Polygonatum odoratum polypeptide as well as screening method and application thereof

Abstract

The invention discloses a polygonatum odoratum polypeptide as well as a screening method and application thereof. The polygonatum odoratum polypeptide comprises one or more of RDLVLY, DDWRKL and DRNLVLY. The screening method comprises the steps of preparation of polygonatum odoratum protein hydrolysate, ultrafiltration separation, anion exchange resin separation, gel column separation and de novo sequencing analysis based on mass spectrometry. The polygonatum odoratum polypeptide can be applied to the fields of oxidation resistance and blood sugar reduction. The screening method is based on an activity-oriented polygonatum odoratum polypeptide separation, purification and identification technical method, and has the advantages of clear components, clear activity, clear mechanism and the like, and meanwhile, three specific polypeptides can be screened from the identified polypeptides through molecular docking screening and cellular level activity verification, so that the screening efficiency is greatly improved. A new thought is provided for further developing polygonatum odoratum polypeptide functional products, and the comprehensive utilization value of polygonatum odoratum is improved.

Description

Technical Field

[0001] The present application relates to the technical field of screening of Polygonatum odoratum polypeptides, and in particular to a Polygonatum odoratum polypeptide and a screening method and application thereof. Background Art

[0002] Polygonatum odoratum is sweet in flavor and slightly cold in nature. It nourishes yin, moistens dryness, promotes fluid production, and quenches thirst. It is commonly used clinically to treat lung and stomach yin damage, dry-heat cough, dry throat and thirst, and internal heat-induced thirst. Polygonatum odoratum also enters the lung and stomach meridians, possessing a sweet, moist, and cooling properties. It can promote fluid production and quench thirst, clear heat and moisten dryness, thereby eliminating the symptoms of thirst. This, by promoting the production of fluids and removing dryness and heat, has been a panacea for the disease, a practice recognized throughout history. Research has shown that the chemically active components of Polygonatum odoratum are primarily macromolecules such as polysaccharides, proteins, and glycoproteins, which have multiple effects, including lowering blood sugar and lipids, regulating immunity, providing antioxidant and fatigue-fatigue benefits, protecting the liver, and improving myocardial function.

[0003] At present, the research on Polygonatum odoratum mainly focuses on the fields of flavonoids, steroidal saponins, sterols, polysaccharides, etc., but there is no mention of obtaining active polypeptide components from Polygonatum odoratum. Summary of the Invention

[0004] The present invention provides a Polygonatum odoratum polypeptide and a screening method and application thereof, so as to solve the technical problem in the prior art that polypeptides in Polygonatum odoratum are difficult to screen.

[0005] To achieve the above object, the technical solution provided by the present invention is as follows:

[0006] In a first aspect of the present invention, a polygonatum polypeptide is provided, wherein the polygonatum polypeptide comprises one or more of RDLVLY, DDWRKL, and DRNLVLY; the molecular structure of RDLVLY is as follows:

[0007]

[0008] The molecular structure of DDWRKL is as follows:

[0009]

[0010] The molecular structural formula of the DRNLVLY is as follows:

[0011]

[0012] The second aspect of the present invention provides a method for screening the above-mentioned Polygonatum odoratum polypeptide, comprising the following steps:

[0013] S1. Crush Polygonatum odoratum, add petroleum ether, soak for several times, and rotary evaporate to obtain a supernatant; filter and dry the supernatant to obtain defatted Polygonatum odoratum; extract the protein component in the defatted Polygonatum odoratum by alkali extraction and acid precipitation to obtain a protein extract;

[0014] S2, hydrolyzing the protein extracts with a plurality of enzymes, then heating and centrifuging them to obtain supernatants to obtain proteolytic solutions, testing the antioxidant activity of the proteolytic solutions, and selecting the proteolytic solutions hydrolyzed with the alkaline protease after comparison;

[0015] S3. The protein hydrolysate selected in step S2 is divided into three sections by ultrafiltration tube, respectively designated as PO-I (<3 KDa), PO-II (3-10 KDa), and PO-III (>10 KDa); the antioxidant activity and hypoglycemic activity of the three sections of the protein hydrolysate are measured, and the protein hydrolysate of the PO-I section is selected after comparison;

[0016] S4. Separating the protease hydrolysate of the PO-I segment by anion chromatography using DEAE cellulose-52 filler, performing gradient elution with water and sodium chloride solutions of different concentrations, collecting fractions, detecting each fraction using a microplate reader, and plotting a fraction elution profile. After combining the solutions based on the elution peaks, three solutions were obtained, designated Poa-3K-I, Poa-3K-II, and Poa-3K-III, respectively. The hypoglycemic and antioxidant activities of the three solutions were determined, and the solution with the highest activity, Poa-3K-III, was selected after comparison.

[0017] S5. Purify the Poa-3K-III by gel filtration chromatography, elute with water, collect fractions, detect each fraction using a microplate reader and draw a fraction elution graph, and combine the solutions according to the elution peaks to obtain purified Poa-3K-III, which is a uniform polypeptide component with optimal hypoglycemic and antioxidant activities;

[0018] S6. The purified Poa-3K-III described in step S5 was subjected to Denovo sequencing analysis based on mass spectrometry to obtain polypeptides with high scores, and then the polypeptides with Keap1-Nrf2 and α-glucosidase inhibitory ability were screened by molecular docking technology, thereby obtaining the Polygonatum odoratum polypeptides RDLVLY, DDWRKL, and DRNLVLY.

[0019] Furthermore, the method for screening out polypeptides with Keap1-Nrf2 inhibitory ability specifically includes: screening out polypeptides with binding sites on 8 key amino acid residues in Keap1 among the high-scoring polypeptides, wherein the key amino acid residues include TYR334, TYR572, TYR525, ASN382, HIS436, ARG415, ARG483, and ARG380.

