A hypoglycemic composite composition, a preparation method thereof and application thereof

Abstract

This invention provides a hypoglycemic compound composition, its preparation method, and its application, belonging to the field of functional product technology. The compound composition uses cornflower-3- O The compound, with α-glucosidase and barley protein as its core components, forms a non-covalently stable complex when combined in a specific mass ratio. This complex exhibits a significant synergistic inhibitory effect on α-glucosidase and α-amylase, more efficiently inhibiting the activity of starch-digesting enzymes, delaying carbohydrate digestion and absorption, and significantly enhancing the hypoglycemic effect. Barley protein, as a carrier of C3G, improves the environmental stability, digestive stability, and bioavailability of C3G. The raw materials of the composite composition provided by this invention are all natural plant resources from the Qinghai-Tibet Plateau, which are widely available, inexpensive, safe to consume, and have no side effects. They can be added as active ingredients to various functional foods and health products, and have good market prospects.

Description

Technical Field

[0001] This invention belongs to the field of functional product technology, specifically relating to a hypoglycemic composite composition, its preparation method, and its application. Background Technology

[0002] Type 2 diabetes is a prevalent metabolic disease. Its core pathological features are insulin resistance and relative insufficiency of pancreatic β-cell function, ultimately leading to an imbalance in glycemic homeostasis. If poorly controlled over a long period, it can easily cause serious complications such as diabetic nephropathy, retinopathy, and cardiovascular disease, significantly reducing patients' quality of life and increasing the medical burden. Studies have confirmed that the pathogenesis of type 2 diabetes is closely related to abnormally elevated activity of amylases (α-glucosidase, α-amylase)—these enzymes rapidly catalyze the hydrolysis of carbohydrates in food into glucose, which is then absorbed by the intestines, leading to a sharp rise in postprandial blood glucose, further exacerbating insulin secretion pressure and insulin resistance. Therefore, inhibiting the activity of α-glucosidase and α-amylase, delaying carbohydrate digestion and absorption, and reducing postprandial blood glucose peaks have become key approaches to assist in regulating blood glucose and improving the condition of type 2 diabetes.

[0003] Currently, commonly used amylase inhibitors in clinical practice are mainly chemically synthesized drugs, such as acarbose and voglibose. Although they can control postprandial blood sugar to some extent, long-term use can easily cause side effects such as gastrointestinal discomfort, intestinal flora imbalance, bloating, and diarrhea. Furthermore, some patients have tolerance issues, limiting their clinical application. With increasing consumer health awareness and focus on the safety of natural products, screening for safe, effective, and low-side-effect amylase inhibitors from natural plants has become a research hotspot in the fields of food nutrition and medicine.

[0004] While existing technologies report that extracts from natural plants have shown potential in lowering blood sugar or inhibiting enzymes, single active ingredients often suffer from limitations such as limited target action and limited efficacy. Developing a composite composition to achieve a synergistic effect of inhibiting amylase digestion and improving insulin resistance, thereby enabling the development of safe and effective blood sugar-lowering products, is of great significance for enriching the application forms of natural blood sugar-lowering active ingredients and meeting the health needs of patients with type 2 diabetes. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a composite composition. This composite composition incorporates cornflower-3- O The combination of glucoside (C3G) and barley protein can significantly enhance the hypoglycemic effect by utilizing their synergistic inhibitory effect. At the same time, the stability and bioavailability of C3G can be significantly enhanced by the carrier effect of barley protein.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides a composite composition comprising, by weight: cornflower-3- O - 1 part glucoside and 0.5-2 parts barley protein; the barley protein includes any one of globulin, albumin, glutenin and prolysin.

[0007] Preferably, the composite composition comprises, by weight, cornflower-3- O - 1 part glucosinolate and 1 part barley protein This invention provides a method for preparing the composite composition described in the above technical solution, comprising the following steps: Cornflower-3- O -After dissolving glucosinolates and barley protein separately to obtain the corresponding solutions, proceed according to cornflower-3- O The mass fractions of glucoside and barley protein were mixed in the solution and incubated to obtain a complex solution; The complex solution was dialyzed and dried to obtain the composite composition.

[0008] Preferably, the temperature for mixed incubation is 4°C; the time for mixed incubation is 2 hours; the mixed incubation is accompanied by stirring; and the stirring speed is 120 r / min.

[0009] Preferably, the dialysate used for dialysis has a sodium chloride concentration of 1% to 10% by mass; and the pH value of the dialysate is 2 to 7.

[0010] Preferably, the dialysis temperature is 4~25℃; the dialysis time is 4h.

[0011] Preferred, Cornflower-3- O - Glucoside and barley protein are dissolved in PBS buffer; the PBS buffer has a molar concentration of 0.01 mol / L and a pH of 7.0~7.4.

[0012] This invention provides the application of the composite composition described in the above-described technical solutions or the composite composition prepared by the preparation method described in the above-described technical solutions in at least one of the following: (1) Increase cornflower-3- O -Stability of glucosides in gastric and / or intestinal fluids; (2) Increase cornflower-3- O -Bioavailability of glucosides.

[0013] The present invention provides the application of the composite composition described in the above technical solution or the composite composition prepared by the preparation method described in the above technical solution in the preparation of products that inhibit starch digestive enzymes.

[0014] This invention provides the application of the composite composition described in the above-described technical solution or the composite composition prepared by the preparation method described in the above-described technical solution in the preparation of hypoglycemic products.

[0015] The beneficial effects of this invention are: This invention provides a composite composition comprising, by weight: cornflower-3- O - 1 part glucoside and 0.5-2 parts barley protein; the barley protein includes any one of globulin, albumin, glutenin, and prolysin. The composite composition uses cornflower-3- O The core components, glucosinolates and barley protein, are combined in a specific mass ratio to form a non-covalently stable complex. This complex exhibits a significant synergistic inhibitory effect on α-glucosidase and α-amylase, more efficiently inhibiting the activity of starch-digesting enzymes, delaying carbohydrate digestion and absorption, and significantly improving the hypoglycemic effect. Barley protein, as a natural carrier of C3G, combines with the complex through hydrogen bonds, hydrophobic interactions, and π-π stacking interactions, enhancing the environmental stability of C3G. Furthermore, during in vitro gastrointestinal digestion, it delays the release of C3G, reduces its degradation, and significantly improves the digestible stability and bioavailability of C3G. Moreover, the raw materials of the composite composition provided by this invention are all natural plant resources from the Qinghai-Tibet Plateau, which are widely available, inexpensive, safe to consume, and have no side effects. They can be added as active ingredients to various functional foods and health products, with a wide range of applications and good market prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The inhibition rate of C3G against BYG and AMY is shown in the graph. Figure 2 Dixon and Cornish-Bowden plots for suppressing BYG and AMY in C3G; Figure 3 The fluorescence spectra of C3G against BYG and AMY are shown. Figure 4 The correlation graph of C3G concentration values ​​for BYG and AMY with their corresponding F0 / F ratios; Figure 5 Molecular docking of C3G with BYG and AMY; Figure 6 The inhibitory effects of Alb, Glo, Gli, and Glu on BYG and AMY; Figure 7Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 19 ... Figure 8 Figures 1-4 show the Dixon and Cornish-Bowden plots of Alb, Glo, Gli, and Glu on AMY; Figures 2-4 show the Dixon plots of Alb, Glo, Gli, and Glu on AMY; Figures 3-4 show the Cornish-Bowden plots of Alb, Glo, Gli, and Glu on AMY. Figure 9 ICs for Alb, Glo, Gli, Glu and BYG and AMY 50 Value and K ic and K iu The correlation between them; AB and CD are the IC50 values ​​of Alb, Glo, Gli, Glu and BYG, AMY, respectively. 50 Value and K ic and K iu Correlation plot; Figure 10 The fluorescence quenching effects of Alb, Glo, Gli, and Glu on BYG and AMY; Figure 11 Stern-Vlomer plots of fluorescence quenching of BYG and AMY by Alb, Glo, Gli, and Glu; Figure 12 KFQ and 1 / K for Alb, Glo, Gli, Glu, BYG, and AMY ic Correlation plot; Figure 13 Molecular docking of Alb, Glo, Gli, and Glu with BYG and AMY, respectively; AD represents molecular docking of Alb, Glo, Gli, and Glu with BYG, respectively; EH represents molecular docking of Alb, Glo, Gli, and Glu with AMY, respectively. Figure 14 The inhibitory effects of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu on BYG and AMY were observed; AD and EH were the inhibitory effects of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu on BYG and AMY, respectively. Figure 15The diagrams show the Dixon and Cornish-Bowden plots of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu against BYG; A, C, E, and G are the Dixon plots of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu against BYG, respectively; B, D, F, and H are the Cornish-Bowden plots of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu against BYG, respectively. Figure 16 The figures are Dixon and Cornish-Bowden plots of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu against AMY; A, C, E, and G are Dixon plots of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu against AMY, respectively; B, D, F, and H are Cornish-Bowden plots of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu against AMY, respectively. Figure 17 The correlation between IC50 values ​​and Kic / Kiu of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu with respect to BYG and AMY is shown; AB and CD are correlation plots of IC50 values ​​and Kic / Kiu of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu with respect to BYG and AMY, respectively. Figure 18 The fluorescence quenching of BYG and AMY by C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu represents the fluorescence quenching of BYG and AMY by C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu, respectively. Figure 19 Stern-Vlomer plots of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu against BYG and AMY; Figure 20 KFQ and 1 / Kic correlation plots of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu with respect to BYG and AMY; Figure 21 Molecular docking of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu to BYG and AMY; AD and EH represent molecular docking of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu to BYG and AMY, respectively. Figure 22X-ray diffraction patterns of C3G, barley protein, and their complexes are shown in Figure 1. A shows the XRD patterns of C3G and four types of barley protein; B shows the XRD pattern of the C3G-protein complex. Figure 23 DSC diagrams of Alb, Glo, Gli, Glu and their complexes; Figure 24 FTIR spectra of C3G with Alb, Glo, Gli, Glu and their complexes; Figure 25 SPR of C3G with Alb, Glo, Gli, and Glu respectively; SPR of C3G with Alb respectively; SPR of C3G with Glo respectively; SPR of C3G with Gli respectively; SPR of C3G with Glu respectively; SPR of C3G with Glu respectively; SPR of C3G with Glu respectively; SPR of C3G with Glu respectively; SPR of C3G with Glu respectively; SPR of C3G with Glu respectively; Figure 26 Molecular docking of C3G with Alb, Glo, Gli, and Glu; A and D represent molecular docking of C3G with Alb, Glo, Gli, and Glu, respectively. Figure 27 The effects of different NaCl concentrations, temperatures, and pH values ​​on the retention rate of C3G were investigated. Figure 28 The effects of Alb, Glo, Gli, and Glu on C3G retention rate were investigated to simulate gastric (A) and small intestinal (B) digestion. Detailed Implementation

[0018] This invention provides a composite composition comprising, by weight: cornflower-3- O - 1 part glucoside and 0.5-2 parts barley protein; the barley protein includes any one of globulin, albumin, glutenin and prolysin.

[0019] The composite composition provided by the present invention comprises, by weight, cornflower-3- O - 1 part of glucoside. This invention relates to the cornflower-3- O - There are no special restrictions on the source of glucosides; they can be obtained from conventional commercial sources or prepared at home.

[0020] Cornflower-3- O Based on the mass fraction of glucoside, the composite composition provided by this invention includes 0.5 to 2 parts of barley protein, which can be 0.5, 1, 1.5, or 2 parts. The barley protein includes any one of globulin, albumin, glutenin, and prolysin. This invention does not specifically limit the source of the barley protein; commercially available products or self-prepared products in the art are acceptable.

[0021] As an optional embodiment of the present invention, the composite composition can be used to coat cornflower-3- O - Glucoside and barley protein can be used separately; cornflower-3- can also be used. O - Use after mixing glucosinolates and barley protein.

[0022] The composite composition provided by this invention uses cornflower-3- O α-Glucoside and barley protein are the core components. When combined in a certain mass ratio, they form a non-covalently stable complex that exhibits a significant synergistic inhibitory effect on α-glucosidase and α-amylase, with an IC50 value of [missing value]. 50 The concentrations were as low as 0.005 mg / mL and 0.064 mg / mL, respectively, demonstrating a significantly better inhibitory effect than C3G or barley protein alone. This resulted in more efficient inhibition of amylase activity, delayed carbohydrate digestion and absorption, and a markedly improved hypoglycemic effect. This invention utilizes barley protein as a natural carrier for C3G. The two bind through hydrogen bonds, hydrophobic interactions, and π-π stacking interactions, effectively encapsulating C3G and enhancing its environmental stability. This composite composition exhibits superior stability under low ion concentration (1% NaCl), low temperature (4℃), and weak acid (pH=2) conditions. Furthermore, it delays the release of C3G and reduces its degradation during in vitro gastrointestinal digestion, significantly improving the digestibility and bioavailability of C3G. Moreover, the raw materials for the composite composition provided by this invention are all natural plant resources from the Qinghai-Tibet Plateau, which are widely available, inexpensive, safe to consume, and have no side effects. They can be added as active ingredients to various functional foods and health products, with a wide range of applications, meeting the dietary needs of different hyperglycemic groups and possessing promising market prospects.

