A black buckwheat polyphenol extract, a preparation method thereof and application thereof in improving sugar tolerance and insulin resistance

Abstract

The present application belongs to the technical field of natural product functional factors and metabolic health intervention, and relates to a black buckwheat polyphenol extract and a preparation method and application thereof. The extraction method adopts microwave light wave ultrasonic extractor assisted extraction to improve the extraction efficiency and polyphenol content. The composition comprises rutin and flos lonicerae aglycone, and further comprises black buckwheat polyphenol extract and pharmaceutically or food acceptable adjuvants. The composition can inhibit the activities of alpha-amylase and alpha-glucosidase by synergy, delay the digestion and absorption of carbohydrates, reduce the postprandial blood glucose peak, improve the insulin resistance induced by high-fat diet, and regulate the expression of liver glucose metabolism related genes. The composition can be used for preparing a medicine, health food or functional food for assisting in reducing blood glucose, and provides a novel, efficient and industrialized technical solution for the application of natural polyphenols in the intervention of abnormal glucose metabolism.

Description

Technical Field

[0001] This invention belongs to the field of natural product functional factors and metabolic health intervention technology, and relates to the application of active substances in black buckwheat. Background Technology

[0002] With changing lifestyles and the increasing Westernization of dietary structures, the incidence of diabetes and related disorders of glucose metabolism continues to rise, and hyperglycemia has become a significant public health issue affecting human health. Long-term hyperglycemia not only leads to impaired pancreatic function but may also further induce various complications such as cardiovascular and cerebrovascular diseases, kidney damage, and retinopathy. Therefore, developing safe, effective, and long-term suitable hypoglycemic products is of great importance. While existing clinically used hypoglycemic drugs such as biguanides and α-glucosidase inhibitors have certain efficacy, long-term use often brings problems such as gastrointestinal discomfort, hypoglycemia risk, and liver and kidney burden, limiting their application in certain populations. Against this backdrop, plant-based active ingredients with natural sources, low toxicity, and multi-target regulatory effects have gradually become a research hotspot.

[0003] Black buckwheat, a plant rich in polyphenols and flavonoids and considered both food and medicine, shows promising application potential in regulating glucose and lipid metabolism. However, existing technologies largely focus on the functional studies of crude extracts or single active ingredients of black buckwheat, with insufficient attention paid to the synergistic effects between different active ingredients. While patent application CN114129621A discloses the use of various buckwheat flavonoids in the preparation of antibacterial or gastritis-preventing drugs or health products, its research focuses on the application of various buckwheat flavonoids in inhibiting *H. pylori* and the inflammation caused by *H. pylori* infection. Furthermore, current research on its mechanism of action remains limited, and its comprehensive mechanisms of action in inhibiting carbohydrate digestive enzyme activity, regulating postprandial blood glucose, influencing insulin resistance, and regulating the expression of genes related to liver glucose metabolism have not yet been fully elucidated.

[0004] Existing extraction methods mostly employ traditional water extraction, alcohol extraction, or maceration, which suffer from low extraction efficiency, long extraction times, high solvent consumption, and loss of active ingredients. Furthermore, single extraction techniques lack sufficient control over the purity and activity stability of the extract, limiting its application in functional foods or pharmaceutical preparations. Therefore, there is an urgent need to develop an efficient, green, and industrially scalable extraction method to obtain a high-purity, multi-component synergistically active, and stable black buckwheat composition, while ensuring its significant effects in regulating postprandial blood glucose and improving abnormal glucose metabolism. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a black buckwheat polyphenol extract, its preparation method, and its applications.

[0006] The technical solution of this invention is implemented as follows:

[0007] In a first aspect, the present invention provides a black buckwheat polyphenol extract, wherein the active substances of the black buckwheat polyphenol extract are mainly rutin and rutin. The two can be used in combination in a preset ratio, preferably a mass ratio of 1-10:1, more preferably 5-8:1, and even more preferably 7:1.

[0008] The total polyphenol content of the black buckwheat polyphenol extract was 6739.59 ng / mg (as a percentage of the dry weight of black buckwheat), of which the rutin content was 4741.22 ng / mg and the pyroside content was 765.11 ng / mg.

[0009] In addition to the aforementioned active ingredients, the composition may further comprise a black buckwheat polyphenol extract and food- or pharmaceutically acceptable excipients to prepare a formulation suitable for consumption or oral administration. This invention provides the application of the black buckwheat polyphenol extract in assisting blood glucose reduction, preferably maintaining at least 80% of the α-amylase and α-glucosidase inhibitory activities under simulated gastrointestinal digestive conditions at pH 6-8 and 37°C. In in vivo experiments, a mouse model of abnormal glucose metabolism induced by a high-fat diet showed that the composition significantly reduced postprandial blood glucose peak and area under the blood glucose curve, improved insulin sensitivity, improved intestinal permeability, and regulated the expression of liver glucose metabolism-related genes.

