Screening method of lipid-lowering peptide from suaeda salsa and target prediction thereof

Abstract

The application discloses a screening method of a lipid-lowering peptide of Suaeda salsa and target point prediction, relates to the technical field of bioactive peptide processing and application, and discloses that the amino acid sequence of the lipid-lowering peptide is GGPSWNVK; the lipid-lowering target points are microsomal triglyceride transfer protein, 3-hydroxy-3-methylglutaryl coenzyme A reductase and proprotein convertase subtilisin 9; the active peptide GGPSWNVK with a clear lipid-lowering target point is screened from Suaeda salsa, stable combination of the active peptide with the three target points of MTP, HMGR and PCSK9 is verified through molecular docking and molecular dynamics simulation, the active peptide has low binding energy and good safety, can effectively improve lipid accumulation and oxidative stress, and provides an important candidate molecule for development of a new lipid-lowering drug, a special medical food and a health product.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide processing and application technology, specifically to a screening method for lipid-lowering peptides from Suaeda salsa and its target prediction. Background Technology

[0002] Hyperlipidemia and related lipid metabolism disorders are metabolic diseases characterized by abnormally elevated levels of blood lipids such as triglycerides, total cholesterol, and low-density lipoprotein cholesterol. Their pathogenesis is closely related to imbalances in lipid absorption, synthesis, transport, and catabolism. Long-term lipid metabolism abnormalities can further induce a series of serious cardiovascular and cerebrovascular complications, such as non-alcoholic fatty liver disease, atherosclerosis, coronary heart disease, and cerebral infarction, posing a significant public health threat to human health.

[0003] Bioactive peptides are small molecule peptides composed of 2 to 20 amino acids. They are easily absorbed, highly safe, and physiologically active, and have important applications in lipid-lowering, antioxidant, anti-inflammatory, and metabolic regulation. Suaedasalsa is a salt-tolerant plant that grows in saline-alkali soils. Its stems and leaves are high in protein and have a well-balanced amino acid composition, making it a high-quality raw material for preparing plant-derived bioactive peptides. Furthermore, it has both edible and medicinal value, being rich in high-quality protein, dietary fiber, and various bioactive components.

[0004] Currently, clinical treatment of hyperlipidemia and lipid metabolism disorders mainly relies on chemically synthesized lipid-lowering drugs, such as statins, fibrates, and cholesterol absorption inhibitors. Existing natural product-related technologies mostly obtain mixed peptides through crude extraction or simple enzymatic hydrolysis of plant proteins for lipid-lowering functional research. However, in the development of lipid-lowering active substances from natural plants, it is not possible to selectively screen single active peptides with well-defined structures and specific lipid-lowering targets from the enzymatic hydrolysis products of *Suaeda salsa*.

[0005] Therefore, there is an urgent need in this field to develop a method for efficiently screening single short peptides with well-defined structures and clear lipid-lowering targets from Suaeda salsa, and to obtain lipid-lowering peptides with application value. Summary of the Invention

[0006] The purpose of this invention is to provide a screening method for lipid-lowering peptides from Suaeda salsa and its target prediction, in order to solve the problem of the lack of single short peptides with clear lipid-lowering activity and targets in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a lipid-lowering peptide of Suaeda salsa, the amino acid sequence of which is GGPSWNVK.

[0008] Furthermore, its lipid-lowering targets are microsomal triglyceride transfer protein, 3-hydroxy-3-methylglutaryl-CoA reductase, and proprotein convertase subtilisin-9.

[0009] A method for screening lipid-lowering peptides from *Suaeda salsa* includes the following steps:

[0010] S1. Using crude protein from Suaeda salsa as raw material, peptide components with a molecular weight of less than 3kDa were obtained through enzymatic hydrolysis, ultrafiltration, and gel chromatography separation.

