A method for improving the inhibitory activity of adenosine deaminase by covalently complexing soybean protein isolate with rosmarinic acid

Abstract

The application discloses a method for improving the inhibitory activity of adenosine deaminase by covalently combining soybean protein isolate and rosmarinic acid. The method improves the structure and function of the compound by covalently combining the protein and the polyphenol. By exploring the functional properties of the compound at different rosmarinic acid addition amounts, the optimal rosmarinic acid addition amount is selected, the stable existence of the compound is ensured, and the properties of rosmarinic acid are improved. The method provides a theoretical basis for exploring the interaction between plant proteins and polyphenols, and proves that the covalent compound of soybean protein and rosmarinic acid has the potential of adenosine deaminase inhibitor.

Description

Technical Field

[0001] The present invention mainly relates to a method for improving adenosine deaminase inhibitory activity by utilizing soy protein isolate and rosmarinic acid in a covalent composite. Background Art

[0002] Gout is a common arthritis, affecting 41 million people worldwide. As consumer living standards and preferences change, the demand for high-purine foods such as seafood and meat is increasing, leading to a rising incidence of gout. Studies have shown that gout is closely linked to hyperuricemia. When uric acid production is increased or uric acid excretion is abnormal, urate crystals deposit in joints and surrounding tissues, activating neutrophils and macrophages, releasing inflammatory factors and matrix metalloproteinases, inducing an inflammatory response and causing acute inflammatory damage to joints and cartilage. Over time, acute gout can become chronic and eventually lead to tophi, which pose a health hazard. Gout is typically prevented by preventing high blood uric acid levels. Lowering uric acid levels typically involves reducing uric acid production or promoting uric acid excretion. The end product, uric acid, is produced by the purine metabolic pathway, and the rate-limiting enzyme in this pathway plays a key role in uric acid production. Inhibiting the activity of this rate-limiting enzyme can effectively reduce the production of uric acid, an important means of preventing gout. Adenosine deaminase is the rate-limiting enzyme in the purine metabolic pathway, and rosmarinic acid (RA) has been reported to alleviate gout by inhibiting its activity.

[0003] RA has low bioavailability due to its low lipophilicity and poor cell membrane permeability. Therefore, improving the lipophilicity and membrane permeability of RA is the key to improving its bioavailability. Based on these characteristics of RA, many studies have established RA encapsulation systems. These systems include chitosan nanoparticles, phospholipid complexes, nanocapsules, and protein complexes. A study used a dynamic intestinal model to study the effect of phospholipid complexes on the bioavailability of rosmarinic acid-phospholipid complex (RA-PLC). The preparation of RA-PLC was confirmed by X-ray diffraction, Fourier transform infrared spectroscopy, partition coefficient determination, and Caco-2 monolayer permeation test. The effect of RA complexation with phospholipids on RA bioaccessibility was successfully evaluated using a dynamic intestinal model system. The results showed that phospholipid complexation reduced the bioaccessibility of RA in the early stage of jejunal digestion and provided more sustained digestion characteristics in the subsequent ileum. A study encapsulated rosmarinic acid and quercetin extracted from sage and coriander in chitosan and characterized them. The results showed that nanoparticles can effectively protect the rapid distribution of rosmarinic acid, but its low encapsulation rate is a problem that needs to be solved at present.

[0004] The combination of protein and polyphenols not only has a high binding rate, but also can avoid the side effects of encapsulating excipients. Some plant proteins can supplement the nutrients needed by the human body and have broad application prospects.

[0005] The present invention improves the structure and function of the complex by covalently binding proteins and polyphenols. By exploring the functional properties of the complex at different rosmarinic acid addition levels, the optimal rosmarinic acid addition level is selected to ensure the stability of the complex while improving the properties of rosmarinic acid. Summary of the Invention

[0006] The present invention utilizes the ability of polyphenols and proteins to covalently bind and change the spatial conformation of proteins, so that after adding different amounts of rosmarinic acid to soy protein isolate, the two can form a stable complex while achieving the purpose of improving the properties of rosmarinic acid.