[0010] Secondly, the present invention provides a method for preparing the above-mentioned black buckwheat polyphenol extract, comprising the following steps: adding black buckwheat powder to an ethanol aqueous solution, extracting the powder, and then rotary evaporating and freeze-drying the resulting extract to obtain the black buckwheat polyphenol extract. The volume percentage of the ethanol aqueous solution is 70%; the solid-liquid ratio of the black buckwheat powder to the ethanol aqueous solution is 1:15.

[0011] Furthermore, the above extraction can be any one of microwave-assisted extraction, light-assisted extraction, and ultrasonic-assisted extraction. Preferably, the power of microwave-assisted extraction is 200-500 W, and the extraction time is 2-10 min; the power of light-assisted extraction is 200-400 W, and the extraction time is 5-15 min; the frequency of ultrasonic-assisted extraction is 20 kHz, the power is 200-1000 W, and the extraction time is 15-30 min.

[0012] Thirdly, the present invention provides the application of the above-mentioned black buckwheat polyphenol extract in the preparation of a drug for type 2 diabetes.

[0013] Fourthly, the present invention provides the application of the above-mentioned black buckwheat polyphenol extract in the preparation of drugs for improving glucose tolerance, insulin resistance or lowering postprandial blood glucose.

[0014] Specifically, the aforementioned black buckwheat polyphenol extract achieves its effect by simultaneously inhibiting the activity of α-amylase and α-glucosidase, thus delaying the digestion and absorption of carbohydrates. The composition of this invention can effectively inhibit the activity of α-amylase and α-glucosidase, delaying the digestion and absorption of starch and disaccharides, thereby reducing the postprandial rise in blood glucose; it can also improve glucose tolerance and increase insulin resistance, thereby improving the body's glucose and lipid metabolism and playing a positive role in maintaining glucose homeostasis.

[0015] The present invention has the following beneficial effects:

[0016] 1. The hypoglycemic composition provided by this invention is based on active ingredients derived from black buckwheat. The raw materials are natural and have high safety, making it suitable for long-term use as a functional food, health food, or adjunctive intervention product. Multiple active ingredients can be combined to produce a synergistic effect, resulting in a hypoglycemic effect superior to that of a single ingredient. It also possesses multiple mechanisms of action, including inhibiting digestive enzymes, improving postprandial blood glucose, and improving insulin resistance, offering more comprehensive health intervention value. Furthermore, the preparation process is simple, and the raw materials are widely available, making it suitable for large-scale production and industrial promotion, with promising market application prospects.

[0017] 2. This invention significantly improves the extraction efficiency and total polyphenol content of rutin and pyrolysis glycosides by employing microwave, light wave and ultrasonic multi-mode assisted extraction technology, thus ensuring the stability and bioavailability of the active ingredients.

[0018] 3. The product of this invention can synergistically inhibit the activity of α-amylase and α-glucosidase, effectively delaying the digestion and absorption of carbohydrates, reducing postprandial blood glucose peak and area under the blood glucose curve, and significantly improving insulin resistance in vivo, while regulating the expression of genes related to liver glucose metabolism. Simultaneously, the composition exhibits high safety and stability, and can be directly used in functional foods, health foods, or pharmaceutical preparations, making it suitable for industrial production and widespread application. This provides a new technical solution for the use of natural polyphenols in the field of assisting in lowering blood sugar and intervening in abnormal glucose metabolism. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1The two extraction methods represent the inhibition rates of α-amylase and α-glucosidase. (A) represents the inhibitory effect of different extraction methods on α-amylase, and (B) represents the inhibitory effect of different extraction methods on α-glucosidase. U represents ultrasonic extraction, L represents optical extraction, M represents microwave extraction, and M+L+U represents microwave-optical-ultrasonic extraction.

[0021] Figure 2 The following diagrams show the molecular docking of rutin, sennaoside, and α-amylase. (A) Molecular docking of rutin and α-amylase; (B) Molecular docking of sennaoside and α-amylase; (C) Molecular docking of rutin and sennaoside dual ligands with α-amylase.

[0022] Figure 3 The following diagrams show the molecular docking of rutin, flavin, and α-glucosidase. (A) Molecular docking of rutin and α-glucosidase; (B) Molecular docking of flavin and α-glucosidase; (C) Molecular docking of rutin and flavin dual ligands with α-glucosidase.