[0023] This invention provides a method for preparing the composite composition described in the above technical solution, comprising the following steps: Cornflower-3- O -After dissolving glucosinolates and barley protein separately to obtain the corresponding solutions, proceed according to cornflower-3- O The mass fractions of glucoside and barley protein were mixed in the solution and incubated to obtain a complex solution; The complex solution was dialyzed and dried to obtain the composite composition.

[0024] This invention will use cornflower-3- O -After dissolving glucosinolates and barley protein separately to obtain the corresponding solutions, proceed according to cornflower-3- O The mass fractions of glucosinolate and barley protein are mixed in the solution to obtain a mixture. As an optional embodiment of the present invention, the cornflower-3- O - Glucosinolate and the barley protein are preferably dissolved separately in PBS buffer to obtain cornflower-3- O- Glucoside solution and barley protein solution; the PBS buffer can have a molar concentration of 0.01 mol / L and a pH value of 7.0~7.4. As an optional embodiment of the present invention, the cornflower-3- O - Glucoside can be prepared at a concentration of 1 mg / mL of cornflower-3- O - Glucoside solution; the barley protein solution can be prepared as a 1 mg / mL barley protein solution. This invention relates to cornflower-3- O -Ultrasonic solubilization is preferred in the preparation of glucosinolate solution and barley protein solution. This yields cornflower-3- O After processing the glucosinolate solution and barley protein solution, the present invention preferably uses cornflower-3- O -The mass ratio of glucosinolates to barley protein will be similar to that of cornflower-3- O - A complex solution is obtained by mixing and incubating a glucosinolate solution and a barley protein solution. As an optional embodiment of the present invention, the incubation temperature can be 4°C; the incubation time can be 2 hours; the incubation can be accompanied by stirring; and the stirring speed can be 120 r / min. The present invention completes the cornflower-3- complex solution through the aforementioned incubation process. O - The self-assembly process of glucosinolates and barley protein involves the two bonding through hydrogen bonds, hydrophobic interactions, and π-π stacking interactions to form a composite composition.

[0025] After obtaining the complex solution, the present invention dialyzes and dries the complex solution to obtain the composite composition. Preferably, the complex solution is added to a dialysis bag for dialysis. In the present invention, the dialysis bag can be a MWCO 6000Da dialysis bag. Preferably, PBS buffer is used as the dialysis solution; the molar concentration of the PBS buffer can be 0.01 mol / L, and the pH value can be 2-7, or 2, 3, 4, 5, 6, or 7. In the present invention, the mass concentration of sodium chloride in the dialysis solution used for dialysis is 1%-10%, or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In the present invention, the dialysis time can be 4 hours; the dialysis temperature can be 4-25°C, or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25°C. This invention primarily removes free ions and unbound small molecules through dialysis. After dialysis, the complex solution in the collection bag is dried. As an optional embodiment of this invention, the drying method can be vacuum freeze-drying; the vacuum freeze-drying temperature can be -50°C; the vacuum freeze-drying time can be 48 hours; the vacuum freeze-drying can be carried out in a vacuum freeze dryer. After drying, this invention yields cornflower-3- O - Glucoside-Barley Protein Complex, also known as Eupatorium fortunei anthocyanin-Barley Protein Complex, or simply Complex.

[0026] The preparation method provided by this invention is simple, green and environmentally friendly, and does not involve any toxic or harmful reagents; the self-assembly process of the complex is carried out under mild weak acid and low temperature conditions, without the need for complex equipment, and is easy to scale up for industrial production; the freeze-dried composition is in powder form, has good flowability, is easy to store, and has a long shelf life.

[0027] This invention provides the application of the composite composition described in the above technical solution or the composite composition prepared by the preparation method described in the above technical solution in at least one of the following: (1) improving cornflower-3- O - Stability of glucosinolates in gastric and / or intestinal fluids; (2) Improvement of cornflower-3- O -Bioavailability of glucosides.

[0028] This invention provides the application of the composite composition described in the above-described technical solutions or the composite composition prepared by the preparation method described in the above-described technical solutions in the preparation of products that inhibit starch digestive enzymes. As an optional embodiment of this invention, the starch digestive enzyme includes α-glucosidase and / or α-amylase. The results of the embodiments of this invention show that, compared with the two substances used alone, the composite composition significantly improves the inhibitory effect on α-glucosidase and α-amylase, and significantly reduces the IC50 of α-glucosidase and α-amylase. 50 The composite composition of this invention achieves a synergistic effect in inhibiting α-glucosidase and α-amylase through the interaction of cyanidin-3-O-glucoside and barley protein.

[0029] This invention provides the application of the composite composition described in the above-described technical solutions or the composite composition prepared by the above-described preparation methods in the preparation of hypoglycemic products. The composite composition provided by this invention can significantly inhibit the activity of amylase, significantly inhibit α-glucosidase and α-amylase, and has a good hypoglycemic effect, and can be used in the preparation of hypoglycemic products.

[0030] This invention provides a product comprising the composite composition described in the above-described technical solutions or the composite composition prepared by the preparation method described in the above-described technical solutions. As an optional embodiment of this invention, the product comprises any one or more of food, pharmaceuticals, and health products. In this invention, the food may be a functional food. As an optional embodiment of this invention, the product comprises any one or more of meal replacement powder, biscuits, pastries, oral liquids, and solid beverages; the dosage form of the product may be any one or more of capsules, tablets, liquid preparations, and granules. As an optional embodiment of this invention, the amount of the composite composition added to the product may be 5% to 20%, or may be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0031] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] BV stands for column bed volume. α-Glucosidase is abbreviated as BYG; α-Amylase is abbreviated as AMY. pNPG stands for nitrophenol-α-D-glucopyranoside. Barley protein is mainly composed of globulin (Glo), albumin (Alb), glutenin (Glu), and gliadin (Gli). The albumin (80% purity), globulin (85% purity), gliadin (85% purity), and glutenin (85% purity) used in the following technical solutions were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0033] In the following technical scheme, all experiments were repeated three times, and the results are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 26.0 software, and P < 0.05 was considered statistically significant. OriginPro 2024 software was used for curve fitting, graph plotting, and calculation of kinetic parameters.

[0034] Example 11. Cornflower-3- O The preparation method of β-glucan (C3G) is as follows: Take 500g of fresh berry fruit, remove impurities, and pulp. The pulping speed is 400r / min and the pulping time is 5min. After pulping, berry fruit pulp is obtained. Add 5L of 65% ethanol aqueous solution to the berry fruit pulp and extract by ultrasonication at 4℃ and 200W for 35min. After extraction, centrifuge at 8000r / min for 10min and collect the supernatant. Load the supernatant onto a pretreated AB-8 macroporous adsorption resin column at a flow rate of 1BV / h. Elute with 3BV of deionized water and 3BV of 30% ethanol aqueous solution to remove impurities, and then elute with 4BV of 60% ethanol aqueous solution at a flow rate of 2BV / h. Collect the eluent, concentrate under reduced pressure at 45℃ to remove ethanol, and freeze-dry at -50℃ to obtain 12.3g of pure C3G with a purity of 96.5%.

[0035] 2. Preparation of barley prolysin: Take 1 kg of barley grains, crush them through an 80-mesh sieve, add 12 L of 75% (v / v) ethanol aqueous solution to the sieve material, and extract at 45℃ with stirring for 2.5 h at a stirring speed of 250 r / min. After extraction, centrifuge at 8000 r / min for 15 min and collect the supernatant. Concentrate under reduced pressure at 45℃ to recover ethanol, transfer the concentrate to a dialysis bag (MWCO 3500 Da), and dialyze with deionized water for 24 h, changing the water every 4 h during dialysis. After dialysis, collect the liquid remaining in the dialysis bag and freeze-dry at -50℃ to obtain 86.5 g of pure barley prolysin with a purity of 88.2% and a molecular weight of 29 kDa.

[0036] Example 2: Determination of the inhibitory effect of single C3G on amylase activity 1. The inhibitory effect of C3G alone on α-glucosidase The C3G concentration gradient was set at 0.021, 0.042, 0.063, 0.084, and 0.105 mg / mL, and the α-glucosidase activity was set at 0.2 U / mL.

[0037] α-Glucosidase inhibition assay: 1 mL of C3G solution of various concentrations was mixed with 1 mL of α-glucosidase enzyme solution and incubated at 4℃ for 15 min. Then, 1 mL of 3 mM pNPG substrate solution was added, and the enzymatic reaction was initiated in a 37℃ water bath. At 0, 3, 6, and 9 min of the reaction, 500 μL of the reaction solution was added to 500 μL of 0.3 M Na2CO3 solution to terminate the reaction. The absorbance at 405 nm was measured using a microplate reader.

[0038] A blank control group and a solvent control group were also set up.

[0039] Inhibition rate ( I ) and half-maximal inhibitory concentration (IC50) 50 The calculation is as follows: I =(1-(v / v0))×100%; In the formula, v is the initial reaction rate when C3G is added, and v0 is the initial reaction rate when C3G is not added.

[0040] A dose-response curve was plotted with C3G concentration on the x-axis and inhibition rate on the y-axis. The half-maximal inhibitory concentration (IC50) was calculated using nonlinear regression fitting. 50 .

[0041] 2. Inhibitory effect of C3G alone on α-amylase The C3G concentration gradient was set at 0.021, 0.042, 0.063, 0.084, and 0.105 mg / mL, and the α-amylase activity was set at 8 U / mL.

[0042] α-Amylase inhibition assay: 1 mL of C3G solution of various concentrations was mixed with 1 mL of α-amylase enzyme solution and incubated at 4℃ for 15 min. Then, 1 mL of 3 mM pNPG substrate solution was added, and the enzymatic reaction was initiated in a 37℃ constant temperature water bath. At 0, 3, 6, and 9 min of the reaction, 500 μL of the reaction solution was added to 500 μL of 0.3 M Na2CO3 solution to terminate the reaction. The absorbance value at 405 nm was measured using a microplate reader.

[0043] A blank control group and a solvent control group were also set up.

[0044] Inhibition rate ( I) and half-maximal inhibitory concentration (IC50) 50 The calculation method is the same as above.

[0045] 3. Kinetic determination of the inhibitory effect of C3G on amylase activity The enzymatic reaction rates were determined under different conditions, with C3G concentrations of 0.021, 0.042, 0.063, 0.084, and 0.105 mg / mL, pNPG solution concentrations of 1, 1.5, 3, 4.5, and 6 mM, α-glucosidase activity of 0.2 U / mL, and α-amylase activity of 8 U / mL. The relationship between the initial reaction rate and substrate concentration was converted into a Lineweaver-Burk double reciprocal plot. Inhibition types were analyzed using Dixon and Cornish-Bowden plots, and kinetic parameters were calculated.

[0046] Competitive inhibition kinetic equation: ; Hybrid inhibition kinetic equation: ; In the formula, v is the initial reaction rate, V max Let i be the maximum reaction rate, i be the concentration of the inhibitor, and a be the... p NPG concentration, K m K represents the Michaelis constant. ic K represents the competitive suppression dissociation constant. iu This represents the non-competitive suppression dissociation constant.

[0047] 4. C3G fluorescence quenching assay for amylase C3G solutions (0.00, 0.04, 0.08, 0.12, 0.16, 0.20 mg / mL) were prepared. 2 mL of each C3G solution was added to 2 mL of α-glucosidase solution (1 U / mL) and α-amylase solution (8 U / mL), respectively. After incubation at 4°C for 15 min, the fluorescence spectra were scanned using a fluorescence spectroscopy scanner. The emission wavelength was set to 295-500 nm, the excitation wavelength to 282 nm, the slit width to 5 nm, and the scan interval to 2 nm. The quenching type and binding parameters were analyzed according to the Stern-Volmer equation.

[0048] ; ; In the formula, F0 and F represent the maximum fluorescence intensity without and with C3G, respectively; K q K is the rate constant for the bimolecular quenching process; FQ [Q] is the fluorescence quenching constant; [Q] is the concentration of C3G; τ0 is the average fluorescence lifetime of the free macromolecule (10 for α-glucosidase).-8 s).

