A method for preparing thyroid immunomodulatory drugs based on selenoprotein-modified astragalus polysaccharide derivatives

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative. The method comprises the following steps: in a first reactor, adding astragalus polysaccharide and maleic anhydride to a Tris-HCl buffer for reaction to obtain olefinated astragalus polysaccharide; in a second reactor, adding food-grade selenoprotein and 2-iminothiolane hydrochloride to a Tris-HCl buffer for reaction to obtain thiolated selenoprotein; pouring the second reactor into the first reactor, adding cysteine ​​to obtain a selenoprotein-modified astragalus polysaccharide derivative; mixing the selenoprotein-modified astragalus polysaccharide derivative, starch, and magnesium stearate, and pressing to obtain the thyroid immunomodulatory drug; the thyroid immunomodulatory drug prepared by the present invention has a positive effect on the immunoregulation of thyroid FRTL-5 cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative. Background Art

[0002] Thyroid disease is a common endocrine disorder whose pathogenesis is closely related to an imbalance in the body's immune regulation. Currently, clinically available medications for thyroid disease have numerous limitations, such as significant side effects and inconsistent therapeutic effects. Therefore, the development of safe and effective thyroid immunomodulatory drugs is of great clinical significance.

[0003] Astragalus polysaccharide (APS) is an active ingredient extracted from the traditional Chinese medicinal herb Astragalus. For example, Chinese invention patent publication number CN119823295B provides an APS extraction method that includes the following steps: initial aqueous extraction, secondary aqueous extraction, reduced pressure concentration, initial alcohol precipitation, secondary alcohol precipitation, and purification. The initial aqueous extraction involves soaking the Astragalus root in a weakly alkaline solution, then heating it to boiling and extracting it. The initial precipitate and primary extract are separated by filtration. APS exhibits various biological activities, including immunomodulatory and antioxidant properties.

[0004] Regarding the applications of astragalus polysaccharides, Chinese invention patent application CN119775447A discloses astragalus polysaccharides processed using different processing techniques and their applications. Four crude astragalus polysaccharides, designated RAPSs, SAPSs, WAPSs, and HAPSs, were extracted from raw and three processed products using a simulated traditional Chinese medicine decoction method. The effects of different processing methods on the structure and bidirectional immunomodulatory activity of astragalus polysaccharides were investigated. Results showed that different processing methods altered the properties and structural characteristics of astragalus polysaccharides, thereby affecting their bidirectional immunomodulatory activity. The four polysaccharides exhibited significant differences in extraction yield, chemical composition, molecular weight, monosaccharide composition, chain conformation and surface morphology, zeta potential and particle size, and immune-enhancing and anti-inflammatory activities, but shared the same typical crystalline structure, primary glycoside residues, and O-glycosidic bonds. Furthermore, within the tested concentration range, the crude astragalus polysaccharide processed with honey exhibited stronger immune-enhancing properties, while the crude astragalus polysaccharide processed with wine exhibited stronger anti-inflammatory activity. This invention provides an important theoretical basis for further revealing the processing mechanism of Astragalus and a scientific basis for the development and efficient utilization of Astragalus polysaccharides in the fields of food and medicine.

[0005] However, astragalus polysaccharides (APS) suffer from low bioavailability and rapid metabolism, which limit their widespread application as a thyroid immunomodulator. Selenoproteins are a class of selenium-rich proteins with unique biological activities and high biocompatibility. Combining selenoproteins with APS is expected to improve the properties of APS and enhance its effectiveness in thyroid immunomodulation. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative. The steps are as follows, calculated by weight:

[0007] S1: In a first reaction kettle, 8-12 parts of astragalus polysaccharide and 2-4 parts of maleic anhydride are added and reacted in 50-60 parts of Tris-HCl buffer at pH 7.5-8.5 to obtain olefinated astragalus polysaccharide;

[0008] S2: In a second reaction vessel, add 8-12 parts of food-grade selenoprotein and 0.03-0.06 parts of 2-iminothiolane hydrochloride, and react in 60-80 parts of Tris-HCl buffer (pH 7.5-8.5) at room temperature for 20-40 minutes to obtain thiolated selenoprotein;

[0009] S3: Pour the contents of the second reaction kettle into the first reaction kettle and mix, add 0.8-2 parts of cysteine, stir and react, dialyze through a dialysis bag to remove unreacted reagents, and freeze-dry to obtain a selenoprotein-modified astragalus polysaccharide derivative;

[0010] S4: Stir and mix 200-300 parts of starch, 1-5 parts of magnesium stearate, and 80-100 parts of the astragalus polysaccharide derivative obtained in step S3, and press them into tablets to obtain a thyroid immunomodulatory drug.

