Linear peptides having dpp-iv inhibitory activity and compositions and uses thereof

Abstract

The application belongs to the technical field of polypeptide and hypoglycemic drugs, and particularly relates to a linear peptide with DPP-IV inhibitory activity, a composition and use. The inventors find six linear short peptides with DPP-IV inhibitory activity, wherein the DPP-IV inhibitory activity of QPEVMG is better, and the linear peptide has good prospects for preparation of hypoglycemic drugs for reducing insulin resistance and good safety.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide and hypoglycemic drug technology, specifically relating to a linear peptide with DPP-IV inhibitory activity, its composition, and its uses. Background Technology

[0002] Human blood glucose regulation involves complex processes such as insulin secretion regulation and glucose consumption. There are dozens of targets related to blood glucose regulation. Currently, a large number of drugs with hypoglycemic activity have been developed targeting different hypoglycemic targets, including small molecule drugs, protein and peptide drugs, etc. Among them, peptide drugs are a class with a significant market advantage. Type 2 diabetes and insulin resistance are closely related to postprandial hyperglycemia, insulin signal transduction disorders, and abnormal hepatic glucose uptake and storage. Dipeptidyl peptidase-IV (DPP-IV) can degrade incretins such as glucagon-like peptide-1, reducing their role in promoting insulin secretion and regulating blood glucose. Therefore, inhibiting DPP-IV activity is one of the important strategies for improving abnormal glucose metabolism.

[0003] However, small molecule drugs, due to their small molecular size, are prone to widespread off-target effects, leading to numerous unpredictable adverse reactions. Most peptide-based hypoglycemic drugs, on the other hand, have peptide chains exceeding 20 amino acids, and many require the attachment of peptide side chains. This large molecular size results in high manufacturing costs and extremely high market prices for these peptide drugs. Developing linear small peptides with good hypoglycemic effects, while lowering the barriers to synthesis technology and reducing the number of components required for drug preparation, is one of the current development directions for peptide-based hypoglycemic drugs. Summary of the Invention

[0004] To address the aforementioned problems, the inventors obtained a large number of peptides during their research on the degradation of animal milk proteins. In screening these peptides, they discovered six short-chain linear peptides with good DPP-IV inhibitory activity. Therefore, the purpose of this invention is to provide a linear peptide with DPP-IV inhibitory activity, a composition thereof, and its uses. This invention includes the following technical solutions:

[0005] A linear peptide with DPP-IV inhibitory activity, wherein the linear peptide is selected from one of the linear peptides with amino acid sequences as shown in SEQ ID NO:1 to SEQ ID NO:6.

[0006] A composition of a linear peptide having DPP-IV inhibitory activity, comprising a pharmaceutically acceptable excipient and a linear peptide having DPP-IV inhibitory activity, wherein the linear peptide having DPP-IV inhibitory activity is selected from at least one of the linear peptides with amino acid sequences as shown in SEQ ID NO:1 to SEQ ID NO:6.

[0007] Preferably, the linear peptide having DPP-IV inhibitory activity includes at least the linear peptide shown in SEQ ID NO:4.

[0008] Preferably, the linear peptide with DPP-IV inhibitory activity is selected from at least two of the linear peptides with amino acid sequences as shown in SEQ ID NO:1 to SEQ ID NO:6. More preferably, the linear peptide with DPP-IV inhibitory activity contains at least one of the linear peptides with amino acid sequences as shown in SEQ ID NO:4, and at least one of the linear peptides shown in SEQ ID NO:5 and SEQ ID NO:6.

[0009] Preferably, the dosage form of the composition is one of an oral formulation or an injectable formulation. More preferably,

[0010] The oral preparation is selected from at least one of powder, tablet, capsule, granule, and oral liquid; the injectable preparation is selected from at least one of subcutaneous injection, intravenous injection, and intramuscular injection.

[0011] The use of the aforementioned linear peptides or compositions having DPP-IV inhibitory activity in the preparation of hypoglycemic drugs. Preferably, the hypoglycemic drug is a hypoglycemic drug that improves insulin resistance.

[0012] Beneficial effects:

[0013] (1) The linear peptide with DPP-IV inhibitory activity of the present invention has good DPP-IV inhibitory activity, which can improve insulin resistance and play a role in alleviating and treating type 2 diabetes.

