A dipeptidyl peptidase iv inhibiting peptide derived from yiren alcohol soluble protein and screening method and application thereof

Abstract

The application provides a coixol soluble protein source dipeptidyl peptidase IV inhibiting peptide and a screening method and application thereof, and belongs to the technical field of natural active peptide extraction. The application takes coixol soluble protein as raw material, adopts a step-by-step enzymolysis mode of papain and alkaline protease, obtains an enzymolysis product with DPP-IV inhibiting activity, collects high-activity components gradually through ultrafiltration, Sephadex G-15 dextran gel chromatography, and finally carries out sequencing through LC-MS / MS mass spectrometry and screening through computer molecular docking technology to obtain two polypeptides. The polypeptide provided by the application can effectively combine with the important target DPP-IV for regulating diabetes, inhibit the activity, and thus achieve the purpose of reducing blood pressure. The inhibiting peptide can become an ideal raw material source of blood sugar reducing drugs.

Description

Technical Field

[0001] This invention belongs to the field of natural active peptide extraction technology, specifically relating to a coix seed prolysin source dipeptidyl peptidase IV inhibitory peptide, its screening method, and its application. Background Technology

[0002] The International Diabetes Federation predicts that the number of people diagnosed with or potentially diagnosed with diabetes will increase by 33.86% in 20 years, with over 90% of them having type 2 diabetes (T2DM). If T2DM is not managed promptly during its "pseudo-diabetes phase," medication is necessary to stabilize blood sugar. However, the adverse reactions of synthetic drugs significantly reduce patients' willingness to take them, making pharmacologically active natural compounds a focus of attention.

[0003] In recent years, research on dietary proteins has increased significantly, and peptides released from their parent protein sequences have shown potent biological functions. In the context of type 2 diabetes mellitus (T2DM) management, inhibiting dipeptidyl peptidase IV (DPP-IV) may be the most ideal means of regulating blood glucose. DPP-IV is a metabolic enzyme that can be expressed on the surface of various cell types and can cleave incretins; inhibiting its activity can prolong the half-life of glucagon-like peptide-1 (GLP-1), stimulating insulin secretion to balance blood glucose levels. Therefore, DPP-IV is a key target for the prevention and treatment of T2DM, and taking DPP-IV inhibitors is a novel therapy based on incretins.

[0004] Numerous studies have emphasized and confirmed the potential of food-derived bioactive peptides as DPP-IV inhibitors. Various peptides with DPP-IV inhibitory activity have been isolated from natural proteins, such as grains, dairy products, meat, and fish. Based on the concept of sustainable food, using plant proteins to replace animal proteins can help mitigate the increase in greenhouse gases; therefore, the exploration and development of new resources has become a new pathway for the production of inhibitory peptides. Coarse grains and legumes contain a large amount of underutilized resources in food industrial production and pharmaceutical development, and these resources can all serve as potential sources of DPP-IV inhibitory peptides.

[0005] Job's tears ( Coix lacryma-jobiJob's tears (L.), also known as coix seed or Job's tears, is listed as a medicinal and edible resource in many countries. As a precious grain that integrates nutrition, health care, and medical treatment, it enjoys the title of "King of the World's Gramineae Plants." It contains abundant protein resources, and the ratio of essential amino acids is close to that of the human body. The content of leucine, which has the effect of "lowering blood sugar," is even higher than that of cordyceps. In recent years, damp heat has been regarded by traditional Chinese medicine as a leading factor in the occurrence and development of type 2 diabetes mellitus (T2DM). The "Shennong's Classic of Materia Medica" records that Job's tears has the effects of promoting diuresis and reducing swelling, strengthening the spleen and removing dampness, clearing heat and draining pus, anti-inflammatory and analgesic, and enhancing immunity. Moreover, Job's tears protein has shown good development potential in lowering blood sugar. Its primary structure contains peptide sequence fragments that may target and regulate key enzymes, hormones, and proteins. Therefore, Job's tears protein, which is high in content and rich in nutrients, may be an excellent source of peptides for intervening in and regulating T2DM.

[0006] Modern medical research emphasizes that Job's tears are favored for their rich content of functional components such as Job's tears oil, Job's tears esters, and Job's tears polysaccharides, which can exert nutritional effects on multiple targets. Currently, the anticancer drug "Kanglaite (KLT)," with Job's tears oil as its main ingredient, has completed Phase III clinical trials approved by the U.S. Food and Drug Administration (FDA) and is being widely promoted. However, in the research and application of Job's tears, the abundant but low-value protein has not received much attention, and there are no reports on the therapeutic effects of Job's tears-derived proteins or peptides on type 2 diabetes. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a dipeptidyl peptidase IV inhibitory peptide derived from coix seed alcohol-soluble protein. In vitro evaluation revealed that it has high inhibitory activity against dipeptidyl peptidase IV, laying the foundation for the preparation of drugs for treating diabetes.

[0008] This invention also provides a method for screening dipeptidyl peptidase IV inhibitory peptides derived from coix seed prolysin. By optimizing the types of biological proteases and enzymatic hydrolysis conditions, peptides with high activity in inhibiting dipeptidyl peptidase IV are screened.

[0009] This invention provides a coix seed prolysin-derived dipeptidyl peptidase IV inhibitory peptide, the amino acid sequence of which is shown in SEQ ID NO:1 and / or SEQ ID NO:2.

[0010] This invention provides a method for screening dipeptidyl peptidase IV inhibitory peptides derived from coix seed prolysin, comprising the following steps: Preparation of coix seed prolysin; The coix seed prolysin was sequentially hydrolyzed with papain and alkaline protease to obtain coix seed protease hydrolysate; Crude peptides with a molecular weight of <1 kDa were separated from the coix seed protein hydrolysate and passed through a Sephadex G-15 gel column to collect the component G3 that showed the best in vitro inhibition of dipeptidyl peptidase IV activity. The component G3 was sequenced by LC-MS / MS, and the peptides with high inhibitory activity against dipeptidyl peptidase IV were selected as the dipeptidyl peptidase IV inhibitory peptides from coix seed prolysin source.

