A method for separating and purifying pyrroloquinoline quinone

Abstract

The invention discloses a separation and purification method of pyrroloquinoline quinone, wherein a fermentation broth rich in pyrroloquinoline quinone is filtered by a combined filter membrane for solid-liquid separation and concentration; hydrochloric acid and sodium chloride are added to the solution, and solid-liquid separation is performed after adjusting the pH and stirring; sodium hydroxide is added to the solid after the buffer solution is dissolved and the pH is adjusted, activated carbon is used for decolorization, and then solid-liquid separation is performed; column chromatography is performed after the liquid is diluted with a buffer solution, and a desorbent with a pH of 7.0-8.5 and a gradient salt concentration is used to desorb to obtain a column chromatography desorption liquid; sodium chloride is added until the product is precipitated, and the mixture is separated after stirring; the solid is dissolved in ultrapure water, ethanol is added, hydrochloric acid is added to adjust to acidity, solid-liquid separation is performed, and solid pyrroloquinoline quinone is obtained after drying. The method does not require the use of a large amount of organic reagents and is green and environmentally friendly; the production cost is low, the separation and purification effect is good, the HPLC purity of the harvested pyrroloquinoline quinone product is> 99%, and the content of each single impurity is < 0.1%, and the quality of the finished product meets the quality requirements of the United States Pharmacopoeia.

Description

Technical Field

[0001] The invention belongs to the technical field of drug separation and purification, and specifically relates to a method for separating and purifying pyrroloquinoline quinone. Background Art

[0002] Pyrroloquinoline quinone (PQQ, also known as 4,5-dihydro-4,5-dioxy-1-hydropyrrole (2,3f)quinone 2,7,9-tricarboxylic acid, Methaxatin) is a coenzyme that can be used by dehydrogenases in the human body and is classified as a B vitamin. PQQ was discovered by researchers in 1979. After being separated and purified from methylotrophic bacteria, studies have shown that it is a coenzyme of dehydrogenases that metabolize methanol in bacteria. It is coupled with the respiratory electron transport chain and plays a role in promoting bacterial metabolism. Some studies have shown that PQQ can regulate epigenetic expression and cell metabolism, play an anti-cancer and anti-aging role, and can be used as a health supplement by taking an appropriate amount (≤20 mg / d) daily.

[0003] Traditional PQQ production methods are mostly chemical synthesis methods, but the disadvantages of multiple synthesis steps, many by-products and high purification costs are difficult to eliminate. The fermentation method for producing PQQ has developed rapidly in recent decades, and due to its cost advantage, it has gradually replaced the chemical synthesis method and become the mainstream production method.

[0004] In view of the current status of PQQ production, the extraction and purification of PQQ from fermentation broth is an indispensable part of PQQ purification research. Many methods for the separation and purification of PQQ have been made public, but these methods either have a long cycle and high production cost; or require the use of a variety of organic reagents, which does not conform to the green and environmentally friendly production concept; although many extraction and purification methods can obtain products with a purity of ≥98%, they cannot guarantee that the products meet the USP quality requirements: purity ≥99%, and the content of each impurity is <0.1%.

[0005] The fermentation process produces a large number of impurities with complex structures and similar physical and chemical properties to the finished product. The limitations of the process technology, equipment and consumables described in the existing patents cannot simultaneously meet the requirements of high yield, high quality and green environmental protection. However, the marketization of pyrroloquinoline quinone products has increasing requirements for quality, cost and environmental protection. Therefore, the technical solutions and methods for separating and purifying high-quality pyrroloquinoline quinone products from fermentation broth rich in pyrroloquinoline quinone need further research to meet the market production requirements for pyrroloquinoline quinone products. Summary of the invention

[0006] In order to solve the problems and shortcomings of the current separation and purification methods of pyrroloquinoline quinone, the present invention provides a separation and purification method of pyrroloquinoline quinone.

[0007] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a method for separating and purifying pyrroloquinoline quinone, comprising the following steps: S1. Using a combined filter membrane to separate solid from liquid and concentrate the fermentation broth rich in pyrroloquinoline quinone; S2, adding hydrochloric acid and sodium chloride to the solution separated in step S1, adjusting the pH to acidic, and performing solid-liquid separation after stirring; S3, adding a buffer to the solid separated in step S2 to dissolve it, then adding sodium hydroxide, adjusting the pH to neutral or weakly alkaline, decolorizing with activated carbon, and then performing solid-liquid separation; S4, the liquid obtained in step S3 is diluted with a buffer solution and then subjected to column chromatography, and desorbed using a desorbent having a pH value of 7.0-8.5 and a gradient salt concentration to obtain a column chromatography desorbent; S5. Add sodium chloride to the column chromatography desorption liquid obtained in step S4 until the product is precipitated, and separate the solid and liquid after stirring; S6. The solid obtained in step S5 is dissolved in ultrapure water, ethanol is added, hydrochloric acid is added to adjust to acidity, solid-liquid separation is performed, and solid pyrroloquinoline quinone is obtained after drying; The chromatography column filler is one of LX-1600, HZ-818, LX-6703, HZ-PQQ, HZ-828, and HZ-835.

