A selenium-containing composition and its application in coronary atherosclerosis

Abstract

The present invention belongs to the field of active peptides, and specifically relates to a selenium-containing composition and an application thereof in coronary atherosclerosis. The selenium-containing composition comprises a selenopeptide chelate, wherein the selenopeptide chelate is prepared by chelating an almond active peptide as shown in SEQ ID NO: 1 with inorganic selenium; the selenopeptide chelate has the effects of significantly reducing the concentrations of TC, TG, HDL-C and LDL-C in serum, having high antioxidant and anti-inflammatory activities, and reducing the area ratio of atherosclerotic plaques, and can be used for treating and / or preventing coronary atherosclerotic diseases.

Description

Technical Field

[0001] The invention belongs to the field of active peptides, and in particular relates to a selenium-containing composition and application thereof in coronary atherosclerosis. Background Art

[0002] Coronary atherosclerosis is a cardiovascular disease that mainly affects the coronary arteries. It is characterized by the deposition of lipids in the arterial wall, which leads to thickening of the arterial wall, reduced elasticity, and narrowing of the vascular lumen. This pathological process not only affects myocardial blood supply, leading to serious consequences such as angina pectoris and myocardial infarction, but also has an increasing incidence rate with changes in lifestyle and aging. Although a variety of drugs have been used to prevent and treat coronary atherosclerosis, long-term use will bring side effects and drug resistance. Therefore, it is necessary to find new natural substances to effectively inhibit the occurrence and development of atherosclerosis.

[0003] Almond active peptides, as a class of small molecule bioactive peptides extracted from almonds, have attracted widespread attention in recent years due to their potential cardiovascular protective effects. Studies have shown that almond active peptides have multiple biological functions such as antioxidant, anti-inflammatory and lipid regulation, and these properties are closely related to the pathogenesis of atherosclerosis. Almond active peptides may play a positive role in the prevention and treatment of atherosclerosis by reducing the production of free radicals, inhibiting the expression of inflammatory factors and reducing the accumulation of low-density lipoprotein cholesterol (LDL-C). In addition, as a natural ingredient, almond active peptides have better biocompatibility and safety than traditional drugs.

[0004] The introduction of selenium chelate further enhances the cardiovascular protective effect of almond active peptides; selenium is an important trace element that has been shown to have powerful antioxidant function and plays a key role in maintaining cardiovascular health; after chelating selenium with almond active peptides, the selenium chelated almond active peptides formed can not only significantly enhance its antioxidant capacity, but also further inhibit oxidative stress and inflammatory response by regulating intracellular signaling pathways, thereby effectively delaying the progression of coronary atherosclerosis; this chelate provides a potential natural treatment option for the prevention and treatment of coronary atherosclerosis. Summary of the invention

[0005] In order to solve the above problems, the present invention first provides a selenium-containing composition and its application in coronary atherosclerosis.

[0006] The first object of the present invention is to provide a selenium-containing composition, which comprises a seleno-peptide chelate, wherein the seleno-peptide chelate is prepared by chelating an almond active peptide as shown in SEQ ID NO: 1 with inorganic selenium.

[0007] The second object of the present invention is to provide an almond active peptide, the amino acid sequence of the almond active peptide is shown in SEQ ID NO:1.

[0008] The third object of the present invention is a method for preparing a seleno-peptide chelate, which is specifically implemented by chelating the almond active peptide shown in SEQ ID NO: 1 with selenium, wherein the chelation reaction uses sodium selenite (Na2SeO3) as a selenium source.

[0009] The fourth object of the present invention is to provide a use of a selenium-containing composition in the preparation of a medicament for treating and / or preventing coronary atherosclerosis.

[0010] Furthermore, the selenium-containing composition comprises a seleno-peptide chelate, wherein the seleno-peptide chelate is prepared by chelating the almond active peptide as shown in SEQ ID NO: 1 with inorganic selenium.