[0014] (2) The linear peptides with DPP-IV inhibitory activity of the present invention are relatively short, and the solid-phase synthesis process is simple and easy to industrialize and promote.

[0015] (3) Cell testing showed that the linear peptide with DPP-IV inhibitory activity of the present invention has good safety. Attached Figure Description

[0016] Figure 1 This is the secondary mass spectrum of the polypeptide SEQ ID NO:1;

[0017] Figure 2 This is the secondary mass spectrum of the polypeptide SEQ ID NO:2;

[0018] Figure 3 This is the secondary mass spectrum of the polypeptide SEQ ID NO:3;

[0019] Figure 4 This is the secondary mass spectrum of the polypeptide SEQ ID NO:4;

[0020] Figure 5 This is the secondary mass spectrum of the polypeptide SEQ ID NO:5;

[0021] Figure 6 This is the secondary mass spectrum of the polypeptide SEQ ID NO:6;

[0022] Figure 7 IC50 of different peptides inhibiting DPP-IV 50 Comparison chart;

[0023] Figure 8 A comparative graph showing the effects of different peptides on the viability of HepG2 cells;

[0024] Figure 9 A comparative graph showing the effects of different peptides on glucose consumption in IR-HepG2 cells;

[0025] Figure 10 A comparative diagram showing the effects of different peptides on glycogen synthesis in IR-HepG2 cells;

[0026] Figure 11 This figure compares the effects of different peptides on the relative activity of DPP-IV in IR-HepG2 cells. Detailed Implementation

[0027] The present invention and its effects are further illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are merely illustrative of the implementation methods and effects of the present invention and are not intended to limit the scope of the invention. After reading the contents of this invention, those skilled in the art can make various simple modifications or alterations to the invention without creative effort, and these equivalent / identical transformations also fall within the protection scope of this invention.

[0028] In the accompanying figures of the following embodiments, A, B, C, D, E, and F represent the polypeptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:4, and SEQ ID NO:6, respectively. The amino acid sequences corresponding to their single-letter codes are as follows: A represents VPGEIVES; B represents PGEIVESL; C represents PEVMG; D represents RELEELN; E represents QPEVMG; and F represents LARELEEL. In the comparison codes and symbols between groups in the figures: different letters indicate significant differences between groups (p<0.05); "*" indicates significant differences compared to the 0 μM treatment.

[0029] Example 1: Fmoc solid-phase chemical synthesis and structural confirmation of six target peptides

[0030] In this embodiment, VPGEIVES, PGEIVESL, PEVMG, RELEELN, QPEVMG, and LARELEEL were selected as target linear peptides. The target peptides were prepared using the conventional Fmoc solid-phase peptide synthesis method. The samples used for DPP-IV inhibitory activity and cell function experiments were all purified synthetic peptides.

[0031] S1 selects 0.5 mmol of the Fmoc-protected amino acid, Wang Resin, corresponding to the first amino acid at the C-terminus of the target polypeptide sequence and adds it to the solid-phase reactor. After swelling the resin with dichloromethane (DCM) for 30 min, the mixture is dried and washed three times with N,N-dimethylformamide (DMF). A 20% (v / v) piperidine (hexahydropyridine) / DMF solution is added and reacted for 5 min. After washing once with DMF, another 20% piperidine / DMF solution is added and reacted for 10 min. After the reaction is complete, the mixture is dried and washed three times with DMF.

[0032] S2 followed the amino acid sequence of the target peptide from C-terminus to N-terminus, alternating between condensation and deprotection reactions using 1.5 mmol of each Fmoc-protected amino acid, condensing the remaining amino acids one by one onto the resin. After each condensation, the free amino group was detected using the ninhydrin method; if the reaction was incomplete, the condensation was repeated. After the last amino acid condensation was complete, the terminal Fmoc was removed using a 20% piperidine / DMF solution in two steps (5 min and 10 min) as described in S1, followed by drying and washing three times with DMF.

[0033] After S3 completes the deprotection of the terminal Fmoc, it is washed three times alternately with DMF and DCM, and then washed with methanol to shrink the resin, thus obtaining a dried peptide-resin.

