White kidney bean polypeptide and application thereof

Abstract

The invention relates to the technical field of polypeptides, and discloses a white kidney bean polypeptide and application thereof. The amino acid sequence of the polypeptide is Asp-Asn-Pro-Phe-Tyr-Phe, and the amino acid sequence of the polypeptide is as shown in the specification. The polypeptide is prepared from white kidney beans through alkaline protease hydrolysis, and experiments prove that the polypeptide has an inhibiting effect on alpha-amylase and alpha-glucosidase, so that the polypeptide can be used for preparing the medicine for reducing blood sugar and can also be used for preparing a health-care product beneficial to maintaining the healthy level of blood sugar.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to a white kidney bean polypeptide and its applications. Background Technology

[0002] Currently, the global incidence of diabetes is rising year by year due to the continuous improvement of people's living standards and unreasonable dietary structure, posing a threat to human health. Imbalance between energy intake and consumption is one of the causes of metabolic diseases such as type II diabetes. Acarbose, as an α-glucosidase inhibitor, has its core side effects directly related to its mechanism of action: by delaying the digestion and absorption of carbohydrates, it often causes gastrointestinal reactions such as bloating, abdominal distension, abdominal pain, and diarrhea. However, serious adverse reactions are rare, but attention should be paid to the risk of rare liver damage and the possibility of hypoglycemia when used in combination with other hypoglycemic drugs.

[0003] White kidney beans are cultivated in all provinces and regions of my country, with Yunnan, Guizhou, and Sichuan having the largest planting areas. Ancient Chinese medical texts record that white kidney beans are sweet and neutral in nature, possessing properties such as warming the middle and lower jiao (digestive system), benefiting the intestines and stomach, stopping hiccups, and nourishing the kidneys and replenishing vital energy. Modern medical research provides increasing evidence that bioactive peptides in plant extracts can positively impact human health through multiple mechanisms. White kidney bean extract can specifically inhibit α-amylase activity in the human body, blocking the hydrolysis and digestion of starch in food, effectively lowering postprandial blood glucose levels and reducing energy intake. Therefore, white kidney beans can be used to develop stable and simple-to-prepare white kidney bean polypeptides with good blood sugar-lowering effects. This not only provides a new raw material for the prevention and auxiliary control of blood sugar levels in diabetic patients but also finds a new source of profit growth for the white kidney bean industry. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the primary objective of this invention is to provide a white kidney bean polypeptide, wherein the amino acid sequence of the white kidney bean polypeptide is Asp-Asn-Pro-Phe-Tyr-Phe(DNPFYF).

[0005] The second objective of this invention is to provide a method for preparing the white kidney bean polypeptide, wherein the white kidney bean polypeptide is prepared from white kidney beans hydrolyzed by alkaline protease (serine protease), and the specific steps are as follows: (1) Dry the white kidney beans, crush them, sieve them, take the bean powder that is sieved, add water to the bean powder and extract it by ultrasound.

[0006] (2) Adjust the solution obtained in step (1) to alkaline, then add alkaline protease for enzymatic hydrolysis to obtain enzymatic hydrolysate. Filter the enzymatic hydrolysate with a filter bag, collect the filtrate, and freeze-dry the filtrate to obtain dried product.

[0007] (3) The dried material is reconstituted and separated using a 10kDa ultrafiltration membrane to collect the components with a molecular weight less than 10kDa.

[0008] (4) A polypeptide with the amino acid sequence DNPFYF can be obtained by performing liquid chromatography-mass spectrometry on components with a molecular weight of less than 10 kDa.

[0009] Preferably, the aperture of the sieve in step (1) is 0.84 mm.

[0010] Preferably, in step (1), the solid-liquid ratio of soybean flour to water is 1:10, in kg:L.

[0011] Preferably, the amount of alkaline protease added in step (2) is 1.1% of the solution mass.

