Pyrroloquinoline quinone disodium salt crystal compound as well as preparation method and application thereof

Abstract

The invention discloses a pyrroloquinoline quinone disodium salt crystal compound as well as a preparation method and application thereof, and belongs to the field of compound crystal forms. The pyrroloquinoline quinone disodium salt crystal compound has diffraction characteristic peaks at least at the following 2 theta angles: 9.7 degrees, 11.4 degrees, 12.3 degrees, 15.1 degrees, 21.0 degrees, 26.7 degrees, 27.4 degrees, 28.5 degrees, 29.1 degrees and 29.6 degrees, wherein the error of the 2 theta angle is + / -0.2 degrees. The pyrroloquinoline quinone disodium salt crystal compound provided by the invention is a novel crystal form, has a single crystal form, and has more excellent stability, higher solubility in water and higher compaction density. Wide application prospects are realized in the fields of medicines, foods, cosmetics and the like.

Description

Technical Field

[0001] The present application relates to the field of compound crystal forms, and in particular to a pyrroloquinoline quinone disodium salt crystal compound and a preparation method and application thereof. Background Art

[0002] Pyrroloquinoline quinone (PQQ) is a coenzyme compound with physiological activities such as antioxidant and mitochondrial generation promotion. Its sodium salt form (such as PQQ disodium salt) is widely used in medicine, functional food and cosmetics due to its ionization characteristics. However, the practical application of pyrroloquinoline quinone disodium salt is limited by its crystal properties, especially the defects in stability, solubility and preparation processing characteristics.

[0003] At present, several patents have disclosed methods for preparing different crystal forms of pyrroloquinoline quinone disodium salt. For example, CN102942567A discloses three methods for preparing pyrroloquinoline quinone disodium salt crystals, namely (A) suspension crystallization method, (B) slow volatilization method and (C) anti-solvent precipitation crystallization method. And three crystal forms are specifically disclosed: Crystal A, Crystal B and Crystal C. Although Crystal A has the lowest hygroscopicity, it still absorbs 2.3% of water at 65% RH; at 80% RH, it absorbs 2.8% of water, which makes it easy to change phases during storage.

[0004] For example, CN117327069A discloses that pyrroloquinoline quinone acid and water are mixed and heated in an alkaline environment, then cooled and the pH is adjusted to 3.4-4.5, and recrystallized to prepare a crystalline form. However, its compaction density is still not high enough to meet the needs of storage, transportation, preparation, etc.

[0005] After analysis, the inventors concluded that the pyrroloquinoline quinone disodium salt crystal in the prior art mainly has the following bottleneck problems: Insufficient stability: Most crystal forms are sensitive to humidity. After absorbing or losing moisture, the crystal lattice collapses, affecting the uniformity of drug content; Limited solubility: The high-density crystalline form has a dissolution rate lower than the pharmacopoeia requirement due to the tight molecular stacking, while the amorphous form is difficult to preserve for a long time due to its metastable characteristics; Poor processing performance: The existing crystalline particles have irregular morphology and low compaction density, which requires the addition of excessive fillers in the direct compression process, increasing the volume and cost of the preparation.

[0006] The above defects severely limit the application of pyrroloquinoline quinone disodium salt in solid preparations (such as tablets and capsules) and transparent liquid products (such as oral liquid and essence). Therefore, the development of a new pyrroloquinoline quinone disodium salt crystal with high stability, rapid dissolution and excellent compaction performance has become a technical problem that needs to be overcome in this field. Summary of the invention

[0007] To solve at least one of the above technical problems, the technical solutions adopted in this application are as follows.

[0008] The first aspect of this application provides a pyrroloquinoline quinone disodium salt crystal compound, which, measured with Cu-Kα rays, has diffraction characteristic peaks at least at the following 2θ angles: 9.7°, 11.4°, 12.3°, 15.1°, 21.0°, 26.7°, 27.4°, 28.5°, 29.1°, 29.6°, where the error of the 2θ angle is ±0.2°.

[0009] In some embodiments of this application, the XRD spectrum of the pyrroloquinoline quinone disodium salt crystal compound is as Figure 2 shown.

[0010] In some embodiments of this application, the TGA spectrum of the pyrroloquinoline quinone disodium salt crystal compound is as Figure 3 shown.