[0034] S4. The peptide-resin mixture was placed in a round-bottom flask, and the lysis buffer was slowly added and stirred at 0 °C. The volume ratio of trifluoroacetic acid (TFA), anisole, phenol, triisopropylsilane, and water in the lysis buffer was 82.5:7.5:5:3:2. After reacting at low temperature for 0.5 h, the mixture was transferred to room temperature and reacted for another 2 h. The lysis buffer was collected by filtration. The lysis buffer was then slowly added to anhydrous ice-cold diethyl ether and stirred continuously. The mixture was filtered to obtain the crude peptide, which was washed three times with ice-cold diethyl ether.

[0035] S5 was purified and separated by high-performance liquid chromatography (HPLC). A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used. Mobile phase A was chromatographically pure acetonitrile, and mobile phase B was ultrapure water containing 0.1% trifluoroacetic acid (v / v). Linear gradient elution was employed, with the proportion of mobile phase A increasing from 5% to 95% within 0–20 min, and correspondingly decreasing the proportion of mobile phase B from 95% to 5%. The detection wavelength was 220 nm, the injection volume was 8 μL, and the flow rate was 1 mL / min.

[0036] The purified synthetic peptides were structurally confirmed using mass spectrometry. Based on the fragmentation patterns of the parent ions of each target peptide, the b-ion and y-ion series were matched to confirm that the amino acid sequences matched the designed sequences. The secondary mass spectra of the six peptides are shown below. Figures 1-6 As shown in Table 1, the basic parameters of the six linear peptides are as follows.

[0037] Table 1. Basic parameters of 6 linear peptides

[0038] The NMR chromatographic data of VPGEIVES, PGEIVESL, PEVMG, RELEELN, QPEVMG, and LARELEEL are as follows:

[0039] (1) VPGEIVES

[0040] 13 C NMR: (101 MHz, DMSO-d6) δ 174.6, 174.5 (Glu-COOH), 172.9, 172.8,172.4, 172.1, 171.9, 171.8, 171.4, 169.7 (backbone C=O), 62.1 (Ser-C β ), 60.6 (Pro-C) α ), 59.4 (Ile-C α ), 59.0, 58.8 (Val-C α ), 55.7 (Ser-C α ), 54.4, 54.2 (Glu-C α ), 47.1 (Pro-C δ ), 43.0 (Gly-C α ), 37.6 (Ile-C β ), 33.8, 33.6 (Glu-C γ ), 31.4, 31.0 (Val-C β), 29.2 (Pro-C β ), 28.6, 28.4 (Glu-C β ), 25.4 (Ile-C γ1 ), 24.7 (Pro-C γ ), 19.7, 19.5, 18.8, 18.6 (Val-CH3), 16.2 (Ile-C γ2 ), 11.8 (Ile-C δ1 )。

[0041] 1 H NMR:(400 MHz, DMSO-d6) δ 8.45–7.60 (m, 6H, backbone NH), 4.50–4.05(m, 7H, H α ), 3.95–3.70 (m, 4H, Gly-H α and Ser-H β ), 3.68–3.42 (m, 2H, Pro-H δ ),2.45–2.20 (m, 4H, Glu-H γ ), 2.18–1.70 (m, Val-H β , Ile-H β , Glu-H β and Pro-H β / H γ ), 1.55–1.20 (m, 2H, Ile-H γ1 ), 1.03–0.79 (m, 18H, Val and Ile methyl H)。

[0042] (2)PGEIVESL

[0043] 13 C NMR:(101 MHz, DMSO-d6) δ 174.6, 174.5 (Glu-COOH), 172.8, 172.7,172.3, 172.1, 171.9, 171.8, 171.3, 169.6 (backbone C=O), 62.2 (Ser-C β ), 60.7(Pro-C α ), 59.4 (Ile-C α ), 58.8 (Val-C α ), 55.7 (Ser-C α), 54.4, 54.2 (Glu-C α ),53.3 (Leu-C α ), 47.1 (Pro-C δ ), 43.0 (Gly-C α ), 41.1 (Leu-C β ), 37.6 (Ile-C β ),33.8, 33.6 (Glu-C γ ), 31.1 (Val-C β ), 29.3 (Pro-C β ), 28.6, 28.4 (Glu-C β ), 25.4(Ile-C γ1 ), 24.9 (Leu-C γ ), 24.7 (Pro-C γ ), 23.1, 21.8 (Leu-CH3), 19.6, 18.7(Val-CH3), 16.2 (Ile-C γ2 ), 11.8 (Ile-C δ1 ).。

