A blood pressure-lowering active peptide from mulberry leaves, its preparation method and application

Abstract

This invention discloses a mulberry leaf antihypertensive active peptide, its preparation method, and its application. Using mulberry leaves as raw material, this invention prepares mulberry leaf albumin. Enzymatic hydrolysis parameters, including hydrolysis time, pH, enzyme-to-substrate ratio, and substrate concentration, were optimized through single-factor and orthogonal experiments, providing optimal hydrolysis conditions for preparing crude ACE-inhibiting peptides. Further purification, peptide sequencing, and virtual screening identified highly active ACE-inhibiting peptides with strong solubility and good ACE-inhibiting activity. The two peptides with the highest activity, VPSCFDLTGK and RLPDFHGL, showed the highest IC50 values. 50 The values ​​were 8.23 ​​μmol / L (8.765 μg / mL) and 23.01 μmol / L (23.58 μg / mL), respectively. These bioactive peptides can act as angiotensin-converting enzyme inhibitory peptides, thereby lowering blood pressure by inhibiting ACE activity.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to a mulberry leaf antihypertensive bioactive peptide, its preparation method, and its application. Background Technology

[0002] Primary hypertension is the most common type of hypertension, accounting for approximately 90%-95%. The pathophysiological factors of hypertension include the sympathetic nervous system, the renin-angiotensin-aldosterone system (RAAS), and the activation of inflammatory mediators. Among these, the RAAS plays a crucial role in regulating blood pressure and fluid balance and is considered a clinical therapeutic target for hypertension. Currently, hypertension can be controlled with medication, primarily synthetic angiotensin-converting enzyme (ACE) inhibitors, receptor blockers, diuretics, calcium channel blockers, and peripheral adrenaline inhibitors. However, these medications often have numerous side effects. Food proteins contain a large number of bioactive peptides, many of which have been shown to have antihypertensive capabilities. Previous studies have identified, isolated, and characterized many peptides with ACE-inhibiting properties. Compared to antihypertensive drugs, food-derived ACE-inhibiting peptides are generally safer. As functional components, food-derived ACE-inhibiting peptides have great potential for treating and preventing hypertension. Currently, antihypertensive peptides mainly originate from animal sources such as eggs, dairy products, livestock, and fish, and plant sources such as grains, nuts, and seeds. Plant protein peptides are widely available and highly active, making them a current research hotspot. Currently, the plant-derived antihypertensive peptides prepared mainly include soybean peptides, rice bran peptides, and nut peptides, while research on leaf protein antihypertensive peptides is relatively limited.

[0003] Mulberry trees, as plants of significant economic value, have a long history of cultivation in my country, with widespread planting areas, abundant resources, and readily available raw materials. In traditional industries, mulberry leaves are mainly used as silkworm feed, resulting in low utilization rates and significant waste. Mulberry leaves contain alkaloids, flavonoids, polysaccharides, polyphenols, and abundant protein, possessing high nutritional value. In traditional Chinese medicine, they are used to lower blood pressure, blood sugar, and blood lipids, improve cardiovascular health, treat fever, and protect the liver. The protein content of mulberry leaves is approximately 19%-27% by dry weight, making them one of the plants with the highest leaf protein content; in fact, the albumin content of mulberry leaves can reach as high as 50%. To maximize resource utilization, there are currently many studies on the bioactivity of mulberry leaves. For example, Chinese patent application CN107119097A uses neutral protease alone or a combination of alkaline protease and neutral protease for enzymatic hydrolysis, and then separates and purifies them using DEAESepharose Fast Flow anion exchange chromatography, Sephadex G-15 gel column chromatography, and RP-HPLC to obtain mulberry leaf immunomodulatory peptides. Chinese patent application CN118830622A grinds mulberry leaves, sterilizes them, inoculates them with a suspension of Aspergillus oryzae spores for fermentation, and then obtains a concentrate by ultrasonic extraction and vacuum concentration. After elution, it is freeze-dried to obtain a polyphenol-rich extract with hypoglycemic function. Chinese patent application CN115998795A uses 80% ethanol solution to prepare mulberry leaf flavonoids with specific in vitro antioxidant activity. Current mulberry leaf-related patents mainly focus on the extraction of its phenolic and alkaloid active substances and its application research on antioxidant and hypoglycemic effects, with few in-depth studies on the antihypertensive activity of mulberry leaf albumin. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, one of the objectives of this invention is to provide a method for preparing mulberry leaf antihypertensive active peptides.

