Trichosanthes kirilowii Maxim seed polypeptide with alpha-glucosidase inhibitory activity and preparation method thereof

Abstract

The invention discloses trichosanthes kirilowii maxim seed polypeptide with alpha-glucosidase inhibitory activity and a preparation method thereof. Four Trichosanthes kirilowii Maxim seed polypeptides with alpha-glucosidase inhibitory activity are obtained through Trichosanthes kirilowii Maxim seed protein preparation, enzymolysis, separation and purification and identification, and the sequences of the four Trichosanthes kirilowii Maxim seed polypeptides are respectively HRQTWD, HANRLP, FGGVGER and SYAPEDR. Molecular docking analysis results show that interaction between the trichosanthes kirilowii maxim seed hydrolytic peptide and amino acid residues of an alpha-glucosidase active pocket is mainly based on hydrogen bonds, electrostatic interaction and hydrophobic interaction, and residues D203, D327, D443, F450, D542 and F575 are key binding sites interacting with the four polypeptides. The trichosanthes kirilowii maxim seed polypeptide with alpha-glucosidase inhibitory activity prepared by the invention can be used as a food-borne hypoglycemic component to be used for developing health-care foods and medicines for reducing blood sugar.

Description

Technical Field

[0001] The invention belongs to the technical field of development of food-derived active peptides, and particularly relates to a trichosanthes seed polypeptide having α-glucosidase inhibitory activity and a preparation method thereof. Background Art

[0002] Diabetes is a metabolic disorder characterized by high blood sugar levels. Its prevalence continues to rise worldwide, and the global incidence is projected to reach 9.9% by 2045. While diabetes typically has no obvious symptoms in its early stages, prolonged illness can lead to a range of complications, including vascular complications, dementia, liver disease, and various cancers, severely impacting patients' health and quality of life. Approximately 90% of patients with diabetes have type 2 diabetes, and controlling postprandial hyperglycemia can effectively stabilize blood sugar levels in patients with type 2 diabetes. Currently, a variety of glucose-lowering drugs are available on the market for controlling postprandial hyperglycemia, such as metformin and thiazolidinediones. However, these drugs are associated with certain side effects. For example, metformin may cause gastrointestinal disturbances, and the combination of thiazolidinediones and insulin therapy may lead to heart failure.

[0003] In recent years, research has revealed that ingredients extracted from natural plants, such as phenols, flavonoids, and peptides, also exhibit promising hypoglycemic effects and are emerging as promising candidates for hypoglycemic drugs. Most of these are inhibitors of α-glucosidase (EC 3.2.1.20). α-glucosidase is a key enzyme in carbohydrate metabolism and a key target for regulating blood sugar. α-glucosidase inhibitory peptides, due to their diverse structures and bioactivities, natural origin, and generally low toxicity, are effective inhibitors for controlling type 2 diabetes.

[0004] A variety of α-glucosidase inhibitory peptides have been isolated from natural plants and food sources. Four α-glucosidase inhibitory peptides, FYNPAAGR, PGVLPVAS, FFVPPSQQ, and FSYNPQAG, were isolated from heat-pressed peanut meal protein hydrolysate. Molecular docking revealed that the peptides occupy the active pocket of α-glucosidase through hydrogen bonding, hydrophobic interactions, salt bridges, and π-stacking, thereby preventing α-glucosidase from forming a complex with the substrate. Furthermore, the inhibitory activity of heat-pressed peanut meal protein hydrolysate against α-glucosidase was stable during heat treatment, pH manipulation, and in vitro gastrointestinal digestion. The α-glucosidase inhibitory peptides TGLGR and SPVI isolated from hemp seed meal exhibited inhibition rates of 30.66% and 28.09%, respectively, at 10 mg / mL. FR and TGLGR exhibited comparable inhibitory activities, with both exhibiting approximately 20% inhibition at 10 mg / mL. IC of α-glucosidase inhibitory peptides LLPLPVLK, SWLRL and WLRL derived from soy protein50 The values ​​were 237.43±0.52, 182.05±0.74 and 165.29±0.74 μmol / L, respectively.

[0005] Trichosanthes kirilowii is a traditional resource in my country, and its roots, stems, leaves, fruits, and seeds can all be used as medicine. Trichosanthes kirilowii seeds are rich in nutrients, mainly containing oils, sterols, triterpenoid saponins, proteins, various amino acids, and 16 inorganic elements such as calcium, iron, zinc, and selenium. Protein is a key nutrient in trichosanthes kirilowii seeds, accounting for approximately one-third of their dry weight. However, current research on trichosanthes kirilowii seed protein focuses primarily on extraction processes, and the hypoglycemic activity and mechanism of trichosanthes kirilowii seed polypeptides remain unclear, hindering the processing and utilization of trichosanthes kirilowii seed protein and the application of its hydrolyzed peptides in the development of products such as hypoglycemic products. Summary of the Invention

[0006] The purpose of the present invention is to provide a trichosanthes seed hydrolyzed peptide with α-glucosidase inhibitory activity and a preparation method thereof.

