A horse milk-derived small molecule peptide and application thereof

Abstract

The application discloses a milk-derived small molecular peptide, which has an amino acid sequence of Phe-Gly-Gly-Leu-Met (FGGLM) and a molecular weight of 523.64 Da. The small molecular peptide has the characteristics of inhibiting the activity of pancrelipases (PL) and reducing the accumulation of triglyceride (TG) content in oleic acid-induced human hepatoma (Hepg2) cells. The small molecular peptide can be applied to the preparation of preparations for treating or assisting in treating hyperlipidemia and obese people, and has the characteristics of simple preparation, suitability for industrial production and market promotion and application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a horse milk-derived small molecule peptide and application thereof. BACKGROUND

[0002] Pancreatic lipase is a kind of enzyme with lipolytic activity, which plays an important role in lipid metabolism. The inhibition and regulation of pancreatic lipase is an ideal target for preventing and treating metabolic diseases such as hyperlipidemia and obesity. Commonly used lipid-lowering drugs in clinical practice include statins, fibrates and cholesterol absorption inhibitors, etc. Although these drugs can effectively regulate lipid abnormalities and reduce plasma cholesterol, they also have many side effects such as rhabdomyolysis and gastrointestinal allergic reactions.

[0003] Bioactive peptides have various metabolic and physiological regulation functions in human body. A variety of lipid-lowering peptides have been isolated from the hydrolysates of whey, soybean and chlorella protein sources, among which the earliest identified sequence is milk-derived protein polypeptide. Therefore, the development of natural lipid-lowering active substances with abundant sources, safety and effectiveness has become a key research direction. SUMMARY

[0004] The present application provides a small molecule peptide derived from fermented horse milk, which has an amino acid sequence of Phe-Gly-Gly-Leu-Met (FGGLM) and a molecular weight of 523.64 Da. The small molecule peptide has pancreatic lipase inhibitory activity and can be used for preparing a pancreatic lipase inhibitor.

[0005] Another object of the present application is to apply the fermented horse milk-derived small molecule peptide to the preparation of an anti-obesity or hyperlipidemia-preventing and treating preparation.

[0006] The components (or effective components) of the preparation according to the present application are the above-mentioned small molecule peptide, and one or more pharmaceutically acceptable excipients can be further added to improve drug absorption effect or facilitate use, so as to be prepared into suitable use dosage forms such as capsules or pills, powders, tablets, granules, oral solutions and injection solutions, i.e. into pharmaceutically suitable use dosage forms. The preparation can be applied to the preparation of a drug for treating or adjuvant treating obesity or preventing and treating hyperlipidemia.

[0007] The object of the present application is achieved by the following scheme:

[0008] 1. Fresh horse milk is sterilized at 65-115 DEG C for 2-15 min, inoculated with lactic acid bacteria and yeast at an inoculation amount of 0.1-10%, and fermented at a temperature of 20-37 DEG C and a shaking speed of 50-300 rpm for 16-90 h; the lactic acid bacteria are Lactobacillus paracasei, and the yeast is Dekkaromyces hetero-thermophilus and Kazachstania unispora.

[0009] 2、Fresh mare milk fermentation is completed, 0.3% (w / v) pepsin is added, the pH of the fermented mare milk is adjusted to 4.0-4.8 with 5M HCl, and the mixture is treated at 30-38℃, 50-100rpm for 2-4h; then the pH is adjusted to 7.0 with 5M NaOH, 0.1% (w / v) trypsin is added, and the mixture is treated at 37℃, 60rpm for 2-4h; then the supernatant is obtained by centrifugation at 4℃, 12000g for 10-15min, and freeze-drying is performed to obtain a freeze-dried powder;