[0005] The second objective of this invention is to provide a mulberry leaf-derived active peptide for lowering blood pressure.

[0006] The third objective of this invention is to provide the application of the above-mentioned mulberry leaf antihypertensive active peptide.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing crude mulberry leaf antihypertensive active peptides includes the following steps:

[0009] Step 1: Wash and dry fresh mulberry leaves, grind them into powder, and sift them to obtain mulberry leaf powder;

[0010] Step 2: After extracting mulberry leaf albumin, dissolve the mulberry leaf albumin in water to prepare a solution with a mass concentration of 0.5% to 5%. Denature the solution at 95 to 100℃ for 5 ± 0.5 min. After cooling, add protease for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme at 95 to 100℃ for 10 ± 1 min to obtain the enzymatic hydrolysate.

[0011] Step 3: After cooling the enzymatic hydrolysate, centrifuge it, take the supernatant and freeze-dry it to obtain crude mulberry leaf antihypertensive active peptide.

[0012] Furthermore, the method for extracting mulberry leaf albumin in step 2 is either water extraction with acid precipitation or water extraction with salt precipitation.

[0013] Furthermore, the amount of water used in step 2 is calculated based on a mulberry leaf albumin mass concentration of 1%.

[0014] Furthermore, the denaturation conditions described in step 2 are: denaturation at 95°C for 5 minutes.

[0015] Furthermore, the enzyme inactivation conditions described in step 2 are: enzyme inactivation at 95°C for 10 minutes.

[0016] Furthermore, the centrifugation conditions described in step 3 are: 5000–10000g, 15–20min.

[0017] Furthermore, the protease is a neutral protease or an alkaline protease.

[0018] Furthermore, the enzymatic hydrolysis conditions are as follows: neutral protease 30–60°C, pH 6–8; or alkaline protease 30–50°C, pH 8–12; and hydrolysis time 0.5–8 h, E / S 2000–10000 U / g.

[0019] Furthermore, the enzymatic hydrolysis conditions are as follows: alkaline protease, 30–50°C, pH 8–12, hydrolysis time 0.5–6 h, enzyme-to-substrate ratio 1%–3.5%, and substrate concentration 0.5%–3%.

[0020] Furthermore, the enzymatic hydrolysis conditions are as follows: alkaline protease, 35–45°C, pH 8–11, hydrolysis time 1–4 h, enzyme-to-substrate ratio 2%–3.5%, and substrate concentration 0.5%–2%.

[0021] Furthermore, the enzymatic hydrolysis conditions are as follows: alkaline protease, 40±2℃, pH 10±0.2, hydrolysis time 2±0.2h, enzyme-to-substrate ratio 2.5±0.2%, and substrate concentration 1±0.1%.

[0022] A crude mulberry leaf antihypertensive active peptidase was obtained by the above preparation method.

[0023] A method for preparing mulberry leaf antihypertensive active peptides includes all the steps of the above-mentioned mulberry leaf antihypertensive active peptide enzymatic hydrolysate and the following steps:

[0024] Step 4: Dissolve the crude mulberry leaf antihypertensive active peptide in water and filter it through a 0.45μm filter membrane. Then, perform ultrafiltration on the filtrate to obtain the component with Mw < 1kDa.

[0025] Step 5: The fraction with Mw < 1 kDa was separated and purified by Sephadex G-15 gel column chromatography. The separated fraction was collected and further identified and analyzed by UPLC-MS / MS to obtain the mulberry leaf antihypertensive active peptide.

[0026] Furthermore, in step 5, the concentration of the Sephadex G-15 gel column chromatography solution is 5–30 mg / mL, the sample volume is 1–5 mL, pure water is used for elution, the elution flow rate is 0.3–0.8 mL / min, and the elution time is 3–6 min / tube.

[0027] Furthermore, in step 5, the concentration of the Sephadex G-15 gel column chromatography solution is 6.8 mg / mL, the sample volume is 2 mL, pure water is used for elution, the elution flow rate is 0.5 mL / min, and the elution time is 3.5 min / tube.