[0049] 5. Molecular docking simulation of C3G on starch digestive enzymes The binding modes of C3G with α-glucosidase and α-amylase were investigated using molecular docking technology. The three-dimensional structure of C3G was obtained from the PubChem database (Compound ID: 441667), and energy minimization (MMFF94 force field) was performed using ChemBio3D Ultra 14.0 software, saving it in mol2 format for later use. The crystal structures of α-glucosidase (PDB ID: 3N04) and α-amylase (PDB ID: 1HNY) were downloaded from the RCSB Protein Data Bank. Crystallization water, existing ligands, and metal ions were removed from the protein structures using PyMOL 2.5.0 software, and polar hydrogen atoms were added. Gasteiger charges were assigned to the proteins using AutoDock Tools 1.5.6 software, and rotatable bonds were assigned to C3G, setting it as a ligand. The gridbox was configured to cover the entire enzyme activity center region: for α-glucosidase, the grid size was 60×60×60 Å, the grid spacing was 0.375 Å, and the center coordinates were referenced to the protoligand position (x=-11.2, y=24.8, z=32.5); for α-amylase, the grid size was 60×60×60 Å, and the center coordinates were set to (x=8.3, y=12.6, z=15.7). Semi-flexible docking was performed using AutoDock 4.2.6 software, with 100 runs and a maximum energy evaluation count of 2.5×10⁻⁶. 6 The docking results are based on binding affinity (K). D The conformations were sorted, and the lowest energy conformation was selected as the optimal binding mode. The interaction map was then plotted using PyMOL and Discovery Studio 2019.

[0050] 6. Data Processing: All experiments were performed in triplicate, and results are expressed as mean ± standard deviation. Excel was used for data processing, and OriginPro 2024 and GraphPad Prism software were used for curve fitting and graph plotting.

[0051] 7. Results and Conclusions (1) The inhibition rates of C3G on α-amylase and α-glucosidase are as follows: Figure 1 As shown in Table 1, the kinetic parameters of C3G suppressing BYG and AMY are shown in Table 1.

[0052] Table 1. Kinetic parameters of C3G inhibiting BYG and AMY

[0053] Note: Data are mean ± standard deviation (n=3); IC 50 The half-maximal inhibitory concentration (IC50) is K. ic K is the competitive suppression dissociation constant. iu The non-competitive suppression dissociation constant; 1 / K ic 1 / K iu This is the association constant; a larger value indicates a stronger binding affinity, and the same applies below.

[0054] From Table 1 and Figure 1 It was found that C3G exhibited significant concentration-dependent inhibitory effects on both α-glucosidase and α-amylase, with the inhibition rate gradually increasing with increasing C3G concentration. The IC50 of C3G on α-glucosidase was... 50 The value was 0.011 ± 0.001 mg / mL, and the IC50 value for α-amylase was... 50 The value was 0.017±0.003 mg / mL, indicating that C3G has a stronger inhibitory activity against α-glucosidase.

[0055] The experimental results are likely closely related to the molecular structure of C3G. The ortho-dihydroxyl structures at the 3' and 4' positions of its B ring and the multiple hydroxyl groups on the glucoside at the 3' position of its C ring can form non-covalent interactions such as hydrogen bonds and hydrophobic interactions with amino acid residues in the enzyme's active site, thereby altering the enzyme's spatial conformation and hindering substrate binding. This result confirms that C3G, as a natural anthocyanin monomer, has significant potential for inhibiting amylase digestion, providing a high-quality candidate ingredient for developing adjunctive hypoglycemic products.

[0056] (2) Kinetic analysis of the inhibition of C3G on amylase digestion C3G suppression of BYG and AMY: Dixon plot and Cornish-Bowden plot, as shown. Figure 2 As shown; A and C are the Dixon plots of C3G against BYG and AMY, respectively; B and D are the Cornish-Bowden plots of C3G against BYG and AMY, respectively.

[0057] From Table 1 and Figure 2 The inhibition kinetic analysis showed that the Dixon and Cornish-Bowden plots of C3G for α-glucosidase and α-amylase intersected at the same point, indicating that its inhibition of both enzymes was a mixed type. From the kinetic parameters, the Kc of C3G for α-glucosidase... ic (0.024±0.009 mg / mL) and K iu The values ​​(0.029 ± 0.002 mg / mL) were significantly lower than the corresponding values ​​for α-amylase (K). ic =0.129±0.009 mg / mL, K iu=0.135±0.021 mg / mL), and 1 / K ic and 1 / K iu A higher value indicates that C3G has a stronger affinity for α-glucosidase and a more significant inhibitory effect. Furthermore, C3G exhibits higher K values ​​for both enzymes. ic All less than K iu 1 / K ic All less than 1 / K iu .

[0058] This result suggests that C3G can bind to both free enzymes and enzyme-substrate ( p The C3G complex interacts with the NPG complex, inhibiting enzyme activity through a dual binding mode. Furthermore, C3G inhibits the K+ binding of both enzymes. ic All less than K iu 1 / K ic All are greater than 1 / K iu This indicates that C3G binds more tightly to the enzyme-substrate complex, further confirming the mixed-type inhibitory effect.

[0059] (3) Mechanism of fluorescence quenching of amylase by C3G The fluorescence spectra of C3G against BYG and AMY are as follows: Figure 3 As shown in Table 2, the fluorescence quenching parameters of C3G for BYG and AMY are shown in Table 3. The correlation between the concentrations of C3G for BYG and AMY and their corresponding F0 / F ratios is shown in Table 2. Figure 4 As shown.

[0060] Table 2. Fluorescence quenching parameters of C3G for BYG and AMY

[0061] Aromatic amino acid residues such as tryptophan (Trp) and tyrosine (Tyr) contained in α-glucosidase and α-amylase molecules have intrinsic fluorescence. Changes in their fluorescence intensity and emission wavelength can reflect the interaction between the inhibitor and the enzyme and changes in enzyme conformation.

[0062] From Table 2 and Figure 3 It was found that with increasing C3G concentration, the intrinsic fluorescence intensity of both enzymes decreased in a concentration-dependent manner, and the maximum emission wavelength showed a slight blue shift. Fluorescence quenching parameters indicated that the quenching rate constants (Kc) of C3G on α-glucosidase and α-amylase... q The values ​​are (33.882±0.074)×10 8 mL·mg -1 ·s -1 and (30.387±0.023)×10 8 mL·mg -1 ·s -1(After unit conversion, they are 1.52 × 10) 12 and 1.37×10 12 M -1 ·s -1 Therefore, it is correct that it is greater than 10 ... 10 M -1 ·s -1 Its quenching type is static quenching. Among them, the fluorescence quenching constant (K0) of C3G for α-glucosidase... FQ =33.882±0.063 mL·mg -1 (Higher than K for α-amylase) FQ Value (30.387±0.033 mL·mg) -1 This indicates that C3G has a stronger binding effect with α-glucosidase, which is consistent with the results of enzyme inhibition experiments and kinetic analysis.

[0063] The above results may be because after C3G binds to the enzyme, the polarity of the microenvironment in which the fluorescent group resides decreases, and the spatial conformation of the enzyme changes. C3G and the enzyme form a stable non-fluorescent complex in the ground state, rather than a simple dynamic collision interaction.

[0064] Depend on Figure 4 Therefore, further analysis of IC is needed. 50 Values ​​and dynamic parameters (K) ic K iu The correlation between IC and α-glucosidase was analyzed, and the linear correlation coefficients were found to be 0.9938 (α-glucosidase) and 0.9981 (α-amylase), respectively, indicating that IC 50 The smaller the value, the better K ic and K iu The smaller the value, the tighter the binding of C3G to the enzyme, and the more significant the inhibitory effect, confirming the consistency between the results of the enzyme inhibition experiment, kinetic analysis, and fluorescence quenching experiment.

[0065] (4) Analysis of C3G on the molecular docking of starch digestive enzymes Molecular docking of C3G with BYG and AMY, such as Figure 5 As shown in Table 3, the molecular docking parameters of C3G with BYG and AMY are shown in Table 3.

[0066] Table 3 Molecular docking parameters of C3G for BYG and AMY

[0067] Molecular docking simulations visually revealed the binding mode and key action sites of C3G with amylase. Figure 5The results showed that C3G can precisely embed into the active site regions of α-glucosidase and α-amylase, forming stable interactions with surrounding key amino acid residues.

[0068] For α-glucosidase (PDB:3N04), C3G interacts with amino acid residues near the active site, including PHE995, TRP985, TYR967, HIS1449, and ARG1206. The main interactions include hydrogen bonds, hydrophobic interactions, π-π stacking interactions, π-σ interactions, and π-alkyl interactions. These interactions collectively stabilize the binding of C3G to the enzyme, preventing the substrate pNPG from entering the active site. For α-amylase (PDB:1HNY), C3G binds to residues in the active site, including TRP58, TRP59, ASP197, GLU233, and HIS299. The inhibitory effect is mainly achieved through hydrogen bonds, hydrophobic interactions, and π-π stacking interactions. TRP58 and TRP59 are key residues in the α-amylase active site; the binding of C3G to these residues directly disrupts the enzyme's catalytic structure. The binding affinity (KD) results (Table 3) show that the binding affinity of C3G to α-glucosidase is -9.2 Kcal / mol, which is lower than that to α-amylase (-8.3 Kcal / mol). This indicates that C3G binds more stably to α-glucosidase and has a more significant inhibitory effect, which is completely consistent with the experimental results mentioned above.

[0069] In summary, the differences in binding characteristics between the two types of enzymes mainly stem from the different amino acid compositions of their active sites. Hydrophobic residues of α-glucosidase dominate multi-component hydrophobic interactions, while α-amylase is centered on hydrogen bonds and π-π stacking interactions.

[0070] Example 3: Study on the inhibitory effect of four types of barley proteins on amylase digestion. 1. Experimental Materials Albumin (80% purity), globulin (85% purity), prolysin (85% purity), glutenin (85% purity): Shanghai Yuanye Biotechnology Co., Ltd.

[0071] 2. Experimental Methods 2.1 Determination of the inhibitory effect of four types of barley protein on amylase activity Four types of barley protein solutions—albumin, globulin, prolysin, and glutenin—were prepared with concentration gradients of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. Following the method described in Example 2, the inhibitory effects of the four proteins on the activities of two amylases (α-glucosidase and α-amylase) were determined. Three replicates were set up for each concentration gradient to ensure the reliability of the experimental results.

[0072] 2.2 Kinetic determination of the inhibitory effect of four types of barley protein on starch digestive enzyme activity Four types of barley protein solutions—albumin, globulin, prolyl, and glutenin—were prepared with concentration gradients of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively. The specific method is described in Example 2.

[0073] 2.3 Determination of fluorescence quenching of four barley proteins on amylase activity Four types of barley protein solutions—albumin, globulin, prolyl, and glutenin—were prepared with concentration gradients of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively. The specific method is described in Example 2.

[0074] 2.4 Determination of Molecular Simulated Docking between Four Barley Proteins and Starch Digestive Enzymes Molecular docking of four barley proteins (albumin, globulin, prolyl, and glutenin) with α-glucosidase and α-amylase was performed using the HADDOCK 2.4 online server. Protein crystal structures were obtained from the RCSB PDB: albumin (PDB ID: 1MID), globulin (PDB ID: 6PYQ), prolyl (PDB ID: 5IFJ), glutenin (PDB ID: 6PX6), α-glucosidase (PDB ID: 3N04), and α-amylase (PDB ID: 1HNY). All water molecules and heteroatoms were removed using PyMOL 2.5.0, and the active residues on the protein surface were predicted using the CPORT tool built into the HADDOCK platform as "active residues" for docking. Docking parameters were set as follows: rigid bulk energy minimization was performed on 1000 structures (it0), followed by semi-flexible simulated annealing on the top 200 structures (it1), and finally, fine optimization was performed on the top 200 structures in an aqueous environment (itw). The docking results were evaluated using the HADDOCK score, which integrates van der Waals energy, electrostatic potential energy, hydrophobic energy, restraints energy, and solvation energy. The cluster with the lowest score was selected as the representative conformation for further analysis, and the free energy was calculated using the PRODIGY server.

[0075] 2.5 Data Processing All experiments were performed in triplicate, and results are expressed as mean ± standard deviation. Data were processed using Excel, and curve fitting and graph plotting were performed using OriginPro 2024 and GraphPad Prism software.

[0076] 3. Results 3.1 Analysis of the inhibitory effect of four types of barley protein on the activity of amylase digestion The inhibitory effects of albumin, globulin, prolysin, and gluten on α-glucosidase and α-amylase activities were systematically evaluated using an in vitro enzyme inhibitory activity evaluation system.

[0077] The kinetic parameters of the inhibition of BYG and AMY by Alb, Glo, Gli, and Glu are shown in Table 4. The inhibitory effects of Alb, Glo, Gli, and Glu on BYG and AMY are as follows: Figure 6 As shown.

[0078] Table 4 Kinetic parameters of Alb, Glo, Gli, and Glu inhibiting BYG and AMY

[0079] From Table 4 and Figure 6 The enzyme kinetics results showed that all four protein components exhibited dose-dependent inhibitory activity towards both enzymes, demonstrating good inhibitory activity. The half-maximal inhibitory concentration (IC50) was calculated by fitting the dose-response curve. 50 The results showed that the IC50 of the four proteins on α-glucosidase was... 50 The values ​​were: albumin 0.233 mg / mL, globulin 0.832 mg / mL, prolysin 0.192 mg / mL, and glutenin 1.250 mg / mL; the IC50 values ​​against α-amylase were... 50 The values ​​were: albumin 0.299 mg / mL, globulin 1.079 mg / mL, prolysin 0.263 mg / mL, and glutenin 1.268 mg / mL. The inhibition rates of albumin, globulin, and prolysin increased with increasing concentration, while the inhibition rate of glutenin decreased with increasing concentration. The inhibitory abilities of the four proteins against both target enzymes were consistent: prolysin > albumin > globulin > glutenin.

