A method for separating and purifying glutathione

Abstract

The present invention provides a separation and purification method of glutathione, which comprises the following steps: adding a solvent to a crude glutathione product dissolved in formic acid, wherein the solvent comprises an alcohol solution of sodium hydroxide, and centrifuging after the crude glutathione product is completely crystallized. When the separation and purification method of the present invention is used to separate and purify glutathione, tripeptides, tetrapeptides, and polypeptide impurities can be effectively removed, and the obtained glutathione has a high yield and high purity, and has low requirements on equipment and process environment, and is more suitable for industrial production.

Description

Technical Field

[0001] The invention relates to a method for separating and purifying propylglutathione. Background Art

[0002] Alanyl Glutamine, chemical name N(2)-L-alanyl-L-glutamine (see formula 1), molecular formula: C8H 15 N3O4, molecular weight: 217.15. This product is white crystalline powder; odorless and tasteless; easily soluble in water and formic acid, insoluble in ethanol, methanol, acetone and ether.

[0003]

[0004] Glutamine is a conditionally essential amino acid. It is an important precursor for the synthesis of nucleic acids, proteins, purines, pyrimidines, etc. in organisms, and is also a regulator of protein synthesis and decomposition. However, glutamine itself cannot be used directly as a drug due to its instability and low water solubility. Only by converting it into a stable derivative can the effect of glutamine on the human body be fully exerted. Peter Furst and others found that alanyl-glutamine (abbreviated as alanyl-glutamine dipeptide) is an ideal substitute for glutamine and developed it as a parenteral nutrition drug in 1995. The drug has the effects of promoting muscle protein synthesis, improving clinical and biochemical indicators of critically ill patients, maintaining intestinal function, maintaining nitrogen balance in the body, and enhancing the immune system. It plays an important role in nutritional support for patients with large-area sudden trauma, and is currently widely used in Europe and the United States.

[0005] The early method for synthesizing glutathione was chemical synthesis, but chemical synthesis has problems such as long steps and more chemical reagents used, which limits its industrial application.

[0006] In recent years, due to the breakthrough in enzyme technology by Japanese scientists Yokozeki and Hara (Yokozeki K., Hara S., Journal of Biotechnology, 2005, 115 (02): 211-220.), the enzymatic synthesis of glutamic acid dipeptide can directly use L-alanine and L-glutamine as substrates to synthesize glutamic acid dipeptide in the next step under the action of enzyme catalysis. Compared with the previous chemical synthesis method, it has achieved a leap forward in technology, cost and safety. However, this technical route will produce tripeptide impurities alanyl alanyl glutamine, tetrapeptide impurities alanyl alanyl alanyl glutamine and other polypeptide impurities that are difficult to remove. In the current process technology, most people use 2 to 3 times the volume of water solvent glutamic acid dipeptide crude product, and then add a large amount of methanol for impurity removal and refining. Chinese patent CN110407914A discloses a separation and purification method of glutathione, that is, the crude product is dissolved in 3 times the amount of water, and then 0.1-50% of acidic substances are added, heated and stirred at 30-80°C, and 6 times the amount of methanol is added for crystallization, and the purified product is dried under reduced pressure at 65°C, with the highest purity reaching 99.83%, and a single impurity is less than 0.1%. This technology uses 12 times the amount of methanol in the crude product, which wastes a lot of resources, has high requirements for equipment and process environment, and has poor impurity removal effect. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a separation and purification method of glutathione in view of the defects of the prior art in removing tripeptides, tetrapeptides, and polypeptide impurities generated when preparing glutathione, such as large waste of resources, high cost, high requirements for equipment and process environment, and poor impurity removal effect. When glutathione is separated and purified by the separation and purification method of the present invention, tripeptides, tetrapeptides, and polypeptide impurities can be effectively removed, the yield of the obtained glutathione is high and the purity is high, and the requirements for equipment and process environment are low, which is more suitable for industrial production.

[0008] After extensive research, the inventor unexpectedly discovered that when formic acid is used as a good solvent for propyl glutathione and crystallization is performed using an alcoholic solution of sodium hydroxide, tripeptides, tetrapeptides, and polypeptide impurities can be effectively removed, and the yield and purity of the obtained propyl glutathione can be effectively improved. In addition, the present invention can further simultaneously use a mixed solvent of an alcoholic solution of sodium hydroxide and a small polar solvent (such as n-heptane, etc.) for crystallization, which can make the crystallization effect better, thereby greatly reducing the amount of related solvents used, thereby reducing costs.