[0028] A mulberry leaf antihypertensive active peptide, the amino acid sequence of which is shown in any of the following sequences: DGFALDLGRN, RLPDFHGL, APGGFRE, VPSCFDLTGK, APGFEGR, FNPGYYDGR, FDRFGP, EFFELFKF, RFDFDPL, YGDFDFGGH, FRGGLR, FDRGSF, HPSPPGSAHRF, FDPNLGGK, PPVHGRL, FDPVGLLPK, SFWDGK, EPFPLLPK, TGGWDFR, PDSFRPK, EPPFLDPK, KPGMFGR, GPGSNPGLK, DFPVGLLPK, TGGGPGFR.

[0029] Furthermore, its amino acid sequence is shown in any of the following sequences: DGFALDLGRN, RLPDFHGL, APGGFRE, VPSCFDLTGK, APGFEGR, FPNPGYYDGR.

[0030] Furthermore, its amino acid sequence is shown in either of the following sequences: RLPDFHGL, VPSCFDLTGK.

[0031] The above-mentioned crude mulberry leaf antihypertensive active peptides or mulberry leaf antihypertensive active peptides are used in the preparation of ACE inhibitors, health products that help maintain healthy blood pressure levels, and / or antihypertensive drugs.

[0032] Furthermore, in the aforementioned applications, the ACE inhibitors, health supplements and / or antihypertensive drugs that help maintain healthy blood pressure levels use the aforementioned mulberry leaf antihypertensive active peptides as active ingredients, and may be supplemented with food- or pharmaceutically acceptable carriers or excipients.

[0033] An ACE inhibitor comprising the aforementioned mulberry leaf antihypertensive active peptide as an active ingredient.

[0034] Furthermore, the ACE inhibitor also contains a pharmaceutically acceptable carrier or excipient.

[0035] A health supplement that helps maintain healthy blood pressure levels contains the aforementioned mulberry leaf blood pressure-lowering active peptide as an active ingredient.

[0036] Furthermore, the health products also contain food-grade carriers or excipients.

[0037] A blood pressure-lowering drug comprising the aforementioned mulberry leaf blood pressure-lowering active peptide as an active ingredient.

[0038] Furthermore, the aforementioned antihypertensive drug also contains pharmaceutically acceptable carriers or excipients.

[0039] This invention first determines the optimal enzymatic hydrolysis conditions through single-factor and orthogonal experiments, then performs enzymatic hydrolysis under these optimal conditions. The resulting peptides are then separated and purified using 10kDa, 3kDa, and 1kDa ultrafiltration membranes and Sephadex G-15 gel columns, respectively. This optimized hydrolysis process is mild and simple, effectively preserving the ACE-inhibiting activity of mulberry leaf peptides and providing optimal enzymatic hydrolysis conditions for preparing mulberry leaf ACE-inhibiting peptides. Furthermore, the preparation of mulberry leaf ACE-inhibiting peptides enhances the medicinal and edible value of mulberry leaves and expands their application range.

[0040] The mulberry leaf ACE inhibitory peptide prepared by this invention has extremely strong solubility and good ACE inhibitory activity. It can be used as a natural antihypertensive agent or health product and is well applied in the food and pharmaceutical industries.

[0041] Specifically, this invention uses mulberry leaves as raw material, preparing mulberry leaf albumin through water extraction and acid precipitation or water extraction and salt precipitation. The parameters of enzymatic hydrolysis time, pH, enzyme-to-substrate ratio, and substrate concentration were optimized through single-factor and orthogonal experiments. Then, ultrafiltration (1kDa-10kDa) and Sephadex G-15 gel column chromatography were used for separation and purification. Twenty-five ACE-inhibiting peptides with potential antihypertensive activity were screened using peptidomics and molecular docking technology. After solid-phase synthesis and ACE inhibitory activity determination, six peptides were finally obtained: DGFALDLGRN, RLPDFHGL, APGGFRE, VPSCFDLTGK, APGFEGR, and FPNPGYYDGR. At a concentration of 2 mg / mL, the ACE inhibition rates of these six peptides were 76.51%, 93.59%, 77.86%, 94.17%, 84.02%, and 89.86%, respectively. The two peptides with the highest activity, VPSCFDLTGK and RLPDFHGL, had IC50 values ​​of 76.51%, 93.59%, 77.86%, 94.17%, 84.02%, and 89.86%. 50 The values ​​were 8.23 ​​μmol / L (i.e., 8.765 μg / mL) and 23.01 μmol / L (i.e., 23.58 μg / mL), respectively. These bioactive peptides can act as angiotensin-converting enzyme (ACE) inhibitory peptides, thereby lowering blood pressure by inhibiting ACE activity.