[0023] Figure 4 The effects of rutin and pyroside synergistically on postprandial blood glucose and in vivo amylase activity in rats were investigated. (A) is the postprandial blood glucose curve of rats; (B) is the area under the blood glucose curve (AUC); (C) is the level of α-amylase activity in the pancreas; (D) is the level of α-amylase activity in serum; (E) is the level of maltase activity in the small intestine; and (F) is the level of sucrase activity in the small intestine.

[0024] Figure 5 The effects of rutin and sennaoside on blood glucose in mice on a high-fat diet are shown in the following figures: (A) HE staining of mouse pancreas; (B) OGTT blood glucose changes in mice; (C) AUC index of mice; (D) pancreatic weight of mice; and (E) plasma INS level of mice.

[0025] Figure 6 Effects of rutin and senna glycoside on the expression of genes related to glucose metabolism in mouse liver; (A) relative expression level of genes related to glycolysis; (B) relative expression level of genes related to gluconeogenesis; (C) relative expression level of genes related to glycogen metabolism; (D) relative expression level of genes related to glucose transport; (E) relative expression level of genes related to β-cell transcription factors. Detailed Implementation

[0026] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0027] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0028] Rats and mice: Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd., License No.: SYXK (Yu) 2025-0012.

[0029] Experimental reagents: Acarbose, rutin, quercetin, naringin, hyperoside, protocatechuic acid standard samples were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; α-glucosidase, α-amylase standards were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; total protein kit (T-P), α-amylase kit, α-glucosidase kit were purchased from Nanjing Jiancheng Bioengineering Institute; endotoxin kit (LPS) was purchased from Xiamen Huijia Bio-Tech Co., Ltd.; hematoxylin-eosin staining kit (HE) was purchased from Beyotime Biotechnology Co., Ltd.; sterile and enzyme-free water, TriQuick total RNA extraction kit, high-specificity qPCR were purchased from Novoprotein Scientific Inc.

[0030] Example 1 <s

[0031] The preparation steps of the black buckwheat polyphenol extract in this example are as follows:

[0032] Take 100 g of black buckwheat powder, add an ethanol-aqueous solution (70%, solid-liquid ratio 1:15), extract with a sonicator under the condition of an ultrasonic power of 300 W for 5 min, filter the extract, then concentrate by rotary evaporation, and freeze-dry to obtain a polyphenol powder. The in vitro α-amylase inhibition rate is 68.17%, and the α-glucosidase inhibition rate is 63.23%.

[0033] Example 2

[0034] The preparation steps of the black buckwheat polyphenol extract in this example are as follows:

[0035] Take 100 g of black buckwheat powder, add an ethanol-aqueous solution (70%, solid-liquid ratio 1:15), perform light-wave assisted extraction under the conditions of a power of 400 W and an extraction time of 10 min, filter the extract, then concentrate by rotary evaporation, and freeze-dry to obtain a polyphenol powder. The in vitro α-amylase inhibition rate is 74.76%, and the α-glucosidase inhibition rate is 72.11%, which is significantly better than extraction with a sonicator.

[0036] Example 3

[0037] The preparation steps of the black buckwheat polyphenol extract in this embodiment are as follows:

[0038] 100 g of black buckwheat powder was added to an ethanol-water solution (70%, solid-liquid ratio 1:15), and microwave-assisted extraction was performed at a frequency of 20 kHz, a power of 600 W, and an extraction time of 20 min. The extract was filtered, concentrated by rotary evaporation, and then freeze-dried to obtain polyphenol powder. The in vitro α-amylase inhibition rate was 76.51%, and the α-glucosidase inhibition rate was 74.23%, significantly better than that of ultrasonic extraction.

[0039] Example 4

[0040] The preparation steps of the black buckwheat polyphenol extract in this embodiment are as follows:

[0041] 100 g of black buckwheat powder was added to an ethanol-water solution (70%, solid-liquid ratio 1:15) and extracted using microwave-assisted extraction under the conditions described in the three examples above. The extract was filtered, concentrated by rotary evaporation, and then freeze-dried to obtain polyphenol powder. The in vitro α-amylase inhibition rate was 84.84%, and the α-glucosidase inhibition rate was 80.32%, significantly better than that of ultrasonic extraction.