[0011] The fifth object of the present invention is to provide an application of almond active peptide in the preparation of a drug for treating and / or preventing coronary atherosclerosis.

[0012] Furthermore, the amino acid sequence of the almond active peptide is shown in SEQ ID NO:1.

[0013] Compared with the prior art, the present invention has at least the following beneficial effects:

[0014] 1. The present invention directly uses almond enzymolysis to obtain almond active peptide for the first time, and separates and purifies it through a G25 chromatographic column to identify its amino acid sequence as HCRMKGALCSTK (SEQ ID NO: 1); further, the present invention uses the almond active peptide shown in SEQ ID NO: 1 to react with selenium to prepare a seleno-peptide chelate;

[0015] 2. The selenopeptide chelate and almond active peptide prepared by the present invention have the effects of significantly reducing the concentrations of TC, TG, HDL-C and LDL-C in serum, having high antioxidant and anti-inflammatory activities, and reducing the area ratio of atherosclerotic plaques, and can be used to treat and / or prevent coronary atherosclerotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Peak diagram of almond enzymatic hydrolysate from group CL-1 passing through G25 chromatographic column;

[0017] Figure 2 Mass spectrum of almond active peptides in group F3;

[0018] Figure 3 Visible-UV spectrum analysis of F3 group almond active peptides after chelation with selenium;

[0019] Figure 4 Effects of almond active peptides and their selenium chelates on the changes of blood lipid levels in mice with coronary atherosclerosis model;

[0020] Figure 5 F3 group almond active peptides and their selenium chelates were used to detect the antioxidant capacity and inflammatory factors of mice with coronary atherosclerosis model;

[0021] Figure 6 Detection of atherosclerotic plaque area in mice with coronary atherosclerosis model using F3 group almond active peptides and their selenium chelates. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0023] Example 1 Preparation of almond active peptides

[0024] Preparation of protease: The protease-producing strain Bacillus pumilus DX-4 was inoculated into LB medium and cultured at 30°C, 200 r / min for 12 h. Then, it was inoculated into enzyme-producing medium (fish meal peptone 1 g / L, beef meal 5 g / L, NaCl 5 g / L, casein 20 g / L) at a 5% inoculum amount and cultured at 30°C, 200 r / min for 30 h. The fermentation broth was centrifuged at 5000×g, 4°C for 30 min. The supernatant was concentrated through an 8KDa filter tube to obtain crude enzyme, which was freeze-dried for later use.

[0025] Enzymatic hydrolysis of almonds: crush the almonds, pass through an 80-mesh sieve to obtain almond powder, defatted with an ethanol-water solution (70% ethanol, volume ratio of 1:3), stirred at room temperature for 30 minutes, repeated twice, and then filtered to obtain defatted almond powder; suspend the defatted almond powder in a 0.1M NaOH solution with a solid-liquid ratio of 1:10, stir and extract for 2 hours, adjust the pH to 4.5, precipitate protein with 0.1M HCl, centrifuge at 4000rpm for 20 minutes, collect the precipitate, wash with distilled water until neutral, and dry to obtain almond protein powder; dissolve the extracted almond protein powder in distilled water, adjust the protein concentration to 5% (w / v), use crude enzyme extracted from Bacillus pumilus DX-4 to hydrolyze at 1.5% (enzyme to substrate protein mass ratio), and adjust the solution to pH 8.5, use 0.1M NaOH or HCl for adjustment, and perform enzymolysis in a constant temperature water bath at 50℃ for 3 hours, during which time, keep stirring evenly; after the enzymolysis is completed, heat the reaction solution to 90℃ and keep warm for 10 minutes to inactivate the enzyme and terminate the enzyme reaction.

