Novel reduced glutathione crystal form and preparation method thereof

Abstract

The invention relates to the technical field of polypeptide compounds, in particular to a novel reduced glutathione crystal form and a preparation method thereof. In an X-ray diffraction pattern of the novel reduced glutathione crystal form, characteristic peaks exist at the positions where 2 theta is equal to 4.9 degrees, 14.7 degrees, 24.6 degrees, 29.6 degrees and 34.7 degrees. The invention aims to provide a novel crystal form of glutathione, and by regulating and controlling a molecular accumulation mode and a hydrogen bond network, the synergistic improvement of solubility and stability is realized, and the hygroscopicity is reduced. The crystal form not only can expand the application potential of glutathione in efficient preparations, but also provides a technical basis for large-scale production through a simplified preparation process. Experimental results show that the novel reduced glutathione crystal form provided by the invention is relatively low in moisture absorption rate, relatively excellent in solubility and relatively excellent in stability under damp and hot conditions.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide compounds, and in particular to a new crystal form of reduced glutathione and a preparation method thereof. Background Art

[0002] Glutathione (GSH), a tripeptide composed of glutamic acid, cysteine, and glycine, is widely present in living organisms and is one of the most important non-enzymatic antioxidants in cells. Its functions include scavenging free radicals, maintaining redox balance, and regulating cellular metabolism, making it valuable in the pharmaceutical, health care, cosmetic, and food industries. For example, in medicine, glutathione is used to treat liver disease, alleviate chemotherapy side effects, and slow the progression of neurodegenerative diseases. In cosmetics, its antioxidant properties are exploited in the development of anti-aging and whitening products.

[0003] However, the physical and chemical properties of glutathione (such as solubility, stability and bioavailability) have a significant impact on its practical application. Currently, commercially available glutathione mainly exists in conventional crystal forms (such as crystal form α), and these known crystal forms have the following limitations:

[0004] 1. Insufficient solubility: The existing crystal form has a low dissolution rate in aqueous or lipid media, resulting in limited oral bioavailability, especially insufficient absorption efficiency in the gastrointestinal tract, which affects the efficacy of the drug.

[0005] 2. Physical stability defects: Conventional crystal forms are easily affected by humidity, temperature or light. Crystal transformation or degradation may occur during storage, resulting in loss of active ingredients and increasing the difficulty and cost of the formulation process.

[0006] Patent application CN116640180A optimizes the solid-state form of glutathione through crystal engineering, improving its performance deficiencies. For example, while certain metastable crystal forms can temporarily increase dissolution rates, they are prone to phase transitions during long-term storage. Meanwhile, while the amorphous form exhibits excellent solubility, stability issues hinder practical application. Therefore, developing a new crystal form that combines high solubility, excellent stability, and process feasibility is crucial to overcoming existing technical bottlenecks. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a new crystal form of reduced glutathione and a preparation method thereof, wherein the new crystal form of reduced glutathione has a low moisture absorption rate, better solubility, and better stability under humid and hot conditions.

[0008] The invention provides a new crystal form of reduced glutathione. In the X-ray diffraction spectrum of the crystal form, characteristic peaks are present at 2θ=4.9°, 14.7°, 24.6°, 29.6° and 34.7°.

[0009] Preferably, the solubility of the new reduced glutathione crystal form at room temperature is 100-110 g / L.

[0010] Preferably, the moisture absorption rate of the new reduced glutathione crystal form at room temperature is 3.2% to 3.8%.

[0011] The present invention also provides a method for preparing the above-mentioned new crystal form of reduced glutathione, comprising the following steps:

[0012] Tetrahydrofuran is added dropwise to a stirred aqueous glutathione solution, and after continued stirring, the temperature is lowered to 3-7°C at a cooling rate of 6-10°C / h for crystallization to obtain a new reduced glutathione crystal form.

[0013] Preferably, the volume ratio of the tetrahydrofuran to the glutathione aqueous solution is 3 to 8:1.

[0014] Preferably, the dripping rate is 300 to 800 mL / h.

[0015] Preferably, the temperature of the glutathione aqueous solution is 20-30°C;

[0016] The mass concentration of the glutathione aqueous solution is 10% to 20%.

[0017] Preferably, the stirring rate is 300-500 rpm.

[0018] Preferably, the crystallization time is 10 to 18 hours.

[0019] Preferably, after the crystallization, the method further comprises:

[0020] The product was filtered, and the powder was washed with tetrahydrofuran and dried.