[0011] S2. The peptide components were sequenced using liquid chromatography-mass spectrometry to obtain candidate short peptides;

[0012] S3. Molecular docking was performed between candidate short peptides and microsomal triglyceride transfer protein, 3-hydroxy-3-methylglutaryl-CoA reductase, and proprotein convertase subtilisin-9 to screen peptides with binding energy below -5 kcal / mol that can enter the target active pocket.

[0013] S4. Perform 100 ns molecular dynamics simulation on the screened peptides, and determine the target lipid-lowering peptide by using the root mean square deviation being stable within 0.3 nm as the criterion.

[0014] Further, the enzymatic hydrolysis step in step S1 uses alkaline protease, and the enzymatic hydrolysis conditions are: pH 8.7, temperature 67℃, enzyme dosage 6600 U / g, and hydrolysis time 2.3 h; ultrafiltration is carried out sequentially using 10 kDa and 5 kDa ultrafiltration membranes, and the filtrate is collected.

[0015] Furthermore, in step S3, molecular docking is performed using Schrödinger software, with the docking box covering the target active pocket region.

[0016] Furthermore, the final amino acid sequence of the lipid-lowering peptide obtained from the screening was GGPSWNVK.

[0017] A method for synthesizing lipid-lowering peptides from Suaeda salsa employs an Fmoc solid-phase synthesis strategy: using 2-chlorotriphenylmethyl chloride resin as a carrier, Fmoc-protected amino acids are sequentially coupled from the C-terminus to the N-terminus according to the GGPSWNVK sequence; the protection is deprotected by trifluoroacetic acid cleavage solution and the resin is cleaved, and the crude peptide is purified by reversed-phase high-performance liquid chromatography to obtain the target lipid-lowering peptide.

[0018] Application of a lipid-lowering peptide from Suaeda salsa in the preparation of lipid-lowering drugs, special medical foods for lipid-lowering, or lipid-lowering health products.

[0019] Compared with existing technologies, this invention provides a screening method for lipid-lowering peptides from Suaeda salsa and its target prediction. The active peptide GGPSWNVK with a clear lipid-lowering target was screened from Suaeda salsa. The stable binding of GGPSWNVK to three targets, MTP, HMGR and PCSK9, was verified by molecular docking and molecular dynamics simulation. It has low binding energy, good safety, and can effectively improve lipid accumulation and oxidative stress, providing an important candidate molecule for the development of new lipid-lowering drugs, special medical foods and health products. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0021] Figure 1 A schematic diagram of molecular docking between the lipid-lowering peptide GGPSWNVK of *Suaeda salsa* and microsomal triglyceride transfer protein (MTP), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), and proprotein convertase subtilisin 9 (PCSK9) provided in an embodiment of the present invention.

[0022] Figure 2 Molecular dynamics simulation stability analysis diagram of the complex of the lipid-lowering peptide GGPSWNVK of Suaeda salsa with three lipid-lowering target proteins: HMGR, PCSK9, and MTP, provided in the embodiments of the present invention;

[0023] Figure 3 The free energy surface (FEL) diagram of the complex of the lipid-lowering peptide GGPSWNVK of Suaeda salsa with three lipid-lowering target proteins, HMGR, MTP and PCSK9, provided in the embodiments of the present invention;

[0024] Figure 4 The reversed-phase high-performance liquid chromatogram of the lipid-lowering peptide GGPSWNVK from Suaeda salsa provided in this embodiment of the invention;

[0025] Figure 5 The mass spectrum of the lipid-lowering peptide GGPSWNVK from Suaeda salsa provided in this embodiment of the invention;

[0026] Figure 6 The effect of the lipid-lowering peptide GGPSWNVK from Suaeda salsa provided in this embodiment of the invention on the survival rate of HepG2 cells;

[0027] Figure 7 The figure shows the effect of the lipid-lowering peptide GGPSWNVK from Suaeda salsa provided in this embodiment of the invention on OA-induced ALT activity in HepG2 cells;

[0028] Figure 8 The figure shows the effect of the lipid-lowering peptide GGPSWNVK from Suaeda salsa provided in this embodiment of the invention on OA-induced AST activity in HepG2 cells;