[0007] To achieve the above objectives and other related objectives, the present invention provides a technical solution: a trichosanthes seed polypeptide having α-glucosidase inhibitory activity, wherein the amino acid sequence of the trichosanthes seed polypeptide is one of the following: HRQTWD, HANRLP, FGGVGER, SYAPEDR.

[0008] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a method for preparing a trichosanthes seed polypeptide having α-glucosidase inhibitory activity, comprising the following steps:

[0009] Step 1: Preparation of crude protein from Trichosanthes seeds

[0010] The seeds of Trichosanthes kirilowii are peeled and crushed to obtain powder, which is then defatted using n-hexane, and then dissolved in alkali and precipitated with acid to obtain crude protein from the seeds of Trichosanthes kirilowii;

[0011] Step 2: Enzymatic hydrolysis of Trichosanthes seeds

[0012] The protein of the trichosanthes seeds is hydrolyzed by using a protease, and the obtained hydrolyzate is subjected to enzyme inactivation treatment and then centrifuged to obtain the supernatant to obtain a crude extract of the trichosanthes seeds polypeptide;

[0013] Step 3: Isolation and purification of Trichosanthes seed polypeptide

[0014] The crude extract of the trichosanthes seed polypeptide is separated and purified by ultrafiltration and liquid chromatography, and a component with a molecular weight of less than 3 kDa is separated based on the difference in molecular weight;

[0015] The components with a molecular weight less than 3 kDa were freeze-dried, then prepared into a solution with water, and separated using a semi-preparative high performance liquid chromatography; the mobile phase was a mixture of solvent A and solvent B; solvent A was water containing 0.1% by mass formic acid, and solvent B was acetonitrile containing 0.1% by mass formic acid;

[0016] Semi-preparative high performance liquid chromatography was used for step-by-step separation, with the mobile phase being a mixture of solvent A and solvent B; the gradient was set as follows: from 0 to 5 minutes, the concentration of solvent B was maintained at 5%; from 5 to 50 minutes, the concentration of solvent B was increased to 100%.

[0017] The preferred technical solution is: the alkali dissolution and acid precipitation method comprises: preparing a solution according to a mass ratio of 1:8-12 between the material and the liquid, adjusting the pH value of the solution to 10.0 with a sodium hydroxide solution, stirring at 50-65°C for 1-3 hours, and centrifuging at a speed of 5000-8000 r / min for 10-20 minutes, taking the supernatant, adjusting the pH value to 3.5 with a hydrochloric acid solution, stirring at 50-65°C for 1-3 hours, and centrifuging at a speed of 5000-8000 r / min for 10-20 minutes, washing the precipitate with water, adjusting the pH value to neutral, and then freeze-drying and refrigerating to obtain the trichosanthes seed crude protein.

[0018] The preferred technical solution is: in step 2, the crude protein of the trichosanthes seeds is prepared into a solution, and then the pH value of the solution is adjusted to 6.0-9.5, and at least one of the following five proteases is added, and the enzyme activity reaches 5000-20000 U / g: alkaline protease, trypsin, neutral protease, flavor protease and papain; the sample solution is subjected to oscillation enzymolysis at 45-65° C. for 2-4 hours, and the hydrolysis degree of the trichosanthes seeds protein is determined by a pH-start method during the enzymolysis process. After the enzymolysis is completed, the enzyme is inactivated to terminate the reaction; the supernatant is collected after centrifugation at a speed of 5000-8000 r / min for 15-30 minutes, and freeze-dried to form a lyophilized powder. The lyophilized powders of the five enzymatic hydrolyses are prepared into a solution, and the α-glucosidase inhibition rate is compared and analyzed.

[0019] The preferred technical solution is: in step 3, the trypsin hydrolyzate of the Trichosanthes seeds is separated by an ultrafiltration tube equipped with 3kDa and 10kDa ultrafiltration membranes, and the hydrolyzate is divided into three components according to the molecular weight: MW < 3kDa, 3-10kDa, and > 10kDa.

[0020] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0021] 1. The present invention discovered new peptides derived from Trichosanthes seeds, among which the peptide sequences with the highest α-glucosidase inhibitory activity were hexapeptide HRQTWD, hexapeptide HANRLP, heptapeptide FGGVGER and heptapeptide SYAPEDR, and their IC50 They were 71.31±1.19, 654.98±24.94, 805.87±25.11 and 1245.22±147.3 μM respectively.

[0022] 2. The present invention uses molecular docking analysis, and the results show that the main interactions between HRQTWD, HANRLP, FGGVGER and SYAPEDR and the amino acid residues of α-glucosidase are hydrogen bonds, electrostatic interactions and hydrophobic interactions, but there are similarities and differences in the specific sites of action.

[0023] 3. The trichosanthes seed polypeptide with α-glucosidase inhibitory activity prepared by the present invention can be used as a food-derived hypoglycemic ingredient for the development of health foods and medicines for the purpose of lowering blood sugar. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Liquid chromatogram of Trichosanthes kirilowii seed polypeptide with Mw<3kDa.