[0042] Example Characterization and Analysis: Analysis of the Polyphenol Composition of Black Buckwheat using Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry

[0043] Take 50 mg of the sample prepared in Example 4 into a centrifuge tube, add 0.5 mL of 80% methanol aqueous solution (containing 0.2% vitamin C), vortex to mix, sonicate at room temperature for 30 min, centrifuge at 12000 rpm for 10 min, take the supernatant, repeat the extraction twice, combine the two supernatants, mix well, dilute several times, and then perform the analysis.

[0044] The liquid chromatography conditions were as follows: A Waters HSS T3 column (100 × 2.1 mm, 1.8 μm) was used for chromatographic separation. The mobile phase consisted of ultrapure water with 0.1% formic acid (phase A) and methanol with 0.1% formic acid (phase B). The flow rate was set to 0.3 mL / min, the column temperature was maintained at 40℃, and the injection volume was 2 μL. The gradient elution program was as follows: at 0 min, A / B was 95:5 (v / v); at 1 min, it was adjusted to 70:30; at 10 min, it became 20:80; at 14 min, it was further adjusted to 5:95 and maintained until 16 min; then at 16.1 min, it was restored to 95:5 and equilibrated to 18 min.

[0045] The mass spectrometry detection conditions were as follows: an electrospray ionization source was used, with sheath gas and auxiliary gas set to 40 arb and 10 arb, respectively; the spray voltage was -2800 V; the ion source temperature was 350℃; and the ion transmission tube temperature was 320℃. Data acquisition was performed in Fullscan-ddMS² mode, with the primary mass spectrometry scan range set to m / z 70-1050.

[0046] The results are shown in Table 1:

[0047] Table 1. Analysis of polyphenol content in black buckwheat using ultra-high performance liquid chromatography-high resolution mass spectrometry.

[0048]

[0049] As shown in Table 1, based on the research logic that high-content components are more likely to exhibit the main effect in the mixed system, rutin, quercetin, styracin, hyperoside, astragaloside, and protocatechuic acid have the highest content. These six polyphenols were selected for subsequent research.

[0050] Application Example 1: Molecular docking analysis of black buckwheat polyphenols and starch digestive enzymes

[0051] The six polyphenols obtained from the characterization analysis in the examples were subjected to molecular docking with amylases. Ligand structures were obtained from the PubChem database (http: / / pubchem.ncbi.nlm.nih.gov / ), and α-amylase (PDB ID: 1b2y) and α-glucosidase (PDB ID: 3a4a) were selected as acceptors for molecular docking from the RCSB PDB database (http: / / www.rcsb.org / ). The molecular docking results were visualized using Discovery Studio 2019 and PyMOL 2.6 software. The optimal binding energy of the protein-ligand complex was calculated using AutoDock Vina 1.2.2, and the binding interface of the protein-ligand complex was systematically analyzed using PLIP and LigPlus.

[0052] like Figure 2As shown in the figure, both rutin and flamingoside exhibit binding activity towards α-amylase, with binding energies of -9.4 and -9.0 kcal / mol, respectively, demonstrating strong binding activity. Rutin forms hydrogen bonds with GLU233 and ASP197, hydrophobic interactions with ALA307 and HIS305, and Pi-Sigma interactions with ILE235 and TRP59. Flamingoside forms hydrogen bonds with THR6 and SER289, hydrophobic interactions with PRO4, and Pi-cationic interactions with ARG252. In the two-ligand docking of α-amylase molecules, the affinity fraction of rutin to α-amylase is -9.3 kcal / mol, and that of flamingoside is -9.1 kcal / mol, both stably binding to the hydrogen bond network through hydrophobic interactions. Specifically, rutin mainly forms multiple hydrogen bonds with polar or charged residues such as GLU, LYS, and HIS, while flamingoside establishes hydrogen bond connections with residues such as SER, ARG, and THR, supplemented by hydrophobic interactions mediated by residues such as PRO. This interaction mode involving different amino acid residues indicates that the two ligands have a certain degree of complementary effects within the same binding region, which is beneficial to enhancing the stability of the overall binding network.

