A method for preparing reduced glutathione alpha-type crystals

Abstract

The invention discloses a method for preparing reduced glutathione α-type crystals, wherein an organic acid or its salt is used as a crystal medium; the crystal medium is added to a solution containing reduced glutathione, and the solution is stirred for crystallization to obtain α-type crystals of reduced glutathione. The method provided by the invention uses an organic acid or its salt as a crystal medium, significantly inhibits the formation of β-type crystals and the transformation of α-type crystals to β-type crystals, can efficiently and stably produce α-type crystals of reduced glutathione, and the reduced glutathione crystals finally obtained have high purity, and the mass fraction can reach more than 99%.

Description

Technical Field

[0001] The invention relates to the field of crystallization, and in particular to a method for preparing reduced glutathione alpha-type crystals. Background Art

[0002] Glutathione (GSH) is an active tripeptide with important physiological functions. It is formed by the condensation of glutamic acid, cysteine ​​and glycine through peptide bonds. Its chemical name is γ-L-glutamyl-L-cysteine-glycine (such as

[0003] (as shown in formula 1).

[0004]

[0005] The relative molecular mass of GSH is 307.33, the melting point is 189-193℃ (decomposition), the crystal is colorless, transparent, and elongated, and the isoelectric point is 5.93. It is soluble in water, dilute alcohol, liquid ammonia, and dimethylformamide, but insoluble in alcohol, ether, and acetone. GSH solid is relatively stable, while the aqueous solution is easily oxidized in the air.

[0006] GSH is an active tripeptide with important physiological functions. It can not only resist the destructive effect of oxidants on protein sulfhydryl groups and maintain the normal biological activity of proteins, but also act as a coenzyme for a variety of enzyme reactions, participate in the tricarboxylic acid cycle and sugar metabolism in the body, and has the functions of detoxification, delaying aging, preventing diabetes and cancer, and eliminating fatigue.

[0007] There are two crystal forms of reduced glutathione, α and β. Among them, α crystals have a higher solubility in water (at 10°C, the solubility in water is about 89g / L; under the same conditions, the solubility of β crystal reduced glutathione in water is only 30g / L). In addition, α crystals are columnar and have larger particles, which are convenient for filtration and drying, and easy to separate from the crystallization mother liquor, so they have higher purity and fewer impurities. β crystals are needle-shaped crystals with very fine particles, which are difficult to separate from the crystallization mother liquor, and are easy to wrap impurities in the mother liquor, resulting in lower purity of the finished product. Because of these characteristics, α crystals are needed in most industrial production.

[0008] The α-crystal form of glutathione is an unstable crystal form relative to the β-crystal form due to its high solubility. Therefore, β-crystals are usually obtained during the crystallization process, and α-crystals are difficult to obtain. Therefore, it is urgent to develop a method for inhibiting the formation of β-crystals in a supersaturated solution of reduced glutathione and achieving selective crystallization to obtain α-crystals.

[0009] The Japanese patent "Method for producing α-type crystals of reduced glutathione and method for preserving the crystals" uses an amino acid-based crystal mediator to obtain α-crystals of glutathione, such as adding aliphatic amino acids such as alanine, proline, etc., sulfur-containing amino acids such as cysteine, etc., aromatic amino acids such as phenylalanine, tryptophan, etc. The amount of the crystal mediator added is about 0.01%-10%. However, if the concentration of this crystal mediator is high, during the crystallization process, as glutathione crystals are formed, the crystal mediator will also precipitate, which increases the content of impurities in the finished product and reduces the purity of glutathione. Therefore, it is necessary to develop a more effective crystallization method for obtaining α crystals to ensure the purity of the glutathione finally obtained. Summary of the invention

[0010] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing α-type crystals of reduced glutathione in view of the deficiencies of the prior art.

[0011] The technical problem that the present invention needs to solve is to provide a method for inhibiting the formation of reduced glutathione β-type crystals.

[0012] In order to solve the above-mentioned first technical problem, the present invention discloses a method for preparing α-type crystals of reduced glutathione using an organic acid or its salt as a crystal medium.

[0013] The organic acid includes but is not limited to any one or a combination of formic acid, acetic acid, propionic acid, sorbic acid and citric acid.

[0014] The salt includes but is not limited to any one or a combination of sodium salt, potassium salt and ammonium salt.

[0015] The method for preparing the α-type crystal of reduced glutathione specifically comprises adding a crystal medium to a solution containing reduced glutathione, stirring and crystallizing, and obtaining the α-type crystal of prototype glutathione.