[0007] The present invention is achieved through the following technical solutions:

[0008] (1) Extraction of soy protein isolate: After defatted soybean meal was crushed through a 60-mesh sieve, 200 g of defatted soybean meal was taken and stirred with 2000 mL of water for 1 h. 2 mol / L sodium hydroxide solution was added to adjust the pH to 8.5. The mixture was then stirred on a magnetic stirrer for 1 h and centrifuged (4000 g, 30 min, 10°C). The supernatant after centrifugation was added with 2 mol / L hydrochloric acid to adjust the pH to 4.5. The mixture was allowed to stand at 4°C overnight and centrifuged for 15 min. The supernatant was removed and the precipitate was washed three times with water. The precipitate was re-dissolved in deionized water and adjusted to pH 7. After dialysis for 48 h, soy protein isolate (SPI) was obtained by freeze-drying. (2) Preparation of protein polyphenol covalent complex: The SPI obtained in step (1) was dissolved in phosphate buffer solution (0.01 M, pH 7.2) to prepare a 20 mg / mL SPI solution. After magnetic stirring at room temperature for 1 h, the solution was completely hydrated in a refrigerator at 4°C overnight. RA (0.05%, 0.1%, 0.2%) was added to the SPI solution and mixed well. The pH of the solution was adjusted to 9.0 with 0.2 mol / L NaOH. The solution was exposed to air and stirred in the dark at room temperature for 2 h; after dialysis for 48 h, the RA-SPI complex powder was obtained by freeze-drying, and the protein without RA under the same treatment was used as the control group; (3) the polyphenol binding rate of the complex in step (2) was determined by the Folin phenol method; (4) the Fourier transform infrared spectrum of the complex in step (2) was determined by the potassium bromide tablet method; (5) simulated gastrointestinal digestion: the complex in step (2) was digested in vitro; 10 mL of a sample with a concentration of 10 mg / mL was taken, the sample was mixed with an equal volume of gastric juice, the pH was adjusted to 2.0, and digested in a 37°C incubator for 1 h. After the digestion was completed, the pH was immediately adjusted to 7.0 to terminate the reaction; the gastric digest and simulated intestinal fluid were mixed evenly in equal volumes, the pH was adjusted to 7.0, the temperature was 37°C, and the enzyme was inactivated by high temperature after digestion for 2 h; (6) the adenosine deaminase inhibitory activity of the sample in step (5) was determined.

[0009] The protein extraction purity is 92.59%.

[0010] The optimal combination amount of the protein and polyphenols is: adding 0.1% RA to SPI, at which time the combination rate is 95.67%.

[0011] The infrared absorption peaks of the protein produced a slight red shift in the amide A band, amide I band and amide II band, indicating that the secondary structure of the complex has undergone a change in content compared with SPI.

[0012] The adenosine deaminase inhibitory activity of the sample after simulated gastrointestinal digestion was the highest when 0.1% RA was added to SPI. 50 It is 69.4μmol / L.

[0013] Compared with existing methods, the method proposed in this paper to improve the structure and function of polyphenol-protein complexes has the following characteristics:

[0014] (1) The preparation method of the RA-SPI complex of the present invention is simple, economical, mild in conditions, and easy to operate.

[0015] (2) The complex prepared by the present invention effectively improves the adenosine deaminase inhibitory activity of post-digestion RA, providing a new idea for the modification of adenosine deaminase inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the technical roadmap of the present invention;

[0017] Figure 2 is the polyphenol binding rate of the complex;

[0018] Figure 3 is the Fourier transform infrared spectrum of the complex;

[0019] Figure 4 is the adenosine deaminase inhibition capacity of the digested sample. DETAILED DESCRIPTION

[0020] The specific embodiments are further described below with reference to the accompanying drawings.

