阅读说明：本技术 一种还原型谷胱甘肽α型晶体的制备方法 (Preparation method of reduced glutathione alpha-type crystal ) 是由 应汉杰 陈勇 周精卫 温庆仕 王骏之 于 2021-10-15 设计创作，主要内容包括：本发明公开了一种还原型谷胱甘肽α型晶体的制备方法,以有机酸或其盐为媒晶剂；向含还原型谷胱甘肽的溶液中加入媒晶剂,搅拌析晶,即得还原型谷胱甘肽的α型晶体。本发明所提供的方法以有机酸或其盐为媒晶剂,显著地抑制了β型晶体的形成和α型晶体向β型晶体的转变,可以高效且稳定地生产还原型谷胱甘肽的α型晶体,且最终获得的还原型谷胱甘肽晶体纯度高,质量分数可达到99％以上。(The invention discloses a preparation method of a reduced glutathione alpha-type crystal, which takes organic acid or salt thereof as a mordant; adding a medium crystal agent into the solution containing the reduced glutathione, stirring and crystallizing to obtain the alpha-type crystal of the reduced glutathione. The method provided by the invention takes the organic acid or the salt thereof as the mordant, remarkably inhibits the formation of beta-type crystals and the transformation of the alpha-type crystals to the beta-type crystals, can efficiently and stably produce the alpha-type crystals of the reduced glutathione, and finally obtains the reduced glutathione crystals with high purity and the mass fraction of more than 99 percent.)

1. A preparation method of reduced glutathione alpha-type crystals is characterized in that organic acid or salts thereof are used as a mordant; adding a medium crystal agent into the solution containing the reduced glutathione, stirring and crystallizing to obtain the alpha-type crystal of the reduced glutathione.

2. The preparation method according to claim 1, wherein the organic acid is any one or a combination of several of formic acid, acetic acid, propionic acid, sorbic acid and citric acid.

3. The method according to claim 1, wherein the salt is any one of a sodium salt, a potassium salt and an ammonium salt.

4. The production method according to claim 1, wherein the concentration of reduced glutathione in the reduced glutathione-containing solution is 100g/L or more.

5. The preparation method according to claim 1, wherein the mass of the mordant is 0.01-1% of the mass of the reduced glutathione-containing.

6. The preparation method according to claim 1, wherein the temperature of stirring crystallization is 15-30 ℃; the stirring and crystallization time is more than 1 h.

7. The preparation method according to claim 1, characterized in that a vehicle crystallization agent is added into the solution containing reduced glutathione, stirred for crystallization, an organic solvent or a mixed solvent of the organic solvent and water is added, and the mixture is stirred for cooling and crystallization, so that reduced glutathione alpha-type crystals are obtained; preferably, the organic solvent is any one or combination of methanol, ethanol, acetone and isopropanol; preferably, the adding rate of the organic solvent or the mixed solvent of the organic solvent and water is less than 20% of the volume of the reduced glutathione-containing solution per hour.

8. The method according to claim 7, wherein the amount of the organic solvent or the mixed solvent of an organic solvent and water is 30% or more by volume of the reduced glutathione-containing solution.

9. The method according to claim 7, wherein the temperature is reduced to below 10 ℃.

10. A method for inhibiting the formation of a reduced glutathione beta-form crystal, characterized in that an organic acid or a salt thereof is used as a mordant.

Technical Field

The invention relates to the field of crystallization, and in particular relates to a preparation method of a reduced glutathione alpha-type crystal.

Background

Glutathione (GSH) is an active tripeptide with important physiological functions, which is formed by condensing glutamic acid, cysteine and glycine through peptide bonds and has the chemical name of gamma-L-glutamyl-L-cysteine-glycine (such as

Shown in formula 1).

GSH has a relative molecular mass of 307.33, a melting point of 189-193 deg.C (decomposition), and the crystal is colorless, transparent, long, columnar, and has an isoelectric point of 5.93. It is soluble in water, dilute alcohols, liquid ammonia and dimethylformamide, but insoluble in alcohols, ethers and acetone. GSH solids are stable, while aqueous solutions are susceptible to oxidation in air.

GSH is an active tripeptide with important physiological functions. It can resist the damage of oxidant to protein sulfhydryl group, maintain normal biological activity of protein, and is also the coenzyme of several enzyme reactions, and can participate in tricarboxylic acid circulation and glycometabolism in vivo, and has the functions of removing toxic material, delaying senility, preventing diabetes and cancer and eliminating fatigue, etc.