[0021] The present invention provides a new crystal form of reduced glutathione, and in the X-ray diffraction pattern of the crystal form, there are characteristic peaks at 2θ=4.9°, 14.7°, 24.6°, 29.6° and 34.7°. The present invention aims to provide a new crystalline form of glutathione, which achieves a synergistic improvement in solubility and stability and reduces hygroscopicity by regulating the molecular stacking mode and hydrogen bond network. This crystal form can not only expand the application potential of glutathione in high-efficiency preparations (such as rapid-release tablets or transdermal drug delivery systems), but its simplified preparation process also provides a technical basis for large-scale production. Experimental results show that the new crystal form of reduced glutathione provided by the present invention has a low moisture absorption rate, better solubility, and better stability under hot and humid conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the XRD pattern of the new crystalline form of reduced glutathione powder of Example 1 of the present invention;

[0023] Figure 2 This is the DSC spectrum of the new crystalline form of reduced glutathione powder of Example 1 of the present invention;

[0024] Figure 3 This is the XRD pattern of commercially available reduced glutathione powder in Comparative Example 1;

[0025] Figure 4 This is the XRD pattern of the reduced glutathione powder prepared in Comparative Example 2;

[0026] Figure 5 The liquid phase spectrum of the powder composition of the reduced glutathione after wet heat treatment in Example 3 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] The invention provides a new crystal form of reduced glutathione. In the X-ray diffraction spectrum of the crystal form, characteristic peaks are present at 2θ=4.9°, 14.7°, 24.6°, 29.6° and 34.7°.

[0029] The new reduced glutathione crystal form has a solubility of 100-110 g / L at room temperature and a moisture absorption rate of 3.2%-3.8% at room temperature, which is 64.15%-71.42% lower than that of the α-crystalline reduced glutathione. Furthermore, the product has a shelf life of over 98% at 60°C and 75% humidity. Therefore, it is less likely to undergo oxidation reactions to form oxidized glutathione under hot and humid conditions, resulting in a more stable storage.

[0030] In some embodiments of the present invention, the moisture absorption rate of the new reduced glutathione crystal with a humidity of 95% is reduced by 68% compared with the α crystal at room temperature.

[0031] The present invention also provides a method for preparing the above-mentioned new crystal form of reduced glutathione, comprising the following steps:

[0032] Tetrahydrofuran is added dropwise to a stirred aqueous glutathione solution, and after continued stirring, the temperature is lowered to 3-7°C at a cooling rate of 6-10°C / h for crystallization to obtain a new reduced glutathione crystal form.

[0033] The dripping rate is 300-800 mL / h;

[0034] The temperature of the glutathione aqueous solution is 20-30°C.

[0035] In some embodiments of the present invention, the mass concentration of the glutathione aqueous solution is 10% to 20%, such as 10%.

[0036] In some embodiments of the present invention, the volume ratio of the tetrahydrofuran to the glutathione aqueous solution is 3 to 8:1, such as 7:1.

[0037] In some embodiments of the present invention, the stirring rate of the stirred glutathione aqueous solution is 300-500 rpm, such as 400 rpm.

[0038] In some embodiments of the present invention, the stirring rate is 300-500 rpm, such as 400 rpm, and the stirring time is 4-6 h, such as 6 h.

[0039] In some embodiments of the present invention, the cooling rate is 8°C / h, and the temperature is reduced to 5°C.

[0040] In some embodiments of the present invention, the crystallization time is 10 to 18 hours.

[0041] In some embodiments of the present invention, after the crystallization, the method further comprises:

[0042] The product was filtered, and the powder was washed with tetrahydrofuran and dried.

[0043] The drying is vacuum drying at a temperature of 40-50°C, such as 45°C.

[0044] In an embodiment of the present invention, the yield of the new reduced glutathione crystal form is 80% to 86%, and the purity is above 99%.

[0045] The present invention has no particular limitation on the sources of the raw materials used above, and they can be generally commercially available.

[0046] To further illustrate the present invention, a new crystal form of reduced glutathione and a preparation method thereof provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] A 100 mL aqueous solution of 10% glutathione was prepared. Seven volumes of tetrahydrofuran (THF) were added dropwise at a rate of 700 mL / h while stirring continuously at 400 rpm, maintaining the temperature at 25°C. The mixture was stirred for 6 hours, then cooled to 5°C at a rate of 8°C / h. Crystallization proceeded for 18 hours, followed by filtration. The powder was washed with THF and then dried under vacuum at 45°C to obtain a new crystalline form of reduced glutathione. Testing revealed an 82.1% yield and 99.1% purity of the new crystalline form of reduced glutathione.

