A small molecule peptide and use thereof

Abstract

The application discloses a small molecule peptide, which has an amino acid sequence of Ser-Pro-Cys-Pro-Met (SPCPM) and a molecular weight of 533.64 Da. The small molecule peptide has the advantages of inhibiting the activity of pancreatic lipase and the inhibition rate of pancreatic lipase is 63.42%. The novel small molecule peptide can reduce the accumulation of triglyceride (TG) content in oleic acid-induced human hepatoma cells (Hepg2) (human hepatoma) and reduce the content of lipid droplets in oleic acid-induced Hepg2 cells. The small molecule peptide can be applied to the preparation of a medicine for treating or assisting in treating obesity, and has the advantages of simple preparation, suitability for industrial production and market promotion and application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a small molecule peptide derived from fermented bovine whey and its applications. Background Technology

[0002] Obesity is a chronic, non-infectious disease that poses a significant threat to human health and requires active treatment. In the 21st century, obesity has become one of the most serious public health problems facing both developed and developing countries, forming a new set of four major socio-medical challenges alongside AIDS, drug abuse, and alcoholism. Due to advancements in science and technology, changes in production methods, and altered dietary structures, the obese population is spreading globally, with its incidence rate increasing year by year. Obesity can cause a series of physiological, psychological, pathological, and neurohumoral regulatory changes in the human body, leading to decreased work capacity and even significantly shortened lifespan. The main danger of obesity lies in its potential to cause serious health consequences, such as inducing cardiovascular disease, diabetes, musculoskeletal disorders, and cancer, and these risks increase with rising body mass index (BMI).

[0003] Currently, over one billion people worldwide suffer from obesity, and it is projected that by 2035, more than 51% of the population will be overweight (27%) or obese (24%), demonstrating the serious threat obesity poses to human health and quality of life. Excessive dietary fat intake is a major contributing factor to obesity. Dietary fat cannot be directly absorbed by the body; it must be hydrolyzed into free fatty acids, monoacylglycerols, and diacylglycerols by pancreatic lipase secreted by the pancreas before being absorbed by intestinal cells. Therefore, inhibiting pancreatic lipase activity is a key control point in preventing the digestion and absorption of dietary fat. Orlistat is an approved weight-loss drug for long-term clinical use, but long-term use can cause side effects such as abdominal pain, bloating, and oily stools. Food-derived pancreatic lipase inhibitory peptides have advantages such as simple production methods, low cost, and few side effects. As people increasingly favor preventative healthcare and dietary therapy, this application has broad market prospects. Summary of the Invention

[0004] This invention provides a small molecule peptide derived from fermented bovine whey. The amino acid sequence of this small molecule peptide is Ser-Pro-Cys-Pro-Met (SPCPM), and its molecular weight is 533.64 Da. It has pancreatic lipase inhibitory activity.

[0005] Another objective of this invention is to apply the above-mentioned fermented milk small molecule peptides in the preparation of anti-obesity or anti-hyperlipidemia preparations.

[0006] The components (or active ingredients) of the formulations described in this invention are the aforementioned small molecule peptides. One or more pharmaceutically acceptable excipients may also be added to improve drug absorption or facilitate use, and to prepare suitable dosage forms, such as capsules or pills, powders, tablets, granules, oral liquids, and injections, etc., that is, to prepare pharmaceutically suitable dosage forms; it can be used to prepare drugs for the treatment or adjuvant treatment of obesity or prevention and treatment of hyperlipidemia.

[0007] The objective of this invention is achieved through the following solution:

[0008] (1) Activation of strains: Lactic acid bacteria and yeast were inoculated into MRS and YPD medium respectively, with an inoculation amount of 1-10%, a fermentation temperature of 20-37℃, a shaking speed of 100-300 rpm, a culture time of 8-24h, and activation for 2-3 generations; the above lactic acid bacteria were Lactobacillus paracasei, and the yeast was Dioscorea heterotrophus;

[0009] (2) Preparation of fermented whey: The whey solution was sterilized at 55-105 °C for 1-30 min. When the temperature dropped to room temperature, activated lactic acid bacteria and yeast were inoculated. The inoculation amount was 1-10%, the fermentation temperature was 20-37 °C, the shaking speed was 100-300 rpm, and the fermentation time was 12-50 h. After fermentation, 0.3% (w / v) pepsin was added to the fermented whey solution, and the pH of the fermented whey solution was adjusted to 4-4.7 with 5M HCl. After treatment at 37 °C and 60 rpm for 2 h, 5M NaOH was added to adjust the pH to 7.0. 0.1% (w / v) trypsin was added and treated at 37 °C and 60 rpm for 2 h to simulate enzymatic hydrolysis in vitro. The supernatant was then centrifuged and freeze-dried to obtain lyophilized powder.