[0009] In order to solve the above technical problems, the present invention provides a method for separating and purifying propylglutathione, which comprises the following steps: adding a solvent to a crude propylglutathione product dissolved in formic acid to crystallize the crude propylglutathione product, wherein the solvent comprises an alcohol solution of sodium hydroxide, and after the crude propylglutathione product is completely crystallized, cooling and centrifuging.

[0010] The inventors have tried to use other acidic solvents during the experiment, but found that it is difficult to achieve the purpose of removing impurities. Only when formic acid is used can the impurities be effectively removed. The inventors have also tried to use an alcohol solution of sodium carbonate for the experiment, but the final effect is not ideal. They have also tried to use sodium methoxide and sodium ethoxide, but the effect is also not ideal, and these reagents are very expensive.

[0011] In the present invention, the complete crystallization generally refers to that the crude propylglutathione is completely precipitated. Those skilled in the art should know that conventional operations in the art can make the crude propylglutathione crystallize and finally be completely precipitated.

[0012] In the present invention, the centrifugation is mainly for discarding the material to obtain the separated and purified propylglutathione product.

[0013] In the present invention, the formic acid can be added to the crude product, and the crude product can also be added to the formic acid, and the order of adding the two can be in no particular order.

[0014] In the present invention, the solvent can only be added to the crude product of propylglutathione (for example, added in the form of drops). The inventors have tried to add the crude product to the solvent, and found that the final separation and purification effect is not good. Therefore, the addition is preferably carried out by dropping.

[0015] In the present invention, the dissolution can be carried out by conventional dissolution methods in the art, such as dissolution by stirring, preferably by stirring at room temperature. The dissolution is usually carried out until the solution is clear (the crude product is completely dissolved), that is, until the solution is clear. The room temperature has the meaning conventionally understood by those skilled in the art, such as about 20 to 25°C.

[0016] In the present invention, the crude glutathione dissolved in formic acid generally refers to dissolving the crude glutathione in formic acid, and the solvent during the dissolution may be only formic acid without other types of solvents such as water. That is, the crude glutathione dissolved in formic acid does not contain water.

[0017] In the present invention, the alcohol solution of sodium hydroxide may be conventional in the art, for example, the mass volume ratio of sodium hydroxide to the alcohol solution may be 5-20%, for example 15%, for example 5-15% or 15-20%, etc.

[0018] In the present invention, the alcohol solution may be preferably a lower alcohol solution. The lower alcohol generally refers to alcohols with shorter carbon chains, such as monohydric alcohols and dihydric alcohols containing less than 6 carbon atoms. The monohydric alcohol generally refers to an alcohol containing only one hydroxyl group in the molecule; the dihydric alcohol generally refers to an alcohol having two hydroxyl groups (-OH); the alcohol solution may be, for example, an ethanol solution, a methanol solution, an isopropanol solution or an ethylene glycol solution.

[0019] In the present invention, the solvent may also include a small polar solvent, which is a straight-chain alkane or its derivatives that are soluble in alcohols but insoluble in water. The derivatives in the straight-chain alkane or its derivatives have the meanings commonly understood by those skilled in the art, and may generally refer to a series of compounds generated by the replacement of hydrogen atoms in straight-chain alkane molecules by other atoms or atomic groups. The small polar solvent may be conventional in the art, for example, one or more selected from n-heptane, n-hexane and methyl tert-butyl ether. When a mixed solvent of an alcoholic solution of sodium hydroxide and a small polar solvent (such as n-heptane, etc.) is further used for crystallization, the crystallization effect can be better, thereby greatly reducing the amount of the relevant solvents and reducing costs. In a preferred embodiment, the solvent is composed of an alcoholic solution of sodium hydroxide and a small polar solvent.

[0020] In the present invention, the alcohols have the meaning commonly understood by those skilled in the art, and generally refer to compounds containing a hydroxyl group bonded to a carbon on a hydrocarbon group or a benzene ring side chain in the molecule, whose functional group is -OH. The alcohols include but are not limited to methanol, ethanol, benzyl alcohol, ethylene glycol, and the like.

[0021] In the present invention, the volume of the low polarity solvent may preferably be 0.5-2.5 times, such as 0.75 times or 1 times, such as 0.75-2.5 times, 0.5-0.75 times or 0.5-1 times the volume of the formic acid.

[0022] In the present invention, the volume ratio of the alcohol solution of sodium hydroxide to the low polarity solvent may preferably be (0.2-1.5):1, such as 0.5:1, 0.75:1 or 1:1, for example, (0.5-1):1, (05-0.75):1, etc.