[0020] Furthermore, in the step S1, the solid-liquid ratio of the polygonatum odoratum to the petroleum ether is 1:5-10; in the alkali extraction and acid precipitation method, the defatted polygonatum odoratum is dissolved in water, and the solid-liquid ratio is 1:9-30.

[0021] Furthermore, in step S2, the multiple enzymes include neutral protease, trypsin, pepsin, alkaline protease and papain; the heating temperature of the protein extract after hydrolysis is 90°C to 100°C, the centrifugal speed is 10000rpm to 12000rpm, and the centrifugal temperature is 15°C to 20°C.

[0022] Furthermore, in the anion chromatography separation process of step S4, ultrapure water, 0.1 mol / L sodium chloride, 0.3 mol / L sodium chloride, 0.5 mol / L sodium chloride, and 1 mol / L sodium chloride were used for elution in sequence, and fractions were collected, and the detection wavelength was set to 280 nm.

[0023] Furthermore, in step S5, Sephadex G-15 dextran gel column is used for separation and purification, ultrapure water is used for elution, and the detection wavelength is set to 280 nm.

[0024] Furthermore, the mass spectrometry-based Denovo sequencing analysis in step S6 is specifically as follows: the purified Poa-3K-III is measured using the NanoDrop method, and then subjected to reductive alkylation treatment, and then the treated sample is analyzed using liquid chromatography-mass spectrometry. The analysis conditions are as follows: a C-18 analytical column, mobile phase A is 0.1% formic acid, mobile phase B is 0.1% formic acid and 80% acetonitrile, and the flow rate is 600 nL / min. A total of 289 active peptides were identified; the peptide sequence of the liquid chromatography-mass spectrometry data was analyzed using the Denovo module of PEAKS Studio (8.5) software.

[0025] Furthermore, during the molecular docking process of step S6, the peptide with a high score is docked with Keap1 protein and α-glucosidase through Discovery Studio.

[0026] The third aspect of the present invention provides the use of the above-mentioned Polygonatum odoratum polypeptide or the Polygonatum odoratum polypeptide screened by the above-mentioned screening method in the preparation of a hypoglycemic drug.

[0027] The odoratum polypeptide provided by the present invention can effectively scavenge hydroxyl radicals and ABTS+ free radicals at low concentrations, and has significant antioxidant efficacy; it has also been demonstrated through in vitro insulin resistance model cell experiments that it can significantly improve glucose consumption and increase antioxidant levels, indicating that the above-mentioned odoratum polypeptide has a significant improvement effect on the insulin cell model.

[0028] The present invention breaks through the technical bottleneck of targeted acquisition of hypoglycemic and antioxidant active peptides with clear structural functions from Polygonatum protein, provides a method for screening Polygonatum polypeptides with excellent activity, establishes an enzymatic hydrolysis process parameter optimization model, and realizes targeted acquisition; at the same time, based on computer-aided design, targeted screening of polypeptide small molecules with clear antioxidant and hypoglycemic functions is carried out, and their antioxidant and hypoglycemic effects are confirmed through in vitro and in vivo experiments.

[0029] This study establishes for the first time a dual inhibition model of Polygonatum odoratum polypeptides acting simultaneously on the KEAP1-Nrf2 pathway and α-glucosidase, breaking through the limitations of traditional single-target screening and emphasizing the dual-target synergistic mechanism and structure-guided design strategy, highlighting the technical advantages. The present study predicts the binding ability of Polygonatum odoratum polypeptides to dual targets through molecular docking.

[0030] The screening method for Polygonatum odoratum polypeptides provided by the present invention is based on an activity-oriented technical method for the separation, purification and identification of Polygonatum odoratum polypeptides, and has the advantages of clear components, clear activities and clear mechanisms. At the same time, the identified polypeptides can be screened out through molecular docking screening and verification of cellular-level activity, and three specific polypeptides can be screened out, which provides new ideas for further developing functional products of Polygonatum odoratum polypeptides and enhances the comprehensive utilization value of Polygonatum odoratum. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 This is a graph showing the antioxidant and blood sugar-lowering effects of six different enzymatic hydrolysates of the protein extract in Example 2 of the present invention;

[0033] Figure 2 Graph showing the antioxidant and hypoglycemic activities of the ultrafiltration fractions separated from the protease hydrolysate in Example 2 of the present invention;

[0034] Figure 3 This is a peak result diagram of PO-I separated by DEAE in Example 2 of the present invention;

[0035] Figure 4 Graph showing the antioxidant and hypoglycemic activities of three solutions of PO-I after DEAE separation in Example 2 of the present invention;

[0036] Figure 5 This is the elution diagram of the fractions after POa-3K-III was purified by G-15 column;

[0037] Figure 6Total ion current chromatogram for peptide sequence identification;

[0038] Figure 7 The molecular structure diagrams of three polypeptides are shown below;

[0039] Figure 8 This is the molecular docking screening result diagram for α-glucosidase and Keap1;

[0040] Figure 9 Secondary mass spectrometry (MS / MS) spectra of peptide identification for RDLVLY, DDWRKL, and DRNLVLY;

[0041] Figure 10 The results of the antioxidant and hypoglycemic activities of three peptides, RDLVLY, DDWRKL, and DRNLVLY, are shown;

[0042] Figure 11 Flow chart for the validation of the in vitro cell model of Polygonatum odoratum peptides;

[0043] Figure 12 The results of the effects of three peptides on HepG2 cell viability are shown;

[0044] Figure 13 Effects of three peptides on glucose consumption ( n=6);

[0045] Figure 14 The results of ROS generation of three peptides in IR-HepG2 cells ( n=3);

[0046] Figure 15 The effects of three peptides on the activity of GSH-Px(A) in IR-HepG2 cells ( n=6);

[0047] Figure 16 The effects of three peptides on the activity of CAT(B) in IR-HepG2 cells ( n=6);

[0048] Figure 17 The effects of three peptides on the activity of MDA(C) in IR-HepG2 cells ( n=6);

[0049] Figure 18 The effects of three peptides on the activity of SOD(D) in IR-HepG2 cells ( n=6). DETAILED DESCRIPTION

[0050] In order to help those skilled in the art better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of this application.