[0011] Preferably, the enzymatic hydrolysis conditions of the papain are: system pH value of 8.5~9.5, enzymatic hydrolysis time of 3~4 h, enzymatic hydrolysis temperature of 60~62℃, and enzyme addition amount of 8780~8790 U / g coix seed prolysin.

[0012] Preferably, the enzymatic hydrolysis conditions of the alkaline protease are: system pH value of 8.5~9.5, enzymatic hydrolysis time of 2~2.5h, enzymatic hydrolysis temperature of 54~56℃, and enzyme addition amount of 11970~11975 U / g coix seed prolysin.

[0013] Preferably, during the enzymatic hydrolysis, the mass percentage of coix seed prolysin is 3.8% to 4.2%.

[0014] Preferably, the method for preparing the coix seed prolysin includes the following steps: The defatted coix seed powder was mixed with an aqueous ethanol solution, dithiothreitol, and an aqueous NaAc solution for extraction. Small molecule impurities in the extract were removed to obtain coix seed prolysin.

[0015] Preferably, the mass-volume ratio of the defatted coix seed powder and the ethanol aqueous solution is 1 g: (5~7) mL; the volume percentage of the ethanol aqueous solution is 75%~85%. The final concentration of the dithiothreitol or NaAc aqueous solution is 0.8%~1.2%.

[0016] Preferably, the method for screening peptides with high inhibitory activity against dipeptidyl peptidase IV is performed using computer simulation technology. The computer simulation technique involves using POCASA 1.1 to predict the binding site of the peptide, using AutoDock Vina 1.1.2 for molecular docking, using the PeptideRanker server for bioactivity prediction, and selecting peptides with low binding energy, activity probability greater than 0.5, and high mass spectrum peak intensity.

[0017] This invention provides a medicament for treating diabetes, comprising the coixol source dipeptidyl peptidase IV inhibitory peptide or the coixol source dipeptidyl peptidase IV inhibitory peptide obtained by the screening method and medically acceptable excipients.

[0018] This invention provides the use of the coix seed prolysin-derived dipeptidyl peptidase IV inhibitory peptide or the coix seed prolysin-derived dipeptidyl peptidase IV inhibitory peptide obtained by the screening method in the preparation of medicaments for the prevention and / or treatment of diabetes.

[0019] This invention provides a dipeptidyl peptidase IV inhibitory peptide derived from coix seed prolysin, with the amino acid sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2. DPP-IV enzyme inhibitory activity assays demonstrate that the two polypeptides (LPFYPN and TFFPQ) protected by this invention exhibit strong DPP-IV inhibitory activity, with an IC50 value of [missing information]. 50 All values ​​were less than 200 μM, among which LPFYPN exhibited the strongest DPP-IV inhibitory activity (IC50). 50 =70.24 μM), which showed a more significant inhibitory effect than dietary peptides derived from egg yolk, sardines, oats, lupins, soybeans, and whey protein. Meanwhile, the binding constants (KA) of the two peptides (LPFYPN and TFFPQ) were 9.16 × 10⁻⁶ and 9.16 × 10⁻⁶, respectively. 3 mol / L, 4.50×10 4 mol / L. Studies have shown that the Xaa–Pro or Xaa–Pro–Yaa structures in the active peptides can competitively bind to the active site of DPP-IV with extremely strong affinity. However, no such structures were found at the N-terminus of TFFPQ, which better explains why LPFYPN has stronger DPP-IV inhibitory activity than TFFPQ in vitro. Furthermore, both inhibitory peptides showed good intestinal absorption (a positive HIA value indicates intestinal absorption ≥30%), and the DPP-IV inhibitory peptide from coix seed extract showed better absorption and transport in normal cells than in cancer cells. Plasma protein binding capacity (PPB) prediction results suggest that both peptides may have good therapeutic effects. Overall, the metabolic transport and absorption capacity of the TFFPQ and LPFYPN inhibitory peptides is relatively superior. The inhibitory peptides provided by this invention can effectively bind to DPP-IV, an important target for regulating diabetes, and inhibit its activity, thereby achieving the purpose of lowering blood sugar. LPFYPN and TFFPQ can be ideal raw material sources for hypoglycemic drugs.

[0020] This invention also provides a method for screening dipeptidyl peptidase IV inhibitory peptides derived from coix seed prolysin. Using coix seed prolysin as the enzymatic hydrolysis material, the types of biological proteases used for hydrolysis are optimized to obtain an enzymatic hydrolysate with good inhibitory effect on dipeptidyl peptidase IV activity. After further locking the crude peptide solution with a molecular weight <1 kDa, it is purified by Sephadex G-15 gel column chromatography. The highly active G3 fraction is selected for further LC-MS / MS sequencing, yielding 44 coix seed polypeptides. Screening for highly active DPP-IV inhibitory peptides yields the results. This screening method is simple to operate and has good reproducibility. Attached Figure Description

[0021] Figure 1 The results are shown in the single enzyme screening results (A) and the single-factor optimization experiment results of papain (BE). Figure 2 Optimization of papain response surface methodology results; Figure 3 The results of screening different types of dual enzymes (A) and the results of single-factor optimization experiments for alkaline protease (BE); Figure 4 To optimize the response surface methodology of alkaline protease in stepwise enzymatic hydrolysis; Figure 5 IC50 of enzymatically hydrolyzed peptides of different molecular weights 50 result; Figure 6 Chromatogram of Sephadex G-15 dextran gel purification; Figure 7 IC for different fractions 50 result; Figure 8 The results are shown in LC-MS / MS analysis; where A is the chromatogram, B is the mass spectrum of LPFYPN peptide, and C is the mass spectrum of TFFPQ peptide. Figure 9 The UV spectrum of the screened inhibitory peptide after binding to DPP-IV; Figure 10 Lineweaver Burk double reciprocal plot of the interaction between the inhibitory peptide and DPP-IV; Figure 11 The results show the docking of the inhibitory peptide with DPP-IV (PDB ID: 1X70). Detailed Implementation

[0022] This invention provides a coix seed prolysin-derived dipeptidyl peptidase IV inhibitory peptide, the amino acid sequence of which is shown in SEQ ID NO:1 (LPFYPN) and / or SEQ ID NO:2 (TFFPQ).