[0010] 3、The freeze-dried powder is dissolved in distilled water to prepare a solution with a concentration of 0.05-5g / mL, the solution is filtered through a cutoff ultrafiltration membrane with a cutoff of 10kDa and 3kDa to obtain a solution containing components with molecular weights >10kDa, 3-10kDa and <3kDa, respectively, and the freeze-dried powders containing different components are collected; the freeze-dried powders containing different components are dissolved in distilled water, and the pancreatic lipase inhibitory activity of each component is detected; the solution with the best inhibitory activity is selected for separation and purification by dextran gel column chromatography and reverse phase high performance liquid chromatography (RP-HPLC); the peak with pancreatic lipase inhibitory activity after RP-HPLC purification is selected for LC-MS identification; the peptide sequence obtained after identification is subjected to activity prediction in BIOPEP, and molecular docking simulation and pancreatic lipase inhibitory activity prediction are performed using α-fold3 and CB-Dock2 databases; the best prediction activity is selected for solid-phase synthesis verification, and finally the small molecule peptide of the application is obtained.

[0011] The small molecule peptide Phe-Gly-Gly-Leu-Met derived from fermented mare milk of the application can significantly inhibit pancreatic lipase activity, improve the accumulation of oleic acid-induced Hepg2 cell triglyceride content, and reduce the accumulation of oleic acid-induced Hepg2 cell lipid droplets; the small molecule peptide of the application can be applied to the preparation of a medicament for alleviating or assisting the treatment of obesity or hyperlipidemia. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The pancreatic lipase inhibitory activity results of the solutions containing different molecular weight substances after ultrafiltration cutoff;

[0013] Figure 2 The separation peak schematic diagram of the component with a molecular weight <3kDa after RP-HPLC separation;

[0014] Figure 3 The pancreatic lipase inhibitory activity detection results of the separation liquid corresponding to the separation peak after RP-HPLC separation;

[0015] Figure 4 The RP-HPLC separation results of the separation liquid in the 35-40min time period;

[0016] Figure 5The HPLC chromatogram of the separated liquid of peak 2;

[0017] Figure 6 The mass spectrum identification chart of the separated liquid of peak 2;

[0018] Figure 7 The molecular docking schematic diagram of the small molecule peptide FGGLM and pancreatic lipase;

[0019] Figure 8 The detection result of the small molecule peptide FGGLM in inhibiting the activity of pancreatic lipase in vitro;

[0020] Figure 9 The result of the effect of the small molecule peptide FGGLM on the activity of Hepg2 cells;

[0021] Figure 10 The effect of the small molecule peptide FGGLM on the accumulation of triglyceride content of Hepg2 cells induced by oleic acid. DETAILED DESCRIPTION

[0022] The present application is further described below by examples, but the protection of the present application is not limited to the content described, and the methods in the present examples are all operated according to the conventional methods if not specially described, and the experimental apparatus, reagents and the like are all obtained from commercial channels if not specially described.

[0023] The pancreatic lipase solution in the following examples is prepared with Tris-HCl buffer, the small molecule peptide solution is prepared with deionized water, and the p-NPB substrate solution is prepared with DMSO;

[0024] After mixing the pancreatic lipase solution and the small molecule peptide solution, preheat at 37℃ for 10 min, then add the p-NPB substrate solution at 37℃ and react for 15-30 min, take out the reaction product, centrifuge at 5000g for 5 min, take the supernatant and detect the absorbance value at 405 nm (experimental group);

[0025] At the same time, set up an experimental blank group: Tris-HCl buffer + small molecule peptide + p-NPB;

[0026] Control group: Tris-HCl buffer + PL + p-NPB, without small molecule peptide;

[0027] Control blank group: Tris-HCl buffer + p-NPB, without small molecule peptide and pancreatic lipase;

[0028] Add Orlistat as a positive control;

[0029] The reaction system is prepared according to Table 1, the experiment needs to be repeated three times, and the inhibition rate is calculated according to the following formula;

[0030] Table 1 Reaction system for determination of pancreatic lipase activity

[0031]

[0032] X 100%;

[0033] Experimental Example 1: Obtaining small molecule peptides

[0034] (1) Fresh mare milk was sterilized at 115°C for 10 min; after three generations of activation, the heteroform of Dekkera, Kazakh single spore yeast and Lactobacillus paracasei were inoculated into the sterilized fresh mare milk at an inoculation ratio of 2% for yeast and 4% for lactic acid bacteria, and then fermented at 37°C and 150 rpm for 48 h;

