Method for purifying reduced glutathione

Abstract

The invention provides a method for purifying reduced glutathione, which comprises the following steps: preparing a glutathione solution to obtain a saturated glutathione solution; titrating a crystallization agent into the saturated glutathione solution, and continuously stirring at a constant speed for a second preset duration to obtain a pre-crystallized glutathione solution; and standing the pre-crystallized glutathione solution until glutathione crystals are separated out, and keeping the glutathione solution in a constant temperature T state in the above processes, T being 30-40 DEG C. Crude glutathione is dissolved at low temperature and constant temperature, so that reduced glutathione is prevented from being oxidized, and the utilization rate of glutathione is guaranteed; by matching the additive solution with the crystallization agent, the purity and the appearance of the recrystallized glutathione are improved, the whole reaction process is mild, the pH value, the temperature range, the pressure and the like of the solution do not need to be adjusted, operation is easy, industrial production is facilitated, and the concept of energy conservation, environmental protection and greenness is met.

Description

Technical Field

[0001] The present application relates to the field of biochemical technology, and in particular to a method for purifying reduced glutathione. Background Art

[0002] Reduced glutathione is a small molecule peptide composed of three amino acids. As an important antioxidant and free radical scavenger in the body, it can participate in biotransformation, thereby converting harmful toxins in the body into harmless substances and then excreting them out of the body. Glutathione can also help maintain a normal immune system. Currently, reduced glutathione has become an important biochemical drug and is widely used in the health care products and food industries. The following reduced glutathione is referred to as glutathione.

[0003] Generally speaking, reduced glutathione is produced as crude glutathione through biological extraction or microbial fermentation. Both the food and pharmaceutical industries require glutathione raw materials with higher purity. Therefore, purifying crude glutathione can not only change the situation in which my country relies on imports of high-purity glutathione APIs, but also has significant social and economic benefits for the industrial production, clinical medicine and food industry applications of glutathione.

[0004] Generally, crude glutathione is dissolved, impurities are removed, recrystallized, washed, and dried to obtain glutathione with higher purity. However, due to different processes and different crystallization agents used, the purity of the glutathione obtained is still not high enough and its application value is low.

[0005] In view of this, it is necessary to design a purification method for reduced glutathione to solve one of the above problems. Summary of the Invention

[0006] The present application provides a method for purifying reduced glutathione, by which high-purity glutathione can be prepared.

[0007] In order to achieve the above objectives, the technical solutions provided by this application are as follows: The present application provides a method for purifying reduced glutathione, which comprises: The glutathione solution is prepared, specifically comprising: obtaining crude glutathione with a weight of m1 and an additive solution with a mass of m2, mixing them in a ratio of m1:m2 of 1:(10-20), ultrasonically vibrating the mixture for a first preset time, filtering to obtain a saturated glutathione solution, and maintaining the saturated glutathione solution at a constant temperature T, wherein T is 30° C. to 40° C.; titrating the crystallization agent into the saturated glutathione solution and continuously stirring at a constant speed for a second preset time to obtain a pre-crystallized glutathione solution; The pre-crystallized glutathione solution is allowed to stand until glutathione crystals are precipitated, and the pre-crystallized glutathione solution is kept at a constant temperature T.

[0008] Furthermore, the constant temperature T is 35°C.

[0009] Furthermore, the additive solution is Tween-80 or phenylalanine.

[0010] Furthermore, the concentration of the additive solution is 0.1% to 0.5%.

[0011] Furthermore, the crystalline agent is ethanol and / or isopropanol.

[0012] Furthermore, if the crystallization agent is ethanol, the volume ratio of ethanol to saturated glutathione solution is (2-6):1; If the crystallization agent is isopropyl alcohol, the volume ratio of isopropyl alcohol to saturated glutathione solution is (6-10):1; If the crystallization agent is a mixture of ethanol and isopropanol, the volume ratio of the mixture of ethanol and isopropanol to the saturated glutathione solution is (4-8):1.

[0013] Furthermore, if the crystallization agent is a mixture of ethanol and isopropanol, the volume ratio of ethanol to isopropanol is (4-8):1.