[0023] In this invention, the IC50 of coix seed prolyl dipeptidyl peptidase IV inhibitory peptides (LPFYPN, TFFPQ) on DPP-IV inhibition... 50 The values ​​were 70.24 μM and 176.87 μM, respectively, indicating higher activity than the peptides obtained from the same batch of sequencing. Although the activity of the coix seed prolysin-derived DPP-IV inhibitory peptide was lower than that of Diprotin A (IC50), it still showed higher activity. 50 =7.09 μM), but its inhibitory effect is more significant compared to food-derived peptides such as egg yolk peptides, sardine peptides, oat peptides, lupin peptides, soybean peptides, and whey protein peptides.

[0024] In this invention, the binding constant K of LPFYPN and TFFPQ is... A They are 9.16×10 3 mol / L, 4.50×10 4 The concentration of mol / L indicates a strong binding affinity between DPP-IV and the inhibitory peptides. Furthermore, both inhibitory peptides provided in this invention are non-toxic and contain sites that can be acted upon by digestive enzymes, but after digestion, they still retain amino acid sequences that inhibit DPP-IV activity, such as FF, PQ, LP, and PN. Using the overall CYP450 inhibition rate as a metabolic prediction indicator, both inhibitory peptides exhibit negative overall CYP450 inhibition rates, indicating that they can enhance the activity of the CYP450 enzyme system and accelerate the metabolism of the inhibitory peptides in vivo. Human intestinal absorption shows that the two inhibitory peptides have good intestinal absorption capacity (a positive HIA value indicates intestinal absorption capacity ≥30%). Simultaneously, both inhibitory peptides have low binding affinity to interfering proteins and superior Fu values, suggesting that the two peptides may have good therapeutic effects. Overall, the predicted results indicate that the TFFPQ and LPFYPN inhibitory peptides have relatively excellent metabolic transport and absorption capabilities, providing theoretical support for their potential as drugs.

[0025] This invention provides a method for screening dipeptidyl peptidase IV inhibitory peptides derived from coix seed prolysin, comprising the following steps: Preparation of coix seed prolysin; The coix seed prolysin was sequentially hydrolyzed with papain and alkaline protease to obtain coix seed protease hydrolysate; Crude peptides with a molecular weight of <1 kDa were separated from the coix seed protein hydrolysate and passed through a Sephadex G-15 gel column to collect the component G3 that showed the best in vitro inhibition of dipeptidyl peptidase IV activity. The component G3 was sequenced by LC-MS / MS, and the peptides with high inhibitory activity against dipeptidyl peptidase IV were selected as the dipeptidyl peptidase IV inhibitory peptides from coix seed prolysin source.

[0026] The present invention first prepares coix seed prolysin.

[0027] In this invention, the method for preparing the coix seed prolysin preferably includes the following steps: The defatted coix seed powder was mixed with an aqueous ethanol solution, dithiothreitol, and an aqueous NaAc solution for extraction. Small molecule impurities in the extract were removed to obtain coix seed prolysin.

[0028] In this invention, the preferred mass-volume ratio of the defatted coix seed powder and the ethanol aqueous solution is 1 g: (5~7) mL, more preferably 1 g: 6 mL. The defatted coix seed powder is preferably prepared by grinding fresh coix seeds after removing impurities, sieving, collecting the undersize, mixing with petroleum ether for extraction, and collecting the precipitate to obtain the defatted coix seed powder. The sieve aperture is preferably ≤80 mesh. The extraction is preferably performed three times. The volume percentage of the ethanol aqueous solution is preferably 75%~85%, more preferably 80%. The final concentration of the dithiothreitol or NaAc aqueous solution is preferably 0.8%~1.2%, more preferably 1.0%.

[0029] In this invention, the extraction method is preferably ultrasonic extraction. The ultrasonic extraction temperature is preferably 38~42℃, more preferably 40℃. The ultrasonic extraction time is preferably 25~35 min, more preferably 30 min. The ultrasonic power is preferably 200~600 W, more preferably 400 W. After extraction, the supernatant is preferably separated by centrifugation. The centrifugation speed is preferably 3000~5000 Rpm, more preferably 4000 Rpm. The centrifugation time is preferably 20~40 min, more preferably 30 min. The centrifugation temperature is preferably 4~25℃, more preferably 4℃. The method for removing small molecule impurities from the extract is preferably low-temperature dialysis. The low-temperature dialysis temperature is preferably 4~6℃, more preferably 4℃. The low-temperature dialysis time is preferably 22~26 h, more preferably 24 h. The pore size of the dialysis bag for low-temperature dialysis is preferably 3500~7000 kDa.

[0030] After obtaining the coix seed prolysin, the present invention sequentially hydrolyzes the coix seed prolysin with papain and alkaline protease to obtain coix seed protease hydrolysate.

[0031] In this invention, during enzymatic hydrolysis, the preferred mass percentage of coix seed prolysin is 3.8% to 4.2%, more preferably 4.0%. The preferred enzymatic hydrolysis conditions for the papain are: a system pH of 8.5 to 9.5, a hydrolysis time of 3 to 4 hours, a hydrolysis temperature of 60 to 62°C, and an enzyme dosage of 8780 to 8790 U / g; more preferably, a system pH of 9.0, a hydrolysis time of 3.4 hours, a hydrolysis temperature of 61°C, and an enzyme dosage of 8785 U / g. Compared with other common proteases (alkaline protease, neutral protease, trypsin, pepsin, and flavor protease, five commercial proteases), the papain yielded the optimal content of inhibitory peptides and exhibited the best DPP-IV inhibition effect of 69.55% in the hydrolysate. The preferred enzymatic hydrolysis conditions for the alkaline protease are: a system pH of 8.5–9.5, a hydrolysis time of 2–2.5 h, a hydrolysis temperature of 54–56 °C, and an enzyme dosage of 11970–11975 U / g; more preferably, a system pH of 9.0, a hydrolysis time of 2–2.5 h, a hydrolysis temperature of 54–56 °C, and an enzyme dosage of 11970–11975 U / g. Compared with other stepwise enzymatic hydrolysis schemes using papain + alkaline protease (papain + trypsin or papain + flavor protease), the stepwise hydrolysis scheme of this invention exhibits the highest DPP-IV inhibition rate of 92.67%.