[0010] (3) Dissolve an appropriate amount of lyophilized powder in deionized water to prepare a solution with a concentration of 5-200 mg / mL. Filter the prepared solution using three ultrafiltration membranes with different molecular weights to obtain liquids with different components. Determine the pancreatic lipase inhibitory activity of each liquid component. Separate and purify the ultrafiltration component with the best inhibitory activity using reverse high-performance liquid chromatography (RP-HPLC). Determine the pancreatic lipase inhibitory activity of the RP-HPLC-purified peak. Then, screen the peptides with the best inhibitory effect on pancreatic lipase activity using LC-MS. Predict whether the identified peptide sequences have biological activity using PetideRanker in BIOWARE. Subsequently, predict pancreatic lipase inhibitory activity using α-flod3. Select the peptide sequence with the best predicted activity for synthesis verification to obtain the small molecule peptide of this invention.

[0011] The fermented whey small molecule peptide sequence disclosed in this invention is Ser-Pro-Cys-Pro-Met, which can significantly inhibit pancreatic lipase activity, improve the accumulation of triglycerides in oleic acid-induced Hepg2 cells, and reduce the accumulation of lipid droplets in Hepg2 cells induced by oleic acid. The small molecule peptide of this invention can be used to prepare drugs to alleviate or assist in the treatment of obesity or hyperlipidemia, which are prevalent diseases. Attached Figure Description

[0012] Figure 1 The results show the inhibitory activity of solutions containing substances of different molecular weights after ultrafiltration on pancreatic lipase.

[0013] Figure 2 This is a schematic diagram of the peak separation of a solution containing components with a molecular weight <3kDa using RP-HPLC.

[0014] Figure 3 The results of pancreatic lipase inhibitory activity assay for solutions containing components with molecular weight <3kDa separated by RP-HPLC;

[0015] Figure 4 This is a schematic diagram of the separation peaks after RP-HPLC separation of the separated solution over a period of 25-30 min.

[0016] Figure 5 The high-performance liquid chromatogram of the separated solution from peak 1;

[0017] Figure 6 The mass spectrometry chromatogram for the separated solution of peak 1 is shown.

[0018] Figure 7 This is a schematic diagram showing the docking results of the small molecule peptide SPCPM with pancreatic lipase.

[0019] Figure 8 The results of in vitro detection of pancreatic lipase activity by the small molecule peptide SPCPM;

[0020] Figure 9 Results of the effect of small molecule peptide SPCPM on Hepg2 cell viability;

[0021] Figure 10 The results show the effect of the small molecule peptide SPCPM on the accumulation of triglycerides in Hepg2 cells induced by oleic acid. Detailed Implementation

[0022] The present invention will be further illustrated by the following examples, but the protection of the present invention is not limited to the content described. Unless otherwise specified, the methods in this embodiment are operated according to conventional methods, and the experimental equipment, reagents and other materials used are obtained from commercial sources unless otherwise specified.

[0023] In the examples below, the pancreatic lipase solution was prepared with Tris-HCl buffer, the small molecule peptide solution was prepared with deionized water, and the p-NPB substrate solution was prepared with DMSO.

[0024] After mixing pancreatic lipase solution and small molecule peptide solution, the mixture was preheated at 37℃ for 10 min, and then p-NPB substrate solution was added and reacted for 5–30 min. The absorbance value was then measured at 405 nm (experimental group).

[0025] A blank control group was also set up: Tris-HCl buffer + small molecule peptide solution + p-NPB;

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

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

[0028] Positive controls were supplemented with orlistat.

[0029] The reaction system was prepared according to Table 1. The experiment was repeated three times, and the inhibition rate was calculated using the following formula.

[0030] Table 1-1 Reaction system for pancreatic lipase activity assay

[0031] Table 1-1 reaction system of pancreatic lipase activity determination

[0032]

[0033] ;

[0034] Example 1: Obtaining Small Molecule Peptides

[0035] (1) Dissolve whey powder in deionized water to prepare a whey solution with a mass volume concentration of 7%. Sterilize at 65℃ for 5 min. After the temperature drops to room temperature, inoculate the activated third-generation Dioscorea opposita and Lactobacillus paracasei into the whey solution at an inoculation ratio of 2% and 4%, respectively. Then ferment at 30℃ and 150 rpm for 48 h.