[0011] Furthermore, in step S1, the reaction temperature is 25-40° C., and the reaction time is 3-5 hours.

[0012] Furthermore, in step S3, the reaction temperature is 40-50° C. and the reaction time is 6-12 hours.

[0013] Furthermore, in step S3, the molecular weight cut-off of the dialysis bag is 3500 Da.

[0014] Furthermore, in step S3, the stirring speed is 100-300 rpm.

[0015] Furthermore, in step S3, the stirring reaction needs to be protected from light.

[0016] Furthermore, in step S4, the stirring and mixing time is 15-30 minutes.

[0017] Furthermore, in step S4, the hardness of the thyroid immunomodulatory drug tablet is 5-10 kg / cm².

[0018] The reaction mechanism of the present invention is as follows:

[0019] Astragalus polysaccharide extraction: The water extraction process is based on the principle of like dissolves like. Astragalus polysaccharides, as polar macromolecules, are soluble in water. Heating promotes the dissolution of astragalus polysaccharides from the medicinal material cells. During alcohol precipitation, ethanol changes the polarity of the solution, reducing the solubility of astragalus polysaccharides and causing them to precipitate.

[0020] Preparation of selenoproteins: Yeast cells are disrupted to release selenoproteins. Different solubility differences among proteins in high salt concentrations are exploited to perform salting-out and preliminary separation of selenoproteins. Ion exchange chromatography is then used to separate the proteins, based on their charge properties, by binding or repelling ion exchangers. Gel filtration chromatography, exploiting differences in diffusion rates within the gel pores due to molecular size, allows for the final separation of the selenoproteins.

[0021] Preparation of selenoprotein-modified astragalus polysaccharide derivatives: Maleic anhydride reacts with astragalus polysaccharide to introduce olefin groups, and 2-iminothiolane hydrochloride reacts with selenoprotein to introduce sulfhydryl groups. In the presence of cysteine, the sulfhydryl and olefin groups undergo a Michael addition reaction, achieving selenoprotein-modified astragalus polysaccharide. Cysteine ​​promotes the reaction.

[0022] Compared with the prior art, the present invention has the following significant effects:

[0023] 1. Enhance drug activity: Selenoproteins themselves possess multiple biological activities, such as antioxidant and immunomodulatory properties. Astragalus polysaccharides also have immunomodulatory effects. The derivatives formed by the combination of the two enhance the thyroid immune regulation effect through synergistic action, increase the biological activity of drugs, and more effectively regulate thyroid-related immune dysfunction.

[0024] 2. Enhanced stability: The astragalus polysaccharide derivatives modified with selenoproteins are more stable in structure. Compared with individual astragalus polysaccharides or selenoproteins, their ability to resist the influence of external environmental factors is enhanced, which is beneficial to the stability of the drug during storage and use, and ensures the continued efficacy of the drug.

[0025] 3. Optimization of the preparation process: The parameters of each step of this preparation process have been optimized, such as the temperature, time and mass fraction of each reactant for the preparation of olefinized astragalus polysaccharide, as well as the reaction temperature, time and mass fraction of each reactant for selenoprotein modification, so that the entire preparation process has a high yield and purity, improves product quality, and the process operation is relatively simple, with good repeatability and industrial application prospects. DETAILED DESCRIPTION

[0026] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following embodiments and comparative examples are described in detail:

[0027] It should be noted that the CAS number of the food-grade selenoprotein in the examples and comparative examples is 15758-45-9, and the CAS number of 2-iminothiolane hydrochloride is 4781-83-3.

