC. Na ion analysis revealed that the mass ratio of pyrroloquinoline quinone to sodium in the crystals was pyrroloquinoline quinone:Na = 1:1.92, indicating that the crystals were pyrroloquinoline quinone disodium salt. X-ray powder diffraction patterns of this crystal form were consistent with those of Example 1.

[0081] Example 6

[0082] 16.5 g of pyrroloquinoline quinone acid and 330 g of water were added to a reaction flask. 13.8 g of sodium bicarbonate was slowly added to the reaction flask at 25-30°C. The temperature was raised to 70-80°C and stirred for 3 hours until the material was substantially completely dissolved. The mixture was then hot filtered to obtain a filtrate. The filtrate was cooled to below 30°C and 3.5 g of dry ice was added to the filtrate with stirring until the pH of the solution was between 3.5 and 4.0. After standing for 2 hours, the solution was filtered to obtain 35.7 g of a solid wet product. After washing with deionized water, the solution was filtered to obtain a solid wet product of pyrroloquinoline quinone disodium salt. This wet product was recrystallized by adding 357 g of purified water. After completion, the solution was cooled to below 30°C and filtered to obtain 33.6 g of a solid wet product of pyrroloquinoline quinone disodium salt. The wet product was dried in an oven to obtain 15.2 g of pyrroloquinoline quinone disodium salt crystal form VI. The vacuum drying temperature was 50°C-60°C (vacuum degree 0.08 MPa). The resulting crystal VI was tested and its purity was 99.9% by HPLC. Na ion analysis revealed that the molar ratio of pyrroloquinoline quinone to sodium in the crystals was pyrroloquinoline quinone:Na = 1:1.94, indicating that the crystals were pyrroloquinoline quinone disodium salt. X-ray powder diffraction patterns of this crystal form were consistent with those of Example 1.

[0083] Example 7

[0084] To a reaction flask, add 18.7g of pyrroloquinoline quinone disodium salt and 380g of water. Slowly add 8.4g of sodium bicarbonate at 25-30°C. Heat to 70-80°C and stir for 3 hours until the material is substantially completely dissolved. Filter hot to obtain a filtrate. Cool the filtrate to below 20°C, introduce CO2 into the filtrate with stirring until the pH reaches between 3.5 and 4.5. After standing for 2 hours, filter to obtain 38.4g of a wet solid. Wash with deionized water and filter to obtain a wet solid of pyrroloquinoline quinone disodium salt. Add 384g of purified water to recrystallize the wet solid. Cool to below 30°C and filter to obtain 36.9g of a wet solid of pyrroloquinoline quinone disodium salt. Dry the wet solid in an oven to obtain 17.3g of crystalline Form VII of pyrroloquinoline quinone disodium salt. The vacuum drying temperature was 55°C-65°C (vacuum degree 0.06 MPa). The resulting crystal VII was tested and its purity was 99.94% by HPLC. Na ion analysis revealed that the mass ratio of pyrroloquinoline quinone to sodium in the crystals was pyrroloquinoline quinone:Na = 1:2.01, indicating that the crystals were pyrroloquinoline quinone disodium salt. The X-ray powder diffraction pattern of this crystal form was consistent with that of Example 1.

[0085] Example 8

[0086] 18.7 g of pyrroloquinoline quinone disodium salt and 380 g of water were added to a reaction flask. 10.6 g of sodium carbonate was slowly added to the reaction flask at 25-30°C. The temperature was raised to 70-80°C and stirred for 3 hours until the material was substantially completely dissolved. The mixture was then hot filtered to obtain a filtrate. The filtrate was cooled to below 20°C and 4.5 g of dry ice was added to the filtrate while stirring. The solution pH was between 3.5 and 4.5. After standing for 2 hours, the solution was filtered to obtain 37.7 g of a solid wet product. After washing with deionized water, the solution was filtered to obtain a solid wet product of pyrroloquinoline quinone disodium salt. This wet product was recrystallized by adding 377 g of purified water. After completion, the solution was cooled to below 30°C and filtered to obtain 36.3 g of a solid wet product of pyrroloquinoline quinone disodium salt. The wet product was dried in an oven to obtain 16.7 g of pyrroloquinoline quinone disodium salt crystal form VIII. The vacuum drying temperature was 55°C-65°C (vacuum degree 0.06 MPa). The resulting crystal VIII was tested and its purity was 99.96% by HPLC. Na ion analysis revealed that the molar ratio of pyrroloquinoline quinone to sodium in the crystals was pyrroloquinoline quinone:Na = 1:1.99, indicating that the crystals were pyrroloquinoline quinone disodium salt. The X-ray powder diffraction pattern of this crystal form was consistent with that of Example 1.