[0032] In this invention, after the coix seed protein hydrolysate is prepared, an enzyme inactivation treatment is preferably performed. The enzyme inactivation method is preferably boiling water bath heating. The boiling water bath heating time is preferably 13-17 min, more preferably 15 min. After enzyme inactivation, the protein hydrolysate is centrifuged, and the supernatant is collected. The centrifugation speed is preferably 3500 g, the centrifugation time is preferably 25 min, and the temperature is preferably 4°C.

[0033] After obtaining the coix seed protein hydrolysate, the present invention separates crude peptides with a molecular weight <1 kDa from the coix seed protein hydrolysate and passes them through a Sephadex G-15 gel column to collect the component G3 that best inhibits dipeptidyl peptidase IV activity in vitro.

[0034] In this invention, the coix seed protein hydrolysate is grouped according to molecular weight, into components with molecular weights of <1 kDa, 1-3 kDa, 3-5 kDa, and >5 kDa. The grouping method preferably involves filtration using a filter membrane of the corresponding molecular weight to collect the target components. The components with molecular weights <1 kDa are preferably collected using a 0.45 μm microporous membrane. The highly active <1 kDa components are selected for further purification. The verification method preferably involves in vitro detection of dipeptidyl peptidase IV activity.

[0035] In this invention, the Sephadex G-15 gel column is preferably equilibrated with distilled water for 24 h before column chromatography. The Sephadex G-15 gel column has a column size of 1 cm × 100 cm. The elution rate is set to 0.5 mL / min, the UV detection wavelength is 220 nm, and elution is performed with distilled water. The eluent is collected from the peak, with each peak collected in the same tube to form a fraction, for a total of four fractions (G1, G2, G3, and G4). The in vitro dipeptidyl peptidase IV inhibitory activity of the four collected eluents is detected, and the results show that fraction G3 has the highest detection activity.

[0036] After obtaining component G3, the present invention performs LC-MS / MS sequencing on component G3 and screens for peptides with high inhibitory activity against dipeptidyl peptidase IV, which are the dipeptidyl peptidase IV inhibitory peptides of coix seed prolysin source.

[0037] In this invention, the LC-MS / MS sequencing preferably utilizes the Easy nLC 1200 chromatography system (ThermoScientific) for chromatographic separation and a Q-Exactive mass spectrometer for DDA (data-dependent acquisition) mass spectrometry analysis. The preferred chromatographic separation conditions are as follows: Buffer: Solution A is a 0.1% formic acid aqueous solution, and Solution B is a mixed solution of 0.1% formic acid, acetonitrile, and water (where acetonitrile is 80%). The column is equilibrated with 100% Solution A. The sample is injected into a trap column (100µm × 20mm, 5µm, C18, Dr. Maisch GmbH) and then subjected to gradient separation using an analytical column (75µm × 150mm, 3µm, C18, Dr. Maisch GmbH) at a flow rate of 300 nl / min; gradient elution is performed using Solution B. The preferred conditions for the mass spectrometry analysis are as follows: analysis time 60 min, detection mode: positive ion, precursor ion scan range: 350-1800 m / z, primary mass spectrometry resolution: 60,000 @ m / z 200, AGC target: 3e6, primary maximum IT: 50 ms. Secondary mass spectrometry analysis of the peptide was performed using the following method: after each full scan, the secondary mass spectra of the 20 highest intensity precursor ions were acquired (MS2 scan), with a secondary mass spectrometry resolution of 15,000 @ m / z 200, AGC target: 1e5, secondary maximum IT: 50 ms, MS2 activation type: HCD, isolation window: 1.6 m / z, and normalized collision energy: 28.

[0038] In this invention, LC-MS / MS sequencing yielded 44 polypeptide sequences. Computer simulation technology was used to select peptides with high inhibitory activity against dipeptidyl peptidase IV. The preferred method for this computer simulation was to use POCASA 1.1 to predict protein binding sites, AutoDock Vina 1.1.2 for molecular docking, and PeptideRanker server for bioactivity prediction, selecting peptides with low binding energies (<-9 kcal / mol), activity probabilities greater than 0.5, and high mass spectrometry peak intensities (>1×10⁻⁶). 7 Three peptides (LPFYPN, TFFPQ, and ATFFPQ) were screened and obtained. The DPP-IV inhibitory activity of the three peptides was determined using in vitro substrate chemistry. The IC50 value of the LPFYPN peptide was calculated. 50 The value was 70.24 μM, and the IC50 value corresponding to the TFFPQ peptide was... 50 The value was 176.87 μM, and the IC50 value corresponding to the ATFFPQ peptide was... 50 The value was greater than 200 μM. Therefore, a peptide with high inhibitory activity was selected for protection.

[0039] Given that the inhibitory peptides screened in this invention have a good inhibitory effect on dipeptidyl peptidase IV, and that dipeptidyl peptidase IV is a key target for the prevention and treatment of diabetes, this invention provides the application of the coix seed prolysin-derived dipeptidyl peptidase IV inhibitory peptide or the coix seed prolysin-derived dipeptidyl peptidase IV inhibitory peptide obtained by the screening method in the preparation of drugs for the prevention and / or treatment of diabetes.