[0029] Figure 9 The figure shows the effect of the lipid-lowering peptide GGPSWNVK from Suaeda salsa provided in this embodiment of the invention on OA-induced LDH activity in HepG2 cells;

[0030] Figure 10 The figure shows the effect of the lipid-lowering peptide GGPSWNVK from Suaeda salsa provided in this embodiment of the invention on OA-induced SOD activity in HepG2 cells;

[0031] Figure 11 The figure shows the effect of the lipid-lowering peptide GGPSWNVK from Suaeda salsa provided in this embodiment of the invention on OA-induced TC content in HepG2 cells;

[0032] Figure 12 The figure shows the effect of the lipid-lowering peptide GGPSWNVK from Suaeda salsa provided in this embodiment of the invention on OA-induced TG content in HepG2 cells;

[0033] Figure 13 The figure shows the effect of the lipid-lowering peptide GGPSWNVK from Suaeda salsa provided in this embodiment of the invention on OA-induced γ-GT activity in HepG2 cells. Detailed Implementation

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0035] Example 1:

[0036] Please see Figure 1-3 This embodiment provides the screening of lipid-lowering peptides from Suaeda salsa.

[0037] crude protein extraction

[0038] The aerial parts of *Suaeda salsa* were collected, washed, dried, and pulverized, then passed through a 60-mesh sieve. Crude protein powder was prepared according to the method disclosed in CN115896221A: 0.04 mol / L phosphate buffer solution was added at a material-to-liquid ratio of 1:13 (w / v), and the mixture was extracted by ultrasonication. After centrifugation, the supernatant was collected, and ammonium sulfate was added to 80% saturation. The mixture was allowed to stand overnight at 4°C, centrifuged, and the precipitate was collected. The precipitate was dialyzed for 24 h and then freeze-dried to obtain crude protein powder from *Suaeda salsa*.

[0039] Enzymatic hydrolysis and enrichment of low molecular weight components

[0040] The crude protein powder was prepared into a 5% (w / v) aqueous solution and enzymatically hydrolyzed with alkaline protease at pH 8.7, temperature 67℃, and enzyme dosage of 6600 U / g for 2.3 h. The hydrolysate was boiled for 10 min to inactivate the enzyme, cooled, and centrifuged (10000 rpm, 10 min). The supernatant was passed through 10 kDa and 5 kDa ultrafiltration membranes sequentially. The filtrate was collected, freeze-dried, and then separated by gel chromatography to obtain peptide fractions with a molecular weight less than 3 kDa.

[0041] Gel chromatography purification

[0042] The lyophilized powder was prepared into a 3 mg / mL solution with ultrapure water and loaded onto a Sephadex G-25 column (1.6 cm × 150 cm). Elution was performed with ultrapure water (20 mL / h), and detection was conducted at 280 nm using UV. Each elution peak was collected, and the inhibition rate against pancreatic lipase (in vitro lipid-lowering activity) was determined. The fraction with the highest activity was then lyophilized for later use.

[0043] Peptide sequence identification

[0044] The active component was separated by reversed-phase high-performance liquid chromatography (RP-HPLC): a C18 column (4.6 × 250 mm, 5 μm), mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-acetonitrile, with gradient elution (5%–50% B, 60 min), a flow rate of 0.8 mL / min, and a detection wavelength of 214 nm. Each chromatographic peak was collected.

[0045] Identification was performed using a Q-Exactive mass spectrometer in positive ion mode, with a scan range of m / z 100-2000. The top 10 strongest fragment ions were collected after each full scan, and the analysis time was 60 min. Sixty-three unique short peptide sequences were successfully identified. The amino acid lengths of these peptides were mainly concentrated in the range of 8 to 13 residues, and their molecular weights were all below 2 kDa.