[0014] Furthermore, if the crystallization agent is ethanol and isopropanol, the titration of the crystallization agent into the saturated glutathione solution includes titrating ethanol in the initial stage of titrating the crystallization agent and titrating isopropanol in the later stage of titrating the crystallization agent, wherein the duration of the initial stage is longer than the duration of the later stage.

[0015] Furthermore, the titration rate of the crystalline agent is 10 drops / min to 20 drops / min.

[0016] Furthermore, the stirring rate of the glutathione solution is 130 r / min to 170 r / min.

[0017] Compared with the related art, the beneficial effects of the present application are: using the reduced glutathione purification method of the present application to dissolve crude glutathione at a low temperature and constant temperature, thereby avoiding oxidation of reduced glutathione and ensuring the utilization rate of glutathione; by matching the addition solution with the crystallization agent, the purity and appearance of the recrystallized glutathione are improved; the entire reaction process is relatively mild, and there is no need to adjust the pH value, temperature range, pressure, etc. of the solution. The operation is simple, which is conducive to industrial production and conforms to the concept of energy conservation, environmental protection and green. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The figure is a flow chart of a purification method of an embodiment of the method for purifying reduced glutathione of the present application.

[0019] Figure 2 This is a liquid chromatogram of glutathione obtained in another embodiment of the purification method of reduced glutathione of the present application.

[0020] Figure 3 yes Figure 2 The peak area and concentration curve of the middle spectrum.

[0021] Figure 4 yes Figure 2 Example of a microscope photo of glutathione.

[0022] Figure 5 This is a photo of crude glutathione under a microscope.

[0023] Figure 6 It is a curve chart showing the purity of glutathione prepared by using Tween-80 as an additive solution at different concentrations. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts should fall within the scope of protection of this application.

[0025] The present application provides a method for purifying reduced glutathione. The purity of the glutathione obtained by the purification method meets the requirements of clinical medicine and the food industry, and has great social and economic benefits.

[0026] like Figure 1 As shown, the purification method includes: first preparing a glutathione solution to dissolve crude glutathione to form a saturated glutathione solution, titrating a crystallization agent used to assist in reducing the solubility of the glutathione solution into the saturated glutathione solution to form a pre-crystallized glutathione solution, and allowing the pre-crystallized glutathione solution to stand until glutathione crystals are precipitated. During the entire purification process, the glutathione solution is maintained at a constant temperature T to prevent glutathione from being oxidized, thereby affecting the purity of glutathione and the purification process.

[0027] Before preparing glutathione solution, prepare an additive solution with a concentration of 0.1%-0.5% for use. For example, to prepare 0.1% Tween-80, since Tween-80 is a viscous liquid, place Tween-80 in a volumetric flask, calculate the mass m0 of Tween-80 in the volumetric flask by the double weighing method, and add pure water to the volumetric flask while stirring until the Tween-80 solution is fixed to 1000ml.

[0028] The preparation of the glutathione solution specifically includes: obtaining crude glutathione with a weight of m1, wherein the crude glutathione is extracted from glutathione fermentation broth and obtained by primary purification, and has a solubility between 3.52g and 5.87g. The purity of the crude glutathione generally cannot meet the requirements of medicine and food, so the crude glutathione needs to be secondary purified.

[0029] Then obtain an additive solution with a mass of m2, and fully mix the crude glutathione and the additive solution in a ratio of m1:m2 of 1:(10~20), match the specific mass m2 of the additive solution according to the solubility of the crude glutathione, and then use ultrasonic vibration to vibrate the mixture of glutathione and the additive solution for a first preset time. The saturated glutathione solution obtained is not easily oxidized. Wherein, the frequency of the ultrasound is 15kHZ, and the first preset time is 1h-3h, which facilitates the more complete dissolution of the crude glutathione and improves the utilization rate of the above-mentioned crude glutathione. Preferably, the first preset time is 2h. The first preset time adopts a suitable time. On the one hand, glutathione can be fully dissolved, and on the other hand, the solution is prevented from being polluted by the environment, thereby affecting the final purity of glutathione.

[0030] In this embodiment, the concentration of the additive solution is 0.1% to 0.5%. The optimal concentration of the additive solution is determined according to the type of additive solution selected. Please refer to the subsequent specific embodiments for details.