[0042] The present invention has the following advantages and effects compared with the prior art:

[0043] 1) This invention optimizes the enzymatic hydrolysis conditions of mulberry leaves and provides the optimal enzymatic hydrolysis conditions for preparing crude ACE inhibitory peptides.

[0044] 2) This invention purified and prepared ACE inhibitory peptides from mulberry leaves, which greatly enriched the research scope of effective components of mulberry leaves and enhanced their application value.

[0045] 3) The ACE inhibitory peptide prepared by this invention has extremely strong solubility and good ACE inhibitory activity.

[0046] 4) This invention identified six highly active ACE inhibitory peptides through polypeptide sequence determination and virtual screening.

[0047] 5) This invention provides a simple and feasible method for preparing mulberry leaf ACE inhibitory peptides.

[0048] 6) The preparation process of this invention does not involve any organic solvents, the operating conditions are mild, the ACE inhibitory activity of mulberry leaf peptides is preserved to the greatest extent, and the safety of the product is guaranteed. Attached Figure Description

[0049] Figure 1The figure shows the results of the study on the effects of hydrolysis time (A), pH of the hydrolysis system (B), enzyme-to-substrate ratio (C), and substrate concentration (D) on the ACE inhibitory activity of the hydrolysis products; different letters represent significant differences in the ACE inhibition rate of the hydrolysis products under different hydrolysis conditions, p < 0.05;

[0050] Figure 2 Figure 1 shows the results of the study on the ACE activity inhibition rate of crude mulberry leaf protein HP and its different ultrafiltration fractions;

[0051] Figure 3 The chromatogram of Sephadex G-15 for HP4;

[0052] Figure 4 Figure showing the results of the ACE activity inhibition rate study of S1 and S2 components obtained by dextran gel separation and purification;

[0053] Figure 5 The figure shows the results of the ACE activity inhibition rate study of the positive control Captopril and six peptides at a concentration of 2 mg / mL;

[0054] Figure 6 IC50 of peptides VPSCFDLTGK(A) and RLPDFHGL(B) 50 Value measurement results graph. Detailed Implementation

[0055] To better understand the present invention, the following description is based on embodiments. However, it should be noted that the embodiments do not constitute a limitation on the scope of protection of the present invention.

[0056] Example 1

[0057] An enzymatic preparation and process optimization of an ACE-inhibiting peptide from mulberry leaves (alkaline protease hydrolysis) includes the following steps:

[0058] (1) Enzymatic preparation and process optimization of crude mulberry leaf ACE inhibitory active peptide: Mulberry leaf albumin was dissolved in water to prepare a 1% (w / v) solution. The protein was denatured at 95℃ for 5 min. After cooling, the pH was adjusted, and alkaline protease (Yuanye Biotechnology) was added. The solution was enzymatically hydrolyzed at 40℃ for a certain time. After enzymatic hydrolysis, the enzyme activity was inactivated at 95℃ for 10 min. After cooling to room temperature, the solution was centrifuged at 8000g for 15 min. The supernatant was dialyzed and desalted using a 100Da dialysis bag, and then freeze-dried to obtain crude mulberry leaf ACE inhibitory active peptide.

[0059] In the enzymatic preparation, parameters such as enzymatic hydrolysis time, pH, enzyme-to-substrate ratio, and substrate concentration were optimized through single-factor experiments and orthogonal experiments, as detailed below:

[0060] 1) Single-factor experiment

[0061] Effect of enzymatic hydrolysis time on the ACE inhibition rate of the enzymatic hydrolysate: Under the conditions of substrate concentration of 1% (w / v), enzyme-to-substrate ratio of 2.5% (w / w), and pH 10, the ACE inhibition rates at enzymatic hydrolysis times of 1 h, 2 h, 3 h, and 4 h were measured (see results). Figure 1 In Figure A), the ACE inhibition rate of the enzymatic hydrolysis product under different enzymatic hydrolysis time conditions showed a trend of first increasing and then decreasing with the extension of enzymatic hydrolysis time. The ACE inhibition rate reached a maximum of 52.93% at 2h of enzymatic hydrolysis. 2h of enzymatic hydrolysis was selected as the optimal enzymatic hydrolysis time for subsequent orthogonal experiments.