[0044] 1 H NMR:(400 MHz, DMSO-d6) δ 8.50–7.55 (m, 7H, backbone NH), 4.52–4.05(m, 7H, H α ), 3.96–3.70 (m, 4H, Gly-H α and Ser-H β ), 3.68–3.42 (m, 2H, Pro-H δ ),2.45–2.20 (m, 4H, Glu-H γ ), 2.18–1.70 (m, Val-H β , Ile-H β , Glu-H β and Pro-H β / H γ ), 1.70–1.20 (m, Ile-H γ1 , Leu-H β and Leu-H γ ), 1.02–0.78 (m, 18H, Ile, Valand Leu methyl H)。

[0045] (3)PEVMG

[0046] 13 C NMR:(101 MHz, DMSO-d6) δ 174.6 (Glu-COOH), 172.8, 172.3, 171.9,171.8, 169.7 (backbone C=O), 60.7 (Pro-C α ), 58.9 (Val-C α ), 54.8 (Met-C α ), 54.4(Glu-C α ), 47.1 (Pro-C δ ), 43.0 (Gly-C α ), 33.8 (Glu-C γ ), 32.0 (Met-C β ), 31.2(Val-C β ), 30.3 (Met-C γ ), 29.3 (Pro-C β ), 28.6 (Glu-C β ), 24.7 (Pro-C γ ), 19.6,18.7 (Val-CH3), 15.2 (Met-SCH3)。

[0047] 1 H NMR:(400 MHz, DMSO-d6) δ 8.45–7.55 (m, 4H, backbone NH), 4.48–4.05(m, 4H, H α ), 3.95–3.72 (m, 2H, Gly-H α ), 3.65–3.42 (m, 2H, Pro-H δ ), 2.62–2.40(m, 2H, Met-H γ ), 2.42–2.20 (m, 2H, Glu-H γ ), 2.12–2.03 (s, 3H, Met-SCH3),2.10–1.65 (m, Val-H β , Met-H β , Glu-H β and Pro-H β / H γ ), 1.00–0.84 (m, 6H, Val-CH3)。

[0048] (4)QPEVMG

[0049] 13 C NMR:(101 MHz, DMSO-d6) δ 174.6 (Glu-COOH), 174.2 (Gln-CONH2),172.8, 172.3, 172.0, 171.8, 171.7, 169.7 (backbone C=O), 60.7 (Pro-C α ), 58.9(Val-C α ), 54.9 (Gln-C α ), 54.8 (Met-C α ), 54.4 (Glu-C α ), 47.1 (Pro-C δ ), 43.0(Gly-C α ), 33.8 (Glu-C γ ), 32.5 (Gln-C γ ), 32.0 (Met-C β ), 31.2 (Val-C β ), 30.3(Met-C γ ), 29.3 (Pro-C β ), 28.9 (Gln-C β ), 28.6 (Glu-C β ), 24.7 (Pro-C γ ), 19.6,18.7 (Val-CH3), 15.2 (Met-SCH3)。

[0050] 1 H NMR:(400 MHz, DMSO-d6) δ 8.50–7.55 (m, 4H, backbone NH), 7.80–6.70(br m, Gln-CONH2), 4.52–4.05 (m, 5H, H α ), 3.95–3.72 (m, 2H, Gly-H α ), 3.66–3.42(m, 2H, Pro-H δ ), 2.62–2.40 (m, 2H, Met-H γ ), 2.45–2.18 (m, 4H, Gln-H γ and Glu-H γ), 2.12–2.03 (s, 3H, Met-SCH3), 2.15–1.65 (m, Gln-H β , Glu-H β , Pro-H β / H γ ,H-wave β and Met-H β ), 1.00–0.84 (m, 6H, Val-CH3)。

