Method for improving activity and stability of cyanidin-based anthocyanin and application thereof

Abstract

This invention provides a method for improving the activity and stability of cyanidyl anthocyanins and its applications, belonging to the field of natural product modification technology. Using cyanidin-3,5-diglucoside as the parent nucleus, this invention introduces an aromatic acyl group into its glycosyl moiety via acylation. This invention achieves a synergistic enhancement of enzyme inhibitory activity and environmental stability through targeted acylation modification. The method provided by this invention is mild, highly selective, and produces high-purity products. The obtained acylated cyanidyl anthocyanins exhibit a high IC50 value against α-glucosidase. 50 The concentration can be as low as 0.006 mg / mL, and it exhibits excellent structural stability under pH 2-12, temperature 4-100℃ and ultraviolet light conditions.

Description

Technical Field

[0001] This invention belongs to the field of natural product modification technology, and particularly relates to a method for improving the activity and stability of cyanidyl anthocyanins and its application. Background Technology

[0002] Anthocyanins are water-soluble flavonoids widely found in plants, possessing various biological activities such as antioxidant, anti-inflammatory, and blood sugar regulation, and have significant application value in functional foods and natural medicines. Among them, cyanidyl anthocyanins have become a research hotspot due to their wide distribution and abundant content. However, natural cyanidyl anthocyanins face two major technical challenges: firstly, their enzyme inhibitory activity is limited, making it difficult to meet the requirements for blood sugar lowering; secondly, their poor environmental stability makes them prone to degradation during processing (high temperature, acid and alkali) and storage (light, oxidation), severely limiting their industrial application.

[0003] In existing technologies, structural modifications of anthocyanins mainly include glycosylation and acylation. While glycosylation can improve stability, it weakens enzyme inhibitory activity due to steric hindrance. Traditional acylation methods suffer from poor reaction selectivity, low product purity, and complex processes, making it difficult to achieve a synergistic improvement in activity and stability. Furthermore, clinically used synthetic starch digestive enzyme inhibitors (such as acarbose) are prone to causing gastrointestinal side effects, while natural anthocyanin inhibitors are increasingly in demand due to their high safety and fewer side effects. Therefore, developing a cyanidyl anthocyanin modification technology that combines high enzyme inhibitory activity with high environmental stability has become a pressing technical problem to be solved in this field. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a method for improving the activity and stability of cyanidyl anthocyanins.

[0005] The second objective of this invention is to provide acylated cyanidyl anthocyanins obtained by the method described above.

[0006] A third objective of this invention is to provide the application of the method or the acylated cyanidyl anthocyanin in the preparation of starch digestive enzyme inhibitors.

[0007] The fourth objective of this invention is to provide the application of the method or the acylated cyanidyl anthocyanin in the preparation of hypoglycemic products.

[0008] The fifth objective of this invention is to provide the application of the method or the acylated cyanidyl anthocyanin in the preparation of highly environmentally stable pigments.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A method for improving the activity and stability of cyanidyl anthocyanins involves using cyanidin-3,5-diglucoside as the parent nucleus and introducing an aromatic acyl group into its glycosyl moiety via an acylation reaction.

[0010] Preferably, the aromatic acyl group is any one of caffeoyl, feruloyl, and benzoyl.

[0011] Preferably, caffeic acid, ferulic acid, or benzoic acid are used as acyl donors, and the acylation reaction is carried out under the catalysis of an enzyme.

[0012] Preferably, the enzyme is immobilized Candida antarcticis enzyme B.

[0013] Preferably, the amount of immobilized Candida antarcticis enzyme B added is 2% to 10% of the total mass of the acyl donor and cyanidin-3,5-diglucoside.

[0014] Preferably, the conditions for enzyme catalysis are: temperature 35~45℃, reaction time 20~35 h, and vacuum degree -85~-90kPa.

[0015] The present invention also provides acylated cyanidyl anthocyanins obtained by the method.

[0016] The present invention also provides the application of the method or the acylated cyanidyl anthocyanin in the preparation of starch digestive enzyme inhibitors.