[0053] Similarly, as Figure 3 As shown in the figure, both rutin and pyroside can form stable complexes with α-glucosidase, with binding energies of -10.5 and -10.5 kcal / mol, respectively, indicating that the above polyphenolic compounds have a strong affinity for α-glucosidase. Regarding the interaction mechanism, the ligands mainly bind to key amino acid residues in the enzyme's active site through hydrogen bonds, hydrophobic interactions, van der Waals forces, Pi-Cation, and Pi-Anion interactions, thereby maintaining the stable conformation of the complex. Specifically, rutin can form multiple hydrogen bonds with residues such as ARG442, ARG315, and ASP242, accompanied by hydrophobic interactions and various π interactions, resulting in a relatively complex binding mode; pyroside forms hydrogen bonds with PRO312, ASP242, and ARG442, supplemented by van der Waals forces and Pi-Anion interactions to enhance binding stability. In the docking of two ligands with α-glucosidase molecules, rutin has an affinity fraction of -10.5 kcal / mol with α-glucosidase, while that of flamingoside is -8.3 kcal / mol. Rutin forms a stable hydrophobic interaction interface with aromatic residues such as TYR and PHE, and constructs a multi-point hydrogen bond network with residues such as ARG and ASP; while flamingoside mainly forms hydrophobic interactions with residues such as TRP and ILE, and forms auxiliary hydrogen bonds through residues such as LYS, ASN, and GLU. The differences in the types and spatial distribution of the interacting residues may allow them to occupy different subregions in the binding pocket, thus forming complementary coverage.

[0054] Overall, rutin tends to enhance binding stability through multi-site hydrogen bonds and aromatic interactions, while flamingoside plays a role in supplementing hydrophobic interactions and expanding the interaction range. The differences between the two in binding sites, active residues, and interaction types provide a structural basis for their synergistic effect, potentially contributing to improved efficiency and stability in target protein regulation. Therefore, rutin and flamingoside may not act independently, but rather enhance the binding capacity and regulatory effect on target proteins through spatial complementarity and the superposition of interaction networks. This finding provides a molecular-level basis for elucidating their potential synergistic pharmacological mechanism.

[0055] Application Example 2: Experimental Analysis of Polyphenol Interactions in Black Buckwheat

[0056] V a V represents the inhibition rate of polyphenol "a" against amylase. b Definition V a Same. V ab This indicates the inhibition rate of starch digestive enzymes after the simultaneous addition of polyphenols a and b. V is calculated based on the Bliss independent model. * The interaction between different polyphenolic components is represented by the Bris cooperativity coefficient Q, where Q = V. ab / V * .

[0057] As shown in Table 2, in the interaction experiments of different polyphenol combinations on the inhibition of α-amylase activity, a synergistic effect was observed in the combination of rutin and hyperoside, with a Q value of 1.17. Additive effects were observed in the combinations of rutin and quercetin, rutin and hyperoside, and hyperoside and hyperoside. The remaining combinations showed antagonistic effects. As shown in Tables 3-6, in the interaction experiments of different polyphenol combinations on the inhibition of α-glucosidase activity, a synergistic effect was observed in the combination of rutin and hyperoside, with a Q value of 1.19. An additive effect was observed in the combination of rutin and quercetin. The remaining combinations showed antagonistic effects.

[0058] Table 2. Interaction analysis of six polyphenols in inhibiting α-amylase activity

[0059]

[0060] Application Example 3: Effects of black buckwheat polyphenol extract on postprandial blood glucose in rats

[0061] The black buckwheat polyphenols and their rutin and pyroside standards extracted in Example 4 were stored at 4°C for later use. Thirty male SD rats (weighing 200±20 g) were selected by the Experimental Animal Management and Use Committee of Henan University of Technology (License No.: HAUTETHI-20251207) and purchased from the Experimental Animal Center of Zhengzhou University. All rats were housed individually in an SPF-grade environment with free access to food and water, and the light / dark cycle was 12 h / 12 ​​h. The subjects were randomly divided into 5 groups (n=6): (1) Control group (Con group, administered physiological saline by gavage); (2) Rutin group (R group, administered 140.70 mg / kg of rutin by gavage); (3) Fire-in-the-flavor glycoside group (N group, administered 22.70 mg / kg of fire-in-the-flavor glycoside by gavage); (4) Rutin + Fire-in-the-flavor glycoside (R+N group, administered 140.70 mg / kg of rutin + 22.70 mg / kg of fire-in-the-flavor glycoside by gavage); (5) Black buckwheat polyphenol extract (BB group, administered 200.00 mg / kg of black buckwheat extract by gavage).