[0023] In the present invention, the volume of the solvent may generally be 1.5-5 times, such as 2 times or 3 times, the volume of the formic acid.

[0024] In the present invention, in the crude glutathione dissolved in formic acid, the mass volume ratio of the crude glutathione to the formic acid may preferably be (0.5-1):1, for example, 1:1.

[0025] In the present invention, the cooling may be conventional in the art, for example, the temperature may be preferably lowered to 5-10°C.

[0026] In the present invention, the crude product of glutathione may be a crude product conventionally prepared in the art, for example, a crude product prepared by an enzyme catalysis method, and the crude product is usually present in the form of glutathione dry matter.

[0027] In a preferred embodiment, the crude glutamine-propionate is obtained by the following method: cooling the mixed glutamine and purified water to 15°C, adjusting the pH value to 9.0, adding enzymes, starting to drip alanine methyl ester hydrochloride aqueous solution, and controlling the pH to maintain at 9.0, adding 30% volume of methanol after the reaction, heating to 60°C, and inactivating. Preferably, post-treatment is also included, which includes the following steps: adding activated carbon for decolorization at 60°C, filtering and then electrodialysis. After removing the solvent by vacuum rotary evaporation, add 1.5 times the volume of water and heat to 60°C, and add methanol for crystallization. Cool to 10°C, centrifuge, and vacuum dry the filter cake at 60°C to obtain the crude glutamine-propionate.

[0028] In a preferred embodiment, the crude product of glutamine-propionate is obtained by the following method: 5 kg of glutamine and purified water are added to the reactor in sequence, stirring is started, the temperature is lowered to 15 ° C, the pH value is adjusted to 9.0, the enzyme is added, 3.5 kg of alanine methyl ester hydrochloride aqueous solution is added, and ammonia water is added to control the pH to maintain at 9.0. After the addition is completed, the reaction end point is detected by HPLC, and after the reaction is completed, 30% of 1.8 kg of methanol is added, and the temperature is raised to 60 ° C for inactivation. Then post-treatment is performed, including: adding activated carbon for decolorization at 60 ° C, filtering and then electrodialysis. After the solvent is removed by vacuum rotary evaporation, 1.5 times the volume of 750g of water is added, the temperature is raised to 60 ° C, and 3.2 kg of methanol is added for crystallization, and the addition is completed in about 2 hours. Cool to 10 ° C, centrifuge, and the filter cake is vacuum dried at 60 ° C to obtain 5.82 kg of crude glutamine-propionate.

[0029] In a preferred embodiment, the separation and purification method comprises the following steps: adding the crude glutathione to formic acid (the mass volume ratio of glutathione to formic acid is preferably 1:1), stirring at room temperature until dissolved, and then dropping a mixed solvent of an alcohol solution of NaOH (e.g., 20% NaOH ethanol solution, 15% NaOH methanol solution, 5% NaOH isopropanol solution or 15% NaOH ethanol solution) and a small polar solvent (e.g., n-heptane or methyl tert-butyl ether), wherein the volume ratio of the mixed solvent to formic acid is preferably (1.5:1) to (3:1), for example, 2:1. After the addition is completed, the temperature is lowered to 5-10°C and centrifuged (to discard the material).

[0030] The product obtained by the above separation and purification method meets the pharmaceutical grade quality standards and can be applied to various industries according to actual needs. For example, it can be added to cell culture medium for cell culture.

[0031] In the present invention, the term “include or include” may mean that in addition to the components listed below, other components are present; or it may mean “consisting of…”, that is, only the components listed below are present without other components.

[0032] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0033] The reagents and raw materials used in the present invention are commercially available.

[0034] The positive and progressive effect of the present invention is that when the separation and purification method of the present invention is used to separate and purify propylglutathione, tripeptide, tetrapeptide, and polypeptide impurities can be effectively removed, the yield of the obtained propylglutathione is high and the purity is greatly improved, and the requirements for equipment and process environment are relatively low, which is more suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The HPLC detection spectrum of crude propylglutathione is shown.

[0036] Figure 2 The HPLC detection spectrum in Example 1 is shown.

[0037] Figure 3 The HPLC detection spectrum in Example 2 is shown.

[0038] Figure 4 The HPLC detection spectrum in Example 3 is shown.

[0039] Figure 5 The HPLC detection spectrum in Example 4 is shown.

[0040] Figure 6 The HPLC detection spectrum in Comparative Example 1 is shown.