[0017] The present invention also provides the application of the method or the acylated cyanidyl anthocyanin in the preparation of hypoglycemic products.

[0018] The present invention also provides the application of the method or the acylated cyanidyl anthocyanin in the preparation of pigments with high environmental stability.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for improving the activity and stability of cyanidyl anthocyanins. An aromatic acyl group is introduced into the glycosyl moiety of cyanidin-3,5-diglucoside via an acylation reaction. This invention achieves a synergistic enhancement of enzyme inhibitory activity and environmental stability through targeted acylation modification. The method provided by this invention is mild, highly selective, and produces high-purity products.

[0020] This invention also provides acylated cyanidyl anthocyanins obtained by the method, and their IC50 values ​​against α-glucosidase. 50 The concentration can be as low as 0.006 mg / mL, and it exhibits excellent structural stability under pH 2~12, temperature 4~100℃ and ultraviolet light conditions. It can be widely used in the preparation of hypoglycemic functional foods, natural pigments and nutritional supplements, and has significant industrialization value and market prospects. Attached Figure Description

[0021] Figure 1 Chromatogram of cornflower-3,5-diglucoside; Figure 2 Chromatogram of cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside; Figure 3 Chromatogram of cyanidin-3-O-(feruloyl)glucoside-5-O-glucoside; Figure 4 Chromatogram of cyanidin-3-O-(benzoyl)glucoside-5-O-glucoside; Figure 5 The inhibitory activity curves of different acylated cyanidyl anthocyanins on α-glucosidase are shown. Figure 6 The graph shows the changes in the retention rate of acylated cyanidyl anthocyanins at pH 2, 4, and 6. Figure 7 The graph shows the changes in the retention rate of acylated cyanidyl anthocyanins at pH 8, 10, and 12. Figure 8 The graph shows the changes in the retention rate of acylated cyanidyl anthocyanins at temperatures of 4℃ and 25℃, respectively. Figure 9 The graph shows the changes in the retention rate of acylated cyanidyl anthocyanins at temperatures of 40℃, 80℃, and 100℃. Figure 10 The graph shows the changes in the retention rate of acylated cyanidyl anthocyanins under sunlight, ultraviolet light, and light-protected conditions. Figure 11 The diagrams show the 3D and 2D representations of the molecular docking of acylated cyanidyl anthocyanin with α-glucosidase; where A represents cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside; B represents cyanidin-3-O-(feruloyl)glucoside-5-O-glucoside; and C represents cyanidin-3-O-(benzoyl)glucoside-5-O-glucoside. Detailed Implementation

[0022] This invention provides a method for improving the activity and stability of cyanidyl anthocyanins, using cyanidin-3,5-diglucoside as the parent nucleus and introducing an aromatic acyl group into its glycosyl moiety through an acylation reaction. The aromatic acyl group is preferably any one of caffeoyl, feruloyl, and benzoyl.

[0023] In this invention, cyanidin-3,5-diglucoside is shown in Formula 1. This invention uses cyanidin-3,5-diglucoside as the parent nucleus and selects caffeic acid, ferulic acid, or benzoic acid as acyl donors for targeted modification. Molecular simulation docking technology confirms that aromatic acyl groups can enhance the interaction network between anthocyanins and key residues (Asp197, Trp271, His478, etc.) in the active site of amylases through π-π stacking, hydrogen bond extension, and hydrophobic anchoring, thereby improving enzyme inhibitory activity. Simultaneously, the acyl groups, through intramolecular cochromaticity and steric hindrance shielding, inhibit nucleophilic attack by water molecules and photo-oxidative chain reactions, significantly enhancing stability.

[0024] Formula 1.