[0035] (2) 0.3% (w / v) pepsin was added to the fermented mare milk solution, the pH of the fermented mare milk was adjusted to 4.30 with 5M HCl, and the solution was treated at 37°C and 60 rpm for 2 h; then the pH value was adjusted to 7.0 with 5M NaOH, 0.1% (w / v) trypsin was added, and the solution was treated at 37°C and 60 rpm for 2 h, and then centrifuged at 4°C and 12000g for 15 min, the supernatant was taken and freeze-dried to obtain a freeze-dried powder;

[0036] (3) The freeze-dried powder was dissolved in deionized water to prepare a 100 mg / mL solution, and the solution was filtered through ultrafiltration membranes with different cut-off values of 10 kDa and 3 kDa to obtain three different components of >10 kDa, 3-10 kDa and <3 kDa, and the three components were freeze-dried using a vacuum freeze dryer and further prepared into a 50 mg / mL solution with deionized water, and the inhibition rate of each component on pancreatic lipase activity was determined, and the results are shown in Figure 1 From the figure, it can be seen that the component solution with a molecular weight of <3 kDa has the best effect on inhibiting pancreatic lipase activity. The component solution was further separated by RP-HPLC, and the results are shown in Figure 2 According to different time periods, the peaks in Figure 2 were divided into 7 parts, and the separated solutions of different time periods were collected and freeze-dried after removing the mobile phase using a rotary evaporator, and each time period was freeze-dried and configured into a 10 mg / mL solution with deionized water, and the inhibition activity of different component solutions on pancreatic lipase was determined, and the results are shown in Figure 3 From the figure, it can be seen that the component with the best effect on inhibiting pancreatic lipase activity is the separated solution of the 35-40 min time period, and the separated solution of the component was further separated by RP-HPLC, and the results are shown in Figure 4, the separated solution of different peaks was collected and freeze-dried after removing the mobile phase by rotary evaporator, and the inhibitory activity of the solution of different peaks on pancreatic lipase was determined. The results showed that the separated solution of peak No. 2 had the best inhibitory effect on pancreatic lipase activity. The high performance liquid chromatogram of the peak is shown in Figure 5 As can be seen from Figure 5 , the liquid phase results showed a single peak, indicating that the separation was completed.

[0037] The chromatographic conditions of RP-HPLC were as follows: injection volume 1000 μL, flow rate 2 mL / min, detection wavelength 215 nm; mobile phase A contained 0.1% (v / v) trifluoroacetic acid (TFA) in deionized water, mobile phase B was acetonitrile (CH3CN or C2H3N) containing 0.1% (v / v) TFA, gradient elution was performed using mobile phases A and B, and the elution program was as follows: 0-5 min, 10% B; 5-40 min, 50% B; 40-50 min, 60% B; 50-60 min, 10% B; 60-70 min, 10% B.

[0038] Example 2: LC-MS identification

[0039] The peak No. 2 component was subjected to LC-MS identification, and the analysis time was 30 min. The detection mode was positive ion. The mass-to-charge ratio of small molecule peptides and small molecule peptide fragments was collected as follows: 10 MS2 scans were performed for each full scan; the mass spectrometry raw file (Raw File) was searched against the corresponding database (Equus caballus) using software Mascot2.2, and finally the protein identification results were obtained.

[0040] See Figure 6 From the figure, the corresponding intensity and mass-to-charge ratio of the corresponding fragments can be obtained, and through database sequence alignment, it is confirmed that the small molecule peptide is FGGLM.

[0041] Experimental Example 3: Molecular docking of small molecule peptide FGGLM and pancreatic lipase

[0042] The three-dimensional model of pancreatic lipase (PL) was downloaded from RCBSPDB protein database, the three-dimensional structure of small molecule peptide was drawn using α-fold3, and NM2 force field was added to minimize the energy. Gold 5.3.0 was used for flexible molecular docking, ligand extraction was performed on the receptor, water molecules were removed, polar hydrogen was added, and Gold score scoring system was used for docking model screening. Moe 2019.10 software was used for visual analysis of the docking molecular complex, and the results are shown in Figure 7As can be seen from the figure, FGGLM forms 2 hydrogen bonds S153 and F78 with PL; 5 hydrophobic bonds F216, P181, V260, I79 and W253, and the total binding energy is -8.1 kcal / mol.

