阅读说明：本技术 一种s-亚硝基谷胱甘肽的制备方法 (Preparation method of S-nitrosoglutathione ) 是由 王平刘 陈涛 秦玉 于 2018-12-25 设计创作，主要内容包括：本发明公开一种S-亚硝基谷胱甘肽的制备方法,包括如下步骤：(1)配制谷胱甘肽水溶液；(2)将一氧化氮气体与空气或氧气的混合气体通入谷胱甘肽水溶液中,室温或冰浴条件下搅拌反应,得到S-亚硝基谷胱甘肽水溶液。本发明直接利用一氧化氮气体为原料,导入谷胱甘肽水溶液中,生成可直接用于终端用途的GSNO水溶液,反应底物转化率高,可达到99％以上,且反应液中GSNO纯度较高；而且,本发明不使用任何其它无机或有机试剂和原料,反应液为纯粹的GSNO水溶液,不含其它金属离子和有机溶剂,该GSNO水溶液可直接用于制备一氧化氮缓释产品,或制得GSNO固体；本发明合成条件温和,合成过程中无需腐蚀性试剂,无固废和液废产生,符合绿色化学的理念。(The invention discloses a preparation method of S-nitrosoglutathione, which comprises the following steps: (1) preparing a glutathione aqueous solution; (2) and introducing the mixed gas of nitric oxide gas and air or oxygen into the glutathione aqueous solution, and stirring for reaction at room temperature or under an ice bath condition to obtain the S-nitrosoglutathione aqueous solution. According to the invention, nitric oxide gas is directly used as a raw material and introduced into a glutathione aqueous solution to generate a GSNO aqueous solution which can be directly used for terminal application, the conversion rate of a reaction substrate is high and can reach more than 99%, and the purity of GSNO in a reaction solution is high; in addition, the invention does not use any other inorganic or organic reagent and raw material, the reaction solution is pure GSNO aqueous solution, does not contain other metal ions and organic solvent, and the GSNO aqueous solution can be directly used for preparing nitric oxide slow release products or preparing GSNO solid; the method has mild synthesis conditions, does not need corrosive reagents in the synthesis process, does not generate solid waste and liquid waste, and accords with the concept of green chemistry.)

1. A preparation method of S-nitrosoglutathione is characterized by comprising the following steps:

(1) preparing a glutathione aqueous solution;

(2) and introducing the mixed gas of nitric oxide gas and air or oxygen into the glutathione aqueous solution, and stirring for reaction at room temperature or under an ice bath condition to obtain the S-nitrosoglutathione aqueous solution.

2. The method for producing S-nitrosoglutathione according to claim 1, wherein the concentration of the aqueous glutathione solution is 0.5mmol/L or more, and the concentration of nitric oxide gas introduced into the aqueous glutathione solution per unit time is 100ppm or more.

3. The method of producing S-nitrosoglutathione according to claim 2, wherein a ratio of a concentration of nitric oxide gas in the glutathione aqueous solution to a concentration of the glutathione aqueous solution per unit time is 1:140 or more.

4. The method of producing S-nitrosoglutathione according to claim 1, wherein in the step (1), glutathione obtained by fermentation with yeast is dissolved in an aqueous solution to obtain an aqueous glutathione solution.

5. The method for preparing S-nitrosoglutathione according to claim 1, wherein the stirring reaction time in step (2) is 4 to 7 hours.

6. The method for producing S-nitrosoglutathione according to claim 1, further comprising a step (3) of freeze-drying the GSNO aqueous solution to obtain a GSNO solid.

Technical Field

The invention relates to a preparation method of S-nitrosoglutathione, belonging to the field of biological medicine.

Background

Nitric Oxide (NO) is a small molecule active substance, which plays an important role in the physiological, biochemical, pathological and pharmacological processes of organisms, can maintain the stability of the intravascular environment and the stability of blood pressure, can be used for treating cardiovascular diseases such as myocardial ischemia and heart failure clinically, has an inhibiting effect on the adhesion between platelets and other cells, can influence the immune system of the organism in many ways, has an anti-inflammatory effect, can prevent the aggregation of white blood cells and stimulate mucus secretion, and can kill pathogens such as bacteria, fungi, parasites and the like.

S-nitrosoglutathione (GSNO), a small molecule nitrosothiol compound widely present in living organisms, is one of the most important donors of NO. GSNO is the main carrier of NO in vivo, and the NO is combined with important antioxidant and free radical scavenger Glutathione (GSH) in vivo to form GSNO which can play a role in storing NO. GSNO has various biological effects in vivo, and can release NO, and has effects of dilating blood vessel, resisting platelet aggregation, etc.; other sulfur-based proteins in cells can also be modified by transferring NO, and the structure and the function of the protein can be regulated and controlled; at the same time, a certain concentration of GSNO has the killing effect on both bacteria and viruses. GSNO has attracted considerable attention as a potential therapeutic drug as a product of glutathione nitrosylation.

Currently, the main synthesis mode of GSNO is to utilize nitrosation of Glutathione (GSH): mixing and stirring high concentration hydrochloric acid (usually 2M or higher), high concentration metal nitrite (such as 3-4M sodium nitrite) and GSH aqueous solution; then, a large amount of acetone is added to continue stirring, a large amount of pink granular GSNO crude products are separated out, and the products are washed by water and ether for many times to remove most of the strong acid and metal inorganic salt raw materials. The method adopts various strong acids as reactants, the synthesized product is complex and is not easy to be directly utilized, and organic solvents are needed, such as acetone for purifying the product and ether for cleaning the product, thereby generating a large amount of organic waste liquid.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems of the existing GSNO synthesis method, the invention provides a preparation method of S-nitrosoglutathione.