[0025] In this invention, cornflower-3,5-diglucoside is preferably a natural product extracted from plants; preferably, it is extracted with acidic ethanol using ultrasound-assisted extraction, wherein the acidic ethanol preferably has a pH of 2.5-3.5, more preferably a pH of 3, the volume fraction of ethanol is preferably 65%-75%, more preferably 70%, the ultrasound extraction temperature is preferably 45-55℃, more preferably 50℃, and the ultrasound extraction time is preferably 1-2 hours, more preferably 1.5 hours; after ultrasound-assisted extraction, a crude extract is obtained, which is preferably subjected to macroporous adsorption resin chromatography to remove impurities, thereby obtaining high-purity cornflower-3,5-diglucoside (purity ≥95%), laying the foundation for subsequent targeted modification.

[0026] In the method of this invention, caffeic acid, ferulic acid, or benzoic acid are preferably used as acyl donors, and the acylation reaction is carried out under the catalysis of an enzyme, wherein the enzyme is immobilized Candida antarcticis enzyme B. The amount of immobilized Candida antarcticis enzyme B added is 2% to 10% of the total mass of the acyl donor and cyanidin-3,5-diglucoside. The temperature for enzyme catalysis of immobilized Candida antarcticis enzyme B is preferably 35 to 45°C, more preferably 40°C; the reaction time for enzyme catalysis is preferably 20 to 35 h, more preferably 24 to 30 h; the enzyme catalysis is preferably a reduced-pressure enzymatic reaction, preferably carried out under a vacuum of -85 to -90 kPa.

[0027] This invention also provides acylated cyanidyl anthocyanins obtained by the method described above. When caffeic acid is used as the acyl donor, cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside is obtained, as shown in Formula 2; when ferulic acid is used as the acyl donor, cyanidin-3-O-(feruloyl)glucoside-5-O-glucoside is obtained, as shown in Formula 3; and when benzoic acid is used as the acyl donor, cyanidin-3-O-(benzoyl)glucoside-5-O-glucoside is obtained, as shown in Formula 4. Acute toxicity tests verified that the LD50 of the acylated anthocyanins of this invention... 50The concentration is >5000mg / kg, which is practically non-toxic and meets the safety standards for food additives.

[0028] Equation 2.

[0029] Formula 3.

[0030] Formula 4.

[0031] This invention also provides the application of the method or the acylated cyanidyl anthocyanin in the preparation of starch digestive enzyme inhibitors. The IC50 of the caffeoyl, ferulic, and benzoyl modified cyanidyl anthocyanins of this invention against α-glucosidase is [not specified]. 50 The values ​​were 0.006, 0.008, and 0.011 mg / mL, respectively, demonstrating its potential as a highly efficient amylase inhibitor.

[0032] The present invention also provides the application of the method or the acylated cyanidyl anthocyanin in the preparation of hypoglycemic products.

[0033] This invention also provides the application of the method or the acylated cyanidyl anthocyanin in the preparation of pigments with high environmental stability. The acylated cyanidyl anthocyanin of this invention exhibits excellent structural stability (retention rate ≥70% after 10 h) under pH 2–12, temperature 4–100℃, and ultraviolet light conditions, demonstrating high environmental stability.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 A method for preparing acylated cyanidyl anthocyanins.

[0036] 1. Preparation of cornflower-3,5-diglucoside, the steps are as follows: Raw material extraction: Take 100 g of purple sweet potato powder, add 500 mL of pH 3 acidic ethanol solution (ethanol volume fraction 70%), and extract with ultrasonic assistance at 50 ℃ for 1.5 h. Filter to obtain crude extract. Purification: The crude extract was loaded onto an AB-8 macroporous adsorption resin column, and impurities were eluted with 3 column volumes of deionized water, followed by elution with 70% ethanol solution. The eluent was collected and concentrated under reduced pressure (45℃, -0.08 MPa) until no alcohol odor remained. The solution was then freeze-dried to obtain 8.2 g of pure cornflower-3,5-diglucoside with a purity of 96.3%. The chromatogram of cornflower-3,5-diglucoside is shown below. Figure 1 As shown.

[0037] 2. Preparation of cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside, the steps are as follows: Acylation modification: Take 5 g of cornflower-3,5-diglucoside, add 60 mL of anhydrous ethanol-dimethyl sulfoxide mixture (the volume ratio of anhydrous ethanol to dimethyl sulfoxide is 4:1), add 6.2 g of caffeic acid, and then add 0.3 g of immobilized Candida antarctica enzyme B (purchased from Shaanxi Jiuan Fusi Biotechnology Co., Ltd., designated as Novozym 435, enzyme activity 5500 U / g).