[0025] Figure 2 The inhibition rate of each trichosanthes seed polypeptide fraction on α-glucosidase after liquid chromatography purification (2 mg / mL); different letters indicate that the inhibition rate of each trichosanthes seed polypeptide fraction on α-glucosidase has significant differences (p < 0.05).

[0026] Figure 3 Inhibition rate of α-glucosidase by purified fractions of Trichosanthes kirilowii seed polypeptide F2 (2 mg / mL); different letters indicate significant differences in the inhibition rate of α-glucosidase among the purified fractions of F2 (p < 0.05).

[0027] Figure 4 Secondary mass spectrum of Trichosanthes kirilowii seed polypeptide.

[0028] Figure 5 Schematic diagram of molecular docking for the interaction between Trichosanthes kirilowii seed polypeptide and α-glucosidase. DETAILED DESCRIPTION

[0029] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in these embodiments.

[0030] See also Figure 1-5 It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for understanding and reading by those familiar with this technology, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no technical significance. Any structural modification, change in proportion or adjustment of size. The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.

[0031] Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. Trichosanthes seeds were purchased from Suqian Sunrise Seed Co., Ltd. Hexane, ethanol, acetonitrile, sodium hydrogen phosphate, acarbose, flavor protease, and neutral protease were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Hydrochloric acid, formic acid, sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide, and sodium carbonate were purchased from Sinopharm Chemical Reagent Co., Ltd. 4-Nitrophenyl-β-D-pyranoglucopyranoside (pNPG) and porcine trypsin were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. α-Glucosidase, alkaline protease, and papain were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Synthetic peptides (HRQTWD, HANRLP, FGGVGER, and SYAPEDR) were from Hefei Guopeptide Co., Ltd.

[0032] PBS (pH = 7.4) is prepared as follows: Dissolve 8g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4 in 800mL of distilled water, adjust the pH to 7.4, and then add water to 1L. To adjust the pH, simply add an appropriate amount of hydrochloric acid or sodium hydroxide solution.

[0033] Example 1: Preparation of Trichosanthes kirilowii seed polypeptides with α-glucosidase inhibitory activity

[0034] (1) Preparation of crude protein from Trichosanthes seeds: The Trichosanthes seeds were peeled and crushed to obtain powder, which was then defatted using n-hexane and dried to obtain defatted Trichosanthes seeds powder. The dried defatted Trichosanthes seeds powder was prepared into a solution at a material-liquid ratio of 1:10, and the pH value of the protein solution was adjusted to 10.0 with 1.0 mol / L sodium hydroxide solution. The solution was stirred at 55°C for 1.5 h and then centrifuged at 6000 r / min for 15 min. The supernatant was collected and the pH value was adjusted to 3.5 with 1.0 mol / L hydrochloric acid solution. The solution was stirred for 1.5 h and then centrifuged at 6000 r / min for 15 min. The precipitate was washed with water, the pH value was adjusted to neutral, and the solution was freeze-dried and stored in a refrigerator to obtain crude protein from Trichosanthes seeds.

[0035] (2) Proteolysis of Trichosanthes seeds: A quantitative amount of Trichosanthes seeds protein was weighed and prepared into a solution of corresponding concentration according to the substrate concentration of 4%. The solution was divided into five groups according to the type of protease added, with three identical samples in each group. That is, three independent experiments were conducted on each sample during the entire experimental process. The types of proteases and the optimal hydrolysis conditions were as follows: alkaline protease (pH 9.0, 55℃), trypsin (pH 8.5, 50℃), neutral protease (pH 7.0, 50℃), flavor protease (pH 7.0, 55℃) and papain (pH 6.5, 55℃). First, the prepared solution was pretreated at 90℃ for 10 minutes in a water bath, and then the protease was added after adjusting to the optimal pH value of the corresponding enzyme. The enzyme dosage was fixed at 6000U / g. After adjusting the shaker to the optimal temperature, the sample solution was shaken and enzymatically digested for 2.5 hours. During this time, a 0.1 mol / L NaOH standard solution was added dropwise to maintain the optimal pH. The degree of hydrolysis of the trichosanthes seed protein was calculated by recording the amount of alkali solution added. After the enzymatic hydrolysis was completed, the sample solution was placed in a 100°C water bath for 10 minutes to inactivate the enzyme. The supernatant was then centrifuged at 6000 rpm for 20 minutes and the resulting lyophilized powder was prepared into 2 mg / mL solutions, and the inhibition rates of α-glucosidase were compared.

[0036] The results in Table 1 show that alkaline protease achieved a hydrolysis degree of 22.23 ± 1.78%, the highest value among all groups. Trypsin followed closely behind, achieving a hydrolysis degree of 21.92 ± 0.69%. The hydrolysis degrees of the remaining three enzymes differed significantly from those of the aforementioned two enzymes. This is because different proteases have different specific cleavage sites for peptide bonds.