[0012] Preferably, the enzymatic hydrolysis conditions in step (2) are 50°C for 3 hours.

[0013] Preferably, the filter bag used in step (2) is 200 mesh.

[0014] The third objective of this invention is to provide an application of Asp-Asn-Pro-Phe-Tyr-Phe white kidney bean polypeptide in the preparation of hypoglycemic drugs.

[0015] The fourth objective of this invention is to provide an application of Asp-Asn-Pro-Phe-Tyr-Phe white kidney bean polypeptide in the preparation of health products that help maintain healthy blood sugar levels.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The average inhibition rates of the polypeptide DNPFYF described in this invention against α-amylase at concentrations of 0.1, 0.2, 0.4, 0.6, and 1 mg / ml were 69.24%, 89.68%, 94.86%, 96.02%, and 97.65%, respectively; and the average inhibition rates against α-glucosidase were 7.33%, 12.70%, 58.38%, 72.63%, and 98.61%, respectively. It also has good water solubility, a long average retention time in the human body, and low acute oral toxicity, and can be used to prepare hypoglycemic drugs.

[0017] (2) The polypeptide described in this invention can be obtained not only through artificial synthesis, but also from white kidney beans, providing a new way to utilize white kidney beans. Attached Figure Description

[0018] Figure 1 The inhibitory activities of components with molecular weight less than 10 kDa and components with molecular weight greater than 10 kDa on α-amylase were measured.

[0019] Figure 2Molecular docking of DNPFYF with α-amylase (PDB:1PIF).

[0020] Figure 3 This represents the molecular docking of DNPFYF with α-glucosidase.

[0021] Figure 4 The inhibitory activity of DNPFYF against α-amylase.

[0022] Figure 5 The inhibitory activity of DNPFYF against α-glucosidase. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1 Preparation of polypeptides from white kidney beans The white kidney beans used in this embodiment are high-quality large white kidney beans from the Yunnan Academy of Agricultural Sciences. The specific steps are as follows: (1) Dry white kidney beans at 60℃ using conventional forced air drying, pulverize them, pass them through a 0.84mm analytical sieve (No. 3 pharmacopoeia sieve), and take the bean powder that passes through the sieve for extraction.

[0025] (2) Remove impurities from soybean flour, add distilled water at a material-to-liquid ratio of 1:10 to obtain a 10% material concentration soybean meal aqueous solution (1 kg soybean flour to 10 L distilled water), extract with ultrasound for 2 h at 50 °C. After ultrasound, adjust the pH of the soybean meal aqueous solution to 10 with NaOH and add alkaline protease. The amount of alkaline protease added is 1.1% of the mass of the soybean meal aqueous solution. Then, hydrolyze at 50 °C for 3 h and inactivate the enzyme in boiling water for 15 min to obtain the enzymatic hydrolysate.

[0026] (3) After the enzymatic hydrolysate obtained in step (2) is cooled to room temperature, it is filtered through a 200-mesh filter bag. The filtrate is collected and freeze-dried under vacuum to obtain the dried product.

[0027] (4) The dried substance was reconstituted with distilled water and then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain two components with molecular weights less than 10 kDa and greater than 10 kDa. The inhibitory activities of the two components on α-amylase and α-amylase were measured, and the results are as follows: Figure 1 As shown, according to Figure 1The results showed that the component with a molecular weight of less than 10 kDa had the strongest inhibitory activity against α-amylase. Therefore, the component with a molecular weight of less than 10 kDa was selected for the subsequent experiments.