[0062] The results are as follows Figure 4 As shown in (A) and (B), by Figure 4 (A) It can be seen that 15 minutes after gavage administration of soluble starch, the blood glucose levels of rats in all groups reached their peak values ​​and then gradually decreased. Throughout the process, the postprandial blood glucose level of group N was consistently higher than that of the other three groups. At time points of 15, 30, 60, 90, and 120 minutes, the blood glucose values ​​of groups R, N, R+N, and BB were significantly lower than those of group Con (p<0.05). Taking the peak value at 15 minutes as an example, the blood glucose values ​​of groups R, N, R+N, and BB were 6.47, 6.67, 5.65, and 5.77 mmol / L, respectively, which were 11.49%, 8.62%, 22.71%, and 21.6% lower than those of group Con (7.31 mmol / L). Notably, groups R+N and BB showed a steeper slope in their blood glucose decline curves after reaching the peak, indicating a stronger inhibitory effect on the digestion and absorption of starch. Figure 4 (B) As can be seen, the AUC analysis further confirmed the above trend. The AUCs of the R group, N group, R+N group, and BB group were 606.56, 622.45, 501.23, and 491.48 mmol·min / L, respectively, which were significantly lower than those of the Con group (663.32 mmol·min / L) (p<0.05). Compared with the Con group, the AUCs of the R group, N group, R+N group, and BB group decreased by 8.56%, 6.16%, 19.17%, and 25.90%, respectively.

[0063] Application Example 4: The synergistic effect of the black buckwheat polyphenol extract (rutin, styracin) prepared in Example 4 on postprandial blood glucose in rats.

[0064] Acute experiment: Mice were acclimatized for 7 days, then fasted for 12 hours according to group assignments, with free access to water during this period. Mice were administered 2 mL of polyphenol solution by gavage according to group assignments. Within 5 minutes after gavage, soluble starch (20% w / v aqueous solution) was administered by gavage at a dose of 2 g / kg body weight. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes after gavage. The area under the blood glucose curve (AUC) was calculated based on the measured blood glucose values. AUC = 0.25 × (0 h blood glucose + 4 × 0.5 h blood glucose + 3 × 2 h blood glucose).

[0065] like Figure 4 As shown in (C) and (D), compared with the Con group, the R+N group and the BB group significantly reduced α-amylase activity in rat pancreatic tissue (p<0.05), and were superior to the R group and the N group. However, no significant difference was found among the groups in serum α-amylase activity, which may be related to the fact that α-amylase is mainly distributed in the pancreas and salivary glands, with a low proportion entering the blood circulation, and the short intervention period.

[0066] Application Example 5: The synergistic effect of the black buckwheat polyphenol extract (rutin, styracin) prepared in Example 4 on amylase digestion in rats.

[0067] Following the postprandial blood glucose test, rats underwent necropsy and blood collection via arterial puncture after anesthesia with ether. Blood samples were allowed to stand at 4°C for 30 min, then centrifuged at 4°C and 4000 rpm for 15 min to separate plasma. The abdominal cavity was dissected along the midline of the rat's abdomen, and small intestinal tissue (jejunum and ileum) and pancreatic tissue were collected. These were stored at -80°C for later use.

[0068] The detection of α-amylase in rat plasma and pancreas, and sucrase and lactase in the small intestine were performed strictly in accordance with the kit instructions.

[0069] The results are as follows Figure 4 As shown in (E) and (F), by Figure 2 It was found that, compared with the Con group, the R+N group and the BB group significantly inhibited the activities of maltase and sucrase (p<0.05). The maltase activities were 308.65 and 328.40 U / mg prot, respectively, and the sucrase activities were 71.97 and 68.62 U / mg prot, respectively, which were also significantly lower than those of the R group and the N group (p<0.05). The experiment confirmed that the combined use of rutin and safflower glycosides, along with black buckwheat polyphenol extract, can synergistically inhibit the activities of pancreatic α-amylase and small intestinal maltase and sucrase, thus delaying carbohydrate digestion and absorption, and effectively reducing postprandial blood glucose.

[0070] Application Example 6: The synergistic effect of black buckwheat polyphenol extracts (rutin, styracin) extracted in Example 4 on pancreatic tissue of mice on a high-fat diet.

[0071] Pancreatic tissue fixed with 1% paraformaldehyde was sequentially dehydrated with 70%, 80%, 90%, 95%, and 100% ethanol, each concentration for 15 min. Subsequently, the tissue was cleared twice with xylene for 30 min each time to thoroughly remove ethanol and improve transparency. The samples were then immersed in paraffin for 2 h, and paraffin-embedded blocks were prepared using an embedding machine. Finally, the embedded samples were pre-cooled on a microtome, and after complete paraffin solidification, they were cut into continuous sections with a thickness of 5 μm. After sectioning, staining and sectioning were performed strictly according to the experimental procedures of the HE staining kit, and the sections were observed under a microscope under magnification. Images of the target samples were acquired using ImageJ software.