[0046] Sixty-three short peptides were systematically screened using molecular docking technology to investigate their effects on ten key targets related to lipid metabolism regulation. These targets included ANGPTL4, CES1, HMGR, HTR2A, HTR2B, HTR2C, MTP, PCSK9, RXRA, and 5-HT4R. Screening results showed that the bioactive peptides primarily bound to three core targets: MTP, PCSK9, and HMGR. Notably, the peptide GGPSWNVK was found to stably bind to all three key targets simultaneously, suggesting a potential role in the synergistic regulation of blood lipids through multiple pathways.

[0047] Crystal structures of three lipid-lowering targets were downloaded from the RCSB: microsomal triglyceride transfer protein (MTP, PDB ID: 6I7S), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR, PDB ID: 1HW9), and proprotein convertase subtilisin 9 (PCSK9, PDB ID: 6U26).

[0048] Molecular docking was performed using Schrödinger software. A three-dimensional structure of the GGPSWNVK peptide was constructed and used as a ligand after energy minimization. The docking box was configured to cover the known active pocket regions of each target site; other parameters used default values.

[0049] The docking results showed that the binding energies of GGPSWNVK with MTP, HMGR, and PCSK9 were -10.324 kcal / mol, -9.307 kcal / mol, and -9.831 kcal / mol, respectively. These binding energies are all far below the set threshold of -5 kcal / mol, indicating that GGPSWNVK has a strong binding affinity to all three targets. Figure 1 As shown, GGPSWNVK forms a stable hydrogen bond network with each target site.

[0050] Interaction analysis showed that GGPSWNVK forms a stable hydrogen bond network with residues such as ASN755, ASN750, GLU559, and ALA865 in the MTP active pocket; it also forms a hydrogen bond network with the corresponding residues in the HMGR active pocket (according to the PDB structure number); and it also forms a hydrogen bond network with the corresponding residues in the PCSK9 active pocket, exhibiting a relatively stable binding mode.

[0051] Molecular dynamics simulation

[0052] Using GROMACS software (version 2020.4), the root mean square deviation (RMSD) of the complexes of GGPSWNVK with the three target sites was calculated under the TIP3P water model and a simulation time of 100 ns. The results showed that the RMSD of each complex stabilized in the range of 0.2–0.3 nm after 20 ns (e.g., ...). Figure 2 As shown in the figure, this indicates that the peptide-target binding mode is stable. Figure 3 The free energy surface analysis results further confirmed the stability of the complex binding conformation.

[0053] Example 2:

[0054] Please see Figure 4-5 This embodiment provides the solid-phase synthesis of the lipid-lowering peptide GGPSWNVK from Suaeda salsa.

[0055] Resin swelling

[0056] Using 2-chlorotriphenylmethyl chloride resin (dichloro resin) as a carrier, 0.1 mmol (approximately 0.2-0.3 g) of resin with a degree of substitution of 0.3-0.8 mmol / g was weighed and placed in a reaction column. 20 mL of dichloromethane (DCM) was added, and the mixture was shaken to swell for 30 min. The DCM was then removed by filtration.

[0057] Loading of the first amino acid

[0058] Add 1.05 times the molar amount of Fmoc-Lys(Boc)-OH protected amino acid (approximately 0.105 mmol), 10 times the molar amount of N,N-diisopropylethylamine (DIEA, approximately 1.0 mmol), and a small amount of N,N-dimethylformamide (DMF) to the swollen resin to dissolve it, and shake to react for 1 h; after the reaction is complete, wash the resin 6 times alternately with DMF and DCM.

[0059] Fmoc deprotection

[0060] Add 20 mL of 20% (v / v) piperidine / DMF solution to the resin, shake for 5 min and then remove the solution. Add another 20 mL of 20% piperidine / DMF solution and shake for 15 min. Remove the reaction solution.

[0061] Post-protection testing

[0062] Take a small amount of resin, wash it three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol, and heat it at 105~110℃ for 5 minutes. If the resin turns dark blue (positive), it indicates that the deprotection is complete and proceed to the next step; if it is colorless, repeat step 2.3.

[0063] washing

[0064] The resin was washed twice with 15 mL each of DMF, methanol, and DMF in sequence.