[0038] Reduced pressure enzymatic reaction: 40℃, -87 kPa, 20 r / min shaking reaction for 30 h; Novozym 435 as catalyst, anhydrous tert-amyl alcohol as medium; after the reaction was completed, the substrate peak area ratio was ≤3% by HPLC monitoring, the reaction was terminated, and the retention time of the target product cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside was about 17.8 min.

[0039] Purification: Novozym 435 was removed by filtration. The filtrate was loaded onto a silica gel column and eluted with ethyl acetate-methanol (ethyl acetate to methanol volume ratio 5:1). The target fraction was collected, and 3 times its volume of ethanol-water mixture (ethanol to water volume ratio 3:1) was added. Crystallization was carried out at 4°C for 14 h. After filtration and drying, 5.8 g of pure cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside was obtained, with a purity of 98.7% and an acylation rate of 92.5%. The chromatogram of cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside is shown below. Figure 2 As shown.

[0040] 3. Preparation of cyanidin-3-O-(feruloyl)glucoside-5-O-glucoside, the steps are as follows: Anhydrous pretreatment: cornflower-3,5-diglucoside and Novozym 435 were vacuum dried at 30 °C for 24 h, and ethyl ferulic acid (≥98%) and anhydrous pyridine were dried using 4 Å molecular sieves for 48 h.

[0041] Reaction system: 50 mg cyanidin-3,5-diglucoside dissolved in 4 mL pyridine; 2.25 g ethyl ferulic acid and 225 mg Novozym 435 added; pyridine added to 5 mL.

[0042] Reduced-pressure enzymatic reaction: 39℃, -85 kPa, 20 r / min for 30 h; HPLC monitoring (product peak: cornflower-3,5-diglucoside, t R ≈18.2 min).

[0043] Termination, extraction, and purification: same as benzoylation; semi-preparative HPLC collection of t R The main peak was approximately 18.2 min in length. After lyophilization, cyanidin-3-O-(feruloyl)glucoside-5-O-glucoside (purity ≥92%) was obtained. The chromatogram of cyanidin-3-O-(feruloyl)glucoside-5-O-glucoside is shown below. Figure 3 As shown.

[0044] 4. Preparation of cyanidin-3-O-(benzoyl)glucoside-5-O-glucoside, the steps are as follows: Anhydrous pretreatment: cornflower-3,5-diglucoside and Novozym 435 were vacuum dried at 30 °C for 24 h; pyridine and methyl benzoate (purity ≥99%) were dried with 4 Å molecular sieves for 48 h.

[0045] Preparation of the reaction system: Add 50 mg of cyanidin-3,5-diglucoside to a 50 mL round-bottom flask and dissolve it in 4 mL of anhydrous pyridine; add 2.0 g of methyl benzoate and 200 mg of Novozym 435; add pyridine to a final volume of 5 mL and seal the flask.

[0046] Reduced-pressure enzymatic reaction: 41℃, -90 kPa, 20 r / min for 24 h; sampling at regular intervals (2, 6, 12, 24 h), conversion monitored by HPLC (detected at 360 nm, product peak: C3,5G-Bz, t R ≈12.5 min).

[0047] Termination and extraction: The reaction solution was cooled to room temperature and filtered to remove enzymes; 10 mL of 0.1% HCl (pH 2.5) was added to the filtrate, and 3 × 15 mL of ethyl acetate was extracted; the organic phases were combined, washed with 2 × 10 mL of saturated brine, dried over anhydrous Na2SO4, and rotary evaporated (35℃) to obtain the crude product.