[0008] In a more preferred embodiment of the present invention, the chromatography column filler is LX-6703 or HZ835.

[0009] In some embodiments of the present invention, in step S1, the combined membrane filtration is sequentially filtered using a ceramic membrane with a pore size of 40-50 nm, an ultrafiltration membrane with a pore size of 8-10 KDa, and a nanofiltration membrane with a pore size of 400-500 Da. In a more preferred embodiment of the present invention, the combined membrane is a ceramic membrane with a pore size of 50 nm, an ultrafiltration membrane with a pore size of 8 KDa, and a nanofiltration membrane with a pore size of 500 Da.

[0010] In some embodiments of the present invention, in step S3, the buffer is 50-60 mmol / L sodium dihydrogen phosphate-sodium hydroxide buffer.

[0011] In some embodiments of the present invention, in step S3, the amount of activated carbon used is 0.5-0.8% w / v, and the decolorization time is 4-5 h.

[0012] In some embodiments of the present invention, in step S4, the desorption of the desorbent with gradient salt concentration is: 3 BV 0.1-0.2 mol / L desorbent, 5 BV 0.3-0.5 mol / L desorbent, and 8-10 BV 0.6-0.7 mol / L desorbent are used in sequence.

[0013] In some embodiments of the present invention, the analytical agent is sodium chloride or sodium sulfate; the analytical agent also contains 50-60 mmol / L of sodium dihydrogen phosphate.

[0014] In some embodiments of the present invention, the analytical agent is 3 BV of 0.1 mol / L sodium chloride or sodium sulfate with a pH of 7.0-8.5, 50 mmol / L sodium dihydrogen phosphate solution, 5 BV of 0.3 mol / L sodium chloride or sodium sulfate with a pH of 7.0-8.5, 50 mmol / L sodium dihydrogen phosphate solution, 8-10 BV of 0.7 mol / L sodium chloride or sodium sulfate with a pH of 7.0-8.5, and 50mmol / L sodium dihydrogen phosphate solution.

[0015] More preferably, 3 BV of 0.1 mol / L sodium chloride at pH=7.0, 50 mmol / L sodium dihydrogen phosphate solution, 5 BV of 0.3 mol / L sodium chloride at pH=7.0, 50 mmol / L sodium dihydrogen phosphate solution, 10 BV of 0.7 mol / L sodium chloride at pH=7.0, 50 mmol / L sodium dihydrogen phosphate solution are used in sequence.

[0016] In some embodiments of the present invention, in step S4, the liquid loading amount for column chromatography is 60-80 g / L.

[0017] In some embodiments of the present invention, the amount of ethanol used in step S6 is 40-50% v / v.

[0018] In some embodiments of the present invention, the concentration of hydrochloric acid in steps S2 and S6 is 3.0-4.0 mol / L; the concentration of sodium hydroxide in S3 is 3.0-4.0 mol / L; and the concentration of sodium chloride in S5 is 1.0-1.3 mol / L.

[0019] In some embodiments of the present invention, the stirring conditions in steps S2 and S5 are 500-600 rpm for 4-5 h.

[0020] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a method for separating and purifying pyrroloquinoline quinone from a fermentation broth containing pyrroloquinoline quinone, which method does not require the use of a large amount of organic reagents, is low-pollution, and is green and environmentally friendly; the separation and purification steps are simple, the production cost is low, and the separation and purification effect is good. When column chromatography is used, the chromatography column filler is one of LX-1600, HZ-818, LX-6703, HZ-PQQ, HZ-828, and HZ-835. In the analysis step after chromatography, when the pH of the analytical agent is 7.0-8.5, the HPLC purity of the harvested pyrroloquinoline quinone product is greater than 99%, and the content of each single impurity is less than 0.1%. Wherein the chromatography column filler is LX-6703 or HZ-835, a good separation and purification effect is achieved, the yield is greater than 80%, and the HPLC purity of the harvested pyrroloquinoline quinone product is greater than 99.8%, and the content of each single impurity is less than 0.1%, and the quality of the finished product meets the quality requirements of the United States Pharmacopoeia (USP). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the liquid phase detection spectrum of pyrroloquinoline quinone in Example 1 of the present invention; Figure 2 is the liquid phase detection spectrum of pyrroloquinoline quinone in Example 4 of the present invention; Figure 3 is the liquid phase detection spectrum of pyrroloquinoline quinone in Example 7 of the present invention; Figure 4 is the liquid phase detection spectrum of pyrroloquinoline quinone in Example 10 of the present invention; Figure 5 This is the liquid phase detection spectrum of pyrroloquinoline quinone in Example 11 of the present invention. DETAILED DESCRIPTION