[0051] 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 people familiar with this technology to understand and read, and are not used to limit the conditions under which this application can be implemented. Therefore, they have no substantive technical significance. Any structural modification, change in proportional relationship or adjustment of size should still fall within the scope of the technical content disclosed in this application without affecting the efficacy and purpose that can be achieved by this application.

[0052] Example 1

[0053] A Polygonatum odoratum polypeptide, comprising one or more of RDLVLY, DDWRKL, and DRNLVLY; the molecular structure of RDLVLY is as follows:

[0054]

[0055] The molecular structure of DDWRKL is as follows:

[0056]

[0057] The molecular structure of DRNLVLY is as follows:

[0058]

[0059] The above-mentioned Polygonatum odoratum polypeptide can effectively scavenge hydroxyl free radicals and ABTS free radicals at low concentrations, and has significant antioxidant effects; it has also been proven through in vitro insulin resistance model cell experiments that it can significantly improve glucose consumption and increase antioxidant levels, indicating that the above-mentioned Polygonatum odoratum polypeptide has a significant improvement effect on the insulin cell model.

[0060] Example 2

[0061] The method for screening the Polygonatum odoratum polypeptide in Example 1 specifically comprises the following steps:

[0062] 1. Sample pretreatment and protein extraction

[0063] The Chinese medicinal material Polygonatum odoratum was crushed, passed through a 60-mesh sieve, 500 g was weighed, and placed in a 2 L beaker. Petroleum ether was added at a solid-liquid ratio of 1:5 (w / v), and the mixture was soaked overnight. The supernatant was removed and repeated several times until the petroleum ether changed from light yellow to colorless. The mixture was filtered and the petroleum ether remaining in the powder was dried in a constant temperature drying oven at 60°C to obtain a defatted Polygonatum odoratum sample.

[0064] The defatted Polygonatum odoratum sample powder was suspended in distilled water (1:10 w / v) and adjusted to pH 10 with 1 M NaOH. The suspension was magnetically stirred at 50°C for 1.5 h and centrifuged at 1000 × g for 10 min at room temperature. The supernatant was collected and extracted twice. The two extracts were combined and the pH was adjusted to 4 with 1 M HCl. The suspension was allowed to stand at 4°C for 24 h and centrifuged at 1000 × g for 10 min. The supernatant was discarded and the precipitate was dissolved with a small amount of water. The solution was then adjusted to neutral with 0.1 M NaOH, dialyzed overnight, and freeze-dried to obtain the protein extract.

[0065] 2. Screening of the best enzyme for enzymatic hydrolysis and extraction of peptides

[0066] Single-enzyme hydrolysis: The protein extract was dispersed in 10% (w / v) distilled water and hydrolyzed using trypsin (pH 7.8, 37.5°C), alkaline protease (pH 8.5, 50°C), neutral protease (pH 7.0, 50°C), papain (pH 7.0, 50°C), and pepsin (pH 2.0, 37.5°C) at a total enzyme dosage of 2% (w / v, 2g enzyme / 100g defatted precipitate). The pH was adjusted to the appropriate pH using 1M NaOH and stirred at 50°C using a magnetic stirrer for 4 hours. After 4 hours, the protein hydrolyzate was heated to 95°C for 10 minutes and centrifuged at 12,000g for 15 minutes. The precipitate was discarded and the supernatant was retained as the protease hydrolyzate. The supernatant of each hydrolysis was lyophilized separately.

[0067] Semi-bionic method (simulated gastrointestinal digestion): Dissolve the freeze-dried powder of Polygonatum odoratum protein in ultrapure water at a ratio of 1:7 w / v, add 2% (w / w) pepsin, adjust the pH to 2 with dilute hydrochloric acid, 37.5°C, 4h, then add 2% (w / w) trypsin, adjust the pH to 7.8 with 1 mol / L NaOH, 37.5°C, 2h, finally boil in water bath for 10min, inactivate, centrifuge, 10000r / min, 15min, obtain the supernatant, freeze-dry, and store at -80°C.

[0068] The antioxidant capacity was detected by adjusting the protein concentration in the range of 0.0-0.5 mg / mL, and the DPPH scavenging activity of the Polygonatum odoratum polypeptides hydrolyzed by the six methods was observed at a concentration of 0.30 mg / mL. It was found that alkaline protease (47.8% ± 0.2%) > trypsin (43.5% ± 0.6%) > neutral protease (36.4% ± 0.9%) > pepsin (33.6% ± 0.7%) > papain (31.1% ± 0.3%) > semi-bionic method (30.2% ± 0.4%), and the EC50 values were 0.37, 0.43, 0.56, 0.42, 0.72, and 0.84 mg / mL, respectively.

[0069] The ABTS+ free radical scavenging activity and hydroxyl scavenging activity of the odoratum polypeptide hydrolyzed with alkaline protease can reach about 80.0% and 96.4%, respectively. Although the effect is comparable to that of the odoratum polypeptide hydrolyzed by other methods, the overall antioxidant effect is better. In addition, the odoratum polypeptide hydrolyzed with alkaline protease showed 27.2%, 40.1%, 42.8%, 57.7%, and 68.1% inhibitory activity against α-glucosidase in a dose-dependent manner at concentrations of 0.08, 0.10, 0.12, 0.19, and 0.33 mg / mL. Specific results are shown in Figure 1 , which is the antioxidant and hypoglycemic effects of 6 different enzymatic hydrolysates of protein extracts ( n=3); A is DPPH scavenging activity, B is ABTS+ free radical scavenging activity, C is hydroxyl radical scavenging activity, and D is α-glucosidase inhibitory activity.