[0037] As shown in Table 2, the degree of proteolysis is not completely positively correlated with the α-glucosidase inhibition rate. In the α-glucosidase inhibition activity experiment, the inhibitory activity of trypsin hydrolyzate is the highest, reaching 35.15 ± 1.98%. Although the hydrolysis degree of papain is the lowest, its inhibition rate can still reach 20.31 ± 7.24%. This is because the recognition effect of these two proteases on substrate specific sites - although limited cleavage sites limit the degree of hydrolysis, it is easier to release highly active inhibitory peptides. Considering that trypsin has good stability in vivo and presents a higher degree of hydrolysis at the same time, the present invention finally selects trypsin hydrolyzate to carry out subsequent experiments.

[0038] Table 1: Degree of protease hydrolysis

[0039]

[0040]

[0041] Table 2: Hypoglycemic activity of enzymatic hydrolysates

[0042] Types of enzymes α-glucosidase inhibition rate / % Alkaline protease <![CDATA[28.23±7.83 bc ]]> Trypsin <![CDATA[35.15±1.98 b ]]> Flavor protease <![CDATA[30.24±1.56 b ]]> Neutral protease <![CDATA[13.93±3.61 d ]]> Papain <![CDATA[20.31±7.24 cd ]]>

[0043] (3) Isolation and purification of Trichosanthes seed polypeptides: The Trichosanthes seed protease hydrolysate obtained in the previous step was fractionated using 3kDa and 10kDa ultrafiltration tubes. It was divided into three fractions based on molecular weight: MW < 3kDa, 3-10kDa, and >10kDa. After the three fractions were collected, concentrated, and freeze-dried, they were prepared into solutions with a concentration of 2 mg / mL, and their inhibitory effects on α-glucosidase activity were compared and analyzed.

[0044] The results in Table 3 show that the α-glucosidase inhibitory activity of the hydrolyzate showed a significant molecular weight dependence. The inhibition rate of the <3 kDa fraction was 27.78 ± 0.28%, significantly higher than that of the other fractions. This is because low-molecular-weight peptides directly act on the active site of α-glucosidase, blocking its substrate binding region by changing the enzyme's spatial conformation. However, for longer-chain and higher-molecular-weight peptides, their binding efficiency to the α-glucosidase active site is significantly reduced due to steric hindrance. Therefore, low-molecular-weight peptides with a molecular weight of less than 3 kDa generally exhibit stronger α-glucosidase inhibitory potential.

[0045] Table 3: Hypoglycemic activity of ultrafiltration peptides

[0046] Components α-glucosidase inhibition rate / % <3 kDa <![CDATA[27.78±0.28 b ]]> 3kDa-10kDa <![CDATA[15.87±0.99 c ]]> >10kDa <![CDATA[14.45±0.27 d ]]>

[0047] Example 2: Further purification of Trichosanthes kirilowii seed polypeptide

[0048] After ultrafiltration, the fraction with Mw < 3 kDa was freeze-dried and then prepared into a solution. Semi-preparative high performance liquid chromatography was used for separation. A 5C18C column (10 μm × 20 mm × 250 mm) was used, and gradient elution was performed using water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B) as the mobile phases. The gradient was set as follows: from 0 to 5 minutes, the concentration of solvent B was maintained at 5%; from 5 to 50 minutes, the concentration of solvent B was increased to 100%. The flow rate was 10 mL / min, the injection volume was 8 mL, and the detection wavelength was 280 nm. Finally, the α-glucosidase inhibition rate of each separated fraction was determined at the same concentration (2 mg / mL).

[0049] The above process of separating the trichosanthes seed polypeptide is carried out according to Figure 1 The fraction was collected in a 5-well buffered saline solution and divided into six subfractions (F1 to F6) according to the elution time, and their inhibition rates on α-glucosidase were tested.

[0050] Depend on Figure 2 The results showed that the inhibition rates of the components were different, among which the F2 component showed the highest α-glucosidase inhibition rate, reaching 60.67±1.85%. Therefore, the F2 component was further separated, such as Figure 1 , the fraction was divided into two subfractions (F2-1, F2-2), and their inhibition rates on α-glucosidase were tested.

[0051] Depend on Figure 3 The results showed that the inhibition rates among subfractions were different, and the F2-2 fraction showed a higher α-glucosidase inhibition rate, reaching 49.33±0%.

[0052] Nano LC-MS / MS was used to sequence the most active F2-2 trichosanthes seed peptide: an appropriate amount of sample was taken and C 18 Desalting was performed using a desalting column. Samples were analyzed by LC-MS / MS equipped with an online nanospray ion source. The system consisted of an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, MA, USA) coupled to an EASY-nano LC1200. A total of 2 μL of sample was loaded onto an Acclaim PepMap C18 analytical column, 75 μm x 25 cm. The sample was separated using a 60-min gradient with a flow rate of 350 nL / min, a column temperature of 40°C, and an electrospray voltage of 2 kV. The gradient started with 2.2% phase B and increased nonlinearly to 50% over 51 minutes, then to 90% over 3.5 minutes, where it was maintained for 5.5 minutes. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 200-1500; resolution: 60,000; Normalized AGC target: 300%; maximum injection time: 25 ms; (2) HCD-MS / MS: resolution: 15,000; Normalized AGC target: 50%; maximum injection time: 22 ms; collision energy: 30%; dynamic exclusion time: 30 s.