[0062] Effect of reaction system pH on ACE inhibition rate of enzymatic hydrolysis products: Under the conditions of substrate concentration of 1% (w / v), enzyme-to-substrate ratio of 2.5% (w / w), and enzymatic hydrolysis time of 2 h, the ACE inhibition rates at pH 8, 9, 10, 11, and 12 were as follows (see results). Figure 1 In Figure B), the ACE inhibition rate of the enzymatic hydrolysis products under different enzymatic hydrolysis pH conditions showed a trend of first increasing and then decreasing with the increase of enzymatic hydrolysis pH. The ACE inhibition rate reached a maximum of 56.14% at the enzymatic hydrolysis pH of 10. The enzymatic hydrolysis pH of 10 was selected as the optimal enzymatic hydrolysis pH for subsequent orthogonal experiments.

[0063] Effect of enzyme-to-substrate ratio on ACE inhibition rate of enzymatic hydrolysis products: Under the conditions of substrate concentration of 1% (w / v), hydrolysis time of 2 h, and pH 10, the ACE inhibition rates at enzyme-to-substrate ratios (ratio of enzyme mass to substrate mass, w / w) of 2%, 2.5%, 3%, and 3.5% are shown in the results. Figure 1 (C). Under different enzyme-to-base ratios, the ACE inhibition rate of the enzymatic hydrolysis products first increased and then decreased with the increase of the enzyme-to-base ratio, reaching a maximum of 55.34% at an enzyme-to-base ratio of 2.5%. Therefore, an enzyme-to-base ratio of 2.5% was selected as the optimal enzymatic hydrolysis time for subsequent orthogonal experiments.

[0064] Effect of substrate concentration on ACE inhibition rate of enzymatic hydrolysate: Under the conditions of an enzyme-to-substrate ratio of 2.5% (w / w), a hydrolysis time of 2 h, and pH 10, the ACE inhibition rates at substrate concentrations (w / v) of 0.5%, 1%, 1.5%, and 2% were as follows (see results). Figure 1 (D). Under different substrate concentrations, the ACE inhibition rate of the enzymatic hydrolysis products first increased and then decreased with increasing substrate concentration. The ACE inhibition rate reached a maximum of 53.13% at a substrate concentration of 1%. Therefore, a substrate concentration of 1% was selected as the optimal enzymatic hydrolysis time for subsequent orthogonal experiments.

[0065] 2) Orthogonal experiment

[0066] Based on the single-factor experiment, L9(3) was adopted. 4 Orthogonal experiments were used to optimize the production process of mulberry leaf ACE inhibitory peptides prepared by alkaline proteolysis. The arrangement of factors and levels is shown in Table 1.

[0067] Based on the results of the single-factor experiments, a four-factor, three-level orthogonal experiment was designed. Independent variables A, B, C, and D represented the four factors: enzymatic hydrolysis time (h), pH value, enzyme-to-substrate ratio (%), and substrate concentration, respectively. The ACE inhibition rate was used as the evaluation index, and the ACE inhibition rates of each treatment group were obtained (see Table 2). The intuitive analysis results in Table 3 show that, by comparing the range R values ​​of the four factors, enzymatic hydrolysis time is the most significant factor affecting ACE inhibition activity. The order of importance of the factors affecting the ACE inhibition rate from largest to smallest is: time (A) > pH (B) > enzyme-to-substrate ratio (C) > substrate concentration (D). From the analysis of variance results (Table 4), factors A (enzymatic hydrolysis time) and B (pH value) have a significant impact on the ACE inhibition rate of crude mulberry leaf peptide HP, while factors C (enzyme-to-substrate ratio) and D (substrate concentration) have no significant impact on the ACE inhibition rate. Based on the above results, A1 (enzyme hydrolysis time of 2 h) and B3 (pH 10) were determined to be the optimal levels. The enzyme-to-substrate ratio and substrate concentration had relatively little effect on the ACE inhibition rate of crude mulberry leaf peptide HP, so C2 (enzyme-to-substrate ratio of 2.5%) and D1 (substrate concentration of 1%) were selected. Therefore, the optimal scheme is A1B3C2D1, i.e., enzyme hydrolysis time 2 h, hydrolysis pH 10, enzyme-to-substrate ratio 2.5%, and substrate concentration 1%. Verification experiments on the optimized enzyme hydrolysis conditions showed that the ACE inhibition activity of the mulberry leaf ACE-inhibiting peptide prepared under the optimal enzyme hydrolysis combination was 56.53%.

