An oligopeptide with α-glucosidase inhibitory activity and its application

Abstract

The present invention discloses an oligopeptide with α-glucosidase inhibitory activity, whose amino acid sequence is Leu-Leu-Val-Leu-Tyr-Tyr-Glu-Tyr. The present invention also discloses its use in the preparation of hypoglycemic drugs. The present invention extracts an oligopeptide with α-glucosidase inhibitory activity from camellia oleifera seed cake, which can achieve a good in vitro inhibition effect of α-glucosidase activity and is of great significance for the development of drugs with hypoglycemic function.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an oligopeptide with alpha-glucosidase inhibitory activity and application thereof. Background Art

[0002] Diabetes is increasingly prevalent worldwide, with the total number of people with diabetes projected to reach 700 million by 2045. According to the International Diabetes Federation (IDF), excessive intake of absorbable monosaccharides can elevate postprandial blood glucose levels, leading to hyperglycemia. Once diagnosed with diabetes, patients require long-term medication to control blood glucose levels. Inhibiting the activity of carbohydrate-digesting enzymes to delay carbohydrate digestion is an effective approach to prevent diabetes or regulate postprandial blood glucose levels. The optimal treatment for diabetes is to maintain appropriate blood glucose levels after meals. α-Glucosidase (GAA), an important carbohydrate hydrolase, plays a key role in converting oligosaccharides and disaccharides into glucose. The resulting monosaccharides are absorbed in the small intestine, leading to elevated blood glucose levels. Therefore, GAA has been recognized as a key target enzyme for the prevention and treatment of type 2 diabetes (T2D), and α-glucosidase inhibitors (AGIs) play an important role in controlling postprandial blood glucose levels and maintaining normoglycemia in diabetic patients. AGIs delay carbohydrate digestion and reduce monosaccharide absorption. Researchers have been working to identify AGIs from organic compounds and natural products, such as acarbose, miglitol, and voglibose. However, these drugs have significant side effects, such as abdominal pain, bloating, increased flatulence, diarrhea, and cramps. Furthermore, these drugs are in high demand, scarce, and expensive. Consequently, a growing number of researchers are searching for more effective α-glucosidase inhibitors from natural products. Summary of the Invention

[0003] The purpose of the present invention is to provide an oligopeptide with α-glucosidase inhibitory activity and its application, so as to solve the deficiencies of the prior art.

[0004] The present invention adopts the following technical solutions:

[0005] In a first aspect, the present invention provides an oligopeptide having α-glucosidase inhibitory activity, the amino acid sequence of which is Leu-Leu-Val-Leu-Tyr-Tyr-Glu-Tyr, abbreviated as LLVLYYEY.

[0006] The second aspect of the present invention provides the use of the above oligopeptide having α-glucosidase inhibitory activity in the preparation of hypoglycemic drugs.

[0007] Beneficial effects of the present invention:

[0008] The present invention extracts an oligopeptide with α-glucosidase inhibitory activity from camellia seed cake, which can achieve a good effect of inhibiting α-glucosidase activity in vitro and is of great significance for the development of drugs with blood sugar lowering function. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 A is the chromatogram of 1KDa-3KDa peptide segment separated by G25 dextran gel chromatography to produce 4 components. Figure 1 B is the α-glucosidase inhibitory activity of the four components.

[0010] Figure 2 This is a diagram showing the binding mode of acarbose to α-glucosidase (2QMJ).

[0011] Figure 3 This is the high performance liquid chromatogram of LLVLYYEY.

[0012] Figure 4 This is the mass spectrum of LLVLYYEY.

[0013] Figure 5 It is a line graph of “concentration-inhibition rate” of LLVLYYEY and acarbose.

[0014] Figure 6 This is a 3D image of the docking results of LLVLYYEY and α-glucosidase (2QMJ).

[0015] Figure 7 This is a graph showing the relationship between the enzymatic reaction rates of different concentrations of LLVLYYEY and different concentrations of α-glucosidase.