[0080] The decreasing inhibition rate of glutenin with increasing concentration may be due to the smaller molecular weight and better diffusivity of albumin and prolysin, while glutenin is a high-molecular-weight aggregate protein tightly bound by intermolecular disulfide bonds and hydrophobic interactions. As concentration increases, intermolecular collisions intensify, rapidly forming large, insoluble clumps or network structures. This results in the binding sites being embedded internally, reducing the effective components and significantly increasing steric hindrance, thus exhibiting the anomalous phenomenon of decreasing inhibition rate with increasing concentration. Furthermore, the differences in inhibition of amylase by the four proteins are determined by the protein molecular structure, the conformation of the enzyme's active site, and the interaction mode between them. Prolysin, due to its small molecular size, high flexibility, and fully exposed active residues, exhibits the best enzyme-binding stability; albumin, with its good water solubility and hydrogen bonding, is next; globulin, due to its compact conformation and weak hydrophobic interactions, has lower inhibitory activity; glutenin, affected by aggregation at high concentrations, has the lowest activity and shows a decreasing trend. The stronger inhibition of α-glucosidase by each protein may be related to the fact that the pocket structure of the enzyme's active site facilitates the binding of small-molecule proteins.

[0081] 3.2 Kinetic analysis of the inhibitory effects of four barley proteins on the activity of amylases Alb, Glo, Gli, and Glu are shown in the Dixon and Cornish-Bowden diagrams for BYG. Figure 7 As shown. Dixon and Cornish-Bowden plots of Alb, Glo, Gli, and Glu against AMY are shown below. Figure 8 As shown. IC50 values ​​and K of Alb, Glo, Gli, Glu, BYG, and AMY. ic and K iu The correlation between them is as follows Figure 9 As shown.

[0082] Through inhibition kinetic experiments, the interaction mechanism between four cereal protein components and α-glucosidase and α-amylase was elucidated in depth. Figures 7-8 As shown, kinetic analysis of α-glucosidase and α-amylase revealed that the Dixon and Cornish-Bowden diagrams of albumin, globulin, and gluten all intersect at a single point, indicating a mixed inhibitory effect on both enzymes. The Dixon diagram of prolysin intersects at a single point, while the Cornish-Bowden diagram shows parallel lines without intersection, indicating competitive inhibition of both enzymes, primarily through competition for the enzyme's active site. Inhibition kinetic parameters (Table 4) show the Kt values ​​of the four proteins for the two enzymes. ic All are greater than K iu .

[0083] This experiment revealed the specific mechanism by which four proteins interact with two starch-digesting enzymes, consistent with the previously mentioned IC... 50The data corroborate each other. Albumin, globulin, and glutenin all exhibit a mixed inhibitory mode, both competitively binding to the enzyme's active site to interfere with substrate binding and inducing conformational changes in the enzyme, thus affecting catalytic efficiency. This is related to the distribution of hydrophilic and hydrophobic residues in multiple regions on their molecular surface, giving them both competitive binding to the enzyme's active site and allosteric regulatory capabilities to bind to allosteric sites, thereby enhancing the inhibitory effect. Prolysin, on the other hand, exhibits competitive inhibitory characteristics. Its small molecule and highly flexible structure makes it easier to precisely bind to the enzyme's active site and seize substrate binding sites, which also explains its IC50 value. 50 The results showed the lowest inhibitory activity and the best inhibitory value. The four proteins exhibited stronger selectivity in inhibiting α-glucosidase. This is essentially because the pocket structure of the α-glucosidase active site is more likely to accommodate small proteins and has a higher binding affinity, while the groove-shaped active site of α-amylase requires greater steric hindrance for ligands, increasing the difficulty of protein binding. The inhibition kinetics indicate differences in the inhibition type and affinity of the four proteins, providing a kinetic basis for the subsequent development of highly efficient natural starch digestion inhibitors.

[0084] Combining C3G, albumin, globulin, prolysin, and gluten to inhibit the IC50 of α-glucosidase and α-amylase 50 IC50 values ​​were plotted for five substances and two amylases. 50 Value and K ic K iu Correlation fitting plot of values ​​( Figure 9 ).Depend on Figure 9 It can be seen that the IC50 values ​​of the five substances for α-glucosidase and α-amylase are... 50 Value and K ic / K iu The linear correlation coefficient (R) of the values 2 The values ​​of IC50 and IC50 were 0.9736 and 0.9829, respectively, indicating that it binds tightly to both enzymes and exhibits good inhibitory activity. This result confirms that IC50... 50 Value and K ic / K iu The correlation can indirectly characterize the binding strength between the inhibitor and the enzyme, and provide data support and theoretical basis for subsequent research on its enzyme inhibition mechanism.

[0085] 3.3 Fluorescence quenching analysis of four barley proteins on the activity of amylase digestion The fluorescence quenching effects of Alb, Glo, Gli, and Glu on BYG and AMY, such as Figure 10 As shown in the figure. Stern-Vlomer plots of fluorescence quenching of BYG and AMY by Alb, Glo, Gli, and Glu are shown in the figure. Figure 11 As shown. Alb, Glo, Gli, Glu vs. BYG and AMY's KFQ and 1 / K ic Correlation plot as follows Figure 12As shown in Table 5, the fluorescence quenching parameters of Alb, Glo, Gli, and Glu for BYG and AMY are shown in Table 5.

[0086] Table 5. Fluorescence quenching parameters of Alb, Glo, Gli, and Glu for BYG and AMY

[0087] Note: Data are mean ± standard deviation (n=3); K FQ K is the quenching constant. q This is the quenching rate constant.

[0088] After adding proteins of different concentration gradients, the intrinsic fluorescence intensity of α-glucosidase and α-amylase decreased with increasing protein concentration. Figure 10 The fluorescence spectrum showed a blue shift. The quenching constant (Kc) was calculated. FQ ) and quenching rate constant (K q (Table 5), and K q All are greater than 2×10 10 M -1 s -1 This indicates that the interaction between the two is mainly static quenching (forming a stable protein-enzyme complex). K FQ The larger the protein, the stronger the quenching effect and enzyme inhibition. The order of the four proteins' effects is: prolysin > albumin > globulin > glutenin. Prolysin has a more stable binding due to its structural advantages and a stronger quenching effect.

[0089] Fluorescence spectroscopy results confirmed the previous conclusions, showing that the four proteins and enzymes formed stable complexes through static quenching, K FQ Value sorting and IC 50 K ic / K iu Consistent. With increasing protein concentration, both enzymes exhibited a blue shift in fluorescence spectra, possibly related to the increased nonpolarity of the tryptophan residue microenvironment and the encapsulation of residues due to protein aggregation. Albumin and prolysin showed more significant quenching and blue shift effects, possibly due to their small molecular weight and high binding efficiency, while globulins and glutenins exhibited lower binding efficiency due to aggregation. Fluorescence spectroscopy confirmed the formation of the protein-enzyme complex and the inhibitory effect of enzyme conformational changes at the conformational level.

[0090] The fluorescence spectral data were fitted using the Stern-Volmer equation to obtain... Figure 11 The fitting results show that the quenching curve exhibits a good linear relationship (R0). 2The result (≈1) indicates that the fluorescence quenching process is not triggered by simple intermolecular dynamic collisions, but rather originates from the binding of the inhibitors (four proteins) to the enzyme molecules to form stable non-fluorescent protein-enzyme complexes. This result further corroborates the mechanism of static quenching and suggests that each target enzyme molecule has approximately one major site on its surface that specifically binds to the protein inhibitor, providing a theoretical basis for the generation of its enzyme inhibitory activity. It also indicates that the four proteins have potential application value as natural starch digestive enzyme inhibitors.

[0091] Depend on Figure 12 It can be seen that the fluorescence quenching constants (KFQ) and the reciprocals of the inhibition constants (1 / Kic) of the four proteins for α-glucosidase and α-amylase all show a good linear correlation. 2 The values ​​reached 0.9920 and 0.9778, respectively, quantitatively reflecting the highly consistent intrinsic relationship between fluorescence quenching and enzyme inhibition effects. This confirms that fluorescence quenching kinetics and enzyme inhibition kinetics can be effectively combined.

[0092] 3.4 Molecular docking simulation of four types of barley protein with starch digestive enzymes The molecular docking parameters of Alb, Glo, Gli, and Glu with BYG and AMY are shown in Table 6. Table 6 Molecular docking parameters of Alb, Glo, Gli, and Glu with BYG and AMY

[0093] Using HADDOCK molecular docking technology, the molecular interactions of albumin, globulin, prolysin, glutenin, and α-glucosidase and α-amylase were simulated. The results are as follows: Figure 13As shown. The docking results indicate that all four proteins can effectively enter the enzyme's catalytic activity pocket and form stable hydrogen bonds with key amino acid residues near the active site. The active amino acids surrounding the four proteins and α-glucosidase are as follows: albumin: THR173, ASP176, GLY170, TRP169, THR483, etc.; globulin: ASP203, ASP199, TRP169, ALA480, LYS240, etc.; prolyl: ASP176, GLY482, GLU481, PHE453, THR78, etc.; glutenin: ASN213, THR173, ASP176, GLY170, etc. The four proteins interacting with α-amylase have the following active amino acids: albumin: TRP344, ARG343, ASP381, CYS378, etc.; globulin: ARG346, PRO345, ARG343, ASP381, etc.; prolyl: SED414, GLY308, GLY306, ASP356, ASP147, etc.; glutenin: PHE286, GLU272, LYS243, ASN150, GLU149, etc. These residues are mostly located in the enzyme's substrate recognition and catalytic regions. By forming hydrogen bonds with these sites, the proteins can alter the enzyme's spatial conformation, hindering substrate binding or the catalytic process, thereby exerting an inhibitory effect.

[0094] Based on this, the binding free energy (ΔG) was used as an indicator to quantitatively evaluate the binding affinity between proteins and enzymes. The results are shown in Table 6: The binding free energies for α-glucosidase were: albumin -8.4 kcal / mol, globulin -8.0 kcal / mol, prolyl -8.8 kcal / mol, and gluten -7.8 kcal / mol; the binding free energies for α-amylase were: albumin -7.7 kcal / mol, globulin -7.2 kcal / mol, prolyl 8.0 kcal / mol, and gluten -6.8 kcal / mol. A smaller ΔG indicates a better inhibitory effect. The inhibitory effects of the four proteins on the enzymes were: prolyl > albumin > globulin > gluten.

[0095] Example 4: Study on the inhibitory effect of C3G-barley protein complex on amylase digestion. 1. Experimental materials: C3G (purity ≥ 95%): Qinghai Lanfeng Biotechnology Co., Ltd.; Albumin (purity 80%), Globulin (purity 85%), Gliadin (purity 85%), Gluten (purity 85%): Shanghai Yuanye Biotechnology Co., Ltd.

[0096] 2. Experimental Methods 2.1 Determination of the inhibitory effect of C3G-barley protein complex on amylase activity The IC50 values ​​of various barley proteins against α-glucosidase and α-amylase were determined according to Example 3. 50 Values ​​were selected for each protein's IC50 value. 50 The midpoint of the value range or close to its EC 50 The levels were specifically: albumin 0.8 mg / mL (approximately its IC50 value for both enzymes). 50 Three times the mean value of 0.266 mg / mL), globulin 1 mg / mL (approximately IC50 value). 50 1.05 times the mean value of 0.956 mg / mL), and prolysin 0.2 mg / mL (approximately IC50). 50 0.88 times the mean of 0.228 mg / mL), gluten 1.2 mg / mL (approximately IC50). 50 (0.92 times the mean 1.309 mg / mL) is intended to ensure that the inhibitory activity contributed by the protein itself in the complex system is within the linear response range of the dose-response curve, facilitating the observation of the interaction between C3G and the protein.

[0097] The C3G solution concentration gradient was 0, 0.021, 0.042, 0.063, 0.084, and 0.105 mg / mL, with α-glucosidase activity of 0.2 U / mL and α-amylase activity of 8 U / mL. The solutions were added in different sequences (①C3G + enzyme → protein; ②C3G + protein → enzyme; ③protein + enzyme → C3G), and incubated at 4°C for 2 hours. Enzyme inhibition experiments were performed according to the method in Example 2.