[0087] Example 9

[0088] 18.7 g of pyrroloquinoline quinone disodium salt and 380 g of water were added to a reaction flask. 4.0 g of sodium hydroxide was slowly added to the reaction flask at 25-30°C. The temperature was raised to 65-75°C and stirred for 3 hours until the material was substantially completely dissolved. The mixture was then hot filtered to obtain a filtrate. The filtrate was cooled to below 20°C and CO2 was introduced into the filtrate with stirring until the pH was between 3.5 and 4.5. After standing for 2 hours, the filtrate was filtered to obtain 37.3 g of a wet solid. After washing with deionized water, the solid was filtered to obtain a wet solid of pyrroloquinoline quinone disodium salt. This wet solid was recrystallized by adding 373 g of purified water. After completion, the solid was cooled to below 30°C and filtered to obtain 35.7 g of a wet solid of pyrroloquinoline quinone disodium salt. The wet solid was dried in an oven to obtain 16.8 g of pyrroloquinoline quinone disodium salt Form IX. The vacuum drying temperature was 55°C-65°C (vacuum degree 0.06 MPa). The resulting crystal IX was tested and its purity was 99.93% by HPLC. Na ion analysis revealed that the mass ratio of pyrroloquinoline quinone to sodium in the crystals was pyrroloquinoline quinone:Na = 1:1.98, indicating that the crystals were pyrroloquinoline quinone disodium salt. The X-ray powder diffraction pattern of this crystal form was consistent with that of Example 1.

[0089] Example 10

[0090] 18.7 g of pyrroloquinoline quinone disodium salt and 380 g of water were added to a reaction flask. 4.0 g of sodium hydroxide was slowly added to the reaction flask at 25-30°C. The temperature was raised to 65-75°C and stirred for 3 hours until the material was substantially completely dissolved. The mixture was then hot filtered to obtain a filtrate. The filtrate was cooled to below 20°C and 3.8 g of dry ice was added to the filtrate with stirring. The solution pH was between 3.5 and 4.5. After standing for 2 hours, the solution was filtered to obtain 36.5 g of a wet solid. The solid was washed with deionized water and filtered to obtain a wet solid of pyrroloquinoline quinone disodium salt. This wet solid was recrystallized by adding 365 g of purified water. After completion, the solid was cooled to below 30°C and filtered to obtain 34.9 g of a wet solid of pyrroloquinoline quinone disodium salt. The wet solid was dried in an oven to obtain 15.8 g of pyrroloquinoline quinone disodium salt crystal form X. The vacuum drying temperature was 55°C-65°C (vacuum degree 0.06 MPa). The resulting crystal X was tested and its purity was 99.91% by HPLC. Na ion analysis revealed that the molar ratio of pyrroloquinoline quinone to sodium in the crystals was 1:1.97, indicating that the crystals were pyrroloquinoline quinone disodium salt. The X-ray powder diffraction pattern of this crystal form was consistent with that of Example 1.

[0091] Comparative Example 1

[0092] Pyrroloquinoline quinone disodium salt was stirred and equilibrated with acetonitrile at 25°C and 50°C for 48 hours, filtered, and air-dried for 10 minutes to obtain pyrroloquinoline quinone disodium salt crystals. The X-ray powder diffraction pattern of the resulting crystals is shown in Figure 4, and the characteristic peaks of the resulting crystals are shown in Table 2. Consistent results were obtained using methanol as the solvent.

[0093] Table 2

[0094] Comparative Example 2

[0095] 60 g of pyrroloquinoline quinone trisodium salt was added to a mixture of 500 mL of water and 500 mL of ethanol. Hydrochloric acid was added with stirring at room temperature to adjust the pH to 3.5. The hydrochloric acid was added dropwise for 2 hours. After the pH stabilized, the mixture was filtered and dried under reduced pressure at room temperature for 16 hours to obtain pyrroloquinoline quinone disodium salt crystals. The X-ray powder diffraction pattern of the resulting crystals is shown in Figure 5, and the characteristic peaks of the resulting crystals are shown in Table 3.