[0072] The results are as follows Figure 5 As shown in (A), the Con group mice exhibited intact islet structures and orderly cell arrangement; the HF group, on the other hand, showed typical compensatory changes, with significantly increased islet volume, disordered internal structure, and vacuolar degeneration in some cells. After intervention, the islet structure of all treatment groups improved, with the R+N and BB groups showing islet morphology close to that of the normal group, more orderly cell arrangement, and reduced vacuolar degeneration.

[0073] Application Example 7: The synergistic effect of black buckwheat polyphenol extracts (rutin, styracin) extracted in Example 4 on oral glucose tolerance in mice on a high-fat diet.

[0074] Mice were fasted for 12 hours before the oral glucose tolerance test (OGTT), during which they were allowed free access to water. Baseline blood glucose levels were then measured via tail vein sampling using a glucometer and recorded as 0 min. Subsequently, mice were administered 20% glucose solution by gavage at a dose of 2 g / kg body weight. Blood glucose levels were measured at 15, 30, 60, 90, and 120 min after administration. The tail tip was disinfected with an alcohol swab before each blood collection, and the AUC was calculated.

[0075] The results are as follows Figure 5 As shown in (B) and (C), the blood glucose levels in all groups of mice peaked at 15 min after gavage and then gradually decreased. Compared with the Con group, the blood glucose levels in the HF group were significantly higher at all time points, and the rate of blood glucose recovery was significantly slower. The OGTT blood glucose levels in all intervention groups were lower than those in the HF group, with the R+N and BB groups showing more significant decreases. AUC analysis showed that the AUC value in the HF group was significantly higher than that in the Con group (p<0.05), and the AUC values ​​in all intervention groups were significantly lower than those in the HF group (p<0.05), indicating that both interventions can effectively improve glucose tolerance impairment induced by a high-fat diet.

[0076] Application Example 8: The synergistic effect of black buckwheat polyphenol extracts (rutin, styracin) extracted in Example 4 on oral glucose tolerance in mice on a high-fat diet.

[0077] Eight weeks after gavage, all experimental animals were fasted for 12 hours, with free access to water. On the day of the experiment, anesthesia was administered using anhydrous ether. After complete anesthesia, blood was collected via orbital sampling and placed in an anticoagulant tube containing sodium heparin. The plasma was collected by centrifugation at 4000 rpm for 15 min at 4 °C and then stored at -80 °C for later use. Subsequent experiments strictly followed the ELASA kit instructions to determine INS levels in the plasma.

[0078] The results are as follows Figure 5 As shown in Figure (E), the plasma INS level in the HF group was significantly higher than that in the Con group (p<0.05), which is consistent with the morphological changes of compensatory enlargement of the islets. All intervention groups showed a decrease compared to the HF group, with the R+N group showing the most significant decrease. This indicates that the combined use of rutin and flavonoids, along with black buckwheat polyphenol extract, can enhance glucose tolerance in high-fat diet mice by protecting islet structure, improving insulin sensitivity.

[0079] Application Example 9: Effects of rutin and flavin on the expression of liver glucose metabolism genes in mice fed a high-fat diet

[0080] RNA extraction was performed using the Trizol method. The steps were as follows: Tissue was homogenized thoroughly in Trizol reagent and incubated at room temperature for 5 min. Then, it was centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was transferred to a new centrifuge tube, and 1 / 5 volume of pre-chilled chloroform (4°C) was added. The mixture was vortexed and incubated at room temperature for 3–5 min, followed by centrifugation at 12000 rpm for 15 min at 4°C. The upper aqueous phase was transferred to an enzyme-free centrifuge tube, and an equal volume of pre-chilled isopropanol was added. The mixture was thoroughly mixed and incubated overnight at -20°C to precipitate RNA. The precipitate was centrifuged at 12000 rpm for 15 min at 4°C, the supernatant was discarded, and 1 mL of pre-chilled 75% ethanol was added to wash the precipitate. The precipitate was then centrifuged for 10 min under the same conditions, and the supernatant was discarded. This washing process was repeated once. The precipitate was then dried at room temperature for approximately 10 min to allow the ethanol to evaporate completely until a translucent RNA precipitate was visible. According to RNA yield, 20-100 μL of DEPC water was added to dissolve the precipitate, and incubated at 56℃ for 10 min to promote dissolution. After incubation, the sample was immediately cooled on ice. One-Drop assay was used to determine the RNA concentration and OD260 / 280 ratio. Samples with OD260 / 280 in the range of 1.8-2.0 were selected for subsequent experiments, and the RNA concentration was uniformly adjusted to 1000 ng / μL using DEPC water. Reverse transcription was then performed according to the kit instructions to transcribe RNA into cDNA. The obtained cDNA samples were stored at -80℃ for later use. Real-time quantitative PCR was performed according to the kit instructions to determine the expression levels of relevant primers. Primer sequences are shown in Table 3. The Ct values ​​of each gene template were determined using an RT-qPCR instrument. β-actin was used as an endogenous reference gene, and relative quantification was performed using the Livak method.