[0011] In some embodiments of this application, the DSC spectrum of the pyrroloquinoline quinone disodium salt crystal compound is as Figure 4 shown.

[0012] The second aspect of this application provides a preparation method of the pyrroloquinoline quinone disodium salt crystal compound according to any one of the first aspect of this application, including the following steps: S1, Add pyrroloquinoline quinone trisodium salt to water, heat to 60 °C, and stir until completely dissolved during this period; S2, Gradually cool down to 10 °C, and during the process of gradient cooling: (1) Dropwise add ethanol to a final concentration of 37 - 42 vt%, (2) Dropwise add hydrochloric acid to adjust the pH to 2.8 - 3.3, S3, Maintain at 10 °C for 3 - 5 h to form crystals; S4, Filter to obtain the wet product of pyrroloquinoline quinone disodium salt, and dry it at 45 - 70 °C to obtain the pyrroloquinoline quinone disodium salt crystal compound.

[0013] In some embodiments of this application, during the gradient cooling, the cooling rate slows down as the temperature decreases. That is to say, during the gradient cooling process, the time from 60 °C to 50 °C is less than the time from 50 °C to 40 °C, and similarly, the time from 50 °C to 40 °C is less than the time from 40 °C to 30 °C, and so on.

[0014] In some specific embodiments of this application, the program of the gradient cooling is as follows: .

[0015] In some other specific embodiments of the present application, the program of gradient cooling is as follows: 。

[0016] Based on the limitation that the cooling rate slows down as the temperature decreases, those skilled in the art can also use other gradient cooling methods.

[0017] The third aspect of the present application provides a composition, which comprises the pyrroloquinoline quinone disodium salt crystal compound according to any one of the first aspect of the present application 。

[0018] In some embodiments of the present application, the composition is selected from one of pharmaceutical compositions, nutritional supplements, food additives, cosmetic compositions, feed additives, and microbial culture synergists.

[0019] The fourth aspect of the present application provides the use of the pyrroloquinoline quinone disodium salt crystal compound according to any one of the first aspect of the present application in the preparation of a composition, the composition is selected from one of pharmaceutical compositions, nutritional supplements, food additives, cosmetic compositions, feed additives, and microbial culture synergists, and the composition is used for at least one of the following uses: (a) Preventing or treating diseases related to oxidative stress, metabolic disorders, neurodegenerative diseases, mitochondrial dysfunction, or aging; (b) Enhancing the energy metabolism, cell proliferation, or microbial growth activity of an organism; (c) Acting as an antioxidant, free radical scavenger, or cytoprotectant; (d) Improving the skin condition, delaying the aging phenotype, or enhancing nutrient absorption; (e) Acting as a functional ingredient to provide antioxidant or metabolic regulation effects in foods, feeds, or cosmetics.

[0020] Compared with the pyrroloquinoline quinone disodium salt crystal in the prior art, the pyrroloquinoline quinone disodium salt crystal compound of the present application has the following beneficial effects: 1) It is a new crystal form and has a single crystal form; 2) It has better stability, with basically no water absorption or loss within 120 h, being easy to store and further process and use; 3) It has higher solubility in water, and the solubility of the crystal compound is at least 8.2% higher than that in the comparative example. Therefore, it is easier to prepare downstream products such as beverages, drugs, and cosmetics, and is more conducive to users' use and absorption; 4) It has a higher compaction density, and the compaction density of the crystal compound is at least 6.6% higher than that in the comparative example. Thus, it can be stored, transported, formulated, pharmaceutically manufactured, and made into cosmetics, etc. with a smaller volume, which is more conducive to further processing and use.