[0098] Sequence ① indicates that C3G is first incubated with the enzyme at 4°C for 15 min, then the protein is added and incubated at 4°C for 2 h, followed by the addition of the substrate; Sequence ② indicates that C3G is premixed with protein and incubated at 4℃ for 15 min, then the enzyme is added, incubated at 4℃ for 2 h, and then the substrate is added; Sequence ③ indicates that the protein is first incubated with the enzyme at 4°C for 15 min, then C3G is added and incubated at 4°C for 2 h, followed by the addition of the substrate.

[0099] 2.2 Kinetic determination of the inhibitory effect of C3G-barley protein complex on amylase activity The solution was prepared according to the method in 2.1 above, and its inhibition kinetics were determined by the method in Example 2.

[0100] 2.3 Assay for fluorescence quenching of amylase by C3G-barley protein complex The solution was prepared according to the method in 2.1 above, and the fluorescence quenching effect of the protein on the activities of α-glucosidase and α-amylase was determined by the method in Example 2.

[0101] 2.4 Simulation of C3G-barley protein complex docking with starch digestive enzymes All crystal structures used were obtained from databases: the C3G crystal structure (CID: 7084-24-4) was obtained from the PubChem database, and the crystal structures of α-glucosidase (PDB: 3N04), α-amylase (PDB: 1HNY), albumin (PDB: 1MID), globulin (PDB: 6PYQ), prolysin (PDB: 5IFJ), and glutenin (PDB: 6PX6) were obtained from the RCSB protein database. PyMOL software was used to preprocess the proteins, removing water molecules and adding hydrogen atoms to construct complete protein structures.

[0102] To preliminarily explore the potential binding modes of C3G-protein complexes with amylases, this study employed a stepwise docking strategy for molecular simulation: First, using Auto Dock software, C3G was simulated to dock with four different barley proteins, resulting in stable conformations of four complexes: C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu. Subsequently, using these complexes as monolithic ligands, HADDOCK software was used to simulate molecular docking with α-glucosidase and α-amylase, respectively, with the binding free energy (ΔG) used as an indicator to evaluate the binding capacity of each system.

[0103] It should be clearly pointed out that the molecular docking method used in this study has the following limitations: (1) The interaction between C3G and protein is a non-covalent dynamic binding. The pre-docked complex is rigidified, ignoring the conformational rearrangement and induced fit effects that may occur during the binding of the complex with the enzyme; (2) In actual solutions, multiple binding modes may exist simultaneously among C3G, protein, and enzyme (such as C3G binding with the enzyme and then binding with the protein, protein binding with the enzyme and then binding with C3G, and the simultaneous formation of ternary complexes, etc.). Stepwise docking cannot fully reflect these complex equilibria; (3) The docking results only provide thermodynamically favorable binding conformations and cannot fully simulate the dynamic molecular recognition process. Therefore, the molecular docking simulation here should be regarded as an exploratory structural prediction, aiming to provide possible molecular-level explanations for the inhibition effects observed in experiments. The relevant conclusions need to be judged comprehensively in conjunction with experimental data.

[0104] 2.5 Data Processing All experiments were performed in triplicate, and results are expressed as mean ± standard deviation. Excel was used for data processing, and OriginPro 2024 and GraphPad Prism software were used for curve fitting and graph plotting.

[0105] 3. Results 3.1 Analysis of the inhibitory activity of C3G-barley protein complex on amylase digestive activity The half-maximal inhibitory concentration (IC50) of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu 50As shown in Table 7, the inhibitory effects of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu on BYG and AMY are as follows. Figure 14 As shown.

[0106] Table 7. Half-maximal inhibitory concentrations (IC50) of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu 50 )

[0107] Note: Single-component control: IC50 of C3G versus BYG 50 The concentration of gluten was 0.011 ± 0.001 mg / mL for gluten and 0.017 ± 0.003 mg / mL for AMY; the IC50 of gluten for BYG was... 50 The concentration was 1.250±0.134 mg / mL for C3G and 1.368±0.114 mg / mL for AMY. The order of addition is as follows: ① indicates C3G is incubated with the enzyme before adding the protein; ② indicates C3G is premixed with the protein before adding the enzyme; ③ indicates the protein is incubated with the enzyme before adding C3G.

[0108] Enzyme inhibition experiments were performed on the C3G-protein complex using three different addition sequences: ① C3G + enzyme → protein; ② C3G + protein → enzyme; ③ protein + enzyme → C3G. The results are as follows: Figure 14 As shown, the four complexes all exhibited inhibitory effects on α-glucosidase and α-amylase. The inhibitory effects of C3G-Alb, C3G-Glo, and C3G-Gli on both enzymes were positively correlated with concentration, with the inhibitory effect increasing with increasing concentration. However, the inhibitory effect of C3G-Glu gradually weakened with increasing concentration at addition order ③. The inhibitory strength of the four complexes against α-glucosidase and α-amylase was ranked as C3G-Gli > C3G-Alb > C3G-Glo > C3G-Glu (Table 7); the inhibitory effect at different addition orders was ranked as ① > ③ > ②. The results indicate that the inhibitory effect of the complexes at all three addition orders was stronger than that of the single substances, confirming that the formation of the C3G-protein complex can enhance the stability of C3G, thereby strengthening its inhibitory effect on both enzymes.

[0109] For IC in Table 7 50 Further analysis of the values ​​showed that, after C3G was combined with four proteins, except for glutenin, the other complexes showed an IC50 value at the addition order ①. 50 The values ​​(0.005-0.008 mg / mL) were all lower than those of C3G alone (0.011 mg / mL), showing a synergistic effect; while the IC50 of the C3G-gluten complex at the addition order ① was lower. 50The value was 0.024 mg / mL, which, although higher than C3G alone, was far lower than gluten alone (1.250 mg / mL), indicating that while the combination of gluten and C3G failed to surpass the inhibitory efficacy of C3G, it still significantly enhanced the inhibitory activity of gluten itself. It is noteworthy that the IC50 of the gluten complex, added in sequence ③ (protein + enzyme → C3G), was... 50 The value (0.374 mg / mL) was significantly higher than that of other addition sequences.

[0110] The possible mechanisms underlying the above phenomena are as follows: Albumin and globulin are both water-soluble proteins with monomeric or aggregated structures. Their surfaces are rich in hydrophilic groups and charges, and their loose, flexible structures provide multiple binding sites when binding to C3G. Through hydrophobic interactions and hydrogen bonds, they form non-covalent, reversible bonds with the benzene ring and phenolic hydroxyl groups of C3G. Glycol-soluble proteins are rich in proline and glutamine, are extremely hydrophobic, and have poor water solubility. They readily form polymers in solution. When binding to C3G, C3G is easily embedded within the protein aggregates, resulting in strong binding but reduced bioavailability. Gluten is a high-molecular-weight protein subunit that forms a fibrous network structure through intermolecular disulfide bonds. Its hydrophobicity is similar to that of prolamins, but it exhibits a higher degree of polymerization and a more complex network structure. When it binds to C3G, it forms a porous, sponge-like structure, deeply embedding C3G within the network. At low concentrations, the network structure is limited, leaving some free C3G exposed. As the concentration increases, the density and volume of the polymerized network increase, with most of the C3G being embedded, creating a steric hindrance effect that hinders its binding to the enzyme's active site, thus reducing the inhibitory effect. The phenomenon observed in gluten addition sequence ③ may be due to the steric hindrance effect created by the large molecular aggregates formed after gluten binds to the enzyme, preventing subsequent binding of C3G to the enzyme's active site. This phenomenon is consistent with the finding in Chapter 3 that gluten self-aggregation at high concentrations leads to a decrease in inhibition rate, further confirming the significant impact of gluten's unique aggregation behavior on its functional activity.

[0111] 3.2 Kinetic analysis of the inhibitory effect of C3G-barley protein complex on amylase activity. Figures 15-16 The inhibition kinetics results shown indicate that the four complexes C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu act on α-glucosidase and α-amylase. The Dixon and Cornish-Bowden plots all intersect, indicating that the four complexes exhibit mixed inhibition of both enzymes. Combined with the specific parameters in Table 8, it can be seen that C3G-Alb has the lowest Kic and Kiu values ​​among the four complexes, indicating its best inhibitory effect.

[0112] The core characteristic of mixed inhibition is that the inhibitor molecule can exert its inhibitory effect through a dual-mode of action. It can specifically bind to the active site of the enzyme molecule, forming an enzyme-inhibitor complex (EI) that inhibits substrate binding to the active site; simultaneously, it can bind to the enzyme-substrate complex (ES) to form an enzyme-substrate-inhibitor complex (ESI), inhibiting the catalytic conversion of the substrate, thus significantly reducing the enzyme's catalytic activity through two pathways. Furthermore, C3G-Alb exhibits the best inhibitory effect, likely because the C3G-Alb complex is more likely to form a structure complementary to the enzyme's active site in terms of spatial conformation. Its hydrophobic distribution and surface charge properties are also more compatible with the microenvironment of the enzyme's active region, thereby promoting hydrophobic interactions, hydrogen bonding, and electrostatic interactions between the complex and the enzyme molecule, significantly enhancing the specificity and stability of their binding. Notably, when the protein is successively replaced by globulin, prolyl, and glutenin, the inhibitory effect of the complex on the enzyme decreases. This indicates that the physicochemical properties of proteins play an important regulatory role in the interaction between the complex and enzyme molecules. Among them, key physicochemical parameters such as protein molecular size, spatial conformation, and hydrophobicity may affect the binding mode of C3G to the protein and the spatial structure of the complex, thereby regulating the recognition and binding efficiency of the complex to the enzyme active site and ultimately affecting its inhibitory activity.

[0113] Table 8. Inhibitory kinetic parameters of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu on BYG and AMY

[0114] Note: Data are mean ± standard deviation (n=3); IC 50 The half-maximal inhibitory concentration (IC50) is K. ic K is the competitive suppression dissociation constant. iu The non-competitive suppression dissociation constant; 1 / K ic 1 / K iu This is the association constant; a larger value indicates a stronger binding affinity.

[0115] To further investigate the inhibitory ability of the complex (IC50) 50 ) and inhibition kinetics (K ic / K iu The intrinsic relationship between them was analyzed using linear regression to quantify the degree of their correlation. Figure 17 ).Depend on Figure 17 It can be seen that the IC50 values ​​of the four complexes for α-glucosidase and α-amylase are... 50 Value and K ic and K iu The values ​​show a significant linear positive correlation, R 2The values ​​were 0.9851 / 0.9821 (α-glucosidase); 0.9988 and 0.9940 (α-amylase), respectively, indicating that the correlation between the two is highly reliable. The inhibitory strength of the complex against α-glucosidase and α-amylase is closely quantitatively correlated with the inhibitor-enzyme binding affinity: IC50 50 The lower the value, the better K ic and K iu The smaller the value, the stronger the inhibitory ability and the higher the binding affinity of the complex. This confirms from a kinetic perspective that the inhibitory effect of the C3G-protein complex mainly depends on its binding ability to the enzyme's active site or allosteric site.

[0116] 3.3 Mechanism of fluorescence quenching of amylase by C3G-barley protein complex Protein molecules contain residues such as tryptophan, phenylalanine, and tyrosine (predominantly tryptophan), and can produce endogenous fluorescence. To elucidate the inhibitory mechanism of the C3G-protein complex on α-glucosidase and α-amylase at the molecular level, fluorescence quenching experiments were further conducted. By measuring the changes in fluorescence intensity before and after the interaction of the complex with the enzymes, fluorescence quenching curves of four complexes on the two enzymes were obtained. Figure 18 ); Plot the Stern-Volmer curve ( Figure 19 And calculate the quenching constant (K) FQ ), quenching rate constant (K) q ) and parameters such as quenching type (Table 9).

[0117] Table 9 shows that the quenching effects of the four complexes on the enzymes were: C3G-Alb (46.168 mg / mL), C3G-Glo (41.081 mg / mL), C3G-Gli (26.919 mg / mL), and C3G-Glu (2.443 mg / mL). The results showed that the intrinsic fluorescence intensity of both enzymes decreased in a concentration-dependent manner with increasing complex concentration. KFQ was obtained through linear fitting, and the expression Kq = K... sv / τ0 (τ0 is the average fluorescence lifetime of biological macromolecules, approximately 10) -8 K calculated by s) q The values ​​are all higher than the maximum diffusion rate constant for dynamic quenching (approximately 2.0 × 10⁻⁶). 10 L·mol -1 ·s -1 This confirms that the fluorescence quenching type is static quenching.

[0118] A larger quenching constant indicates a stronger quenching effect of the quencher on the enzyme. The quenching effect of the complexes is C3G-Alb > C3G-Glo > C3G-Gli > C3G-Glu. All four complexes exhibit static quenching, meaning that the complexes and enzyme molecules form stable ground-state complexes through non-covalent interactions such as hydrogen bonds, hydrophobic interactions, and van der Waals forces. As the complex concentration increases, the maximum fluorescence emission wavelength of the enzyme exhibits a blue shift, suggesting changes in the microenvironment of fluorophores such as tryptophan residues or energy transfer, leading to a decrease in fluorescence intensity. This directly confirms the existence of specific molecular binding between the complexes and the enzyme, rather than simple physical collisions.