[0048] Semi-preparative HPLC purification: Column: C18, 5 μm, 10 × 250 mm; Mobile phase: A (0.1% formic acid in water), B (acetonitrile); Gradient: 0→5 min 15% B; 5→20 min 15%→35% B; 20→25 min 35%→80% B; Flow rate: 3.0 mL / min; Detection wavelength: 520 nm (anthocyanin) + 360 nm (benzoyl); Collect the main peak (t R (≈12.5 min), rotary evaporation to remove acetonitrile, lyophilization to obtain cyanidin-3-O-(benzoyl)glucoside-5-O-glucoside (purity ≥95%). The chromatogram of cyanidin-3-O-(benzoyl)glucoside-5-O-glucoside is shown below. Figure 4 As shown.

[0049] The acute toxicity test was conducted, following the limit method in GB 15193.3-2014 "National Food Safety Standard Acute Oral Toxicity Test". SPF-grade Kunming mice (half male and half female, weighing 18-22 g) were selected and fasted for 12 hours before the experiment (water was allowed). The three acylated anthocyanin samples prepared in this example were diluted with ultrapure water to the maximum dosing concentration (5000 mg / kg·bw) and administered orally to the experimental group mice (n=20, half male and half female) once, while the control group received an equal volume of ultrapure water. The mice were observed for 14 consecutive days after administration, and the symptoms of poisoning, weight changes, and mortality were recorded. After the experiment, the mice were sacrificed, and dissection was performed to observe any obvious abnormalities in the major organs. The median lethal dose (LD50) was calculated using the modified Kohl's method. 50 The acylated anthocyanin median lethal dose (LD50) of the present invention. 50 The concentration is >5000mg / kg, which is practically non-toxic and meets the safety standards for food additives.

[0050] Example 2 Enzyme inhibition activity assay.

[0051] Using the cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside (C3G(caffeoyl)5G), cyanidin-3-O-(feruloyl)glucoside-5-O-glucoside (C3G(Feruloyl)5G), and cyanidin-3-O-(benzoyl)glucoside-5-O-glucoside (C3G(Benzoyl)5G) prepared in Example 1 as raw materials, the inhibitory activity of different raw materials on α-glucosidase was tested.

[0052] The inhibitory activity against α-glucosidase was determined using the pNPG method. Acetylated anthocyanin samples at different concentration gradients were prepared, with acarbose as a positive control and unacylated cyanidin-3,5-diglucoside (prepared in Example 1) as a negative control. Each concentration was measured in triplicate, following the method of Zhang et al. (Zhang J, Syn L, Dong Y, et al. Chemical compositions and α-glucosidase inhibitory effects of anthocyanidins from blueberry, blackcurrant and blue honeysuckle fruits[J]. Food Chem, 2019, 299:125102.). The acylated anthocyanin concentrations were 0.01, 0.04, 0.08, 0.12, 0.16, and 0.20 mg / mL, with an α-glucosidase activity of 0.2 U / mL. Take 1 mL of each reaction solution and place it at 4℃ for 15 min. Add 1 mL of 3 mM p-nitro-α-D-glucopyranoside (pNPG) solution and place at 37℃ to start the enzyme reaction. At time points of 0, 3, 6, and 9 min, add 600 µL of the reaction solution to 600 µL of 0.3 M Na2CO3 solution to terminate the reaction. Measure the absorbance of the solution at 405 nm using an ELISA reader. Parameters are calculated according to formulas (1) and (2).

[0053] I= ×100 (1) In the formula, v and v 0 The initial reaction rates are shown with and without each anthocyanin.

[0054] The inhibitory effect of each anthocyanin on α-amylase was determined using IC50 assay. 50 IC indicates. 50 The value is calculated according to formula (2): (2) In the formula, [I] represents the concentration of each anthocyanin, while I max This represents the maximum inhibition rate.

[0055] The results are as follows Figure 5 As shown, cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside has an IC50 value for α-glucosidase. 50 The IC50 of cyanidin-3-O-(feruloyl)glucoside-5-O-glucoside against α-glucosidase was 0.006 mg / mL.50 =0.008 mg / mL, IC50 of cyanidin-3-O-(benzoyl)glucoside-5-O-glucoside against α-glucosidase 50 =0.011 mg / mL, IC50 of unacylated cyanidin-3,5-diglucoside against α-glucosidase 50 =0.045 mg / mL. Positive control acarbose IC50 against α-glucosidase. 50 =0.028 mg / mL.