[0028] Example 1

[0029] A method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative, the operating steps of which are as follows:

[0030] S1: In a first reaction kettle, 8 g of astragalus polysaccharide and 2 g of maleic anhydride were added and reacted in 50 g of Tris-HCl buffer at pH 7.5 to obtain olefinated astragalus polysaccharide;

[0031] S2: In a second reaction vessel, 8 g of food-grade selenoprotein and 0.03 g of 2-iminothiolane hydrochloride were added and reacted in 60 g of Tris-HCl buffer (pH 7.5) at room temperature for 20 minutes to obtain thiolated selenoprotein;

[0032] S3: The contents of the second reaction kettle were poured into the first reaction kettle and mixed, 0.8 g of cysteine ​​was added, and the mixture was stirred for reaction. The mixture was dialyzed through a dialysis bag to remove unreacted reagents, and the mixture was freeze-dried to obtain a selenoprotein-modified astragalus polysaccharide derivative.

[0033] S4: 80 g of the astragalus polysaccharide derivative obtained in step S3 is taken, mixed with 200 g of starch and 1 g of magnesium stearate, and pressed into tablets to obtain a thyroid immunomodulatory drug.

[0034] Wherein, the reaction temperature in step S1 is 25° C. and the reaction time is 3 hours.

[0035] In step S3, the reaction temperature is 40° C., the reaction time is 6 hours, the molecular weight cutoff of the dialysis bag is 3500 Da, the stirring speed is 100 rpm, and the stirring reaction needs to be protected from light.

[0036] The stirring and mixing time in step S4 is 15 minutes, and the hardness of the prepared thyroid immunomodulatory drug tablet is 5 kg / cm².

[0037] Example 2

[0038] A method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative, the operating steps of which are as follows:

[0039] S1: In a first reaction kettle, 9 g of astragalus polysaccharide and 3 g of maleic anhydride were added and reacted in 53 g of Tris-HCl buffer at pH 8 to obtain olefinated astragalus polysaccharide;

[0040] S2: In a second reaction vessel, 9 g of food-grade selenoprotein and 0.04 g of 2-iminothiolane hydrochloride were added and reacted in 65 g of Tris-HCl buffer (pH 8) at room temperature for 25 minutes to obtain thiolated selenoprotein;

[0041] S3: The contents of the second reaction kettle were poured into the first reaction kettle and mixed, 1.2 g of cysteine ​​was added, and the mixture was stirred for reaction. The mixture was then dialyzed through a dialysis bag to remove unreacted reagents, and freeze-dried to obtain a selenoprotein-modified astragalus polysaccharide derivative.

[0042] S4: Take 85 g of the astragalus polysaccharide derivative obtained in step S3, mix it with 240 g of starch and 2 g of magnesium stearate, and press it into tablets to obtain a thyroid immunomodulatory drug.

[0043] Wherein, the reaction temperature in step S1 is 30° C. and the reaction time is 4 hours.

[0044] In step S3, the reaction temperature is 45° C., the reaction time is 8 hours, the molecular weight cutoff of the dialysis bag is 3500 Da, the stirring speed is 200 rpm, and the stirring reaction needs to be protected from light.

[0045] The stirring and mixing time in step S4 is 20 minutes, and the hardness of the prepared thyroid immunomodulatory drug tablet is 6 kg / cm².

[0046] Example 3

[0047] A method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative, the operating steps of which are as follows:

[0048] S1: In a first reaction kettle, 11 g of astragalus polysaccharide and 3 g of maleic anhydride were added and reacted in 58 g of Tris-HCl buffer at pH 8 to obtain olefinated astragalus polysaccharide;

[0049] S2: In a second reaction vessel, 11 g of food-grade selenoprotein and 0.05 g of 2-iminothiolane hydrochloride were added and reacted in 75 g of Tris-HCl buffer (pH 8) at room temperature for 35 minutes to obtain thiolated selenoprotein;

[0050] S3: The contents of the second reaction kettle were poured into the first reaction kettle and mixed, 1.6 g of cysteine ​​was added, and the mixture was stirred for reaction. The mixture was then dialyzed through a dialysis bag to remove unreacted reagents, and freeze-dried to obtain a selenoprotein-modified astragalus polysaccharide derivative.

[0051] S4: Take 95 g of the astragalus polysaccharide derivative obtained in step S3, stir and mix it with 280 g of starch and 4 g of magnesium stearate, and press it into tablets to obtain a thyroid immunomodulatory drug.

[0052] Wherein, the reaction temperature in step S1 is 35° C. and the reaction time is 4 hours.

[0053] In step S3, the reaction temperature is 45° C., the reaction time is 11 hours, the molecular weight cutoff of the dialysis bag is 3500 Da, the stirring speed is 200 rpm, and the stirring reaction needs to be protected from light.