[0065] amino acid condensation

[0066] Following the target sequence GGPSWNVK from C-terminus to N-terminus, add 3 times the resin molar amount of the next Fmoc-protected amino acid, 3 times the resin molar amount of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), dissolve in a small amount of DMF, then add 10 times the resin molar amount of DIEA, and shake for 30 min.

[0067] Wash after condensation

[0068] The resin was washed twice with 15 mL each of DMF, methanol, and DMF in sequence.

[0069] Post-condensation testing

[0070] Take a small amount of resin, wash it three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol, and heat it at 105~110℃ for 5 minutes. If the resin is colorless (negative), the condensation is complete; if it is blue, repeat step 2.6 for a second condensation.

[0071] peptide chain elongation

[0072] Repeat the above steps (Fmoc deprotection ~ detection after condensation) to sequentially couple the remaining amino acids until the complete sequence is completed: Gly-Gly-Pro-Ser-Trp-Asn-Val-Lys.

[0073] Peptide resin shrinkage

[0074] After the last amino acid coupling was completed, the mixture was washed three times with DMF, three times with DCM, and three times with methanol, and finally dried to obtain a dry peptide resin.

[0075] Cutting and side chain deprotection

[0076] Prepare 15 mL of cutting solution with a volume ratio of trifluoroacetic acid (TFA):water:1,2-ethylenedithiol (EDT):triisopropylsilane (TIS) = 94.5:2:2.5:1. Add the above peptide resin to the cutting solution and incubate at 30°C with shaking for 2 h. After the reaction is complete, purge most of the TFA with nitrogen gas, slowly add the remaining liquid to pre-cooled diethyl ether, centrifuge, discard the supernatant, wash the precipitate 6 times with diethyl ether, and evaporate to dryness at room temperature to obtain the crude peptide.

[0077] purification

[0078] The crude peptide was dissolved in acetonitrile / water (containing 0.1% TFA) and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). Chromatographic conditions: C18 column (4.6 × 250 mm, 5 μm); mobile phase A: 0.1% TFA-water; mobile phase B: 0.1% TFA-acetonitrile; gradient elution (5%–50% B, 30 min); flow rate: 1.0 mL / min; detection wavelength: 214 nm. The target peak was collected and lyophilized to obtain the refined peptide GGPSWNVK with a purity ≥95%.

[0079] like Figure 4 As shown, the HPLC chromatogram indicates good purity of the main peak; identification by mass spectrometry (ESI-MS) ( Figure 5 The measured molecular weight is consistent with the theoretical molecular weight (843.93 Da).

[0080] Example 3:

[0081] Please see Figure 6-13 This embodiment provides verification of cellular lipid-lowering activity.

[0082] Experimental replication and statistical methods

[0083] All experiments were independently repeated three times, and data are expressed as mean ± standard deviation (SD). One-way ANOVA was performed using GraphPad Prism 8.0, and Tukey's post-hoc test was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0084] Cell Culture and Model Establishment

[0085] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. When the cells reached 80% confluence, they were treated with 0.5 mM oleic acid (OA) for 24 h to establish a lipid accumulation model.

[0086] Grouping and Dosing

[0087] This experiment employed a dose-response design, treating cells with different concentrations of GGPSWNVK. The experiment consisted of six groups, grouped according to a treatment concentration gradient: 0 (blank control), 25, 50, 100, 150, and 200 μg / ml. Figure 6 As can be seen, the cell survival rate in each dose group was >90%, indicating that GGPSWNVK has no significant cytotoxicity.

[0088] The following groups were set up: a normal control group (without OA treatment), a model group (with OA treatment), a positive control group (simvastatin 10 μM), and low, medium, and high dose groups of GGPSWNVK (100, 150, and 200 μM). OA was added simultaneously with drug administration, and the incubation was carried out for a total of 24 h.

[0089] Indicator Testing

[0090] Cell supernatants were collected, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and gamma-glutamyl transferase (γ-GT) were measured. Cells were lysed, and the intracellular levels of triglycerides (TG), total cholesterol (TC), and superoxide dismutase (SOD) were measured. All kits were purchased from Nanjing Jiancheng Biotechnology Institute.