[0028] (5) Analyze the peptide mass spectrometry information of the fractions with less than 10 kDa obtained in step (4) using liquid chromatography-mass spectrometry (HPLC-MS). Chromatographic conditions: pre-column: 150 μm id × 50 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm; analytical column: 150 μm id × 170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9 μm; column temperature: 30℃; mobile phase A: water containing 0.1% formic acid; mobile phase B: 80% acetonitrile containing 0.1% formic acid; flow rate: 0.60 mL / min. The elution gradients were as follows: 0-2 min (4-8% B), 2-35 min (8-28% B), 35-55 min (28-40% B), 55-56 min (40-95% B), 56-66 min (95% B). Mass spectrometry conditions: Primary mass spectrometry parameters were as follows: resolution 70000, AGC target: 3e6, maximum injection time 100 ms, scan range 100 to 1500 mass-to-charge ratio. Secondary mass spectrometry parameters were as follows: resolution 17500, AGC target: 1e5, maximum injection time 50 ms, TopN: 20, NCE / stepped NCE: 28, positive ion mode (ESI+), spray voltage 3.2 kV, capillary temperature 350 °C, desiccator temperature 350 °C. Mass spectrometry data were analyzed using PeaksStudio 8.0 software in the database: uniprotkb_taxonomy_Phaseolus vulgaris L. Target proteins were retrieved, and de novo sequencing was used to analyze the components of the identified peptide fragments. Among the 4996 identified peptides, fragments meeting both "Total Hydrophobicity < -4" and "-10lgP > 15" were simultaneously screened, yielding 1303 fragments. PeptideRanker (…) was then used to analyze these fragments. http: / / distilldeep.ucd.ie / PeptideRanker / Bioactivity prediction scoring was used to retain 18 peptides with a score >0.8, which have potential biological activity, as shown in Table 1.

[0029] Table 1 lists peptides with potential biological activity after screening for “Total Hydrophobicity < -4” and “-10lgP > 15”. The α-amylase inhibitory activity and α-glucosidase inhibitory activity of the peptides shown in Table 1 were evaluated using the computer bioinformatics tool BIOPEP-UWM (https: / / biochemia.uwm.edu.pl / ), and the amino acid sites that produced the inhibitory effect were identified. The results are shown in Table 2, in which one peptide showed inhibitory activity against both α-amylase and α-glucosidase.

[0030] Table 2 Example 2 Predictive properties of absorption, distribution, metabolism, excretion, and toxicity of the four peptides obtained in Example 1 (ADMET) First, the amino acid sequences of the peptides shown in Table 2 were converted into the Simplified Molecular Input Line Canonical System (SMILES). AdmetSAR (http: / / lmmd.ecust.edu.cn / admetsar1) was used to predict the ADME properties of the screened peptides. Peptides with good absorption, good metabolic performance, and low (or non-toxic) toxicity were further analyzed through molecular docking. The screening results are shown in Table 3. According to Table 3, the screened peptides all exhibit good solubility, long average retention time in the human body, good metabolic performance, and low acute oral toxicity. Table 3 shows the ADME characteristic prediction of the screened peptides using SMILES codes. Example 3 The polypeptide DNPFYF, which had the highest bioactivity prediction score in Example 2, was selected and molecularly docked with α-amylase and α-glucosidase, respectively. The molecular docking method was as follows: the 3D structure of the polypeptide was constructed using Chem Draw 20.0, and energy was minimized and converted to "mol2" format. The crystal structure of α-amylase was obtained from the RCSB protein database (http: / / www.rcsb.org), PDBID 1PIF, and the crystal structure of α-glucosidase (UniprotID: P53051) was obtained by searching (https: / / www.uniprot.org / ). Subsequently, the receptor proteins (α-amylase and α-glucosidase) were dehydrated, impurity removed, and hydrogenated using PyMol 3.0.4 and AutoDockTools 1.5.7, and then exported as files as receptors. Molecular docking was performed using AutoDock Vina. All results were visualized and analyzed using PyMol and LigPlot. The optimal binding conformation of the peptide with α-amylase and α-glucosidase was predicted based on the binding energy score, and the interactions between the peptide and the active sites of α-amylase and α-glucosidase were constructed. The results are shown in Table 4. DNPFYF established four hydrogen bonds with α-amylase (Trp59 (2.5 Å), Gln63 (2.1 Å, 2.7 Å, 2.0 Å)) and five hydrogen bonds with α-glucosidase (Asn417 (2.3 Å), Glu421 (2.2 Å; only hydrogen bonds smaller than 5 Å are shown in the 3D diagram)). This peptide also formed one and four hydrophobic interactions with α-amylase and α-glucosidase, respectively. Table 4 shows the interactions between DNPFYF and α-amylase; molecular docking is shown in [reference needed]. Figure 2 Table 5 shows the interaction between DNPFYF and α-glucosidase; molecular docking is shown in [reference needed]. Figure 3 .