[0022] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0024] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods. It should be noted that the following embodiments do not constitute a limitation on the protection scope of the present invention.

[0025] The following is the high performance liquid phase detection method used in the examples (determined in accordance with the General Rules 0512 of Part IV of the Chinese Pharmacopoeia 2020): Mobile phase for HPLC: Phase A: Weigh 2.28 g of potassium dihydrogen phosphate trihydrate and 4.84 g of tetrabutylammonium bromide, add 800 mL of water and ultrasonically dissolve, adjust the pH to 7.4 with phosphoric acid, and dilute to 1000 mL with water; Phase B: acetonitrile; Phase A / Phase B: 0-25 min: 75 / 25; 25-48 min: from 75 / 25 to 79 / 21; 48-50 min: from 79 / 21 to 75 / 25; 50-55 min: 75 / 25; Chromatographic column: octadecylsilane bonded silica filler or high pH resistant chromatographic column of equivalent specifications, 4.6×150 mm, 5μm; Chromatographic conditions: flow rate 1 mL / min, column temperature 30 °C, detector wavelength 254 nm.

[0026] Example 1 (1) The fermentation broth containing 2.0 g / L of pyrroloquinoline quinone was filtered through a 50 nm pore size ceramic membrane, an 8 KDa ultrafiltration membrane, and a 500 Da nanofiltration membrane for solid-liquid separation and concentration, so that the solid impurities in the solution were separated; (2) Add 4.0 mol / L hydrochloric acid to the solution obtained in step 1) to adjust the pH to 2.5, and add solid sodium chloride to a final concentration of 1.3 mol / L. Stir at 600 rpm for 4 h and then centrifuge or filter; (3) The solid obtained in step 2) is dissolved in 50 mmol / L sodium dihydrogen phosphate-sodium hydroxide buffer to a pyrroloquinoline quinone concentration of 8 g / L, and then 4.0 mol / L sodium hydroxide is added to adjust the pH to 7.0. The solid is decolorized using activated carbon at a dosage of 0.5% w / v for 4 h, and then centrifuged or filtered. (4) The liquid obtained in step 3) was diluted with 50 mmol / L sodium dihydrogen phosphate-sodium hydroxide buffer to a concentration of 6 g / L of pyrroloquinoline quinone, and then subjected to HZ-16SS column chromatography with a sample volume of 60 g / L. 3 BV of 0.1 mol / L sodium chloride at pH=7.0, 50 mmol / L sodium dihydrogen phosphate solution, 5 BV of 0.3 mol / L sodium chloride at pH=7.0, 50 mmol / L sodium dihydrogen phosphate solution, 10 BV of 0.7 mol / L sodium chloride at pH=7.0, and 50 mmol / L sodium dihydrogen phosphate solution were used for desorption to obtain a total of 18 BV of column chromatography desorption liquid; (5) Add sodium chloride to the column chromatography desorption obtained in step 4) to a final concentration of 1.3 mol / L, stir at 600 rpm for 4 h, and then centrifuge or filter; (6) The solid obtained in step 5) was dissolved in ultrapure water to a concentration of 15 g / L pyrroloquinoline quinone, 40% v / v ethanol was added, and 4.0 mol / L hydrochloric acid was added to adjust the pH to 3.0. The mixture was centrifuged or filtered, and dried to obtain solid pyrroloquinoline quinone with a yield of 86.8% and an HPLC purity of 99.71%. The single impurity content in the HPLC spectrum was 0.169% at RT = 6.897 min, which did not meet the USP quality requirements.

[0027] Example 2 The difference from Example 1 is that the filler used in the column chromatography in step 4) is LX-1600, and the other operations are the same. The yield is 56.3%, the HPLC purity is 99.24%, and the maximum single impurity content is 0.081%, which meets the USP quality requirements.