[0049] The X-ray diffraction pattern of the new crystalline form of reduced glutathione powder obtained in Example 1 was measured by Cu-Kα ray, as shown in Table 1 and Figure 1 shown.

[0050] Table 1 X-ray diffraction results of new crystalline form of reduced glutathione powder

[0051]

[0052]

[0053] Figure 1 This is the XRD pattern of the new crystalline powder of reduced glutathione in Example 1 of the present invention.

[0054] from Figure 1 As can be seen from Table 1, the new crystal form of reduced glutathione has characteristic peaks at 2θ=4.9°, 14.7°, 24.6°, 29.6° and 34.7°.

[0055] Figure 2 The DSC spectrum of the new crystalline form of reduced glutathione powder of Example 1 of the present invention is shown in FIG. Figure 2 It can be seen that the new crystal form of reduced glutathione has an endothermic peak at 202°C.

[0056] Example 2

[0057] A 100 mL aqueous solution of 13% glutathione was prepared. Five volumes of tetrahydrofuran (THF) were added dropwise at a rate of 500 mL / h, maintained at 20°C, under continuous stirring at 350 rpm. The mixture was stirred for 5.5 hours, then cooled to 5°C at a rate of 8°C / h. Crystallization proceeded for 15 hours, followed by filtration. The powder was washed with THF and dried under vacuum at 45°C to obtain a new crystalline form of reduced glutathione. Testing revealed an 83.7% yield and 99.2% purity of the new crystalline form of reduced glutathione.

[0058] Example 3

[0059] A 100 mL aqueous solution of 15% glutathione was prepared. Four volumes of tetrahydrofuran (THF) were added dropwise at a rate of 400 mL / h, maintained at 30°C, under continuous stirring at 360 rpm. The mixture was stirred for 5 hours, then cooled to 5°C at a rate of 8°C / h. Crystallization was allowed to proceed for 10 hours, followed by filtration. The powder was washed with THF and then dried under vacuum at 45°C to obtain a new crystalline form of reduced glutathione. Testing revealed an 86% yield and 99.3% purity of the new crystalline form of reduced glutathione.

[0060] Example 4

[0061] To a 100 mL aqueous solution of 20% glutathione was prepared, stirred continuously at 300 rpm and maintained at 27°C. Three volumes of tetrahydrofuran were rapidly added dropwise at a rate of 300 mL / h. The mixture was stirred for 4 hours, then cooled to 5°C at a rate of 8°C / h. Crystallization proceeded for 10 hours, followed by filtration. The powder was washed with tetrahydrofuran and then dried under vacuum at 45°C to obtain a new crystalline form of reduced glutathione. Testing revealed an 84.2% yield and 99.3% purity of the new crystalline form of reduced glutathione.

[0062] Comparative Example 1

[0063] The reduced glutathione in Comparative Example 1 is a commercially available product (purchased from Jincheng Pharmaceutical). Figure 3 The XRD pattern of the commercially available reduced glutathione powder described in Comparative Example 1 is shown in FIG. Figure 3 It can be seen that the characteristic peaks 2θ of the commercially available reduced glutathione powder are 6.3°, 12.6°, 13.8°, 22.3°, 30.1°, and 31.8°, confirming that it is an α-crystalline form. After testing, the mass content of the reduced glutathione is 98%.

[0064] Comparative Example 2

[0065] Reduced glutathione was prepared according to the method of Example 1 of patent application document CN108129550B, such as Figure 4 As shown, Figure 4 The XRD pattern of reduced glutathione powder was obtained as Comparative Example 2. Figure 4 It can be seen that the X-ray powder diffraction spectrum of the reduced glutathione has characteristic peaks at 4.8°, 14.7°, 21.3°, 25.2°, and 30.5° in 2θ.

[0066] Hygroscopicity comparison test:

[0067] 25 new 20 mL glass bottles were selected, placed in a 105 ° C oven to dry to constant weight, and numbered; after the reduced glutathione of Examples 1 to 4 and Comparative Examples 1 to 2 were dried to constant weight, 1 g of powder was filled into each glass bottle and placed in a constant temperature and humidity chamber (room temperature, 95% humidity); the samples were weighed every 24 h until the samples were constant weight. The results are shown in Table 2.

[0068] Table 2 Comparison of moisture absorption rate of reduced glutathione of Examples 1 to 4 and Comparative Example 1

[0069]

[0070] Table 2 (Continued from Table 1)

[0071]

[0072] As can be seen from Table 2, the hygroscopicity of the new reduced glutathione crystal form of the present invention is significantly lower than that of the commercially available product, and is not much different from the β crystal form in Comparative Example 2, and is more stable in an environment with high humidity.