[0031] As a preferred embodiment of the present application, the additive solution is Tween-80, which is polyoxyethylene sorbitan monooleate-80. Tween-80 can improve the dissolution rate and stability of crude glutathione based on its surfactant properties and intermolecular interactions; Tween-80 micelles can prevent their aggregation or precipitation by regulating the polarity of the microenvironment, reduce the surface tension of the solution, improve the wettability of water molecules to glutathione, and further promote the dispersion and dissolution of glutathione. The non-ionic properties of Tween-80 avoid charge conflicts and do not cause precipitation.

[0032] In this embodiment, when Tween-80 is used as the additive solution, the preferred concentration is 0.3%.

[0033] As another preferred embodiment of the present application, the additive solution is L-phenylalanine. The unique properties of the L-phenylalanine can destroy the aggregation between glutathione molecules, thereby promoting the dissolution of glutathione, and can increase and reduce the probability of its oxidation, so that the recrystallization reaction is sufficient. Preferably, the concentration of L-phenylalanine as the additive solution is 0.1%-0.3%.

[0034] After the ultrasonic vibration is performed for a first preset time, the glutathione solution is filtered to remove the crude glutathione or impurities that are not completely dissolved, thereby obtaining a saturated glutathione solution. The glutathione solution is now in a clean and saturated state, which is conducive to the subsequent precipitation of high-purity glutathione crystals.

[0035] In the above process, the glutathione solution is always maintained at a constant temperature T, wherein T is 30°C to 40°C; preferably, the constant temperature T is 35°C, the glutathione solution will not be oxidized, and the utilization rate of glutathione is improved. Moreover, at this constant temperature, there is no need to adjust the pH value of the glutathione solution, which simplifies the purification process.

[0036] After obtaining a saturated glutathione solution, the crystallization agent is titrated into the saturated glutathione solution, and the solution is continuously stirred at a uniform speed for a second preset time to obtain a pre-crystallized glutathione solution. During the entire stirring process, the glutathione solution is still maintained at a constant temperature T to prevent glutathione from being oxidized. The second preset time is 4 to 6 hours, preferably, the second preset time is 5 hours.

[0037] In the present application, the crystallization agent is ethanol and / or isopropanol, that is, the crystallization agent can be ethanol, isopropanol, or both ethanol and isopropanol, which are described below. Wherein, ethanol or isopropanol are used as pure solutions and do not need to be diluted.

[0038] As another preferred embodiment of the present application, the crystalline agent is ethanol, that is, ethanol is titrated into a saturated glutathione solution. The ethanol can reduce the saturation of the glutathione solution and prevent the glutathione solution from being oxidized.

[0039] In this embodiment, the volume ratio of the saturated glutathione solution to the total amount of ethanol is 1:(2-6). Preferably, the volume ratio of the saturated glutathione solution to the total amount of ethanol is 1:4, that is, the total amount of ethanol titrated into the saturated glutathione solution is 4 times the volume of the glutathione solution, so that the ethanol is fully mixed with the glutathione solution, which is conducive to inducing the formation of rod-shaped crystal nuclei, thereby improving the purity of the glutathione crystals.

[0040] In another preferred embodiment of the present application, the crystallization agent is isopropyl alcohol. Isopropyl alcohol has a strong hydrophobic effect, promoting molecular aggregation and reducing the saturation of the glutathione solution. In this embodiment, the addition of isopropyl alcohol by titration can control the rate of isopropyl alcohol titration, facilitate sufficient mixing of the glutathione solution and isopropyl alcohol, control the growth rate of rod-shaped crystal nuclei, and thereby improve the purity of the glutathione crystals.

[0041] In this embodiment, the volume ratio of the saturated glutathione solution to the total amount of isopropanol is 1:(6-10). Preferably, the volume ratio of the saturated glutathione solution to the total amount of isopropanol is 1:8, and the total amount of isopropanol titrated into the saturated glutathione solution is 4 times the volume of the glutathione solution.

[0042] As another preferred embodiment of the present application, the crystallization agent is a mixture of ethanol and isopropanol, that is, ethanol and isopropanol are mixed in a volume ratio of (4-8):1. The mixed crystallization agent can reduce the co-precipitation of impurities through synergistic action. The gentle precipitation of ethanol reduces amorphous impurities, and the strong hydrophobicity of isopropanol increases the growth rate of glutathione crystals. Moreover, the mixed use can reduce the amount of isopropanol used and reduce costs while maintaining a high efficiency of glutathione crystal precipitation.