[0054] The stirring and mixing time in step S4 is 25 minutes, and the hardness of the prepared thyroid immunomodulatory drug tablet is 8 kg / cm².

[0055] Example 4

[0056] A method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative, the operating steps of which are as follows:

[0057] S1: In a first reaction kettle, 12 g of astragalus polysaccharide and 4 g of maleic anhydride were added and reacted in 60 g of Tris-HCl buffer at pH 8.5 to obtain olefinated astragalus polysaccharide;

[0058] S2: In a second reaction vessel, 12 g of food-grade selenoprotein and 0.06 g of 2-iminothiolane hydrochloride were added and reacted in 80 g of Tris-HCl buffer (pH 8.5) at room temperature for 40 minutes to obtain thiolated selenoprotein;

[0059] S3: The contents of the second reaction kettle were poured into the first reaction kettle and mixed, 2 g of cysteine ​​was added, and the mixture was stirred for reaction. The mixture was dialyzed through a dialysis bag to remove the unreacted reagent, and the mixture was freeze-dried to obtain a selenoprotein-modified astragalus polysaccharide derivative.

[0060] S4: Take 100 g of the astragalus polysaccharide derivative obtained in step S3, mix it with 300 g of starch and 5 g of magnesium stearate, and press it into tablets to obtain a thyroid immunomodulatory drug.

[0061] Wherein, the reaction temperature in step S1 is 40° C. and the reaction time is 5 hours.

[0062] In step S3, the reaction temperature is 50° C., the reaction time is 12 hours, the molecular weight cutoff of the dialysis bag is 3500 Da, the stirring speed is 300 rpm, and the stirring reaction needs to be protected from light.

[0063] The stirring and mixing time in step S4 is 30 minutes, and the hardness of the prepared thyroid immunomodulatory drug tablet is 10 kg / cm².

[0064] Comparative Example 1

[0065] A method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative, the operating steps of which are as follows:

[0066] S1: After mixing 8 g of astragalus polysaccharide and 8 g of food-grade selenoprotein, 0.8 g of cysteine ​​was added. After stirring and reacting in a first reactor, the mixture was dialyzed through a dialysis bag to remove unreacted reagents and freeze-dried to obtain a selenoprotein-modified astragalus polysaccharide derivative;

[0067] S2: 80 g of the astragalus polysaccharide derivative obtained in step S1 is mixed with 200 g of starch and 1 g of magnesium stearate, and the mixture is pressed into tablets to obtain a thyroid immunomodulatory drug.

[0068] In step S1, the reaction temperature is 40° C., the reaction time is 6 hours, the molecular weight cutoff of the dialysis bag is 3500 Da, the stirring speed is 100 rpm, and the stirring reaction needs to be protected from light.

[0069] The stirring and mixing time in step S2 is 15 minutes, and the hardness of the prepared thyroid immunomodulatory drug tablet is 5 kg / cm².

[0070] Comparative Example 2

[0071] A method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative, the operating steps of which are as follows:

[0072] S1: In a first reaction kettle, 8 g of astragalus polysaccharide and 2 g of maleic anhydride were added and reacted in 50 g of Tris-HCl buffer at pH 7.5 to obtain olefinated astragalus polysaccharide;

[0073] S2: 8 g of food-grade selenoprotein was poured into the first reactor and mixed, 0.8 g of cysteine ​​was added, and the mixture was stirred for reaction. The mixture was then dialyzed through a dialysis bag to remove unreacted reagents, and freeze-dried to obtain a selenoprotein-modified astragalus polysaccharide derivative.

[0074] S3: 80 g of the astragalus polysaccharide derivative obtained in step S2 is mixed with 200 g of starch and 1 g of magnesium stearate, and the mixture is pressed into tablets to obtain a thyroid immunomodulatory drug.

[0075] Wherein, the reaction temperature in step S1 is 25° C. and the reaction time is 3 hours.

[0076] In step S2, the reaction temperature is 40° C., the reaction time is 6 hours, the molecular weight cutoff of the dialysis bag is 3500 Da, the stirring speed is 100 rpm, and the stirring reaction needs to be protected from light.

[0077] The stirring and mixing time in step S3 is 15 minutes, and the hardness of the prepared thyroid immunomodulatory drug tablet is 5 kg / cm².