[0096] Table 3

[0097] Comparative Example 3

[0098] 1.5 g of pyrroloquinoline quinone trisodium salt was added to 100 mL of water and dissolved under stirring at 30°C to obtain an aqueous solution. Filter the resulting filtrate, slowly add acetic acid to the aqueous solution while stirring, adjust the pH to 3.0, and crystallize to obtain a solid suspension. The solid was filtered, washed with water until neutral, and then dried under reduced pressure at 50°C for 24 hours to obtain pyrroloquinoline quinone disodium salt crystals. The X-ray powder diffraction spectrum of the resulting crystals is shown in Figure 6, and the characteristic peaks of the resulting crystals are shown in Table 4.

[0099] Table 4

[0100] Experimental example

[0101] 1. Hygroscopicity test:

[0102] Experimental method: Take about 5g of sample (taken from the products prepared in Examples 1-10 and Comparative Examples 1-3, respectively) and put it into a watch glass. The setting conditions of the constant temperature and humidity chamber are 30℃ and 75%RH. The weighed sample (calculated as W) is quickly placed in a constant temperature and humidity chamber with a temperature of 30℃ and a humidity of 75% for 48h and 96h to fully absorb moisture. After absorbing moisture for 48h, take out the material in the watch glass and weigh it as W1; after absorbing moisture for 96h, take out the material in the watch glass and weigh it as W2. The formula for calculating moisture absorption rate is: Moisture absorption rate (%) = (final mass - initial mass) / initial mass × 100%. The results are shown in Table 5.

[0103] Table 5

[0104] As can be seen from Table 5, the disodium salt crystals of Comparative Examples 1, 2 and 3 have a certain degree of hygroscopicity, while the disodium salt crystals provided by the present invention have almost no hygroscopicity in a high humidity environment, thus making it more widely applicable.

[0105] 2. Powder compaction density test

[0106] The compaction density of solid powder is measured by a powder compaction density meter. The unit volume of the powder in the container is measured after compaction under specified conditions. The unit is g / cm 3 The compacted density of a crystalline compound is an important physical parameter that describes the solid-state density of a crystalline compound. It is affected by the crystal structure and chemical composition, and is related to its structural characteristics, porosity, molecular arrangement, etc. It can reflect the dense packing state inside the crystal.

[0107] The specific detection methods are as follows:

[0108] (1) Open the powder compaction density meter and start the test to clear the mold. Set the fixed parameters as follows: constant pressure time 10s, mass 1g, vacuum time 10s, pressure 1 ton.

[0109] (2) After zeroing, weigh the sample accurately and place it in the mold (taken from the products prepared in Examples 1-10 and Comparative Examples 1-3) for analysis and testing. When adding the sample to the mold, first stack the weighing paper and pour the sample into the bottom as much as possible without sticking to the mold wall. When placing the mold, evacuate the air for about 10 seconds (to prevent pore clogging during compaction) and then place the mold in the experimental chamber.

[0110] (3) Start the compaction density test and obtain the compaction density data. After the instrument automatically completes the test, replace the demoulding mold, clean the mold, and then test the next sample.

[0111] (4) The sample data tested are shown in Table 6 below.

[0112] Table 6

[0113] Table 6 shows the compacted density of the pyrroloquinoline quinone disodium salt crystals prepared in various examples and comparative examples. According to the results, the compacted density of the pyrroloquinoline quinone disodium salt crystals prepared in Examples 1-10 of the present invention is significantly higher than that of the existing disodium salt crystal form, which shows that the pyrroloquinoline quinone disodium salt crystals of the present invention have the following advantages: 1) good stability, a large compacted density means that the crystal structure is more compact and relatively more stable, and is less likely to undergo polymorphic transformation, which is beneficial to ensuring the efficacy; 2) good fluidity, a large compacted density can improve the fluidity of the powder, which is beneficial to the transportation and mixing of the industrial production line; 3) good compressibility, and high-density crystals can obtain better The advantages of the present invention are as follows: 1) good compressibility, which is beneficial to the preparation, and higher hardness can be obtained after compression; 2) good shape retention, the high-density crystal structure is compact, and the preparations, especially tablets, are not easy to break or disintegrate after forming; good preparation uniformity, which is beneficial to the uniform dispersion of different ingredients and avoids drug separation; 3) high tableting yield, under the same pressure conditions, high compression density can increase the number of tablets per unit time, thereby improving production efficiency; 4) good granulation performance, high density is conducive to improving the efficiency of wet granulation; 5) smaller volume required in the preparation process, better mechanical processing performance in the preparation process, and higher compaction density can reduce the size of the capsule, which has the advantage of being easy to take.