Reduced glutathione exists in two crystal forms, alpha and beta respectively. Wherein, the alpha crystal has higher solubility in water (the solubility in water is about 89g/L at 10 ℃, and the solubility of beta crystal form reduced glutathione in water is only 30g/L under the same conditions). In addition, the alpha crystal is columnar, has larger particles, is convenient for suction filtration and drying, and is easy to separate from the crystallization mother liquor, so the purity is higher, and the impurities are less. The beta crystal is needle crystal, the particles are very fine, the separation from the crystallization mother liquor is difficult, and impurities in the mother liquor are easily wrapped, so that the purity of the finished product is low. Because of these characteristics, it is desired to obtain α crystals in most industrial processes.

The alpha crystal form of glutathione is an unstable crystal form compared with the beta crystal form due to high solubility. Therefore, beta crystals are generally obtained during the crystallization process, and alpha crystals are difficult to obtain. Therefore, there is an urgent need to develop a method for suppressing the formation of β crystals in a supersaturated solution of reduced glutathione and realizing selective crystallization to obtain α crystals.

Japanese patent "method for producing alpha-form crystals of reduced glutathione" and method for preserving said crystals "uses a mordant of an amino acid to obtain alpha-crystals of glutathione, for example, aliphatic amino acids such as alanine, proline and the like, sulfur-containing amino acids such as cysteine and the like, aromatic amino acids such as phenylalanine, tryptophan and the like are added. The addition amount of the mordant is about 0.01-10%. However, if the concentration of the mordant is higher, the mordant can be separated out along with the formation of glutathione crystals in the crystallization process, so that the content of impurities in the finished product is increased, and the purity of the glutathione is reduced. Therefore, it is required to develop a more efficient crystallization method for obtaining α crystals to ensure the purity of the finally obtained glutathione.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of the prior art and provides a preparation method of alpha-type crystals of reduced glutathione.

The technical problem to be solved by the invention is to provide a method for inhibiting the formation of reduced glutathione beta-type crystals.

In order to solve the first technical problem, the invention discloses a preparation method of alpha-type crystals of reduced glutathione, which takes organic acid or salts thereof as a mordant.

Wherein, the organic acid includes but is not limited to any one or combination of several of formic acid, acetic acid, propionic acid, sorbic acid and citric acid.

Wherein, the salt includes any one or a combination of several of sodium salt, potassium salt and ammonium salt.

The preparation method of the alpha-type crystal of the reduced glutathione comprises the steps of adding a mordant into a solution containing the reduced glutathione, stirring and crystallizing to obtain the alpha-type crystal of the prototype glutathione.

Wherein the solvent of the reduced glutathione-containing solution is water.

The preparation method of the solution containing the reduced glutathione comprises the steps of adding the reduced glutathione into water, and dissolving to obtain the solution containing the reduced glutathione.

Wherein the dissolution is promoted by any one or a combination of the following:

(i) heating up;

(ii) adjusting the pH value to 6-7;

(iii) and (4) stirring.

In the aspect (i), the temperature-raising heating is heating at 50 ℃ or lower.

In the mode (ii), the adjustment of the pH to 6 to 7 is preferably performed by adjusting the pH to 6 to 7 with sodium hydroxide or an aqueous ammonia solution.

In the mode (ii), preferably, the pH is adjusted to 6 to 7, and the pH is adjusted to 2.5 to 3.0 after the dissolution of the reduced glutathione.

Wherein, the pH value is preferably adjusted to 2.5-3.0 by hydrochloric acid, sulfuric acid, phosphoric acid and the like.

Wherein the concentration of reduced glutathione in the reduced glutathione-containing solution is 100g/L or more.

Wherein, when the concentration of the reduced glutathione in the solution containing the reduced glutathione is less than 200g/L, before or after the addition of the mordant, the concentration is concentrated to the concentration of the reduced glutathione of more than 200g/L, preferably more than 400 g/L.

Wherein the mass of the mordant is 0.01-1% of the mass of the reduced glutathione.

Wherein, seed crystals are added in the stirring and crystallization process.

Wherein the seed crystal is an alpha-type prototype glutathione crystal.

Wherein the variety is added at 15-30 ℃; preferably, the seed crystals are added at 20-25 ℃; further preferably, the temperature of the seed crystal can be naturally reduced, and can also be program temperature control.

Wherein the addition amount of the seed crystal is 0.2-1.3g/100g of reduced glutathione; preferably, the seed crystal is added in an amount of 0.5-1g/100g reduced glutathione.

Wherein the temperature of stirring crystallization is 15-30 ℃.