[0041] Figure 7 The process route in the embodiment of the present invention is shown. DETAILED DESCRIPTION

[0042] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0043] In the following examples, the synthesis process of the crude alanine dipeptide is as follows: glutamine (5 kg) and purified water are added to the reactor in sequence, stirring is started, the temperature is lowered to 15°C, the pH value is adjusted to 9.0, α-amino acid ester acyltransferase (purchased from Beijing Biolab Technology Co., Ltd.) is added, and alanine methyl ester hydrochloride aqueous solution (3.5 kg) is added dropwise, and ammonia water is added to control the pH to maintain at 9.0. After the addition is completed, the reaction end point is detected by HPLC, and after the reaction is completed, 30% (volume percentage) methanol (1.8 kg) is added, and the temperature is raised to 60°C for inactivation.

[0044] Post-treatment: add activated carbon for decolorization at 60°C, filter and perform electrodialysis. After removing the solvent by vacuum rotary evaporation, add 1.5 times the volume of water (750g) and raise the temperature to 60°C, add methanol (3.2kg) dropwise for crystallization, and the addition is completed in about 2h. Cool down to 10°C, centrifuge, and vacuum dry the filter cake at 60°C to obtain 5.82kg of crude glutathione.

[0045] Example 1

[0046] The process route in the embodiment is as follows Figure 7 As shown, specifically: take 100g of the crude product and dissolve it in 1 volume of formic acid (100ml), stir at room temperature (room temperature refers to 20-25℃), and after it is dissolved, add a mixed solvent of 100ml of 20% NaOH ethanol solution and 100ml of n-heptane to crystallize the crude product of propyl glutathione. After the crystallization is complete, cool to 10℃. Centrifuge and discard the material, vacuum dry at 60℃, crush and sieve to obtain a total of 92.3g of the refined finished product propyl glutathione, with a yield of 92.3%. After refining, the removal of impurities such as tripeptides and polypeptides is detected by liquid phase. The crude product HPLC results are shown in Figure 1 As shown in Table 1, the content of propyl glutathione is 87.66%, of which the content of tripeptide is 6.4% and the content of tetrapeptide is 1.91%. After the refining process, the content of propyl glutathione is increased to 99.82%. The tripeptide, tetrapeptide impurities and other impurities have been removed. The HPLC results are shown in Figure 2 and Table 2. The formic acid soluble residual test content is 0.03%, and the ethanol soluble residual test content is 0.02%, both of which meet the quality requirements of pharmaceutical grade. The results are shown in Table 8.

[0047] Table 1 HPLC test results of crude glutathione

[0048]

[0049]

[0050] Table 2 HPLC test results in Example 1

[0051]

[0052] Example 2

[0053] Dissolve 100g of crude product in 1 volume of formic acid (100ml), stir at room temperature (room temperature refers to 20-25℃), and after it is dissolved, add 50ml of 15% NaOH methanol solution and 250ml of n-hexane mixed solvent to crystallize the crude product of propyl glutathione. After the crystallization is complete, cool to 10℃. Centrifuge and discard the material, vacuum dry at 60℃, crush and sieve to obtain a total of 92.3g of refined finished propyl glutathione, with a yield of 92.3% and a purity of 99.90%. HPLC results are shown in Figure 3 and Table 3. The formic acid soluble residual detection content is 0.03%, and the methanol soluble residual detection content is 0.03%, both of which meet the quality requirements of pharmaceutical grade. The results are shown in Table 8.

[0054] Table 3 HPLC test results in Example 2

[0055]

[0056] Example 3

[0057] Dissolve 100g of crude product in 1 volume of formic acid (100ml), stir at room temperature (room temperature refers to 20-25℃), and after it is dissolved, add a mixed solvent of 150ml of 5% NaOH isopropanol solution and 50ml of n-heptane to crystallize the crude product of propyl glutathione. After the crystallization is complete, cool to 10℃. Centrifuge and discard the material, vacuum dry at 60℃, crush and sieve to obtain a total of 88.0g of refined finished propyl glutathione, with a yield of 88.0% and a purity of 99.77%. HPLC results are shown in Figure 4 and Table 4. The formic acid soluble residual detection content is 0.02%, and the isopropanol soluble residual detection content is 0.03%, both of which meet the quality requirements of pharmaceutical grade. The results are shown in Table 8.