[0070] In conclusion, the POP obtained by alkaline protease hydrolysis has the best antioxidant and hypoglycemic activities.

[0071] 3. Ultrafiltration to obtain Polygonatum odoratum polypeptides of different molecular weights

[0072] Polygonatum odoratum polypeptides obtained by alkaline protease (pH = 8.5, 50 ° C) were separated into three fractions by 3kDa and 10kDa ultrafiltration tubes. The specific steps are as follows: Polygonatum odoratum polypeptide freeze-dried powder was accurately weighed and dissolved in ultrapure water to prepare Polygonatum odoratum protein hydrolysate. Ultrafiltration centrifuge tubes with molecular weight cut-off (MWCO) of 10kDa and 3kDa were used for fractionation, centrifuged at 4500r / min for 30min, and the retentate was diluted and washed three times. The resulting fractions PO-I (<3kDa), PO-II (3-10kDa), and PO-III (>10kDa) were collected and freeze-dried, and the hypoglycemic and antioxidant activities of each fraction were determined.

[0073] The antioxidant activity of each fraction at a concentration of 0.15 mg / mL was observed. PO-I exhibited 54.7% ± 1.3% DPPH scavenging, 53.3% ± 4.1% ABTS+ scavenging, and 66.0% ± 0.7% OH- scavenging activities, respectively. PO-II exhibited 50.0% DPPH scavenging, 17.1% ± 0.7% ABTS+ scavenging, and 46.6% ± 0.9% OH- scavenging activities, respectively. PO-III exhibited 25.5% ± 0.2% DPPH scavenging, 4.3% ± 0.2% ABTS+ scavenging, and 46.2% ± 2.1% OH- scavenging activities, respectively. EC50 values for the antioxidant and hypoglycemic activities of the Polygonatum odoratum polypeptide fractions are shown in Table 1.

[0074] Table 1 EC50 values of antioxidant and hypoglycemic activities of isolated components of Polygonatum odoratum polypeptide

[0075]

[0076] Combined with Table 1, it can be seen that the EC50 values of PO-I for DPPH free radical scavenging activity and OH- scavenging activity are 0.15 mg / mL and 0.08 mg / mL, respectively, while those of vitamin C are 0.03 mg / mL and 0.55 mg / mL, respectively. When the hypoglycemic activity of each part was observed at a concentration of 0.4 mg / mL, it was found that the glucosidase inhibition rates of PO-I, PO-II, and PO-III were 60.4% ± 0.9%, 55.0% ± 4.6%, and 35.7% ± 1.7%, respectively. Combined with the EC50 values in Table 1, it can be seen that PO-I (<3KDa) is the best. Specific results are shown in Figure 2 , which is the antioxidant and hypoglycemic activity of the ultrafiltration fraction of the protease hydrolysate, A is ABTS + B is the free radical scavenging activity, C is the hydroxyl group scavenging activity, and D is the α-glucosidase inhibitory activity.

[0077] 4. Anion exchange resin separation

[0078] PO-I (<3 kDa) was further isolated for antioxidant activity using anion exchange chromatography using DEAE-52 filler. The following steps were performed: 100 g of DEAE-52 powder was weighed, added to 10 volumes of ultrapure water, stirred, and allowed to swell at room temperature for 72 hours. Unsettled particles were removed from the upper layer, and the column was loaded into a 1.6 cm × 60 cm glass chromatography column. The column was allowed to stand for 1 hour, and then eluted with ultrapure water until equilibrium was reached. PO-I was added to a small amount of ultrapure water to prepare a sample solution. The solution was filtered through a 0.45 μm microporous filter with a 3 mL sample volume at a flow rate of 1 mL / min. A gradient of elution was performed using ultrapure water, 0.1 M NaCl, 0.3 M NaCl, 0.5 M NaCl, and 1 M NaCl solutions, followed by fraction collection. 5 mL fractions were collected, and the absorbance of each fraction was measured at 280 nm. A fraction elution profile was plotted. According to the elution peaks in the fraction elution diagram, the fractions were combined to obtain three solutions, which were named POa-3K-I, POa-3K-II, and POa-3K-III. The peak results of PO-I separated by DEAE were referred to Figure 3 .

[0079] The above solutions were freeze-dried separately, and the hypoglycemic and antioxidant activities of each fraction were measured. At a concentration of 0.03 mg / mL, the POa-3K-III fraction demonstrated DPPH scavenging activity of 91.9% ± 3.3%, ABTS+ scavenging activity of 61.2% ± 3.4%, and OH- scavenging activity of 99.5% ± 0.1%, with a glucosidase inhibition rate of 86.2% ± 0.5%. The POa-3K-III fraction reached its EC50 value more quickly than POa-3K-I and POa-3K-II. The EC50 value is the concentration at which 50% of free radicals are scavenged.