[0021] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it used to limit the scope of the present application. Other features of the present application will become readily understood through the following description. Description of the Drawings

[0022] By referring to the accompanying drawings and reading the following detailed description, the above and other objects, features, and advantages of the exemplary embodiments of the present application will become readily understandable. In the drawings, several embodiments of the present application are shown in an exemplary rather than restrictive manner, where: Figure 1 It is the high performance liquid chromatography (HPLC) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Example 1 of the present application; Figure 2 It is the X-ray diffraction (XRD) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Example 1 of the present application; Figure 3 It is the thermogravimetric analysis (TGA) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Example 1 of the present application; Figure 4 It is the differential scanning calorimetry (DSC) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Example 1 of the present application; Figure 5 It is the high performance liquid chromatography (HPLC) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Example 2 of the present application; Figure 6 It is the X-ray diffraction (XRD) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Example 2 of the present application; Figure 7 It is the high performance liquid chromatography (HPLC) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Example 3 of the present application; Figure 8 It is the X-ray diffraction (XRD) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Example 3 of the present application; Figure 9 It is the X-ray diffraction (XRD) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Comparative Example 1 of the present application; Figure 10 It is the X-ray diffraction (XRD) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Comparative Example 2 of the present application; Figure 11 It is the X-ray diffraction (XRD) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Comparative Example 3 of the present application; Figure 12 It is the X-ray diffraction (XRD) spectrum of the pyrroloquinoline quinone disodium salt crystal obtained in Comparative Example 4 of the present application; Figure 13This is the X-ray diffraction (XRD) pattern of the pyrroloquinoline quinone disodium salt crystal obtained under the conditions of being stored in the dark and sealed at 100 °C for 24 h in the stability test of this application; Figure 14 This is the high performance liquid chromatography (HPLC) detection pattern of the pyrroloquinoline quinone disodium salt crystal obtained under the conditions of being stored in the dark and sealed at 100 °C for 24 h in the stability test of this application; Figure 15 This is the X-ray diffraction (XRD) pattern of the pyrroloquinoline quinone disodium salt crystal obtained under the conditions of being stored in the dark and sealed at 100 °C for 48 h in the stability test of this application; Figure 16 This is the high performance liquid chromatography (HPLC) detection pattern of the pyrroloquinoline quinone disodium salt crystal obtained under the conditions of being stored in the dark and sealed at 100 °C for 48 h in the stability test of this application; Figure 17 This is the X-ray diffraction (XRD) pattern of the pyrroloquinoline quinone disodium salt crystal obtained under the conditions of being stored in the dark and sealed at 100 °C for 120 h in the stability test of this application; Figure 18 This is the high performance liquid chromatography (HPLC) detection pattern of the pyrroloquinoline quinone disodium salt crystal obtained under the conditions of being stored in the dark and sealed at 100 °C for 120 h in the stability test of this application. Detailed implementation mode

[0023] In order to make the technical problems, technical solutions and beneficial effects solved by this application clearer, the following further elaborates on this application in combination with examples.

[0024] The following examples are used here to demonstrate the preferred implementation schemes of this application. Those skilled in the art will understand that the technologies disclosed in the following examples represent the technologies discovered by the inventor that can be used to implement this application, and thus can be regarded as the preferred schemes for implementing this application. However, those skilled in the art should understand according to this specification that many modifications can be made to the specific embodiments disclosed here, and still obtain the same or similar results, without departing from the spirit or scope of this application.

[0025] Those skilled in the art will realize or can understand through routine tests many equivalent technologies of many specific implementation schemes of the inventions described here. These equivalents will be included in the claims.

[0026] In the embodiments of this application, the analysis instruments and test conditions are as follows: 1. X-ray diffraction (XRD): Instrument model: Bruker D8 advance; Target: Cu-Kα (30KV, 10mA); Scanning range: 3° - 40° (2θ value); Scanning step size: 0.02°; Scanning step diameter: 0.1 s.

[0027] 2. Thermogravimetric analysis (TGA): The test gas atmosphere is nitrogen. The specific test temperature range (°C) is 30 - 500, and the heating rate is 10 °C / min.

[0028] 3. Differential scanning calorimetry (DSC): From 10 °C / min to 250 °C, non-sealed pan.

[0029] 4. High performance liquid chromatography (HPLC) method (determined according to General Chapter 0512 of the Fourth Part of Chinese Pharmacopoeia 2020) Mobile phase of HPLC: Phase A: Weigh 2.28 g of dipotassium hydrogen phosphate trihydrate and 4.84 g of tetrabutylammonium bromide, add 800 mL of water and dissolve by ultrasonic wave, adjust the pH to 7.4 with phosphoric acid, and dilute to 1000 mL with water; Phase B: Acetonitrile; Phase A / Phase B: 75 / 25 for 0 - 25 min; from 75 / 25 to 79 / 21 for 25 - 48 min; from 79 / 21 to 75 / 25 for 48 - 50 min; 75 / 25 for 50 - 55 min; Chromatographic column: Octadecylsilyl bonded silica gel packing or chromatographic column of the same specification resistant to high pH, 4.6 * 150 mm, 5 μm; Chromatographic conditions: Flow rate 1 mL / min, column temperature 30 °C, detector wavelength 254 nm.