[0119] Table 9. Fluorescence quenching parameters of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu for BYG and AMY

[0120] Note: Data are mean ± standard deviation (n=3); K FQ K is the quenching constant. q This is the quenching rate constant.

[0121] Figure 20 The quenching constants (Ka) of four complexes on amylase digestive enzymes. FQ The correlation analysis between α-glucosidase and the competitive association constant (1 / Kic) showed a significant linear positive correlation. The R-values ​​for α-glucosidase and α-amylase were also analyzed. 2 The values ​​were 0.9766 and 0.9768, respectively, indicating that the fluorescence quenching properties and inhibition kinetics can synergistically explain the inhibition mechanism.

[0122] K FQ The higher the fluorescence intensity, the stronger the static binding of the complex to the enzyme, and the stronger its binding ability to the enzyme's active site. This correlation provides spectroscopic evidence for the inhibition kinetics. The core mechanism is that fluorescence quenching reflects the specific binding of the complex to the enzyme's active site and surrounding regions. This binding can hinder substrate binding and may induce enzyme conformational changes, thereby regulating enzyme catalytic activity, consistent with the previous conclusions.

[0123] 3.4 Analysis of C3G-protein complex docking with amylase molecules To further elucidate the molecular mechanism by which the C3G-protein complex inhibits α-glucosidase and α-amylase, a stepwise docking strategy was employed to simulate the binding modes of four complexes (C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu) to the two enzymes. Figure 21 This serves as an exploratory structural prediction. The results show that the C3G-protein complex can specifically interact with α-glucosidase and α-amylase through hydrogen bonds.

[0124] Specifically, when docking with α-glucosidase, C3G-Alb can bind key amino acids such as GLU355, GLU322, and LYS358 around its active site; C3G-Glo can bind active amino acids such as THR173, LYS212, and ASN210; C3G-Gli can bind active amino acids such as TYR115, ASN61, and ASP47; and C3G-Glu can bind active amino acids such as TYR361, ARG370, and GLU355. When docked with α-amylase, C3G-Alb can bind active amino acids such as ARG389, CYS378, and TRP382; C3G-Glo can bind active amino acids such as TRP357, ASN53, and SER108; C3G-Gli can bind active amino acids such as ASN364, TYR52, and LYS368; and C3G-Glu can bind active amino acids such as THR143, SER145, and GLU149.

[0125] Table 10 shows the molecular docking simulation parameters, indicating that all C3G-protein complexes could stably bind to the active pockets and entry points of both enzymes. The binding free energies of the four complexes for α-glucosidase were ranked as follows: C3G-Alb (-9.1 Kcal / mol) > C3G-Glo (-8.9 Kcal / mol) > C3G-Gli (-8.4 Kcal / mol) > C3G-Glu (-7.6 Kcal / mol); and for α-amylase, the ranking was: C3G-Alb (-8.8 Kcal / mol) > C3G-Glo (-8.6 Kcal / mol) > C3G-Gli (-8.1 Kcal / mol) > C3G-Glu (-7.4 Kcal / mol). This ranking corresponds to the experimentally measured IC50 values. 50 Value and inhibition kinetic binding constant (K) ic / K iu The identical sequencing provides a possible molecular-level explanation for the differences in the inhibitory effects of the complexes.

[0126] Table 10 Molecular docking parameters of C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu for BYG and AMY

[0127] It is important to emphasize that the above molecular docking results are only exploratory structural predictions. Since the binding between C3G and the protein is non-covalent and dynamic, this study rigidified the pre-docked C3G-protein complex before docking with the enzyme, neglecting potential conformational rearrangements and induced fit effects during enzyme binding. Furthermore, in real solutions, various complex binding modes may exist among C3G, protein, and enzyme (e.g., C3G binds to the enzyme first, then the protein; or vice versa), and stepwise docking cannot fully reflect these dynamic equilibria. Therefore, Figure 21 The demonstrated binding mode should be considered as one of several possible conformations, and the ranking of binding free energies needs to be combined with experimental data (IC). 50 K ic / K iu K FQ A comprehensive assessment is needed. In the future, molecular dynamics simulations could be considered to study the dynamic binding process of ternary complexes under flexible conditions, in order to obtain a more reliable understanding of the molecular mechanisms.

[0128] Example 5: Interaction between C3G and barley protein 1. Experimental Materials and Instruments C3G (purity ≥95%): Qinghai Lanfeng Biotechnology Co., Ltd.; Albumin (purity 80%), Globulin (purity 85%), Glycol (purity 85%), Gluten (purity 85%), Dialysis Bag (MWCO 6000 Da): Shanghai Yuanye Biotechnology Co., Ltd.; Artificial Small Intestinal Fluid, Artificial Gastric Fluid: Regen Biotechnology Co., Ltd.; Potassium Chloride (KCl), Analytical Grade: Tianjin Tianli Chemical Reagent Co., Ltd.

[0129] HH-4 type constant temperature magnetic stirring water bath: Changzhou Tianrui Instrument Co., Ltd.; LabXXRD-6100 type X-ray diffractometer, IR-Affinity-1 type Fourier transform infrared spectrometer: Shimadzu Corporation, Japan; Discovery X3 type differential scanning calorimeter (DSC): TA Instruments, USA; BioTek EPOCH2 microplate reader: Agilent Technologies, USA; Biacore T200 instrument: Cytiva, USA.

[0130] 2. Experimental Methods 2.1 Structural Characterization of the C3G-Barley Protein Complex (1) XDR characterization of the C3G-barley protein complex Preparation of C3G-protein complexes: Weigh 2 mg of each of the four proteins (albumin, globulin, prolyl, and glutenin) and an equal mass of C3G, and dissolve them in 5 mL of 0.01 mol / L PBS buffer (pH=7.0). Mix and shake in the dark for 2 h (i.e., at 4°C and 120 r / min, magnetically stirred in the dark for 2 h). Add the mixture to a MWCO 6000 Da dialysis bag and dialyze in 0.01 mol / L PBS buffer (pH=7.0) in the dark for 4 h. Collect the solution in the bag and freeze-dry it to obtain the four C3G-protein complexes.

[0131] Take an appropriate amount of C3G-protein complex sample and place it in the sample holder. After thorough grinding, flatten it and measure it under the conditions of 40 kV and 30 mA. The diffraction angle (2θ) range is 5°~45°, the scanning speed is 4° / min, and the step size is 0.05°.

[0132] (2) DSC characterization of the C3G-barley protein complex The C3G-protein complex was prepared according to method (1). 5 mg of sample was accurately weighed and placed in a DSC aluminum crucible, which was sealed with a perforated aluminum cap. An empty aluminum crucible of the same specification was used as a blank reference. The temperature was then programmed to rise from 20 °C to 250 °C at a rate of 10 °C / min. The DSC thermogram was obtained by recording the correspondence between the heat flow signal (y-axis) and the temperature (x-axis).

[0133] (3) FITR characterization of the C3G-barley protein complex The C3G-protein complex was prepared according to method (1). Fourier transform infrared spectroscopy was used to determine the lyophilized C3G, the single protein, and the C3G-protein complex. Sample processing employed the potassium bromide pelleting method. 5 mg of sample was accurately weighed and mixed with potassium bromide at a ratio of 1:50. After thorough grinding and uniform mixing, the mixture was pelleted. The detection wavenumber range was 400-40000 cm⁻¹. -1 The resolution is 4cm. -1 .

[0134] (4) SPR analysis of the C3G-barley protein complex The molecular interactions between C3G and target proteins (albumin, globulin, prolysin, and glutenin) were quantitatively analyzed using a Biacore T200 instrument. Purified proteins were used as ligands and immobilized on the surface of a Series SCMS sensor chip using a standard amine coupling chemical method. Using the C3G-protein complex as the analyte, a 0-100 μM concentration gradient was prepared and dissolved in HBS-EP+ running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P2O, pH 7.4), and injected into the chip surface at a flow rate of 30 μL / min. The binding and dissociation processes were monitored in real time. After each run, the sensor surface was regenerated using 10 mM glycine-HCl buffer (pH 2.0). The obtained sensor maps were subjected to dual-reference subtraction using Biacore evaluation software and fitted to a 1:1 Langmuir binding model to calculate the binding rate constant (k). a ), dissociation rate constant (K) d and equilibrium dissociation constant (K) D Dynamic parameters such as )

[0135] (5) Molecular docking simulation of C3G-barley protein complex To elucidate the binding mechanism of C3G with albumin, globulin, prolysin, and glutenin, molecular docking was performed using AutoDock 4.2.6 software. The three-dimensional structure preprocessing of C3G was the same as before. The crystal structures of the four proteins (PDB IDs as before) were processed using PyMOL 2.5.0 to remove water molecules, add hydrogen, and assign Gasteiger charges. Then, AutoDock Tools 1.5.6 was used to define docking boxes covering the entire protein structure (box size 80×80×80 Å, grid spacing 0.5 Å). Docking results were sorted according to binding free energy, and the lowest energy conformation was selected as the final binding mode. LigPlot+v.2.2 and Discovery Studio 2019 were used to analyze hydrogen bonding, hydrophobic interactions, π-π stacking, and other interaction types, and the equilibrium dissociation constant (K0) was calculated. D ).

[0136] 2.2 Effect of processing conditions on the stability of C3G-barley protein complex (1) Effect of ion concentration on the stability of C3G-barley protein complex Weigh 5 mg of C3G and an equal volume of protein, dissolve in 5 mL of buffer, mix well, and incubate at 4°C for 2 h (4°C, 120 rpm, magnetic stirring in the dark for 2 h). Then transfer to dialysis bags and place in dialysis buffers containing 1%, 5%, and 10% NaCl, respectively. Use 0.01 mol / L PBS buffer (pH 7.0) for dialysis, and magnetically stir for 4 h. Measure the C3G content inside and outside the dialysis bag using a microplate reader at 520 nm and 700 nm wavelengths, respectively. Calculate the protein adsorption rate for C3G after subtracting the blank background.

[0137] (2) Effect of temperature on the stability of C3G-barley protein complex 5 mg of C3G and 5 mg of protein solution were dissolved in 5 mL of buffer (0.01 mol / L PBS buffer at pH 7.0) and mixed thoroughly. The solution was then placed at 4 °C for 2 h (4 °C, 120 r / min, magnetic stirring in the dark for 2 h). After dialyzing with 0.01 mol / L PBS buffer at pH 7.0 for 4 h at temperatures of 4, 25, and 55 °C, the C3G content inside and outside the dialysis bag was measured. The adsorption rate of protein on C3G was calculated after subtracting the blank background.

[0138] (3) Effect of pH on the stability of C3G-barley protein complex 5 mg of C3G and 5 mg of protein solution were dissolved in 5 mL of buffer (0.01 mol / L PBS buffer at pH 7.0) and mixed thoroughly. The solution was then placed at 4°C for 2 h (4°C, 120 r / min, magnetic stirring in the dark for 2 h). After dialyzing with 0.01 mol / L PBS buffer for 4 h at pH 2, 7, and 9, the C3G content inside and outside the dialysis bag was measured. The adsorption rate of protein on C3G was calculated after subtracting the blank background.

[0139] 2.3 Effects of the gastrointestinal digestive environment on the stability of the C3G-barley protein complex Preparation of C3G-protein complex: 10 mg of C3G and 10 mg of target protein were dissolved in 10 mL of 0.01 mol / L PBS buffer (pH 7.4), respectively. The mixture was then incubated at 4°C in the dark on a shaker (120 r / min) for 2 h. The mixture was then transferred to a 3500 Da dialysis bag and dialyzed with PBS of the same specification for 4 h (with one buffer change). After freeze-drying (-50°C, 0.01 MPa, 48 h), the complex powder was obtained and stored at 4°C in the dark. For subsequent experiments, the complex solution was prepared by dissolving the complex powder in PBS of the same specification.

[0140] In vitro gastric digestion simulation: 5 mL of a 5 mg / mL complex solution was mixed with 5 mL of fresh artificial gastric juice (containing 0.23 g / L pepsin, 1.62 g / L NaCl, pH=1.2), placed in a 3500 Da dialysis bag, and then placed in 400 mL of preheated (37℃) pH=1.2 PBS release medium. The mixture was incubated at 37℃ in the dark with shaking at 120 r / min for 1, 2, and 4 hours. At each time point, the solutions inside and outside the bag were centrifuged, and the C3G content was measured. The C3G retention rate was calculated using the following formula: ; Where: C0: the concentration of C3G in the dialysis bag at the initial stage of digestion (t=0) (mg / mL or μg / mL); C in : Concentration of free C3G in the dialysis bag after digestion time t (mg / mL or μg / mL); C out : The concentration of unbound C3G outside the dialysis bag after digestion time t (mg / mL or μg / mL).