[0040] The present invention also provides a medicament for treating diabetes, comprising the coixol source dipeptidyl peptidase IV inhibitory peptide or the coixol source dipeptidyl peptidase IV inhibitory peptide obtained by the screening method and medically acceptable excipients.

[0041] The present invention does not impose any special restrictions on the dosage form of the drug; any drug dosage form containing polypeptide active ingredients well known in the art can be used.

[0042] The following detailed description, in conjunction with embodiments, illustrates a coix seed prolysin-derived dipeptidyl peptidase IV inhibitory peptide, its screening method, and its application. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0043] Example 1 Screening of two proteases for stepwise enzymatic hydrolysis and optimization of process conditions ① The defatted coix seed powder and 80% ethanol were mixed evenly in a ratio of 1:6 (w / v). DTT with a concentration of 1 mg / ml and NaAc solution with a concentration of 3 mol / L were added sequentially, with each added at 1%. The mixture was then extracted by ultrasonication at 40℃ for 30 min. The supernatant was collected by centrifugation, and deionized water was added to it in equal proportion. The mixture was dialyzed at low temperature for 24 h and then freeze-dried to obtain coix seed prolysin powder for later use.

[0044] ② Coix seed prolysin was dispersed in deionized water to prepare a protein suspension with a mass concentration of 4%. Six commercial proteases (6000 U / g each): alkaline protease (pH 9, 50℃), neutral protease (pH 7, 50℃), papain (pH 8, 60℃), trypsin (pH 8, 37℃), pepsin (pH 2, 37℃), and flavor protease (pH 7, 53℃) were added to the suspension. Enzymatic hydrolysis was performed on the suspension under optimal conditions for each enzyme for 2.5 h. After the reaction, the enzymes were inactivated by boiling in a water bath for 15 min. After cooling, the pH was adjusted to 7, and the suspension was centrifuged (4℃, 3500 μL / min). (g, 25 min), collect the supernatant.

[0045] The results of the single enzyme screening are shown below. Figure 1 Data from Figure A shows that the papain hydrolysate exhibited the best DPP-IV inhibition effect (69.55%), with a relatively high peptide content. While not significantly different from the alkaline protease hydrolysate, it was significantly higher than the neutral protease hydrolysate. This may be because the papain hydrolysis temperature range is closer to the denaturation temperature of coix seed prolysin, altering its self-assembly structure and exposing the hydrolytic sites, thus allowing the protease to exert its hydrolytic effect. Furthermore, alkaline conditions can increase protein solubility and improve substrate utilization during hydrolysis, resulting in a higher peptide content and better DPP-IV inhibitory activity. Therefore, temperature and pH are speculated to be important factors in producing hydrolysates with DPP-IV inhibitory activity. Thus, papain was selected as the main enzyme for the first-step hydrolysis step for single-factor and response surface methodology evaluation.

[0046] Optimal enzymatic hydrolysis conditions for papain were optimized by adjusting four factors: hydrolysis temperature (40-65℃), hydrolysis time (0.5-5.5 h), pH value (5-10), and enzyme dosage (4000-14000 U / g). The effects of these factors on the peptide content and in vitro DPP-IV inhibition rate of the coix seed alcohol-soluble protein hydrolysate in a one-step enzymatic hydrolysis were investigated. Results are attached. Figure 1 BE in China.

[0047] Based on the results of single-factor experiments, a Box-Benhnken optimization experiment was designed with pH, ​​enzymatic hydrolysis time, temperature, and enzyme addition amount as independent variables, and DPP-IV inhibition rate and peptide content as response values. The experimental design method is shown in Table 1 below.

[0048] Table 1. Single-enzyme enzymatic hydrolysis response surface experimental design

[0049] See results Figure 2 Tables 2 and 3.

[0050] Table 2. Analysis of variance of the DPP-IV inhibition regression model in the one-step enzymatic hydrolysis experiment.

[0051] Table 3. Analysis of variance results of the peptide content regression model in the one-step enzymatic hydrolysis experiment.

[0052] The results showed that the three-dimensional surface plot of the interaction between various factors and the multivariate regression analysis fit table of the model equations showed that the regression model had a significant P-value and the lack of fit P-value was not significant, indicating that the model had a high degree of fit. The obtained regression equation is as follows. Combining the two response values, the optimized response surface parameters for one-step enzymatic hydrolysis of papain are: pH 9, 3.4 h, 61 ℃, 8785 U / g.

[0053] Y1=-469.13+10.57A+49.59B+11.63C+8.83×10 -3 D - 1.49AB - 0.54AC + 1.12 × 10 - 3 AD - 0.34BC + 1.93 × 10 -4 BD-1.41×10 -5 CD+1.38A 2 -2.64B 2 -0.047C 2 -1.06×10 -6 D 2 Y2=-122.32+10.51A+5.25B+2.36C+2.79×10 -4 D - 0.24AB + 0.028AC + 1.03 × 10 -4 AD+4.62×10 -3 BC + 1.15 × 10 -4 BD+1.80×10 -5 CD-0.73A 2 -0.63B 2 -0.022C2 -1.54×10 -7 D 2 ③ Considering the special self-assembly structure of coix seed prolysin, single protease hydrolysis has limiting factors. Stepwise enzymatic hydrolysis can increase cleavage sites, promote the hydrolysis range, and increase the probability of preparing highly active small peptides. Therefore, this invention selects alkaline protease, trypsin or flavor protease, which have relatively good effects in one-step enzymatic hydrolysis experiments, as the second-step hydrolytic enzymes.

[0054] The stepwise enzymatic hydrolysis process is as follows: First, papain is added and reacted for 3.4 h (pH 9, 61℃, 8785 U / g). Second, alkaline protease (pH=9, 50℃, 6000 U / g); or trypsin (pH=8.0, 37℃, 6000 U / g); or flavor protease (pH=7, 53℃, 6000 U / g) is added and reacted for 1–7 h. The enzymes are then inactivated, the mixture is cooled, the pH is adjusted, and the supernatant is collected by centrifugation.