[0030] 5. High performance liquid chromatography charged aerosol detector (HPLC - CAD) Chromatographic column: C6 4.0 * 150 mm; Flow rate: 0.9 mL / min; Column temperature: 35 °C; Injection volume: 25 μL; Running time: Not less than 3 times the sodium retention time; Eluent: 4.6 mM HNO3 solution.

[0031] 6. Karl Fischer volumetric titration method Take 0.1 g of the test sample, weigh accurately, and carry out according to the standard operating procedure of Karl Fischer volumetric titration method; Calculation formula: W = (F × V - drift value × t) / (m × 1000) × 100% Wherein, W represents the moisture content of the test sample (%); F represents the titration degree of the Karl Fischer reagent (mg / mL); V represents the volume of the Karl Fischer reagent consumed by the test sample (mL); m represents the weight of the test sample (g); t represents the test time (s).

[0032] For other experimental methods in the following examples, if not otherwise specified, they are all conventional methods. For the instruments and equipment used in the following examples, if not otherwise specified, they are all conventional laboratory instruments and equipment; for the test materials used in the following examples, if not otherwise specified, they are all obtained by purchasing from a conventional biochemical reagent store.

[0033] Example 1 Preparation and Detection of Pyrroloquinoline Quinone Disodium Salt Crystal 1. Preparation of Pyrroloquinoline Quinone Disodium Salt Crystal Add 25 g of the crude pyrroloquinoline quinone trisodium salt solid to 1 L of water, heat from room temperature to 60 °C, and stir until completely dissolved during this process. Slowly cool down to 10 °C in a gradient manner, and the specific cooling procedure is shown in Table 1.

[0034] Table 1 Cooling Gradient Table

[0035] While cooling, slowly add ethanol dropwise until the final concentration reaches 40 vt% and adjust the pH of the crystallization mother liquor to 3.0 with 4.0 mol / L hydrochloric acid, and complete the dropping process within 1.5 h. After the crystallization process is completed, filter the crystallization system to obtain the wet pyrroloquinoline quinone disodium salt, and dry it at 60 °C for 4 h to obtain the pyrroloquinoline quinone disodium salt crystal compound.

[0036] 2. Detection of Pyrroloquinoline Quinone Disodium Salt Crystal Compound The purity of the pyrroloquinoline quinone disodium salt crystal compound detected by high performance liquid chromatography (HPLC) is 99.936%; the sodium ion proportion detected by high performance liquid chromatography charged aerosol detector (HPLC-CAD) for the pyrroloquinoline quinone disodium salt crystal compound is 12.1%; the moisture content of the pyrroloquinoline quinone disodium salt crystal compound detected by the Karl Fischer volumetric titration method is 8.8%.

[0037] Based on the above detection results, it is calculated that this crystal form is a crystal compound of pyrroloquinoline quinone disodium salt dihydrate.

[0038] The HPLC chromatogram of this crystal compound is shown in Figure 1 ; the XRD chromatogram is shown in Figure 2 ; the TGA chromatogram is shown in Figure 3 ; the DSC chromatogram is shown in Figure 4 . According to Figures 1 to 4 Comprehensive analysis of the detection results and the relevant chromatograms of the existing pyrroloquinoline quinone disodium salt crystal compounds, it is concluded that this crystal form compound has the following characteristics: 1) Preferential height of the main XRD peak: A single crystal form shows a few strong peaks, with lower intensities and uniform distribution of secondary peaks.

[0039] 2) Rational segmentation of TGA weight loss: The weight loss at low temperature conforms to the characteristics of hydrates, and the weight loss at high temperature is related to salt decomposition.

[0040] 3) Single exothermic peak in DSC: The exothermic peak corresponds to the melting or decomposition of the crystal form.