[0114] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A pyrroloquinoline quinone disodium salt crystalline compound, which has diffraction characteristic peaks at least at the following 2θ angles measured by Cu-Kα rays: 8.279, 16.599, 18.722, 19.136, 22.558, 23.219, 25.621, 26.061, 27.26, 29.721, 30.28, 33.579, 37.581, 38.819, 42.143, 47.499, wherein the error of the 2θ angle is ±0.2°.

2. The pyrroloquinoline quinone disodium salt crystalline compound according to claim 1, characterized in that: Its X-ray powder diffraction pattern is shown in Figure 1.

3. The pyrroloquinoline quinone disodium salt crystalline compound according to claim 1, characterized in that: It loses weight twice in the temperature range of 80-400℃.

4. The pyrroloquinoline quinone disodium salt crystalline compound according to claim 1, characterized in that: Its decomposition peak temperature is 308.8℃.

5. The pyrroloquinoline quinone disodium salt crystalline compound according to claim 1, characterized in that: Its TG / DTG curve is shown in Figure 2.

6. A method for preparing the pyrroloquinoline quinone disodium salt crystalline compound according to any one of claims 1 to 5, comprising: After mixing pyrroloquinoline quinone acid and water, add alkali at 25-30°C, heat to 70-80°C, and filter after complete dissolution; cool the filtrate to below 30°C, adjust the pH to 3.4-4.5 under stirring, stop the reaction, let stand, and filter to obtain a solid wet product, add purified water for recrystallization, cool to below 30°C, filter, and dry to obtain the product.

7. A method for preparing the pyrroloquinoline quinone disodium salt crystalline compound according to any one of claims 1 to 5, characterized in that: The method comprises: mixing pyrroloquinoline quinone disodium salt and water, adding alkali at 25-30°C, heating to 70-80°C, and filtering after complete dissolution; cooling the filtrate to below 20°C, adjusting the pH to 3.4-4.5 under stirring, stopping the reaction, standing and filtering to obtain a solid wet product, adding purified water for recrystallization, cooling to below 30°C, filtering and drying to obtain the solid wet product.

8. The method according to claim 6 or 7, characterized in that: The base is selected from sodium hydroxide, sodium bicarbonate and sodium carbonate.

9. The method according to claim 6 or 7, characterized in that: The pH was adjusted by adding dry ice or bubbling carbon dioxide.

10. The method according to claim 6 or 7, characterized in that: The drying temperature is 50-70°C and the vacuum degree is 0.06-0.08Mpa.

11. A product comprising the pyrroloquinoline quinone disodium salt crystalline compound according to any one of claims 1 to 5.

12. The product according to claim 11, characterized in that The product is a medicine or a food.

13. The product according to claim 12, characterized in that The food is a beverage.

14. [Corrected 19.08.2024 in accordance with Rule 26] Use of the crystalline compound of disodium salt of pyrroloquinoline quinone according to any one of claims 1 to 5 or a composition comprising the crystalline compound of disodium salt of pyrroloquinoline quinone according to any one of claims 1 to 5 in the preparation of a product having at least one of the following functions: (1) Promote microbial growth; (2) Promote plant growth; (3) Antioxidant; (4) Protect the heart from oxidative damage; (5) Prevent and treat liver damage; (6) Promote nerve growth and protect the nervous system; (7) prevent acetaldehyde poisoning; and (8) Chelating metal ions.

15. The use according to claim 14, characterized in that: The anti-oxidation refers to at least one function selected from the group consisting of scavenging free radicals, activating antioxidant enzymes, increasing the synthesis of antioxidant substances, and inhibiting lipid peroxidation.

16. The use according to claim 14, characterized in that The products include: food additives, foods containing the food additives, cosmetic additives, cosmetics containing the cosmetic additives, health products, feed additives, feed containing the feed additives, drug carriers and chemical analysis reagents.

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