[0043] In this embodiment, the ratio of the total volume of the saturated glutathione solution to the total volume of ethanol and isopropanol is 1:(4-8). Preferably, the ratio of the total volume of the saturated glutathione solution to the total volume of ethanol and isopropanol is 1:6.

[0044] As another preferred embodiment of the present application, the crystallization agents are ethanol and isopropanol. Specifically, ethanol is added in the initial stage of addition to gradually reduce the solubility of the glutathione solution and induce the formation of rod-shaped crystal nuclei; isopropanol is added in the later stage of addition to enhance the precipitation ability of the glutathione solution and improve the crystallization yield. The duration of the initial stage is longer than that of the later stage, which is conducive to controlling the uniform growth of the rod-shaped crystal nuclei of glutathione.

[0045] In this embodiment, the use of ethanol and isopropanol as crystallization agents can accurately adjust the polarity gradient of the solvents, balance the crystallization rate and quality of the glutathione crystals, and help optimize the crystal morphology and purity of the purified glutathione. Moreover, the mixed use can reduce the amount of isopropanol used and reduce costs while maintaining a high efficiency of glutathione crystal precipitation.

[0046] Furthermore, the titration rate of the crystallization agent is 10 drops / min to 20 drops / min. By gradually adding the crystallization agent, the supersaturation of the solution can be precisely controlled, thereby avoiding rapid precipitation to form an amorphous solid, inducing orderly rod-shaped crystal nuclei and crystal growth, thereby improving the purity of the recrystallized glutathione and the transmittance in the 430nm band.

[0047] Furthermore, the stirring rate of the glutathione solution is 130 r / min to 170 r / min, and controlling the stirring rate is beneficial to the shape of the subsequent glutathione crystals.

[0048] It is understandable that the additive solutions and crystallization reagents in the above embodiments can be cross-matched to achieve the purpose of purifying crude glutathione, and are all within the scope of protection of this application.

[0049] After obtaining the pre-crystallized glutathione solution, the pre-crystallized glutathione solution is allowed to stand until glutathione crystals are precipitated, and the pre-crystallized glutathione solution is kept at a constant temperature T. In order to fully crystallize the pre-crystallized glutathione solution, the pre-crystallized glutathione solution is allowed to stand for a third preset time, which is 10 hours to 14 hours, preferably 12 hours.

[0050] As an example of a preferred embodiment of the present application, Tween-80 with a concentration of 0.3% is selected for dissolving crude glutathione, and after ultrasonic vibration for 2 hours, a saturated glutathione solution is obtained by filtration. Isopropanol is titrated to the bubbled glutathione solution at a titration rate of 15 drops / min. The total volume of the titrated isopropanol is 4 times the volume of the glutathione solution. During the titration of isopropanol, the mixture is stirred at a stirring rate of 150r / min for 5 hours to obtain a pre-crystallized glutathione solution, and then the pre-crystallized glutathione solution is allowed to stand for 12 hours, which is conducive to the sufficient precipitation of crystals of the glutathione solution. During the above process, the glutathione solution is maintained at a constant temperature of 35 ° C to prevent glutathione oxidation.

[0051] The recrystallized glutathione crystals were subjected to liquid chromatography detection, such as Figure 2 As shown in FIG, the liquid chromatogram of glutathione crystals prepared with Tween-80 at a concentration of 0.3% as the additive and isopropanol as the crystallization agent, wherein the test conditions are: mobile phase PBS, that is, phosphate buffer prepared with KH2PO4 solution and K2HPO4 solution: acetonitrile = 99:1, pH = 2.5, flow rate is 1.0 mL / min, and detection wavelength is 210 nm.

[0052] Use the standard solution to make a standard curve, as shown in the attached Figure 3 As shown, according to Figure 2 The glutathione concentration (g / mL) in the sample was calculated using the peak area of the glutathione sample and the standard curve, and then multiplied by the sample volume (mL) to obtain the amount of glutathione in the sample; the purity of glutathione was calculated by dividing the amount of glutathione by the amount weighed when preparing the sample.