[0051] (5)RELAY

[0052] 13 C NMR:(101 MHz, DMSO-d6) δ 174.6, 174.5, 174.4 (Glu-COOH), 173.3(Asn-CONH2), 172.9, 172.8, 172.7, 172.4, 172.3, 171.6, 171.1 (backbone C=O), 157.2 (Arg-C ζ ), 54.6 (Arg-C α ), 54.4, 54.3, 54.2 (Glu-C α ), 53.4, 53.2 (Leo-C α ),52.8 (Asn-C α ), 41.2 (Arg-C δ ), 41.1, 40.9 (Leo-C β ), 37.1 (Asn-C β ), 33.8, 33.7,33.6 (Glu-C γ ), 29.5 (Arg-C β ), 28.7, 28.6, 28.5 (Glu-C β ), 25.3 (Arg-C γ ), 24.9,24.8 (Leo-C γ ), 23.2, 22.9, 21.8, 21.6 (Leu-CH3).

[0053] 1H NMR:(400 MHz, DMSO-d6) δ 8.60–7.50 (m, 6H, backbone NH), 8.30–7.00(br m, Arg guanidinium NH), 7.80–6.70 (br m, Asn-CONH2), 4.60–4.05 (m, 7H,H α ), 3.30–3.08 (m, 2H, Arg-H δ ), 2.90–2.55 (m, 2H, Asn-H β ), 2.45–2.18 (m, 6H,Glu-H γ ), 2.10–1.70 (m, Glu-H β and Arg-H β ), 1.75–1.30 (m, Arg-H γ , Leu-H β andLeu-H γ ), 0.98–0.78 (m, 12H, Leu-CH3)。

[0054] (6)LARELEEL

[0055] 13 C NMR:(101 MHz, DMSO-d6) δ 174.6, 174.5, 174.4 (Glu-COOH), 172.9,172.8, 172.7, 172.4, 172.3, 172.2, 171.8, 171.5 (backbone C=O), 157.2 (Arg-C ζ ), 54.6 (Arg-C α ), 54.4, 54.3, 54.2 (Glu-C α ), 53.4, 53.3, 53.2 (Leu-C α ), 50.4(Ala-C α ), 41.2 (Arg-C δ ), 41.1, 41.0, 40.9 (Leu-C β ), 33.8, 33.7, 33.6 (Glu-C γ ),29.5 (Arg-C β ), 28.7, 28.6, 28.5 (Glu-C β ), 25.3 (Arg-C γ), 24.9, 24.8, 24.7 (Leu-C γ ), 23.2, 23.1, 23.0, 21.9, 21.8, 21.7 (Leu-CH3), 18.1 (Ala-CH3).

[0056] 1 H NMR: (400 MHz, DMSO-d6) δ 8.55–7.50 (m, 7H, backbone NH), 8.30–7.00 (br m, Arg guanidinium NH), 4.52–4.02 (m, 8H, H α ), 3.30–3.08 (m, 2H, Arg-H δ ), 2.45–2.18 (m, 6H, Glu-H) γ ), 2.10–1.70 (m, Glu-H β and Arg-H β ), 1.75–1.35 (m,Arg-H γ Leu-H β and Leu-H γ ), 1.38–1.20 (d, 3H, Ala-CH3), 0.98–0.76 (m, 18H, Leu-CH3).

[0057] Example 2: DPP-IV in vitro inhibitory activity IC 50 Measurement

[0058] This example demonstrates the in vitro inhibition of DPP-IV activity by different peptides using IC50. 50 Determination and Results. The purified polypeptide was prepared according to the method in Example 1.

[0059] Take 25 μL of sample solutions of different concentrations and mix them with 25 μL of 1.5 mM Gly-Pro-pNA substrate (dissolved in 0.1 M Tris-HCl buffer, pH 8.0), and pre-incubate at 37 ℃ for 10 min; then add 50 μL of DPP-IV enzyme solution (40 U / mL, dissolved in 0.1 M Tris-HCl buffer, pH 8.0), and continue the reaction at 37 ℃ for 60 min; finally, add 100 μL of 1 M sodium acetate buffer (pH 4.0) to terminate the reaction, and measure the absorbance at 405 nm. The DPP-IV inhibition rate was calculated using formula (1), where C is the absorbance of the control group (using buffer instead of the sample solution, with all other conditions the same, reflecting the maximum enzyme activity), C0 is the absorbance of the control blank (using buffer instead of the sample solution and enzyme solution, with all other conditions the same, deducting background absorption caused by spontaneous hydrolysis of the substrate), S is the absorbance of the sample group (including the complete reaction system of sample, enzyme and substrate), and S0 is the absorbance of the sample blank (including sample and substrate, using buffer instead of enzyme solution, deducting the background absorption caused by the sample's own color and non-enzymatic hydrolysis of the substrate). The IC50 was calculated based on the concentration-dependent inhibition curve. 50 value.