[0021] A method for improving the inhibitory activity of adenosine deaminase by covalently combining soy protein isolate with rosmarinic acid, characterized in that the method comprises the following steps: (1) extracting soy protein isolate: grinding defatted soybean meal through a 60-mesh sieve, taking 200 g of defatted soybean powder and stirring with 2000 mL of water for 1 h, adding 2 mol / L sodium hydroxide solution to adjust the pH to 8.5, then stirring on a magnetic stirrer for 1 h and centrifuging (4000 g, 30 min, 10° C.); taking the supernatant after centrifugation and adding 2 mol / L hydrochloric acid to adjust the pH to 4.5, standing at 4° C. overnight, centrifuging for 15 min, removing the supernatant, washing the precipitate with water three times, re-dissolving with deionized water and adjusting the pH to 7, dialyzing for 48 h, and obtaining soy protein isolate (SPI) by freeze-drying; (2) preparing a protein polyphenol covalent complex: dissolving the SPI extracted in step (1) in a phosphate buffer solution (0.01 M, pH 7.2) to prepare a SPI solution with a concentration of 20 mg / mL. After magnetic stirring at room temperature for 1 hour, the solution was completely hydrated in a refrigerator at 4°C overnight. RA (0.05%, 0.1%, and 0.2%) was added to the SPI solution and mixed thoroughly. The pH of the solution was adjusted to 9.0 with 0.2 mol / L NaOH. The solution was exposed to air and stirred in the dark at room temperature for 2 h; after dialysis for 48 h, the RA-SPI complex powder was obtained by freeze-drying, and the protein without RA under the same treatment was used as the control group; (3) the polyphenol binding rate of the complex in step (2) was determined by the Folin phenol method; (4) the Fourier transform infrared spectrum of the complex in step (2) was determined by the potassium bromide tablet method; (5) simulated gastrointestinal digestion: the complex in step (2) was digested in vitro; 10 mL of a sample with a concentration of 10 mg / mL was taken, the sample was mixed with an equal volume of gastric juice, the pH was adjusted to 2.0, and digested in a 37°C incubator for 1 h. After the digestion was completed, the pH was immediately adjusted to 7.0 to terminate the reaction; the gastric digest and simulated intestinal fluid were mixed evenly in equal volumes, the pH was adjusted to 7.0, the temperature was 37°C, and the enzyme was inactivated by high temperature after digestion for 2 h; (6) the adenosine deaminase inhibitory activity of the sample in step (5) was determined.

[0022] The protein extraction purity is 92.59%.

[0023] The optimal combination amount of the protein and polyphenols is: adding 0.1% RA to SPI, at which time the combination rate is 95.67%.

[0024] The infrared absorption peaks of the protein produced a slight red shift in the amide A band, amide I band and amide II band, indicating that the secondary structure of the complex has undergone a change in content compared with SPI.

[0025] The adenosine deaminase inhibitory activity of the sample after simulated gastrointestinal digestion was the highest when 0.1% RA was added to SPI. 50 It is 69.4μmol / L.

[0026] Example 1

[0027] (1) Preparation of protein-polyphenol covalent complex: SPI was dissolved in phosphate buffer solution (0.01 M, pH 7.2) to prepare a 20 mg / mL SPI solution. After magnetic stirring at room temperature for 1 h, the solution was completely hydrated in a refrigerator at 4°C overnight. 0.05% RA was added to the SPI solution and mixed evenly. The pH of the solution was adjusted to 9.0 with 0.2 mol / L NaOH. The solution was exposed to air and stirred in the dark at room temperature for 2 h; after dialysis for 48 h, the RA-SPI complex powder was obtained by freeze-drying.

[0028] (2) The content of RA in the complex was determined by the Folin phenol method: Standard curve drawing: 0.5 mL of RA standard solution of different concentrations was taken into a centrifuge tube, 2.5 mL of 0.2 mol / L Folin phenol reagent was added, and the mixture was reacted in the dark for 5 minutes. Then 2 mL of 7.5% Na2CO3 was added and the mixture was reacted in the dark for 2 hours. Deionized water was used as a blank and its absorbance was measured at 760 nm. The sample was prepared into a 2 mg / mL solution and its absorbance was measured. The RA content in the sample was calculated based on the standard curve. The results showed that the binding rate at this time was 87.37%;

[0029] (3) Fourier transform infrared spectroscopy was determined using the potassium bromide tablet method. The freeze-dried covalent complex was ground into powder, mixed with pre-dried potassium bromide at a ratio of 1:100, ground into powder, and tableted. The scanning conditions were set as follows: spectral range 4000-500 cm -1 , scanning times 32 times, resolution 4cm -1 The wavelength is plotted as the horizontal axis and the absorbance is plotted as the vertical axis. -1 ) It can be seen that the absorption peak intensity of the complex becomes stronger, and compared with the protein group, a slight red shift occurs from 3273.56 cm -1 Redshifted to 3276.63 cm -1 Amide I band (1700-1600cm -1 The absorption peak of the complex red-shifted from 1629.93 cm -1 Redshifted to 1630.09 cm -1 Amide II band (1600-1500cm -1 ) has a red shift from 1515.24 cm -1 Redshifted to 1525.21 cm -1 ;