[0058] Table 4 Example 3 HPLC test results

[0059]

[0060] Example 4

[0061] Dissolve 2kg of crude product in 1 volume of formic acid (2L), stir at room temperature (room temperature refers to 20-25℃), and wait until it is dissolved. Add 1.5L of 15% NaOH ethanol solution and 1.5L of n-heptane as a mixed solvent to crystallize the crude product of propyl glutathione. After the crystallization is complete, cool to 10℃. Centrifuge and discard the material, vacuum dry at 60℃, crush and sieve to obtain a total of 1.814kg of refined finished propyl glutathione, with a yield of 90.7% and a purity of 99.76%. HPLC results are shown in Figure 5The formic acid soluble residual test content is 0.04%, and the ethanol soluble residual test content is 0.03%, both of which meet the quality requirements of pharmaceutical grade. The results are shown in Table 8.

[0062] Table 5 Example 4 HPLC test results

[0063]

[0064]

[0065] In summary, the above refining process can solve the problem that polypeptides are difficult to remove, and the refining process steps are simple and easy to operate. The solvents used are cheap and easy to obtain, and the production cost is low. The product purity is above 99%, the impurity removal effect is good, and it has good industrial use value.

[0066] Comparative Example 1 (Refer to the refining method of CN110407914A)

[0067] Dissolve 100g crude product in 3 times volume of purified water (200ml), add 30% monohydrate citric acid, heat to 60℃ and stir, keep warm for 30min, add 6 times volume of methanol for crystallization, add 1800ml methanol dropwise to crystallize the crude product of propylglutathione. After the crystallization is complete, cool to 10℃. Centrifuge and discard, vacuum dry at 65℃, crush and sieve to obtain the refined finished product propylglutathione, totaling 90.01g, with a yield of 90% and a purity of 91.59%. HPLC results are shown in Figure 6 and Table 7. Compare it with Example 2, see Table 6: The purity of Example 2 is 99.90%, which is 91.59% of that of Example 1. Comparison of impurities: No tripeptide or tetrapeptide was detected in Example 2, while the tripeptide impurity in the comparative example was 4.81% and the tetrapeptide impurity was 0.8%. We can draw the following conclusion: The impurity removal effect of the refining method provided in this example is significantly better than that of the refining method of CN110407914A.

[0068] Table 6 Comparison of results between Example 2 and Comparative Example 1

[0069] Sample name Tripeptide Tetrapeptide Methanol soluble residue purity Yield Example 2 Not detected Not detected 0.03 99.90％ 92.3％ Comparative Example 1 4.81％ 0.8％ 0.2 91.59％ 90％

[0070] Table 7 Comparative Example 1 HPLC test results

[0071]

[0072] Table 8 Residue detection:

[0073]

[0074]

Claims

1. A method for separating and purifying propylglutathione, characterized in that: It includes the following steps: Adding a solvent to the crude propyl glutathione dissolved in formic acid, wherein the solvent is a mixed solvent of an alcohol solution of sodium hydroxide and a small polar solvent, and centrifuging after the crude propyl glutathione is completely crystallized; The mass volume ratio of sodium hydroxide in the alcohol solution of sodium hydroxide to the alcohol solution is 5-20%; The alcohol solution is ethanol solution, methanol solution or isopropanol; The low polarity solvent is selected from one or more of n-heptane and n-hexane; The volume of the low polarity solvent is 0.5-2.5 times the volume of the formic acid; The volume ratio of the alcohol solution of sodium hydroxide to the low polarity solvent is (0.2-1.5):1; In the crude glutathione dissolved in formic acid, the mass volume ratio of the crude glutathione to the formic acid is (0.5-1):

1.

2. The separation and purification method according to claim 1, wherein The dissolving is carried out by stirring; and / or, the crude glutathione dissolved in formic acid does not contain water; And / or, the adding is performed by dropwise addition.

3. The separation and purification method according to claim 1, wherein The dissolution is carried out by stirring at room temperature.

4. The separation and purification method according to claim 1, wherein The mass volume ratio of sodium hydroxide in the alcoholic solution of sodium hydroxide to the alcoholic solution is 15%.

5. The separation and purification method according to claim 1, wherein The volume of the low polar solvent is 0.75 times or 1 times the volume of the formic acid.

6. The separation and purification method according to claim 1, wherein The volume ratio of the alcohol solution of sodium hydroxide to the low-polarity solvent is 0.5:1, 0.75:1 or 1:

1.

7. The separation and purification method according to claim 1, wherein The mass volume ratio of the crude propylglutathione to the formic acid is 1:

1.

8. The separation and purification method according to claim 1, wherein The cooling is to cool to 5-10°C.

9. The separation and purification method according to claim 1, wherein The crude product of propylglutathione is a crude product prepared by enzyme catalysis.

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