[0028] Example 3 The difference from Example 1 is that the filler used in the column chromatography in step 4) is HZ-818, and the other operations are the same. The yield is 65.7%, the HPLC purity is 99.37%, and the maximum single impurity content is 0.073%, which meets the USP quality requirements.

[0029] Example 4 The difference from Example 1 is that the filler used in the column chromatography in step 4) is LX-6703, and the other operations are the same. The yield is 82.4%, the HPLC purity is 99.88%, and the maximum single impurity content is 0.064%, which meets the USP quality requirements.

[0030] Example 5 The difference from Example 1 is that the filler used in the column chromatography in step 4) is HZ-PQQ, and the other operations are the same. The yield is 65.3%, the HPLC purity is 99.32%, and the maximum single impurity content is 0.087%, which meets the USP quality requirements.

[0031] Example 6 The difference from Example 1 is that the filler used in the column chromatography in step 4) is HZ-828, and the other operations are the same. The yield is 76.4%, the HPLC purity is 99.62%, and the maximum single impurity content is 0.079%, which meets the USP quality requirements.

[0032] Example 7 The difference from Example 1 is that the filler used in the column chromatography in step 4) is HZ-835, and the other operations are the same. The yield is 83.2%, the HPLC purity is 99.93%, and the maximum single impurity content is 0.051%, which meets the USP quality requirements.

[0033] Example 8 Different from Example 7, the desorbent used in the column chromatography in step 4) is 3 BV of 0.1 mol / L sodium sulfate at pH=7.0, 50 mmol / L sodium dihydrogen phosphate solution, 5 BV of 0.3 mol / L sodium sulfate at pH=7.0, 50 mmol / L sodium dihydrogen phosphate solution, 10 BV of 0.7 mol / L sodium sulfate at pH=7.0, 50 mmol / L sodium dihydrogen phosphate solution for desorption to obtain a total of 18 BV column chromatography desorbent, and the rest of the operations are the same. The yield is 80.4%, the HPLC purity is 99.76%, and the single impurity content is up to 0.072%, which meets the USP quality requirements.

[0034] Example 9 Different from Example 7, the desorbent used in the column chromatography in step 4) is 3 BV of 0.1 mol / L sodium chloride at pH=7.0, 50 mmol / L sodium dihydrogen phosphate solution, 5 BV of 0.3 mol / L sodium chloride at pH=7.0, 50 mmol / L sodium dihydrogen phosphate solution, 8 BV of 0.7 mol / L sodium chloride at pH=7.0, 50 mmol / L sodium dihydrogen phosphate solution for desorption to obtain a total of 16 BV column chromatography desorbent, and the rest of the operations are the same. The yield is 78.7%, the HPLC purity is 99.79%, and the single impurity content is up to 0.074%, which meets the quality requirements of the United States Pharmacopoeia USP.

[0035] Example 10 Different from Example 7, the desorbent used in the column chromatography in step 4) is 3 BV of 0.1 mol / L sodium chloride at pH=9.0, 50 mmol / L sodium dihydrogen phosphate solution, 5 BV of 0.3 mol / L sodium chloride at pH=9.0, 50 mmol / L sodium dihydrogen phosphate solution, 10 BV of 0.7 mol / L sodium chloride at pH=9.0, 50 mmol / L sodium dihydrogen phosphate solution for desorption to obtain a total of 18 BV column chromatography desorbent, and the rest of the operations are the same. The yield is 83.6%, the HPLC purity is 99.60%, and the single impurity content in the HPLC spectrum at RT=6.899 min is 0.151%, which does not meet the USP quality requirements.

[0036] Embodiment 11 Different from Example 8, the desorbent used in the column chromatography in step 4) is 3 BV of 0.1 mol / L sodium sulfate at pH=9.0, 50 mmol / L sodium dihydrogen phosphate solution, 5 BV of 0.3 mol / L sodium sulfate at pH=9.0, 50 mmol / L sodium dihydrogen phosphate solution, 10 BV of 0.7 mol / L sodium sulfate at pH=9.0, 50 mmol / L sodium dihydrogen phosphate solution for desorption to obtain a total of 18 BV column chromatography desorbent, and the rest of the operations are the same. The yield is 85.3%, the HPLC purity is 99.77%, and the single impurity content in the HPLC spectrum at RT=7.085 min is 0.136%, which does not meet the USP quality requirements.

[0037] The separation and purification results in Examples 1 to 11 are shown in Table 1.