[0060] DPP-IV inhibition rate (%) = [1-(S-S0) / (C-C0)]×100 (1)

[0061] like Figure 7 As shown, all six synthetic peptides exhibited DPP-IV inhibitory activity, but significant differences in IC50 values ​​were observed among different sequences. The IC50 of QPEVMG was 41.57 ± 1.86 μM, significantly lower than the other synthetic peptides (p < 0.05); the IC50 values ​​of LARELEEL and RELEELN were 150.20 ± 7.12 μM and 159.53 ± 8.66 μM, respectively; the IC50 values ​​of VPGEIVES and PGEIVESL were 227.73 ± 3.40 μM and 251.17 ± 5.78 μM, respectively; and the IC50 value of PEVMG was 375.13 ± 13.12 μM. These results indicate that the DPP-IV inhibitory activity of synthetic peptides is closely related to their amino acid sequence composition, and QPEVMG possesses the potential for further validation of its cellular function.

[0062] Example 3: Evaluation of HepG2 cell viability and determination of effective concentration

[0063] This example demonstrates the results of in vitro assays to determine the effects of different peptides on HepG2 cell viability. The purified peptides were prepared according to the method described in Example 1.

[0064] The effect of different concentrations of peptides on the viability of HepG2 cells was detected using the CCK-8 assay. HepG2 cells were seeded in 96-well plates and cultured at 37 ℃ in a 5% CO2 incubator until adherence and approximately 80% confluence. Then, 0, 25, 50, 100, 200, 400, 600, 800, and 1000 μM of the corresponding peptides were added for 24 h. Subsequently, 10 μM of CCK-8 solution was added to each well, and the reaction was carried out in the dark for 30 min. The absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated according to the kit instructions. The results are shown below. Figure 8 As shown.

[0065] Figure 8 The results showed that the effects of different peptides on HepG2 cell viability exhibited concentration-related characteristics. Most synthetic peptides did not cause significant cytotoxicity (higher than the IC50 for DPP-IV inhibition) in the 25–400 μM range, and cell viability was mostly maintained above 90%. Cell viability in some low- and medium-concentration treatment groups was significantly higher than in the 0 μM group (p<0.05). When the concentration increased to 600 μM and above, cell viability decreased in some peptide treatment groups. Based on the cell viability results, subsequent IR-HepG2 insulin resistance model experiments selected 100, 200, and 400 μM as treatment concentrations to balance cell safety and concentration gradient.

[0066] Example 4: Effect of synthetic peptides on glucose consumption in IR-HepG2 cells

[0067] This example demonstrates the results of in vitro assays to determine the effects of different peptides on glucose consumption in IR-HepG2 cells. The purified peptides were prepared according to the method described in Example 1.

[0068] HepG2 cells were loaded at 5 × 10⁻⁶ 3 Cells / mL were seeded in 96-well plates and cultured for 24 h until cell adhesion. Then, the cells were starved for 12 h using serum-free medium. Subsequently, control, model, and positive control groups (1 mM metformin combined with 10 μM sitagliptin) and different peptide treatment groups were established. The model, positive control, and peptide treatment groups were induced with insulin resistance (IR) using medium containing 18 mM glucosamine. The peptide treatment groups received the corresponding concentration of peptide, and cultured for another 24 h. After culture, the glucose concentration in the medium was measured according to the glucose assay kit instructions, and calculated as "glucose consumption = glucose content in the control group - glucose content in the treatment group".