[0053] Tandem mass spectra were analyzed using PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The database used was the uniprot-Trichosanthes kirilowii (version 2023, 259 entries) database, with a "none" digestion setting. The search parameters included a fragment ion mass tolerance of 0.02 Da, a precursor mass tolerance of 10 ppm, and variable modifications: oxidation (M) 15.99 and deamidation (NQ) 0.98. Proteins were considered to contain at least one unique peptide; peptides were considered to have a cardinal value of -101 gP ≥ 20.

[0054] Through the above steps, 6 candidate peptides were prioritized from 227 candidate peptides according to their specificity and synthetic feasibility. The mass spectra are shown in Figure 2. Figure 4 The comprehensive structural and physicochemical information of these peptides were analyzed using the PepDraw visualization tool (https: / / www2.tulane.edu / ~biochem / WW / PepDraw / ), and their amino acid sequences, molecular weights, and hydrophobicities are shown in Table 4.

[0055] It is generally believed that suitable candidate peptides for α-glucosidase inhibitors have a molecular weight of less than 2000Da and contain 2-9 amino acids. Short peptides with low molecular weight can more effectively penetrate into the catalytic region of the enzyme due to their compact structure, and therefore have higher inhibitory activity. As shown in Table 4, the molecular weights of the first four polypeptides are all less than 2000Da and are less than 9 amino acids in length. This structural feature is consistent with the polypeptides with α-glucosidase inhibitory activity recorded in previous studies, such as IQAEGGLT from quinoa and SWLRL from soybeans. Therefore, it can be considered that HRQTWD (SEQ ID NO.1), HANRLP (SEQ ID NO.2), FGGVGER (SEQ ID NO.3), and SYAPEDR (SEQ ID NO.4) are candidate peptides for α-glucosidase inhibitors with great potential.

[0056] The hydrophobicity and terminal amino acids of the peptide also influence its inhibitory activity against α-glucosidase by regulating enzyme interactions. The higher the hydrophobicity, the stronger the α-glucosidase inhibitory activity through hydrophobic binding. HRQTWD (SEQ ID NO. 1), HANRLP (SEQ ID NO. 2), FGGVGER (SEQ ID NO. 3), and SYAPEDR (SEQ ID NO. 4) have a hydrophobicity of 12-18 kcal / mol, similar to the known inhibitory peptides ERFQ, VHHK, and EKFMKE from osmanthus seeds, suggesting similar efficacy.

[0057] Table 4: Candidate Trichosanthes kirilowii seed polypeptide sequences

[0058] Serial number sequence length Molecular weight (Da) Hydrophobicity (kcal / mol) 1 HRQTWD 6 841.3820 +14.61 2 HANRLP 6 706.3864 +12.28 3 FGGVGER 7 720.3544 +14.62 4 SYAPEDR 7 836.3652 +17.37 5 PRINVQLFIDSI 12 1413.7955 +8.79 6 LGANVGSAQGPTGLGK 16 1425.7552 +16.96

[0059] Note: The amino acid abbreviations in the sequence are: A-Alanine (Ala); D-Aspartic acid (Asp); E-Glutamic acid (Glu); P-Proline (Pro); H-Histidine (His); K-Lysine (Lys); F-Phenylalanine (Phe); Y-Tyrosine (Tyr); L-Leucine (Leu); Q-Glutamine (Gln); I-Isoleucine (Ile); R-Arginine (Arg); N-Asparagine (Asn); W-Tryptophan (Trp); T-Threonine (Thr); V-Valine (Val); S-Serine (Ser); G-Glycine (Gly).

[0060] Example 3: Verification Experiment

[0061] According to the results obtained from mass spectrometry analysis, the α-glucosidase inhibitory activities of HRQTWD (SEQ ID NO. 1), HANRLP (SEQ ID NO. 2), FGGVGER (SEQ ID NO. 3), and SYAPEDR (SEQ ID NO. 4) were compared.

[0062] As shown in Table 5, the IC values ​​of HRQTWD (SEQ ID NO. 1), HANRLP (SEQ ID NO. 2), FGGVGER (SEQ ID NO. 3) and SYAPEDR (SEQ ID NO. 4) were 50 The concentration ranged from 70 to 1400 μM, which was more potent than known α-glucosidase inhibitory peptides such as AGGFR, LDLQR, and LDNFR. These results confirmed the presence of peptides with hypoglycemic activity in the F2-2 fraction and clearly demonstrated that trypsin effectively released peptides with potential α-glucosidase inhibitory ability from Trichosanthes seed proteins.