[0041] Through comprehensive analysis, the test results support the single crystal form of the compound.

[0042] Example 2 Preparation and detection of pyrroloquinoline quinone disodium salt crystals 1. Preparation of pyrroloquinoline quinone disodium salt crystals Add 25 g of crude pyrroloquinoline quinone trisodium salt solid to 1 L of water, heat from room temperature to 60 °C, and stir until completely dissolved during this process. Gradually cool down to 10 °C slowly, and the specific cooling procedure is shown in Table 2.

[0043] Table 2 Cooling gradient table

[0044] While cooling, slowly add ethanol dropwise to a final concentration of 40 vt% and adjust the pH of the crystallization mother liquor to 3.0 with 4.0 mol / L hydrochloric acid, and complete the dropping process within 1.5 h. After the crystallization process is completed, filter the crystallization system to obtain the wet product of pyrroloquinoline quinone disodium salt, and obtain the pyrroloquinoline quinone disodium salt crystal compound after drying at 60 °C for 4 h.

[0045] 2. Detection of pyrroloquinoline quinone disodium salt crystal compound The purity of the pyrroloquinoline quinone disodium salt crystal compound detected by high performance liquid chromatography (HPLC) is 99.604%; the sodium ion ratio of the pyrroloquinoline quinone disodium salt crystal compound detected by high performance liquid chromatography charged aerosol detector (HPLC-CAD) is 11.9%; the water content of the pyrroloquinoline quinone disodium salt crystal compound detected by Karl Fischer volumetric titration method is 8.7%.

[0046] Calculated from the above test results, the crystal form is a crystal compound of pyrroloquinoline quinone disodium salt dihydrate.

[0047] The HPLC chromatogram of this crystal compound is shown in Figure 5 ; the XRD chromatogram is shown in Figure 6 . According to Figures 5 to 6 Comprehensive analysis of the test results and the test chromatograms obtained in Example 1 shows that this crystal form compound is the same as that obtained in Example 1.

[0048] Example 3 Preparation and detection of pyrroloquinoline quinone disodium salt crystals 1. Preparation of pyrroloquinoline quinone disodium salt crystals Add 25 g of crude pyrroloquinoline quinone trisodium salt solid to 1 L of water, heat the mixture from room temperature to 60 °C, and stir until completely dissolved. Rapidly cool the solution to 10 °C within 1.5 h. While cooling, slowly add ethanol dropwise to a final concentration of 40 vt% and adjust the pH of the crystallization mother liquor to 3.0 with 4.0 mol / L hydrochloric acid. The dropping process should be completed within 1.5 h. After the crystallization process is completed, filter the crystallization system to obtain wet pyrroloquinoline quinone disodium salt. Dry it at 60 °C for 4 h to obtain the crystalline compound of pyrroloquinoline quinone disodium salt.

[0049] 2. Detection of the crystalline compound of pyrroloquinoline quinone disodium salt The purity of the crystalline compound of pyrroloquinoline quinone disodium salt detected by high performance liquid chromatography (HPLC) is 99.601%; the proportion of sodium ions in the crystalline compound of pyrroloquinoline quinone disodium salt detected by high performance liquid chromatography with charged aerosol detector (HPLC-CAD) is 11.9%; the moisture content of the crystalline compound of pyrroloquinoline quinone disodium salt detected by Karl Fischer volumetric titration is 8.7%.

[0050] Calculated from the above detection results, the crystal form is a crystalline compound of pyrroloquinoline quinone disodium salt dihydrate.

[0051] The HPLC chromatogram of this crystalline compound is shown in Figure 7 ; the XRD pattern is shown in Figure 8 . According to Figures 7 to 8 Comprehensive analysis of the detection results and the detection spectra obtained in Example 1 shows that this crystalline compound is different from that obtained in Example 1, with a more disordered crystal form and not a single crystal form.

[0052] Comparative Example 1 Prepare the crystalline pyrroloquinoline quinone disodium salt according to the method of Example 1 in the Chinese invention patent publication text CN102942567A: Take the pyrroloquinoline quinone disodium salt raw material and stir it in acetonitrile at 25 °C for 5 d to reach equilibrium. Then filter the solution respectively, and dry the solid part in air for 10 min to obtain the crystalline pyrroloquinoline quinone disodium salt.