[0053] like Figure 4As shown, the glutathione crystals prepared with a concentration of 0.3% and an additive of Tween-80 and a crystallization agent of isopropyl alcohol are rod-shaped crystals, and Figure 5 The crystals of crude unpurified glutathione are irregular, that is, through the synergistic effect of the additive solution and the crystallization agent, the amorphous crystals of the crude product can be transformed into uniform needle-shaped regular crystals.

[0054] like Figure 6 As shown, different concentrations of Tween-80 and isopropanol matched the obtained glutathione concentrations, from Figure 6 It can be seen that 0.1% to 0.5% concentration of Tween-80 as an additive solution can produce a glutathione solution with higher purity.

[0055] The glutathione crystals obtained by matching the additive solution and the crystallization reagent are tested and calculated, and the purity obtained is shown in Table 1: Table 1:

[0056] As can be seen from Table 1, matching different additive solutions with crystallization reagents can produce glutathione crystals with higher purity, meeting the requirements of the medical and food industries.

[0057] In summary, the method for purifying reduced glutathione of the present application dissolves crude glutathione at a low and constant temperature to prevent oxidation of reduced glutathione, thereby ensuring the utilization rate of glutathione; by matching the addition solution with the crystallization agent, the purity and appearance of the recrystallized glutathione are improved. The entire reaction process is relatively mild and does not require adjustment of the pH value, temperature range, pressure, etc. of the solution. The operation is simple, conducive to industrial production, and in line with the concept of energy conservation, environmental protection, and green.

[0058] It should be understood that although this specification is described according to embodiments, not every embodiment contains only one independent technical solution. This narrative style of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0059] The series of detailed descriptions listed above are merely specific descriptions of feasible embodiments of the present application and are not intended to limit the scope of protection of the present application. Any equivalent embodiments or changes that do not deviate from the technical spirit of the present application should be included in the scope of protection of the present application.

Claims

1. A method for purifying reduced glutathione, characterized in that: The purification method comprises: The glutathione solution is prepared, specifically comprising: obtaining crude glutathione with a weight of m1 and an additive solution with a mass of m2, wherein the additive solution is Tween-80 or phenylalanine, and the concentration of the additive solution is 0.1% to 0.5%; Mixing the mixture in a ratio of m1:m2 of 1:(10-20) and ultrasonically vibrating for a first preset time, filtering to obtain a saturated glutathione solution, and maintaining the saturated glutathione solution at a constant temperature T, wherein T is 30°C to 40°C; titrating the crystallization agent into the saturated glutathione solution and continuously stirring at a constant speed for a second preset time to obtain a pre-crystallized glutathione solution; The pre-crystallized glutathione solution is allowed to stand until glutathione crystals are precipitated, and the pre-crystallized glutathione solution is kept at a constant temperature T.

2. The method for purifying reduced glutathione according to claim 1, wherein The constant temperature T is 35℃.

3. The method for purifying reduced glutathione according to claim 1, wherein The crystalline agent is ethanol and / or isopropyl alcohol.

4. The method for purifying reduced glutathione according to claim 3, wherein If the crystallization agent is ethanol, the volume ratio of ethanol to saturated glutathione solution is (2-6):1; If the crystallization agent is isopropyl alcohol, the volume ratio of isopropyl alcohol to saturated glutathione solution is (6-10):1; If the crystallization reagents are ethanol and isopropanol, the volume ratio of the total volume of ethanol and isopropanol to the saturated glutathione solution is (4-8):

1.

5. The method for purifying reduced glutathione according to claim 3, wherein If the crystallization agent is a mixture of ethanol and isopropanol, the volume ratio of ethanol to isopropanol is (4-8):

1.

6. The method for purifying reduced glutathione according to claim 3, wherein If the crystallization agent is ethanol and isopropanol, the titration of the crystallization agent into the saturated glutathione solution includes titrating the ethanol in the initial stage of titrating the crystallization agent and titrating the isopropanol in the later stage of titrating the crystallization agent, wherein the duration of the initial stage is longer than the duration of the later stage.

7. The method for purifying reduced glutathione according to any one of claims 1 to 6, wherein: The titration rate of the crystalline drug is 10 drops / min to 20 drops / min.

8. The method for purifying reduced glutathione according to any one of claims 1 to 6, wherein: The glutathione solution is stirred at a stirring rate of 130 r / min to 170 r / min.

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