[0063] Table 5: Half inhibition concentration (IC50) of each synthetic peptide 50 )

[0064]

[0065]

[0066] Example 4: Analytical docking of Trichosanthes kirilowii seed polypeptide and α-glucosidase

[0067] Human maltase-glucoamylase (PDB ID: 2QMJ) was selected as a structural template for molecular docking studies with HPEPDOCK. The site configuration used the software's pre-set configuration to position the binding site coordinates around the acarbose ligand. The optimal docking conformation was determined through a comprehensive evaluation of computational affinity metrics and subsequent empirical structural verification using molecular testing.

[0068] Binding affinity was assessed using a docking score, with lower scores indicating stronger predicted binding. As shown in Table 6, HRQTWD (SEQ ID NO. 1) had the lowest docking score of -201.792, followed by HANRLP (SEQ ID NO. 2), FGGVGER (SEQ ID NO. 3), and SYAPEDR (SEQ ID NO. 4). These results indicate that HRQTWD exhibits the strongest binding affinity to α-glucosidase.

[0069] Table 6: Peptide ranking and docking scores

[0070] Serial number sequence Docking score 1 HRQTWD -201.792 2 HANRLP -178.792 3 FGGVGER -175.261 4 SYAPEDR -168.425

[0071] Representative docking models of each peptide with α-glucosidase (PDB ID: 2QMJ) are shown in Figure 2. Figure 5 The analysis showed that the main interactions between peptides and enzymes include hydrogen bonds, electrostatic forces, and hydrophobic interactions. The specific sites of interaction on the peptides and enzymes where secondary bonds exist are shown in Table 7.

[0072] In terms of hydrogen bonding, all tested peptides exhibited unique hydrogen bonding configurations when interacting with α-glucosidase (PDB ID: 2QMJ), and these hydrogen bonding interactions significantly impacted α-glucosidase inhibitory activity. Specifically, H1R2Q3T4W5D6 (SEQ ID NO. 1) formed six hydrogen bonds with the enzyme, R2 formed two hydrogen bonds with D327, Q3 formed single hydrogen bonds with D203, S448, and N449, and W5 formed one hydrogen bond with T544. H1A2N3R4L5P6 (SEQ ID NO. 2) exhibited nine hydrogen bonds, including two with T544, two with Q603, and one with Y605, R4 formed one hydrogen bond each with D327 and D443, and two with T205. F1G2G3V4G5E6R7 (SEQ ID NO.3) forms 6 hydrogen bonds, of which F1 forms 1 bond each with D203 and D542, R7 forms 1 bond with P206, and 3 bonds with T544. S1Y2A3P4E5D6R7 (SEQ ID NO.4) contains the most hydrogen bonds, with a total of 10, of which S1 forms 1 bond with D203, Y2 forms 1 bond with D443 and D237, E5 forms 2 and 1 bond with T205 and N207, respectively, D6 forms 1 bond each with N207 and N209, and R7 forms 1 bond each with T546 and D549. These findings demonstrate the key role of hydrogen bonds in promoting the binding of polypeptides to α-glucosidase. Each polypeptide exhibits a different bonding pattern, which determines their different α-glucosidase inhibition strength.

[0073] In terms of electrostatic and hydrophobic interactions, R2 in H1R2Q3T4W5D6 (SEQ ID NO. 1) forms a salt bridge with D327, with D443 contributing to this electrostatic interaction. Furthermore, W5 forms hydrophobic interactions with A576 and L577. R4 in H1A2N3R4L5P6 (SEQ ID NO. 2) forms salt bridges with D327 and D443, while A2 forms a hydrophobic interaction with F450. F1 in F1G2G3V4G5E6R7 (SEQ ID NO. 3) forms a salt bridge with D203 and also forms hydrophobic interactions with W406, Y299, and F575. R7 forms a salt bridge with D549, and V4 forms hydrophobic interactions with A576 and L577. In S1Y2A3P4E5D6R7 (SEQ ID NO. 4), S1 and R7 form salt bridges with D203 and D549, respectively, while Y2 forms hydrophobic interactions with W406, Y299, and F575, and A3 forms a hydrophobic interaction with A576. These analysis results are consistent with the conclusions reported in relevant literature, indicating that electrostatic and hydrophobic interactions are the primary factors affecting the α-glucosidase inhibitory properties of the peptide.