[0080] As shown in Table 1, in the DPPH free radical scavenging assay, the EC50 value of POa-3K-III was 0.01156 mg / mL, that of POa-3K-I was 0.01396 mg / mL, and that of POa-3K-II was 0.01811 mg / mL. In the ABTS+ free radical scavenging assay, the EC50 value of POa-3K-III was 0.02722 mg / mL, that of POa-3K-I was 0.04081 mg / mL, and that of POa-3K-II was 0.05938 mg / mL. In the hydroxyl radical scavenging assay, the EC50 values of POa-3K-I, POa-3K-II, and POa-3K-III were 0.006920 mg / mL, 0.008169 mg / mL, and 0.005448 mg / mL, respectively. In the α-glucosidase inhibition assay, the EC50 values of POa-3K-I, POa-3K-II, and POa-3K-III were 0.04253 mg / mL, 0.09890 mg / mL, and 0.02422 mg / mL, respectively. It can be seen that compared with unpurified PO-I, except for the ABTS+ free radical scavenging rate which increased by 5 times, the other inhibitory activities increased by more than 10 times (see Table 1 and Figure 4 Therefore, POa-3K-III was selected and further purified using Sephadex G-15 (3.6×150 cm). Figure 4 The antioxidant and hypoglycemic activities of the three solutions of PO-I after separation by DEAE; A represents DPPH scavenging activity, B represents ABTS scavenging activity + free radical scavenging activity, C represents hydroxyl radical scavenging activity, and D represents α-glucosidase inhibitory activity.

[0081] 5. Gel filtration chromatography purification

[0082] POa-3K-III was further purified by gel filtration chromatography. The specific steps include: weighing 100g of Sephadex G-15 gel powder, adding 10 times the volume of ultrapure water, stirring evenly, swelling at room temperature for 72h, removing the unprecipitated particles on the upper layer, loading into a 3.6cm×150cm glass chromatography column, letting it stand for 1h, and eluting with ultrapure water to equilibrium. Add a small amount of ultrapure water to POa-3K-III to prepare a sample solution, filter with a 0.45μm microporous filter membrane, load 3mL, flow rate of 0.5mL / min, use ultrapure water as eluent, collect one fraction every 3mL, measure the absorbance of each fraction at 280nm, and draw a fraction elution graph. According to the elution peak row in the figure, after combining the various parts, a single peak was obtained (see Figure 5 ), POa-3K-III was lyophilized for amino acid sequence identification.

[0083] 6. Sequencing and identification of Polygonatum odoratum polypeptides

[0084] The purified POa-3K-III was selected for de novo peptide sequence identification. The specific steps include: first using the NanoDrop method to determine the peptide concentration, then taking an appropriate amount of sample for reductive alkylation, then desalting the sample, and using liquid chromatography-mass spectrometry (LC-MS / MS) analysis. The analysis conditions are shown in Table 2, and the ion current chromatogram is shown in Figure 6 .

[0085] Table 2 Liquid chromatography condition settings

[0086]

[0087] The de novo sequencing analysis of LC-MS / MS data was performed using the de novo module of Peaks Studio (8.5) software. The main parameters were set as follows: average local confidence (ALC)>90, parent mass error tolerance (Parent Mass Error Tolerance): 5.0 ppm, fragment ion mass tolerance (Fragemnt Mass Error Tolerance): 0.02 Da, maximum variable PTM per peptide: 3, and the Unipro database was searched, which contains 5086 Paeoniaostii protein entries, to analyze the raw data.

[0088] The peptide sequences after mass spectrometry were screened according to the above conditions. A total of 289 active peptides were identified by LC-MS / MS. In the BIOPEP data (http: / / www.uwm.edu.pl / biochemia / index.php / en / biopep), possible bioactive peptides were searched and screened. The peptide sequence was used to draw the peptide structure using the software ChemDraw Professional, and then the structure energy was minimized in the software Chem3D and saved in MDL SDfile (sdf) format. The molecular formula structures of the three peptides can be found in Figure 7 .

[0089] Molecular docking technology selected human KEAP1-NRF2 (PDB ID: 2FLU) and α-glucosidase, and the ligand was a polypeptide from Polygonatum odoratum, and molecular docking was performed to screen candidate peptides. Before docking, the KEAP1-NRF2 (PDB ID: 2FLU) and α-glucosidase structures were processed by the protein preparation program to add hydrogen, minimize energy, and meet other program requirements. At the same time, the ligand energy was minimized before docking analysis. Based on whether its binding energy is lower than the positive control (the positive controls are Nrf2-like peptide and acarbose), and whether it can simultaneously bind to the key 8 amino acid residues in Keap1 (TYR334, TYR572, TYR525, ASN382, HIS436, ARG415, ARG483, ARG380), etc., a total of 3 peptides that meet the conditions were finally screened, namely DDWRKL, RDLVLY and DRNLVLY. The screening results are shown in Figure 8 From top to bottom, the figure shows the binding energy screening, the 8 amino acid residue binding screening and the peptide screening of the two proteins due to ligands. Figure 9 The amino acid sequences of the peptides are shown in Table 3. The following synthesis and activity verification experiments will be conducted on these three peptides.

[0090] Table 3 Amino acid sequences of peptides of three peptides

[0091] Peptide Sequence DDWRKL Asp-Asp-Trp-Arg-Lys-Leu RDLVLY Arg-Asp-Leu-Val-Leu-Tyr DRNLVLY Asp-Arg-Asn-Leu-Val-Leu-Tyr

[0092] Example 3

[0093] In Example 2, three peptides were screened, namely DDWRKL, RDLVLY, and DRNLVLY. These three peptides were synthesized and tested for their hypoglycemic and antioxidant activities in vitro. The three peptides in this example were synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. and identified as the three peptides listed above.

[0094] The hypoglycemic and antioxidant activities of the above-mentioned peptides were evaluated using DPPH free radical scavenging ability and α-glucosidase inhibitory activity experiments.

[0095] The three Polygonatum odoratum polypeptide monomers were accurately weighed and dissolved in pure water to prepare a 0.1 mg / mL Polygonatum odoratum polypeptide stock solution, which was then diluted 5-fold, 100-fold, 500-fold, and 1000-fold to obtain the test sample.