Wherein the stirring and crystallization time is more than 1 h; preferably, the stirring crystallization time is more than 1.5 h; more preferably, the stirring crystallization time is 2 hours or more.

Wherein after stirring and crystallizing, most of alpha-type crystals of the reduced glutathione are precipitated.

Preferably, the preparation method of the alpha-type crystal of the reduced glutathione comprises the steps of adding a mordant into a solution containing the reduced glutathione, stirring for crystallization, adding an organic solvent or a mixed solvent of the organic solvent and water, stirring, cooling and crystallizing to obtain the prototype glutathione alpha-type crystal.

Wherein, the organic solvent includes but is not limited to any one or combination of methanol, ethanol, acetone and isopropanol.

Wherein the organic solvent or the mixed solvent of the organic solvent and water can be added by feeding, such as adding by a peristaltic pump.

Wherein the adding 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 the reduced glutathione.

Wherein the addition amount of the organic solvent or the mixed solvent of the organic solvent and water is more than 30% of the volume of the reduced glutathione-containing solution.

Wherein the temperature reduction is to be reduced to below 15 ℃; preferably, the temperature is reduced to below 10 ℃; further preferably, the temperature is reduced to 5-10 ℃.

Wherein the stirring, cooling and crystallizing time is more than 0.5 h; preferably, the stirring, cooling and crystallizing time is more than 1 h; further preferably, the stirring, cooling and crystallizing time is more than 0.5 h; preferably, the stirring, cooling and crystallizing time is more than 1.5 h; more preferably, the stirring, cooling and crystallization time is more than 2 h.

And after the stirring, cooling and crystallization are finished, stopping stirring, and carrying out suction filtration and drying on the alpha-type crystal of the prototype glutathione to obtain the alpha-type crystal finished product of the prototype glutathione.

The alpha-type crystal of the prototype glutathione has an obvious columnar structure, has good sedimentation performance in a crystallization tank, is obvious in layering, is easy for subsequent suction filtration and drying, and has high dissolution rate in water at normal temperature and higher solubility than beta-type crystal.

In order to solve the second technical problem, the invention discloses a method for inhibiting the formation of reduced glutathione beta-type crystals, which takes organic acid or salt thereof as a mordant.

Wherein, the organic acid includes but is not limited to any one or combination of several of formic acid, acetic acid, propionic acid, sorbic acid and citric acid.

Wherein, the salt includes any one or a combination of several of sodium salt, potassium salt and ammonium salt.

The invention adopts the organic acid mechanism: because the solubility of alpha crystal of glutathione is high (10 ℃, 89g/L) and the solubility of beta crystal is low (10 ℃, 30g/L), the stability of the beta crystal is higher, and the beta crystal is easily obtained in the crystallization process. According to the invention, a certain retardation effect is brought by adding the organic acid, organic acid molecules are adsorbed on the surface of the beta crystal to prevent glutathione elementary molecules from entering crystal lattices, so that the formation of the beta crystal form in the crystallization process is inhibited, and the transformation of the beta crystal form to the alpha crystal is promoted.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the method provided by the invention takes the organic acid or the salt thereof as the mordant, remarkably inhibits the formation of beta-type crystals and the transformation of the alpha-type crystals to the beta-type crystals, can efficiently and stably produce the alpha-type crystals of the reduced glutathione, and finally obtains the reduced glutathione crystals with high purity and the mass fraction of more than 99 percent.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is an X-ray diffraction chart of an alpha-form crystal powder of a reduced glutathione product obtained in example 1.

FIG. 2 shows the appearance of the finished alpha-form crystals of reduced glutathione obtained in example 1 under a microscope.

FIG. 3 is a HPLC chart of glutathione as a finished product.

FIG. 4 shows the microscopic appearance of glutathione crystals obtained by adding L-cysteine as a habit modifier.

Fig. 5 shows the results of powder X-ray diffraction analysis of glutathione β crystals.

Detailed Description

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

The reduced glutathione content in the glutathione raw material described in the following examples was 98%.

In the following examples, the stirring was carried out at a rotational speed of 150rpm unless otherwise specified.

The 95% ethanol described in the examples below was a 95 vt% ethanol aqueous solution.