[0069] like Figure 9As shown, the glucose consumption in the control group was 2.50±0.08 mM, while that in the model group decreased to 0.90±0.05 mM, indicating a reduction in glucose utilization in HepG2 cells after glucosamine induction. The glucose consumption in the positive control group was 2.40±0.07 mM, indicating that the model evaluation system had good responsiveness. Compared with the model group, all six peptides increased glucose consumption in IR-HepG2 cells to varying degrees at 100, 200, or 400 μM. Specifically, at 100 μM, the glucose consumption in the QPEVMG treatment group was 1.86±0.09 mM; at 200 μM, the glucose consumption increased to 2.12±0.08 mM; and at 400 μM, the glucose consumption in the LARELEEL and QPEVMG treatment groups were 2.15±0.08 mM and 1.98±0.08 mM, respectively. QPEVMG maintains a high glucose consumption level within the range of 100–400 μM, making it a preferred peptide for improving glucose utilization in IR-HepG2 cells.

[0070] Example 5: Effects of synthetic peptides on glycogen synthesis in IR-HepG2 cells

[0071] This example demonstrates the results of in vitro assays to determine the effects of different peptides on glycogen synthesis in IR-HepG2 cells. The purified peptides were prepared according to the method described in Example 1.

[0072] HepG2 cells were fed at a rate of 1×10⁻⁶ 5 Cells / mL were seeded in 12-well plates and cultured for 24 h. Following the same principles as the glucose consumption experiment (Example 4), model construction, grouping, and peptide treatment were performed. After treatment, intracellular glycogen content was measured using a glycogen assay kit to evaluate the effect of synthetic peptides on glycogen synthesis. The results are as follows: Figure 10 As shown.

[0073] like Figure 10As shown, the glycogen synthesis level in the control group was 0.71±0.02 mg / mg Pro, while it decreased to 0.39±0.02 mg / mg Pro in the model group; the positive control group was 0.65±0.02 mg / mg Pro. Compared with the model group, all six peptides increased glycogen synthesis in IR-HepG2 cells to varying degrees at 100, 200, or 400 μM. Specifically, at 100 μM, the glycogen synthesis level in the QPEVMG treatment group was 0.60±0.02 mg / mg Pro, and the glycogen synthesis levels in the VPGEETS, PGEIVESL, and RELEELN treatment groups were also significantly higher than those in the model group (p<0.05); at 200 μM, the glycogen synthesis level in the QPEVMG group increased to 0.62±0.02 mg / mg Pro; and at 400 μM, the glycogen synthesis level in the QPEVMG group was 0.56±0.02 mg / mg Pro. QPEVMG maintains a high level of glycogen synthesis in the range of 100~400 μM, and can be regarded as a preferred peptide to promote glycogen synthesis in IR-HepG2 cells.

[0074] Example 6 Effect of synthetic peptides on the relative activity of DPP-IV in IR-HepG2 cells

[0075] This example demonstrates the results of determining the effect of different peptides on the relative activity of IR-HepG2 DPP-IV in vitro. The purified peptides were prepared according to the method in Example 1.

[0076] HepG2 cells were loaded at 5 × 10⁻⁶ 3 Cells / mL were seeded in 96-well plates and cultured for 24 h until cell adhesion. Then, the cells were starved for 12 h using serum-free medium. Subsequently, control, model, and positive control groups (metformin combined with sitagliptin, as in Example 4) and different peptide treatment groups were set up. The model, positive control, and synthetic peptide treatment groups were induced with insulin resistance using medium containing 18 mM glucosamine. The peptide treatment groups received the corresponding concentration of synthetic peptide. After 24 h of culture, cell supernatant was collected. 20 μL of cell supernatant, 30 μL of Tris-HCl buffer (pH 8.0), and 50 μL of Gly-Pro-pNA substrate solution (final concentration 500 μM) were added sequentially to each well of the 96-well plate. The reaction was carried out at 37 ℃ in the dark for 1 h, and the absorbance of each well was measured at 405 nm using a microplate reader. The relative content of DPP-IV was calculated as "DPP-IV relative content (%) = OD...". 样品组 / OD 空白对照组 Calculated as ×100%, where OD 样品组The absorbance values ​​are those of the model group, positive control group, or groups treated with different concentrations of peptides; OD 空白对照组 The absorbance value of the culture supernatant of HepG2 cells without modeling treatment represents the basic DPP-IV enzyme activity level of the cells.