[0096] 1. Polygonatum odoratum polypeptide inhibits α-glucosidase activity

[0097] 20 μL of various concentrations of Polygonatum odoratum polypeptide solutions were placed in a 96-well plate. 2.5 μL of 10 U / mL α-glucosidase (0.1 mol / L phosphate buffer, pH 6.8) was added. The plates were incubated at 37°C for 15 minutes. The reaction was initiated by adding 10 μL of 10 mM p-NPG (0.1 mol / L phosphate buffer, pH 6.8). The reaction was continued at 37°C for 20 minutes. The reaction was then terminated by adding 150 μL of 1 M Na₂CO₃ solution.

[0098] The absorbance was measured at 405 nm using a microplate reader. The results were calculated according to the following formula. The results showed that at a concentration of 50 mg / mL, RDLVLY had an α-glucosidase inhibition rate of 98.58%, DDWRKL had an α-glucosidase inhibition rate of 98.56% ± 0.3%, and DRNLVLY had a α-glucosidase inhibition rate of 98.53% ± 0.04%. In the α-glucosidase inhibition experiment, the EC50 value of RDLVLY was 3.065 mg / mL, the EC50 value of DDWRKL was 11.95 mg / ml, the EC50 value of DRNLVLY was 0.0001809 mg / mL, and the EC50 value of acarbose was 0.0002177 mg / mL. Acarbose is a glucosidase inhibitor, and the EC50 value of DRNLVLY for α-glucosidase inhibition is lower than that of acarbose. The results are as follows Figure 10 As shown in A.

[0099] α-glucose enzyme inhibition activity % = (ab) / a × 100%

[0100] Note: a is the blank group; b is the sample group.

[0101] 2. DPPH free radical scavenging ability

[0102] For the sample group, 50 μL of various concentrations of Polygonatum odoratum polypeptide solution was added to a 96-well plate, followed by 150 μL of DPPH solution (prepared in methanol to a concentration of 0.1 mg / mL). Mix thoroughly and incubate at room temperature in the dark for 30 minutes. Methanol was used in place of the DPPH solution in the sample control group, while ultrapure water was used in place of the DPPH solution in the blank group. After the reaction, the absorbance (A) was measured at 517 nm using a microplate reader. Activity was calculated according to the following formula.

[0103] The results showed that the DPPH scavenging activity of RDLVLY at a concentration of 50 mg / mL could reach 29.67%±2.07%, the DPPH scavenging activity of DDWRKL at a concentration of 50 mg / ml could reach 62.67%±2.3%, and the DPPH scavenging activity of DRNLVLY at a concentration of 50 mg / mL could reach 10.95%±2.15%. In the DPPH free radical scavenging experiment, the EC50 value of RDLVLY was 131.2 mg / mL, the EC50 value of DDWRKL was 21.05 mg / mL, and the EC50 value of DRNLVLY was 805.6 mg / mL. Figure 10 As shown in B.

[0104] DPPH scavenging activity (%) = (Ac + Ab - As) / Ac × 100%

[0105] Note: As is the sample group, Ab is the sample control group, and Ac is the blank group.

[0106] Example 4

[0107] In Example 2, three peptides were screened and obtained: DDWRKL, RDLVLY, and DRNLVLY. These three peptides were synthesized and used in an in vitro cellular insulin resistance model experiment to verify the antioxidant and hypoglycemic efficacy of the monomeric peptides. The three peptides in this example were purchased from Nanjing Jiepeptide Biotechnology Co., Ltd.

[0108] The antioxidant and hypoglycemic effects of Polygonatum odoratum peptides were verified using an in vitro insulin resistance (IR) cell model. Using HepG2 cells as a carrier, an optimal IR-HepG2 cell model was established to explore the effect of Polygonatum odoratum peptides on glucose consumption in IR-HepG2 cells. Oxidative stress indicators were detected to verify its antioxidant effect, and the inhibitory and antioxidant effects of Polygonatum odoratum peptides on insulin-resistant HepG2 cells were evaluated. The flow chart is shown in the figure. Figure 11 .

[0109] 1. CCK-8 assay to determine the effect of drugs on HepG2 cell proliferation

[0110] The effects of the anti-oxidative and hypoglycemic activities of the IR-HepG2 cells were verified by insulin-induced oxidative stress model. 5HepG2 cells at a density of 100 μg / mL were seeded in a 96-well plate and divided into a blank control group, a CON group, and a metformin drug group (10, 100, 1000 μM), with 4 replicates. After 24 h, the culture medium in the 96-well plate was discarded, and phenol red-free DMEM medium was added to the blank control group and CON group. 10, 100, and 1000 μM metformin solution were added to the RDLVLY drug group (0.1, 0.5, 1, 5, 10, 50, 100, and 150 μM), DDWRKL drug group (0.1, 0.5, 1, 5, 10, 50, 100, and 150 μM), and metformin group, respectively. The cells were cultured for 24 h in the dark, and CCK-8 dilution solution was added (CCK-8:DMEM ratio of 1:9). The cells were cultured for another 1 h, and the absorbance was measured at 450 nm.

[0111] Compared with the Control group, when HepG2 cells were stimulated with 10, 100, and 1000 μM metformin for 24 hours, the cell viability of each concentration was greater than 90% and there was no significant difference; when the concentration of RDLVLY, DDWRKL, and DRNLVLY was 150 μM, there was a significant difference (P<0.01), and the cell viability of other concentrations was greater than 80% and there was no significant difference, indicating that metformin, RDLVLY, DDWRKL, and DRNLVLY had no toxic effect on HepG2 cells ( Figure 12 ), where A is metformin; B is RDLVLY; C is DRNLVL; and D is DDWRKL. Note: **P<0.01 compared with the Control group ( n=6).