[0074] In summary, these four peptides formed distinct secondary interactions with α-glucosidase, enhancing their α-glucosidase inhibitory activity. Previous studies have shown that primary binding sites involved in inhibiting α-glucosidase activity typically contain amino acid residues such as arginine, tyrosine, and aspartic acid, with aspartic acid playing a key role in the catalytic process. This study identified residues D203, D327, D443, F450, D542, and F575 as key binding sites for these four peptides. D203, D327, and D542 are also the primary binding sites for the exemplary α-glucosidase inhibitor acarbose, while F450 and F575 are common hydrophobic interaction sites for several α-glucosidase inhibitory peptides. Furthermore, residues D327 and D443, located within the enzyme's active pocket, frequently form hydrogen bonds or salt bridge interactions with HRQTWD (SEQ ID NO. 1), HANRLP (SEQ ID NO. 2), and SYAPEDR (SEQ ID NO. 4). The arginine residues present in HRQTWD (SEQ ID NO.1) and HANRLP (SEQ ID NO.2) showed significant interactions with both D327 and D443. Similar binding patterns were observed in other α-glucosidase inhibitory peptides, such as PPSPRP from Bacillus subtilis fermented laver, LDLQR and AGGFR from wheat germ, and ERFQ from Osmanthus fragrans seeds, which may be the reason why HRQTWD (SEQ ID NO.1) and HANRLP (SEQ ID NO.2) have higher α-glucosidase inhibitory activity. Although FGGVGER (SEQ ID NO.3) did not show strong interactions with these two key residues, its abundant hydrophobic contact sites may contribute to its enhanced α-glucosidase inhibitory activity.

[0075] Table 7: Interaction sites and secondary bond types between peptides and proteins

[0076]

[0077]

[0078] Example 5: A Trichosanthes seed polypeptide having α-glucosidase inhibitory activity and its preparation method

[0079] A trichosanthes seed polypeptide with α-glucosidase inhibitory activity is composed of the following amino acid residues: HRQTWD.

[0080] A trichosanthes seed polypeptide with α-glucosidase inhibitory activity is composed of the following amino acid residues: HANRLP.

[0081] A trichosanthes seed polypeptide with α-glucosidase inhibitory activity is composed of the following amino acid residues: FGGVGER.

[0082] A trichosanthes seed polypeptide with α-glucosidase inhibitory activity is composed of the following amino acid residues: SYAPEDR.

[0083] A method for preparing a trichosanthes seed polypeptide having α-glucosidase inhibitory activity comprises the following steps:

[0084] Step 1: Preparation of crude protein from Trichosanthes seeds

[0085] The seeds of Trichosanthes kirilowii are peeled and crushed to obtain powder, and then defatted with n-hexane, and then dissolved in alkali and precipitated with acid to obtain the crude protein of the seeds of Trichosanthes kirilowii;

[0086] Step 2: Enzymatic hydrolysis of Trichosanthes seeds

[0087] The trichosanthes seed protein was enzymatically hydrolyzed with alkaline protease, trypsin, flavor protease, neutral protease and papain, and the obtained enzymatic hydrolysates were inactivated and centrifuged to obtain the supernatant to obtain a crude extract of the trichosanthes seed polypeptide;

[0088] Step 3: Isolation and purification of Trichosanthes seed polypeptide

[0089] The trypsin hydrolyzate of the trichosanthes seeds is separated and purified by ultrafiltration and liquid chromatography, and components with a molecular weight of less than 3 kDa are separated based on the difference in molecular weight;

[0090] The components with a molecular weight less than 3 kDa were freeze-dried, then prepared into a solution with water, and separated using a semi-preparative high performance liquid chromatography; the mobile phase was a mixture of solvent A and solvent B; solvent A was water containing 0.1% by mass formic acid, and solvent B was acetonitrile containing 0.1% by mass formic acid;

[0091] Semi-preparative high performance liquid chromatography was used for step-by-step separation, with the mobile phase being a mixture of solvent A and solvent B; the gradient was set as follows: from 0 to 5 minutes, the concentration of solvent B was maintained at 5%; from 5 to 50 minutes, the concentration of solvent B was increased to 100%.

[0092] The preferred technical solution is: in step 1, protein is extracted from the defatted powder of Trichosanthes kirilowii seeds using an alkali dissolution and acid precipitation method.

[0093] The preferred technical solution is: the dried trichosanthes seed powder is configured into a solution according to a material-liquid ratio of 1:8-12, the pH value of the solution is adjusted to 10.0 with a 1.0 mol / L sodium hydroxide solution, the solution is stirred at 50-65°C for 1-3 hours, and then centrifuged at a speed of 5000-8000 r / min for 10-20 minutes, the supernatant is taken, the pH value is adjusted to 3.5 with a 1.0 mol / L hydrochloric acid solution, the solution is stirred at 50-65°C for 1-3 hours, and the solution is centrifuged at a speed of 5000-8000 r / min for 10-20 minutes, the precipitate is washed with water, the pH value is adjusted to neutral, and then the solution is freeze-dried and refrigerated to obtain trichosanthes seed crude protein.

[0094] The preferred technical solution is: in step 2, the trichosanthes seed protein is prepared into a solution, and then the pH value of the solution is adjusted to 6.0-9.5, and the following five proteases are added to make the enzyme activity reach 5000-20000 U / g: alkaline protease, trypsin, neutral protease, flavor protease and papain, the sample solution is subjected to oscillation enzymolysis for 2-4 hours under the conditions of 45-65° C. and constant optimal pH value, the pH-start method is used to determine the hydrolysis degree of the trichosanthes seed protein during the enzymolysis process, and the enzyme is inactivated after the enzymolysis is completed to terminate the reaction; the supernatant is collected after centrifugation at a speed of 5000-8000 r / min for 15-30 minutes, and the supernatant is freeze-dried to form a freeze-dried powder, the freeze-dried powders of the five enzymatic hydrolyses are prepared into a solution, and the α-glucosidase inhibition rate is compared and analyzed.