[0016] Figure 8 A is the Lineweaver-Burk plot of LLVLYYEY inhibition of α-glucosidase, Figure 8 B is the LLVLYYEY concentration and slope graph, Figure 8 C is the LLVLYYEY concentration and 1 / V max picture. DETAILED DESCRIPTION

[0017] The present invention will be further explained below in conjunction with the examples and drawings. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0018] Example 1 Screening of oligopeptides with α-glucosidase inhibitory activity

[0019] First, the camellia seeds were shelled and then cold-pressed and degreased using a hydraulic oil press at room temperature, 50 MPa, and 40 minutes to obtain camellia seed cake. The resulting camellia seed cake was then pulverized and passed through a 60-mesh sieve to obtain camellia seed cake powder. The camellia seed cake powder was mixed with petroleum ether at a material-liquid ratio of 1:5 (g / mL), stirred and extracted at room temperature at 500 rpm for 2 hours, then filtered at room temperature for three extractions. The residue was collected and dried at 45°C for 12 hours to obtain defatted camellia seed cake powder. The defatted camellia seed cake powder was mixed with 80 v / v% ethanol solution at a material-liquid ratio of 1:10 (g / mL), stirred and extracted at 40°C at 500 rpm for 1 hour, then filtered at room temperature for two extractions. The residue was collected and dried at 45°C to a moisture content of less than 4 wt%. The powder was then passed through a 60-mesh sieve to obtain defatted and saponin-free camellia seed cake powder, which was refrigerated at 4°C for later use.

[0020] Camellia oleifera protein was extracted by alkali extraction and acid precipitation method: the defatted and saponin-free camellia seed powder obtained above was prepared into a solution with distilled water at a solid-liquid ratio of 1:20 (g / mL), the pH was adjusted to 10.0 with 1.0 mol / L NaOH solution, and the mixture was stirred and extracted at 50°C and 500 r / min for 2 h. After cooling to room temperature, the mixture was centrifuged at 4°C and 4000 r / min for 15 min, the supernatant was taken, the pH was adjusted to 4.5 with 1.0 mol / L HCl solution, and the mixture was allowed to stand for 2 h. The precipitate was taken, washed with distilled water until neutral, and then freeze-dried at -80°C for 24 h to obtain camellia oleifera protein, which was stored at -20°C for later use.

[0021] A certain amount of camellia oil seed protein was weighed and added with distilled water according to the substrate concentration of 1% w / v (g / mL) to prepare a camellia oil seed protein solution. The solution was denatured in a 95°C water bath for 10 min and then cooled to room temperature. The pH was adjusted to 7.1 with 1.0 mol / L NaOH solution. 4200 U / g (based on the mass of camellia oil seed protein) of composite protease was added (the composite protease used was the composite protease sold by Shanghai Yuanye Biotechnology Co., Ltd., product number S10155, mainly including endoprotease produced by Bacillus subtilis), and enzymolysis was carried out at 50°C and 300 r / min with stirring for 3.95 h. After the enzymatic hydrolysis is completed, the enzyme is inactivated in a 95°C water bath for 10 minutes and then cooled to room temperature. The mixture is centrifuged at 4°C and 8000 r / min for 20 minutes. The supernatant is taken and filtered through a 0.45 μm filter membrane. Ultrafiltration is then performed using 3KDa and 1KDa MWCO ultrafiltration membranes to collect the filtrate with a molecular weight of 1KDa-3KDa peptides. The filtrate is freeze-dried at -80°C for 24 hours and stored at -20°C for later use.

[0022] The 1KDa-3KDa peptide solution was prepared into 5 mg / mL with deionized water and chromatographed on G25 dextran gel to obtain 4 components ( Figure 1A), the α-glucosidase inhibitory activity of the four components was determined using the same method as in Example 2. The results showed that the α-glucosidase inhibitory activity of the F4 component was significantly higher than that of the other components ( Figure 1 B), collect the fraction F4 solution, freeze-dry it at -80°C for 24 h, and store it at -20°C for later use.