[0141] In vitro enteric digestion simulation: Take 5 mL of the 5 mg / mL complex solution and mix it with 5 mL of fresh artificial intestinal fluid (containing 1.0 g / L trypsin, 6.8 g / L KH2PO4, pH=7.5). Place the mixture into a 3500 Da dialysis bag and put it into 400 mL of PBS release medium preheated to 37℃ (pH=7.5). Incubate at 37℃ in the dark with constant temperature shaking (120 r / min) for 1, 2, and 4 h. At each time point, take the solution inside and outside the bag, centrifuge it, and determine the C3G content. Calculate the C3G retention rate using the same formula as above.

[0142] All the above experiments were set up with 3 independent replicates, each using a separate reagent, a separately prepared complex, and a separate digestion system, with consistent operating conditions. The experimental results are expressed as the mean ± standard deviation of the 3 sets of data to ensure that the results are accurate and reliable.

[0143] 2.3 Data Processing All experiments were performed in triplicate, and results are expressed as mean ± standard deviation. Excel was used for data processing, and OriginPro 2024 and GraphPad Prism software were used for curve fitting and graph plotting.

[0144] 3. Results 3.1 Structural Feature Analysis of the C3G-Barley Protein Complex (1) XRD characterization of the C3G-barley protein complex To elucidate the interaction mechanism between C3G and albumin, globulin, prolysin, and glutenin, XRD was used to analyze each component, and the results are as follows: Figure 22 As shown. By Figure 22As seen in A, albumin, globulin, prolysin, and glutenin all exhibit characteristic peaks at 2θ of 10° and 20°. When C3G forms a complex with the protein ( Figure 22 (B) , sharp diffraction peaks appear near 2θ of 30° and 45°.

[0145] XRD crystallinity reflects the degree of ordered arrangement of a substance; higher crystallinity indicates a more ordered structural arrangement. In the XRD patterns, the diffraction peaks at 2θ of 10° and 20° correspond to the α-helix and β-sheet structures of proteins, indicating that α-helices and β-sheets exist in the secondary structures of all four proteins, with β-sheets being the predominant structure. Among them, prolysins and glutenins show even higher diffraction peaks at 2θ≈20°, which is a typical characteristic of partially crystalline or amorphous protein structures, suggesting that although their molecular chains exhibit some order, they are predominantly amorphous, and their spatial structure is mainly maintained by hydrogen bonds and hydrophobic interactions. C3G, on the other hand, shows multiple sharp and high-intensity diffraction peaks in the 2θ range of 10°-20°, indicating that C3G is a highly crystalline substance, with its molecules forming a regular and ordered crystal structure through strong hydrogen bonds and π-π stacking interactions.

[0146] After the complex was formed, sharp diffraction peaks appeared near 30° and 45° at 2θ. This is presumably because C3G is dispersed amorphously within the complex matrix, leading to a reconstruction of the ordered structure and the formation of new crystalline phases or ordered aggregates. Furthermore, the phenolic hydroxyl groups, glycosidic bonds, and aromatic rings in the C3G molecule can form hydrogen bonds or hydrophobic interactions with hydrophobic amino acid residues such as phenylalanine and leucine in the protein molecule, thereby driving structural changes and enhancing intermolecular stability. Notably, the diffraction peaks of the C3G complexes with different proteins show subtle differences, reflecting a close relationship between the protein molecular structure, spatial conformation, and amino acid composition and the complex's structural characteristics. The increased structural order of the C3G-protein complex can reduce the impact of environmental factors such as oxidation and degradation on C3G, improving its stability in food or biological systems; simultaneously, changes in the crystallinity of the complex also regulate the functional properties of the protein.

[0147] (2) DSC characterization of the C3G-barley protein complex The DSC parameters of Alb, Glo, Gli, Glu and their complexes are shown in Table 11.

[0148] Table 11 DSC parameters of Alb, Glo, Gli, Glu and their complexes

[0149] DSC, as a classic method for characterizing the thermal denaturation behavior of materials, can be obtained by measuring the denaturation temperature (T). dKey parameters such as denaturation enthalpy (ΔH) directly reflect the changes in the thermal stability of C3G and proteins during their interaction.

[0150] like Figure 23 As shown in Table 11, all single proteins exhibit a distinct broad denaturation endothermic peak within the temperature range of 50-90℃, with the prolysin showing the highest T peak. d The highest T value indicates the strongest thermal stability; glutenin's T... d The lowest value indicates a relatively loose spatial structure. When the protein binds to C3G to form a complex, the protein's denaturation endothermic peak changes significantly, T... d The overall trend is downward, indicating that the binding of C3G to proteins can perturb the protein conformation and reduce its thermal stability. From the perspective of denaturation enthalpy (ΔH), the absolute values ​​of ΔH for C3G-Alb, C3G-Glo, and C3G-Gli are significantly increased, suggesting that the above three proteins interact with C3G mainly through a mild surface binding mode, forming a relatively stable complex. Conversely, the ΔH of C3G-Glu is slightly decreased, indicating that C3G has the most significant perturbation effect on the structure of glutenin, and the degree of binding between the two is deeper. The strong interaction disrupts the hydrogen bond and hydrophobic interaction network inside glutenin, leading to a decrease in its structural stability.

[0151] (3) FTIR characterization of the C3G-barley protein complex FTIR spectra of C3G with Alb, Glo, Gli, Glu and their complexes are shown below. Figure 24 As shown.

[0152] FTIR can visually reflect the functional group environment and chemical bond interactions of molecules through changes in the position, intensity, and shape of characteristic absorption peaks, thereby elucidating the binding mode of C3G to proteins and the impact of their interaction on protein secondary structure. The characteristic FTIR absorption peaks of proteins are mainly concentrated in the amide I band (1600-1700 cm⁻¹). -1 ), Amide II band (1500-1600 cm) -1 ) and amide III band (1200-1300 cm) -1 The amide I band corresponds to the C=O stretching vibration of the peptide bond, and its peak position and shape changes directly reflect the protein's secondary structure (α-helix: 1650-1660 cm⁻¹). -1 β-fold: 1620–1640 cm -1 The change of amide band (I-I) is the core characteristic band for characterizing protein conformational changes; the amide II band originates from the coupling of NH bending vibration and CN stretching vibration, and its intensity change can reflect the hydrogen bonding between protein molecules; the amide III band is related to CN stretching vibration and NH bending vibration, and can help characterize changes in protein secondary structure.

[0153] Figure 24The FTIR spectra of C3G, albumin, globulin, prolysin, glutenin, and their corresponding C3G-protein complexes are presented. By comparing the differences in characteristic absorption peaks of each component, the interaction mechanism between C3G and the four proteins can be elucidated. Figure 24 It can be seen that C3G is between 3200-3600 cm. -1 A broad and strong absorption peak appeared at the amide I band, and all four proteins showed obvious characteristic absorption peaks: albumin (1636 cm⁻¹). -1 ), globulin (1630 cm) -1 ), prolysin (1632 cm) -1 ), gluten (1636 cm) -1 Its β-sheet structure accounts for a higher proportion. After C3G forms complexes with four proteins, the FTIR spectra change: C3G shows changes in the 3200-3600 cm⁻¹ range. -1 The intensity of the characteristic absorption peak of the phenolic hydroxyl group at the position was significantly reduced and the peak position was slightly shifted.

[0154] The C3G absorption peak corresponds to the stretching vibration of the phenolic hydroxyl group (-OH), indicating the presence of numerous hydrogen bonds between its molecules. The amide I bands of the four proteins all showed varying degrees of shift, indicating a change in the proportion of β-sheet structures, suggesting that C3G binding to the proteins affects their secondary structures. After C3G formed complexes with the four proteins, the FTIR spectra changed: C3G showed changes in the 3200-3600 cm⁻¹ range. -1 The intensity of the characteristic absorption peak of the phenolic hydroxyl group at the phenolic hydroxyl group was significantly reduced and the peak position was slightly shifted, indicating that the phenolic hydroxyl group formed new hydrogen bonds with the amino and hydroxyl groups in the protein molecule, resulting in a weakening of the intermolecular hydrogen bonding. In addition, the absorption peak intensities of the amide II band of the complex were all reduced, suggesting that the binding of C3G to the protein disrupted the internal hydrogen bond network of the protein and weakened the coupling effect of NH bending vibration and CN stretching vibration, further confirming the existence of a specific interaction between the two.

[0155] The FTIR characteristic peak changes of different C3G-protein complexes showed subtle differences: the amide I band of C3G-Glu showed the largest shift, and the characteristic absorption peak of C3G was weakened the most significantly, indicating that glutenin and C3G are most deeply bound, which corresponds to the result of the previous DSC analysis that C3G has the most significant perturbation on the glutenin structure; this indicates that there are differences in the interaction mode and strength between C3G and the four proteins, and this difference is closely related to the protein molecular structure, spatial conformation and amino acid composition.

[0156] (4) SPR analysis of C3G-barley protein complex The dynamic binding process of C3G to protein molecules was quantitatively characterized in real time using surface plasmon resonance (SPR) experiments. Figure 25It can be seen that albumin, globulin, and prolysin exhibit rapid binding and dissociation characteristics after C3G injection, consistent with a typical reversible binding pattern; however, the binding behavior of glutenin with C3G differs somewhat from the other three proteins. Figure 25 When G and H are injected into C3G, the response signal rises slowly, taking a long time to reach the plateau phase; during the dissociation phase, the signal drops rapidly, and the dissociation curves at different concentrations exhibit non-overlapping characteristics, indicating that the binding process may involve multi-site interactions or be accompanied by conformational changes. Its K... D The values ​​were: albumin (9.16±0.52)×10 -7 M, glutenin (1.70±0.34)×10 -6 M, globulin (2.15±0.31)×10 -6 M, prolysin (5.82±0.43)×10 -6 M, the affinity order is albumin ≈ glutenin > globulin > prolysin, which is basically consistent with the previous experimental results.

[0157] It is worth noting that the effect of gluten in the SPR results is slightly different from that in the previous article. This may be because: (1) The binding mode is different: DSC and FTIR detect the overall interaction (including hydrogen bonding, hydrophobic interaction, etc.) after C3G is mixed with gluten, which reflects the fact of complex formation; while SPR detects the dynamic binding-dissociation process, which is more sensitive to the binding rate. As a high molecular weight aggregate protein (molecular weight 35-40 kDa, and easy to form multimers), the binding of gluten with C3G may involve a complex process involving multiple sites and multiple stages. (2) The distinction between binding strength and stability: DSC and FTIR confirm that C3G and gluten can form a complex and change the protein conformation, but the SPR results show that this binding may occur in a multi-site, low-affinity manner, and dissociate rapidly after binding, with low overall binding stability. (3) Immobilization effect: In the SPR experiment, gluten is immobilized on the chip surface by amine coupling, which may change its native conformation or block some binding sites, while DSC and FTIR are measured in solution, which can better reflect the native state of the protein. The above analysis suggests that the interaction between C3G and glutenin may be a "multi-mode binding," which can be further verified in the future using methods such as steady-state fluorescence titration and isothermal titration calorimetry (ITC).

[0158] Table 12 Surface plasmon resonance parameters of C3G with Alb, Glo, Gli, and Glu, respectively

[0159] Note: K D The value is the equilibrium dissociation constant; the smaller the value, the higher the affinity.

[0160] (5) Molecular docking simulation of C3G-barley protein complex Molecular docking simulations were performed to elucidate the binding mechanism of C3G to proteins, and the results are as follows: Figure 26 As shown, C3G is embedded in the hydrophobic pocket of albumin, mainly through synergistic binding via hydrogen bonds (CYS48), π-σ interactions (ILE85, LEU51, VAL47), and π-alkyl interactions (LYS52, ILE81, VAL77); the binding of C3G-Glo includes hydrogen bonds (ASP65, ASN82, SER18), π-σ interactions (MEI139, LEU40, LEU171), π-π stacking (PHE67), and π-alkyl interactions (LEU34, MEI107, ILE141); C3G-Gli mainly relies on hydrogen bonds (GLU1, ASP116, GLY119, GLN120, LEU4) and π-alkyl interactions (ARG51); C3G-Glu is bound through hydrogen bonds (GLN38, GLY108) and π-σ interactions (VAL89). The predicted binding free energy (ΔG) ordering based on molecular docking is: C3G-Glu (-8.4 Kcal / mol) > C3G-Gli (-8.1 Kcal / mol) > C3G-Glo (-7.7 Kcal / mol) > C3G-Alb (-7.1 Kcal / mol). This order differs from the affinity ordering determined by SPR experiments (albumin ≈ glutenin > globulin > prolamins).