[0078] Comparative Example 3

[0079] A method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative, the operating steps of which are as follows:

[0080] S1: In a second reaction vessel, 8 g of food-grade selenoprotein and 0.03 g of 2-iminothiolane hydrochloride were added and reacted in 60 g of Tris-HCl buffer (pH 7.5) at room temperature for 20 minutes to obtain thiolated selenoprotein;

[0081] S2: 8 g of astragalus polysaccharide was poured into a second reaction kettle and mixed, 0.8 g of cysteine ​​was added, and after stirring for reaction, the mixture was dialyzed through a dialysis bag to remove the unreacted reagent, and the mixture was freeze-dried to obtain a selenoprotein-modified astragalus polysaccharide derivative;

[0082] S3: 80 g of the astragalus polysaccharide derivative obtained in step S2 is mixed with 200 g of starch and 1 g of magnesium stearate, and the mixture is pressed into tablets to obtain a thyroid immunomodulatory drug.

[0083] In step S2, the reaction temperature is 40° C., the reaction time is 6 hours, the molecular weight cutoff of the dialysis bag is 3500 Da, the stirring speed is 100 rpm, and the stirring reaction needs to be protected from light.

[0084] The stirring and mixing time in step S3 is 15 minutes, and the hardness of the prepared thyroid immunomodulatory drug tablet is 5 kg / cm².

[0085] In the present invention, the thyroid immunomodulatory drug activity assays of Examples 1-4 and Comparative Examples 1-3 were conducted using thyroid FRTL-5 cells as the research subjects. Cell viability was determined using the MTT assay. Changes in interleukin-6 and tumor necrosis factor-α levels were measured using an enzyme-linked immunosorbent assay to assess the effects of the thyroid immunomodulatory drugs on cellular immune regulation. The specific test results are shown in Table 1.

[0086] Table 1 Test results of Examples 1 to 4 and Comparative Examples 1 to 3

[0087]

[0088] Through the data analysis of the above Examples 1 to 4 and Comparative Examples 1 to 3, the thyroid immunomodulatory drug prepared by the present invention has a positive effect on the immunomodulation of thyroid FRTL-5 cells.

[0089] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative, characterized in that: The operation steps are as follows, calculated by mass: S1: In a first reaction kettle, 8-12 parts of astragalus polysaccharide and 2-4 parts of maleic anhydride are added and reacted in 50-60 parts of Tris-HCl buffer at pH 7.5-8.5 to obtain olefinated astragalus polysaccharide; S2: In a second reaction vessel, 8-12 parts of food-grade selenoprotein and 0.03-0.06 parts of 2-iminothiolane hydrochloride were added, and the mixture was reacted in 60-80 parts of Tris-HCl buffer (pH 7.5-8.5) at room temperature for 20-40 minutes to obtain thiolated selenoprotein; S3: Pour the contents of the second reaction kettle into the first reaction kettle and mix, add 0.8-2 parts of cysteine, stir for reaction, dialyze through a dialysis bag to remove unreacted reagents, and freeze-dry to obtain a selenoprotein-modified astragalus polysaccharide derivative; S4: 200-300 parts of starch, 1-5 parts of magnesium stearate and 80-100 parts of the astragalus polysaccharide derivative obtained in step S3 are stirred and mixed, and pressed into tablets to obtain a thyroid immunomodulatory drug.

2. The method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative according to claim 1, characterized in that: In step S1, the reaction temperature is 25-40° C., and the reaction time is 3-5 hours.

3. The method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative according to claim 1, characterized in that: In step S3, the reaction temperature is 40-50° C. and the reaction time is 6-12 hours.

4. The method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative according to claim 1, characterized in that: In step S3, the molecular weight cut-off of the dialysis bag is 3500 Da.

5. The method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative according to claim 1, characterized in that: In step S3, the stirring speed is 100-300 rpm.

6. The method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative according to claim 1, characterized in that: In step S3, the stirring reaction needs to be protected from light.

7. The method for preparing a thyroid immunomodulatory drug based on a selenoprotein-modified astragalus polysaccharide derivative according to claim 1, characterized in that: In step S4, the stirring and mixing time is 15-30 minutes.

8. The method for preparing thyroid immunomodulatory drugs based on selenoprotein-modified astragalus polysaccharide derivatives according to claim 1, characterized in that: In step S4, the tablet hardness of the thyroid immunomodulatory drug is 5-10 kg / cm².

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