[0023] The F4 component was identified using LC-MS / MS technology to obtain the sequence information of the component.

[0024] Virtual screening

[0025] 52 peptides with denovo scores greater than 90 were selected and used The Glide module in Maestro software was used for virtual screening. The target protein, the receptor protein α-glucosidase (PDB ID: 2QMJ), was processed using the Protein Preparation Wizard module to remove crystal water, add missing hydrogen atoms, and repair missing bond information. Finally, the receptor protein (2QMJ) was energy minimized and the geometry structure optimized. The OPLS3e force field was used to constrain the receptor protein (2QMJ), and all peptide molecules were prepared according to the default settings of the LigPre module. When screening in the Glide module, the prepared receptor protein file (processed 2QMJ file) was imported, and the Glide grid module was used to generate a docking grid file centered on the original ligand (acarbose, positive control). XP scoring was used to screen out peptides with higher scores using the Glide module. During the screening process, acarbose was used as a positive control to determine the binding mode of the original ligand (acarbose, positive control) and the active site (acarbose and 2QMJ action site) ( Figure 2 The in vitro activity of the peptide molecules with higher scores was verified, and an oligopeptide with potential α-glucosidase inhibitory activity was screened out. The amino acid sequence is Leu-Leu-Val-Leu-Tyr-Tyr-Glu-Tyr, abbreviated as LLVLYYEY, and the molecular weight is 1074.6Da.

[0026] The toxicity and stability of LLVLYYEY were predicted by Toxin Pred and Expasy. The results showed that LLVLYYEY was non-toxic and stable (stability index 14.75).

[0027] The oligopeptides provided by the present invention can be derived from camellia seed cake hydrolysate or obtained by solid phase synthesis. The LLVLYYEY used in the following Examples 2 and 3 was obtained by solid phase synthesis (Nanjing GenScript Biotechnology Co., Ltd.), and the HPLC and mass spectra are shown in FIG. Figure 3 and Figure 4 shown.

[0028] Example 2 Detection of α-glucosidase inhibitory activity of LLVLYYEY

[0029] The solid-phase synthesized oligopeptide LLVLYYEY was selected and oligopeptide solutions with concentrations of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.25 mg / mL and 1.5 mg / mL were prepared using 0.1 M PBS, pH 6.8. The in vitro inhibitory activity of the oligopeptide was determined using α-glucosidase as the research object.

[0030] Determination method: 40 μL PBS (0.1 M, pH 6.8), 80 μL 0.2 U / mL α-glucosidase (prepared in 0.1 M, pH 6.8 PBS) and 40 μL oligopeptide solution (different concentrations of oligopeptide solutions were prepared in 0.1 M, pH 6.8 PBS) were added to the wells of a 96-well ELISA plate in sequence. After mixing, the mixture was incubated at 37°C for 15 min. Then, 80 μL 1.5 mM p-nitrophenyl-α-D-pyranoglucoside (pNPG, prepared in 0.1 M, pH 6.8 PBS) was added. After mixing, the mixture was incubated at 37°C for 20 min. 150 μL 0.2 M Na2CO3 solution was added to terminate the reaction. The absorbance at 405 nm was then detected. The experiment was repeated 3 times and the average value was taken.

[0031] The positive control groups were composed of 40 μL of acarbose solution (prepared in 0.1 M PBS, pH 6.8) of the same concentration (i.e., 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.25 mg / mL, and 1.5 mg / mL) instead of the oligopeptide solution, and the rest of the operations were the same as above.

[0032] The formula for the inhibition rate of α-glucosidase activity is: I% = [1-(A1-A2) / (A3-A4)] × 100%; where: A1 is the absorbance value of the sample solution group, A2 is the background absorbance value of the sample solution group measured by replacing the α-glucosidase solution with PBS, A3 is the absorbance value of the blank control group measured by replacing the sample solution with PBS solution, and A4 is the background absorbance value of the blank control group measured by replacing the sample solution with PBS solution and replacing the α-glucosidase solution with PBS solution.