[0055] See results Figure 3 A. The results show that the enzymatic hydrolysate produced by the combination of papain and alkaline protease exhibits the highest inhibitory activity against DPP-IV. From Figure 3 As can be seen from Figure A, the product treated with papain, after successive enzymatic hydrolysis by alkaline protease, trypsin, and flavor protease, significantly increased the peptide content, and the DPP-IV inhibitory activity increased by 24.27%-33.57%. It is worth noting that... Figure 3 Data shows that excessive enzymatic digestion can cause the active sequence of the obtained peptide to break down, thereby reducing its DPP-IV inhibitory activity.

[0056] Based on the fact that sequential hydrolysis with papain and alkaline protease yields the highest efficiency, an optimization experiment was conducted to optimize the dual enzymatic hydrolysis conditions using papain and alkaline protease. Single-factor experiments were performed on four factors: hydrolysis temperature (40–65℃), hydrolysis time (1–6 h), pH value (6–11), and enzyme dosage (4000–14000 U / g). The results are shown in […]. Figure 3 B~E.

[0057] Based on the results of single-factor experiments, a Box-Benhnken optimization experiment was designed with pH, ​​hydrolysis time, temperature, and enzyme dosage as independent variables, and DPP-IV inhibition rate and peptide content as response values ​​to investigate the effects of various factors in stepwise enzymatic hydrolysis on the peptide content and in vitro DPP-IV inhibition rate of the coix seed alcohol-soluble protein hydrolysate. The experimental design method is shown in Table 4 below.

[0058] Table 4. Response surface methodology for dual-enzyme digestion

[0059] Based on the interaction effect of independent variables on two response variables (see...) Figure 4 The optimal conditions for continuous enzymatic hydrolysis of alkaline protease were determined by f-test and p-value evaluation (Tables 5 and 6): 2.01 h, 55℃, pH 9, and enzyme dosage of 11974.73 U / g. The stepwise enzymatic hydrolysis response surface regression equation is as follows: Y3=8.69-5.55A+4.70B+1.55C-2.98×10-3D+0.04AB-0.07AC+3.87×10-5AD- 0.06BC-1.70×10-4BD+3.68×10-5CD+0.37A2-0.04B2-0.01C2+6.61×10-8D2 Y4=152.18-4.83A-0.12B-0.30C+9.96×10-5D Under stepwise enzymatic hydrolysis conditions, the theoretical DPP-IV inhibition rate of the coix seed alcohol-soluble protein hydrolysate was 93.01%, and the theoretical peptide content was 10.29 mg / mL. The actual results were 92.67% and 9.91 mg / mL, respectively, which were close to the predicted values.

[0060] Table 5. Analysis of variance of the regression model for DPP-IV inhibition rate in the stepwise enzymatic digestion experiment.

[0061] Table 6. Analysis of variance of the peptide content regression model in the stepwise enzymatic hydrolysis experiment.

[0062] Example 2 A method for isolating and identifying DPP-IV inhibitory peptides from coix seed extract. DPP-IV inhibitory peptides were generated by stepwise enzymatic hydrolysis of coix seed prolysin using papain and alkaline protease. Fresh Job's tears were cleaned of impurities and ground into powder using a grinder, then passed through an 80-mesh sieve. Petroleum ether (W:V = 1:5, g:mL) was added to the Job's tears powder and stirred in a fume hood for 5 hours, changing the ether three times until the supernatant was clear. The ether was then poured off, and the defatted Job's tears powder was air-dried. The defatted Job's tears powder and 80% ethanol were mixed uniformly at a ratio of 1:6 (w / v). DTT (1 mg / mL) and NaAc solution (3 mol / L) were added sequentially, each at 1%. The mixture was then ultrasonically extracted at 40℃ for 30 minutes. The supernatant was collected by centrifugation, and deionized water was added in an equal proportion. The mixture was dialyzed at low temperature for 24 hours and then freeze-dried to obtain Job's tears prolysin powder. The prolysin was dispersed in deionized water to prepare a protein suspension with a mass concentration of 4%. First, papain was used for enzymatic hydrolysis under the following conditions: hydrolysis time 3.4 h, hydrolysis temperature 61 ℃, pH 9, and enzyme dosage 8785 U / g. Then, alkaline protease was added for stepwise enzymatic hydrolysis under the following conditions: hydrolysis time 2.01 h, hydrolysis temperature 55 ℃, pH 9, and enzyme dosage 11974.73 U / g. After the reaction, the enzyme was inactivated by boiling in a water bath for 15 min. After cooling to room temperature (25 ℃), the pH was adjusted to 7, and the mixture was centrifuged (4 ℃, 3500 μL / g). g Collect the supernatant after 25 min.

[0063] (3) The coix seed prolysin peptides were fractionated by ultrafiltration, and the highly active <1 kDa fractions were selected for further purification. Specifically, the supernatant was collected using a micro ultrafiltration device to collect components with molecular weights of <1 kDa, 1-3 kDa, 3-5 kDa, and >5 kDa, respectively. The crude peptide solution with a molecular weight of <1 kDa and high inhibitory activity was selected and freeze-dried. The resulting powder was used as the sample for the next step of separation and purification.

[0064] (4) The coix seed prolysin peptides were purified by Sephadex G-15 dextran gel electrophoresis, and the highly active G3 fraction was selected for further sequencing. Specifically, 1 ml of crude peptide solution with a concentration of 10 mg / mL was prepared from the ultrafiltered sample (filtered through a 0.45 μm microporous membrane) and added to a Sephadex G-15 gel column equilibrated with distilled water for 24 h. The column was filled using a wet-filling method (column size 1 cm × 100 cm). The elution rate was set to 0.5 mL / min, and the UV detection wavelength was 220 nm. Elution was performed with distilled water to obtain four fractions: G1, G2, G3, and G4. The fraction with the best inhibitory activity (IC50) was collected. 50 =0.38 mg / mL), used for the next step of identification.