[0036] (2) Add 0.3% (w / v) pepsin to the fermented whey solution, adjust the pH of the whey solution after fermentation to 4.30 with 5 mol / L hydrochloric acid, and shake at 37℃ and 60 rpm for 2 h; add 5 mol / L sodium hydroxide to adjust the pH to 7.0, add 0.1% (w / v) trypsin and perform in vitro enzymatic hydrolysis simulation at 37℃ and 60 rpm for 2 h, and then centrifuge at 4℃ and 12000g for 15 min to collect the supernatant and obtain lyophilized powder using freeze-drying technology;

[0037] (3) Dissolve the lyophilized powder from step (2) in deionized water to obtain a 100 mg / mL solution. Filter the component using two ultrafiltration membranes with different cut-off values ​​of 10 kDa and 3 kDa to obtain three different components: > 10 kDa, 3-10 kDa, and < 3 kDa. Freeze-dry the three components using a vacuum freeze dryer and further prepare a 50 mg / mL solution with deionized water. Determine the inhibition rate of each component on pancreatic lipase activity.

[0038] See results Figure 1 ,from Figure 1 It was found that the solution containing a molecular weight <3kDa showed the best inhibitory effect on pancreatic lipase activity. This solution was further separated using RP-HPLC, and the results are as follows: Figure 2 As shown, according to different time periods Figure 2 The peak was divided into five parts. Separates from different time periods were collected, and after removing fluidity using a rotary evaporator, they were freeze-dried. The freeze-dried components from each time period were then prepared into 10 mg / mL solutions using deionized water. The inhibitory activity of different component solutions on pancreatic lipase was measured. The results are shown in [Figure Number]. Figure 3 As shown in the figure, the fraction 2 (25-30 min) fragment showed the best inhibitory effect on pancreatic lipase activity. This fraction was further separated using RP-HPLC, and the results are shown in the figure. Figure 4 After collecting the separated solutions of different peaks and removing the mobile phase using a rotary evaporator, the solutions were freeze-dried. The inhibitory activity of the solutions of different peaks on pancreatic lipase was determined. The separated solution of peak 1 showed an inhibitory effect on pancreatic lipase activity. The high-performance liquid chromatogram of peak 1 is shown in [reference needed]. Figure 5 ,from Figure 5 The liquid chromatography results show a single peak, indicating that purification is complete.

[0039] The chromatographic conditions for RP-HPLC were as follows: injection volume 1000 μL, flow rate 2 mL / min, detection wavelength at 215 nm; mobile phase A was deionized water containing 0.1% (v / v) trifluoroacetic acid (TFA), and mobile phase B was acetonitrile (CH3CN or C2H3N) containing 0.1% (v / v) TFA. Gradient elution was performed using mobile phases A and B, with the following elution program: 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.

[0040] Experimental Example 2: LC-MS Identification

[0041] The fraction of peak 1 was identified by LC-MS, analysis time: 30 min. Detection mode: positive ion. The mass-charge ratio of small peptides and small peptide fragments was acquired using the following method: 10 fragment spectra were acquired for each full scan (MS2 scan); the raw mass spectrometry file was retrieved from the relevant database (Equus caballus) using Mascot 2.2 software, and finally, the protein identification results were obtained.

[0042] See Figure 6 The corresponding intensity and charge-to-mass ratio of the fragments can be obtained from the figure. Sequence alignment with the database confirms that the small molecule peptide sequence is SPCPM.

[0043] Experimental Example 3: Molecular docking of small molecule peptide SPCPM with pancreatic lipase

[0044] A three-dimensional model of pancreatic lipase (PL) was downloaded from the RCBSPDB protein database. The 3D structure of the small peptide was plotted using Alphafold 3, and an NM2 force field was added to minimize its energy. The small peptide SPCPM was docked with pancreatic lipase using CBDOCK, yielding a total binding energy of -7.7 Kcal / mol. The key amino acid residues and interaction forces between SPCPM and pancreatic lipase were determined. Figure 7 As shown in the figure, the small molecule peptide SPCPM forms a hydrophobic interaction with PL at binding sites V21C, I20A, I20C, and Q220C.