[0053] Detect the X-ray powder diffraction pattern of the obtained crystal (see Figure 9 ), which is consistent with the crystal form A of pyrroloquinoline quinone prepared in Example 1 of CN102942567A and different from the peak appearance in Example 1 of this application.

[0054] Comparative Example 2 Prepare pyrroloquinoline quinone disodium salt crystals according to the method of Example 4 in the Chinese invention patent publication text CN112125899A: Take 15 g of pyrroloquinoline quinone trisodium salt and dissolve it in 1 L of water. Heat and stir at 75 °C until completely dissolved to obtain an aqueous solution; slowly add sulfuric acid to the above aqueous solution under heating and stirring at 75 °C to adjust the pH value to 3.0 to obtain an acidic aqueous solution; the time for dropping sulfuric acid is 50 min. Cool the acidic aqueous solution to 25 °C, stand for crystallization for 12 h to obtain crude crystals, filter, and dry in vacuo at 80 °C to obtain pyrroloquinoline quinone disodium salt crystals.

[0055] Detect the X-ray powder diffraction pattern of the obtained crystals (see Figure 10 ), which is consistent with the crystal form of pyrroloquinoline quinone disodium salt prepared by the method of Example 4 in CN112125899A, and is different from the peak appearance of Example 1 of this application.

[0056] Comparative Example 3 Prepare pyrroloquinoline quinone disodium salt crystals according to the method of Example 2 in the Chinese invention patent publication text CN117777147A: Take 40 g of pyrroloquinoline quinone disodium salt and add it to 1 L of water. Heat to 60 °C, adjust the pH of the obtained solution to 9.0, stir until the solid is completely dissolved, then cool to 5 °C, slowly add isopropanol to make the volume fraction of isopropanol reach 50%. At this time, a solid is formed. Adjust the pH of the suspension to 4 with hydrochloric acid. After the pH is relatively stable, filter. Dry the filter residue at 50 °C for 20 h to obtain pyrroloquinoline quinone disodium salt crystals.

[0057] Detect the X-ray powder diffraction pattern of the obtained crystals (see Figure 11 ), which is consistent with the crystal form of pyrroloquinoline quinone disodium salt prepared by the method of Example 2 in CN117777147A, and is different from the peak appearance of Example 1 of this application.

[0058] Comparative Example 4 Prepare pyrroloquinoline quinone disodium salt crystals according to the method of Example 1 in the Chinese invention patent publication text CN117327069A: Add 50 g of pyrroloquinoline quinone acid to 1 L of water. Slowly add 21.21 g of sodium hydroxide to the reaction flask at 30 °C, then raise the temperature to 80 °C and stir for 2 h until completely dissolved. Cool to below 30 °C, and while stirring, pass CO2 into the filtrate to adjust the pH to 4.0. Stand for 2 h and then filter by suction. Add the obtained wet product to 1 L of water for recrystallization. Cool to below 30 °C, filter by suction, and dry to obtain pyrroloquinoline quinone disodium salt crystals. Detect the X-ray powder diffraction pattern of the obtained crystals (see Figure 12 ), which is consistent with the crystal form of pyrroloquinoline quinone disodium salt prepared in Example 1 of CN117327069A, and is different from the peak appearance of Example 1 of this application.

[0059] Experimental Example 1. XRD Pattern Comparison Experimental Method: Compare the XRD diffraction peaks of the crystals prepared in Example 1 with those in Comparative Examples 1 - 4. The comparison results are shown in Table 3.

[0060] Table 3 XRD Diffraction Peaks of Each Pyrroloquinoline Quinone Disodium Salt Crystal

[0061] The data comparison in Table 3 shows that the crystals obtained in Example 1 and Example 2 have significantly different peak emergence situations from other crystal forms, and they are new crystal forms of pyrroloquinoline quinone disodium salt.

[0062] 2. Stability Test The sample obtained in Example 1 of this application was stored in the dark and sealed at -20 °C. Samples were taken after intervals of 3, 6, 9, and 12 months for XRD and HPLC tests, and the spectra were compared with the initial spectra; the sample obtained in Example 1 of this application was stored in the dark and sealed at 100 °C. Samples were taken after intervals of 24 h, 48 h, 72 h, and 120 h for XRD and HPLC tests, and the spectra were compared with the initial spectra. The comparison results are shown in Table 4. The changed XRD and HPLC spectra are shown in Figures 13 to 18 .