[0056] The above results indicate that the three acylated cyanidyl anthocyanins prepared in this invention exhibit significantly better inhibitory activity against α-glucosidase than the unacylated cyanidin-3,5-diglucoside (p<0.01). Among them, the caffeinated product (cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside) showed the most significant activity enhancement, with an approximately 7.5-fold increase in inhibitory activity against α-glucosidase. Furthermore, the inhibitory activity of all three acylated products is superior to that of the commonly used clinical drug acarbose, demonstrating their potential as highly efficient amylase inhibitors.

[0057] Example 3 Stability determination.

[0058] Using cyanidin (Cy), cyanidin-3-O-glucoside (Cy3Glu), cyanidin-3-O-galactoside (Cy3Gal), cyanidin-3-O-arabinoside (Cy3Ara), cyanidin-3,5-diglucoside (C3G5G), and cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside (C3G(caffeoyl)5G), cyanidin-3-O-(feruloyl)glucoside-5-O-glucoside (C3G(Feruloyl)5G), and cyanidin-3-O-(benzoyl)glucoside-5-O-glucoside (C3G(Benzoyl)5G) prepared in Example 1 as raw materials, the stability of different raw materials was tested.

[0059] Following the method of Zhao et al. (Zhao WP, Zhao CY, Shan HY, et al. Degradation rules of anthocyanin in different environmental factors and food matrices[J]. J LightInd, 2023, 38(2):41-47.), the absorbance values ​​of cyanidyl anthocyanin solutions with different structures under different external factors were measured at 525 nm, and the retention rate (%) of anthocyanins was calculated according to formula (3): (3) In the formula, A t The absorbance of anthocyanins after different treatment times is denoted as A0, where A0 is the initial absorbance before anthocyanin treatment, and t is the treatment time (h).

[0060] (1) pH stability: Acylated anthocyanin samples were dissolved in buffer solutions of pH 2, 4, 6, 8, 10 and 12, respectively, and placed at 25°C for 10 h to determine the retention rate.

[0061] The results show that ( Figures 6-7 At pH 2-8, the retention rates of the acylated cyanidyl anthocyanins prepared by this invention are all ≥80%, at pH 10, the retention rates are all ≥75%, and at pH 12, the retention rates are all ≥70%; indicating that the acylated cyanidyl anthocyanins of this invention have excellent pH stability, which is significantly better than that of unacylated anthocyanins (retention rate ≤40%).

[0062] (2) Temperature stability: The samples were treated at 4℃, 25℃, 40℃, 80℃ and 100℃ for 10 h respectively, and the retention rate was measured.

[0063] The results show that ( Figure 8 and Figure 9 The retention rates were ≥95% at 4℃ and 25℃ for 10 h, ≥90% at 40℃, ≥78% at 80℃, and ≥72% at 100℃, indicating that the acylated cyanidyl anthocyanins prepared by this invention have good thermal stability and can adapt to high-temperature processing conditions in food processing.

[0064] (3) Light stability: Ultraviolet light: Take an equal amount of the acylated cyanidyl anthocyanin to be tested and dispense it into colorless transparent sealed glass bottles. Each bottle has the same filling volume. Set up 3 parallel samples for each group. Place the sample under an ultraviolet lamp (365 nm, 15 cm distance). The ambient temperature is uniformly controlled at 25 ℃. Irradiate continuously for 10 h and determine the retention rate. Light protection: For the UV group, the same batch of samples was used. Equal amounts of the acylated cyanidyl anthocyanins to be tested were dispensed into colorless, transparent, sealed glass bottles, with consistent filling volume in each bottle. Three parallel samples were set up for each group. All samples were placed in a constant-temperature experimental cabinet, completely shielded from light (inner layer of black light-blocking cloth + light-blocking cabinet body, no visible or ultraviolet light could enter), with the ambient temperature uniformly controlled at 25℃. The samples were left to stand for 10 hours, consistent with the UV irradiation group, and the retention rate was measured.