[0081] Table 3 Primer Sequences

[0082]

[0083] The results are as follows Figure 6As shown in the figure, the gene expression results of the glycolysis pathway showed that, compared with the Con group, the mRNA expression levels of all glycolysis genes in the liver of mice in the HF group were significantly downregulated (p<0.05); compared with the HF group, the expression levels of both the R+N group and the BB group were significantly upregulated (p<0.05), indicating that the intervention effectively restored the liver's glucose oxidation and utilization capacity. The gene expression results of the gluconeogenesis pathway showed that the mRNA expression levels of G6PC and PCK1 in the liver of the HF group were significantly higher than those in the Con group (p<0.05); the expression levels of both the R+N group and the BB group were significantly downregulated, and the expression levels of the R+N group and the BB group were significantly lower than those of the single-drug groups (p<0.05), indicating that the intervention effectively inhibited the overactivated gluconeogenesis pathway. The results of gene expression analysis of the glycogen metabolism pathway showed that, compared with the Con group, the expression of GYS2 and UGP2 in the liver was significantly downregulated and the expression of PYGL was significantly upregulated in the HF group (p<0.05). In all intervention groups, GYS2 and UGP2 were significantly upregulated and PYGL was significantly downregulated (p<0.05), with the R+N and BB groups showing the most significant improvement, indicating that the intervention restored the dynamic balance between hepatic glycogen synthesis and breakdown. The results of glucose transport gene expression analysis showed that the expression levels of GLUT2 and GLUT4 mRNA in the liver of the HF group were significantly downregulated compared with the Con group (p<0.05); the R+N and BB groups showed the most significant upregulation (p<0.05), indicating that the intervention can improve hepatic glucose sensing capacity and peripheral tissue glucose uptake efficiency. The results of β-cell transcription factor gene expression showed that, compared with the Con group, the expression of PDX1 and MAFA in the pancreas was significantly downregulated and the expression of FOXO1 was significantly upregulated in the HF group; PDX1 and MAFA were significantly upregulated and FOXO1 was significantly downregulated in each intervention group (p<0.05), with the R+N group showing the most significant improvement, indicating that the intervention can improve the inhibition of β-cell transcription factors by a high-fat diet.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A black buckwheat polyphenol extract, characterized in that: The active substances in the black buckwheat polyphenol extract are rutin and pyroside.

2. The black buckwheat polyphenol extract according to claim 1, characterized in that: The mass ratio of rutin to pyroside is 1-10:

1.

3. The black buckwheat polyphenol extract according to claim 1, characterized in that: The mass ratio of rutin to pyroside is 5-8:

1.

4. The method for preparing the black buckwheat polyphenol extract according to any one of claims 1-3, characterized in that, The steps are as follows: add black buckwheat powder to an ethanol aqueous solution, extract, and then evaporate and freeze-dry the resulting extract to obtain black buckwheat polyphenol extract.

5. The method for preparing black buckwheat polyphenol extract according to claim 4, characterized in that: The volume percentage of the ethanol-water solution is 70%; the solid-liquid ratio of the black buckwheat powder to the ethanol-water solution is 1:

15.

6. The method for preparing black buckwheat polyphenol extract according to claim 5, characterized in that: The extraction is any one or more of microwave-assisted extraction, light-assisted extraction, and ultrasonic-assisted extraction.

7. The method for preparing black buckwheat polyphenol extract according to claim 6, characterized in that: The microwave-assisted extraction has a power of 200-500 W and an extraction time of 2-10 min; the light wave-assisted extraction has a power of 200-400 W and an extraction time of 5-15 min; the ultrasonic-assisted extraction has a frequency of 20 kHz, a power of 200-1000 W, and an extraction time of 15-30 min.

8. The use of the black buckwheat polyphenol extract according to any one of claims 1-3 in the preparation of a drug for type 2 diabetes.

9. The use of the black buckwheat polyphenol extract according to any one of claims 1-3 in the preparation of drugs for improving glucose tolerance, insulin resistance or lowering postprandial blood glucose.

10. The application according to claim 8 or 9, characterized in that: The black buckwheat polyphenol extract is obtained by simultaneously inhibiting the activity of α-amylase and α-glucosidase, thereby delaying the digestion and absorption of carbohydrates.

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