[0031] Table 4. Interactions between DNPFYF and α-amylase Table 5. Interactions between DNPFYF and α-glucosidase Example 4 Artificial synthesis of DNPFYF and assay of its inhibitory activities on α-amylase and α-glucosidase The solid-phase synthesis method was used, and the synthesis sequence from the C-terminus to the N-terminus of the sequence (i.e., phenylalanine-tyrosine-phenylalanine-proline-asparagine-aspartic acid) was followed. The specific steps are summarized as follows: (1) Initiation and coupling: After the resin is swollen, the C-terminal amino acid of the α-amino group of the first amino acid protected by the Fmoc group (DIEA diisopropylethylamine) is linked to the resin through its carboxyl group.

[0032] (2) Deprotection: Remove the Fmoc protecting group of the newly attached amino acid with a decapping solution (such as N,N-dimethylformamide containing piperidine) to expose the free amino group.

[0033] (3) Activation and coupling of the next amino acid: The next Fmoc protected amino acid is pre-activated in solution with an activator (such as HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate)) and a base (DIEA (N,N-diisopropylethylamine)).

[0034] (4) Add the activation solution to the resin so that the carboxyl group of the new amino acid reacts with the exposed amino group on the resin to form a peptide bond.

[0035] (5) Detection and capping: The coupling was verified to be complete using the triketone assay. Unreacted amino groups were capped with an acetic anhydride / pyridine mixture to prevent the formation of byproducts with deleted sequences.

[0036] (6) Loop: Repeat steps (2) to (5) until the target sequence is completed.

[0037] (7) Final cleavage and purification: After synthesis, the complete peptide chain was cleaved from the resin using a strong acid cleavage buffer (the volume ratio of trifluoroacetic acid, ethylenedithiol, triisopropylsilane, and distilled water in the strong acid cleavage buffer was 95:2:2:1), while simultaneously removing all side chain protecting groups. The filtrate containing the product was poured into cold diethyl ether to precipitate the crude peptide. After centrifugation and washing, the crude product was obtained. The crude product was purified to 98% using high performance liquid chromatography.

[0038] The prepared peptides were prepared into active peptide solutions with concentrations of 0.1, 0.2, 0.4, 0.6, and 1 mg / mL using PBS buffer. The α-amylase inhibitory activity of the peptide DNPFYF was determined using the DNS colorimetric method. The results are as follows: Figure 4 As shown in the figure, at concentrations of 0.1-1 mg / ml, the α-amylase inhibitory activity of peptide DNPFYF was significantly higher than that of acarbose. The α-glucosidase inhibitory activity of peptide DNPFYF was determined spectrophotometrically based on the p-nitrophenol colorimetric reaction, and the results are as follows. Figure 5 As shown in the figure, at a concentration of 1 mg / ml, the α-glucosidase inhibitory activity of the polypeptide DNPFYF is comparable to that of acarbose.

[0039] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A white kidney bean polypeptide, characterized in that: The amino acid sequence of the white kidney bean polypeptide is Asp-Asn-Pro-Phe-Tyr-Phe.

2. The use of the white kidney bean polypeptide of claim 1 in the preparation of hypoglycemic drugs.

3. The use of the white kidney bean polypeptide of claim 1 in the preparation of health products that help maintain healthy blood sugar levels.

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