[0030] (4) Determination of adenosine deaminase inhibitory activity of digested samples: Prepare 100 μL adenosine solution (0.5 mM) and 200 μL digested sample solution, then add 800 μL phosphate buffer (0.01 M, pH 7.2) to the system and incubate at 37°C for 5 minutes. Then add 100 μL (0.128 U·mL-1) ADA solution and incubate at 37°C for 30 minutes. Subsequently, 12% perchloric acid was added to the reaction system to terminate the reaction. The inosine content was determined by high performance liquid chromatography. The chromatogram was recorded at 248 nm, with 70% ethanol as the control group; the results showed that the IC 50 The IC value of RA is 78.19 μmol / L. 50 The adenosine deaminase inhibitory activity of the complex was 87.89 μmol / L, which was 11.04% higher than that of RA alone.

[0031] Example 2

[0032] (1) Preparation of protein-polyphenol covalent complex: SPI was dissolved in phosphate buffer solution (0.01 M, pH 7.2) to prepare a 20 mg / mL SPI solution. After magnetic stirring at room temperature for 1 h, the solution was completely hydrated in a refrigerator at 4°C overnight. 0.1% RA was added to the SPI solution and mixed evenly. The pH of the solution was adjusted to 9.0 with 0.2 mol / L NaOH. The solution was exposed to air and stirred in the dark at room temperature for 2 h; after dialysis for 48 h, the RA-SPI complex powder was obtained by freeze-drying.

[0033] (2) The content of RA in the complex was determined by the Folin phenol method: Standard curve drawing: 0.5 mL of RA standard solution of different concentrations was taken into a centrifuge tube, 2.5 mL of 0.2 mol / L Folin phenol reagent was added, and the mixture was reacted in the dark for 5 minutes. Then 2 mL of 7.5% Na2CO3 was added and the mixture was reacted in the dark for 2 hours. Deionized water was used as a blank and its absorbance was measured at 760 nm. The sample was prepared into a 2 mg / mL solution, and its absorbance was measured. The RA content in the sample was calculated based on the standard curve. The results showed that the binding rate at this time was the highest, which was 95.67%;

[0034] (3) Fourier transform infrared spectroscopy was determined using the potassium bromide tablet method. The freeze-dried covalent complex was ground into powder, mixed with pre-dried potassium bromide at a ratio of 1:100, ground into powder, and tableted. The scanning conditions were set as follows: spectral range 4000-500 cm -1 , scanning times 32 times, resolution 4cm -1 The wavelength is plotted as the horizontal axis and the absorbance is plotted as the vertical axis. -1 ) It can be seen that the absorption peak intensity of the complex becomes stronger, and compared with the protein group, a slight red shift occurs from 3273.56 cm-1 Redshift to 3277.98 cm -1 Amide I band (1700-1600cm -1 The absorption peak of the complex red-shifted from 1629.93 cm -1 Redshifted to 1630.62 cm -1 Amide II band (1600-1500cm -1 ) has a red shift from 1515.24 cm -1 Redshifted to 1525.39 cm -1 ;

[0035] (4) Determination of adenosine deaminase inhibitory activity of digested samples: Prepare 100 μL adenosine solution (0.5 mM) and 200 μL digested sample solution, then add 800 μL phosphate buffer (0.01 M, pH 7.2) to the system and incubate at 37°C for 5 minutes. Then add 100 μL (0.128 U·mL-1) ADA solution and incubate at 37°C for 30 minutes. Subsequently, 12% perchloric acid was added to the reaction system to terminate the reaction. The inosine content was determined by high performance liquid chromatography. The chromatogram was recorded at 248 nm, with 70% ethanol as the control group; the results showed that the IC 50 The IC value of RA is 69.4 μmol / L. 50 The adenosine deaminase inhibitory activity of the complex was 87.89 μmol / L, which was 21.04% higher than that of RA alone.

[0036] Example 3

[0037] (1) Preparation of protein-polyphenol covalent complex: SPI was dissolved in phosphate buffer solution (0.01 M, pH 7.2) to prepare a 20 mg / mL SPI solution. After magnetic stirring at room temperature for 1 h, the solution was completely hydrated in a refrigerator at 4°C overnight. 0.2% RA was added to the SPI solution and mixed evenly. The pH of the solution was adjusted to 9.0 with 0.2 mol / L NaOH. The solution was exposed to air and stirred in the dark at room temperature for 2 h; after dialysis for 48 h, the RA-SPI complex powder was obtained by freeze-drying.