The technical scheme is as follows: the preparation method of S-nitrosoglutathione comprises the following steps:

(1) preparing a glutathione aqueous solution;

(2) and introducing the mixed gas of nitric oxide gas and air or oxygen into the glutathione aqueous solution, and stirring for reaction at room temperature or under an ice bath condition to obtain the S-nitrosoglutathione aqueous solution.

Wherein the concentration of the prepared glutathione aqueous solution is more than or equal to 0.5 mmol/L; correspondingly, the concentration of the nitric oxide gas introduced into the glutathione aqueous solution in unit time is more than or equal to 100 ppm. The concentration is the lowest concentration at which a better conversion rate can be obtained by reacting NO gas with glutathione aqueous solution. When the concentration of NO introduced into the GSH solution is less than 100ppm, the reaction efficiency and conversion rate are low. The concentration of nitric oxide gas introduced into the glutathione aqueous solution per unit time is the average gas concentration of NO introduced into the glutathione aqueous solution per minute.

Preferably, the ratio of the concentration of the nitric oxide gas introduced into the glutathione aqueous solution to the concentration of the glutathione aqueous solution in unit time is more than or equal to 1:140, and at the moment, the substrate conversion rate can reach more than 90%. The amount of air or oxygen in the mixed gas is not limited, and a trace amount of air can also promote the reaction of NO and the glutathione aqueous solution.

The glutathione aqueous solution can be prepared by the existing modes of fermentation, chemical synthesis, enzymatic synthesis and the like; glutathione obtained by yeast fermentation is preferably dissolved in the aqueous solution to obtain glutathione aqueous solution, and the raw materials from fermentation can be directly used for terminal products such as cosmetics, health products and the like.

Preferably, in the step (2), stirring and reacting for 4-7 h at room temperature or under an ice bath condition. The ice bath condition is a conventional ice bath condition, namely an ice bath at 0-4 ℃, the reaction is carried out at room temperature or under the ice bath condition, the conversion rate of the substrate is different, and the conversion rate of the reaction substrate is higher under the ice bath condition.

The preparation method of S-nitrosoglutathione can also comprise a step (3), and GSNO solid can be obtained by freeze drying the GSNO aqueous solution prepared in the step (2).

Has the advantages that: (1) according to the invention, nitric oxide gas is directly used as a raw material and introduced into a glutathione aqueous solution to generate a GSNO aqueous solution and a solid which can be directly used for terminal application, the conversion rate of a reaction substrate is high and can reach more than 99%, and the purity of GSNO in a reaction solution is high; (2) the invention simplifies the preparation process, reactants are only nitric oxide gas and glutathione, no other inorganic and organic reagents and raw materials are used, the reaction solution is pure GSNO aqueous solution, no other metal ions and organic solvents are contained, the GSNO aqueous solution can be directly used for preparing nitric oxide slow release products, or GSNO solid is obtained by freeze drying and other modes; (3) the method has mild synthesis conditions, only needs ice bath and normal pressure, does not need corrosive reagents in the synthesis process, does not generate solid waste and liquid waste, and accords with the concept of green chemistry.

Drawings

FIG. 1 is a graph of the reaction conversion for the preparation of GSNO in example 1;

FIG. 2 shows the release stability of the GNSO solution prepared in example 1;

FIG. 3 is a graph of the reaction conversion for the preparation of GSNO in example 2;

FIG. 4 is a graph of the reaction conversion for the preparation of GSNO in example 3;

FIGS. 5(a) and 5(b) are a reaction progress diagram and a conversion diagram of GSNO prepared from GSH under room temperature and ice bath conditions, respectively, in example 5;

FIGS. 6(a) and 6(b) are a graph showing the reaction progress and conversion rate of GSNO produced from GSH of different concentrations in example 6;

fig. 7 shows the storage stability of the GNSO solution prepared in example 6.

Detailed Description

The technical scheme of the invention is further explained by combining the attached drawings.

In the following examples, the quantitative determination method of S-nitrosoglutathione was as follows, and the conversion rate of the substrate was determined by this quantitative determination method.

16.8mg, 8.4mg, 4.2mg, 2.1mg and 1.05mg of S-nitrosoglutathione (GSNO) were weighed, respectively, dissolved in 1mL of ultrapure water, shaken up to obtain GSNO aqueous solutions having concentrations of 50, 25, 12.5, 6.25 and 3.125mmol/L, diluted 80-fold, numbered 1 to 5 GSNO aqueous solutions having different concentrations, and blanked with the aqueous solutions, and the absorbance values of the solutions in the respective EP tubes were measured at 334nm, as shown in Table 1 below.

TABLE 1 UV absorbance of GSNO aqueous solutions of different concentrations

According to the data in table 1, a standard curve of the numerical relationship between the concentration of the GSNO aqueous solution and the absorbance value is drawn with the concentration of the GSNO aqueous solution as ordinate and the absorbance value as abscissa. In the subsequent examples, the concentration and content of GSNO in the reaction solution can be calculated from the standard curve by measuring the absorbance of the reaction solution.

In examples 1 to 6 described below, the "NO gas concentration per unit time introduced into the glutathione aqueous solution" means the average gas concentration of NO introduced into the glutathione aqueous solution per minute.