[0161] The possible reasons for this difference are: (1) The difference between molecular docking and SPR detection principles: Molecular docking calculates the binding free energy of the thermodynamically most stable static binding conformation, reflecting the "theoretical maximum affinity"; while SPR measures the comprehensive performance of the dynamic binding-dissociation process (including binding rate and dissociation rate), reflecting the "actually achievable affinity". For albumin, although the static binding free energy prediction is low, its high rapid binding rate and moderate dissociation rate make it exhibit the highest net affinity in dynamic equilibrium. (2) The difference in protein structure source: The crystal structure used for docking comes from different species: albumin (maize PDB: 1MID), globulin (pea PDB: 6PYQ), prolysin (wheat PDB: 5IFJ), and glutenin (rice PDB: 6PX6), which have different amino acid sequence homology with barley protein (albumin homology is about 85%, and glutenin homology is about 72%), which may lead to a systematic deviation in the prediction of binding free energy. (3) Specific aggregation behavior of gluten: Molecular docking simulates the binding of monomeric gluten to C3G, while gluten exists in an immobilized form in the SPR experiment, which may form aggregates or undergo conformational changes. In solution, gluten easily forms polymers through intermolecular disulfide bonds and hydrophobic interactions, resulting in the embedding of some C3G binding sites or increased steric hindrance, and the actual binding affinity is lower than the predicted value of the monomeric state. This explanation is consistent with the observation in Chapter 3 that the inhibition rate decreases due to self-aggregation of gluten at high concentrations. (4) Limitations of docking algorithm: The AutoDock scoring function has different adaptability to different protein structures. The binding of albumin to C3G mainly depends on hydrophobic interactions (π-σ, π-alkyl), while gluten mainly depends on hydrogen bonds. The scoring function may overestimate the contribution of hydrogen bonds and underestimate the contribution of hydrophobic interactions, resulting in a lower (more negative) predicted value of gluten binding free energy. In summary, the discrepancies between molecular docking and SPR results primarily stem from the fundamental difference between static theoretical predictions and dynamic experimental measurements, as well as the combined influence of multiple factors such as protein structure origin, aggregation state, and algorithm adaptability. The results of the two methods should be considered complementary rather than contradictory: molecular docking reveals potential binding sites and force types, while SPR reflects actual dynamic binding behavior. Future research could employ molecular dynamics simulations to examine the dynamic binding process of complexes under flexible conditions, thereby gaining a more comprehensive understanding of the molecular mechanisms.

[0162] (6) Integration analysis of results from multiple methods Based on the combined results of XRD, DSC, FTIR, SPR, and molecular docking, the interaction between C3G and four types of barley proteins exhibits the following characteristics: (1) Albumin: The results from multiple methods are highly consistent, showing high affinity and rapid reversible binding, mainly forming stable complexes through hydrogen bonds and hydrophobic interactions; (2) Globulins and prolysins: The binding affinity is moderate, and the interaction strength is between that of albumin and glutenin; (3) Glutenin: XRD, DSC, and FTIR all show that there is a clear interaction between C3G and glutenin. After the complex is formed, the crystal structure changes, the thermal stability decreases, the secondary structure rearranges, and the binding mode is rapid dissociation. This incomplete consistency of results from multiple methods precisely reflects the complexity of the interaction between glutenin and C3G: As a high molecular weight aggregate protein, the binding of glutenin to C3G may involve a multi-site, multi-stage synergistic process, rather than a simple single-site reversible binding. Therefore, when analyzing results related to glutenin, it is necessary to synthesize evidence from multiple methods to avoid the one-sidedness of conclusions from a single method.

[0163] 3.2 Effect of processing conditions on the stability of C3G-protein complex The effects of different environmental conditions (NaCl, temperature, pH) on the stability of four C3G-protein complexes (C3G-Alb, C3G-Glo, C3G-Gli, and C3G-Glu) were investigated. The results are as follows: Figure 27 As shown.

[0164] The effect of NaCl concentration (1%, 5%, 10%) on the stability of the complex is as follows: Figure 27 As shown in Figure A, the retention rate of C3G in all four complexes gradually decreased with increasing NaCl concentration. At the same concentration, the adsorption capacity of the four proteins for C3G showed significant differences (P<0.05), with albumin showing the best adsorption and glutenin the worst. At 1% NaCl, C3G-Alb had the highest retention rate (38.36%), while C3G-Glu had the lowest (21.86%). At 10% NaCl concentration, the retention rate of C3G-Alb dropped to around 10%, and C3G-Glu dropped below 5%. High ionic strength weakens the binding of C3G to proteins, possibly because NaCl... + Cl - The shielding of the electrostatic interaction between the two affects the arrangement of water molecules and weakens the hydrophobic interaction, making it difficult to maintain the binding site.

[0165] The effect of temperature on the stability of the complex is as follows: Figure 27As shown in Figure B, within the temperature range of 4-55℃, the stability of the complex continuously decreased with increasing temperature, and there were significant differences in the adsorption of C3G by the four proteins at the same temperature (P<0.05). The retention rate of C3G by the protein at 4℃ was significantly higher than that at 25℃ and 55℃. This indicates that high temperature leads to irreversible denaturation of the protein, degradation of polyphenols, and causes the expansion of the protein's secondary and tertiary structures, altering the binding sites and reducing the binding capacity.

[0166] The effect of pH (2, 7, 9) on the stability of the complex is as follows: Figure 27 As shown in Figure C, there were significant differences in the adsorption of C3G by the four proteins at the same pH (P<0.05). The adsorption rate of the complex was relatively high under acidic conditions, and gradually decreased with increasing pH. This may be because hydrophobic interactions are dominant and electrostatic repulsion is minimal near the isoelectric point of the protein under acidic conditions, while increasing pH changes the charge state and structure of the protein, enhancing the electrostatic repulsion between C3G and the protein, weakening hydrogen bonding, and reducing the adsorption rate.

[0167] In summary, the C3G-Alb complex exhibits better stability than the other three under different environmental conditions. Low ionic strength, low temperature, and weakly acidic or neutral conditions are more conducive to maintaining the stability of the C3G-protein complex.

[0168] 3.3 Stability of C3G-protein complex and C3G release characteristics during gastrointestinal digestion To assess the bioavailability of the C3G-protein complex in the gastric and small intestinal environments, an in vitro simulated digestion model was used to analyze the changes in C3G content after 1, 2, and 4 hours of digestion. The results are as follows: Figure 28 As shown.

[0169] Depend on Figure 28 As shown in Figure A, there were significant differences in the C3G retention rates of the four C3G-protein complexes in the gastric environment (P<0.05). The highest retention rate was observed at 1 hour of digestion, indicating that the proteins provided the strongest protection for C3G in the early stages of gastric digestion. With prolonged digestion, the retention rate gradually decreased, and at 4 hours, the retention rates of all four complexes were below 10%. This is mainly due to the strong acidity of gastric juice inducing conformational changes in the proteins, disrupting hydrogen bonds and hydrophobic interactions, and the pepsin hydrolyzing the protein backbone and destroying the complex binding sites, leading to C3G exposure and degradation.

[0170] Depend on Figure 28 As shown in Figure B, the retention rate of the C3G complex also exhibited significant differences in the simulated small intestinal environment (P<0.05), and the trend was consistent with that in the stomach (decreasing over time), but the retention rate was lower in the small intestine. The C3G-Alb retention rate was higher than the other three, indicating that the protein's retention of C3G is related to the protein type. Furthermore, some proteins are broken down during gastric digestion, and trypsin in the small intestine further hydrolyzes proteins, further disrupting the complex structure and accelerating the release of C3G.

[0171] Example 6 A composite composition, by weight, comprising cornflower-3- O - 1 part glucoside and 1 part barley prolysin.

[0172] Cornflower-3- O The preparation method of the composite composition using glucoside and barley prolysin is as follows: C3G pure product and barley prolysin pure product are dissolved separately in 0.01 mol / L PBS buffer (pH=7.0) to prepare a 1 mg / mL solution. 100 mL of each solution is mixed and magnetically stirred at 120 r / min in the dark for 2 h at 4℃ to allow for complete self-assembly through non-covalent interactions, resulting in a composite solution. The composite solution is transferred to a MWCO 6000 Da dialysis bag and dialyzed at 4℃ for 4 h with PBS buffer (pH=2.0, 0.01 mol / L) containing 1% NaCl to remove free ions and unbound small molecules. The composite solution in the bag is collected and freeze-dried at -50℃ for 48 h to obtain the composite composition powder.

[0173] Example 7 A composite composition, by weight, comprising cornflower-3- O - 1 part glucoside and 1 part glutenin. Preparation method is the same as in Example 6.

[0174] Example 8 A composite composition, by weight, comprising cornflower-3- O - 1 part glucosinolate and 1 part globulin. Preparation method is the same as in Example 6.

[0175] Example 9 A composite composition, by weight, comprising cornflower-3- O - 1 part glucoside and 1 part albumin. Preparation method is the same as in Example 6.

[0176] Example 10 Application of the composite composition in meal replacement powder in Example 6 A hypoglycemic meal replacement powder, by weight percentage, comprises: 10% of the composite composition of Example 6, 60% highland barley flour, 20% oat flour, 5% inulin, and 5% xylitol; all raw materials are mixed evenly, passed through an 80-mesh sieve, and packaged into 20g / bags to obtain the hypoglycemic meal replacement powder. This meal replacement powder has good mixing properties and a pleasant taste. After consumption, it can effectively delay starch digestion and reduce postprandial blood sugar fluctuations.

[0177] Example 11 Application of the composite composition in hard capsules in Example 6 A blood sugar-lowering capsule is formulated as follows: the composite composition powder of Example 6 is mixed evenly with microcrystalline cellulose at a mass ratio of 2:1, and then filled into hard capsules using a capsule filling machine. Each capsule contains 200mg of the composite composition, thus obtaining a blood sugar-lowering capsule. This capsule is convenient to take, has high bioavailability, and is suitable for daily health maintenance by people with high blood sugar.

[0178] Comparative Example 1 Unreconstituted free C3G (purity ≥95%) was placed under different environmental conditions (ionic strength: 1%, 5%, 10% NaCl; temperature: 4℃, 25℃, 55℃; pH: 2, 7, 9) and dialyzed with 0.01mol / L PBS buffer for 4h. The retention rate of C3G was determined by HPLC, and the results are shown in Table 13.

[0179] Table 13 Retention rate of C3G under different environmental conditions (%)

[0180] In summary, the addition of barley protein to the composite composition provided by this invention can significantly improve cornflower-3- O - The stability of glucosinolates and the retention rate in gastrointestinal fluids, the composite composition through barley protein and cornflower-3- O α-Glucosides can significantly inhibit α-glucosidase activity and reduce the IC50 of α-glucosidase through synergistic effects. 50 It has a value that can synergistically enhance the effect of lowering blood sugar.

[0181] The composite composition provided by this invention is a hypoglycemic composition of berry anthocyanins and barley protein. The raw materials are widely available, the preparation process is simple, green and environmentally friendly, and no complicated equipment is required, which can realize large-scale industrial production. The composition has a significant effect on inhibiting the activity of starch digestive enzymes, has high stability and bioavailability, and is safe for consumption. It can be added to a variety of functional foods and health products, and has good industrial application value and market prospects.

[0182] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composite composition, characterized in that, By weight, it includes: cornflower-3- O - 1 part glucoside and 0.5-2 parts barley protein; the barley protein includes any one of globulin, albumin, glutenin and prolysin.

2. The composite composition according to claim 1, characterized in that, By weight, the composite composition comprises: cornflower-3- O - 1 part glucosinolate and 1 part barley protein.

3. The method for preparing the composite composition according to claim 1 or 2, characterized in that, Includes the following steps: Cornflower-3- O -After dissolving glucosinolates and barley protein separately to obtain the corresponding solutions, proceed according to cornflower-3- O The mass fractions of glucoside and barley protein were mixed in the solution and incubated to obtain a complex solution; The complex solution was dialyzed and dried to obtain the composite composition.

4. The preparation method according to claim 3, characterized in that, The temperature for mixed incubation is 4°C; the incubation time is 2 hours; the incubation is accompanied by stirring; and the stirring speed is 120 r / min.

5. The preparation method according to claim 3, characterized in that, The dialysate used for dialysis has a sodium chloride concentration of 1% to 10% by mass; the pH value of the dialysate is 7 to 7.

4.

6. The preparation method according to claim 3 or 5, characterized in that, The dialysis temperature is 4~25℃; the dialysis time is 4h.

7. The preparation method according to claim 3, characterized in that, Cornflower-3- O - Glucoside and barley protein were dissolved in PBS buffer, respectively; The PBS buffer has a molar concentration of 0.01 mol / L and a pH value of 2.

0.

8. The use of the composite composition according to claim 1 or 2 or the composite composition prepared by the preparation method according to any one of claims 3 to 7 in at least one of the following: (1) Increase cornflower-3- O -Stability of glucosides in gastric and / or intestinal fluids; (2) Increase cornflower-3- O -Bioavailability of glucosides.

9. The use of the composite composition according to claim 1 or 2 or the composite composition prepared by the preparation method according to any one of claims 3 to 7 in the preparation of a product that inhibits starch digestive enzymes.

10. The use of the composite composition according to claim 1 or 2 or the composite composition prepared by the preparation method according to any one of claims 3 to 7 in the preparation of hypoglycemic products.

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