[0063] Table 4 Stability Experiment of the New Crystal Form of Pyrroloquinoline Quinone Disodium Salt in Example 1 of this Application

[0064] As can be seen from the data in Table 4, the crystal structure of the pyrroloquinoline quinone disodium salt prepared in Example 1 of this application remains stable under the condition of being stored in the dark and sealed at -20 °C within 12 months; it remains stable within 24 h under the accelerated condition of being stored in the dark and sealed at 100 °C. The pyrroloquinoline quinone disodium salt crystal prepared in Example 1 of this application is easy to store and further process and use. Its crystal form and purity will not change within 12 months under the condition of being stored in the dark and sealed at -20 °C; its crystal form and purity will not change within 24 h under the condition of being stored in the dark and sealed at 100 °C.

[0065] 3. Hygroscopicity Test For each pyrroloquinoline quinone disodium salt crystal prepared in Example 1 and Comparative Examples 1 - 4 of this application, after weighing the mass of each sample, it was placed in a constant temperature and humidity environment of 25 °C and 50% RH for 120 h. Then, the samples were taken out again to weigh their masses, and the moisture absorption rate was calculated as = (mass at 120 h - mass at 0 h) / mass at 0 h × 100%. The water absorption comparison after 120 h is shown in Table 5.

[0066] Table 5 Hygroscopicity Comparison of Each Pyrroloquinoline Quinone Disodium Salt Crystal

[0067] As can be seen from Table 5, the pyrroloquinoline quinone disodium salt crystals prepared in Example 1 and Example 2 of this application are basically non-hygroscopic, less likely to absorb or lose water compared with the existing disodium salt crystal forms, and are easy to store. The pyrroloquinoline quinone disodium salt crystals prepared in Example 3 have worse hygroscopicity than those prepared in Example 1 and Example 2.

[0068] 4. Solubility test Experimental method: Referring to the "Solubility Determination Method" in the general rules of the "Chinese Pharmacopoeia", the relevant solutions of the pyrroloquinoline quinone disodium salt crystals prepared in Example 1 of this application and Comparative Examples 1 to 4 were prepared by the shaking flask method (25 °C ± 0.5 °C, shaking for 24 h to reach equilibrium), and their saturated concentrations were measured by HPLC. The solubility comparison is shown in Table 6.

[0069] Table 6 Comparison of the solubilities of various pyrroloquinoline quinone disodium salt crystals

[0070] As can be seen from Table 6, the solubilities of the pyrroloquinoline quinone disodium salt crystals prepared in Example 1 and Example 2 of this application in water at 25 °C are higher than those of the existing pyrroloquinoline quinone disodium salt crystal compounds, and are at least 8.2% higher than the solubilities of the crystal compounds in the comparative examples. The solubility of the pyrroloquinoline quinone disodium salt crystals prepared in Example 3 in water at 25 °C is worse than that of the pyrroloquinoline quinone disodium salt crystals prepared in Example 1 and Example 2. Therefore, the pyrroloquinoline quinone disodium salt crystals prepared in Example 1 and Example 2 of this application are more suitable for the preparation of downstream products such as beverages, drugs, and cosmetics, and are more conducive to users' use and absorption.

[0071] 5. Compaction density test Experimental method: Set the parameters of the powder compaction density meter as constant pressure time 10 s, vacuum time 10 s, and pressure 1 t. Take 1 g of each of the pyrroloquinoline quinone disodium salt crystals prepared in Example 1 of this application and Comparative Examples 1 to 4, and use the powder compaction density meter to measure the compaction density of each sample respectively. The compaction density comparison is shown in Table 7.