[0026] Ultrafiltration and identification of almond hydrolysate: The almond hydrolysate was centrifuged at 8000 rpm for 15 minutes, the supernatant was collected, and filtered using a microporous filter membrane (0.45 μm) to remove the residue to obtain a clear almond peptide solution; the almond peptide solution was ultrafiltered through membranes with molecular weights (MW) of 3 KDa and 6 KDa, respectively, and the retained and permeated solutions of each membrane were collected to obtain three component solutions with different molecular weights, namely ultrafiltration component-1 (CL-1, less than 3 KDa), ultrafiltration component-2 (CL-2, 3 to 6 KDa) and ultrafiltration component-3 (CL-3, greater than 6 KDa), and the three components were freeze-dried for later use.

[0027] Antioxidant and anti-inflammatory experiments of various components of almond enzymatic hydrolysate: The experimental animals were 8-week-old male ApoE- / - mice (20-25g), which were randomly divided into 4 groups, with 10 mice in each group: normal control group (NC), high-fat model group (Model), almond active peptide CL-1 group (CL-1, 100 mg / kg·d), almond active peptide CL-2 group (CL-2, 100 mg / kg·d) and almond active peptide CL-3 group (CL-3, 100 mg / kg·d), atorvastatin group (4 mg / kg·d). The mice were kept under 22±2℃, humidity 50%-60%, 12-hour light-dark cycle, free access to food and water. During the experiment, the CL-1, CL-2 and CL-3 groups were given corresponding doses of almond active peptides by gavage for 12 weeks. At the end of the experiment, the serum and tissues of mice were collected to detect oxidative stress markers including superoxide dismutase (SOD) and malondialdehyde (MDA), as well as inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The results are shown in Table 1.

[0028] Table 1 Antioxidant and anti-inflammatory activities of various components of almond enzymatic hydrolysate

[0029]

[0030] Note: Compared with the normal group a P<0.05, b P<0.01; compared with the model group c P<0.05, d P<0.01

[0031] The results in Table 1 show that compared with other groups, the almond enzymatic hydrolysate in group CL-1 can significantly enhance the antioxidant capacity by increasing the activity of antioxidant enzymes (SOD) and reducing the content of lipid peroxidation products (MDA); at the same time, the almond enzymatic hydrolysate in group CL-1 can also significantly reduce the levels of inflammatory factors (IL-6 and TNF-α), showing excellent anti-inflammatory effects, that is, the almond enzymatic hydrolysate in group CL-1 has potential high antioxidant and high anti-inflammatory activity.

[0032] Separation and identification of almond active peptides: The almond hydrolysate of group CL-1 was separated using Sephadex G-25 gel. The specific steps included gel swelling and equilibrium, sample loading, elution and segmented collection. 0.02M phosphate buffer (pH 7.0) was used as the eluent in the experiment. The flow rate was controlled at 1-2mL / min, and the elution peak was detected by 280nm UV absorption. The loading volume did not exceed 10% of the column bed volume. The components were determined by collecting the eluents of each segment and analyzing the UV absorption peaks. Figure 1 .

[0033] Figure 1 The results of separating the almond hydrolysate of the CL-1 group by the G25 chromatographic column according to the molecular size are shown, that is, CL-1 separates 5 components of almond active peptides (5 peaks). According to the previous method for detecting the antioxidant and anti-inflammatory activities of the components of the almond hydrolysate, the 5 components separated by CL-1 were simultaneously detected and analyzed, as shown in Table 2.

[0034] Table 2 Antioxidant and anti-inflammatory activities of each peak of almond active peptides

[0035]

[0036] Note: Compared with the normal group a P<0.05, b P<0.01; compared with the model group c P<0.05, d P<0.01

[0037] The results in Table 2 show that the almond active peptides in group F3 have the effect of increasing the activity of antioxidant enzymes (SOD) and reducing the content of lipid peroxidation products (MDA); at the same time, the almond active peptides in group F3 can also significantly reduce the levels of inflammatory factors (IL-6 and TNF-α), that is, the almond active peptides in group F3 have both high antioxidant and high anti-inflammatory activities.