[0038] (2) The content of RA in the complex was determined by the Folin phenol method: Standard curve drawing: 0.5 mL of RA standard solution of different concentrations was taken into a centrifuge tube, 2.5 mL of 0.2 mol / L Folin phenol reagent was added, and the mixture was reacted in the dark for 5 minutes. Then 2 mL of 7.5% Na2CO3 was added and the mixture was reacted in the dark for 2 hours. Deionized water was used as a blank and the absorbance was measured at 760 nm. The sample was prepared into a 2 mg / mL solution and its absorbance was measured. The RA content in the sample was calculated based on the standard curve. The results showed that the binding rate was the highest at this time, which was 89.24%;

[0039] (3) Fourier transform infrared spectroscopy was determined using the potassium bromide tablet method. The freeze-dried covalent complex was ground into powder, mixed with pre-dried potassium bromide at a ratio of 1:100, ground into powder, and tableted. The scanning conditions were set as follows: spectral range 4000-500 cm -1 , scanning times 32 times, resolution 4cm -1 The wavelength is plotted as the horizontal axis and the absorbance is plotted as the vertical axis. -1 ) It can be seen that the absorption peak intensity of the complex becomes stronger, and compared with the protein group, a slight red shift occurs from 3273.56 cm -1 Redshifted to 3275.16 cm -1 Amide I band (1700-1600cm -1 ) The absorption peak of the complex red-shifted from 1629.93 cm -1 Redshifted to 1631.00 cm -1 Amide II band (1600-1500cm -1 ) has a red shift from 1515.24 cm -1 Redshifted to 1521.49 cm -1 ;

[0040] (4) Determination of adenosine deaminase inhibitory activity of digested samples: Prepare 100 μL adenosine solution (0.5 mM) and 200 μL digested sample solution, then add 800 μL phosphate buffer (0.01 M, pH 7.2) to the system and incubate at 37°C for 5 minutes. Then add 100 μL (0.128 U·mL-1) ADA solution and incubate at 37°C for 30 minutes. Subsequently, 12% perchloric acid was added to the reaction system to terminate the reaction. The inosine content was determined by high performance liquid chromatography. The chromatogram was recorded at 248 nm, with 70% ethanol as the control group; the results showed that the IC 50 The IC value of RA is 79.29 μmol / L. 50 The adenosine deaminase inhibitory activity of the complex was 87.89 μmol / L, which was 9.81% higher than that of RA alone.

Claims

1. A method for preparing a covalent complex of soy protein isolate and rosmarinic acid, characterized in that: The method comprises the following steps: (1) extracting soy protein isolate: crushing defatted soybean meal through a 60-mesh sieve, taking 200 g of defatted soybean powder and stirring it with 2000 mL of water for 1 hour, adding 2 mol / L sodium hydroxide solution to adjust the pH to 8.5, stirring it on a magnetic stirrer for 1 hour, and centrifuging it at 4000×g and 10°C for 30 minutes. The supernatant after centrifugation was added with 2 mol / L hydrochloric acid to adjust the pH to 4.5, and after standing at 4°C overnight and centrifuging for 15 min, the supernatant was removed and the precipitate was washed with water three times. After redissolving in deionized water, the pH was adjusted to 7. After dialysis for 48 h, soy protein isolate (SPI) was obtained by freeze-drying. (2) Preparation of protein polyphenol covalent complex: The SPI extracted in step (1) was dissolved in 0.01 M phosphate buffer solution with a pH of 7.2 to prepare a SPI solution with a concentration of 20 mg / mL. After magnetic stirring at room temperature for 1 h, the solution was completely hydrated in a refrigerator at 4°C overnight. 0.05%, 0.1%, and 0.2% rosmarinic acid (RA) were added to the SPI solution, respectively, and mixed evenly. The pH of the solution was adjusted to 9.0 with 0.2 mol / L NaOH. The solution was exposed to air, stirred in the dark for 2 h at room temperature, dialyzed for 48 h, and freeze-dried to obtain RA-SPI complex powder.

2. A method for preparing a covalent complex of soy protein isolate and rosmarinic acid according to claim 1, characterized in that, The purity of the protein extracted in step (1) is 92.59%.

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