[0033] The results showed that (Table 1 and Figure 5 ), LLVLYYEY has good α-glucosidase inhibitory activity.

[0034] Table 1

[0035]

[0036] Example 3 Molecular docking analysis

[0037] The crystal structure of α-glucosidase (2QMJ) was downloaded from the RCSB PDB database (http: / / www.rcsb.org / ). The crystal structure of the receptor protein α-glucosidase (2QMJ) was highly accurate and had no missing key residues. It was used as the target protein and molecular docking was performed using the Autodock Vina program to clarify the active site. The downloaded receptor protein (2QMJ) was dehydrated and hydrogenated using the Autodock Tool 1.5.6 software. The active center of 2QMJ was then defined. The active center of 2QMJ was X: -20.83, Y: -6.71, and Z: -5.25; and the size of the active pocket was adjusted to include the entire receptor protein (2QMJ). The results showed that the binding energy of LLVLYYEY for docking with α-glucosidase was -9.335 Kcal / mol, and it bound to the main amino acid residues of α-glucosidase, namely GLU-661, ARG-653, ARG-730, and GLY-732 ( Figure 6 ).

[0038] Example 4 Inhibition Mechanism Analysis

[0039] Reversibility analysis of inhibition

[0040] 40 μL of oligopeptides of different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mg / mL) (prepared in 0.1 M PBS, pH 6.8) and 80 μL of α-glucosidase of different concentrations (0, 2, 4, 6, 8 μg / mL) (prepared in 0.1 M PBS, pH 6.8) were mixed, and the concentration of substrate pNPG was fixed at 2.5 mM (prepared in 0.1 M PBS, pH 6.8) for incubation. The experimental method was the same as described in Example 2. The different enzyme concentrations [E] were plotted against the enzymatic reaction rate V (ΔOD / t) ( Figure 7 ), the enzymatic reaction rate was fitted into a straight line as the enzyme concentration changed. As the concentration of the oligopeptide increased, the enzymatic reaction rate gradually decreased, the slope of the straight line decreased and passed through the origin, which indicated that LLVLYYEY had reversible inhibition on α-glucosidase.

[0041] Inhibition type analysis

[0042] Different concentrations (0, 0.5, 0.75, 1 mg / mL) of oligopeptide solutions (prepared in 0.1 M, pH 6.8 PBS) were mixed with 6 μg / mL of α-glucosidase (prepared in 0.1 M, pH 6.8 PBS), and then incubated with different concentrations of substrate pNPG (0.8, 1, 2, 3, 4 mM, prepared in 0.1 M, pH 6.8 PBS). The experimental method was the same as described in Example 2. The Linerweave-Burk double reciprocal method was used for plotting ( Figure 8 A) With the inverse of pNPG concentration 1 / [S] as the abscissa and the inverse of the enzymatic reaction rate 1 / V as the ordinate, straight lines were drawn at different concentrations to determine the inhibition type. Taking the oligopeptide concentration as 0 mg / mL as the standard, as the oligopeptide concentration increased, the intercept of the straight line intersecting the Y axis increased, i.e., the corresponding maximum enzymatic reaction rate V max The value also increases, and the straight lines fitted with different oligopeptide concentrations intersect at the X-axis, which is the Michaelis constant K m unchanged, indicating that the inhibition type of oligopeptide on α-glucosidase is non-competitive inhibition. In addition, the slope (Slope) ( Figure 8 B) and 1 / V max (min / △OD)( Figure 8 C) The oligopeptide concentration I was plotted twice, and a simple straight line was obtained in each fitting, indicating that there is a single inhibitory site or a single inhibitory-like site on α-glucosidase.

Claims

1. An oligopeptide having α-glucosidase inhibitory activity, characterized in that: Its amino acid sequence is Leu-Leu-Val-Leu-Tyr-Tyr-Glu-Tyr.

2. Use of the oligopeptide having α-glucosidase inhibitory activity according to claim 1 in the preparation of hypoglycemic drugs.

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