[0045] Experimental Example 4: Determination of the inhibitory activity of the small molecule peptide SPCPM on pancreatic lipase

[0046] The small peptide sequence was synthesized by Shanghai Sangon Biotech Co., Ltd., with a purity ≥98%. The obtained small peptide was then subjected to gradient dissolution to obtain small peptide solutions of 1, 2, 3, 4, and 5 mg / mL. The inhibitory effect of different concentrations of small peptide solutions on pancreatic lipase activity was detected. The results are shown in [Figure Number]. Figure 8 The small molecule peptide SPCPM inhibited pancreatic lipase by 63.42% at a concentration of 3 mg / mL.

[0047] Experimental Example 3: Functional Evaluation of Small Molecule Peptide SPCPM

[0048] (1) Establishment of an oleic acid (OA)-induced hyperlipidemia model in HepG2 cells

[0049] HepG2 cells were revived in DMEM high-glucose medium supplemented with 20% FBS and 1% penicillin-streptomycin; they were then cultured at 37 ℃ in a 5% CO2 incubator until confluence reached 80%, digested with 0.25% trypsin, and passaged at a ratio of 1:3, with the medium being changed every 24 h; after HepG2 cells were plated, the original medium supernatant was discarded, and a high-fat model was induced in DMEM medium containing 0.5 mmol / L oleic acid;

[0050] Model group (DC): HepG2 cells were cultured in DMEM medium containing 0.5 mmol / L oleic acid;

[0051] Negative control group (NC): HepG2 cells were cultured in DMEM medium without 0.5 mmol / L oleic acid;

[0052] Small molecule peptide group: HepG2 cells were cultured in DMEM medium containing 0.5 mmol / L oleic acid with different concentrations of small molecule peptides (1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL);

[0053] Positive control group (PC): HepG2 cells were cultured in DMEM medium containing 0.5 mmol / L oleic acid and 25 μmol / L orlistat;

[0054] All groups underwent further experiments after the cells adhered to the culture vessel for 24 hours.

[0055] (2) The effect of SPCPM small molecule peptides on cell viability was detected by CCK8 assay.

[0056] HepG2 cells were digested with trypsin and then suspended in DMEM medium containing 10% FB. Cells were then cultured at a rate of 4 × 10⁻⁶ cells / year. 3The cells were seeded into 96-well plates. To reduce the influence of edge effects, the wells were filled with phosphate-buffered saline (PBS). The cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. The original culture medium was discarded, and then small molecule peptide solutions with concentrations of 1-5 mg / mL were added to each well. A control (NT) without added small molecule peptide solution was also included. After culturing for 24 hours at 37°C in a 5% CO2 incubator, 10 μL of CCK8 reagent was added to each well, and the cells were cultured for 1 hour. The absorbance was measured at OD450. Four auxiliary wells were included in each group as controls. The results are shown below. Figure 9 As shown in the figure, the small molecule peptide SPCPM has no toxic side effects on HepG2 cells.

[0057] (3) Effect of small molecule peptide SPCPM on triglyceride accumulation in HepG2 cells

[0058] HepG2 cells cultured and plated to 80% confluence were digested with trypsin and then suspended in DMEM medium containing 10% FBS. Cells were then cultured at a concentration of 2.5 × 10⁻⁶ cells / mL. 4 Add the cells to 96-well plates and perform group experiments according to step (1). Incubate the cells in a cell culture incubator at 37℃ and 5% CO2 for 24 h. Discard the original culture medium, wash the cells twice with PBS, add trypsin to digest the cells in each group, and centrifuge them under the following conditions: 4℃, 3000 rpm, 5 min. Add 200 μL of cell lysis buffer to each group and lyse on ice for 30 min. After lysis, centrifuge at 4℃ and 14000 rpm for 15 min and collect the supernatant. Determine the triglyceride content according to the steps of the Nanjing Jiancheng Biotechnology Co., Ltd. triglyceride assay kit. Determine the protein concentration of the precipitate using the Beyotime BCA protein assay kit. Perform 3 replicates for each group. See the results below. Figure 10 As shown in the figure, the small molecule peptide SPCPM can significantly improve the accumulation of triglycerides in oleic acid-induced hyperlipidemic HepG2 cells.

Claims

1. A small molecule peptide derived from fermented bovine whey, with the amino acid sequence Ser-Pro-Cys-Pro-Met.

2. The use of the small molecule peptide according to claim 1 in the preparation of drugs for treating obesity or hyperlipidemia.

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