[0077] like Figure 11 As shown, with the relative DPP-IV activity of the normal control group as 100%, the model group increased to 120.32±2.45%, indicating an increase in DPP-IV-related enzyme activity in the supernatant of IR-HepG2 cells after glucosamine induction; the positive control group decreased to 79.78±2.35%. After peptide treatment, the relative DPP-IV activity decreased to varying degrees compared to the model group. The 100, 200, and 400 μM QPEVMG treatment groups showed activity levels of 92.81±2.70%, 87.46±2.95%, and 83.84±3.55%, respectively, showing a gradual decreasing trend with increasing concentration. Based on the changes in different sequences and concentrations, QPEVMG can be considered a preferred peptide for reducing the relative DPP-IV activity in IR-HepG2 cells.

[0078] Example 7: Preferred Peptides and Evaluation

[0079] Based on the results of Examples 2-6, a comprehensive evaluation suggests that QPEVMG can be considered a preferred polypeptide of the present invention. Firstly, the IC50 of QPEVMG's in vitro DPP-IV inhibitory activity is 41.57±1.86 μM, significantly lower than other synthetic peptides (p<0.05). Secondly, in HepG2 cells, QPEVMG did not show significant cell viability inhibition within the range of 100-400 μM. Thirdly, in the IR-HepG2 cell model, QPEVMG increased glucose consumption and glycogen synthesis at 100, 200, and 400 μM. Fourthly, QPEVMG reduced the relative activity of DPP-IV to 83.84±3.55% at 400 μM.

[0080] Besides QPEVMG, LARELEEL also showed certain advantages at some concentrations, such as higher glucose consumption at 400 μM and lower relative DPP-IV activity at 200 μM; RELEELN also showed a certain improvement trend in glycogen synthesis and relative DPP-IV activity. The above-mentioned synthetic peptides can be used as single active ingredients or combined with QPEVMG depending on the product form and target of action.

[0081] In actual product development, QPEVMG can be used alone or in combination with one or more of VPGEIVES, PGEIVESL, PEVMG, RELEELN, and LARELEEL. The composition can be prepared as powder, tablet, capsule, granule, oral liquid, fermented milk, milk beverage, solid beverage, nutrition bar, or other edible or pharmaceutical dosage forms.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A linear peptide with DPP-IV inhibitory activity, characterized in that, The linear peptide is selected from one of the linear peptides whose amino acid sequences are shown in SEQ ID NO:1 to SEQ ID NO:

6.

2. A composition of a linear peptide having DPP-IV inhibitory activity, comprising a pharmaceutically acceptable excipient and a linear peptide having DPP-IV inhibitory activity, characterized in that, The linear peptide with DPP-IV inhibitory activity is selected from at least one of the linear peptides with amino acid sequences as shown in SEQ ID NO:1 to SEQ ID NO:

6.

3. The composition of a linear peptide having DPP-IV inhibitory activity according to claim 2, characterized in that, The linear peptide having DPP-IV inhibitory activity includes at least the linear peptide shown in SEQ ID NO:

4.

4. The composition of a linear peptide having DPP-IV inhibitory activity according to claim 2, characterized in that, The linear peptide with DPP-IV inhibitory activity is selected from at least two of the linear peptides with amino acid sequences as shown in SEQ ID NO:1 to SEQ ID NO:

6.

5. The composition of a linear peptide having DPP-IV inhibitory activity according to claim 4, characterized in that, The linear peptide with DPP-IV inhibitory activity contains at least one of the linear peptides with the amino acid sequence shown in SEQ ID NO:4; and at least one of the linear peptides shown in SEQ ID NO:5 and SEQ ID NO:

6.

6. The composition of a linear peptide having DPP-IV inhibitory activity according to claim 2, characterized in that, The formulation of the composition is either an oral formulation or an injectable formulation.

7. The composition of a linear peptide having DPP-IV inhibitory activity according to claim 6, characterized in that, The oral preparation is selected from at least one of powder, tablet, capsule, granule, and oral liquid.

8. The composition of a linear peptide having DPP-IV inhibitory activity according to claim 6, characterized in that, The injectable formulation is selected from at least one of subcutaneous injection formulations, intravenous injection formulations, and intramuscular injection formulations.

9. Use of a composition of a linear peptide having DPP-IV inhibitory activity as described in claim 1 or any one of claims 2 to 8 in the preparation of a hypoglycemic drug.

10. The use according to claim 9, characterized in that, The hypoglycemic drug is one that improves insulin resistance.

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