[0068] Table 1L9(3) 4 Enzymatic hydrolysis orthogonal experiment factor level table

[0069]

[0070] Table 2. Orthogonal experimental design and results

[0071]

[0072]

[0073] Table 3. Intuitive Analysis of Orthogonal Models

[0074]

[0075] Table 4. Analysis of Variance for Orthogonal Models

[0076]

[0077] (2) Isolation and purification of mulberry leaf ACE inhibitory active peptides: The crude mulberry leaf ACE inhibitory peptides obtained by enzymatic hydrolysis under the optimal enzymatic hydrolysis conditions in (1) were dissolved in deionized water to prepare a solution with a concentration of 1 mg / mL, and filtered through a 0.45 μm filter membrane; 10 mL of the filtrate was passed through 10 kDa, 3 kDa, and 1 kDa ultrafiltration membranes respectively, and 4 fractions were collected, namely HP1 (Mw > 10 kDa), HP2 (3 kDa < Mw < 10 kDa), HP3 (1 kDa < Mw < 3 kDa), and HP4 (Mw < 1 kDa). After freeze-drying, they were dissolved in water and subjected to ACE inhibitory activity determination (the results are as shown in Figure 2 ). It can be seen that ultrafiltration has a significant effect on improving the ACE inhibitory rate of the crude mulberry leaf peptides HP. Among them, the ACE inhibitory rate of HP4 is the highest, reaching 74.19%, which is 20.07% higher than that of HP.

[0078] The fraction HP4 with the highest activity was separated and purified by Sephadex G-15 gel column chromatography. The sample loading concentration was 6.8 mg / mL, the sample loading volume was 2 mL, and it was eluted with pure water at an elution flow rate of 0.5 mL / min and 3.5 min / tube. The separated fractions S1 and S2 ( Figure 3 ) were collected for freeze-drying and the ACE inhibitory activity was determined (the results are as shown in Figure 4 ). The fraction with the highest activity is S2, which is 78.22%, and it is the mulberry leaf ACE inhibitory peptide.

[0079] (3) Determination of polypeptide sequence: The polypeptide fraction S2 obtained in (2) was subjected to sequence identification by UPLC-MS / MS. The polypeptide sample was dissolved in 5% acetonitrile (containing 0.1% formic acid), filtered through a 0.22 μm filter membrane, and then detected by UPLC-MS / MS. The data collected was processed by MM File Conversion software to convert it into an MGF format file, and was matched with the NCBI-Morus alba-Taxid3498 database using Mascot Distiller v2.4.2.0 software (Matrix Science, Inc., Boston, MA) (http: / / www.matrixscience.com) to obtain the peptide sequence. The analytical instrument is a liquid chromatography-mass spectrometry system composed of an Easy-nLC 1200 ultra-high performance liquid tandem Orbitrap Eclipse DDA high-resolution mass spectrometer. Chromatographic column: Acclaim PepMap RSLC C18 chromatographic column ( 2 μmol / L, 0.075 mm × 150 mm, nanoViper); Mobile phase A: Milli-Q water (0.1% FA); Mobile phase B: 80% acetonitrile (0.1% FA); Flow rate: 300 nL·min⁻¹; Elution program: 0–5 min 5% B, 5–50 min 5%–38% B, 50–52 min 38%–95% B, 52–60 min 30%–35% B, 45–50 min 35%–90% B, 50–55 min 90%–90% B, 55–56 min 90%–5% B, 56–65 min 5%–5% B. Peptides separated by UPLC were directly analyzed online in MS. Peptides unmodified by functional gene groups and with a peak area greater than 1 × 10⁻⁶ were selected from the sequenced peptides. 5 A total of 6,341 polypeptide sequences were identified.

[0080] Mass spectrometry conditions were as follows: Mass spectrometry system: Q Exactive (Thermo Scientific). Stage 1: Resolution, 70000; AGC target, 3e6; Maximum IT, 100ms; Scan range: 350-1800m / z. Stage 2: Resolution, 17500; AGC target, 5e4; Maximum IT, 120ms; TopN, 20; NCE / stepped NCE, 30.