[0095] The preferred technical solution is: in step 3, an ultrafiltration tube equipped with 3kDa and 10kDa ultrafiltration membranes is used to separate the trypsin hydrolyzate of Trichosanthes seeds, and the hydrolyzate is divided into three components according to molecular weight: MW < 3kDa, 3-10kDa, and > 10kDa.

[0096] A composition having α-glucosidase inhibitory activity, characterized by comprising the polypeptides HRQTWD, HANRLP, FGGVGER, and SYAPEDR, and further comprising a stabilizer, an emulsifier, and a flavoring agent.

[0097] The above description is only used to explain the preferred embodiments of the present invention and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A trichosanthes seed polypeptide having α-glucosidase inhibitory activity, characterized in that: The amino acid sequence of the Trichosanthes kirilowii seed polypeptide is one of the following: HRQTWD, HANRLP, FGGVGER, SYAPEDR.

2. A method for preparing the trichosanthes seed polypeptide having α-glucosidase inhibitory activity according to claim 1, characterized in that: The following steps are involved: Step 1: Preparation of crude protein from Trichosanthes seeds The seeds of Trichosanthes kirilowii are peeled and crushed to obtain powder, which is then defatted using n-hexane, and then dissolved in alkali and precipitated with acid to obtain crude protein from the seeds of Trichosanthes kirilowii; Step 2: Enzymatic hydrolysis of Trichosanthes seeds The protein of the trichosanthes seeds is hydrolyzed by using a protease, and the obtained hydrolyzate is subjected to enzyme inactivation treatment and then centrifuged to obtain the supernatant to obtain a crude extract of the trichosanthes seeds polypeptide; Step 3: Isolation and purification of Trichosanthes seed polypeptide The crude extract of the trichosanthes seed polypeptide is separated and purified by ultrafiltration and liquid chromatography, and a component with a molecular weight of less than 3 kDa is separated based on the difference in molecular weight; The components with a molecular weight less than 3 kDa were freeze-dried, then prepared into a solution with water, and separated using a semi-preparative high performance liquid chromatography; the mobile phase was a mixture of solvent A and solvent B; solvent A was water containing 0.1% by mass formic acid, and solvent B was acetonitrile containing 0.1% by mass formic acid; Semi-preparative high performance liquid chromatography was used for step-by-step separation, with the mobile phase being a mixture of solvent A and solvent B; the gradient was set as follows: from 0 to 5 minutes, the concentration of solvent B was maintained at 5%; from 5 to 50 minutes, the concentration of solvent B was increased to 100%.

3. The method for preparing the trichosanthes seed polypeptide having α-glucosidase inhibitory activity according to claim 2, wherein: The alkali dissolution and acid precipitation method comprises the following steps: preparing a solution according to a material-liquid ratio of 1:8-12 by mass, adjusting the pH value of the solution to 10.0 with a sodium hydroxide solution, stirring at 50-65°C for 1-3 hours, and then centrifuging at a speed of 5000-8000 r / min for 10-20 minutes, taking the supernatant, adjusting the pH value to 3.5 with a hydrochloric acid solution, stirring at 50-65°C for 1-3 hours, and then centrifuging at a speed of 5000-8000 r / min for 10-20 minutes, washing the precipitate with water, adjusting the pH value to neutral, and then freeze-drying and refrigerating to obtain trichosanthes seed crude protein.

4. The method for preparing the trichosanthes seed polypeptide having α-glucosidase inhibitory activity according to claim 2, wherein: In step 2, the crude protein of the trichosanthes seeds is prepared into a solution, and then the pH value of the solution is adjusted to 6.0-9.5, and at least one of the following five proteases is added, and the enzyme activity reaches 5000-20000 U / g: alkaline protease, trypsin, neutral protease, flavor protease and papain; the sample solution is subjected to oscillation enzymolysis at 45-65° C. for 2-4 hours, and the hydrolysis degree of the trichosanthes seeds protein is determined by a pH-start method during the enzymolysis process. After the enzymolysis is completed, the enzyme is inactivated to terminate the reaction; the supernatant is collected after centrifugation at a speed of 5000-8000 r / min for 15-30 minutes, and the supernatant is freeze-dried to form a lyophilized powder. The lyophilized powders of the five enzymatic hydrolyses are prepared into a solution, and the α-glucosidase inhibition rate is compared and analyzed.

5. The method for preparing the trichosanthes seed polypeptide having α-glucosidase inhibitory activity according to claim 2, wherein: In step 3, the trypsin hydrolyzate of the Trichosanthes seeds is separated by an ultrafiltration tube equipped with 3kDa and 10kDa ultrafiltration membranes, and is divided into three components according to molecular weight: MW < 3kDa, 3-10kDa, and > 10kDa.

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