[0073] Solubility comparison test:

[0074] Fifteen new 20 mL glass bottles were selected, placed in a 105 ° C oven to dry to constant weight, and numbered; after drying the reduced glutathione of Examples 1 to 4 and Comparative Examples 1 to 2 to constant weight, 4 to 5 g were taken in a glass bottle, the ambient temperature was controlled to 25 ° C, 2 mL of water was added and stirred to dissolve until the powder in the system was insoluble, and after filtering with a 0.22 μm aqueous filter membrane, the filtrate was sampled and the glutathione concentration was detected by liquid phase. The solubility results are shown in Table 3.

[0075] Table 3 Comparison of the solubility of reduced glutathione in Example 3 and Comparative Example 1

[0076]

[0077]

[0078] Table 3 (Continued Table 1)

[0079]

[0080] As can be seen from Table 3, the solubility of the new reduced glutathione crystal form of the present invention is not much different from that of the commercially available product, or even better; it is better than the β crystal form in Comparative Example 2.

[0081] Storage stability test under hot and humid conditions:

[0082] Select two new 20mL glass bottles, place them in a 105℃ oven to dry to constant weight, and number them; after drying the reduced glutathione of Example 3 and Comparative Example 1 to constant weight, take 2-3g into a glass bottle, transfer it to an incubator at a temperature of 60℃ and a humidity of 75% and let it stand for 2 months. Then, apply liquid phase detection to compare the composition of the powder before and after. The liquid phase detection spectrum is as follows: Figure 5 As shown, Figure 5 The liquid phase spectrum of the powder composition of the reduced glutathione after wet heat treatment in Example 3 of the present invention and Comparative Example 1.

[0083] from Figure 5 It can be seen that the commercially available product of Comparative Example 1 underwent a certain oxidation reaction under hot and humid conditions, producing 5.28% (mass content) of oxidized glutathione, while the reduced glutathione of the present invention was relatively more stable, producing only 1.54% (mass content) of oxidized glutathione.

[0084] At the same time, the oxidation of the reduced glutathione of Examples 1, 2, and 4 after wet heat treatment was detected according to the above method. The results showed that Example 1 produced 1.62% (mass content) of oxidized glutathione, Example 2 produced 1.48% (mass content) of oxidized glutathione, and Example 4 produced 1.67% (mass content) of oxidized glutathione. Comparative Example 2 produced 2.51% (mass content) of oxidized glutathione.

[0085] Experimental results show that compared to the most common commercially available α-crystalline form of glutathione, the present invention's crystal form has comparable solubility and only one-third the hygroscopicity of the α-crystalline form. It is less susceptible to oxidation reactions to form oxidized glutathione under hot and humid conditions, resulting in more stable storage. Furthermore, the present invention's crystal form has a simple preparation process, facilitating large-scale production.

[0086] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A new crystal form of reduced glutathione, wherein the X-ray diffraction pattern of the crystal form has characteristic peaks at 2θ=4.9°, 14.7°, 24.6°, 29.6° and 34.7°.

2. The new crystal form of reduced glutathione according to claim 1, characterized in that The solubility of the new reduced glutathione crystal form at room temperature is 100-110 g / L.

3. The new crystal form of reduced glutathione according to claim 1, characterized in that The moisture absorption rate of the new reduced glutathione crystal form at room temperature is 3.2% to 3.8%.

4. A method for preparing the new crystal form of reduced glutathione according to any one of claims 1 to 3, comprising the following steps: Tetrahydrofuran is added dropwise to a stirred aqueous glutathione solution, and after continued stirring, the temperature is lowered to 3-7°C at a cooling rate of 6-10°C / h for crystallization to obtain a new reduced glutathione crystal form.

5. The preparation method according to claim 4, characterized in that The volume ratio of the tetrahydrofuran to the glutathione aqueous solution is 3 to 8:

1.

6. The preparation method according to claim 4, characterized in that The dripping rate is 300-800 mL / h.

7. The preparation method according to claim 4, characterized in that The temperature of the glutathione aqueous solution is 20-30° C.; The mass concentration of the glutathione aqueous solution is 10% to 20%.

8. The preparation method according to claim 4, characterized in that The stirring speed is 300-500 rpm.

9. The preparation method according to claim 4, characterized in that The crystallization time is 10 to 18 hours.

10. The preparation method according to claim 4, characterized in that After the crystallization, the method further comprises: The product was filtered, and the powder was washed with tetrahydrofuran and dried.

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