[0065] (5) Sequencing of G3 components using LC-MS / MS The G3 fraction (desalted) was separated chromatographically using an Easy nLC 1200 system (Thermo Scientific). Buffer solutions: Solution A was a 0.1% formic acid aqueous solution, and Solution B was a mixture of 0.1% formic acid, acetonitrile, and water (acetonitrile comprising 80%). The column was equilibrated with 100% Solution A. The sample was injected into a trap column (100µm × 20mm, 5µm, C18, Dr. Maisch GmbH) and then subjected to gradient separation on an analytical column (75µm × 150mm, 3µm, C18, Dr. Maisch GmbH) at a flow rate of 300 nl / min. Gradient elution was performed using Solution B. After peptide separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive mass spectrometer (Thermo Scientific). The analysis time was 60 min. Detection mode: positive ion; precursor ion scan range: 350-1800 m / z; primary mass spectrometry resolution: 60,000 m / z 200; AGC target: 3e6; primary maximum IT: 50 ms. Secondary mass spectrometry analysis of peptides was performed using the following method: after each full scan, the secondary mass spectra of the 20 highest intensity precursor ions were acquired (MS2 scan). Secondary mass spectrometry resolution: 15,000 m / z 200; AGC target: 1e5; secondary maximum IT: 50 ms; MS2 activation type: HCD; isolation window: 1.6 m / z; normalized collision energy: 28. A total of 44 coix seed peptides were identified, as detailed in Table 7.

[0066] Table 7 Information on 44 Coix Seed Polypeptides

[0067] (6) Using molecular docking technology, highly active DPP-IV inhibitory peptides were screened from the 44 peptides identified in (4), mainly including the following steps: The 44 peptide structures were constructed using ChemBioDraw Ultra 14.0, and the peptide energy was minimized using ChemBio3D Ultra 14.0. The DPP-IV (PDB ID: 1X70) protein model was used. The target 3D structure of DPP-IV obtained from the PDB file was prepared by removing water of crystallization and the original ligand using Discovery Studio. Energy optimization and target correction by adding hydrogen atoms were achieved using AutoDocktools. During docking, the ligand was used for flexible docking, while the protein was used for rigid docking. POCASA1.1 was used to predict protein binding sites, and AutoDock Vina1.1.2 was used for molecular docking. Protein-related parameters were set as follows: center_x = -15.9, center_y = 56.7, center_z = 27.2; search space: size_x: 50, size_y: 50, size_z: 50 (interval of 0.375 Å per grid point), exhaustiveness: 10, and other parameters were left at default settings. PyMol was used to analyze the interactions between active peptide-enzymes and to assess the final fraction of stable peptide-enzyme complexes.

[0068] Based on molecular docking binding energy (< -9 kcal / mol) and peptide mass spectrum peak intensity (> 1 × 10⁻⁶), 7 Based on the scores from the PeptideRanker server (PeptideRanker (ucd.ie)) (activity prediction probability > 0.5), three target peptides, LPFYPN, TFFPQ, and ATFFPQ, were finally selected (Table 8). The DPP-IV inhibitory activity of the three peptides was verified after solid-phase synthesis, and the highly active inhibitory peptide was selected for protection.

[0069] Table 8 shows the information of three target peptides selected.

[0070] (7) Ultraviolet spectral scanning DPP-IV was diluted to 0.03 U / mL with Tris-HCl buffer (100 mM, pH 8.0). 1 mL of enzyme solution was gradually added to peptide solutions of different concentrations (Q, 10 μL). After equilibration for 5 min, the absorbance values ​​A0 at wavelengths of 190–400 nm were recorded at 25 °C. The initial absorbance value of the enzyme was recorded as A. A Lineweaver-Burk double reciprocal curve was plotted between (A0-A)⁻¹ and [Q]⁻¹ to determine the binding constant KA between the peptide and DPP-IV. The specific formula I is as follows: Formula I The binding constant K of peptides (LPFYPN, TFFPQ) A They are 9.16×10 3 mol / L, 4.50×10 4 mol / L.

[0071] Example 3 The specific steps for DPP-IV enzyme inhibitory activity assay (in vitro substrate chemistry method) are as follows: 40 μl of sample at a certain concentration and 50 μl of substrate Gly-Pro-p-nitroanilide (0.4 mmol / L) were added to each well of a 96-well plate and incubated at 37 °C for 10 min. Then, 10 μl of DPP-IV (0.05 units / ml) was added to initiate the reaction. After reacting at 37 °C for 60 min, 100 μl of acetate-sodium acetate buffer (1 mol / L, pH=4) was added to terminate the reaction, and the absorbance was measured at 405 nm. The sample blank group required replacing the sample with Tris-HCl buffer, and the negative blank group required replacing both the sample and DPP-IV enzyme with Tris-HCl buffer. The IC50 of DPP-IV was... 50 The values ​​will be plotted as a function of the logarithm of sample concentration and the percentage of inhibition rate, and the sample concentration at which half of DPP-IV is inhibited will be calculated according to Formula II. Here, AS, ASB, AC, and ACB represent the absorbance of the sample group, sample blank group, negative group, and negative blank group, respectively.

[0072] Formula II Figure 5 and Figure 7 IC50 values ​​representing the DPP-IV inhibitory activity of each component during ultrafiltration fractionation and Sephadex G-15 dextran gel purification processes, respectively. 50 value; Figure 6 Chromatogram of Sephadex G-15 dextran gel purification.

[0073] As shown in Table 9, two peptides (LPFYPN and TFFPQ) exhibited strong DPP-IV inhibitory activity, with their IC50 values ​​being [missing information]. 50 All values ​​were less than 200 μM. Among them, LPFYPN exhibited the strongest DPP-IV inhibitory activity (IC50). 50 =70.24μM), although the activity of the inhibitory peptide is not as high as that of Diprotin A (IC50). 50 =7.09 μM), but its inhibitory effect is more significant compared to food-derived peptides such as egg yolk peptides, sardine peptides, oat peptides, lupin peptides, soybean peptides, and whey protein peptides.