[0016] Wherein, the solvent of the solution containing reduced glutathione is water.

[0017] The method for preparing the solution containing reduced glutathione is as follows: adding reduced glutathione into water, dissolving the water, and obtaining the solution containing reduced glutathione.

[0018] Wherein, the dissolution is promoted by any one or a combination of the following methods:

[0019] (i) heating;

[0020] (ii) adjusting the pH to 6-7;

[0021] (iii) stirring.

[0022] In the embodiment (i), the heating is performed at a temperature of 50° C. or lower.

[0023] In mode (ii), the pH is adjusted to 6-7 preferably by using sodium hydroxide or ammonia solution to adjust the pH to 6-7.

[0024] In the method (ii), preferably, the pH is adjusted to 6-7, and the pH is adjusted to 2.5-3.0 after the reduced glutathione is dissolved.

[0025] Wherein, the pH value is adjusted to 2.5-3.0 preferably by using hydrochloric acid, sulfuric acid, phosphoric acid or the like to adjust the pH value to 2.5-3.0.

[0026] Wherein, the concentration of reduced glutathione in the reduced glutathione-containing solution is greater than 100 g / L.

[0027] When the concentration of reduced glutathione in the solution containing reduced glutathione is less than 200 g / L, the solution is concentrated to a concentration of reduced glutathione of 200 g / L or more, preferably 400 g / L or more, before or after adding the crystal mediator.

[0028] Wherein, the mass of the crystal mediator is 0.01%-1% of the mass of reduced glutathione.

[0029] Wherein, seed crystals are added during the stirring crystallization process.

[0030] Wherein, the seed crystal is an α-type prototype glutathione crystal.

[0031] Wherein, the variety is added at 15-30°C; preferably, the seed crystals are added at 20-25°C; further preferably, the temperature at which the seed crystals are added can be natural cooling or programmed temperature control.

[0032] Wherein, the added amount of the seed crystals is 0.2-1.3 g / 100 g reduced glutathione; preferably, the added amount of the seed crystals is 0.5-1 g / 100 g reduced glutathione.

[0033] Wherein, the temperature of the stirring crystallization is 15-30°C.

[0034] Preferably, the stirring and crystallization time is more than 1 h; preferably, the stirring and crystallization time is more than 1.5 h; further preferably, the stirring and crystallization time is more than 2 h.

[0035] Wherein, after the stirring crystallization, most of the α-type crystals of reduced glutathione are precipitated.

[0036] Preferably, the method for preparing the α-type crystal of reduced glutathione is to add a crystal medium to a solution containing reduced glutathione, stir and crystallize, add an organic solvent or a mixed solvent of an organic solvent and water, stir and cool and crystallize, so as to obtain the prototype glutathione α-type crystal.

[0037] The organic solvent includes but is not limited to any one or a combination of methanol, ethanol, acetone and isopropanol.

[0038] The organic solvent or the mixed solvent of the organic solvent and water can be added by flow addition, such as by using a peristaltic pump.

[0039] Wherein, the addition rate of the organic solvent or the mixed solvent of the organic solvent and water is less than 20% / h of the volume of the solution containing reduced glutathione.

[0040] Wherein, the added amount of the organic solvent or the mixed solvent of the organic solvent and water is more than 30% of the volume of the solution containing reduced glutathione.

[0041] Preferably, the cooling is to below 15°C; preferably, the cooling is to below 10°C; further preferably, the cooling is to 5-10°C.

[0042] Preferably, the stirring and cooling crystallization time is more than 0.5h; preferably, the stirring and cooling crystallization time is more than 1h; further preferably, the stirring and cooling crystallization time is more than 0.5h; preferably, the stirring and cooling crystallization time is more than 1.5h; further preferably, the stirring and cooling crystallization time is more than 2h.

[0043] After the stirring, cooling and crystallization is completed, the stirring is stopped, and the α-type crystals of the prototype glutathione are filtered and dried to obtain the finished product of the α-type crystals of the prototype glutathione.

[0044] The α-type crystals of the prototype glutathione have a distinct columnar structure, good sedimentation performance in a crystallization tank, obvious stratification, easy subsequent filtration and drying, and a fast dissolution rate in water at room temperature, and a higher solubility than β-crystals.

[0045] In order to solve the second technical problem mentioned above, the present invention discloses a method for inhibiting the formation of reduced glutathione β-type crystals, using an organic acid or a salt thereof as a crystal mediator.