[0081] (4) Using molecular docking technology, 25 peptides with potential antihypertensive activity were screened from the 6341 peptides identified in (3). The results of their bio-predicted activity, water solubility and binding affinity are shown in Table 5. The six peptides with the highest potential activity are DGFALDLGRN, RLPDFHGL, APGGFRE, VPSCFDLTGK, APGFEGR, and FPNPGYYDGR (Table 5).

[0082] Table 5. Biopredicted activity, water solubility, and binding affinity

[0083]

[0084]

[0085] (5) Peptide synthesis: The antihypertensive peptide with the highest potential activity screened in (4) was synthesized by FMOC solid phase synthesis method and desalted to achieve a purity of ≥98%. The synthesis was entrusted to Sangon Biotech (Shanghai) Co., Ltd.

[0086] (6) ACE inhibitory activity assay

[0087] 1) Preparation of reaction solution

[0088] A certain amount of sample was dissolved in sodium phosphate buffer, filtered, and the solution was prepared to the corresponding concentration. 15 μL of ACE solution was added to the sample tube and the blank tube, 30 μL of ACE inhibitory peptide sample solution was added to the sample tube, and 30 μL of buffer was added to the blank tube. After incubation at 37°C for 10 min, 50 μL of HCl solution was added, and the reaction was carried out at 37°C for 1 h. Finally, 150 μL of 1.0 mol / L HCl solution was added to stop the reaction, and the reaction solution was obtained as shown in Table 6 below.

[0089] Table 6 Preparation of reaction solution

[0090]

[0091] 2) Chromatographic conditions: Column: ECOSIL C18 (260 mm × 4.6 mm, 5 μm); Mobile phase A: H₂O (containing 0.05% TFA, v / v); Mobile phase B: Acetonitrile (containing 0.05% TFA, v / v). Chromatographic conditions: Acetonitrile:pure water = 25:75; Flow rate: 1 mL / min. -1 Detection wavelength: 228 nm; Column temperature: 30℃; Injection volume: 10 μL. Elution program: 0-10 min, 5% B-60% B; 10-12 min, 60% B; 12-13 min, 5% B.

[0092] 3) Result Calculation

[0093] HHL rapidly decomposes under the catalysis of ACE to produce hippuric acid (Hip) and a dipeptide (His-Leu, HL). Hippuric acid has a maximum absorption at 228 nm. When an ACE inhibitor sample is added, the activity of ACE enzyme is inhibited, and the amount of hippuric acid produced decreases. Therefore, the activity of the ACE inhibitor can be evaluated by measuring the amount of hippuric acid produced using high-performance liquid chromatography (HPLC). The inhibition rate of ACE activity is calculated using the formula: R = (A0 - B) / A0 × 100%, where: R: the inhibition rate of ACE by the ACE inhibitory peptide sample (%); B: the peak area of ​​hippuric acid in the group with added ACE inhibitory peptide; A0: the peak area of ​​hippuric acid in the blank tube. When the inhibition rate is 50%, the concentration of the inhibitory peptide is the half-maximal inhibitory concentration (IC50). 50 .

[0094] The results showed that at a concentration of 2 mg / mL, the inhibition rates of these six peptides against ACE were 76.51%, 93.59%, 77.86%, 94.17%, 84.02%, and 89.86%, respectively. Among them, the two peptides with the highest activity, VPSCFDLTGK and RLPDFHGL, had IC50 values ​​of 76.51%, 93.59%, 77.86%, 94.17%, 84.02%, and 89.86%. 50 The values ​​were 8.23 ​​μmol / L (i.e., 8.765 μg / mL) and 23.01 μmol / L (i.e., 23.58 μg / mL), respectively. Figure 6 ).

[0095] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A mulberry leaf antihypertensive active peptide, characterized in that: Its amino acid sequence is shown in either of the following sequences: RLPDFHGL, VPSCFDLTGK.

2. The use of the mulberry leaf antihypertensive active peptide described in claim 1 in the preparation of health products that help maintain healthy blood pressure levels or in the preparation of antihypertensive drugs.

3. A health supplement that helps maintain healthy blood pressure levels, characterized in that: It contains the mulberry leaf antihypertensive active peptide as described in claim 1 as an active ingredient.

4. A blood pressure-lowering drug, characterized in that: It contains the mulberry leaf antihypertensive active peptide as described in claim 1 as an active ingredient.

Images