[0038] Identification of active peptides from almonds of group F3: The active peptides from almonds of group F3 were purified using a solid phase extraction (SPE) column; the purified samples were concentrated using a vacuum concentrator and dissolved in an appropriate amount of 0.1% formic acid aqueous solution for LC-MS / MS analysis; a C18 reverse phase column (Thermo Scientific Accucore aQ, 2.1×100 mm, 2.6 μm) was used; phase A: 0.1% formic acid aqueous solution. Phase B: 0.1% formic acid-acetonitrile solution; the injection volume was 5 μL, and a high-resolution quadrupole tandem mass spectrometer (Thermo Scientific QExactive) was used; the mass spectrum of active peptides from group F3 is shown in Figure 2 .

[0039] Figure 2 The results showed that the amino acid sequence of the F3 group almond active peptide was HCRMKGALCSTK (SEQ ID NO: 1), and its molecular weight was 1532.81 Da.

[0040] Example 2 Preparation and Identification of Almond Active Peptide Selenium Chelate

[0041] The F3 group almond active peptides were chelated with selenium, and sodium selenite (Na2SeO3) was used as a selenium source. In a pH 7.0 buffer (phosphate buffer), the F3 group almond active peptide solution was mixed with sodium selenite at a mass ratio of 3:1, and the volume of the reaction system was 100 mL. The chelation reaction was carried out at 25°C, the stirring speed was 300rpm, and the reaction time was set to 2 hours. After the reaction, the reaction solution was dialyzed (dialysis bag with a molecular weight cutoff of 500Da) to remove free selenium. The dialysis time was 24 hours, the dialysate was deionized water, and the dialysate was replaced every 4 hours. The dialyzed sample was freeze-dried to obtain the F3 group almond active peptide selenium chelate powder, which was stored at -20°C.

[0042] The selenium content was determined by 3,3′-diaminobenzidine colorimetry. The selenium chelation rate was calculated according to formula (1):

[0043] Selenium chelation rate (%) = (C1 / C2) × 100% (1)

[0044] Where: C1 is the total amount of selenium in the almond active peptide selenium chelate g; C2 is the total amount of selenium in the reaction system g; after calculation, the selenium chelation rate is 76 + 0.36%, which meets the requirements of subsequent experiments.

[0045] Visible-UV spectral analysis: In order to evaluate the spectral characteristics of the F3 group almond active peptides after chelation with selenium, a UV-visible spectrophotometer was used to perform UV spectral analysis; the sample solution was prepared as follows: the freeze-dried F3 group almond active peptide selenium chelate and the F3 group pure almond active peptide were dissolved in deionized water and the concentration was adjusted to 1 mg / mL. The test was carried out at room temperature (25°C) with a measurement wavelength range of 200nm to 400nm; the UV spectrum was measured using a UV-visible spectrophotometer (UV-2600, Shimadzu, Japan) using a quartz cuvette (1cm light path); the scanning rate was set to 200nm / min, the bandwidth was 2nm, and deionized water was used for baseline correction; by comparing the absorption spectra of pure almond active peptides and their chelates, the changes in the chelate in the 200nm to 300nm region were observed, see Figure 3 .

[0046] Figure 3The results showed that the F3 group almond active peptide selenium chelate had obvious absorption peaks near 240nm and 290nm, indicating that selenium interacted with the carboxyl, amino or thiol groups in the peptide; in contrast, the absorption intensity of the F3 group almond active peptide in this position band was weaker. The enhancement of this absorption peak indicated that selenium had successfully combined with the peptide and changed its electronic transition characteristics, further proving the effectiveness of the chelation reaction.

[0047] Example 3 Experiment on the treatment of coronary atherosclerosis model mice with almond active peptides and selenium chelates thereof

[0048] Healthy male ApoE- / - mice (8 weeks old, weighing 20-25 g) were selected. This strain of mice is an ideal choice for the coronary atherosclerosis model and can naturally produce atherosclerotic lesions. The mice were adaptively fed for 1 week before the experiment.