[0065] Sunlight: The same batch of samples as the light-avoidance group and the ultraviolet group were used, and the same amount was dispensed into transparent sealed glass bottles of the same specifications. The bottles were placed on sunny windowsills indoors to avoid direct sunlight and local high temperatures. The ambient temperature was 25 ℃, and the samples were exposed to indoor diffused sunlight and intermittent natural light without any additional ultraviolet light source. The samples were placed at a uniform distance of 15 cm from the windowsill, which was consistent with the distance parameters of the ultraviolet lamp. The samples were continuously irradiated for 10 h, and the retention rate was measured.

[0066] The results show that ( Figure 10 After irradiation with ultraviolet light (365 nm) for 10 h, the retention rate of acylated cyanidin anthocyanins prepared in this invention was 75%, while the retention rate of unacylated cyanidin-3,5-diglucoside under the same conditions was only 38%, indicating that acylation modification significantly enhances the photostability of anthocyanins.

[0067] Example 4 Preparation of hypoglycemic functional beverages.

[0068] 0.5 g of cyanidin-3-O-(caffeoyl)glucoside-5-O-glucoside prepared in Example 1 was added to 1000 mL of deionized water and stirred to dissolve. Then, 50 g of sucrose, 2 g of citric acid, and 10 g of a composite stabilizer (sucrose, low-concentration sodium chloride (0.08%), and ascorbyl palmitate mixed in a mass ratio of 85:10:5) were added. The mixture was stirred until homogenized (10000 r / min), sterilized (85℃, 15 min), cooled, and bottled to obtain a hypoglycemic functional beverage. After storage at room temperature and protected from light for 6 months, the anthocyanin retention rate was ≥85% (initial anthocyanin absolute content: 0.4935 g, anthocyanin residue after 6 months: 0.4195 g), and there was no significant change in sensory quality.

[0069] Example 5 Molecular docking simulations were performed using AutoDock Vina software, with human α-glucosidase (PDB ID: 3A4A) as the acceptor model. PyMOL software was used to simulate the docking of different cyanidyl anthocyanins with α-glucosidase. The molecular structures of different cyanidyl anthocyanins were plotted using ChemDraw 20.0, and initial energy minimization was performed using the MM2 force field (5000 iterations, energy convergence criterion 0.01 kcal / (mol·Å)). The crystal structure of α-glucosidase (PDB ID: 3N04) was obtained from a protein database, and the preprocessing steps were as follows: water molecules, ligands, and redundant atoms were removed; polar hydrogen atoms were added and side chains were deleted; charge was distributed using the AMBER14 force field; and energy minimization was performed using the Conjugate gradient algorithm (maximum 10000 iterations, convergence threshold 0.001 kcal / (mol·Å)) to ensure protein structural stability. Docking region definition: Referring to the structural and functional studies of α-glucosidase, Asp214, Glu276, and Asp349 were identified as key catalytic residues. The docking center coordinates were defined as X=22.5 Å, Y=35.1 Å, Z=28.3 Å, with a docking box size of 30 Å × 30 Å × 30 Å. A Lamarckian genetic algorithm was used for docking search (population size 100, iterations 1000 generations, crossover probability 0.8, mutation probability 0.02). Ten docking conformations were generated for each ligand, and the optimal conformation was selected based on binding free energy. After docking, the 2D molecular docking diagram was exported using Discovery Studio 2019 Client software for interaction mode analysis. The molecular docking results showed that the binding modes of the three acylation products to the α-glucosidase active site were significantly different. (1) Caffeine acylation products ( Figure 11 In step A): The caffeoyl group forms a tight planar stack with the Thr265 residue in the α-glucosidase active pocket via π-π stacking, with a spacing of 3.5 Å. Simultaneously, the glycosyl moiety of the parent nucleus forms hydrogen bonds (2.8 Å) with the key catalytic residue Asp307 and a double hydrogen bond network (2.7 Å and 2.9 Å) with the Lys412 residue. The binding affinity is -9.8 kcal / mol. This synergistic mechanism of "π-π stacking + multiple hydrogen bonds" allows the caffeoylated product to be firmly anchored to the enzyme active site, thus exhibiting optimal enzyme inhibitory activity (α-glucosidase IC50). 50 =0.006 mg / mL, the lowest among the three.