[0046] The organic acid includes but is not limited to any one or a combination of formic acid, acetic acid, propionic acid, sorbic acid and citric acid.

[0047] The salt includes but is not limited to any one or a combination of sodium salt, potassium salt and ammonium salt.

[0048] The mechanism of the organic acid of the present invention is that since the solubility of α crystal of glutathione is high (10°C, 89g / L), while the solubility of β crystal is low (10°C, 30g / L), the stability of β crystal is higher, and the β crystal is easily obtained during the crystallization process. The present invention brings a certain blocking effect by adding organic acid, and the organic acid molecules are adsorbed on the surface of the β crystal, which hinders the glutathione base molecules from entering the crystal lattice, thereby inhibiting the formation of β crystal during the crystallization process and promoting the transformation of β crystal to α crystal.

[0049] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0050] The method provided by the present invention uses an organic acid or its salt as a crystal medium, significantly inhibits the formation of β-type crystals and the transformation of α-type crystals to β-type crystals, can efficiently and stably produce α-type crystals of reduced glutathione, and the reduced glutathione crystals finally obtained have high purity, and the mass fraction can reach more than 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0052] Figure 1 The figure is the X-ray diffraction pattern of the α-type crystal powder of the reduced glutathione product obtained in Example 1.

[0053] Figure 2 The morphology of the finished α-type crystal of reduced glutathione obtained in Example 1 under a microscope.

[0054] Figure 3 This is the HPLC chart of finished glutathione.

[0055] Figure 4 The morphology of glutathione crystals under a microscope obtained by adding L-cysteine ​​as a crystal mediator.

[0056] Figure 5 This is the result of powder X-ray diffraction analysis of glutathione β crystals. DETAILED DESCRIPTION

[0057] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0058] The reduced glutathione content in the glutathione raw material described in the following examples is 98%.

[0059] In the following examples, unless otherwise specified, the stirring speed is 150 rpm.

[0060] The 95% ethanol described in the following examples is a 95vt% ethanol aqueous solution.

[0061] Example 1: Crystallization of Glutathione - Acetic Acid as Crystallization Agent

[0062] Dissolve 150g of glutathione raw material in 1L of pure water, accelerate dissolution by stirring and water bath (45-47°C), then add 0.5mL of acetic acid, and then increase the concentration of glutathione to 450g / L by vacuum concentration (50°C). Transfer the concentrated solution to a 1L crystallizer, cool it to 25°C naturally under constant stirring, add 0.75g of α-type glutathione crystals as seed crystals, continue stirring for 2 hours, so that most of the α-type glutathione crystals are precipitated. Then use a peristaltic pump to flow into the crystallizer with 95% ethanol at a flow rate of 40mL / h, and the total addition amount is 120mL. After the addition of alcohol is completed, the temperature is lowered to 5°C through the jacket of the crystallizer, and maintained at this temperature for 2 hours, then the stirring is stopped, the glutathione slurry in the crystallizer is released, the crystals are filtered, and placed in a vacuum drying oven at 45°C for drying, and 126g of α-glutathione crystals are obtained, with a yield of 84% and a purity of 99.5%; the X-ray diffraction pattern of the obtained finished product is as follows Figure 1 The diffraction data are shown in Table 1, and the morphology under the microscope is shown in Figure 2 As shown, the HPLC diagram of the sample is as follows Figure 3 shown.

[0063] Table 1 Diffraction data of α-glutathione crystal product

[0064]

[0065]

[0066]

[0067] Example 2: Crystallization of Glutathione - Sodium Acetate as Crystallization Agent

[0068] 100g of glutathione raw material was dissolved in 500mL of pure water, and then 0.6g of sodium acetate was added, and then the glutathione solution was concentrated to 250mL by vacuum concentration, and the concentrated solution was transferred to a 1L crystallizer, the speed of the agitator was 150rpm, and it was naturally cooled to 20°C, and 0.5g of α-type prototype glutathione crystal was added, and then 95% ethanol was added dropwise at a flow rate of 40mL / h, and a total of 250mL was added. Then the temperature of the crystallizer was cooled to 10°C within 2 hours, and maintained at this temperature for 2 hours, and then the stirring was stopped, and the glutathione slurry in the crystallizer was released, and the crystal was filtered and dried to obtain 91g of α-type glutathione crystal finished product, with a yield of 91% and a purity of 99%.