[0049] Forty ApoE- / - mice were randomly divided into five groups, with eight mice in each group:

[0050] Blank control group (NC group): normal diet;

[0051] Model control group (Model group): high-fat diet (1.25% cholesterol + 15% triglyceride) to induce coronary atherosclerosis;

[0052] F3 group almond active peptide group (F3 group): on the basis of high-fat diet, the F3 group was given almond active peptide, 100 mg / kg body weight per day, for 8 consecutive weeks;

[0053] F3 group almond active peptide selenium chelate treatment group (selenium chelate group): on the basis of high-fat diet, F3 group was given almond active peptide selenium chelate, 100 mg / kg body weight per day, for 8 consecutive weeks;

[0054] Atorvastatin group (Ator group): On the basis of high-fat diet, atorvastatin was given at 4 mg / kg body weight per day for 8 consecutive weeks.

[0055] Mice were raised under standard experimental conditions, with a temperature of 22±2℃, a humidity of 60-70%, and a 12h light-dark cycle. Mice in each group were allowed to drink water and eat freely. The F3 group and the selenium chelate group were given active peptides and their selenium chelates by gavage every day, and the Ator group was given atorvastatin by gavage every day; the blank control group and the model control group were gavaged with an equal volume of normal saline.

[0056] Blood lipid test: At the end of the experiment, blood was collected through the tail vein, and the serum was separated to detect the concentrations of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Figure 4 .

[0057] Figure 4 The results showed that compared with the blank control group, the serum TC (P<0.05), TG (P<0.05), HDL-C (P<0.01) and LDL-C (P<0.01) levels of the mice in the model group were increased; compared with the model group, the serum TC, TG, HDL-C and LDL-C levels of the F3 group, selenium chelate group and atorvastatin group were significantly decreased, and there were significant differences; at the same time, the lipid-lowering effect of the selenium chelate group was better than that of the F3 group, and was comparable to that of the atorvastatin group.

[0058] Antioxidant capacity and inflammatory factor detection: The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were measured to evaluate the level of antioxidant stress; the concentrations of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum were detected by ELISA method, see Figure 5 .

[0059] Figure 5 The results showed that the selenium chelate group also had the effect of increasing the activity of antioxidant enzymes (SOD), reducing the content of lipid peroxidation products (MDA), and reducing the expression levels of inflammatory factors IL-6 and TNF-α. The selenium chelate group had high antioxidant and anti-inflammatory activity at a level comparable to that of the atorvastatin group and the F3 almond active peptide group.

[0060] Detection of atherosclerotic plaque area: After the experiment, samples were collected from the abdominal aorta, and the aortic plaque area was detected by Oil Red O staining, and the plaque ratio was calculated using image analysis software. Figure 6 .

[0061] Figure 6 The results showed that compared with the model group, the F3 group, selenium chelate group and atorvastatin group could significantly reduce the area proportion of atherosclerotic plaques, and the selenium chelate group was significantly better than the F3 group and atorvastatin group in reducing atherosclerotic plaques.

[0062] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A selenium-containing composition, characterized in that The composition comprises a seleno-peptide chelate, wherein the seleno-peptide chelate is prepared by chelating an almond active peptide as shown in SEQ ID NO: 1 with inorganic selenium.

2. An almond active peptide, characterized in that: The amino acid sequence of the almond active peptide is shown in SEQ ID NO:

1.

3. A method for preparing a selenopeptide chelate, characterized in that: The almond active peptide shown in SEQ ID NO: 1 is subjected to a chelation reaction with selenium, wherein sodium selenite (Na2SeO3) is used as a selenium source in the chelation reaction.

4. Use of the composition according to claim 1 in preparing a medicament for treating and / or preventing coronary atherosclerosis.

5. Use of the almond active peptide according to claim 2 in the preparation of a medicament for treating and / or preventing coronary atherosclerosis.

Images