[0072] Table 7 Comparison of the densities of various pyrroloquinoline quinone disodium salt crystals

[0073] As can be seen from Table 7, the tap density of the pyrroloquinoline quinone disodium salt crystals prepared in Example 1 and Example 2 of this application is higher than that of the existing pyrroloquinoline quinone disodium salt crystals, and the tap density of the crystal compound in the comparative example is increased by at least more than 6.6%. The tap density of the pyrroloquinoline quinone disodium salt crystals prepared in Example 3 is less than that of the pyrroloquinoline quinone disodium salt crystals prepared in Example 1 and Example 2. Therefore, the pyrroloquinoline quinone disodium salt crystals of Example 1 and Example 2 of this application can be stored, transported, formulated, used in pharmaceuticals, cosmetics, etc. with a smaller volume than the existing disodium salt crystals, which is more conducive to further processing and use in the next step.

[0074] In summary, the pyrroloquinoline quinone disodium salt crystals prepared in Example 1 and Example 2 of this application are the same new crystal form. Compared with the existing pyrroloquinoline quinone disodium salt crystals, the advantages are as follows: 1) The crystal form of the compound is single; 2) It has better stability, and there is basically no water absorption and loss within 120 h, which is easy to store and further process and use; 3) It has higher solubility in water, and the solubility of the crystal compound in the comparative example is increased by at least more than 8.2%. Therefore, it is easier to prepare downstream products such as beverages, drugs, and cosmetics, and is more conducive to users' use and absorption; 4) It has a higher tap density, and the tap density of the crystal compound in the comparative example is increased by at least more than 6.6%. Thus, it can be stored, transported, formulated, used in pharmaceuticals, cosmetics, etc. with a smaller volume, which is more conducive to further processing and use in the next step.

[0075] All documents mentioned in this application are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A pyrroloquinoline quinone disodium salt crystalline compound, measured by Cu-Kα radiation, characterized in that: The pyrroloquinoline quinone disodium salt crystalline compound has diffraction characteristic peaks at least at the following 2θ angles: 9.7°, 11.4°, 12.3°, 15.1°, 21.0°, 26.7°, 27.4°, 28.5°, 29.1°, and 29.6°, 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 XRD spectrum is shown in Figure 2.

3. The pyrroloquinoline quinone disodium salt crystalline compound according to claim 1, characterized in that: Its TGA spectrum is shown in Figure 3.

4. The pyrroloquinoline quinone disodium salt crystalline compound according to claim 1, characterized in that: Its DSC spectrum is shown in Figure 4.

5. The method for preparing the pyrroloquinoline quinone disodium salt crystalline compound according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1, adding pyrroloquinoline quinone trisodium salt into water, heating to 60 °C, stirring until completely dissolved; S2, gradient cooling to 10 ℃, and during the gradient cooling process: (1) Add ethanol dropwise to a final concentration of 37~42vt%. (2) Add hydrochloric acid to adjust the pH to 2.8~3.

3. S3, maintain 10 °C for 3-5 h to form crystals; S4, filtering to obtain the wet product of pyrroloquinoline quinone disodium salt, and drying at 45-70° C. to obtain the pyrroloquinoline quinone disodium salt crystalline compound.

6. The preparation method according to claim 5, characterized in that: In the gradient cooling, the cooling speed becomes slower as the temperature decreases.

7. The preparation method according to claim 6, characterized in that: The procedure of the gradient cooling is as follows: 。 8. A composition comprising the pyrroloquinoline quinone disodium salt crystalline compound according to any one of claims 1 to 4.

9. The composition according to claim 8, characterized in that The composition is selected from one of a pharmaceutical composition, a nutritional supplement, a food additive, a cosmetic composition, a feed additive and a microbial culture enhancer.

10. Use of the pyrroloquinoline quinone disodium salt crystalline compound according to any one of claims 1 to 4 in preparing a composition, characterized in that: The composition is selected from one of a pharmaceutical composition, a nutritional supplement, a food additive, a cosmetic composition, a feed additive and a microbial culture enhancer, and the composition is used for at least one of the following purposes: (a) preventing or treating conditions associated with oxidative stress, metabolic disorders, neurodegenerative diseases, mitochondrial dysfunction or aging; (b) enhancing the energy metabolism, cell proliferation or microbial growth activity of organisms; (c) act as antioxidants, free radical scavengers or cytoprotectants; (d) improving skin condition, delaying aging phenotypes, or enhancing nutrient absorption; (e) As functional ingredients in food, feed or cosmetics to provide antioxidant or metabolic regulatory effects.

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