[0112] 2. Effects of different drugs on glucose consumption (GC) in IR-HepG2 cell supernatant

[0113] Figure 13 The effects of different concentrations of RDLVLY, DDWRKL, and DRNLVLY on glucose consumption. Note: Compared with the Control group, # P<0.05, ## P<0.01; compared with the Mod group, * P<0.05, **P < 0.01. HepG2 cells were seeded at a density of 3 × 105 cells / mL in 24-well plates (500 μL of cell solution per well) and divided into three groups: CON group, Model group, RDLVLY drug group (10, 50, 100 μM), DDWRKL drug group (10, 50, 100 μM), DRNLVLY drug group (10, 50, 100 μM), and positive control group (metformin: 100 μM), with three replicates. After 24 hours, the culture medium in the 24-well plate was replaced with phenol red-free DMEM medium, and the cells were starved overnight. Phenol red-free DMEM medium was then added to the blank control group and CON group, and 10 μL of phenol red-free DMEM medium was added to the drug group and Model group, respectively. - 6 Cells were incubated with a 100-mol / L insulin solution (prepared in phenol red-free DMEM) for 24 hours. Phenol red-free DMEM without fetal bovine serum was then added to the CON and Model groups. Different concentrations of drugs were added to each drug group. After 24 hours of incubation, the cell supernatant was collected. Glucose content was determined according to the instructions of the glucose assay kit, and glucose consumption (GC) of the supernatant was calculated based on the glucose content.

[0114] from Figure 13 Compared with the control group, the GC content of IR-HepG2 cells was significantly decreased (P < 0.01), indicating that the insulin resistance model was successfully established. GC content increased with 10 μM RDLVLY, and both 50 μM and 100 μM RDLVLY treatments significantly increased GC content (P < 0.05). GC content also increased significantly with 10 μM, 50 μM, and 100 μM DDWRKL and DRNLVLY (P < 0.05), with the effect of 100 μM DRNLVLY being more pronounced. This suggests that RDLVLY, DDWRKL, and DRNLVLY can alleviate a certain degree of insulin resistance by improving hepatocyte glucose utilization, thereby lowering blood glucose levels.

[0115] 3. Effects on intracellular ROS generation

[0116] Immunofluorescence experiments were used to verify the effect of Polygonatum odoratum polypeptide on ROS generation in IR-HepG2 cells. Figure 14 Note: Compared with the Control group, # P<0.05, ## P<0.01; compared with the Mod group, * P<0.05, **P < 0.01. DCFH-DA was diluted 1:1000 in serum-free DMEM medium to a final concentration of 10 μmol / L. Slides were seeded into 24-well plates and treated with drugs. After 24 hours of culture, the cell culture medium was removed and 500 μL of the diluted DCFH-DA was added to the plates and incubated in a 37°C, 5% CO2 incubator for 20 minutes. The cells were then rinsed 2-3 times with PBS to fully remove any DCFH-DA that had not entered the cells. Fluorescence images were acquired using a Nikon A1R+ (Ti2-E) laser confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Fluorescence intensity and area were analyzed using Image J software.

[0117] The results showed that the green fluorescence area of HepG2 cells in the CON group was smaller and the brightness was lower; after the IR-HepG2 cell model was established, the fluorescence density and brightness of HepG2 cells in the Mod group increased significantly, indicating that the ROS generation in the HepG2 cell IR model increased; compared with the CON group, the ROS generation in the Mod group increased significantly (P<0.01), indicating that IR-HepG2 suffered obvious oxidative damage.

[0118] After 24 hours of treatment with 10μM, 50μM, and 100μM RDLVLY, DDWRKL, and DRNLVLY in the IR-HepG2 cell model, the fluorescence density and intensity of the HepG2 cells gradually decreased with increasing concentration, and the amount of ROS generated gradually decreased, indicating that RDLVLY, DDWRKL, and DRNLVLY can improve the oxidative damage of IR-HepG2 cells in a concentration-dependent manner within the 10-100μM concentration range. RDLVLY, DDWRKL, and DRNLVLY inhibited the production of ROS in a dose-dependent manner. Among them, except for 10μM RDLVLY, the ROS generation of the other Polygonatum odoratum peptide groups was significantly reduced compared with the Mod group (P<0.01), and the effect of 100μM DRNLVLY was the most significant.

[0119] 4. Effects on intracellular GSH-Px, CAT, MDA and SOD activities

[0120] The levels of antioxidant enzymes GSH-Px, CAT, MDA, and SOD in HepG2 cells were further measured. GSH-Px, CAT, MDA, and SOD activities were measured using kits according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute). After treatment, cells were cultured for 24 hours, the supernatant discarded, and the cells were rinsed 2-3 times with PBS. The cell pellet was then removed by purging with 200 μL of PBS and collected in a 1.5 mL EP tube. The cells were then placed at -80°C and repeatedly freeze-thawed to lyse the cells. The cells were then centrifuged at 10,000 g for 10 minutes at 4°C. The total protein concentration in the supernatant was determined using a BCA protein assay kit.

[0121] The results are as follows Figures 15 to 18 As shown (Note: Compared with the Control group, # P<0.05, ## P<0.01; compared with the Mod group, * P<0.05, ** Compared with the CON group, the Mod group significantly reduced the activities of GSH-Px, CAT, and SOD (P<0.01), and the MDA content was significantly increased (P<0.01), indicating that IR-HepG2 cells suffered obvious oxidative damage. After adding 10μM, 50μM, and 100μM of RDLVLY, DDWRKL, and DRNLVLY to the IR-HepG2 cell model for 24 hours, the activities of GSH-Px, CAT, and SOD increased, and the MDA activity decreased. Compared with the Mod group, the GSH-Px and CAT activities of 100 μM RDLVLY, DDWRKL, and DRNLVLY were significantly increased (P<0.01), the MDA activity of 50 μM RDLVLY, DDWRKL, and DRNLVLY was significantly decreased (P<0.01), and the SOD activity of 100 μM DDWRKL and DRNLVLY was significantly increased (P<0.01), among which the effect of 100 μM DRNLVLY was more obvious.