[0043] Example 4: Detection of inhibitory activity of small molecule peptide FGGLM on pancreatic lipase

[0044] The small molecule peptide sequence was synthesized by Shanghai Shenguo, with a purity of ≥98%, and the obtained small molecule peptide was gradient dissolved to obtain 1, 2, 3, 4, 5 mg / mL small molecule peptide solutions. The inhibitory effect of different concentrations of small molecule peptide solutions on pancreatic lipase activity was detected; the results are shown in Table 1. Figure 8 The inhibitory rate of small molecule peptide FGGLM on pancreatic lipase was 75.95% at a concentration of 5 mg / mL.

[0045] Experimental Example 5: Functional evaluation of small molecule peptide FGGLM

[0046] (1) Establishment of oleic acid (OA) -induced HepG2 cell high-fat model

[0047] After the HepG2 cells were resuscitated with DMEM high-sugar medium added with 20% FBS and 1% penicillin-streptomycin, they were cultured in a 37 °C, 5% CO2 carbon dioxide incubator to a confluence of 80%. The cells were digested with 0.25% trypsin and subcultured at a ratio of 1:3. The medium was replaced every 24 h. After the HepG2 cells were plated, the original culture supernatant was discarded, and the high-fat model was induced with DMEM medium containing 0.5 mmol / L oleic acid;

[0048] The model group (DC) was cultured with DMEM medium containing 0.5 mmol / L oleic acid;

[0049] The negative control group (NC) was cultured with DMEM medium without 0.5 mmol / L oleic acid;

[0050] The small molecule peptide group was cultured with different concentrations of small molecule peptide (1 mg, 2 mg, 3 mg, 4 mg, 5 mg) and DMEM medium containing 0.5 mmol / L oleic acid;

[0051] The positive control group (PC) was cultured with DMEM medium containing 0.5 mmol / L oleic acid and 25 μmol / L orlistat (Orlistat) ;

[0052] All groups were subjected to subsequent experiments after being cultured for 24 h;

[0053] (2) CCK-8 method to detect the effect of FGGLM small molecule peptide on cell activity

[0054] The HepG2 cells cultured and plated to 80% were digested with trypsin, and then a cell suspension was prepared with DMEM medium containing 10% FBS. The cells were seeded in 96-well plates at a concentration of 1.0 x 10 5 The small molecule peptide solution was added to the 96-well plates at a concentration of 1-5 mg / mL, and a control (NT) without the addition of the small molecule peptide was set up. The cells were incubated at 37°C in a cell incubator with 5% CO2 for 24 h. Then 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in the cell incubator for 4 h. The absorbance value was detected at OD 450 Each group was repeated three times, and the results are shown in Figure 9 It can be seen from the figure that the small molecule peptide FGGLM has no toxic side effects on HepG2 cells. Figure 9

[0055] (3) Effect of small molecule peptide FGGLM on accumulation of triglyceride content in HepG2 cells

[0056] The HepG2 cells cultured and plated to 80% were digested with trypsin, and then a cell suspension was prepared with DMEM medium containing 10% FBS. The cells were seeded in 96-well plates at a concentration of 1.0 x 10 5 The small molecule peptide solution was added to the 96-well plates at a concentration of 1-5 mg / mL, and a control (NT) without the addition of the small molecule peptide was set up. The cells were incubated at 37°C in a cell incubator with 5% CO2 for 24 h. Then 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in the cell incubator for 4 h. The absorbance value was detected at OD Figure 10 It can be seen from the figure that the small molecule peptide FGGLM can significantly improve the accumulation of triglyceride content in oleic acid-induced high-fat HepG2 cells.​

Claims

1. A small molecule peptide derived from horse milk, the amino acid sequence of which is Phe-Gly-Gly-Leu-Met.

2. The use of the horse milk-derived small molecule peptide according to claim 1 in the preparation of drugs for anti-obesity or prevention and treatment of hyperlipidemia.

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