[0070] (2) Ferula acylated products ( Figure 11In the B group: the ferulic yl group also forms a π-π stack with the Trp271 residue (spacing 3.7 Å), but due to steric hindrance caused by the introduction of the methoxy group, the stacking distance is slightly larger than that of the caffeoyl group; in terms of hydrogen bonding, it only forms single hydrogen bonds with Asp197 and His478 (bond lengths of 2.9 Å and 3.0 Å, respectively). These differences result in a slightly lower binding affinity (-9.2 kcal / mol) than the caffeoylated product, and correspondingly, a slightly weaker enzyme inhibitory activity (α-glucosidase IC50). 50 =0.008 mg / mL).

[0071] (3) Benzoylation products ( Figure 11 In the C group: Although the benzoyl group forms a π-π stack (3.8 Å spacing) with the Trp271 residue, it lacks a phenolic hydroxyl group and cannot form additional hydrogen bond interactions with surrounding residues; it binds to the enzyme's active pocket solely through hydrophobic interactions. Therefore, it has the lowest binding affinity (-8.7 kcal / mol) and the weakest enzyme inhibitory activity (α-glucosidase IC50). 50 =0.011 mg / mL), but still significantly better than unacylated anthocyanins (IC50). 50 =0.045 mg / mL) and positive control acarbose (IC50) 50 =0.028 mg / mL).

[0072] The binding energy of unacylated cyanidin-3,5-diglucoside is only -6.5 kcal / mol, indicating that acylation modification significantly enhances the binding affinity to the enzyme.

[0073] In summary, Figure 11 The molecular docking results are in high agreement with the enzyme inhibitory activity data of Example 2, fully revealing the core technical principle of this invention: by directionally introducing aromatic acyl groups with different structures, the interaction mode between anthocyanins and the active site of starch digestive enzymes can be regulated. Among them, the caffeoyl gene has both π-π stacking ability and phenolic hydroxyl hydrogen bond donor characteristics, which can achieve optimal enzyme inhibitory activity. This provides solid molecular-level evidence to support the technical effect of "directional acylation modification synergistically improving enzyme inhibitory activity and environmental stability".

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for improving the activity and stability of cyanidyl anthocyanins, characterized in that, Using cornflower-3,5-diglucoside as the parent nucleus, aromatic acyl groups are introduced into its glycosyl moiety through an acylation reaction.

2. The method according to claim 1, characterized in that, The aromatic acyl group is any one of caffeoyl, feruloyl, and benzoyl.

3. The method according to claim 1, characterized in that, Acylation reaction is carried out under the catalysis of enzymes using caffeic acid, ferulic acid, or benzoic acid as acyl donors.

4. The method according to claim 3, characterized in that, The enzyme is immobilized Candida antarcticis enzyme B.

5. The method according to claim 4, characterized in that, The amount of immobilized Candida antarcticis enzyme B added was 2% to 10% of the total mass of the acyl donor and cyanidin-3,5-diglucoside.

6. The method according to claim 3, characterized in that, The conditions for enzyme catalysis are: temperature 35~45℃, reaction time 20~35 h, and vacuum degree -85~-90 kPa.

7. Acylated cyanidyl anthocyanins obtained by the method according to any one of claims 1 to 6.

8. The use of the method according to any one of claims 1 to 6 or the acylated cyanidyl anthocyanin according to claim 7 in the preparation of starch digestive enzyme inhibitors.

9. The application of the method according to any one of claims 1 to 6 or the acylated cyanidyl anthocyanin according to claim 7 in the preparation of hypoglycemic functional products.

10. The use of the method according to any one of claims 1 to 6 or the acylated cyanidyl anthocyanin according to claim 7 in the preparation of pigments with high environmental stability.

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