[0122] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Polygonatum odoratum polypeptide, characterized in that: The Polygonatum odoratum polypeptide includes one or more of RDLVLY, DDWRKL, and DRNLVLY; The molecular structural formula of the RDLVLY is as follows: The molecular structure of DDWRKL is as follows: The molecular structural formula of the DRNLVLY is as follows:

2. The method for screening the Polygonatum odoratum polypeptide according to claim 1, characterized in that: The following steps are involved: S1. Grind Polygonatum odoratum, add petroleum ether and soak for several times, and then rotary evaporate to obtain a supernatant; The supernatant is filtered and dried to obtain defatted Polygonatum odoratum; the protein component in the defatted Polygonatum odoratum is extracted by alkali extraction and acid precipitation to obtain a protein extract; S2, hydrolyzing the protein extracts with a plurality of enzymes, then heating and centrifuging them to obtain supernatants to obtain proteolytic solutions, testing the antioxidant activity of the proteolytic solutions, and selecting the proteolytic solutions hydrolyzed with the alkaline protease after comparison; S3. The protein hydrolysate selected in step S2 is divided into three sections by ultrafiltration tube, respectively designated as PO-I (<3 KDa), PO-II (3-10 KDa), and PO-III (>10 KDa); the antioxidant activity and hypoglycemic activity of the three sections of the protein hydrolysate are measured, and the protein hydrolysate of the PO-I section is selected after comparison; S4. Separating the protease hydrolysate of the PO-I segment by anion chromatography using DEAE cellulose-52 filler, performing gradient elution with water and sodium chloride solutions of different concentrations, collecting fractions, detecting each fraction using a microplate reader, and plotting a fraction elution profile. After combining the solutions based on the elution peaks, three solutions were obtained, designated Poa-3K-I, Poa-3K-II, and Poa-3K-III, respectively. The hypoglycemic and antioxidant activities of the three solutions were determined, and the solution with the highest activity, Poa-3K-III, was selected after comparison. S5. Purify the Poa-3K-III by gel filtration chromatography, elute with water, collect fractions, detect each fraction using a microplate reader and draw a fraction elution graph, and combine the solutions according to the elution peaks to obtain purified Poa-3K-III, which is a uniform polypeptide component with optimal hypoglycemic and antioxidant activities; S6. The purified Poa-3K-III described in step S5 was subjected to Denovo sequencing analysis based on mass spectrometry to obtain polypeptides with high scores, and then the polypeptides with Keap1-Nrf2 and α-glucosidase inhibitory ability were screened by molecular docking technology, thereby obtaining the Polygonatum odoratum polypeptides RDLVLY, DDWRKL, and DRNLVLY.

3. The method for screening Polygonatum odoratum polypeptide according to claim 2, wherein: The method for screening out polypeptides with Keap1-Nrf2 inhibitory ability specifically includes: screening out polypeptides with binding sites with 8 key amino acid residues in Keap1 among the high-scoring polypeptides, wherein the key amino acid residues include TYR334, TYR572, TYR525, ASN382, HIS436, ARG415, ARG483, and ARG380.

4. The method for screening Polygonatum odoratum polypeptide according to claim 2 or 3, characterized in that: In the step S1, the solid-liquid ratio of the odoratum to the petroleum ether is 1:5-10; in the alkali extraction and acid precipitation method, the defatted odoratum is dissolved in water, and the solid-liquid ratio is 1:9-30.

5. The method for screening Polygonatum odoratum polypeptide according to claim 2 or 3, characterized in that: In step S2, the multiple enzymes include neutral protease, trypsin, pepsin, alkaline protease and papain; the heating temperature of the protein extract after hydrolysis is 90°C to 100°C, the centrifugal speed is 10000rpm to 12000rpm, and the centrifugal temperature is 15°C to 20°C.

6. The method for screening Polygonatum odoratum polypeptide according to claim 2 or 3, characterized in that: During the anion chromatography separation process in step S4, ultrapure water, 0.1 mol / L sodium chloride, 0.3 mol / L sodium chloride, 0.5 mol / L sodium chloride, and 1 mol / L sodium chloride are used for elution in sequence, and fractions are collected. The detection wavelength is set to 280 nm; and / or, In step S5, a Sephadex G-15 dextran gel column is used for separation and purification, and ultrapure water is used for elution. The detection wavelength is set at 280 nm.

7. The method for screening Polygonatum odoratum polypeptide according to claim 2 or 3, characterized in that: The mass spectrometry-based Denovo sequencing analysis in step S6 is specifically as follows: the purified Poa-3K-III is measured using the NanoDrop method, followed by reduction and alkylation treatment, and then the treated sample is analyzed using liquid chromatography-mass spectrometry. The analysis conditions are: a C-18 analytical column, mobile phase A is 0.1% formic acid, mobile phase B is 0.1% formic acid and 80% acetonitrile, and the flow rate is 600 nL / min. A total of 289 active peptides were identified; the LC-MS data were analyzed for peptide sequences using the Denovo module of PEAKS Studio (8.5) software.

8. The method for screening Polygonatum odoratum polypeptide according to claim 2 or 3, characterized in that: During the molecular docking process in step S6, the peptide with a high score is docked with the Keap1 protein and α-glucosidase through Discovery Studio.

9. Use of the Polygonatum odoratum polypeptide according to claim 1 or the Polygonatum odoratum polypeptide obtained by screening according to any one of claims 2 to 8 in the preparation of a hypoglycemic drug.

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