[0091] Figure 7-9 The results showed that ALT, AST, and LDH levels were significantly reduced; Figure 10 The results showed a significant increase in SOD activity; Figure 11-12 The results showed a decrease in TC and TG levels; Figure 13The results showed decreased γ-GT activity. Compared with the model group, the medium and high doses of GGPSWNVK significantly reduced ALT, AST, γ-GT, LDH, TG, and TC levels (P<0.05 or P<0.01) and increased SOD activity. In the positive control group (simvastatin 10 μM), TG and TC levels decreased to 42.3% and 51.6% of those in the model group, respectively, and ALT, AST, γ-GT, and LDH were also significantly reduced, while SOD activity was significantly increased. The lipid-lowering effect of the high-dose GGPSWNVK group (200 μM) was comparable to that of the positive control group (P>0.05). These results indicate that this lipid-lowering peptide can effectively improve oleic acid-induced lipid accumulation and oxidative stress in HepG2 cells.

[0092] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A lipid-lowering peptide from Suaeda salsa, characterized in that, Its amino acid sequence is GGPSWNVK.

2. The lipid-lowering peptide of *Suaeda salsa* according to claim 1, characterized in that, Its lipid-lowering targets are microsomal triglyceride transfer protein, 3-hydroxy-3-methylglutaryl-CoA reductase, and proprotein convertase subtilisin-9.

3. A method for screening the lipid-lowering peptides of *Suaeda salsa* as described in claim 1, characterized in that, Includes the following steps: S1. Using crude protein from Suaeda salsa as raw material, peptide components with a molecular weight of less than 3 kDa were obtained through enzymatic hydrolysis, ultrafiltration, and gel chromatography separation. S2. The peptide components were sequenced using liquid chromatography-mass spectrometry to obtain candidate short peptides; S3. Molecular docking was performed between candidate short peptides and microsomal triglyceride transfer protein, 3-hydroxy-3-methylglutaryl-CoA reductase, and proprotein convertase subtilisin-9 to screen peptides with binding energy below -5 kcal / mol that can enter the target active pocket. S4. Perform 100 ns molecular dynamics simulation on the screened peptides, and determine the target lipid-lowering peptide by using the root mean square deviation being stable within 0.3 nm as the criterion.

4. The method for screening lipid-lowering peptides from *Suaeda salsa* according to claim 3, characterized in that, The enzymatic hydrolysis step described in step S1 uses alkaline protease, and the enzymatic hydrolysis conditions are: pH 8.7, temperature 67℃, enzyme dosage 6600 U / g, and hydrolysis time 2.3 h; ultrafiltration is carried out sequentially using 10 kDa and 5 kDa ultrafiltration membranes, and the filtrate is collected.

5. The method for screening lipid-lowering peptides from *Suaeda salsa* according to claim 3, characterized in that, In step S3, molecular docking was performed using Schrödinger software, with the docking box covering the target active pocket region.

6. The method for screening lipid-lowering peptides from *Suaeda salsa* according to claim 3, characterized in that, The final amino acid sequence of the lipid-lowering peptide obtained from the screening was GGPSWNVK.

7. A method for synthesizing the lipid-lowering peptide of *Suaeda salsa* according to claim 1, characterized in that, The Fmoc solid-phase synthesis strategy was adopted: using 2-chlorotriphenylmethyl chloro resin as a carrier, Fmoc-protected amino acids were sequentially coupled from the C-terminus to the N-terminus according to the GGPSWNVK sequence; the protection was removed by trifluoroacetic acid cleavage solution and the resin was cleaved, and the crude peptide was purified by reversed-phase high-performance liquid chromatography to obtain the target lipid-lowering peptide.

8. The use of the lipid-lowering peptide of Suaeda salsa as described in claim 1 in the preparation of lipid-lowering drugs, lipid-lowering special medical foods, or lipid-lowering health products.

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