Example 1: glutathione crystals-acetic acid as a mordant

150g of glutathione raw material was dissolved in 1L of purified water, dissolution was accelerated by stirring and water bath (45-47 ℃), then 0.5mL of acetic acid was added, and the concentration of glutathione was increased to 450g/L by vacuum concentration (50 ℃). Transferring the concentrated solution into a 1L crystallizing tank, naturally cooling to 25 ℃ under continuous stirring, adding 0.75g of alpha-type glutathione crystals as seed crystals, and continuously stirring for 2 hours to separate out most of the alpha-type glutathione crystals. Subsequently, 95% ethanol was added to the crystallizer by means of a peristaltic pump at a flow rate of: 40mL/h, and the total addition amount is 120 mL. After the alcohol is added, cooling to 5 ℃ through a jacket of a crystallizing tank, maintaining for 2 hours at the temperature, stopping stirring, discharging glutathione crystal slurry in the crystallizing tank, performing suction filtration on the crystals, and drying in a vacuum drying oven at 45 ℃ to obtain a finished product of alpha-type glutathione crystals of 126g, wherein the yield is 84%, and the purity is 99.5%; the resulting finished product has an X-ray diffraction pattern as shown in FIG. 1, diffraction data as shown in Table 1, a microscopic morphology as shown in FIG. 2, and an HPLC profile of the sample as shown in FIG. 3.

TABLE 1 diffraction data for alpha-glutathione crystal preparations

Example 2: glutathione crystal-sodium acetate as a mordant

Dissolving 100g of glutathione raw material in 500mL of pure water, adding 0.6g of sodium acetate, concentrating the glutathione solution to 250mL in a vacuum concentration mode, transferring the concentrated solution to a 1L crystallizing tank, naturally cooling to 20 ℃ at the rotating speed of a stirrer of 150rpm, adding 0.5g of alpha-type prototype glutathione crystals, dropwise adding 95% ethanol at the flow rate of 40mL/h, and adding 250mL in total. And then cooling the temperature of the crystallization tank to 10 ℃ within 2 hours, maintaining the temperature for 2 hours, stopping stirring, discharging glutathione crystal slurry in the crystallization tank, performing suction filtration on the crystals, and drying to obtain 91g of alpha-type glutathione crystal finished products, wherein the yield is as follows: 91%, purity is: 99 percent.

Example 3: glutathione crystal-sodium citrate as medium crystal agent

200g of glutathione raw material was dissolved in 1L of pure water by heating and stirring at 45 ℃ followed by addition of 2g of sodium citrate. After stirring and dissolving, transferring the prepared solution to a 2L crystallizing tank, and gradually cooling the temperature of the solution to 25 ℃ within 2 hours under continuous stirring, then adding 2g of alpha-type prototype glutathione crystals into the crystallizing tank as seed crystals, stirring for 1 hour, and then adding 95% ethanol into the crystallizing tank at the speed of 150mL/h through a peristaltic pump, and adding 1.5L in total. And then, carrying out gradient cooling on the temperature program of the solution to 5 ℃ in a jacket cooling mode, maintaining the temperature for more than 2 hours, stopping stirring, discharging the solution into a tank, collecting crystal slurry, carrying out suction filtration on the crystal slurry, and drying to obtain 160g of finished alpha-type glutathione crystals with the yield of 80% and the purity of 99%.

Comparative example 1: glutathione crystal-L-cysteine as a mordant

150g of glutathione raw material is dissolved in 1L of pure water, the dissolution is accelerated by stirring and water bath (45-47 ℃), then 0.5g L-cysteine is added, and the glutathione solution is concentrated by vacuum concentration (50 ℃). It was found that when the concentration was increased to about 260g/L, a white precipitate began to precipitate out of the solution. After further concentration, a large amount of precipitate was precipitated. The precipitated precipitate was filtered and dried to find a very fine glutathione β crystal, and a photograph thereof under a microscope is shown in fig. 4, and the result of X-ray diffraction analysis of the crystal powder is shown in fig. 5. Therefore, it is difficult to produce alpha-glutathione crystals by the method of adding L-cysteine (the amount of addition is 0.33% of glutathione).

Comparative example 2: crystallization of glutathione without mordant

20g of glutathione raw material (purchased from Jincheng medicine, alpha crystal, the content of glutathione is 98%) is dissolved in 200mL of pure water, and the glutathione raw material is completely dissolved by heating and stirring, and then is cooled to 10 ℃ under continuous stirring, a large amount of precipitate is separated out in the process, and the precipitate is observed under a microscope to be fine beta crystal. Thus, without the addition of a habit modifier, beta crystals are obtained.

The invention provides a method and a method for preparing a reduced glutathione alpha crystal, and a plurality of methods and ways for realizing the technical scheme, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of improvements and decorations can be made without departing from the principle of the invention, and the improvements and decorations should also be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.