[0069] Example 3: Crystallization of glutathione - sodium citrate as a crystallization medium

[0070] By heating and stirring at 45°C, 200g of glutathione raw material is dissolved in 1L of pure water, and then 2g of sodium citrate is added. After stirring and dissolving, the prepared solution is transferred to a 2L crystallization tank, and under constant stirring, the temperature of the solution is programmed to cool to 25°C within 2 hours, and then 2g of α-type prototype glutathione crystals are added to the crystallization tank as seeds. After stirring for 1 hour, 95% ethanol is added to the crystallization tank at a speed of 150mL / h by a peristaltic pump, and a total of 1.5L is added. Then, the temperature of the solution is programmed to cool to 5°C by a jacket cooling method, and maintained at this temperature for more than 2 hours, then the stirring is stopped, the tank is lowered, the slurry is collected, the crystals are filtered and dried, and 160g of α-type glutathione crystals are obtained, with a yield of 80% and a purity of 99%.

[0071] Comparative Example 1: Crystallization of Glutathione - L-Cysteine ​​as a Crystallization Agent

[0072] Dissolve 150g of glutathione raw material in 1L of pure water, accelerate the dissolution by stirring and water bath (45-47℃), then add 0.5g of L-cysteine, and concentrate the glutathione solution by vacuum concentration (50℃). It was found that when the glutathione concentration reached about 260g / L, a white precipitate began to precipitate in the solution. After further concentration, a large amount of precipitate precipitated. After the precipitate was filtered and dried, it was found that it was mainly very fine glutathione β crystals, which were obtained under a microscope as shown in the following picture. Figure 4 The crystal powder X-ray diffraction analysis results are shown in Figure 5 Therefore, it is difficult to prepare α-glutathione crystals by adding L-cysteine ​​(the added amount is 0.33% of glutathione).

[0073] Comparative Example 2: Crystallization of glutathione without a crystal mediator

[0074] 20g of glutathione raw material (purchased from Jincheng Pharmaceutical, α crystal, glutathione content of 98%) was dissolved in 200mL of pure water, and completely dissolved by heating and stirring, and then cooled to 10°C under continuous stirring. During this process, a large amount of precipitate was precipitated, which was finely broken β crystals under microscope observation. Therefore, if no crystal mediator is added, β crystals are obtained.

[0075] The present invention provides a method and idea for preparing reduced glutathione α-type crystals. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing reduced glutathione α-type crystals, characterized in that: An organic acid or its salt is used as a crystal medium; the crystal medium is added to a solution containing reduced glutathione, stirred for crystallization, an organic solvent or a mixed solvent of an organic solvent and water is added, stirred, cooled and crystallized, and α-type crystals of reduced glutathione are obtained; The organic acid is any one or a combination of acetic acid and citric acid; The mass of the crystal mediator is 0.01%-1% of the mass of reduced glutathione; The stirring crystallization temperature is 15-30°C; the stirring crystallization time is more than 1h; The organic solvent is any one or a combination of methanol, ethanol, acetone and isopropanol; The rate of adding the organic solvent or the mixed solvent of the organic solvent and water is less than 20% / h of the volume of the solution containing reduced glutathione.

2. The preparation method according to claim 1, characterized in that: The salt is any one of a sodium salt, a potassium salt and an ammonium salt.

3. The preparation method according to claim 1, characterized in that: The concentration of reduced glutathione in the reduced glutathione-containing solution is above 100 g / L.

4. The preparation method according to claim 1, characterized in that: The amount of the organic solvent or the mixed solvent of the organic solvent and water added is more than 30% of the volume of the solution containing reduced glutathione.

5. The preparation method according to claim 1, characterized in that: The cooling is to cool the temperature to below 10°C.

6. A method for inhibiting the formation of reduced glutathione β-type crystals, characterized in that: An organic acid or its salt is used as a crystal medium; the crystal medium is added to a solution containing reduced glutathione, stirred for crystallization, an organic solvent or a mixed solvent of an organic solvent and water is added, stirred, cooled and crystallized, and α-type crystals of reduced glutathione are obtained; The organic acid is any one or a combination of acetic acid and citric acid; The mass of the crystal mediator is 0.01%-1% of the mass of reduced glutathione; The stirring crystallization temperature is 15-30°C; the stirring crystallization time is more than 1h; The organic solvent is any one or a combination of methanol, ethanol, acetone and isopropanol; The rate of adding the organic solvent or the mixed solvent of the organic solvent and water is less than 20% / h of the volume of the solution containing reduced glutathione.

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