[0074] Table 9. IC50 of the three peptides screened in this invention 50value

[0075] Example 4 1. Structural analysis of peptides LPFYPN and TFFPQ and their interaction with DPP-IV like Figure 9 As shown, the secondary mass spectra of LPFYPN and TFFPQ were obtained by LC-MS / MS mass spectrometry analysis in step (5) of Example 2.

[0076] The effect of the active peptide on the electronic activity range of DPP-IV was investigated using UV spectroscopy in step (7) of Example 1 to clarify the mechanism of their interaction. The UV spectra of DPP-IV under different concentrations of LPFYPN and TFFPQ are shown below. Figure 5 As shown, the absorption peaks at 220 nm (carbon-oxygen double bonds and aromatic hydrocarbons) all exhibit a red shift (~3 nm) and a significant hyperchromic effect. This phenomenon becomes more pronounced with increasing active peptide concentration, indicating an interaction between the three coixol-producing prolysin inhibitory peptides and DPP-IV, with the presence of hydrogen bonds or conjugation. This results in a wider active range for electrons during energy transitions, shifting the main absorption peaks towards longer wavelengths. The Lineweaver Burk double reciprocal curves of the interactions between LPFYPN and TFFPQ with DPP-IV are shown below. Figure 10 The fitting calculation yielded binding constants KA of 9.16 × 10⁻⁶. 3 mol / L, 4.50×10 4 The concentration of mol / L is on the order of magnitude, indicating a strong binding force between DPP-IV and the inhibitory peptide.

[0077] 2. Molecular docking of peptides LPFYPN and TFFPQ with DPP-IV (PDB ID: 1X70) like Figure 11 As shown, the C-terminal amino acid residues (Gln and Pro) of TFFPQ form hydrogen bonds with Tyr547 and Tyr631 in the S1 pocket of the DPP-IV enzyme and participate in an extended entropy effect network, namely, binding with the Tyr666 residue of the enzyme (S1 pocket) through hydrophobic interactions. Although the carboxyl ends of the two are the same, the number of associated hydrogen bonds differs greatly. TFFPQ also involves Asn710, Tyr662, and Ser630 residues in the S1 pocket of the DPP-IV enzyme, forming hydrogen bonds with lengths of 3.09 Å, 3.10 Å, and 2.69 Å, respectively.

[0078] LPFYPN interacts with DPP-IV enzymes primarily through hydrogen bonding and hydrophobic interactions. LPFYPN forms hydrogen bonds with Gln553, His740, Asn710, and Arg125 of DPP-IV, with bond lengths of 3.40 Å, 3.14 Å, 3.06 Å, and 2.79 Å, respectively. It also exhibits hydrophobic interactions with Ser552, Tyr547 (S1 pocket), Trp629 (S1 pocket), Trp627, Tyr666, and Tyr662. Furthermore, studies have shown that the Xaa–Pro or Xaa–Pro–Yaa structures in the active peptide can competitively bind to the active site of DPP-IV with extremely strong affinity. However, these structures were not found at the N-terminus of TFFPQ, which further explains why LPFYPN exhibits stronger DPP-IV inhibitory activity in vitro than TFFPQ.

[0079] Example 5 1. Prediction of absorption, metabolism, transport and toxicity of peptides LPFYPN and TFFPQ The activity, toxicity, absorption, transport, and metabolism of the active peptides were calculated using servers such as ADMET, ToxinPred, SwissTargetPrediction, and PeptideRanker (see Tian Wenhui, Sun Liping, Zhang Cui, et al. Virtual screening and activity analysis of dipeptidyl peptidase-IV inhibitory peptides from Sorghum Kafirin[J]. Journal of Agricultural and Food Chemistry, 2022, 70(6): 2010–2017.).

[0080] As shown in Table 10, neither of the two DPP-IV inhibitory peptides of coix seed prolysin is toxic. They contain sites that can be acted upon by digestive enzymes, but after digestion, amino acid sequences that inhibit DPP-IV activity, such as FF, PQ, LP, and PN, still exist. The metabolic prediction index was the comprehensive inhibition rate of CYP450. CYP450 is mainly found in the liver and is a key enzyme in the metabolic process. The prediction results showed that the comprehensive inhibition rate of CYP450 for both inhibitory peptides was negative, indicating that they can enhance the activity of the CYP450 enzyme system and accelerate the metabolic rate of the inhibitory peptides in vivo. Human intestinal absorption showed that the two inhibitory peptides had good intestinal absorption capacity (a positive HIA value indicates intestinal absorption capacity ≥30%). The predicted results of Caco-2 permeability and MDCK permeability show that the Caco-2 permeability of the two DPP-IV inhibitory peptides is moderate, while the MDCK permeability is better, indicating that the DPP-IV inhibitory peptides in coix seed prolysin are absorbed and transported better in normal cells than in cancer cells. The low probability of blood-brain barrier penetration (BBB) ​​for both inhibitory peptides indicates that this pathway is not their primary mechanism of action. Plasma protein binding capacity (PPB) expresses their ability to bind to proteins; the predicted results showed low binding to interfering proteins and favorable Fu values, suggesting that the two inhibitory peptides may have good therapeutic effects. Overall, the predicted results indicate that the TFFPQ and LPFYPN inhibitory peptides have relatively excellent metabolic transport and absorption capabilities, providing theoretical support for their potential as drugs.

[0081] Table 10. Predicted absorption, metabolism, transport, and toxicity of the two screened peptides.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A dipeptidyl peptidase IV inhibitory peptide derived from coix seed prolysin, characterized in that, The amino acid sequence is as shown in SEQ ID NO:

1.

2. A drug for treating diabetes, characterized in that, It includes the coix seed prolysin source dipeptidyl peptidase IV inhibitory peptide of claim 1 and medically acceptable excipients.

3. The use of the coix seed prolysin-derived dipeptidyl peptidase IV inhibitory peptide according to claim 1 in the preparation of a medicament for treating type 2 diabetes.

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