[0038] Table 1 Separation and purification results in Example 1 to Example 11

[0039] The results showed that in the analytical step after chromatography, the pH of the analytical agent affected the content of the single impurity of the product; column chromatography was used, and the column filler was one of LX-1600, HZ-818, LX-6703, HZ-PQQ, HZ-828, and HZ-835, and in the analytical step after chromatography, when the pH of the analytical agent was 7.0-8.5, the HPLC purity of the pyrroloquinoline quinone product obtained was >99%, and the content of each single impurity was <0.1%. Among them, the chromatographic column filler was LX-6703 or HZ-835, and a good separation and purification effect was achieved, with a yield of >80%. At the same time, the HPLC purity of the pyrroloquinoline quinone product obtained was >99.8%, and the content of each single impurity was <0.1%. The quality of the finished product met the quality requirements of the United States Pharmacopoeia (USP).

[0040] When the pH of the analytical agent is 9.0, although a high yield and high purity can be obtained, the content of a single impurity is >0.1%, and the quality of the finished product does not meet the quality requirements of the United States Pharmacopoeia (USP).

[0041] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A method for separating and purifying pyrroloquinoline quinone, characterized in that: The steps include: S1. Using a combined membrane filtration to separate solid from liquid and concentrate the fermentation broth rich in pyrroloquinoline quinone; S2, adding hydrochloric acid and sodium chloride to the solution separated in step S1, adjusting the pH to acidic, and performing solid-liquid separation after stirring; S3, adding a buffer to the solid separated in step S2 to dissolve it, then adding sodium hydroxide, adjusting the pH to neutral or weakly alkaline, decolorizing with activated carbon, and then performing solid-liquid separation; S4, the liquid obtained in step S3 is diluted with a buffer solution and then subjected to column chromatography, and desorbed using a desorbent having a pH value of 7.0-8.5 and a gradient salt concentration to obtain a column chromatography desorbent; S5. Add sodium chloride to the column chromatography desorption liquid obtained in step S4 until the product is precipitated, and separate the solid and liquid after stirring; S6. The solid obtained in step S5 is dissolved in ultrapure water, ethanol is added, hydrochloric acid is added to adjust to acidity, solid-liquid separation is performed, and the obtained solid is dried to obtain solid pyrroloquinoline quinone; The chromatography column filler is one of LX-1600, HZ-818, LX-6703, HZ-PQQ, HZ-828, and HZ-835.

2. The method for separating and purifying pyrroloquinoline quinone according to claim 1, characterized in that: In the step S1, the combined membrane filtration is to sequentially use a ceramic membrane with a pore size of 40-50 nm, an ultrafiltration membrane with a pore size of 8-10 KDa, and a nanofiltration membrane with a pore size of 400-500 Da for filtration.

3. The method for separating and purifying pyrroloquinoline quinone according to claim 1, characterized in that: In step S3, the buffer is 20-50 mmol / L sodium dihydrogen phosphate-sodium hydroxide buffer.

4. The method for separating and purifying pyrroloquinoline quinone according to claim 1, characterized in that: In step S3, the amount of activated carbon used is 0.5-0.8% w / v, and the decolorization time is 4-5 h.

5. The method for separating and purifying pyrroloquinoline quinone according to claim 1, characterized in that: In the step S4, the desorption of the desorbent with gradient salt concentration is: 3 BV 0.1-0.2 mol / L desorbent, 5 BV 0.3-0.5 mol / L desorbent, and 8-10 BV 0.6-0.7 mol / L desorbent are used in sequence.

6. The method for separating and purifying pyrroloquinoline quinone according to claim 5, characterized in that: The analytical agent is sodium chloride or sodium sulfate; the analytical agent also contains 50-60 mmol / L of sodium dihydrogen phosphate.

7. The method for separating and purifying pyrroloquinoline quinone according to claim 1, characterized in that: In step S4, the liquid loading amount for column chromatography is 60-80 g / L.

8. The method for separating and purifying pyrroloquinoline quinone according to claim 1, characterized in that: In step S6, the amount of ethanol used is 40-50% v / v.

9. The method for separating and purifying pyrroloquinoline quinone according to any one of claims 1 to 8, characterized in that: The concentration of hydrochloric acid in steps S2 and S6 is 3.0-4.0 mol / L; the concentration of sodium hydroxide in S3 is 3.0-4.0 mol / L; and the concentration of sodium chloride in S5 is 1.0-1.3 mol / L.

10. The method for separating and purifying pyrroloquinoline quinone according to any one of claims 1 to 8, characterized in that: The stirring conditions in steps S2 and S5 are 500-600 rpm for 4-5 h.

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